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Introduction to Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

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

 

This leads into a series of presentations and the metabolic imbalance central to findings of endocrine, metabolic, inflammatory, immune diseases and cancer.  All of this has been a result of discoveries based on the methods of study of genomiocs, proteomics, transcriptomics, and metabolomics that have preceded this.  In some cases there has been the use of knockout methods. The completion of the human genomic and other catalogues have been instrumental in the past few years.  In all cases there has been a thorough guidance by a biological concept of mechanism based on gene expression, metabolic disturbance, signaling pathways, and up- or down- regulation of metabolic circuits.  It is interesting to recall that a concept of metabolic circuits was not yet formulated at the time of the mid 20th century physiology, except perhaps with respect to the coagulation pathways, and to some extent, glycolysis, gluconeogenesis, the hexose monophosphate shunt, and mitochondrial respiration, which were linear strings of enzyme substrate reactions that intersected and that had flow restraints not then understood as to the complexity we now appreciate.  We did know the importance of cytochrome c, the adenine and pyridine nucleotides, and the energy balance.  Electron microscopy had opened the door to understanding the mechanism of contraction of skeletal muscle and myocardium, but it also opened the door to understanding kidney structure and function, explaining the “mesangium”.  The first cardiac maker was discovered by Arthur Karmen in the serum alanine and aspartate aminotransferases, with a consequent differentiation between hepatic and myocardial damage.  This was followed by lactic dehydrogenase and the H- and M-type isoenzymes in the 1960s, and in the next decade, by the MB-isoenzyme of creatine kinase.  Troponins T and then I would not be introduced until the mid 1980s, and they have become a gold standard for the diagnosis of myocardial infarction.

In the 1980s we also saw the development of antiplatelet therapy that rapidly advanced interventional cardiology.  But advances in surgical as well as medical intervention also proceeded as the understanding of the lipid metabolism was opened by the work of Brown and Goldstein, and UTSW Medical Campus, and major advances in treatment came at Baylor and UT Medical Center in Houston, and at the Cleveland Clinic.  The next important advance came with the discovery of nitric oxide synthase role in endothelium and oxidative stress.  The field of endocrinology saw advances as well for a solid period of 30 years in a comparable period for the adrenals, thyroid, and pituitary glands, and for the understanding of the male and female sex hormones, and discoveries in breast, ovarian, and prostate cancer.  There were cancer markers, such as, CA125 and CA15-3, and PSA.  This had more of an impact on timely surgical intervention, and if not that, post surgical followup.  Despite a long time into the war on cancer, introduced by President Lynden Johnson, the fundamental knowledge needed was not sufficient.  In the meantime, there were advances in the treatment of diabetes, with eventual introduction of the insulin pump for type I diabetes.  The problem of Type 2 DM increased in prevalence, reaching into the childhood age group, with ascendent obesity.  An epidemiological pattern of disease comorbidities was emergent.  Our population has aged out, and with it we are seeing an increase in dementias, especially Alzheimer’s disease.  But the knowledge of the brain has lagged far behind.

What follows is a series of chapters that address what has currently been advanced with repect to the alignment of our knowledge of the last decade and pharmacetical discovery.  Pharmaceuticals were suitable for bacterial infections until the 1990s, when we saw the rise of resistance to penicillins and Vancomycin, and we had issues with gram negative enterobacter, salmonella, and E. coli strains.  That has been and is a significant challenge.  The elucidation of the gut microbiome in recent years will help to relieve this problem.  The problem of the variety and different aggressive types of cancer has been another challenge.  The door has been opened to better diagnostic tools with respsct to imaging and targeted biomarkers for localization.  I am not dealing with imaging, which is not the subject here.

HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection

From http://www.pnas.org –      Sep 3, 2013 4:24 PM

Experimental and computational evidence suggests that HLAs preferentially bind

  • conserved regions of viral proteins, a concept we term “targeting efficiency,” and
  • that this preference may provide improved clearance of infection in several viral systems.

To test this hypothesis, T-cell responses to A/H1N1 (2009)

  • were measured from peripheral blood mononuclear cells
  • obtained from a household cohort study performed during the 2009–2010 influenza season.

We found that HLA targeting efficiency scores

  • significantly correlated with IFN-γ
    enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

A further population-based analysis found that

  • the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,
  • were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
  • are common in certain indigenous populations in which
  • increased pH1N1 morbidity has been reported.

HLA efficiency scores and HLA use are associated with

  • CD8 T-cell magnitude in humans after influenza infection.

The computational tools used in this study may be useful predictors of

  • potential morbidity and identify immunologic differences of new variant influenza strains
  • more accurately than evolutionary sequence comparisons.

Population-based studies of the relative frequency of these alleles

  • in severe vs. mild influenza cases might advance clinical practices
  • for severe H1N1 infections among genetically susceptible populations.

A deeper look into cholesterol synthesis

By Swathi Parasuraman

The human body needs cholesterol to maintain membrane fluidity, and

  • it acts as a precursor molecule for several important biochemical pathways.

Its regulation requires strict control, as it can cause problems if it’s produced in excess. In 1964, Konrad Bloch received a Nobel Prize for his work elucidating the mechanisms of cholesterol synthesis. His work

  • eventually contributed to the discovery of statins, drugs used today to lower blood cholesterol levels.

The biosynthesis of cholesterol is a complex process with more than 20 steps. One of the first enzymes is

  • 3-hydroxy-3-methylglutaryl-CoA reductase, also known as HMGCR, the main target of statins.

As links between intermediates in cholesterol synthesis and various diseases are being discovered continually, more information about the regulatory role of the post-HMGCR pathway is needed.

In a recent minireview in The Journal of Biological Chemistry, Laura Sharpe and Andrew Brown of the University of New South Wales describe

  • multiple ways various enzymes other than HMGCR
  • are implicated in the modulation of cholesterol synthesis.

One such enzyme is squalene monooxygenase, which, like HMGCR, can be destroyed

  • by the proteasome when cholesterol levels are high.

The minireview also explains how pathway intermediates

  • can have functions distinct from those of cholesterol.

For example, intermediate 7-dehydrocholesterol usually is converted to cholesterol by the enzyme DHCR7

  • but is also a vitamin D precursor.

To synthesize the enzymes necessary to make cholesterol,

  • SREBPs, short for sterol regulatory element binding proteins, have special functions.

Along with transcriptional cofactors, they activate gene expression

  1. in response to low sterol levels and, conversely,
  2. are suppressed when there is enough cholesterol around.

Additionally, SREBPs control production of

  • nicotinamide adenine dinucleotide phosphate, or NADPH,
  • which is the reducing agent required to carry out the different steps in the pathway.

Lipid carrier proteins also can facilitate cholesterol synthesis. One example is SPF, or supernatant protein factor,

  1. which transfers substrate from an inactive to an active pool or
  2. from one enzyme site to another.

Furthermore, translocation of several cholesterogenic enzymes

  • from the endoplasmic reticulum to other cell compartments can occur under various conditions,
  • thereby regulating levels and sites of intracellular cholesterol accumulation.

Immunology in the gut mucosa:

20 Feb 2013 by Kausik Datta, posted in Immunology, Science (Nature)

The human gut can be the scene for devastating conditions such as inflammatory bowel disease,

  • which arises through an improperly controlled immune response.

The gut is often the body’s first point of contact with microbes; every mouthful of food is accompanied by a cargo of micro-organisms that go on to encounter the mucosa, the innermost layer of the gut. Most microbes are destroyed by the harsh acidic environment in the stomach, but a hardy few make it through to the intestines.

The intestinal surface is covered with finger-like protrusions called villi,

whose primary function is the absorption of nutrients.

These structures and the underlying tissues

  • host the body’s largest population of immune cells.

Scattered along the intestinal mucosa are

  • dome-like structures called Peyer’s Patches.

These are enriched in lymphoid tissue, making them key sites for

  • coordinating immune responses to pathogens,
  • whilst promoting tolerance to harmless microbes and food.

The villi contain a network of blood vessels to transport nutrients from food to the rest of the body. Lymphatics

  • from both the Peyer’s Patches and the villi
  • drain into the mesenteric lymph nodes.

Within the villi is a network of loose connective tissue called the lamina propria, and

  • at the base of the villi are the crypts which host the stem cells that replenish the epithelium.

The epithelium together with its overlying mucus forms

  • a barrier against microbial invasion.

A mix of immune cells including T- and B-lymphocytes, macrophages, and dendritic cells are

  • embedded within the matrix of the Peyer’s Patches, .

A key function of the Peyer’s Patch is the sampling of antigens present in the gut. The Peyer’s Patch has a thin mucous layer and specialized phagocytic cells, called M-cells, which

  • transport material across the epithelial barrier via a process called transcytosis.

Dendritic cells extend dendrites between epithelial cells to sample antigens that are then

  • broken down and used for presenting to lymphocytes.

Sampling antigens in this way typically results in so-called tolerogenic activation, where

  • the immune system initiates an anti-inflammatory response.

With their cargo of antigens, these Dendritic Cells then

  • traffic to the T-cell zones of the Peyer’s Patch.

Upon encounter with specific T-cells, the Dendritic Cells

  • convert them into an immunomodulatory cell called regulatory T-cell or T-reg.

Defects in the function of these cells are associated with

  • inflammatory bowel disease in both animals and humans.

These T-regs migrate to lamina propria of the villi via the lymphatics. Here, the T-regs

  • secrete a molecule called Interleukin (IL)-10,
  • which exerts a suppressive action on immune cells within the lamina propria
  • and upon the epithelial layer itself.

IL10 is, therefore, critical in maintaining immune quiescence

  • and preventing unnecessary inflammation.

However, a breakdown in this process of immune homeostasis results in gut pathology and

  • when this occurs over a prolonged period and in an uncontrolled manner,
  • it can lead to inflammatory bowel disease.

Chemical, mechanical or pathogen-triggered barrier disruption

  • coupled with particular genetic susceptibilities may all combine to set off inflammation.

Epithelium coming into contact with bacteria

  • is activated, leading to bacterial influx.

Alarm molecules released by the epithelium

  • activates immune cells, and T-regs in the vicinity
  • scale down their IL10 secretion to enable an immune response to proceed.

Dendritic cells are also activated by this environment, and

  • start to release key inflammatory molecules,
  • such as IL6, IL12, and IL23.

Effector T-cells also appear on the scene and

  • these coordinate an escalation of the immune response
  • by secreting their own inflammatory molecules,
  • Tumor Necrosis Factor (TNF)-α, Interferon (IFN)-γ and IL17.

Soon after the effector T-cells are arrived, a voracious phagocyte called a neutrophil is recruited. Neutrophils are critical for the clearance of the bacteria. One weapon in the neutrophil armory is

  • the ability to undergo self-destruction.

This leaves behind a jumble of DNA saturated with enzymes, called the Neutrophil Extracellular Trap.

Although this can effectively destroy the bacterial invaders

  • and plug any breaches in the epithelial wall,
  • it also causes collateral damage to tissues.

Slowly the tide begins to turn and the bacterial invasion is repulsed. Any remaining neutrophils die off,

  • and are cleared by macrophages.

Epithelial integrity is restored by replacement of damaged cells with new ones from the intestinal crypts. Finally T-regs are recruited once again to calm the immune response.

Targeting the molecules involved in gut pathology is leading to

  • effective therapies for inflammatory bowel disease.

Notes:

T- and B-lymphocytes, Macrophages, and Dendritic Cells: These are all important immune effector cells. Macrophages and Dendritic cells are primary defence cells that can eat up (‘phagocytosis’) microbes and destroy them; they also can present parts of these microbes to lymphocytes. T-lymphocytes or T-cells help B-lymphocytes or B-cells recognize the antigen and form antibodies against it. Other types of T-cells can themselves kill microbes. All these cells also secrete various chemical substances, called cytokines and chemokines, which act as molecular messengers in recruiting various immune cells, coordinating and fine-tuning the immune response. Some of these cytokines are called Interleukins, shortened to IL.

Anti-inflammatory response: A type of immune response in which molecular messengers are used to scale down heavy-handed immune cell activity and switch off processes that recruit immune cells. This helps the body recognize and selectively tolerate beneficial substances such as commensalic microbes that live in the gut.

Neutrophils: These are highly versatile immune effector cells. Usually, they are one of the first cells recruited to the site of infection or tissue damage via message spread by molecular messengers. Neutrophils can themselves elaborate cytokines and chemokines, and have the ability to directly kill microbes.

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53-MDM2 interaction.

J Soares, et al. Eur J Pharm Sci 10/2014; http://dx.doi.org:/10.1016/j.ejps.2014.10.006

An appealing target for anticancer treatment is

  • the p53 tumor suppressor protein.

This protein is inactivated in half of human tumors

  • due to endogenous negative regulators such as MDM2.

Therefore, restoring the p53 activity through

  • the inhibition of its interaction with MDM2
  • is considered a valuable therapeutic strategy
  • against cancers with a wild-type p53 status.

We report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams

  • and the evaluation of their biological effects as p53-MDM2 interaction inhibitors.

Using a yeast-based screening assay, two oxazoloisoindolinones,

  • were identified as potential p53-MDM2 inhibitors.

The molecular mechanism of oxazoloisoindolinone 3a validated

  • in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53(+/+)) and
  • in its isogenic derivative without p53 (HCT116 p53(-/-)).

we demonstrated that oxazoloisoindolinone 3a exhibited

  • a p53-dependent in vitro antitumor activity through
  • induction of G0/G1-phase cell cycle arrest and apoptosis.

The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported

  • by the occurrence of PARP cleavage only in p53-expressing HCT116 cells.

Oxazoloisoindolinone 3a led

  • to p53 protein stabilization
  • to the up-regulation of p53 transcriptional activity &
  • increased expression levels of several p53 target genes,
  • as p21, MDM2, BAX and PUMA,
  • in p53(+/+) but not in p53(-/-) HCT116 cells.

the ability of oxazoloisoindolinone 3a to block the p53-MDM2 interaction in HCT116 p53(+/+) cells was confirmed by co-immunoprecipitation.

molecular docking analysis of the interactions

  • between the compounds and MDM2 revealed that
  • oxazoloisoindolinone 3a binds to MDM2.

this work adds the oxazoloisoindolinone scaffold to the activators of a wild-type p53-pathway with promising antitumor activity.

it may open the way to the development of

  • a new class of p53-MDM2 interaction inhibitors.

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

Sanu Shameer, et al. Nucleic Acids Research10/2014;
http://dx.doi.org/10.1093/nar/gku944

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome.

Metabolic network databases are important in allowing us to

  • contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments.

Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes

  • the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan
  • responsible for human and animal African trypanosomiasis.

In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have implemented a network

  • representation of the information through MetExplore,

yielding a novel environment in which to visualise the metabolism of this important parasite.

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