Posts Tagged ‘Crohn’s disease’

Diarrheas – Bacterial and Nonbacterial

Writer and Curator: Larry H. Bernstein, MD, FCAP 



Diarrheas are one of the common problems of societies worldwide. However, the prevalence of cause of the diarrhea may be different depending on location, water quality, food source, age, and psychological stress factors.

Microbial and Parasitic Diseases

A Systematic Review on Neglected Important Protozoan Zoonoses

Yibeltal Muhie Mekonen and Simenew Keskes Melaku
Int. J. Adv. Res. Biol.Sci. 2(1): (2015): 53–65

Infectious protozoan parasites are transmitted to humans through several routes, including contaminated food and water, inadequately treated sewage/sewage products, and livestock and domestic pet handling. Several enteric protozoa cause severe morbidity and mortality in both humans and animals worldwide. In developed settings, enteric protozoa are often ignored as a cause of diarrheal illness due to better hygiene conditions, and as such, very little effort is used toward laboratory diagnosis. Although these protozoa contribute to the high burden of infectious diseases, estimates of their true prevalence are sometimes affected by the lack of sensitive diagnostic techniques to detect them in clinical and environmental specimens. Despite recent advances in the epidemiology, molecular biology, and treatment of protozoan illnesses, gaps in knowledge still exist, requiring further research. There is evidence that climate-related changes will contribute to their burden due to displacement of ecosystems and human and animal populations, increases in atmospheric temperature, flooding and other environmental conditions suitable for transmission, and the need for the reuse of alternative water sources to meet growing population needs. This review discusses the common enteric protozoa from a public health perspective, highlighting their epidemiology, modes of transmission, prevention and control and epidemiological pictures in Ethiopia. It also discusses the potential impact of climate changes on their epidemiology and the issues surrounding waterborne transmission and suggests a multidisciplinary approach to their prevention and control.

Approximately 60 percent of all human pathogens are zoonoses of microbes that are naturally transmitted between animals and humans. Neglect of their control persists because of a lack of information and awareness about their distribution, a lack of suitable tools and managerial capacity for their diagnosis, and a lack of appropriate and sustainable strategies for their prevention and control. Furthermore, many of the most affected countries have poor or non-existent veterinary public health infrastructures. This situation has marginalized control of zoonoses to the gap between veterinary responsibilities and medical needs, generating a false perception that their burden and impact on society are low. As a result, neither the human and animal health resources nor the research needed for their control are available spawning a category of non zoonotic diseases (Choffnes and Relman, 2011).

The neglected tropical diseases (NTDs) are the most common conditions affecting the poorest 500 million people living in sub-Saharan Africa (SSA), and together produce a burden of disease that may be equivalent to up to one-half of SSA’s malaria disease burden and more than double that caused by tuberculosis (Hotez and Kamath, 2009).Starting with an initial set of 13-15 diseases, there are now over 40 helminth, protozoal, bacterial, viral, fungal and ectoparasitic infections covered under the brand-name of the neglected tropical diseases. Gaps in our understanding of the epidemiology and control of many of the neglected tropical diseases remain, which calls for additional funding for innovative research (Jürg et al, 2012).

The health and socioeconomic impacts of zoonotic parasitic and related food-borne diseases are growing continuously and increasingly being felt most particularly by developing countries. Apart from causing human morbidity and mortality, they hamper agricultural production, decrease availability of food, and create barriers to international trade (Solaymani-Mohammadi and Petri, 2006). The problem of zoonoses has spread from predominantly restricted rural areas into regional and, in some cases, worldwide epidemics. This is due to the great changes of the previous decades, especially the increasing urbanization, most of which is inadequate planned. In addition, large movements of populations, opening up of badly needed new areas for food production, the increasing trade in meat, milk and other products of animal origin, the increasing number and speed of vehicles, and even tourism have contributed to expanding the impact of zoonotic diseases. The challenges of food-borne, waterborne, and zoonotic protozoan diseases associated with climate change are expected to increase, with a need for active surveillance systems, some of which have already been initiated by several developed countries. However, very little effects are attempting in the developing world which actually are the main victims.

The prevalence rates are generally higher in immunodeficient compared to immune-competent patients. However, most studies on prevalence have been carried out in developed countries where the laboratory and clinical infrastructure are more easily available. Protozoan pathogens and HIV interact in their host, modifying the immunopathology of disease and complicating therapeutic intervention. Disease prevalence and distribution and population movements impact greatly on HIV/protozoan parasite co-infections (Andeani et al, 2012).

In Ethiopia there are little reports regarding protozoan zoonoses. However, there are still reports from clinics and hospitals where these diseases are becoming major issues of concern. This review will examine published data on the neglected protozoan pathogens in Ethiopia and analyses their current importance to public health.

Important but Neglected Protozoan Zoonoses Dealt in this Critical Review

  • Amebiasis

This disease is caused by a single cell protozoan parasite called Entamoeba  spp. (E. histolytica, E. polecki). Invasive amebiasis is one of the world most prevalent and fatal infectious diseases. Around 500 million people are infected worldwide while 75,000 die of the disease annually. Behind malaria and schistosomiasis, amebiasis ranks third on the list of parasitic causes of death worldwide. The infection is common in developing countries and predominantly affects individuals with poor socioeconomic conditions, non hygienic practices, and malnutrition (Stanley, 2003).

A number of survey and routine diagnosis in Ethiopia indicate that amebiasis is one of the most widely distributed diseases. In a countrywide survey of amebiasis in 97 communities, the overall prevalence of Entamoeba histolytica infections, as measured by rate of cyst-passers, in schoolchildren and non-school communities were 15.0% and 3.5%, respectively (Erko et al, 1995). A study conducted on the prevalence of Entamoeba histolytica/dispar among children in Legedini, Adada and Legebira, Dire-Dawa administrative region was 33.7% (Dawit, 2006 Unpublished MSc Thesis).

  • Giardiasis

Giardiasis is caused by Giardia lamblia (also known as Giardia duodenalis or G. intestinalis) is a unicellular, flagellated intestinal protozoan parasite of humans isolated worldwide and is ranked among the top 10 parasites of man (Farthing and Kelly, 2005). Its occurrence is worldwide (Figure 1) and prevalence very high in areas with poor sanitation and in institutions. Human infections usually originate from other humans but may result from contact with dogs, cats, rodents, beavers, or nonhuman primates. The prevalence of the disease varies from 2% to 5% in developed to 20% to 30% in developing countries. The variation in prevalence might be attributed to factors such as the geographical area, the urban or rural setting of the society, the age group composition and the socio-economical conditions of the study subject.

Risk of disease caused by Giarda species

Risk of disease caused by Giarda species

Risk of disease caused by Giarda species with different degrees Source: Esch and Petersen (2013)

According to Birrie and Erko (1995) based on a countrywide survey of giardiasis, the overall prevalence among school children and residents were 8.9% and 3.1%, respectively and that of the non-school children were 4.4%. Recent report indicates that the prevalence of Giardia lamblia among diarrhea patients referred to EHNRI (Ethiopian Health and Nutrition Research Institute) was 8.6%. In a study conducted in South Western Ethiopia, the prevalence of Giardiasis was 13.7%. A study conducted for the determination of Prevalence of Giardiasis and Cryptosporidiosis among children in relation to water sources in selected Village of Pawi Special District in Benishangul-Gumuz Region, Northwestern Ethiopia showed that out of the 384 children examined, 102 for giardiasis.

  • Leishmaniasis

Leishmaniasis is an ancient disease caused by protozoans from the Leishmania genus and transmitted by the bite of a sand fly. It has four subtypes of varying severity, which include cutaneous and visceral infections. Cutaneous infection results in the formation of disfiguring lesions which frequently occur on the face, arms and legs. Lesions may last anywhere from a few weeks to over a year; secondary lesions may also occur years after the initial lesion has healed. Visceral cases can result in anemia, fever, debility and death if left untreated.

About 20 species of Leishmania infect mammals and many of them can cause human leishmaniasis. Motile infective forms of the parasite (metacyclic promastigotes with a long free flagellum) develop in the guts of competent sand fly vectors, which inoculate them into mammalian skin. Infections can spread, often via the lymphatic system, to cause secondary dermal lesions with forms and tissue tropisms in humans that show some parasite species specificity. Leishmaniasis can visceralize (for example Leishmania (Leishmania) tropica, which normally causes Oriental sore), but only two species of the subgenus Leishmania routinely do so, and these are the causative agents of most human visceral leishmaniasis (VL) worldwide.

Global burden of Leishmania

Global burden of Leishmania

Global burden of Leishmania as adapted from the “Leshimaniases and Leishmania HIV co-infection” WHO fact sheet No. 116, May 2000

The New World visceral leishmaniasis is in Latin America and southern United States. Of course the visceral form also is common in Asia, Africa, Europe and Latin America. Both VL and CL are important endemic vector‐borne diseases in Ethiopia. The Federal Ministry of Health (FMoH) estimates the annual burden of VL to be between 4,500 and 5,000 cases (FMoH Ethiopia, 2006 unpublished). Known VL endemic foci are in the arid southwest, and the Humera and Metema lowlands in the north‐west. About 2-12% of all visceral leishmaniasis cases involve HIV coinfections underlines the synergic aspect of both diseases; such proportions may reach 40%, as in Humera, northwest Ethiopia (WHO, 2007), where coinfections have increased two-fold in the last decade (Andreani et al, 2012).

  • Cryptosporidiosis

The causes of this disease are Cryptosporidium spp. (C. parvum, possibly others). In humans, abdominal pain, nausea, watery diarrhea lasting 3-4 days. In immune-deficient or immune-suppressed people, the disease is severe, with persistent diarrhea (6-25 evacuations per day) and malabsorption of nutrients. In normal persons the disease is self-limiting. In immune-compromised individuals, disease is severe and case fatality rate may be high. In animals normally a clinical disease can be seen only among young neonates. In ruminants, gastroenteritis and diarrhea are common.

  • Toxoplasmosis

Toxoplasmosis is among the global major zoonotic diseases and the third leading cause of food-related deaths in the USA. It is caused by Toxoplasma gondii, an Apicomplexa protozoan parasite, with cats as the definitive host. Cats are considered the key in the transmission of Toxoplasma gondii to humans because they are the only hosts that can excrete the environmentally resistant oocysts in their feces.

Human seroprevalece of Toxoplasma gondii

Human seroprevalece of Toxoplasma gondii

Human seroprevalece of Toxoplasma gondii.  Esch and Petersen (2013)

The clinical impact of zoonotic enteric protozoan infections is greatest in the developing world where inadequate sanitation, poor hygiene and proximity to zoonotic reservoirs, particularly companion animals and livestock are greatest. In such circumstances, it is not surprising that infections with more than one species of enteric protozoan are common, and in fact single infections are rare.

Impact of animal disease on human health

Impact of animal disease on human health

Impact of animal disease on human health

The protozoan zoonoses circulating in Ethiopia are major burden on public health and wellbeing. The magnitude and scope of this burden varies for each of the protozoan parasites discussed in this manuscript. Apart from causing human morbidity and mortality, they hamper agricultural production, decrease availability of food, and create barriers to international trade. It is generally believed that although these parasitic infections are distributed worldwide, their prevalence is higher in developing compared to developed countries. However, the relative importance of zoonotic infections especially in developing countries has not been studied in detail including. These protozoan zoonoses are the most neglected but very important in terms of human health and veterinary concerns. The main share belongs to cryptosporidiosis; giardiasis, toxoplasmosis, leishmaniasis and amebiasis are some of the major protozoan zoonoses.

Clinical Microbiology: Past, Present, and Future

Henry D. Isenberg
J Clin Microbiol, Mar. 2003; 41(3):917–918–918.2003

During the last two decades of the 19th century, a plethora of bacteria were isolated and designated etiological agents of human infectious diseases. As with many instances at the interface between cause and effective therapy, the further characterization of these alleged pathogens remained in the hands of a few devoted investigators until drugs with therapeutic potential became available. This vague period before the advent of proper cures for infections explains the shadowy origin of clinical or diagnostic microbiology. But, as R. Porter has stated, “history should be rooted in detail and as messy as life itself”; this is an undeniable description of the history of clinical microbiology, long the stepchild, frequently denied legitimacy, among the many siblings that constitute the science of microbiology. Yet the practice of clinical microbiology is the application of knowledge gained to the betterment of the human condition, the goal of clinical microbiologists.

The advances in the grouping and typing of streptococci, salmonellae, and shigellae, the separation of Staphylococcus aureus on the basis of the coagulase reaction, and the growing awareness of the need for safe water and uncontaminated food items established the need for laboratories to assume these responsibilities. It was only logical that microbiology should join endeavors such as chemistry, hematology, and serology under the rubric of clinical pathology. Differential media especially designed to sequester species increased dramatically during Word War II; military hospitals developed clinical microbiology sections devoted not only to recognizing agents endangering the health of troops in camps, in battle, and in foreign environments but also to assessing the responses of certain of the microorganisms isolated to several sulfonamides and that hitherto unknown agent, penicillin. The subsequent explosion of antimicrobial agents—streptomycin, chloramphenicol, tetracyclines, and erythromycin—suggested to the reigning powers of medical facilities that clinical microbiologists could be phased out, since infectious disease would disappear before the onslaught of agents discovered through human ingenuity.

In the interim, cotton plugs gave way to Bakelite, polypropylene, glass, metal, and plastic closures; in-house medium preparation was relieved in part by the beginnings of commercially manufactured ready-to-use media especially for mycobacteria and antimicrobial susceptibility testing. Alcohol, Bunsen, and Tyril burners were replaced by microincinerators, eventually followed by disposable loops and transfer needles. The prescient wisdom of hospital boards soon was shattered by the genetic versatility of the microbial world, dramatically demonstrated by the pandemic of S. aureus 80/81 in the late 1950s and early 1960s and the emergence of gram-negative rods that demonstrated the superiority of the bacterial physiology over the commercially prepared secondary microbial metabolites that initially appeared so promising. To be sure, the tug of war between antimicrobial agents—natural and synthetic—and the microorganisms continues unabated, with signs that the evolutionary potential of the microbial world will succeed in the long run.

Since the 1960s, numerous ingenious innovations have been introduced. Molecular biology techniques promise to revolutionize the diagnosis of infectious disease—to date a promise still in its infancy.  Systems approaches began to replace the single test tube with but one substrate. Perhaps the first was double sugar iron agar for the recognition of so-called enteric pathogens, followed by triple sugar iron agar and the next tentative shortcut, the r/b tube. Rollender and Beckford, the inventors of the r/b tube, must be credited with initiating manufacturers’ efforts to teach laboratory staffs the vagaries and problems of new system approaches. Shortly thereafter, the API system was introduced in the United States, bringing a novel numerical approach first to the identification of Enterobacteriaceae (enteric – gut bacteria) and then to that of several other categories of microorganisms. Similarly, the Roche Enterotube used fewer reaction substrates to decrease the time needed to identify isolates to the species level; initially it was used for members of the Enterobacteriaceae and eventually for other microbial representatives. All systems eventually addressed yeasts and nutritionally demanding bacteria, obviating the multiple-tube approaches in use.

Clinical microbiologists are acutely aware of the constantly emerging intruders into the intimate human biosphere. These agents appear as the traditional scourges of humanity are brought under control. But the application of antimicrobial agents to the food chain, cosmetics, and over-the-counter medications, and the advances in medical science, sparing individuals afflicted with a variety of diseases but accompanied by impaired immunity—all these factors have combined to increase nosocomial infections, placing the medical facility at the very apex of the selective-pressure pyramid. The selection results in colonization by microbiota with a minority of antimicrobial-tolerant or -resistant constituents; administration of antimicrobial therapy converts these organisms to a majority. These selected prokaryotes and eukaryotes,
along with the emerging viruses, coccidia, yeasts, and molds, pose a dynamic challenge to the clinical microbiologist and promise a continued need for her or his services. But these challenges must be met by the expansion of technical skills brought to bear on the changing nature of the challenging microbiota and the willingness of clinical microbiologists
to adopt and practice evolving technologies, to gain knowledge in addition to information, and to remain in the forefront of innovation and invention.

Gut microbiota: next frontier in understanding human health and development of biotherapeutics

Satya Prakash, L Rodes, M Coussa-Charley, C Tomaro-Duchesneau
Biologics: Targets and Therapy 2011:5 71–86

The human gastrointestinal tract houses a huge microbial ecosystem, the gut microbiota. This intestinal ecosystem is partially responsible for maintaining human health. However, particular changes in the ecosystem might contribute to the development of certain diseases. With this in mind, there is a need for an exhaustive review on the functions of the gut microbiota, occurrence of gut dysbiosis (alteration of the microbiota), mechanisms by which intestinal bacteria can trigger development of disease, how this ecosystem can be exploited for understanding human health, development of biotherapeutics, expert opinion on current biotherapeutics, and future perspectives. This review presents a descriptive and comprehensive analysis on “the good, the bad, and the ugly” of the gut microbiota, and methods to study these and their modulation of human health.

The gut microbiota is a remarkable asset for human health. As a key element in the development and prevention of specific diseases, its study has yielded a new field of promising biotherapeutics. This review provides comprehensive and updated knowledge of the human gut microbiota, its implications in health and disease, and the potentials and limitations of its modification by currently available biotherapeutics to treat, prevent and/ or restore human health, and future directions. Homeostasis of the gut microbiota maintains various functions which are vital to the maintenance of human health. Disruption of the intestinal ecosystem equilibrium (gut dysbiosis) is associated with a plethora of human diseases, including autoimmune and allergic diseases, colorectal cancer, metabolic diseases,

and bacterial infections. Relevant underlying mechanisms by which specific intestinal bacteria populations might trigger the development of disease in susceptible hosts are being explored across the globe. Beneficial modulation of the gut microbiota using biotherapeutics, such as prebiotics, probiotics, and antibiotics, may favor health-promoting populations of bacteria and can be exploited in development of biotherapeutics. Other technologies, such as development of human gut models, bacterial screening, and delivery formulations e.g., microencapsulated probiotics, may contribute significantly in the near future. Therefore, the human gut microbiota is a legitimate therapeutic target to treat and/or prevent various diseases. Development of a clear understanding of the technologies needed to exploit the gut microbiota is urgently required.

Seven bacterial divisions constitute the gut microbiota, i.e., Firmicutes, Bacteroides, Proteobacteria, Fusobacteria, Verrucomicrobia, Cyanobacteria, and Actinobacteria, with Firmicutes and Bacteroides being the most abundant species. Bacterial communities exhibit quantitative and qualitative variations along the length of the gastrointestinal tract due to host factors (e.g., pH, transit time, bile acids, digestive enzymes, and mucus), nonhost factors (eg, nutrients, medication, and environmental factors), and bacterial factors (e.g., adhesion capacity, enzymes, and metabolic capacity).

Until recently, the analysis of bacterial ecosystems was performed by growth on defined media, which has some limitations because this method is labor-intensive and, more importantly, only 80% of stool bacteria can be cultivated. As a consequence, new molecular techniques have been developed. In terms of qualitative measurements of the microbiota, techniques such as fingerprinting (denaturing gradient gel electrophoresis), terminal restriction fragment length polymorphism, ribosomal intergenic spacer analysis, and 16S ribosomal RNA sequencing are widely used. Specifically, genome sequencing has provided tremendous information in the microbial world, spearheading technologies such as microarrays. New automated parallel sequencing technologies, based on the 16S ribosomal RNA gene present in all prokaryotes, can offer a cost-effective solution for rapid sequencing and identification of bacterial species of the gut.

Essential metabolic functions

Metabolic functions of the gut microbiota include production of vitamin, amino acid synthesis, and bile acid biotransformation. Bile acid biotransformations, performed by microbial enzymes, have implications for cholesterol and glucose metabolism. Importantly, the microbiome provides biochemical pathways required for the fermentation of nondigestible substrates and endogenous mucus. Through fermentation, bacterial growth is stimulated, producing short-chain fatty acids and gases. The major short-chain fatty acids produced are acetate, butyrate, and propionate. Other bacterial end products include lactate, ethanol, succinate, formate, valerate, caproate, isobutyrate, 2-methyl-butyrate,
and isovalerate. Bacterial fermentation is present in the cecum and colon, where the short-chain fatty acids are absorbed, stimulating the absorption of salts and water.

Ensures protection

Pathogen displacement or “colonization resistance” is an accepted function of the gut microbiota. Commensal organisms prevent pathogenic colonization by competing for attachment sites and nutrients, and also through the production and secretion of antimicrobials. Those mechanisms are relevant for reducing the level of lipopolysaccharides, peptidoglycans, bacterial CpG-DNA motifs, and superantigens, which can all be detrimental to the host. The indigenous microbiota is also essential for development of the immune system. Short-chain fatty acids, such as butyrate, may exert potent immunomodulatory effects by suppressing nuclear factor-kB activation and/or by acting on G-coupled receptors, as demonstrated with acetate. These concepts illustrate a dynamic relationship between the immune system and the microbiota. The intestinal mucosa averts threats by signaling to the innate immune system through pattern recognition receptors, such as toll-like receptors. Pattern recognition receptors recognize and bind to specific microbial macromolecules, referred to as microbial-associated molecular patterns. These include lipopolysaccharide, flagellin, peptidoglycan, and N-formylated peptides.

Structural and histological function

The microbiota ensures intestinal structure and function. Firstly, the mucus layer, which reflects the balance between mucus secretion and bacterial degradation, constitutes an obstacle to the uptake of antigens and proinflammatory molecules. Secondly, some bacterial communities may strengthen the barrier at the level of the tight junctions, ie, protein clusters that form a barrier between the lumen and the lamina propria. Moreover, the gut microbiota is involved in cell and tissue development. Butyrate regulates cell growth and differentiation, inhibiting transformed cell growth while encouraging reversion of cells from a neoplastic to a non-neoplastic phenotype. Most of the structural and morphological development of the gut contributes to and manages the gut bacterial system.

Dysbiosis is a state in which the microbiota becomes altered as a consequence of an alteration in the composition of the microbiota, a change in bacterial metabolic activity, and/or a shift in local distribution of communities. Many factors can alter the gastrointestinal ecosystem, including antibiotics, psychological and physical stresses, radiation, altered peristalsis, and dietary changes. At present, the focus is on the description of dysbiosis in a plethora of human disorders.

  • Autoimmune disease

Autoimmune diseases occur when the body’s immune system attacks and destroys healthy cells and tissues, as is the case in type 1 diabetes mellitus, celiac disease, inflammatory bowel diseases, and allergic asthma. Most often, the immune response is initiated by unknown factors. Alteration of the gut microbiota as a result of modern lifestyles is an attractive hypothesis to explain the rise in prevalence of celiac disease, type 1 diabetes mellitus, and inflammatory bowel diseases.

Celiac disease is an inflammatory disease of the small intestine that is triggered and maintained by the storage proteins of wheat, barley, and rye. Studies have investigated the composition of the microbiota in patients with celiac disease. Fecal samples from patients with celiac disease had reduced the proportions of Bifidobacterium, Clostridium histolyticum, Clostridium lituseburense, Faecalibacterium prausnitzii, and increased proportions of Bacteroides/Prevotella.

Type 1 diabetes mellitus, characterized by insulin deficiency resulting from immune-mediated destruction of pancreatic β cells, is thought to be triggered by environmental factors in genetically susceptible individuals. Given that antibiotics prevented type 1 diabetes mellitus in biobreeding diabetes-prone rats and in nonobese diabetic mice, alteration of the microbiota has been associated with progression of type 1 diabetes mellitus. Evidence shows that bacterial communities from biobreeding diabetes-prone and diabetes-resistant rats differ, marked by a higher abundance of Lactobacillus and Bifidobacterium in diabetes-resistant rats.

Inflammatory bowel diseases include ulcerative colitis and Crohn’s disease. Crohn’s disease is characterized by patchy and transmural inflammation that may affect any part of the gastrointestinal tract, while ulcerative colitis is a chronic episodic inflammatory condition that involves only the large bowel. There is evidence that species belonging to the normal gut microbiota are involved in the etiology and/or maintenance of inflammatory processes. Reduced microbial diversity, increased Bacteroidetes and Enterobacteriaceae, and decreased Firmicutes were all observed in patients with inflammatory bowel diseases.

  • Irritable bowel syndrome

Irritable bowel syndrome is characterized by abdominal pain, bloating, and changes in bowel habit, in the absence of any overt mucosal abnormality. Observations have directed attention towards the gut microbiota, identifying a postinfectious variant of the syndrome, ie, evidence that antibiotics induced a reduction in the microbiota which may be a risk factor, and the proposal that some patients may have bacterial overgrowth in the small bowel.49 Studies have demonstrated that patients with irritable bowel syndrome have fewer intestinal Bifidobacteria, Collinsella aerofaciens, Coprococcus eutactus, and Clostridium cocleatum, and an increase in Veillonella and Enterobacteriaeae.

  • Bacterial infection

It is well established that a disruption in the commensal microbiota increases susceptibility to enteric infections. Antibiotic-treated mice are particularly useful for studying colitis induced by Salmonella spp, Shigella spp, and E. coli infections. In addition, in murine Citrobacter rodentium infections, pathogen colonization is associated with a reduced total density and a relative increase in γ-Proteobacteria. Furthermore, elderly patients with C. difficile-associated diarrhea demonstrate reduced numbers of Bacteroides, Prevotella, and Bifidobacteria, and a greater diversity of facultative species, ie, Lactobacilli and Clostridia. The evidence suggests an association between disruption of the gut microbiota and bacterial infections, further accentuating the dysbiosis.

Altered composition of the human gastrointestinal ecosystem can lead to physiological changes in the intestinal environment, disrupting the functions of the microbiota and having serious consequences for human health.

  1. Altered gut microbiota may trigger serious immune deregulation
  2. Specific gut dysbiosis can engender metabolic endotoxemia
  3. Bacterial infection might be promoted by gut dysbiosis
  4. Abnormal bacterial metabolite levels may trigger cancer

An altered microbial balance in the gut can lead to A) an increase in immune mediated disorders and B) chronic low-grade inflammation.

A mechanism based on the triggering of the host’s immune defenses was elucidated using models of C. rodentium (mimicking diarrheal pathogen associated inflammation), Campylobacter jejuni infection, and chemically and genetically induced models of intestinal inflammation are used for altered microbiota investigations. An overgrowth of Enterobacteriaceae was observed in all models, indicating that inflammation induced microbiota changes support colonization by aerotolerant bacteria.

Many etiological bacterial mechanisms have been hypothesized to promote carcinogenesis. Amongst those, hydrogen sulfide, a product of bacterial sulfate reduction, appears to be linked to the incidence of chronic disorders, such as ulcerative colitis and colorectal cancer. Because DNA strand breaks are associated with mutation and promotion of carcinogenesis, bacterial hydrogen sulfide may be responsible for the induction of mutations in the development of sporadic colorectal cancer.

  • Gut microbiota alters energy and lipid metabolism

Reared mice have more body and gonadal fat than germ-free mice, despite reduced chow consumption. The increase in fat was accompanied with increased fasting glucose and insulin levels and an insulin-resistant state.

– Prebiotics

Prebiotics are “nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon, and thus improves host health”. A prebiotic should not be hydrolyzed by human intestinal enzymes, but selectively fermented by bacteria, benefiting the host.

The relationship between health and the gastrointestinal system is established. Due to the inherent plasticity of microbiota, one can consider exploiting it to develop biotherapeutics.

Gut Microbiota Regulates Bile Acid Metabolism by Reducing the Levels of Tauro-beta-muricholic Acid, a Naturally Occurring FXR Antagonist

Sama I. Sayin, Annika Wahlstrom, Jenny Felin, Sirkku Jantti, et al.
Cell Metabolism  Feb 5, 2013; 17, 225–235

Bile acids are synthesized from cholesterol in the liver and further metabolized by the gut microbiota into secondary bile acids. Bile acid synthesis is under negative feedback control through activation of the nuclear receptor farnesoid X receptor (FXR) in the ileum and liver. Here we profiled the bile acid composition throughout the enterohepatic system in germfree (GF) and conventionally raised (CONV-R) mice.

We confirmed a dramatic reduction in muricholic acid, but not cholic acid, levels in CONV-R mice. Rederivation of Fxr-deficient mice as GF demonstrated that the gut microbiota regulated expression of fibroblast growth factor 15 in the ileum and cholesterol 7α-hydroxylase (CYP7A1) in the liver by FXR-dependent mechanisms. Importantly, we identified tauroconjugated β- and α-muricholic acids as FXR antagonists. These studies suggest that the gut microbiota not only regulates secondary bile acid metabolism but also inhibits bile acid synthesis in the liver by alleviating FXR inhibition in the ileum.


Diet rapidly and reproducibly alters the human gut microbiome.

Lawrence A David, Corinne F Maurice, Rachel N Carmody, et al.
Nature 12/2013;

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles

Honing in on enteric fever

Lyle R Mckinnon And Quarraisha Abdool Karim
eLife 2014;3:e03545.

Enteric fever, also known as typhoid, is a disease that affects about 22 million people and causes about 200,000 deaths every year, according to conservative estimates. Enteric fever is spread by bacteria belonging to the Salmonella genus, with two sub-species—Salmonella Typhi and Salmonella Paratyphi A—being responsible for most cases of the disease. And although the number of cases of enteric fever has fallen significantly over recent decades, there is a clear need for a diagnostic test for Salmonella that is rapid, affordable and accurate. Moreover, it is important to be able to distinguish between enteric fever caused by Salmonella Typhi and enteric fever caused by Salmonella Paratyphi A in order to ensure that the correct drugs are prescribed and to combat the development of antibiotic resistance.

The application of metabolomics is relatively new in infectious diseases research compared to the application of genomics and proteomics. Despite this, screening the metabolome in blood plasma has identified useful prognostic profiles of several diseases, including sepsis.  Using a combination of gas chromatography and mass spectrometry, Näsström et al. identified 695 distinct peaks that were associated with different metabolites: from these they selected six peaks that had significantly different heights in the three groups of patients. This meant that they were able to tell if the patient had S. Typhi, S. Paratyphi A, or neither. That this mass spectrometric analysis was able to distinguish two Salmonella groups that share many similarities is remarkable. Moreover, in addition to its diagnostic potential, this new approach might also provide insights into the antigenic and physiological differences between the two strains.

Clostridium difficile colitis results from a disturbance of the normal bacterial flora of the colon, colonization by C difficile, and the release of toxins that cause mucosal inflammation and damage. Antibiotic therapy is the key factor that alters the colonic flora. C difficile infection (CDI) occurs primarily in hospitalized patients.

Essential update: CDC promotes improving inpatient antibiotic prescribing to reduce drug resistance and increase patient safety In a CDC analysis of data regarding antibiotic prescribing in hospitalized patients, Fridkin and colleagues estimated that a 30% reduction in use of broad-spectrum antibiotics would result in a 26% reduction in C difficile infections (CDIs).[1, 2] In addition, improvement in physician antibiotic prescribing habits from overuse and incorrect use would also help to reduce antibiotic resistance.

The authors recommend the following[2] :

Promptly initiate antibiotics for a presumed infection, but first obtain any recommended cultures.

Document and specify the drug’s indication, dose, and expected duration of use in the patient’s medical chart.

Reassess the patient within 48 hours based on test results and patient examination; adjust the antibiotic regimen (dose, duration) and/or the agent, or end the antibiotic treatment, as needed.

Signs and symptoms

Symptoms of C difficile colitis often include the following:

Mild to moderate watery diarrhea that is rarely bloody

Cramping abdominal pain



Physical examination may reveal the following in patients with the disorder:

Fever: Especially in more severe cases


Lower abdominal tenderness

Rebound tenderness: Raises the possibility of colonic perforation and peritonitis

Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered questions

Charlotte R. Grant, Rodrigo Liberal, Giorgina Mieli-Vergani, et al.
Autoimmunity Reviews 14 (2015) 105–116

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.
Role of dendritic cells in the initiation, progress and modulation of systemic autoimmune diseases

Juan Pablo Mackern-Oberti, Carolina Llanos, Fabián Vega, et al.
Autoimmunity Reviews 14 (2015) 127–139

Dendritic cells (DCs) play a key role in the activation of the immune response against pathogens, as well as in the modulation of peripheral tolerance to self-antigens (Ags). Furthermore, an imbalance in the activating/inhibitory receptors expressed on the surface of DCs has been linked to increased susceptibility to develop autoimmune diseases underscoring their immunogenicity potential. It has been described that modulation of activating or inhibitory molecules expressed by DCs, such as CD86, TLRs, PDL-1 and FcγRs, can define the immunogenic phenotype. On the other hand, T cell tolerance can be achieved by tolerogenic DCs, which have the capacity of blocking undesired autoimmune responses in several experimental models, mainly by inducing T cell anergy, expansion of regulatory T cells and limiting B cell responses. Due to the lack of specific therapies to treat autoimmune disorders and the tolerogenic capacity of DCs shown in experimental autoimmune disease models, autologous tol-DCs are a potential therapeutic strategy for fine-tuning the immune system and reestablishing tolerance in human autoimmune diseases. New advances in the role of DCs in systemic lupus erythematosus (SLE) pathogenesis and the identification of pathogenic self-Ags may favor the development of novel tol-DC based therapies with amajor clinical impact. In this review, we discuss recent data relative to the role of DCs in systemic autoimmune pathogenesis and their use as a therapy to restore tolerance.

T cell subsets and their signature cytokines in autoimmune and inflammatory diseases

Itay Raphael, Saisha Nalawade, Todd N. Eagar, Thomas G. Forsthuber
Cytokine xxx (2014) xxx–xxx

CD4+ T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful.

Irritable Bowel Syndrome and Gluten Sensitivity Without Celiac Disease: Separating the Wheat from the Chaff

Biesiekierski JR, Newnham ED, Irving PM, et al. Gluten causes gastrointestinal symptoms in subjects without celiac disease: a double-blind randomized placebo controlled trial. Am J Gastroenterol 2011;106:508–514.

Courtney C. Ferch, William D. Chey
Gastroenterology 2012; 142:664–673

Over the past several years, there has been increasing discussion concerning the topic of gluten sensitivity as a cause of irritable bowel syndrome (IBS) symptoms in patients for whom celiac disease has been excluded. Biesiekierski et al performed a double-blind, placebo-controlled, dietary rechallenge trial to better understand the role of gluten ingestion in the development of gastrointestinal (GI) and non-GI symptoms in patients diagnosed with IBS. This study included a sample of 34 patients diagnosed with IBS by the Rome III criteria who had experienced symptom improvement with a gluten-free diet for 6 weeks before study enrollment. Celiac disease had been excluded in all study participants by either a negative HLADQ2/HLA-DQ8 haplotype or a normal duodenal biopsy. Patients with potentially important confounders such as cirrhosis, inflammatory bowel disease, nonsteroidal anti-inflammatory drug ingestion, or excessive alcohol use were excluded from the study.

Upon completion of the study period, it was found that a significantly greater proportion of patients in the gluten group compared with the gluten-free group answered “no” to the primary outcome question (68% vs 40%; P < .001). Compared with the gluten group, those who remained gluten free also reported significant improvements in pain (P < .016), bloating (P < .031), satisfaction with stool consistency (P <.024), and tiredness (P < .001), but showed no significant differences in wind (P < .053) or nausea (P < .69). The results of celiac antibodies at baseline and after the dietary intervention were similar. Intestinal permeability as measured by urine lactulose-to-rhamnose ratio was also unchanged by the dietary intervention. Fecal lactoferrin levels were persistently undetectable in all but 1 patient during the treatment period. High-sensitivity C-reactive protein levels remained normal before and after the dietary intervention. There were no differences in the likelihood of symptomatic response in those with and without HLA-DQ2 and HLA-DQ8 alleles, arguing against undiagnosed celiac disease as a cause for symptom response to a gluten-free diet.

The authors felt that these data support the existence of non–celiac-associated gluten sensitivity. They concluded that gluten is indeed associated with overall IBS symptoms, bloating, dissatisfaction with stool consistency, abdominal pain, and fatigue in a subset of patients.

A recent meta-analyses of studies from around the world found that patients with IBS symptoms were significantly more likely to have celiac disease than controls. (Arch Intern Med 2009;169:65165– 65168). As such, the American College of Gastroenterology Task Force has recommended that routine serologic screening for celiac sprue be pursued in patients with diarrhea-predominant IBS and IBS with a mixed bowel pattern (grade 1B recommendation; Am J Gastroenterol 2009;104[Suppl 1]:S1–S35). Although much of the recent discussion around the potential role of food in IBS symptoms has focused on celiac disease, it is important to note that data from the available US studies have not shown a significantly greater risk for celiac disease among patients with IBS symptoms and no warning signs (Am J Gastroenterol 2008;103[Suppl 1]:S472; Gastroenterology 2011;141:1187–1193). A recent prospective study from the United States reported a 0.4% prevalence of biopsy-proven celiac disease in 492 patients with IBS symptoms and 458 asymptomatic persons undergoing colonoscopy for colorectal cancer screening or surveillance (Gastroenterology 2011;141:1187–1193). Although not significantly different, it is interesting that 7.3% of the IBS group and 4.8% of controls had 1 abnormal celiac serology test result (adjusted odds ratio, 1.49; 95% confidence interval, 0.76 – 0.90; P =.25). Thus, this study suggests that the likelihood of an abnormal immunologic response to gluten is orders of magnitude more common than biopsy-proven celiac disease in IBS patients and controls from the United States. It has been suggested that ~20% of the general population reports symptoms in association with the ingestion of gluten. Such patients have been said to suffer from “gluten sensitivity.”

It is also interesting to consider the potential effects of food on gut immune function beyond celiac disease. There is emerging evidence to suggest that immune activation and/or low-grade inflammation may play a role in the pathogenesis of IBS (GI Clin North Am 2011;40:65–85). The data are currently conflicting, but alternations in the number of mast cells in close proximity to afferent neurons, mucosal lymphocytes, and certain pro-inflammatory or anti-inflammatory cytokines have been identified in a subset of patients with IBS. It is not difficult to envision that alterations in the gut immune system could occur as a consequence of an acute GI infection in a genetically susceptible individual. However, it is interesting to speculate that other environmental factors, such as an altered gut microbiota, physical or emotional abuse, stress, or food, might result in abnormal gut immune function translating clinically into IBS symptoms.

A better understanding of how differences in gut immune function, the microbiome, and fermentation might influence the development of IBS symptoms in association with the ingestion of gluten are all deserving of further investigation. The study by Biesiekierski et al is the first randomized, controlled trial to suggest that nonceliac IBS patients might benefit from a gluten-free diet. Although these results are certainly intriguing and hypothesis generating, they require validation in larger, randomized, controlled trials in other parts of the world. What is clear and important for providers to understand is that gluten sensitivity is here to stay and significantly more likely for them to encounter in day-to-day practice than celiac disease.

No Effects of Gluten in Patients With Self-Reported Non-Celiac Gluten Sensitivity After Dietary Reduction of Fermentable, Poorly Absorbed, Short-Chain Carbohydrates

Jessica R. Biesiekierski, Simone L. Peters, Evan D. Newnham, et al.
Gastroenterology 2013;145:320–328

Background & Aims: Patients with non-celiac gluten sensitivity (NCGS) do not have celiac disease but their symptoms improve when they are placed on gluten-free diets. We investigated the specific effects of gluten after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates (fermentable, oligo-, di-, monosaccharides, and polyols [FODMAPs]) in subjects believed to have NCGS. Methods: We performed a double-blind crossover trial of 37 subjects (aged 2461 y, 6 men) with NCGS and irritable bowel syndrome (based on Rome III criteria), but not celiac disease. Participants were randomly assigned to groups given a 2-week diet of reduced FODMAPs, and were then placed on high-gluten (16 g gluten/d), low-gluten (2 g gluten/d and 14 g whey protein/d), or control (16 g whey protein/d) diets for 1 week, followed by a washout period of at least 2 weeks. We assessed serum and fecal markers of intestinal inflammation/injury and immune activation, and indices of fatigue. Twenty-two participants then crossed over to groups given gluten (16 g/d), whey (16 g/d), or control (no additional protein) diets for 3 days. Symptoms were evaluated by visual analogue scales. Results: In all participants, gastrointestinal symptoms consistently and significantly improved during reduced FODMAP intake, but significantly worsened to a similar degree when their diets included gluten or whey protein. Gluten-specific effects were observed in only 8% of participants. There were no diet-specific changes in any biomarker. During the 3-day rechallenge, participants’ symptoms increased by similar levels among groups. Gluten-specific gastrointestinal effects were not reproduced. An order effect was observed. Conclusions: In a placebo controlled, cross-over rechallenge study, we found no evidence of specific or dose-dependent effects of gluten in patients with NCGS placed diets low in FODMAPs.

Infection, inflammation, and the irritable bowel syndrome

Spiller, K. Garsed
Digestive and Liver Disease 41 (2009) 844–849

Infectious diarrhea is one of the commonest afflictions of mankind. Worldwide most of the burden, about 1 billion cases a year, is seen in children <5 years old, the vast majority in the developing world in communities where access to clean water and adequate sanitation is restricted. Here a child can expect to have 6–7 episodes per year compared to 1–2 in the developed world. Following recovery from an episode of gastroenteritis (GE) the vast majority of healthy adults and children develop some degree of immunity to the organism responsible and return to normal functioning. However 7–31% develop post-infectious irritable bowel syndrome (PI-IBS). The proportion of unselected IBS that is post-infectious varies from 6 to 17% in the USA and Europe but whether this differs in the developing world is unknown, though previous enteric infection is a known risk factor for IBS in Southern China.

This review will compare the epidemiology of infectious diarrhea in the developing and developed world and the link between mucosal inflammation and the development of IBS symptoms. The available evidence suggests that the acquisition of immunity in early childhood reduces the severity of subsequent gastroenteritis in adulthood. Since these are known risk factor for developing PI-IBS we hypothesize that this may underlie some of the regional differences in the incidence of both infection and IBS.

Gastrointestinal infection is ubiquitous worldwide though the pattern of infection varies widely. Poor hygiene and lack of piped water is associated with a high incidence of childhood infection, both viral and bacterial. However in developed countries bacterial infection is commoner in young adults. Studies of bacterial infections in developed countries suggest 75% of adults fully recover, however around 25% have long lasting changes in bowel habit and a smaller number develop the irritable bowel syndrome (IBS). Whether the incidence is similar in developing countries is unknown. Post-infective IBS (PI-IBS) shares many features with unselected IBS but by having a defined onset allows better definition of risk factors. These are in order of importance: severity of initial illness, smoking, female gender and adverse psychological factors. Symptoms may last many years for reasons which are unclear. They are likely to include genetic factors controlling the immune response, alterations in serotonin signaling, low grade mucosal inflammation maintained by psychological stressors and alterations in gut microbiota. As yet there are no proven specific treatments, though 5HT3 receptor antagonists, anti-inflammatory agents and probiotics are all logical treatments which should be examined in large well-designed randomized placebo controlled trials.

There are three key questions. Firstly is the incidence of IBS less in the developing world, secondly is the incidence increasing with the adoption of a western urban life style and finally is the disease itself different? The answer to all three is probably yes though interpretation of cross-cultural surveys is fraught with problems relating to the imprecise translation of questions into different cultures. Initial reports from small uncontrolled studies suggested that IBS was very uncommon and predominantly affected a subpopulation who pursued a “western life style”. More recent and robust work gives a range of values for prevalence from very low in Iran and India with just 5.8 and 4.2% respectively, to values in developed Asian countries that are generally lower but not dissimilar to those seen in the west. The key factors associated with rapid westernization that underlie this increase in numbers is unclear but could include the effect of improved hygiene, increased overcrowding, stress and changes in diet. The best evidence comes from studies in which the same populations have been studied over a number of years as has been done in Singapore where after a decade of steady industrial growth the prevalence of IBS has risen from 2.3% to 8.6%.

This raises is a most important question – why should these differences occur? It is clear that major differences in the epidemiology of gut infection exist between the west and the developing world. This is illustrated by Campylobacter jejuni enteritis, which causes a shorter, less severe illness in childhood than in adulthood, which is when most Europeans and North Americans are infected. The greater degree of inflammation which adults experience may increase the risk of developing subsequent PI-IBS which might partly account for the higher prevalence of IBS in the westernized nations.

Worldwide the average number of episodes of infection annually per person is 3. A poorly nourished child living in cramped conditions without access to sewerage and running water will have 8 or more infections in the first year of life, most frequently with enteric bacteria and parasites whereas a child in better sanitary conditions would have less infections and these would be more likely to be viral in origin. Even in England an estimated 1 in 5 people per year have an episode of diarrhea in the community adding up to 9.4 million cases in total a year, largely unreported since only 1 in 30 present to their doctor. It seems here that viral infections predominate in the very young, with bacterial infection particularly Campylobacter spp. being most common in adolescence and early adulthood. PCR analysis of stool in the same study showed that Norovirus and Rotavirus were the commonest pathogens detected across all age groups. Campylobacter spp. were most commonly found in age group 30–39 (16% compared to 6.7% of those aged 1–4).

Infectious diarrhea results from either an increase in fluid and electrolyte secretion, predominantly in the small intestine, or a decrease in absorption which can involve both the small and large bowel. During a diarrheal illness these two mechanisms frequently co-exist. Enterotoxins from Vibrio cholerae or enterotoxigenic E. coli induce profuse secretion while decreased intestinal absorption can be induced by mucosal injury caused by enteroinvasive organisms (e.g., Salmonella, Shigella, and Yersinia spp.). These invasive infections injure cells and excite an immune response and activate enteric nerves and mast cells resulting in an acute inflammatory infiltrate with the release of pro-inflammatory mediators and stimulation of secretion. Clinically the patient will have an acutely inflamed mucosa with ulceration and bleeding.

Campylobacter jejuni produces a range of toxins including cytolethal distending toxin, that first produces a secretory diarrhea in the small intestine in the early part of the illness after which there is invasion of the distal ileum and colon to produce an inflammatory ileocolitis, which can extend all the way to the rectum. The disease is less severe in developing countries than in developed countries, with watery stool, fever, abdominal pain, vomiting and dehydration predominating as opposed to the severe abdominal pain, weight loss, fever and bloody stool that is seen more frequently in infections in the west. Infants usually have milder disease with less fever and pain, which in some cases is due to immunity acquired during previous infection. The reasons for these differences between the developed and developing world are unclear.

The composition of the resident intestinal microbiota is highly variable between individuals but relatively stable for each individual, though IBS patients showamore unstable microbiota. This instability may be due to antibiotic therapy or alterations in diet, both of which are commoner in IBS. Patients given antibiotics are 4 times more likely than untreated controls to report bowel symptoms 4 months later, and antibiotic use is a risk factor for developing IBS with an adjusted OR of 3.70 (1.80–7.60). Antibiotic use increases the incidence of post-infective functional diseases following both Salmonella enteritidis and travellers’ diarrhea, in whom antibiotic treatment gave a relative risk of developing PI-IBS of 4.1 (1.1–15.3) compared with those not receiving treatment.

During acute infectious diarrhea there is a decrease in anaerobes. Mice infected with Citrobacter rodentium or C. jejuni or subjected to a chemically induced colitis show significant reduction in the total numbers of microbiota, which is mainly due to activation of the host immune response and only to a much lesser degree by bacterial factors. This loss of anaerobes is associated with a depletion in short chain fatty acids and an increase in the pH of the stool allowing overgrowth of other organisms which may contribute to disturbed bowel function.

The study of patients with PI-IBS has yielded many new insights for several reasons. Firstly the patients are a more homogenous group than unselected IBS, most having diarrhea with fewer psychological problems than unselected IBS. Secondly the direction of causation is easier to ascertain as they represent a “natural experiment”, with subjects “randomized” to receive an infection, thus producing an unbiased study group. Finally onset of symptoms on a clearly defined date in a previously well patient provides an opportunity to examine the prior host and bacterial factors that predispose to developing IBS.

The severity of injury is mediated not only by factors related to the infecting organism but also by the host’s own immune response which develops in early life and declines in old age. However little is known about the incidence of PI-IBS in the pediatric population and whether it is different to the condition seen in adults. Functional bowel disorders are common in children, with IBS affecting 14% of high school and 6% of middle school patients in a US community study and are classified according to the main complaints made by parents or children rather than in an organ-specific way. This makes comparisons with the adult population difficult however a single recent study reports a very high incidence of postinfectious symptoms in 88 children with positive bacterial stool culture results presenting to a single institution. These had a 36% prevalence of functional gastrointestinal disorders compared to 11% in age- and sex-matched healthy controls. This is much higher than most adult studies with the exception of the Walkerton outbreak. Unlike adults, female gender is not a risk factor for PI-IBS in children suggesting the gender effect depends on hormonal and/or psychosocial factors rather than being genetic.

Despite uncertainty about PI-IBS in childhood we do know that age in adulthood does have an effect on the likelihood of developing PI-IBS. A meta-analysis indicates that patients who develop PI-IBS are slightly younger and one study showed increasing age was protective with age >60 years giving a relative risk of PI-IBS of 0.36 (0.1–0.09) though not all studies have shown this.

Why should this inflammation persist in some and not others? As we have already discussed adverse life events, anxiety and epression may play a part however less psychological morbidity is seen in PI-IBS than IBS indicating the presence of other factors which predispose to an exaggerated or prolonged inflammatory response.  These factors might be genetic since a larger proportion of IBS patients have the high producing heterozygous TNF-α G/A polymorphism at position-308 than controls. Some PI-IBS patients were contained in this study but too few to examine as a subgroup. This study did not confirm an earlier finding of a decrease in the presumed immunoregulatory high IL-10 producing phenotype in IBS.

Although it is likely from animal work that infection does alter the gut microbiota there is no data on this in PI-IBS. There is some indirect evidence that altered microbiota may be important in IBS since fecal serine protease activity, which may be of bacterial origin, is increased in D-IBS. This is of great interest because these proteases can increase visceral sensitivity in rats, acting via the protease activated receptor-2 (PAR-2) group of receptors found in the mucosa and enteric nerves.

A recent small randomized placebo controlled trial of Mesalazine suggested this could reduce mast cell numbers and improve symptoms, a finding which needs repeating with larger numbers. Given the increase in 5HT availability and the effectiveness of 5HT3 receptor antagonists in animal studies and in unselected IBS-D patients a trial of a 5HT3 receptor antagonist would also be logical.

Gut motility and enteroendocrine secretion

Tongzhi Wu, Christopher K Rayner, Richard L Young and Michael Horowitz
Current Opinion in Pharmacology 2013, 13:928–934
The motility of the gastrointestinal (GI) tract is modulated by complex neural and hormonal networks; the latter include gut peptides released from enteroendocrine cells during both the interdigestive and postprandial periods. Conversely, it is increasingly recognised that GI motility is an important determinant of gut hormone secretion, in that the transit of luminal contents influences the degree of nutrient stimulation of enteroendocrine cells in different gut regions, as well as the overall length of gut exposed to nutrient. Of particular interest is the relationship between gallbladder emptying and enteroendocrine secretion. The inter-relationships between GI motility and enteroendocrine secretion are central to blood glucose homeostasis, where an understanding is fundamental to the development of novel strategies for the management of diabetes mellitus.

Enteroendocrine cells account for release of more than 30 known peptides, including motilin and ghrelin during the interdigestive period, and cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) after meals. The latter are key mediators of the shift from an interdigestive to a postprandial GI motor pattern. Conversely, the delivery of luminal contents to be sensed by enteroendocrine cells in various gut regions is dependent on GI motor activity.

During the interdigestive period, both the stomach and small intestine undergo a cyclical motor pattern — the ‘migrating motor complex (MMC)’ — consisting of a quiescent phase (~40 min, phase I), a phase of irregular contraction (~50 min, phase II), and a period of maximum contraction (5–10 min, phase III). The MMC migrates from the stomach (or proximal small intestine) to the terminal ileum, and acts to sweep small intestinal contents (including bile, digestive juice and indigestible debris) towards the large intestine. Phase III of the MMC is also associated with spontaneous gallbladder emptying.

The cyclical occurrence of MMC activity during the interdigestive state closely parallels the secretion of motilin, and to a lesser degree, ghrelin. Increases in plasma motilin concentrations follow immediately each episode of spontaneous gallbladder emptying, while after phase III there is a decrease in motilin. The latter might be associated with the relative absence of luminal content due to the ‘house-keeping’ effect of phase III. Patients with gallstones have defective gallbladder emptying and lack the cyclical profile of motilin concentrations and exhibit a reduced frequency of phase III activity.

GI motility has a major impact on enteroendocrine secretion; conversely, enteroendocrine hormones play a pivotal role in the regulation of interdigestive and postprandial GI motility. The significance of these interrelationships is increasingly recognized as being central to the regulation of postprandial glycemia. Slowing gastric emptying and intestinal transit, accelerating gallbladder emptying and intestinal exposure to bile acids, and stimulating postprandial enteroendocrine hormones, all represent novel therapeutic approaches for the management of type 2 diabetes.

Enteroendocrine cell types revisited

Maja S Engelstoft, Kristoffer L Egerod, Mari L Lund and Thue W Schwartz
Current Opinion in Pharmacology 2013, 13:912–921

The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa.

Go with the flow — membrane transport in the gut

Editorial overview, David T Thwaites
Current Opinion in Pharmacology 2013, 13:843–846

The primary function of the gastrointestinal tract is the assimilation of nutrients from diet. The final stages of digestion and almost all absorption take place in the small intestine and, to a lesser extent, the large intestine. The intestinal epithelium is the single layer of polarized, differentiated cells that lines the wall of the intestine. It sits at the interface between the outside world and the internal environment of the human body. It is across this epithelial barrier that all essential nutrients, vitamins, electrolytes and fluid are absorbed. Many toxins and waste products can be secreted directly across the intestinal epithelium or excreted through the biliary route. The gastrointestinal tract is of great interest to the pharmacologist, and the pharmaceutical industry beyond, because most patients, if given the opportunity, would choose to take medication orally rather than have it delivered by any other route. In addition, many drugs and metabolites are lost from the body by active secretion from the intestine and liver. Thus, the intestinal epithelium is a major target for clinical intervention to improve bioavailability and modulate gut function.

To allow net transport in either the absorptive or secretory direction, the polarised cells in the small intestine (enterocytes), large intestine (colono-cytes) and liver (hepatocytes) express a distinct set of membrane transport proteins in their apical and basolateral membrane domains. Each epithelial cell type mediates net solute and ion movement through the coordinated activity of an array of membrane transport proteins (primary active transporters or pumps, secondary active cotransporters or antiporters, and channels).

Chloride channel-targeted therapy for secretory diarrheas

Jay R Thiagarajah and AS Verkman
Current Opinion in Pharmacology 2013, 13:888–894

Secretory diarrheas caused by bacterial and viral enterotoxins remain a significant cause of morbidity and mortality. Enterocyte Cl channels represent an attractive class of targets for diarrhea therapy, as they are the final, rate-limiting step in enterotoxin-induced fluid secretion in the intestine. Activation of cyclic nucleotide and/or Ca2+ signaling pathways in secretory diarrheas increases the conductance of Cl channels at the enterocyte luminal membrane, which include the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl channels (CaCCs). High-throughput screens have yielded several chemical classes of small molecule CFTR and CaCC inhibitors that show efficacy in animal models of diarrheas. Natural-product diarrhea remedies with Cl channel inhibition activity have also been identified, with one product recently receiving FDA approval for HIV-associated diarrhea.

The intestinal epithelium consists of villi and crypts, with absorption occurring mainly in villi and secretion in crypts. Fluid absorption in the small intestine is driven by the luminal Na+/H+ exchanger (NHE3), Na+-glucose cotransporter (SGLT1), and Cl/HCO3 exchanger (DRA)(Figure 1, not shown). As in all epithelia the electrochemical driving force is established by a basolateral Na+K+-ATPase pump. The pro-absorptive solute transporters are constitutively active, though they can be modulated by second-messengers including cAMP and Ca2+. NHE3, SGLT1 and DRA are thus potential membrane transporter targets to increase intestinal fluid absorption. In the colon, fluid absorption is also facilitated by the epithelial Na+ channel (ENaC) and short-chain fatty acid (scfa) transporters (SMCT1).

Intestinal signal pathways controlling fluid secretion. Not shown. (a) Signaling pathways in CFTR activation by bacterial enterotoxins. Cholera toxin and heat stable enterotoxin (STa) bind to membrane receptors (GM1 —ganglioside receptor, guanylin receptor) causing increases in cyclic nucleotides (cAMP, cGMP) and neurotransmitters, resulting in CFTR activation. EC — enterochromaffin cells, 5-HT — 5-hydroxytryptamine, VIP — vasoactive intestinal peptide, ENS — enteric nervous system. (b) Signaling pathways in CaCC activation by rotavirus. Rotavirus releases NSP4 (non-structural protein 4), which causes elevation of cytoplasmic Ca2+ either: directly via binding to a membrane receptor (integrin α1β2); via neuropeptide galanin; or through activation of enteric nerves. Gal1-R — galanin 1 receptor. (c) Cross-talk between Ca2+ and cAMP pathways in intestinal epithelial cells. Epac — exchange protein directly activated by cAMP, PDE — phosphodiesterase, AC —adenylate cyclase, CaSR — calcium sensing receptor.

Natural-product ClS channel inhibitors Natural products have been identified with antidiarrheal efficacy in humans and a putative mechanism of action involving Cl channel inhibition. Crofelemer, a heterogeneous proanthocyanidin oligomer extracted from the bark latex of South American tree Croton lechleri, was approved recently for HIV-associated diarrhea following clinical trials showing efficacy in reducing the number and severity of diarrhea episodes. Whether CaCC inhibition by crofelemer can explain its efficacy in HIV-associated diarrhea is unclear.

Following a natural product screen that identified tannic acid as a general CaCC inhibitor, we found that red wines containing polyphenolic gallotannins fully inhibited intestinal CaCC without effect on CFTR. In recent follow-up work, we generated an alcohol-free red wine extract with potent CaCC inhibition activity, and showed its efficacy in a neonatal mouse model of rotaviral diarrhea (unpublished data). The wine extract inhibited intestinal Ca2+-activated Cl current and fluid secretion without affecting rotaviral infection of intestinal epithelial cells. CaCC inhibition may account for anecdotal reports of antidiarrheal action of red wines. Motivated by the possibility that known herbal antidiarrheal remedies might act by Cl channel inhibition, we recently screened a selection of diarrhea remedies from sources worldwide and identified a commonly used Thai herbal remedy that fully inhibited both CFTR and CaCC (unpublished observations). The herbal remedy showed efficacy in mouse models of cholera and rotaviral diarrhea.

Clinical relevance of drug efflux pumps in the gut

Shingen Misaka, Fabian Muller and Martin F Fromm
Current Opinion in Pharmacology 2013, 13:847–852

Important export pumps expressed in the apical membrane of enterocytes are P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2). They are believed to be a crucial part of the bodies’ defense mechanisms against potentially toxic, orally administered xenobiotics. In particular P-gp and BCRP also limit the bioavailability of drugs. Inhibition of these intestinal export pumps by concomitantly administered drugs leads to increased plasma concentrations, whereas induction can reduce absorption of the substrate drugs and decrease plasma concentrations. The role of polymorphisms in genes encoding for these transporters will also be discussed. Taken together this review will focus on the role of intestinal export pumps using selected examples from clinical studies in humans.

P-gp (gene: ABCB1) is a protein consisting of two homologous halves, each containing six transmembrane helices and one nuclear-binding domain. The protein expression of P-gp has been shown to increase from proximal to distal parts of the intestine. P-gp generally tends to transport hydrophobic, amphipathic or cationic compounds. Clinically important P-gp substrates include anticancer agents, cardiovascular drugs and immunosuppressants. It is of note that most of the listed drugs are also substrates of CYP3A4, and thus intestinal P-gp and intestinal CYP3A4 efficiently collaborate to enhance the removal of their substrates. ABCB1 mRNA expression is regulated by several nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), thyroid hormone receptor and vitamin D receptor (VDR).

Human intestinal P-gp limits bioavailability of drugs and induction and inhibition of intestinal P-gp are important mechanisms underlying drug–drug interactions in humans. Direct evidence for these processes in humans was largely generated using studies in healthy volunteers, who received P-gp drug substrates with negligible drug metabolism such as digoxin and talinolol. Further work is required regarding the importance of intestinal P-gp for drug disposition and drug–drug interactions for the majority of P-gp substrates, which are also metabolized, for example, by intestinal and hepatic CYP3A4, since inducers or inhibitors of P-gp frequently also affect CYP3A4 expression or function. For intestinal BCRP and intestinal MRP2, so far only a limited number of examples with specific drugs exist, which indicate their clinical importance in humans.

Gastrointestinal HCO3 S transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways

Ursula E Seidler
Current Opinion in Pharmacology 2013, 13:900–908

The concept of a protective alkaline gastric and duodenal mucus layer is a century old, yet it is amazing how much new information on HCO3 transport pathways has emerged recently, made possible by the extensive utilization of gene deleted and transgenic mice and novel techniques to study HCO3  transport. This review highlights recent findings regarding the importance of HCO3  for mucosal protection of duodenum and other gastrointestinal epithelia against luminal acid and other damaging factors. Recently, methods have been developed to visualize HCO3  transport in vivo by assessing the surface pH in the mucus layer, as well as the epithelial pH. New information about HCO3  transport pathways, and emerging concepts about the intricate regulatory network that governs duodenal HCO3 secretion are described, and new perspectives for drug therapy discussed.

The lack of HCO3 ions in the pancreatic secretions of children with cystic fibrosis was recognized in the 1960s and the significance for impaired mucus release discussed. It is now evident that CFTR expression is essential for HCO3  secretion in most gastrointestinal epithelia, such as the esophagus, the small intestine, the biliary tract, and the pancreatic ducts, as well as the reproductive tract and the airways. The low pH in the acinar-ductal unit after release of the zymogen granules needs to be quickly neutralized to prevent acinar damage. Similarly, the bile ducts need a ‘biliary HCO3 umbrella’ to keep toxic bile acids ionized and thereby membrane-impermeable, and the esophagus needs HCO3  secretion to protect the epithelial surface from acid reflux, and this is possibly mediated also by CFTR-dependent mechanisms. HCO3 is essential for the release and proper expansion of mucin molecules. CF patients and CFTR-deficient mice have impaired lipid absorption, which in mice has been experimentally linked with the duodenal HCO3 deficit. Thus the HCO3 secretory defect of cystic fibrosis patients is closely linked to many of the pathophysiological GI manifestations of CF.

Fluid and electrolyte secretion in the inflamed gut: novel targets for treatment of inflammation-induced diarrhea

Melanie G Gareau and Kim E Barrett
Current Opinion in Pharmacology 2013, 13:895–899

Diarrheal disease can occur in the context of both inflammatory and infectious challenges. Inflammation can result in changes in ion transporter expression or simply mislocalization of the protein. In addition to development of diarrhea, an altered secretory state can lead to changes in mucus secretion and luminal pH. Bacterial infection can lead to subversion of host cell signaling, leading to transporter mislocalization and hyposecretion, promoting bacterial colonization. Novel therapeutic strategies are currently being developed to ameliorate transporter defects in the setting of inflammation or bacterial infection including, for example, administration of probiotics and fecal microbiota transplantation. This review will highlight recent findings in the literature detailing these aspects of ion transport in the inflamed gut.

Inflammatory diarrhea can occur in many different pathological conditions including IBD, comprising Crohn’s disease (CD) and ulcerative colitis (UC). The resulting inflammation triggers production of cytokines, including TNFα and IFNϒ, that can modulate ion transporters directly, including Na+K+ATPase and Na+H+ exchanger (NHE)-1 (SLC9A1), and decrease barrier function. Inflammation can activate several potential mechanisms that can underlie diarrheal symptoms via distinct pathways.

The presence of immune cells, such as T cells, results in the production of cytokines that can inhibit Na+ absorption, activate Cl secretion, and cause mucosal barrier dysfunction, resulting in diarrhea. In the IL-10 deficient mouse model of colitis, inflammation is characterized by T cells and macrophages, and high levels of pro-inflammatory cytokines, including TNFα. This was accompanied by dysfunctional NHE3 (SLC9A3) transport activity in the absence of overall changes in gene expression and protein localization. A decrease in expression of PDZ adaptor proteins (NHERF2 and PDZK1 scaffolding proteins), which modulate NHE3 activity by regulating transporter interactions and signal transduction, was also observed.

Ion transporters and their regulatory mechanisms represent potential therapeutic targets for the treatment of inflammatory diarrhea. Probiotics, live microorganisms provided in adequate amounts to confer a benefit on the host beyond their inherent nutrition, have been demonstrated to provide a beneficial effect in various GI diseases, including diarrhea. Acute administration of Lactobacillus acidophilus to Caco-2 cells in vitro and to mice in vivo increased DRA expression. Administration of Bifidobacterium breve, but not Lactobacillus rhamnosus or Eubacterium rectale, to HT29 cells down-regulated both Ca2+ (carbachol [CCh]) and cAMP (FSK) mediated Cl secretion. This effect by B. breve was not seen at the expense of monolayer integrity or tight junction dysfunction, occurred downstream of Ca2+ mobilization and was hypothesized to occur via CFTR based on the observation that a CFTR inhibitor could block the effects of CCh. In contrast, administration of the probiotic strain Enterococcus faecium was able to improve intestinal barrier function in piglets, as measured by mannitol flux rates, whereas prostaglandin E2-induced short circuit current was increased, suggesting an increased secretory state.

Differing degrees of susceptibility to infection with C. rodentium within different strains of mice have been well established and characterized; however the precise mechanisms involved are not well defined. A decrease in DRA was found in C3H and FVB mice, which succumb to C. rodentium infection, compared to resistant C57BL/6 mice. It was recently demonstrated that gavaging C3H mice with the colonic microbiota of C57BL/6 mice, following antibiotic administration, could transfer the protection against death following infection with C. rodentium in C3H mice. Survival was accompanied with restoration of DRA gene expression and other transporters that are known to be involved in protection from diarrhea. While this is extremely preliminary, fecal microbiota transplant may serve as an alternative in a subset of cases of infectious diarrhea, separate from the well-established data on C. difficile.

Phospholipids are increasingly being recognized for their signaling roles in addition to their traditional roles in cell structure. Lysophosphatidic acid (LPA) is a naturally occurring glycerophospholipid that can serve as a signaling molecule via binding to its G-protein coupled receptors LPA1, LPA2, and LPA3. In colonic Caco-2 cells, administration of LPA for 24 hours induced DRA expression via LPA2, increasing its Cl/HCO3exchange activity via a PI3 kinase pathway. The ability of LPA to increase ion transporter activity in the setting of inflammation or infection needs to be tested directly, but the findings at least potentially suggest that LPA may serve as a useful anti-diarrheal agent. Studies in bronchial epithelial cells suggest that LPA can also ameliorate lipopoly-saccharide-induced barrier dysfunction, suggesting a similar effect may be present in the intestinal tract. The ability of LPA to increase migration and proliferation of intestinal epithelial cells, however, would warrant some concerns with long-term administration and would need to be carefully assessed.

Intestinal ion transporters represent a valid physiological target for limiting inflammatory and infectious diarrhea. Their ability to regulate both water secretion and absorption allows bidirectional mechanisms to be exploited, creating a wide range of possible therapeutic targets.

Discovery and Development of Antisecretory Drugs for Treating Diarrheal Diseases

Jay R. Thiagarajah, Eun–A Ko, L Tradtrantip, M Donowitz, and A. S. Verkman
Clinical Gastroenterology and Hepatology 2014;12:204–209

Diarrheal diseases constitute a significant global health burden and are a major cause of childhood mortality and morbidity. Treatment of diarrheal disease has centered on the replacement of fluid and electrolyte losses using oral rehydration solutions. Although oral rehydration solutions have been highly successful, significant mortality and morbidity due to diarrheal disease remains. Secretory diarrheas, such as those caused by bacterial and viral enterotoxins, result from activation of cyclic nucleotide and/or Ca2+ signaling pathways in intestinal epithelial cells, enterocytes, which increase the permeability of Cl channels at the lumen-facing membrane. Additionally, there is often a parallel reduction in intestinal Na+ absorption. Inhibition of enterocyte Cl channels, including the cystic fibrosis transmembrane conductance regulator and Ca2-activated Cl channels, represents an attractive strategy for antisecretory drug therapy. High-throughput screening of synthetic small-molecule collections has identified several classes of Cl channel inhibitors that show efficacy in animal models of diarrhea but remain to be tested clinically. In addition, several natural product extracts with Cl channel inhibition activity have shown efficacy in diarrhea models. However, a number of challenges remain to translate the promising bench science into clinically useful therapeutics, including efficiently targeting orally administered drugs to enterocytes during diarrhea, funding development costs, and carrying out informative clinical trials. Nonetheless, Cl channel inhibitors may prove to be effective adjunctive therapy in a broad spectrum of clinical diarrheas, including acute infectious and drug-related diarrheas, short bowel syndrome, and congenital enteropathies.

Cl- channels as targets for therapy of secretory diarrheas

Cl- channels as targets for therapy of secretory diarrheas

Cl channels as targets for therapy of secretory diarrheas. This diagram of fluid secretory mechanism in enterocytes lining intestinal crypts and villi illustrates active Cl transport from the blood or submucosa to the intestinal lumen facilitated by luminal membrane CFTR and CaCC channels.

Natural products represent a potentially attractive source of antidiarrheal therapeutics, because they are generally inexpensive and have the potential for rapid translation to the clinic. In addition, there is a long history of anecdotal evidence of efficacy of various antidiarrheal remedies in many parts of the world.

A number of hurdles remain in the translation of antidiarrheal drug candidates to widely used therapy. Although a number of compounds have been advanced through preclinical testing in murine models, new high throughput model systems of enterocyte fluid secretion, such as human intestinal enteroids, or genetically tractable systems, such as zebrafish, warrant development to identify novel compounds and antidiarrheal drug targets. A major translational roadblock, however, is the difficulty in designing and funding informative clinical trials.

Barriers to diarrheal drug development in developing countries include the need for very low manufacture cost, high stability in hot and humid environments, and obtaining funding to support commercial development of new chemical entities with relatively low profit potential.

For drugs targeting the enterocyte extracellular surface, an additional challenge is convective washout in which secreted fluid in intestinal crypts washes away inhibitor drugs. A mathematical model of intestinal convection-diffusion concluded that in severe secretory diarrheas, such as cholera, the antisecretory efficacy of an orally administered, surface-targeted inhibitor requires high inhibitor affinity to its target (low nanomolar Kd) to obtain sufficiently high luminal inhibitor concentration (>100-fold Kd), and sustained high luminal inhibitor concentration or slow inhibitor dissociation. Washout is a significant concern for small-molecule CFTR glycine hydrazides, such as iOWH032, and potentially for several of the natural product agents.

Current and emerging therapies in irritable bowel syndrome: from pathophysiology to treatment

Joseph Y. Chang and Nicholas J. Talley
Trends in Pharmacological Sciences 31 (2010) 326–334

Irritable bowel syndrome is a common functional gastrointestinal disorder with characteristic symptoms of abdominal pain/discomfort with a concurrent disturbance in defecation. It accounts for a significant healthcare burden, and symptoms may be debilitating for some patients. Traditional symptom-based therapies have been found to be ineffective in the treatment of the entire syndrome complex, and do not modify the natural history of the disorder. Although the exact etiopathogenesis of IBS is incompletely understood, recent advances in the elucidation of the pathophysiology and molecular mechanisms of IBS have resulted in the development of novel therapies, as well as potential future therapeutic targets. This article reviews current and emerging therapies in IBS based upon: IBS as a serotonergic disorder; stimulating intestinal chloride channels; modulation of visceral hypersensitivity; altering low-grade intestinal inflammation; and modulation of the gut microbiota.

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder characterized by abdominal pain or discomfort that is associated with disturbances in defecation; bloating is common, and the symptoms are not estimates for North America being 10–15%. Only a minority seek care for their symptoms, but IBS has a dramatic impact on patients and utilization of healthcare resources. It is estimated that IBS accounts for 3.5 million physician visits annually in the USA, and is associated with annual direct costs of $1.6 billion and indirect costs of $19.2 billion; patients with IBS consistently report lower health-related quality of life (HRQOL).

Serotonin, or 5-hydroxytryptamine (5-HT), is a neurotransmitter which is largely stored in the enterochromaffin cells of the gut and plays a critical part in the motility, sensation, and secretion of the GI tract. There is growing evidence that a serotonergic mechanism may be involved in the pathophysiology of IBS. Some of the notable findings include: increased postprandial levels of circulating 5-HT in subjects with diarrhea-predominant IBS (D-IBS); D-IBS subjects were observed to have elevated platelet-depleted plasma 5-HT levels in fasting and fed states; the mucosal 5-Hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased in subjects with constipation-predominant IBS (C-IBS); and a lack of increase in plasma 5-HT levels after meal ingestion in those with C-IBS. These findings suggested that a subset of IBS may be a disorder centered on the serotonin disequilibrium, with 5-HT excess responsible for symptoms of D-IBS and insufficient release of 5-HT in the circulation being responsible for the features of C-IBS. However, not all studies support this disease model.

Given the possible role of serotonin in IBS, several 5-HT receptor-modulating agents have been developed as disease-specific therapeutic agents. The 5-HT3 antagonist alosetron has been shown in multiple randomized clinical trials as well as meta-analyses to be an effective agent in the treatment of D-IBS with improvements in global IBS symptoms, relief of abdominal pain, improvement of the consistency and frequency of bowel movements, and reduced fecal urgency. Furthermore, alosetron has been reported to inhibit intestinal secretion, delay colonic transit time, increase colonic compliance in response to distention, and have central effects that result in its beneficial effects on sensation in IBS.

Current and emerging therapies in irritable bowel syndrome

Serotonergic mechanisms·       Alosetron

·       Tegaserod

·       Prucalopride

Chloride channelsActivators

·       Lubiprostone

·       Linaclotide


·       Crofelemer

Visceral hypersensitivityTricyclic antidepressants (TCAs)

·       Selective serotonin reuptake inhibitors (SSRIs)

·       ϒ-Aminobutyric acid analog (pregabalin)

K-opioid receptor agonists

·       Asimadoline

Corticotropin-releasing factor (CRF) receptor antag

Modulation of immune activation and inflammation·       5-aminosalicylic acid

·       Corticosteroids ?

Modulating intestinal floraProbiotics

·       Bifidobacteria



·       Rifaximin

Fiber supplementation·       Psyllium
Antispasmodics·       Hyoscine

·       Cimetropium

·       Pinaverium

·       Peppermint oil

Alternative therapiesDietary factors and modification

·       Food elimination diet (based on IgG antibodies)

·       Low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) diet

·       Gluten-free diet

Agonists to 5-HT4 receptors have been found to be effective in the treatment of C-IBS. 5-HT4 receptor agonists accelerate intestinal transit in the small intestine and colon. Tegaserod is an aminoguanidine indole and selective partial agonist of the 5-HT4 receptor that has been shown to provide improvements in global IBS symptoms and improve constipation in female C-IBS patients. Reports have supported the efficacy of tegaserod in CIBS in terms of global symptom improvement as well as improvement of constipation.

The GI tract contains numerous chloride channels that have an integral role in the transport and secretion of fluids. Type-2 chloride channels (ClC-2) have been investigated with respect to their role in C-IBS and constipation. The ClC-2 channel is an a-helical transmembrane protein located on the apical cell membrane of the intestines, is highly selective for chloride ions, and is involved in the transport and secretion of fluids as well as maintaining cellular membrane potential.  Activation of ClC-2 channels through second messenger induced phosphorylation causes an efflux of chloride ions into the lumen of the GI tract, which results in a subsequent efflux of sodium ions due to isoelectric balance. It is the efflux of sodium that results in the efflux of water into the lumen due to the maintenance of isotonic neutrality through the paracellular pathway. This resulting increase in intestinal secretion and fluid volume has been of interest in the development of chloride channel-directed therapies for C-IBS and constipation.

TCAs and SSRIs have been of interest in the treatment of IBS for their modulation of hyperalgesia and not for their psychotropic effects. TCAs have been demonstrated to be effective in the treatment of neuropathic pain, whereas SSRIs have been suggested to enhance the effectiveness of endogenous pain inhibition systems, and both have been effective the in treatment of various chronic pain disorders. Despite the analgesic effects of these agents, some authors have cited the lack of evidence based on well designed large clinical trials of these agents in IBS as reason for caution.

Gammaa Aminobutyric acid (GABA) analog: pregabalin

Pregabalin is a novel second-generation α2δ ligand that is structurally related to ϒ-aminobutyric acid (GABA). It has been shown to be effective in the treatment of inflammatory and neuropathic pain. Its precise mechanism of action is incompletely understood because it does not appear to have GABA-related functional activity or metabolites; it is believed to decrease depolarization-induced calcium influx at nerve terminals, and thereby inhibit release of excitatory neurotransmitters by acting on the α2δ auxiliary proteins associated with voltage-gated calcium channels. Its potential role in IBS is based upon a recent study demonstrating normalization of rectal distension sensory thresholds in IBS patients with rectal hypersensitivity. Placebo-controlled trials of pregabalin for IBS are currently ongoing.

Potential advances in the visceral modulation of IBS have been seen through studies of the role of opiate receptors in visceral pain. Specifically, peripheral K-opioid receptor agonists are of great interest because they are involved in the inhibition of noxious stimuli from the gut and are devoid of many of the adverse side effects (e.g. constipation, opioid dependence) seen in other opioid agonists that bind to µ receptors; K receptors are found most abundantly in the stomach and colon and in the brain. Asimadoline, a novel selective K-opioid receptor agonist, may be promising in the treatment of IBS. Its low blood–brain barrier permeability and low distribution in the central nervous system (CNS) suggest that its analgesic effects are mediated by reduction of excitability of nociceptors on peripheral nerve endings. Human pharmacodynamics studies of asimadoline demonstrated attenuation of visceral sensation without affecting gut motor function, a decrease in satiation and postprandial fullness independent of effects on gastric volume, and attenuation of pain intensity to colonic distension in IBS subjects. These findings led to the investigation of the possible role of asimadoline in IBS.


Read Full Post »

Reporter: Aviva Lev-Ari, PhD, RN


The healing element is also the enemy – an enigma probed by Hebrew University Lautenberg Center researchers

April 3, 2013

Jerusalem – The same factor in our immune system that is instrumental in enabling us to fight off severe and dangerous inflammatory ailments is also a player in doing the opposite at a later stage, causing the suppression of our immune response.

Why and how this happens and what can be done to mediate this process for the benefit of mankind is the subject of an article published online in the journal Immunity by Ph.D. student Moshe Sade-Feldman and Professor Michal Baniyash of the Lautenberg Center for General and Tumor Immunology at The Hebrew University Faculty of Medicine.
Chronic inflammation poses a major global health problem and is common to different pathologies — such as autoimmune diseases (diabetes, rheumatoid arthritis, lupus and Crohn’s), chronic inflammatory disorders, chronic infections (HIV, leprosy, leishmaniasis) and cancer. Cumulative data indicate that at a certain stage of each of these diseases, the immune system becomes suppressed and results in disease progression.
In their previous work, The Hebrew University researchers had shown that in the course of chronic inflammation, unique immune system cells with suppressive features termed myeloid derived suppressor cells (MDSCs) are generated in the bone marrow and migrate into the body’s organs and blood, imposing a general immune suppression.
A complex network of inflammatory compounds persistently secreted by the body’s normal or cancerous cells support MDSC accumulation, activation and suppressive functions. One of these compounds is tumor necrosis factor-a (TNF-a), which under acute immune responses (short episodes), displays beneficial effects in the initiation of immune responses directed against invading pathogens and tumor cells.
However, TNF-a also displays harmful features under chronic responses, as described in pathologies such as rheumatoid arthritis, psoriasis, type II diabetes, Crohn’s disease and cancer, leading to complications and disease progression. Therefore, today several FDA- approved TNF-a blocking reagents are used in the clinic for the treatment of such pathologies.
What has remained unclear until now, however, is just how TNF-a plays its deleterious role in manipulating the host’s immune system towards the generation of a suppressive environment.
In their work, The Hebrew University researchers discovered the mechanisms underlying the TNF-a  function, a key to controlling this factor and manipulating it, perhaps, for the benefit of humans.  Using experimental mouse models, they showed unequivocally how TNF-a is critical in the induction of immune suppression generated during chronic inflammation. The TNF-a was seen to directly affect the accumulation and suppressive function of MDSCs, leading to an impaired host’s immune responses as reflected by the inability to respond against invading pathogens or against developing tumors.
Further, the direct role of how TNF-a works in humans was mimicked by injecting the FDA-approved anti-TNF-a drug, etanercept, into mice at the exacerbated stage of an inflammatory response, when MDSC accumulation was observed in the blood. The etanercept treatment changed the features of MDSCs and abolished their suppressive activity, leading to the restoration of the host’s immune function.
Taken together, the results show clearly how the TNF-a-mediated inflammatory response, whether acute or chronic, will dictate its beneficial or harmful consequence on the immune system. While during acute inflammation TNF-a is vital for immediate immune defense against pathogens and clearance of tumor cells, during chronic inflammation — under conditions where the host is unable to clear the pathogen or the tumor cells — TNF-a is harmful due to the induction of immune suppression.
These results, providing new insight into the relationship between TNF-a and the development of an immune suppression during chronic inflammation, may aid in the generation of better therapeutic strategies against various pathologies when elevated TNF-a and MDSC levels are detected, as seen, for example, in tumor growths.


Read Full Post »

Breakthrough Digestive Disorders Research: Conditions affecting the Gastrointestinal Tract.

Reporter: Aviva Lev-Ari, PhD, RN


Forthcoming Electronic Book on

Metabolism and MetabolOMICS, 2013

Larry H. Bernstein, MD, FCAP and Ritu Saxena, Ph.D., Editors

Book will cover innovations in

  • Digestive Disorders GENOMICS,
  • Pharmaco-Therapy for gut infalmmation,
  • Genetic Immunology,
  • Enzymatic-therapy,
  • Bacterial infection in the gut and pharmaco-therapies
  • Cancer Biology and Therapy

of the following most common digestive disorders today

In the meantime, we are sharing the encouraging news, that is, that the symptoms of digestive disorders can be alleviated, and often completely eliminated, with the right combination of medication, dietary changes, exercise, weight loss, stress reduction and surgery.

It’s all detailed in an important new research report from Johns Hopkins — rated #1 of America’s best hospitals for 21 consecutive years 1991-2011 by U.S. News & World Report.

The 2013 Johns Hopkins Digestive Disorders White Paper

Johns Hopkins Digestive Disorders White Paper

Your Digestive Expert, H. Franklin Herlong, M.D. Adjunct Professor of MedicineJohns Hopkins University School of Medicine

The expertise you need, in language you can understand and use

In The 2013 Johns Hopkins Digestive Disorders White Paper, you will discover exciting advances and the most useful, current information to help you prevent or treat conditions affecting the digestive tract.

You’ll find a thorough overview of what the medical field knows about upper and lower digestive tract disorders (including everything from gastroesophageal reflux disease [GERD] to peptic ulcers, and irritable bowel syndrome to colorectal polyps) and conditions that affect the liver, gallbladder and pancreas.

You will learn how to prevent these diseases and, when symptoms arise, the best ways for you and your doctor to diagnose and treat them. The Johns Hopkins White Papers redefine the term “informed consumer.” In The 2013 Johns Hopkins Digestive Disorders White Paper, specialists from Johns Hopkins University School of Medicine report in depth on the latest digestive disorders prevention strategies and treatments. Thousands of Americans rely on Johns Hopkins expertise to help them manage their digestive disorders.

In The 2013 Johns Hopkins Digestive Disorders White Paper you’ll get a thorough overview of what the medical field knows about the most common digestive disorders today. You’ll find a wealth of news you can use about:

  • Celiac disease
  • Constipation
  • Crohn’s disease
  • Diarrhea
  • Diverticulosis and diverticulitis
  • Gallstones
  • Gastritis
  • GERD
  • Hiatal hernia
  • Irritable bowel syndrome
  • Ulcerative colitis
  • Ulcers

and more…

Timely Information Backed by Johns Hopkins Resources and Expertise

The symptoms of digestive disorders can be alleviated, and often completely eliminated, with the right combination of medication, dietary changes, exercise, weight loss, stress reduction and, as a last resort, surgery.

Learning as much as possible about the causes, effects and treatments for your digestive disorder is the first step toward living a fuller life with minimal discomfort and physical limitations.

The 2013 Johns Hopkins Digestive Disorders White Paper is designed to help you ensure the best outcome. Use what you learn to help you:

  • Recognize and respond to symptoms and changes as they occur.
  • Communicate effectively with your doctor, ask informed questions and understand the answers.
  • Make the right decisions, based on an understanding of the newest drugs, the latest treatments and the most promising research.
  • Take control over your condition and act out of knowledge rather than fear.

Tips for optimal digestive health

  • Maybe It’s Not “Just Heartburn”: Occasional heartburn can be treated with over-the-counter antacids. But if you have any of these symptoms, talk to your doctor to rule out more serious problems.
  • Should You Try Probiotics? Evidence is mounting that these “friendly bacteria” can help treat many digestive problems, such as IBS and Crohn’s disease. See how they work and are used, and whether they might relieve your gastrointestinal issues.
  • New Ways to Look Inside: The benefits and drawbacks of patient-friendly imaging tools including the “video pill” and virtual colonoscopy. How do state-of-the-art tools compare with established diagnostic exams?
  • Making Friends with Fiber: Getting enough dietary fiber is an easy way to prevent or treat a wide variety of digestive complaints. See which foods deliver the most fiber.
  • How to Avoid a Foodborne Illness: Follow these guidelines to choose, store, prepare and serve food in ways that minimize the health risks that result in 76 million infections and 325,000 hospitalizations annually.


Read Full Post »

Nitric Oxide, Platelets, Endothelium and Hemostasis (Coagulation Part II)

Curator: Larry H. Bernstein, MD, FCAP 

Subtitle: Nitric oxide and hemostatic mechanisms.  Part II.

Summary: This is the second of a coagulation series on

Treating the diverse effects of NO on platelets, the coagulation cascade, and protein-membrane interactions with low flow states, local and systemic inflammatory disease, oxidative stress, and hematologic disorders.  It is highly complex as the distinction between intrinsic and extrinsic pathways become blurred as a result of  endothelial shear stress, distinctly different than penetrating or traumatic injury.  In addition, other factors that come into play are also considered.

Please refer to Part I. Coagulation Pathway

The workhorse tests of the modern coagulation laboratory, the prothrombin time (PT) and the activated partial thromboplastin time (aPTT), are the basis for the published extrinsic and intrinsic coagulation pathways.  This is, however, a much simpler model than one encounters delving into the mechanism and interactions involved in hemostasis and thrombosis, or in hemorrhagic disorders.

We first note that there are three components of the hemostatic system in all vertebrates:

  • Platelets,
  • vascular endothelium, and
  • plasma proteins.

The liver is the largest synthetic organ, which synthesizes

  • albumin,
  • acute phase proteins,
  • hormonal and metal binding proteins,
  • albumin,
  • IGF-1, and
  • prothrombin, mainly responsible for the distinction between plasma and serum (defibrinated plasma).

Role of vascular endothelium.

I have identified the importance of prothrombin, thrombin, and the divalent cation Ca 2+ (1% of the total body pool), mention of heparin action, and of vitamin K (inhibited by warfarin).  Endothelial functions are inherently related to procoagulation and anticoagulation. The subendothelial matrix is a complex of many materials, most important related to coagulation being collagen and von Willebrand factor.

What about extrinsic and intrinsic pathways?  Tissue factor, when bound to factor VIIa, is the major activator of the extrinsic pathway of coagulation. Classically, tissue factor is not present in the plasma but only presented on cell surfaces at a wound site, which is “extrinsic” to the circulation.  Or is it that simple?

Endothelium is the major synthetic and storage site for von Willebrand factor (vWF).  vWF is…

  • secreted from the endothelial cell both into the plasma and also
  • abluminally into the subendothelial matrix, and
  • acts as the intercellular glue binding platelets to one another and also to the subendothelial matrix at an injury site.
  • acts as a carrier protein for factor VIII (antihemophilic factor).
  • It  binds to the platelet glycoprotein Ib/IX/V receptor and
  • mediates platelet adhesion to the vascular wall under shear. [Lefkowitz JB. Coagulation Pathway and Physiology. Chapter I. in Hemostasis Physiology. In ( ???), pp1-12].

Ca++ and phospholipids are necessary for all of the reactions that result in the activation of prothrombin to thrombin. Coagulation is initiated by an extrinsic mechanism that

  • generates small amounts of factor Xa, which in turn
  • activates small amounts of thrombin.

The tissue factor/factorVIIa proteolysis of factor X is quickly inhibited by tissue factor pathway inhibitor (TFPI).The small amounts of thrombin generated from the initial activation feedback

  • to create activated cofactors, factors Va and VIIIa, which in turn help to
  • generate more thrombin.
  • Tissue factor/factor VIIa is also capable of indirectly activating factor X through the activation of factor IX to factor IXa.
  • Finally, as more thrombin is created, it activates factor XI to factor XIa, thereby enhancing the ability to ultimately make more thrombin.

The reconceptualization of hemostasis 

The common theme in activation and regulation of plasma coagulation is the reduction in dimensionality. Most reactions take place in a 2D world that will increase the efficiency of the reactions dramatically. The localization and timing of the coagulation processes are also dependent on the formation of protein complexes on the surface of membranes. The coagulation processes can also be controlled by certain drugs that destroy the membrane binding ability of some coagulation proteins – these proteins will be lost in the 3D world and not able to form procoagulant complexes on surfaces.

Assembly of proteins on membranes – making a 3D world flat

• The timing and efficiency of coagulation processes are handled by reduction in dimensionality

– Make 3 dimensions to 2 dimensions

• Coagulation proteins have membrane binding capacity

• Membranes provide non-coagulant and procoagulant surfaces

– Intact cells/activated cells

• Membrane binding is a target for anticoagulant drugs

– Anti-vitamin K (e.g. warfarin)

Modern View

It can be divided into the phases of initiation, amplification and propagation.

  • In the initiation phase, small amounts of thrombin can be formed after exposure of tissue factor to blood.
  • In the amplification phase, the traces of thrombin will be inactivated or used for amplification of the coagulation process.

At this stage there is not enough thrombin to form insoluble fibrin. In order to proceed further thrombin  activates platelets, which provide a procoagulant surface for the coagulation factors. Thrombin will also activate the vital cofactors V and VIII that will assemble on the surface of activated platelets. Thrombin can also activate factor XI, which is important in a feedback mechanism.

In the final step, the propagation phase, the highly efficient tenase and prothrombinase complexes have been assembled on the membrane surface. This yields large amounts of thrombin at the site of injury that can cleave fibrinogen to insoluble fibrin. Factor XI activation by thrombin then activates factor IX, which leads to the formation of more tenase complexes. This ensures enough thrombin is formed, despite regulation of the initiating TF-FVIIa complex, thus ensuring formation of a stable fibrin clot. Factor XIII stabilizes the fibrin clot through crosslinking when activated by thrombin.

Platelet Aggregation

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3′:5′-phosphodiesterase were determined during the aggregation of human blood platelets with

  • thrombin, ADP,
  • arachidonic acid and
  • epinephrine.

[Aggregation is dependent on an intact release mechanism since inhibition of aggregation occurred with adenosine, colchicine, or EDTA.  (Herman GE, Seegers WH, Henry RL. Autoprothrombin ii-a, thrombin, and epinephrine: interrelated effects on platelet aggregation. Bibl Haematol 1977;44:21-7)].

  1. The platelet guanylate cyclase activity during aggregation depends on the nature and mode of action of the inducing agent.
  2. The membrane adenylate cyclase activity during aggregation is independent of the aggregating agent and is associated with a reduction of activity and
  3. Cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release.

The role of platelets in arterial thrombosis

Formation of a thrombus on a ruptured plaque is the product of a complex interaction between coagulation factors in the plasma and platelets.

  • Tissue factor (TF) released from the subendothelial tissue after endothelial damage induces a cascade of activation of coagulation factors ultimately leading to the formation of thrombin.
  • Thrombin cleaves fibrinogen to fibrin, which assembles into a mesh that supports the platelet aggregates.

The Platelet

The platelets are …

  • anucleated,
  • discoid shaped cell fragments
  • originating from megakaryocytes
  •  fragmented as they are released from the bone marrow

Whether they can in circumstances be developed at extramedullary sites (liver sinusoid) is another matter. They have a lifespan of 7-10 days.  Of special interest is:

  • They have a network of internal membranes forming a dense tubular system and the open canalicular system (OCS).
  • The plasma membrane is an extension of the OCS, thereby greatly increasing the surface area of the platelet.
  • The dense tubular system is comparable to the endoplasmatic reticulum in other cell types and is the main storage place of the majority of the platelet’s Ca2+.

Three types of secretory granules exist in platelets:

  • the dense granules
    •  In the dense granules serotonin
    • adenosine diphosphate (ADP) and
    • Ca2+ are stored.
    • a-granules contain
      • P-selectin,
      • fibrinogen,
      •  thrombospondin,
      • Von Willebrand Factor,
      • platelet factor 4 and
      • platelet derived growth factor
      • lysosomes.

Circulating platelets are kept in a resting state by endothelial cell derived

  • prostacyclin (PGI2) and
  • nitric oxide (NO).

PGI2 increases cyclic adenosine monophosphate (cAMP), the most potent platelet inhibitor.

Contact activation

The major regulator of the activation of the contact system is the plasma protease inhibitor, C1-INH, which inhibits activated fXII, kallikrein and fXIa. In addition, α2-macroglobulin is an important inhibitor of kallikrein and α1-antitrypsin for fXIa. Factor XII also converts the fXI to an active enzyme, fXIa, which, in turn, converts fIX to fIXa, thereby activating the intrinsic pathway of coagulation.


Several agonists can activate platelets;

  • ADP,
  • collagen,
  • thromboxane A2 (TxA2),
  • epinephrin,
  • serotonine and
  • thrombin,

which lead to activation previously referred to:

  • platelet shape change is
  • followed by aggregation and
  • granule secretion.

Upon activation the discoid shape changes into a spherical form.

Activation of platelets is increased by two positive feedback loops

  1. arachidonic acid is cleaved from phospholipids and transformed by cyclooxygenase

(COX) to prostaglandin G2 and H2,

  • followed by the formation of TxA2, a potent platelet agonist.

2.   the secretion of ADP by the dense granules,

  • resulting in activation of the ADP receptor P2Y12.

This causes inhibition of cyclic AMP and sustained aggregation.


The integrin receptor αIIbβ3 plays a vital role in platelet aggregation. The platelet agonists

  • induce a conformational change of the αIIbβ3 receptor and
  • exposition of binding domains for fibrinogen and von Willebrand Factor.

This allows cross-linking of platelets and the formation of aggregates.

In addition to shape change and aggregation, the membranes of the α- and dense granules fuse with the membranes of the OCS. This causes the release of their contents and the transportation of proteins embedded in their membrane to the plasma membrane.

This complex interaction between

  • endothelial cells
  • clotting factors
  • platelets and
  • other factors and cells

can be studied in both in vitro and in vivo model systems. The disadvantage of in vitro assays is that it studies the role of a certain protein or cell in isolation. Given the large number of participants and the complex interactions of thrombus formation there is need to study thrombosis and hemostasis in intact living animals, with all the components important for thrombus formation – a vessel wall and flowing blood – present.

Endothelial Damage and Role as “Primer”

  • Endothelial injury changes the permeability of the arterial wall.
  • This is followed by an influx of low-density lipoprotein (LDL).
  • This elicits an inflammatory response in the vascular wall.
  • Monocytes and T-cells bind to the endothelial cells promoting increased migration of the cells into the intima layer
  • The monocytes differentiate into macrophages, which take up modified lipoproteins and transform them into foam cells.
  • Concurrent with this process macrophages produce cytokines and proteases.

This is a vicious circle of lipid driven inflammation that leads to narrowing of the vessel’s lumen without early clinical consequences. Clinical manifestations of more advanced atherosclerotic disease are caused by destabilization of an atherosclerotic plaque formed as described.

  • The first recognizable lesion of the stable atherosclerotic plaque is the fatty streak, which consists of the above described foam cells and T-lymphocytes in the intima.
  • Further development of the lesion leads to the intermediate lesion, composed
  • of layers of macrophages and smooth muscle cells.
  • A more advanced stage is called the vulnerable plaque.
    • It has a large lipid core that is covered by a thin fibrous cap.
    • This cap separates the lipid contents of the plaque from the circulating blood.
    • The vulnerable plaque is prone to rupture, resulting in the formation of a thrombus on the site of disruption or the thrombus can be superimposed on plaque erosion without signs of plaque rupture.

The formation of a superimposed thrombus on a disrupted atherosclerotic plaque in the lumen of the artery leads to

  • an acute occlusion of the vessel
  • hypoxia of the downstream tissue.

Depending on the location of the atherosclerotic plaque this will cause a myocardial infarction, stroke or peripheral vascular disease.

Endothelial regulation of coagulation

The endothelium attenuates platelet activity by releasing

  • nitric oxide and
  • prostacyclin.

Several coagulation inhibitors are produced by endothelial cells.

Endothelium-derived TFPI (on its surface) is rapidly released into circulation after heparin administration, reducing the pro-coagulant activities of TF-fVIIa. Endothelial cells also secrete heparin-sulphate, a glycosaminoglycan which catalyzes anti-coagulant activity of AT. Plasma AT binds to heparin-sulphate located on the luminal surface and in the basement membrane of the endothelium. Thrombomodulin is another endothelium-bound protein with anti-coagulant and anti-inflammatory functions. In response to systemic pro-coagulant stimuli, tissue-type plasminogen activator (tPA) is transiently released from the Weibel-Palade bodies of endothelial cells to promote fibrinolysis. Downstream of the vascular injury, the complex of TF-fVIIa/fXa is inhibited by TFPI. Plasma (free) fXa and thrombin are rapidly neutralized by heparan-bound AT. Thrombin is also taken up by endothelial surface-bound thrombomodulin.

The protein C pathway works in hemostasis to control thrombin formation in the area surrounding the clot. Thrombin, generated via the coagulation pathway, is localized to the endothelium by binding to the integral membrane protein, thrombomodulin (TM). TM by occupying exosite I on thrombin, which is required for fibrinogen binding and cleavage, reduces thrombin’s pro-coagulant activities. TM bound thrombin  on the endothelial cell surface is able to cleave PC producing activated protein C (APC), a serine protease.  In the presence of protein S, APC inactivates FVa and FVIIIa. The proteolytic activity of APC is regulated predominantly by a protein C inhibitor.

Fibrinolytic pathway

Fibrinolysis is the physiological breakdown of fibrin to limit and resolve blood clots. Fibrin is degraded primarily by the serine protease, plasmin, which circulates as plasminogen. In an auto-regulatory manner, fibrin serves as both the co-factor for the activation of plasminogen and the substrate for plasmin. In the presence of fibrin, tissue plasminogen activator (tPA) cleaves plasminogen producing plasmin, which proteolyzes the fibrin. This reaction produces the protein fragment D-dimer, which is a useful marker of fibrinolysis, and a marker of thrombin activity because fibrin is cleaved from fibrinogen to fibrin.

Nitric Oxide and Platelet Energy Production

Nitric oxide (NO) has been increasingly recognized as an important intra- and intercellular messenger molecule with a physiological role in

  • vascular relaxation
  • platelet physiology
  • neurotransmission and
  • immune responses.

In vitro NO is a strong inhibitor of platelet adhesion and aggregation. In the blood stream, platelets remain in contact with NO that is permanently released from the endothelial cells and from activated macrophages. It  has been suggested that the activated platelet itself is able to produce NO. It has been proposed that the main intracellular target for NO in platelets is soluble cytosolic guanylate cyclase. NO activates the enzyme. When activated, intracellular cGMP elevation inhibits platelet activation. Further, elevated cGMP may not be the sole factor directly involved in the inhibition of platelet activation.

The reaction mechanism of Nitric oxide synthase

The reaction mechanism of Nitric oxide synthase (Photo credit: Wikipedia)

Platelets are fairly active metabolically and have a total ATP turnover rate of about 3–8 times that of resting mammalian muscle. Platelets contain mitochondria which enable these cells to produce energy both in the oxidative and anaerobic pathways.

  • Under aerobic conditions, ATP is produced by aerobic glycolysis which can account for 30–50% of total ATP production,
  • by oxidative metabolism using glucose and glycogen (6–11%), amino-acids (7%) or free fatty acids (20–40%).

The inhibition of mitochondrial respiration by removing oxygen or by respiratory chain blockers (antimycin A, cyanide, rotenone) results in the stimulation of glycolytic flux. This phenomenon indicates that in platelets glycolysis and mitochondrial respiration are tightly functionally connected. It has been reported that the activation of human platelets by high concentration of thrombin is accompanied by an acceleration of lactate production and an increase in oxygen consumption.

The results (in porcine platelets) indicate that:

  • NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase
  • The inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.

Porcine blood platelets stimulated by collagen produce more lactate. This indicates that both glycolytic and oxidative ATP production supports platelet responses, and blocking of energy production in platelets may decrease their responses. It is well established that platelet responses have different metabolic energy (ATP) requirements increasing in the order:

  • Aggregation
  • < dense and alfa granule secretion
  • < acid hydrolase secretion.

In addition, exogenously added NO (in the form of NO donors) stimulates glycolysis in intact porcine platelets. Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected, this indicates the stimulatory effect of NO on glycolysis in intact platelets may be produced by non-functional mitochondria.

Can this be the case?

  • NO donors are able to inhibit both mitochondrial respiration and platelet cytochrome oxidase.
  • Interestingly, the concentrations of NO donors inhibiting mitochondrial respiration and cytochrome oxidase were similar to those stimulating glycolysis in intact platelets.

Studies have shown that mitochondrial complex I is inhibited only after a prolonged (6–18 h) exposure to NO and

  • This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex.
  • Further studies are needed to establish whether long term exposure of platelets to NO affects Mitochondrial complexes I and II.

Comparison of the concentrations of SNAP and SNP affecting cytochrome oxidase activity and mitochondrial respiration with those reducing the platelet responses indicates that NO does not reduce platelet aggregation through the inhibition of oxidative energy production. The concentrations of the NO donors inhibiting platelet secretion, mitochondrial respiration and cytochrome oxidase were similar. Thus, the platelet release reaction strongly depends on the oxidative energy production, and  in porcine platelets NO inhibits mitochondrial energy production at the step of cytochrome oxidase.

Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production.

Key words: glycolysis, mitochondrial energy production, nitric oxide, porcine platelets.
Abbreviations: NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicyllamine; SNP, sodium nitroprusside.

[M Tomasiak, H Stelmach, T Rusak and J Wysocka.  Nitric oxide and platelet energy metabolism.  Acta Biochimica Polonica 2004; 51(3):789–803.]

Nitric Oxide and Platelet Adhesion

The adhesion of human platelets to monolayers of bovine endothelial cells in culture was studied to determine the role of endothelium-derived nitric oxide in the regulation of platelet adhesion. The adhesion of unstimulated and thrombin-stimulated platelets, washed and labelled with indium-111, was lower in the presence than in the absence of bradykinin or exogenous nitric oxide. The inhibitory action of both bradykinin and nitric oxide was abolished by hemoglobin, but not by aspirin, and was potentiated by superoxide dismutase to a similar degree. It appears that the effect of bradykinin is mediated by the release of nitric oxide from the endothelial cells, and that nitric oxide release contributes to the non-adhesive properties of vascular endothelium.

(Radomski MW, Palmer RMJ, Moncada S.   Endogenous Nitric Oxide Inhibits Human Platelet Adhesion to Vascular Endothelium. The Lancet  1987 330; 8567(2): 1057–1058.

1 The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined to determine whether release of NO accounts for the inhibition of platelet aggregation attributed to EDRF.

2 Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10-100 nM) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium-derived relaxing factor (EDRF).

3 In the absence of indomethacin, stimulation of the cells with bradykinin (1-3 nM) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation.

4 EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619.

5 A reciprocal potentiation of both the anti- and the disaggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells.

6 It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.

(Radomski MW, Palmer RMJ & Moncada S. The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmac 1987; 92: 639-646.


Factor Xa–Nitric Oxide Signaling

Although primarily recognized for maintaining the hemostatic balance, blood proteases of the coagulation and fibrinolytic cascades elicit rapid cellular responses in

  • vascular
  • mesenchymal
  • inflammatory cell types.

Considerable effort has been devoted to elucidate the molecular interface between protease-dependent signaling and pleiotropic cellular responses. This led to the identification of several membrane protease receptors, initiating intracellular signal transduction and effector functions in vascular cells. In this context, thrombin receptor activation

  • generated second messengers in endothelium and smooth muscle cells,
  • released inflammatory cytokines from monocytes, fibroblasts, and endothelium, and
  • increased the expression of leukocyte-endothelial cell adhesion molecules.

Similarly, binding of factor Xa to effector cell protease receptor-1 (EPR-1) participated in

  • in vivo acute inflammatory responses,
  • platelet and brain pericyte prothrombinase activity, and
  • endothelial cell and smooth muscle cell signaling and proliferation.

Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1–2.5 mgyml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective.

A factor Xa inter-epidermal growth factor synthetic peptide L83FTRKL88(G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective.

Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-NG-nitroarginine methyl ester (LNAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55–3.1 ngyml) in a reaction

  • inhibited by L-NAME and by the
  • inter-epidermal growth factor peptide Leu83–Leu88 but
  • unaffected by hirudin.
We observe that incubation of HUVEC monolayers with factor Xa which resulted in a concentration-dependent release of NO, as determined by cGMP accumulation in these cells, was inhibited by the nitric oxide synthase antagonist L-NAME.

Catalytically inactive DEGR-factor Xa or TAP-treated factor Xa failed to stimulate NO release by HUVEC.

To determine whether factor Xa-induced NO release could also modulate acute phase/inflammatory cytokine gene expression we examined potential changes in IL-6 release following HUVEC stimulation with factor Xa. HUVEC stimulation with factor Xa resulted in a concentration-dependent release of IL-6.

The specificity of factor Xa-induced cytokine release was investigated. Factor Xa-induced IL-6 release from HUVEC was quantitatively indistinguishable from that obtained with tumor necrosis factor-a or thrombin stimulation. This response was abolished by heat denaturation of factor Xa.

Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, and was unaffected by subsequent addition of tissue factor pathway inhibitor (TFPI). These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and IL-6 cytokine gene expression.

Here, we find that factor Xa induces the release of endothelial cell NO

  • regulating vasorelaxation in vivo and acute response cytokine gene expression in vitro.

This pathway requires a dual step cascade, involving

  • binding of factor Xa to EPR-1 and
  • a secondary as yet unidentified protease activated mechanism.

This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.

In summary, these investigators have identified a signaling pathway centered on the ability of factor Xa to rapidly stimulate endothelial cell NO release. This involves a two-step cascade initiated by catalytic active site-independent binding of factor Xa to its receptor, EPR-1, followed by a second step of ligand dependent proteolysis.

(Papapetropoulos A, Piccardoni P, Cirino G, Bucci M, et al. Hypotension and inflammatory cytokine gene expression triggered by factor Xa–nitric oxide signaling. Proc. Natl. Acad. Sci. USA. Pharmacology. 1998; 95:4738–4742.)

Platelets and liver disease

Thrombocytopenia is a marked feature of chronic liver disease and cirrhosis. Traditionally, this thrombocytopenia was attributed to passive platelet sequestration in the spleen. More recent insights suggest an increased platelet breakdown and to a lesser extent decreased platelet production plays a more important role. Besides the reduction in number, other studies suggest functional platelet defects. This platelet dysfunction is probably both intrinsic to the platelets and secondary to soluble plasma factors. It reflects not only a decrease in aggregability, but also an activation of the intrinsic inhibitory pathways. (Witters P, Freson K, Verslype C, Peerlinck K, et al. Review article: blood platelet number and function in chronic liver disease and cirrhosis. Aliment Pharmacol Ther 2008; 27: 1017–1029).

The shortcomings of the old Y-shaped model of normal coagulation are nowhere more apparent than in its clinical application to the complex coagulation disorders of acute and chronic liver disease. In this condition, the clotting cascade is heavily influenced by numerous currents and counter-currents resulting in a mixture of pro- and anticoagulant forces that are themselves further subject to change with altered physiological stress such as super-imposed infection or renal failure.

Multiple mechanisms exist for thrombocytopenia common in patients with cirrhosis besides hypersplenism and expected altered thrombopoietin metabolism. Increased production of two important endothelial derived platelet inhibitors

  • nitric oxide and
  • prostacyclin

may contribute to defective platelet activation in vivo. On the other hand, high plasma levels of vWF in cirrhosis appear to support platelet adhesion.

Reduced levels of coagulation factors V, VII, IX, X, XI, and prothrombin are also commonly observed in liver failure. Vitamin K–dependent clotting factors (II, VII, IX, X) may be defective in function as a result of decreased  y-carboxylation (from vitamin K deficiency or intrinsically impaired carboxylase activity). Fibrinogen levels are decreased with advanced cirrhosis and in patients with acute liver failure.

A hyperfibrinolytic state may develop when plasminogen activation by tPA is accelerated on the fibrin surface. Physiologic stress including infection may be key in tipping this process off through increased release of tPA.  Not uncommonly, laboratory abnormalities in decompensated cirrhosis come to resemble disseminated intravascular coagulation (DIC). Relatively stable platelet levels and characteristically high factor VIII levels distinguish this process from DIC as does the absence of uncompensated thrombin generation. The features of both hyperfibrinolysis and DIC are often evident in the decompensated liver disease patient, and the term “accelerated intravascular coagulation and fibrinolysis” (AICF) has been proposed as a way to encapsulate the process under a single heading. The essence of AICF can be postulated to be the result of formation of a fibrin clot that is more susceptible to plasmin degradation due to elevated levels of tPA coupled with inadequate release of PAI to control tPA and lack of a-2 plasmin inhibitor to quench plasmin activity and the maintenance of high local concentrations of plasminogen on clot surfaces despite lower total plasminogen production. These normally balanced processes become pronounced when disturbed by additional stress such as infection.

Normal hemostasis and coagulation is now viewed as primarily a cell-based process wherein key steps in the classical clotting cascade

  • occur on the phospholipid membrane surface of cells (especially platelets)
  • beginning with activation of tissue factor and factor VII at the site of vascular breach
    •  which produces an initial “priming” amount of thrombin and a
    • subsequent thrombin burst.

Coagulation and hemostasis in the liver failure patient is influenced by multiple, often opposing, and sometimes changing variables. A bleeding diathesis is usually predominant, but the assessment of bleeding risk based on conventional laboratory tests is inherently deficient.

(Caldwell SH, Hoffman M, Lisman T, Gail Macik B, et al. Coagulation Disorders and Hemostasis in Liver Disease: Pathophysiology and Critical Assessment of Current Management. Hepatology 2006;44:1039-1046.)

Bleeding after Coronary Artery bypass Graft

Cardiac surgery with concomitant CPB can profoundly alter haemostasis, predisposing patients to major haemorrhagic complications and possibly early bypass conduit-related thrombotic events as well. Five to seven percent of patients lose more than 2 litres of blood within the first 24 hours after surgery, between 1% and 5% require re-operation for bleeding. Re-operation for bleeding increases hospital mortality 3 to 4 fold, substantially increases post-operative hospital stay and has a sizeable effect on health care costs. Nevertheless, re-exploration is a strong risk factor associated with increased operative mortality and morbidity, including sepsis, renal failure, respiratory failure and arrhythmias.

(Gábor Veres. New Drug Therapies Reduce Bleeding in Cardiac Surgery. Ph.D. Doctoral Dissertation. 2010. Semmelweis University)

Hypercoagulable State in Thalassemia

As the life expectancy of β-thalassemia patients has increased in the last decade, several new complications are being recognized. The presence of a high incidence of thromboembolic events, mainly in thalassemia intermedia patients, has led to the identification of a hypercoagulable state in thalassemia. Patients with thalassemia intermedia (TI) have, in general, a milder clinical phenotype than those with TM and remain largely transfusion independent. The pathophysiology of TI is characterized by extravascular hemolysis, with the release into the peripheral circulation of damaged red blood cells (RBCs) and erythroid precursors because of a high degree of ineffective erythropoiesis. This has also been recently attributed to severe complications such as pulmonary hypertension (PHT) and thromboembolic phenomena.

Many investigators have reported changes in the levels of coagulation factors and inhibitors in thalassemic patients. Prothrombin fragment 1.2 (F1.2), a marker of thrombin generation, is elevated in TI patients. The status of protein C and protein S was investigated in thalassemia in many studies and generally they were found to be decreased; this might be responsible for the occurrence of thromboembolic events in thalassemic patients.

The pathophysiological roles of hemolysis and the dysregulation of nitric oxide homeostasis are correlated with pulmonary hypertension in sickle cell disease and in thalassemia. Nitric oxide binds soluble guanylate cyclase, which converts GTP to cGMP, relaxing vascular smooth muscle and causing vasodilatation. When plasma hemoglobin liberated from intravascularly hemolyzed sickle erythrocytes consumes nitric oxide, the balance is shifted toward vasoconstriction. Pulmonary hypertension is aggravated and in sickle cell disease, it is linked to the intensity of hemolysis. Whether the same mechanism contributes to hypercoagulability in thalassemia is not yet known.

While there are diverse factors contributing to the hypercoagulable state observed in patients with thalassemia. In most cases, a combination of these abnormalities leads to clinical thrombosis. An argument has been made for the a higher incidence of thrombotic events in TI compared to TM patients  attributed to transfusion for TM. The higher rate of thrombosis in transfusion-independent TI compared to polytransused TM patients suggests a potential role for transfusions in decreasing the rate of thromboembolic events (TEE). The reduction of TEE in adequately transfused patients may be the result of decreased numbers of pathological RBCs.

(Cappellini MD, Musallam KM,  Marcon A, and Taher AT. Coagulopathy in Beta-Thalassemia: Current Understanding and Future Prospects. Medit J Hemat Infect Dis 2009; 1(1):22009029.
DOI 10.4084/MJHID.2009.0292.0),  ISSN 2035-3006.)

Microvascular Endothelial Dysfunction

Severe sepsis, defined as sepsis associated with acute organ dysfunction, results from a generalized inflammatory and procoagulant host response to infection. Coagulopathy in severe sepsis is commonly associated with multiple organ dysfunction, and often results in death. The molecule that is central to these effects is thrombin, although it may also have anticoagulant and antithrombotic effects through the activation of Protein C and induction of prostacyclin. In recent years, it has been recognized that chemicals produced by endothelial cells play a key role in the pathogenesis of sepsis. Thrombomodulin on endothelial cells coverts Protein C to Activated Protein C, which has important antithrombotic, profibrinolytic and anti-inflammatory properties. A number of studies have shown that Protein C levels are reduced in patients with severe infection, or even in inflammatory states without infection. Because coagulopathy is associated with high mortality rates, and animal studies have indicated that therapeutic intervention may result in improved outcomes, it was rational to initiate clinical studies.

Considering the coagulation cascade as a whole, it is the extrinsic pathway (via TF and thrombin activation) rather than the intrinsic pathway that is of primary importance in sepsis. Once coagulation has been triggered by TF activation, leading to thrombin formation, this can have further procoagulant effects, because thrombin itself can activate factors VIII, IX and X. This is normally balanced by the production of anticoagulant factors, such as TF pathway inhibitor, antithrombin and Activated Protein C.

It has been recognized that endothelial cells play a key role in the pathogenesis of sepsis, and that they produce important regulators of both coagulation and inflammation. They can express or release a number of substances, such as TF, endothelin-1 and PAI-1, which promote the coagulation process, as well as other substances, such as antithrombin, thrombomodulin, nitric oxide and prostacyclin, which inhibit it.

Protein C is the source of Activated Protein C. Although Protein C is a biomarker or indicator of sepsis, it has no known specific biological activity. Protein C is converted to Activated Protein C in the presence of normal endothelium. In patients with severe sepsis, the vascular endothelium becomes damaged. The level of thrombomodulin is significantly decreased, and the body’s ability to convert Protein C to Activated Protein C diminishes. Only when activated does Protein C have antithrombotic, profibrinolytic and anti-inflammatory properties.

Blood Coagulation (Thrombin) and Protein C Pat...

Blood Coagulation (Thrombin) and Protein C Pathways (Blood_Coagulation_and_Protein_C_Pathways.jpg) (Photo credit: Wikipedia)

Coagulation abnormalities can occur in all types of infection, including both Gram-positive and Gram-negative bacterial infections, or even in the absence of infection, such as in inflammatory states secondary to trauma or neurosurgery. Interestingly, they can also occur in patients with localized disease, such as those with respiratory infection. In a study by Günther et al., procoagulant activity in bronchial lavage fluid from patients with pneumonia or acute respiratory distress syndrome was found to be increased compared with that from control individuals, with a correlation between the severity of respiratory failure and level of coagulant activity.

Severe sepsis, defined as sepsis associated with acute organ dysfunction, results from a generalized inflammatory and procoagulant host response to infection.  Once the endothelium becomes damaged, levels of endothelial thrombomodulin significantly decrease, and the body’s ability to convert Protein C to Activated Protein C diminishes. The ultimate cause of acute organ dysfunction in sepsis is injury to the vascular endothelium, which can result in microvascular coagulopathy.

(Vincent JL. Microvascular endothelial dysfunction: a renewed appreciation of sepsis pathophysiology.
Critical Care 2001; 5:S1–S5.

Endothelial Cell Dysfunction in Severe Sepsis

During the past decade a unifying hypothesis has been developed to explain the vascular changes that occur in septic shock on the basis of the effect of inflammatory mediators on the vascular endothelium. The vascular endothelium plays a central role in the control of microvascular flow, and it has been proposed that widespread vascular endothelial activation, dysfunction and eventually injury occurs in septic shock, ultimately resulting in multiorgan failure. This has been characterized in various models of experimental septic shock. Now, direct and indirect evidence for endothelial cell alteration in humans during septic shock is emerging.

The vascular endothelium regulates the flow of nutrient substances, diverse biologically active molecules and the blood cells themselves. This role of endothelium is achieved through the presence of membrane-bound receptors for numerous molecules, including proteins, lipid transporting particles, metabolites and hormones, as well as through specific junction proteins and receptors that govern cell–cell and cell–matrix interactions. Endothelial dysfunction and/or injury with subendothelium exposure facilitates leucocyte and platelet aggregation, and aggravation of coagulopathy. Therefore, endothelial dysfunction and/or injury should favour impaired perfusion, tissue hypoxia and subsequent organ dysfunction.

Anatomical damage to the endothelium during septic shock has been assessed in several studies. A single injection of bacterial lipopolysaccharide (LPS) has long been demonstrated to be a nonmechanical technique for removing endothelium. In endotoxic rabbits, observations tend to demonstrate that EC surface modification occurs easily and rapidly, with ECs being detached from the internal elastic lamina with an indication of subendothelial oedema.  Proinflammatory cytokines increase permeability of the ECs, and this is manifested approximately 6 hours after inflammation is triggered and becomes maximal over 12–24 hours as the combination of cytokines exert potentiating effects. Endothelial physical disruption allows inflammatory fluid and cells to shift from the blood into the interstitial space.

In sepsis

  • ECs become injured, prothrombotic and antifibrinolytic
  • They promote platelet adhesion
  • They promote leucocyte adhesion and inhibit vasodilation

An important point is that EC injury is sustained over time. In an endotoxic rabbit model, we demonstrated that endothelium denudation is present at the level of the abdominal aorta as early as after several hours following injury and persisted for at least 5 days afterward. After 21 days we observed that the endothelial surface had recovered. The de-endothelialized surface accounted for approximately 25% of the total surface.

Thrombomodulin and protein C activation at the microcirculatory level.

The endothelial cell surface thrombin (Th)-binding protein thrombomodulin (TM) is responsible for inhibition of thrombin activity. TM, when bound to Th, forms a potent protein C activator complex. Loss of TM and/or internalization results in Th–thrombin receptor (TR) interaction. Loss of TM and associated protein C activation represents the key event of decreased endothelial coagulation modulation ability and increased inflammation pathways.
( Iba T, Kidokoro A, Yagi Y: The role of the endothelium in changes in procoagulant activity in sepsis. J Am Coll Surg 1998; 187:321-329. Keywords: ATIII, antithrombin III; NF-κ, nuclear factor-κB; PAI,plasminogen activator inhibitor).

In order to test the role of the endothelial-derived relaxing factors NO and PGI2, we investigated, in dogs, the influence of a combination of NG-nitro-L-arginine methyl ester (an inhibitor of NO synthesis) and indomethacin (an inhibitor of PGI2 synthesis). In these dogs treated with indomethacin plus NG-nitro-L-arginine methyl ester, the severity of the oxygen extraction defect was lower than that observed in the deoxycholate-treated dogs, suggesting that other mediators and/or mechanisms may be involved in microcirculatory control during hypoxia. One of these mediators or mechanisms could be related to hyperpolarization. Membrane potential is an important determinant of vascular smooth muscle tone through its influence on calcium influx via voltage-gated calcium channels. Hyperpolarization (as well as depolarization) has been shown to be a means of cell–cell communication in upstream vasodilatation and microcirculatory coordination. It is important to emphasize that intercell coupling exclusively involves ECs.

Interestingly, it was recently shown that sepsis, a situation that is characterized by impaired tissue perfusion and abnormal oxygen extraction, is associated with abnormal inter-EC coupling and reduction in the arteriolar conducted response.  An intra-organ defect in blood flow related to abnormal vascular reactivity, cell adhesion and coagulopathy may account for impaired organ oxygen regulation and function. If specific classes of microvessels must or must not be perfused to achieve efficient oxygen extraction during limitation in oxygen delivery, then impaired vascular reactivity and vessel injury might produce a pathological limitation in supply. In sepsis, the inflammatory response profoundly alters circulatory homeostasis, and this has been referred to as a ‘malignant intravascular inflammation’ that alters vasomotor tone and the distribution of blood flow among and within organs. These mechanisms might coexist with other types of sepsis associated cell dysfunction. For example, data suggest that endotoxin directly impairs oxygen uptake in ECs and indicate the importance of endothelium respiration in maintaining vascular homeostasis under conditions of sepsis.

Consistent with the hypothesis that alteration in endothelium plays a major in the pathophysiology of sepsis, it was observed that chronic ecNOS overexpression in the endothelium of mice resulted in resistance to LPS-induced hypotension, lung injury and death . This observation was confirmed by another group of investigators, who used transgenic mice overexpressing adrenomedullin  – a vasodilating peptide that acts at least in part via an NO-dependent pathway. They demonstrated resistance of these animals to LPS-induced shock, and lesser declines in blood pressure and less severe organ damage than occurred in the control animals. It might therefore be of importance to favour ecNOS expression and function during sepsis. The recent negative results obtained with therapeutic strategies aimed at blocking inducible NOS with the nonselective NOS inhibitor NG-monomethyl-L-arginine in human septic shock further confirm the overall importance of favoring vessel dilatation.

(Vallet B. Bench-to-bedside review: Endothelial cell dysfunction in severe sepsis: a role in organ dysfunction?  Critical Care 2003; 7(2):130-138 (DOI 10.1186/cc1864). (Print ISSN 1364-8535; Online ISSN 1466-609X).

Thrombosis in Inflammatory Bowel Disease

An association between IBD and thrombosis has been recognized for more than 60 years. Not only are patients with IBD more likely to have thromboembolic complications, but it has also been suggested that thrombosis might be pathogenic in IBD.

Coagulation Described.  See Part I. (Cascade)

Endothelial injury exposes TF, which forms a complex with factor VII.  This complex activates factors X and, to a lesser extent, IX. TFPI prevents this activation progressing  further; for coagulation to progress, factor Xa must be produced via factors IX and VIII. Thrombin, generated by the initial production of factor Xa, activates factor VIII and, through factor XI, factor IX, resulting in further activation of factor X. This positive feedback loop allows coagulation to proceed. Fibrin polymers are stabilized by factor XIIIa. Activated proteins CS (APCS) together inhibit factors VIIIa and Va, whereas antithrombin (AT) inhibits factors VIIa, IXa, Xa, and XIa. Fibrinolysis balances this system through the action of plasmin on fibrin. Plasminogen activator inhibitor controls the plasminogen activator-induced conversion of plasminogen to plasmin.

Inflammation and Thrombotic Processes Linked

Although interest has recently moved away from the proposal that ischemia is a primary cause of IBD, it has become increasingly clear that inflammatory and thrombotic processes are linked.  A vascular component to the pathogenesis of CD was first proposed only a year after Crohn et al. described the condition.  Subsequently, in 1989, a series of changes comprising vascular injury, focal arteritis, fibrin deposition, arterial occlusion, and then microinfarction or neovascularization was proposed as a possible pathogenetic sequence in CD.  In this study, resin casts of the intestinal vasculature showed changes ranging from intravascular fibrin deposition to complete thrombotic occlusion. Furthermore, the early vascular changes appeared to precede mucosal changes, suggesting that they were more likely to cause rather than result from the pathologic features of CD. Subsequent studies showed that intravascular fibrin deposition occurred at the site of granulomatous destruction of mesenteric blood vessels, and positive immunostaining for platelet glycoprotein IIIa occurred in fibrinoid plugs of mucosal capillaries in CD. In addition, intracapillary thrombus has been identified in biopsies from inflamed rectal mucosa from patients with CD. When combined with evidence of ongoing intravascular coagulation in both active and quiescent CD, the above data point toward a thrombotic element contributing to the pathogenesis of CD.

Not only are many different prothrombotic changes described in association with IBD, but they can also have multiple causes. Hyperhomocysteinemia, for example, is known to predispose to thrombosis, and patients with IBD are more likely to have hyperhomocysteinemia than control subjects. Hyperhomocysteinemia in IBD might be due to multiple possible causes, such as deficiencies of vitamin B12 as a result of terminal ileal disease or resection; B6, which is commonly reduced in IBD.  A vegan diet can’t be discarded either because of seriously deficient methyl donors (S-adenosyl methionine).

The realization that platelets are not only prothrombotic but also proinflammatory has stimulated interest in their role in both the pathogenesis and complications of IBD. The association between thrombocytosis and active IBD was first described more than 30 years ago. More recent observations link decreased or normal platelet survival to IBD-related thrombocytosis, possibly due to increased thrombopoiesis. This in turn could be driven by an interleukin-6 –induced increase in thrombopoietin synthesis in the liver. Spontaneous in vitro platelet aggregation occurs in platelets isolated from 30% of patients with IBD but not in platelets from control subjects. Moreover, collagen, arachidonic acid, ristocetin, and ADP-induced platelet activation are more marked in platelets from patients with active IBD than in those from healthy volunteers.

The roles of activated platelets and PLAs in mucosal inflammation. Activated platelets can interact with other cells involved in the inflammatory response either through direct contact or through the release of soluble mediators. Activated platelets interact directly with activated vascular endothelium, causing the latter to express adhesion molecules and release inflammatory and chemotactic cytokines.

Platelet activation might be pathogenic in IBD in several ways. Platelet activation might increase platelet aggregation, hence increasing the likelihood of thrombus formation at sites of vascular injury, for example, within the mesenteric circulation. P-selectin is the major ligand for leukocyte-endothelial interaction and is responsible for the rolling of platelets, leukocytes, and PLAs on vascular endothelium. Moreover, platelets adherent to injured vascular endothelium support leukocyte adhesion via P-selectin, an effect that could contribute to leukocyte emigration from the vasculature into the lamina propria in patients with IBD. In addition, P-selectin is the major platelet ligand for platelet-leukocyte interaction, which in turn causes both leukocyte activation and further platelet activation.

Platelet-Leukocyte Aggregation

Recently, studies showing that platelets and leukocytes that circulate together in aggregates (PLA) are more activated than those that circulate alone have generated interest in the role of PLA in various inflammatory and thrombotic conditions. PLA numbers are increased in patients with ischemic heart disease, systemic lupus erythematosus and rheumatoid arthritis, myeloproliferative disorders, and sepsis and are increased by smoking.

We have recently shown that patients with IBD have more PLAs than both healthy and inflammatory control subjects (patients with inflammatory arthritides).  As with platelet activation, there was no correlation with disease activity, suggesting that increased PLA formation might be an underlying abnormality. PLAs could contribute to the pathogenesis of IBD in a number of ways. As previously mentioned, TF is key to the initiation of thrombus formation. TF has recently been demonstrated on the surface of activated platelets and in platelet-derived microvesicles. Interaction between neutrophils and activated platelets or microvesicles vastly increases the activity of “intravascular” TF.


It is becoming increasingly apparent that thrombosis and inflammation are intrinsically linked. Hence the involvement of thrombotic processes in the pathogenesis of IBD, although perhaps not as the primary event, seems likely. Indeed, with the recently mounting evidence of the role of activated platelets and of their interaction with leukocytes in the pathogenesis of IBD, it seems even more probable that thrombosis plays some role in the pathogenic process.

(Irving PM, Pasi KJ, and Rampton DS. Thrombosis and Inflammatory Bowel Disease. Clinical Gastroenterology and Hepatology 2005;3:617–628. PII: 10.1053/S1542-3565(05)00154-0.)

Bleeding in Patients with Renal Insufficiency

Approximately 20–40% of critically ill patients will have renal insufficiency at the time of admission or will develop it during their ICU stay, depending on the definition of renal insufficiency and the case mix of the ICU. Such patients are also predisposed to bleeding because of uremic platelet dysfunction, typically multiple comorbidities, coagulopathies and frequent concomitant treatment with antiplatelet or anticoagulant agents.

The impairment in hemostasis in uremic patients is multifactorial and includes physiological defects in platelet hemostasis, an imbalance of mediators of normal endothelial function and frequent comorbidities such as vascular disease, anemia and the frequent need for medical interventions required to treat such comorbidities. Physiologic alterations in uremia include:

  • decreased platelet glycoprotein IIb–IIIa binding to both von Willebrand factor (vWf) and fibrinogen, causing an impairment in platelet aggregation;
  • increased prostacyclin and nitric oxide production, both potent inhibitors of platelet activation and vasoconstriction; and
  • decreased levels of platelet adenosine diphosphate (ADP) and serotonin, causing an impairment in platelet secretion.

In addition to other factors, small peptides containing the RGD (Arg-Gly-Asp) sequence of amino acids have been shown to be inhibitors of platelet aggregation that act by competing with vWf and fibrinogen for binding to the glycoprotein IIb–IIIa receptor.


ICU patients have dynamic risks of thrombosis and bleeding. Invasive procedures may require temporary interruption of anticoagulants. Consequently, approaches to thromboprophylaxis require daily reevaluation.

(Cook DJ, Douketis J, Arnold D, and Crowther MA. Bleeding and venous thromboembolism in the critically ill with emphasis on patients with renal insufficiency. Curr Opin Pulm Med 2009;15:455–462.)


I have covered a large amount of material on one of the most complex systems in medicine, and still not comprehensive, with a sufficient dash of repetition.  The task is to have some grasp of the cell-mediated imbalances inherent if coagulation and bleeding disorders.  The key points are:

  • inflammation and oxidative stress invariably lurk in the background
  • the Y-shaped model with an extrinsic, intrinsic, and common pathway has no basis in understanding
  • the current model is based on a cell-mediated concept of endothelial damage and platelet-endothelial interaction
  • the model has 3 components: Initiation, Amplification, Propagation
  • NO and prostacyclin have key roles in the process
  • The plasma proteins involved are in the serine-protease class of enzymes
  • The conversion of Protein C to APC has a central role as anti-coagulant

Part II goes into organ aystem abnormalities that are all related to impairment of the Nitric Oxide balance and dual platelet-endothelial roles.

Part III will explore therapeutic targets and opportunities.

Read Full Post »

Ulcerative colitis

Ulcerative colitis (Photo credit: Wikipedia)

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis

Reporter: Larry Bernstein, MD

This is an overview of a recently published article about a new treatment for ulcerative colitis. It also reviews the use of a class of drug in inflammatory conditions, and introduces the problem of sepsis.

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis.
WJ Sandborn, S Ghosh, J Panes, I Vranic, C Su, for the Study A3921063 Investigators
N Engl J Med 2012; 367:616-624 August 16, 2012


Ulcerative colitis  is a chronic inflammatory disease of the colon that belongs to a group of diseases lumped together as Inflammatory Bowel Disease (IBD). There is a distinction to be made between Crohn’s disease, which may be limited to the small intestine (regional enteritis), the terminal ileum, or a portion of the transverse colon, and ulcerative colitis.

In ulcerative colitis the inflammation is limited to the mucosa and submucosa, but in Crohn’s disease there is a deep penetration of the intestinal wall (fistula) that may extend to the peritoneum causing abscess, scarring, peritonitis and possibly volvulus, obstruction and gangrenous bowel, which necessitate surgical resection. IBD tends to occur in children and young adults, repeats in families, and requires dietary management (fluid intake, Metamucil, restriction of fiber) . It is characterized by abdominal pain, diarrhea, bleeding, weight loss, and episodic fever, but also may be associated with joint pain.
Conservative medical treatment focuses on suppressing the immune response using 5-ASA, azathioprine, 6-mercaptopurine. If severe, biologic therapy is used to treat patients with severe Crohn’s disease that does not respond to any other types of medication, such as a TNF (tumor necrosis factor) inhibitor which can have secondary effects, and they are not universally effective. The importance of immunity can’t be understated, it involves a large portion of immune system and primitive Toll-like receptors (TLRs) that trigger signaling pathways. TLRs represent an important mechanism by which the host detects a variety of microorganisms that colonize in the gut. Endothelial and epithelial cells, and resident macrophages are potent producers of inflammatory cytokines, interleukins, IL-1, IL-6, and TNF-α, which are distinguished from another set that is treated in this study. In addition, there is a balance that has to be achieved between suppression and upregulation in treatment, which is referred to as immunomodulation.
The opposite of immunosuppression is upregulation It is cental to recent advances in chemotherapy of melanolma, small cell carcinoma and NSCCL of lung, and treatment resistant prostate cancer. An example is ipilimumab, whic upregulates cytotoxic T-cells to destroy cancer cells, but it has runaway destructive effects on the GI tract.

This study investigates the use of tofacitinib (CP-690,550), an oral inhibitor of Janus kinases 1, 2, and 3 with in vitro functional specificity for kinases 1 and 3 over kinase 2, which is expected to block signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. These cytokines are integral to lymphocyte activation, function, and proliferation.

The mechanism of drug action

Jak 1 and 3 inhibitor, which is targeted at blocking signaling involving gamma chain–containing cytokines including interleukins 2, 4, 7, 9, 15, and 21. The result would be to block signaling involving (gamma chains)–suppressing “lymphokines” 2, 4, 7, 9, 15, and 21. The lymphocyte pool is regional, being the antibody mediated immune system of the Bursa of Fabricius (B-lymphocytes, as opposed to the thymic derived T-cells) that form the largest immune organ extending the length of the intestines and the stomach.  The family transmission suggests an epigenetic event.

  • Gastrointestinal Tract
  • Oropharynx – Tonsils
  • Distal small intestine (ilieum) – Peyer’s Patches
  • Appendix, cecum

However, this classification of the lymphocytes has much greater complexity than I indicate.  The so called B-cells have receptors that recognize foreign antigen, but the T-cells have similar receptors and are tied to both the innate and the adaptive immune response.  Lymphocytes are the predominant cells of the immune system, but macrophages and plasma cells are present also.  Lymphocytes circulate, alternating between the circulatory blood stream and the lymphatic channels.  The end result of the immune reaction is the production of specific antibodies and antigen-reactive cells. These cells are called lymphocytes and are found in the blood and in the lymphoid system.

See Appendix

Trial features: double-blind, placebo-controlled, phase 2 trial; Patients were randomly assigned to receive tofacitinib at a dose of 0.5 mg, 3 mg, 10 mg, or 15 mg or placebo twice daily for 8 weeks.
Study goal: evaluated the efficacy of tofacitinib in 194 adults with moderately to severely active ulcerative colitis.

Primary outcome: a clinical response at 8 weeks, defined as an absolute decrease from baseline in the score on the Mayo scoring system for assessment of ulcerative colitis activity (possible score, 0 to 12, with higher scores indicating more severe disease) of 3 or more and a relative decrease from baseline of 30% or more with an accompanying decrease in the rectal bleeding subscore of 1 point or more or an absolute rectal bleeding subscore of 0 or 1.
Results and conclusion: The primary outcome, clinical response at 8 weeks, occurred in 32%, 48%, 61%, and 78% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.39), 3 mg (P=0.55), 10 mg (P=0.10), and 15 mg (P<0.001), respectively, as compared with 42% of patients receiving placebo.
Clinical remission (defined as a Mayo score ≤2, with no subscore >1) at 8 weeks occurred in 13%, 33%, 48%, and 41% of patients receiving tofacitinib at a dose of 0.5 mg (P=0.76), 3 mg (P=0.01), 10 mg (P<0.001), and 15 mg (P<0.001), respectively, as compared with 10% of patients receiving placebo. Three patients treated with tofacitinib had an absolute neutrophil count of less than 1500.
Patients with moderately to severely active ulcerative colitis treated with tofacitinib were more likely to have clinical response and remission than those receiving placebo. (Funded by Pfizer; number, NCT00787202.)
Commentary: The study is only phase 2, and it is also limited to disease of the descending colon. The next phase will be necessary to determine the effect on a larger population at the selected dose, and will be necessary to determine both the size of the effect and identify unexpected adverse effects. We also have to keep in mind that the success of the study would limit the treatment to a subset of patients with IBD.

Efficacy of Proposed Treatment:

  • it is effective at about 40% remission for 8 weeks compared to 10% for placebo, or an adjusted actual 30% for 8 weeks.
  • A much larger study needs to be done to see how well the dose holds up, as well as the dosing interval. There are two factors that will affect the t1/2 of the drug so that 1/2 dose could be replaced at the end of t1/2.
  • The dose of 15 mg was no better for clinical response.
  • I would think that the next trial might give a loading dose of 15 mg, and then 7 mg (better that 3 mg) would be replaced every t1/2.  But this is more complicated than usual.

I identified two steps, not one direct effect.

  • The inhibitor has to balance the production rate versus the removal rate of the T-cell population. The drug itself is not measured, only the effect. I know that albumin, the liver produced protein, has a half-life of removal of 21 days. Platelets are short shelf-life as well as rapid turnaround in plasma.
  •  I don’t know what is the local production and removal rate of lymphocytes in the gut. That would be the key determinant for dosing.

The following may shed some light on what has been discussed:

Common characteristics of the lymphoid system.

  • The lymphoid system involves organs and tissues where lymphocytic cells originate as lymphocyte precursors that mature and differentiate, and either lodge in the lymphoid organs or move throughout the body.
  • Precursor cells originate in the yolk sac, liver, spleen, or bursa of Fabricius (or its mammalian equivalent, the bone marrow) in an embryo or fetus.
  • Stem cells from bone marrow or embryonic tissues are deposited and mature into lymphocytes in the central or primary lymphoid organs, which include the thymus and the bursa or bone marrow. Upon maturation, the lymphocytes undergo further maturation toward immunocompetence and production of immunoglobulins or sensitized lymphocytes.

Adaptive immunity has 2 main classes:

  • Antibody-mediated – B Lymphocyte
  • Cell-mediated – T Lymphocyte

Lymph follicles are our point of reference:

  • Organized concentrations of Lymphocytes
  • No capsule, covered by epithelia
  • Nodules are unit structure seen in a node
  • Oval concentrations in meshwork of reticular cells

If pathogens initially evade constitutive defenses, they may yet be attacked by more specific inducible defenses. The inducible defenses are so-called because they are induced upon primary exposure to a pathogen or one of its products. The inducible defenses must be triggered in a host, take time to develop, and are a function of the immune response. The type of resistance thus developed in the host is called acquired immunity.

Three important features of the immunological system relevant to host defense and/or “immunity are:

1. Specificity. An antibody or reactive T cell will react specifically with the antigen that induced its formation; it will not react with other antigens. Generally, this specificity is of the same order as that of enzyme-substrate specificity or receptor-ligand specificity.

  • The specificity of the immune response is explained on the basis of the clonal selection hypothesis: during the primary immune response, a specific antigen selects a pre-existing clone of specific lymphocytes and stimulates exclusively its activation, proliferation and differentiation.

2.  Memory. The immunological system has a “memory”.

  • Once the immunological response has reacted to produce a specific type of antibody or reactive T cell, it is capable of producing more of the antibody or activated T cell more rapidly and in larger amounts.

3. Tolerance. An animal generally does not undergo an immunological response to its own (potentially-antigenic) components.

  • The animal is said to be tolerant, or unable to react to its own potentially-antigenic components.

Gene expression – CD28 signal transduction , λδ T repertoire and antigen reactivity

Efficient lymphokine gene expression appears to require both T-cell antigen receptor (TCR) signal transduction and an uncharacterized second or costimulatory signal. CD28 is a T-cell differentiation antigen that can generate intracellular signals that synergize with those of the TCR to increase T-cell activation and interleukin-2 (IL-2) gene expression.

  • These investigators examined the effect of CD28 signal transduction on granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and gamma interferon (IFN-gamma) promoter activity.
  • Stimulation of CD28 in the presence of TCR-like signals increases the activity of the GM-CSF, IL-3, and IFN-gamma promoters by three- to sixfold.
  • As previously demonstrated for the IL-2 promoter, the IL-3 and GM-CSF promoters contain distinct elements of similar sequence which specifically bind a CD28-induced nuclear complex.
  • Mutation of the CD28 response elements in the IL-3 and GM-CSF promoters abrogates the CD28-induced activity without affecting phorbol ester- and calcium ionophore-induced activity.
  • These studies indicate that the TCR and CD28-regulated signal transduction pathways, coordinately regulate the transcription of several lymphokines, and the influence of CD28 signals on transcription is mediated by a common complex.

Fraser JD, Weiss A.  Regulation of T-cell lymphokine gene transcription by the accessory molecule CD28. Mol Cell Biol. 1992 Oct;12(10):4357-63.

These investigators looked at the relevance λδ T repertoire and the antigen reactivity of clones isolated from CSF in multiple sclerosis (MS).

  • they found an increased percentage of V delta 1+ cells as compared to peripheral blood of the same donors.
  • Phenotypic analysis of cells from MS CSF with V gamma- and V delta-specific monoclonal antibodies (mAb) showed that the V delta 1 chain is most frequently associated with gamma chains belonging to the V gamma 1 family.
  • Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both delta and gamma genes indicating polyclonal expansion. gamma delta clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines.
  • Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the TH0-like phenotype.
  • Remarkably, these tumor-reactive gamma delta cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient.
  • in the CSF there is a skewed TCR gamma delta repertoire and suggest that gamma delta cells reacting against brain-derived antigens might have been locally expanded.

Nick S, Pileri P, Tongiani S, Uematsu Y, Kappos L, De Libero G. T cell receptor gamma delta repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive gamma delta clones. Eur J Immunol. 1995 Feb;25(2):355-63. PMID: 1328852 [PubMed – indexed for MEDLINE] PMCID: PMC360359 Free PMC Article

B Cells and T Cells:  Addendum…/B/B_and_Tcells.htmlShareAIDS; Building the T-cell Repertoire; Gamma/Delta T Cells … T cells specific for this structure (i.e., with complementary TCRs) bind the B cell and; secrete lymphokines that: … Each chain has a variable (V) region and a constant (C) region.

Although mature lymphocytes all look pretty much alike, they are extraordinarily diverse in their functions. The most abundant lymphocytes are:

  • B lymphocytes (often simply called B cells) and
  • T lymphocytes (likewise called T cells).
  • B cells are produced in the bone marrow.
  •  The precursors of T cells are also produced in the bone marrow but leave the bone marrow and mature in the thymus (which accounts for their designation).
  • Each B cell and T cell is specific for a particular antigen. What this means is that each is able to bind to a particular molecular structure.

The specificity of binding resides in a receptor for antigen:

  • the B cell receptor (BCR) for antigen and
  • the T cell receptor (TCR) respectively.

Both BCRs and TCRs share these properties:

  • They are integral membrane proteins.
  • They are present in thousands of identical copies exposed at the cell surface.
  • They are made before the cell ever encounters an antigen.
  • They are encoded by genes assembled by the recombination of segments of DNA.

How antigen receptor diversity is generated.

  • They have a unique binding site.
  • This site binds to a portion of the antigen called an antigenic determinant or epitope.
    The binding, like that between an enzyme and its substrate depends on complementarity of the surface of the receptor and the surface of the epitope.
  • The binding occurs by non-covalent forces (again, like an enzyme binding to its substrate).

Successful binding of the antigen receptor to the epitope, if accompanied by additional signals, results in:

  • stimulation of the cell to leave G0 and enter the cell cycle.
  • Repeated mitosis leads to the development of a clone of cells bearing the same antigen receptor; that is, a clone of cells of the identical specificity.

BCRs and TCRs differ in:

  • their structure;
  • the genes that encode them;
  • the type of epitope to which they bind.

heavy (H) plus kappa (κ) or lambda (λ) chains for BCRs;

alpha (α) and beta (β) or gamma (γ) and delta (δ) chains for TCRs)

……is encoded by several different gene segments.

The genome contains a pool of gene segments for each type of chain. Random assortment of these segments makes the largest contribution to receptor diversity.

There are two types of T cells that differ in their TCR:

alpha/beta (αβ) T cells. Their TCR is a heterodimer of an alpha chain with a beta chain. Each chain has a variable (V) region and a constant (C) region. The V regions each contain 3 hypervariable regions that make up the antigen-binding site. [Link]

gamma/delta (γδ) T cells. Their TCR is also a heterodimer of a gamma chain paired with a delta chain.

The discussion that follows now concerns alpha/beta T cells. Gamma/delta T cells, which are less well understood, are discussed at the end [Link].

The TCR (of alpha/beta T cells) binds a bimolecular complex displayed at the surface of some other cell called an antigen-presenting cell (APC).

Most of the T cells in the body belong to one of two subsets. These are distinguished by the presence on their surface of one or the other of two glycoproteins designated:

  • CD8+ T cells bind epitopes that are part of class I histocompatibility molecules. Almost all the cells of the body express class I molecules.
  • CD4+ T cells bind epitopes that are part of class II histocompatibility molecules. Only specialized antigen-presenting cells express class II molecules.

These include:

  • dendritic cells
  • phagocytic cells like macrophages and
  • B cells!

Building the T-cell Repertoire

T cells have receptors (TCRs) that bind to antigen fragments nestled in MHC molecules. But,

  • all cells express class I MHC molecules containing fragments derived from self proteins;
  • many cells express class II MHC molecules that also contain self peptides.

This presents a risk of the T cells recognizing these self-peptide/self-MHC complexes and mounting an autoimmune attack against them. Fortunately, this is usually avoided by a process of selection that goes on in the thymus (where all T cells develop).


FDA approves Abbott Humira as Ulcerative Colitis therapy
PBR Staff Writer Published 01 October 2012
The USFDA has approved Abbott’s Humira (adalimumab) for the treatment of adult patients with moderate to severe Ulcerative Colitis (UC) when certain other medicines have not worked well enough.
Humira, which works by inhibiting tumour necrosis factor-alpha (TNF-alpha), was previously approved for the treatment of moderate to severe Crohn’s disease.

Abbott Global Pharmaceutical Research and Development senior vice president John Leonard said, “Since the first FDA approval of HUMIRA in late 2002, Abbott has continued to investigate the medication in multiple conditions with the goal of bringing this treatment option to more patients who may benefit from it.”

The approval was based on the data from two phase 3 studies, ULTRA 1 and ULTRA 2, both of which enrolled adult patients who had moderately to severely active UC despite concurrent or prior treatment with immunosuppressants.  This should have special significance in view of the past history, which may be explainable, but also keep in mind the serious risks of complications.

It is worthy of comment that anti-TNF treatment was previously rejected in trials for use in sepsis leading to Multiple Organ Dysfunction Syndrome and cardiovascular collapse (shock).  More recently an anti-Factor Xa drug, Xygris,  to prevent hypercoagulability only in severe sepsis was withdrawn.

Anti TNF for sepsis

1.   In a group of patients with elevated interleukin-6 levels, the mortality rate was 243 of 510 (47.6%) in the placebo group and 213 of 488 (43.6%) in the afelimomab group. Using a logistic regression analysis, treatment with afelimomab was associated with an adjusted reduction in the risk of death of 5.8% (p = .041) and a corresponding reduction of relative risk of death of 11.9%. Mortality rates for the placebo and afelimomab groups in the interleukin-6 test negative population were 234 of 819 (28.6%) and 208 of 817 (25.5%), respectively. In the overall population of interleukin-6 test positive and negative patients, the placebo and afelimomab mortality rates were 477 of 1,329 (35.9%)and 421 of 1,305 (32.2%), respectively.

Panacek EAMarshall JCAlbertson TEJohnson DH, at al.  Efficacy and safety of the monoclonal anti-tumor necrosis factor antibody F(ab’)2 fragment afelimomab in patients with severe sepsis and elevated interleukin-6 levelsCrit Care Med. 2004 Nov;32(11):2173-82.

2. No survival benefit was found for the total study population, but patients with increased circulating TNF concentrations at study entry appeared to benefit by the high dose anti-TNF antibody treatment. Increased interleukin (IL)-6 levels predicted a fatal outcome (p =.003), but TNF levels were not found to be a prognostic indicator. TNFlevels were higher (206.7 +/- 60.7 vs. 85.9 +/- 26.1 pg/mL; p <.001) and outcome was poor (41% vs. 71% survival; p =.007) in patients who were in shock at study entry when compared with septic patients not in shock.

Fisher CJ JrOpal SMDhainaut JFStephens S, et al. Influence of an anti-tumor necrosis factor monoclonal antibody on cytokine levels in patients with sepsis. The CB0006 Sepsis Syndrome Study Group.  Critical Care Medicine [1993, 21(3):318-327] (PMID:8440099)

3.  Large clinical trials involving anti-TNF-alpha MAb have proven to be less conclusive and less successful than clinicians had hoped. The International Sepsis Trial (INTERSEPT), reported by Cohen and Carlet,[14] was designed to assess the safety and efficacy of Bay x 1351, a murine MAb to recombinant human TNF-alpha in patients with sepsis. The INTERSEPT trial was an international, multicenter trial involving 564 patients, 420 of whom were in septic shock. The main study end point — 28-day survival — showed no significant benefit for the treatment group vs controls. Prospectively, the researchers identified 2 secondary variables: shock reversal and frequency of organ failure. Post-28-day survival, treatment groups showed a more rapid reversal of shock compared with placebo, as well as a significant delay in time to first organ failure. The researchers concluded that the anti-TNF-alpha antibody may have a role as adjunctive therapy, but that such a putative role requires more in the way of clinical trial confirmation.

In the TNF-alpha MAb Sepsis Study Group trial, also called the North American Sepsis Trial I (NORASEPT I), Abraham and associates[15] evaluated the efficacy and safety of an anti-TNF-alpha MAb in the treatment of patients with sepsis syndrome. A total of 994 patients in 31 hospitals were enrolled in a randomized, prospective, multicenter, double-blind, placebo-controlled clinical trial. Patients were stratified into shock/nonshock subgroups, then randomized to receive a single infusion of 15 mg/kg of anti-TNF-alpha MAb, 7.5 mg/kg of anti-TNF-alpha MAb, or placebo. The researchers found that among all infused patients, there was no difference in mortality among those receiving therapy and those on placebo. In septic shock patients (n = 478), however, there was a trend toward a reduction in all-cause mortality, which was most evident 3 days after infusion. At day 3, 25 of 162 patients treated with the 15 mg/kg dose died; 22 of 156 treated with 7.5 mg/kg died, but 44 of 160 placebo-treated patients died (15 mg/kg: 44% mortality reduction vs placebo, P = .01; 7.5 mg/kg: 48% reduction vs placebo, P = .004). However, at day 28, the reduction in mortality of shock patients was not significant for either dose of the anti-TNF-alpha MAb relative to placebo.

All studies of MAb against TNF in septic patients and found an absolute risk reduction of 3.5%. The most recently published clinical trial found an absolute reduction in mortality of 3.7%.

Of note, therapy with MAb against TNF has been proven efficacious for treatment of rheumatoid arthritis and is approved by the US Food and Drug Administration for this purpose.

New directions in research on severe sepsis. Human trials with TNF alpha.  Medscape.

4. Why the poor results with sepsis?

This would be sufficient for another discussion.  That can be left for another day.


Sepsis syndrome, or sepsis, is an adverse systemic response to infection that includes fever, rapid heartbeat and respiration, low blood pressure and organ dysfunction associated with compromised circulation.

LPS is a major constituent of Gram-negative bacterial cell walls (see section 3-0) and is essential for membrane integrity. The portion of LPS that causes shock is the innermost and most highly conserved phosphoglycolipid, lipid A. Lipid A is a phosphoglycolipid consisting of a core hexosamine disaccharide with ester- and amide-linked acylated fatty acid tails arranged in either asymmetric or symmetric arrays that anchor the structure in the membrane. It acts by potently inducing inflammatory responses that are life-threatening when systemic, and is known as bacterial endotoxin.  Mice deficient in any of the LPS receptor components are more
susceptible to Gram-negative bacterial infection but, at the same time, are less susceptible to the sepsis syndrome.

TLRs have a lethal function in the septic shock syndrome. The physiological function of signaling through phagocyte TLRs is to induce the release of the cytokines TNF, IL-1, IL-6, IL-8 and IL-12 and trigger the inflammatory response, which is critical to containing bacterial infection in the tissues. However, if infection disseminates in the blood, the widespread activation of phagocytes in the bloodstream is catastrophic. Increase in the numbers of circulating neutrophils, or neutrophilia, is driven by effects of colony stimulating factors, such as G-CSF.

Time course of sepsis. The clinical manifestations of sepsis are manifested by successive waves of the serum cytokine cascade. In humans injected with purified LPS, TNF rises almost immediately and peaks at 1.5 h; the sharp decline of TNF may be due to modulation by its soluble receptor sTNFR. A second wave of cytokines that peaks at 3 h activates the acute-phase response
in the liver, the systemic pituitary response (via IL-6 and IL-1), and the activation and chemotaxis of neutrophils (via IL-6, IL-8 and  G-CSF). Neutrophil activation results in the release of lactoferrin from neutrophil secondary granules; the activation of endothelial procoagulants with the rise of tissue plasminogen activator (t-PA). Pituitary-derived adrenocorticotropic hormone (ACTH)  and migration inhibition factor (MIF) peak at 5 h and coincide with peak levels of the regulatory cytokines IL-Ra and IL-10 that counteract the release or activity of inflammatory cytokines. Diffuse endothelial activation is shown by the appearance of soluble E-selectin that peaks at about 8 h and remains elevated for several days.

Susceptibility to LPS Toxicity in Gene Knockout Mice

High LPS; Low LPS/D-Gal



LPS recognition

phagocyte function

caspase 1
The proteins encoded by the deleted genes are listed. SR-A is scavenger receptor A; Hck and Fgr are Src-family kinases with an essential role in integrin-mediated migration of neutrophils out of the bloodstream.

The Immune Response to Bacterial InfectionSepsis Syndrome: Bacterial Endotoxin
Chapter 9-3.  2007. p 232-233. New Science Press Ltd

Related articles

Read Full Post »


Reporter: Aviva Lev-Ari, PhD, RN
Aug. 15, 2012

Crohn’s disease driven by inflammation – not genetics, reports study

diagram of intestinal inflammation

A new study shows that inflammation drives dysbiosis, Gram negative proliferation and E. coli invasion.

Inflammation — not genetic susceptibility — drives the growth of intestinal bacteria and invasive E. coli linked to Crohn’s disease (CD), reports a new Cornell study.

Scientists have long wondered about the role of bacteria in CD. Recent studies have shown marked changes in the composition of the intestinal bacteria in people with CD, leading researchers to ask: Are microbial abnormalities a direct consequence of genetic abnormalities linked to Crohn’s and precede and initiate inflammation, or does intestinal inflammation bring on the bugs?

Inflammation, in fact, drives microbial imbalances (dysbiosis) and the proliferation of a specific type of E. coli that is adherent, invasive and found in the ileum, reported Cornell researchers July 31 in PLoS (7[7]). And genetics, they said, do play a role in determining the threshold and magnitude of dysbiosis in response to acute inflammation induced by environmental triggers.

This study also reports that a common therapy directed against intestinal inflammation decreases dysbiosis. In addition, the study found that the lack of a receptor that helps recruit T cells, which are needed for cell-mediated immunity, to the gut also decreases inflammation and dysbiosis, offering a new option for therapeutic intervention.

“Today, remission is our mission,” said Kenneth Simpson, professor of small animal medicine at Cornell’s College of Veterinary Medicine and principal investigator. “Crohn’s disease is a highly complex condition that finds its strength in the combination of negatives: environmental factors, genetic mutations and immune system malfunctions. Ultimately, there may be a cure. Until then, we need to find ways to relieve suffering.”

CD is a chronic debilitating inflammatory bowel disease that involves a complex interaction of host genes, the immune system, the intestinal microbiome and the environment. Afflicting more than half a million people in North America, CD can trigger mild to severe diarrhea, fever, fatigue, anemia, reduced appetite and weight loss.

To mirror the complex nature of the disease, Simpson’s team designed a study that incorporated inflammatory triggers related to relapse of CD and ileal inflammation. Unlike previous studies that have focused on colonic or fecal dysbiosis, the team focused on ileal dysbiosis, which is prevalent in 70 percent of CD cases. Also novel to this study, the team used a variety of contemporary techniques to generate a comprehensive picture of the composition and spatial distribution of the ileal microbiome. Particular attention was paid to pinpointing the number, pathotype and location of E. coli associated with intestinal inflammation in people, dogs and mice.

“Our findings clearly demonstrate that inflammation drives ileal dysbiosis and proliferation of CD-associated adherent invasive E. coli. Further, in the context of a patient with Crohn’s, we found that the host genotype and therapeutically blocking inflammation both impact the onset and extent of ileal dysbiosis. These novel findings are of high relevance to Crohn’s disease.”

The investigation leveraged the knowledge and resources of researchers in the labs of Erik Denker, Dwight Bowman and Sean McDonough labs. Building on findings in patients with Crohn’s disease evaluated by Dr. Ellen Scherl’s group at Weill Cornell Medical College, this collaboration shed new light on this debilitating disease.

“It appears that we harbor our own powder keg,” said Simpson. “The bacteria are already seeded. It’s what controls the relative balance between the different species of bacteria and their numbers, relative proportions, our ability to deal with them, and the cross-talk between the bacteria and host that is important.”

This work was supported by NewYork-Presbyterian Hospital/Weill Cornell Medical Center, the Jill Roberts Center for Inflammatory Bowel Disease and the National Institutes of Health.



Read Full Post »