Funding, Deals & Partnerships: BIOLOGICS & MEDICAL DEVICES; BioMed e-Series; Medicine and Life Sciences Scientific Journal – http://PharmaceuticalIntelligence.com
Robotically Driven System Could Reduce Cost of Discovering Drugs
Reporter: Irina Robu, PhD
However, their approach had only been tested using synthetic or previously acquired data, the team’s current model builds on this by letting the computer choose which experiments to do. The experiments were then carried out using liquid-handling robots and an automated microscope.
A total of 9,216 experiments were done, each consisting of acquiring images for a given cell clone in the presence of a given drug. The challenge for the algorithm was to learn how proteins were affected in each of these experiments, without performing all of them.
The originality of this work was to identify new phenotypes on its own as part of the learning process. To do this, it clustered the images to form phenotypes. The phenotypes were used to form a predictive model, so the learner could estimate the outcomes of unmeasured experiments. The basis of the model was to identify different sets of proteins that responded similarly to sets of drugs, so that it could predict the trend in the unmeasured experiments. The learner repeated the process for a total of 30 rounds, completing 2,697 out of the 9,216 possible experiments. As it progressively performed the experiments, it identified more phenotypes and more patterns in how sets of proteins were affected by sets of drugs.
Using an assortment of calculations, the team determined that the algorithm was able to learn a 92% accurate model for how the 96 drugs affected the 96 proteins, from only 29% of the experiments conducted.
Blood cells are divided into three groups: the red blood cells (erythrocytes), the white blood cells (leukocytes), and the blood platelets (thrombocytes). The white blood cells are subdivided into three broad groups: granulocytes, lymphocytes, and monocytes.
Blood cells do not originate in the bloodstream itself but in specific blood-forming organs, notably the marrow of certain bones. In the human adult, the bone marrow produces all of the red blood cells, 60–70 percent of the white cells (i.e., the granulocytes), and all of the platelets. The lymphatic tissues, particularly the thymus, the spleen, and the lymph nodes, produce the lymphocytes (comprising 20–30 percent of the white cells). The reticuloendothelial tissues of the spleen, liver, lymph nodes, and other organs produce the monocytes (4–8 percent of the white cells). The platelets, which are small cellular fragments rather than complete cells, are formed from bits of the cytoplasm of the giant cells (megakaryocytes) of the bone marrow.
In the human embryo, the first site of blood formation is the yolk sac. Later in embryonic life, the liver becomes the most important red blood cell-forming organ, but it is soon succeeded by the bone marrow, which in adult life is the only source of both red blood cells and the granulocytes. Both the red and white blood cells arise through a series of complex, gradual, and successive transformations from primitive stem cells, which have the ability to form any of the precursors of a blood cell. Precursor cells are stem cells that have developed to the stage where they are committed to forming a particular kind of new blood cell.
In a normal adult the red cells of about half a liter (almost one pint) of blood are produced by the bone marrow every week. Almost 1 percent of the body’s red cells are generated each day, and the balance between red cell production and the removal of aging red cells from the circulation is precisely maintained.
Because of the inability of erythrocytes (red blood cells) to divide to replenish their own numbers, the old ruptured cells must be replaced by totally new cells. They meet their demise because they don’t have the usual specialized intracellular machinery, which controls cell growth and repair, leading to a short life span of 120 days.
This short life span necessitates the process erythropoiesis, which is the formation of red blood cells. All blood cells are formed in the bone marrow. This is the erythrocyte factory, which is soft, highly cellar tissue that fills the internal cavities of bones.
Erythrocyte differentiation takes place in 8 stages. It is the pathway through which an erythrocyte matures from a hemocytoblast into a full-blown erythrocyte. The first seven all take place within the bone marrow. After stage 7 the cell is then released into the bloodstream as a reticulocyte, where it then matures 1-2 days later into an erythrocyte. The stages are as follows:
Hemocytoblast, which is a pluripotent hematopoietic stem cell
Common myeloid progenitor, a multipotent stem cell
Unipotent stem cell
Pronormoblast
Basophilic normoblast also called an erythroblast.
Polychromatophilic normoblast
Orthochromatic normoblast
Reticulocyte
These characteristics can be seen during the course of erythrocyte maturation:
The size of the cell decreases
The cytoplasm volume increases
Initially there is a nucleus and as the cell matures the size of the nucleus decreases until it vanishes with the condensation of the chromatin material.
Low oxygen tension stimulates the kidneys to secrete the hormone erythropoietin into the blood, and this hormone stimulates the bone marrow to produce erythrocytes.
Rarely, a malignancy or cancer of erythropoiesis occurs. It is referred to as erythroleukemia. This most likely arises from a common myeloid precursor, and it may occur associated with a myelodysplastic syndrome.
There are various types of white blood cells (WBCs) that normally appear in the blood: neutrophils (polymorphonuclear leukocytes; PMNs), band cells (slightly immature neutrophils), T-type lymphocytes (T cells), B-type lymphocytes (B cells), monocytes, eosinophils, and basophils. T and B-type lymphocytes are indistinguishable from each other in a normal slide preparation. Any infection or acute stress will result in an increased production of WBCs. This usually entails increased numbers of cells and an increase in the percentage of immature cells (mainly band cells) in the blood. This change is referred to as a “shift to the left” People who have had a splenectomy have a persistent mild elevation of WBCs. Drugs that may increase WBC counts include epinephrine, allopurinol, aspirin, chloroform, heparin, quinine, corticosteroids, and triamterene. Drugs that may decrease WBC counts include antibiotics, anticonvulsants, antihistamine, antithyroid drugs, arsenicals, barbiturates, chemotherapeutic agents, diuretics and sulfonamides. (Updated by: David C. Dugdale, III, MD)
Note that the mature forms of the myeloid series (neutrophils, eosinophils, basophils), all have lobed (segmented) nuclei. The degree of lobation increases as the cells mature.
The earliest recognizable myeloid cell is the myeloblast (10-20m dia) with a large round to oval nucleus. There is fine diffuse immature chromatin (without clumping) and a prominant nucleolus.
The cytoplasm is basophilic without granules. Although one may see a small golgi area adjacent to the nucleus, granules are not usually visible by light microscopy. One should not see blast cells in the peripheral blood.
The promyelocyte (10-20m) is slightly larger than a blast. Its nucleus, although similar to a myeloblast shows slight chromatin condensation and less prominent nucleoli. The cytoplasm contains striking azurophilic granules or primary granules. These granules contain myeloperoxidase, acid phosphatase, and esterase enzymes. Normally no promyelocytes are seen in the peripheral blood.
At the point in development when secondary granules can be recognized, the cell becomes a myelocyte.
Myelocytes (10-18m) are not normally found in the peripheral blood. Nucleoli may not be seen in the late myelocyte. Primary azurophilic granules are still present, but secondary granules predominate. Secondary granules (neut, eos, or baso) first appear adjacent to the nucleus. In neutrophils this is the “dawn” of neutrophilia.
Metamyelocytes (10-18m) have kidney shaped indented nuclei and dense chromatin along the nuclear membrane. The cytoplasm is faintly pink, and they have secondary granules (neutro, eos, or baso). Zero to one percent of the peripheral blood white cells may be metamyelocytes (juveniles).
Segmented (segs) or polymorphonuclear (PMN) leukocytes (average 14 m dia) are distinguished by definite lobation with thin thread-like filaments of chromatin joining the 2-5 lobes. 45-75% of the peripheral blood white cells are segmented neutrophils.
Large progenitor cells in the bone marrow called megakaryocytes (MKs) are the source of platelets. MKs release platelets through a series of fascinating cell biological events. During maturation, they become polyploid and accumulate massive amounts of protein and membrane. Then, in a cytoskeletal-driven process, they extend long branching processes, designated proplatelets, into sinusoidal blood vessels where they undergo fission to release platelets.
Acute myeloid leukemias with recurrent genetic abnormalities:
AML with t(8;21)(q22;q22); RUNX1-RUNX1T1
AML with inv(16)(p13.1;q22) or t(16;16)(p13.1;q22); CBF&beta-MYH11
Acute promyelocytic leukemia with t(15;17)(q22;q12); PML/RAR&alpha and variants
AML with t(9;11)(p22;q23); MLLT3-MLL
AML with t(6;9)(p23;q34); DEK-NUP214
AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1
AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1
AML with mutated NPM1*
AML with mutated CEBPA*
* provisional
Acute myeloid leukemia with myelodysplasia related changes
Therapy related acute myeloid leukemia
Alkylating agent related
Topoisomerase II inhibitor related (some maybe lymphoid)
Acute myeloid leukemia not otherwise categorized:
AML minimally differentiated (M0)
AML without maturation (M1)
AML with maturation (M2)
Acute myelomonocytic leukemia (M4)
Acute monoblastic and monocytic leukemia (M5a, M5b)
Acute erythroid leukemia (M6)
Acute megakaryoblastic leukemia (M7)
Acute basophilic leukemia
Acute panmyelosis with myelofibrosis
Myeloid Sarcoma
Myeloid proliferations related to Down syndrome:
Transient abnormal myelopoeisis
Myeloid leukemia associated with Down syndrome
Blastic plasmacytoid dentritic cell neoplasm:
Acute leukemia of ambiguous lineage:
Acute undifferentiated leukemia
Mixed phenotype acute leukemia with t(9;22)(q34;q11.2); BCR-ABL1
Mixed phenotype acute leukemia with t(v;11q23); MLL rearranged
Mixed phenotype acute leukemia, B/myeloid, NOS
Mixed phenotype acute leukemia, T/myeloid, NOS
Mixed phenotype acute leukemia, NOS, rare types
Other acute leukemia of ambiguous lineage
References: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue (IARC, 2008), Discovery Medicine 2010, eMedicine
Acute lymphocytic leukemia
General
=================================================================
WHO classification system includes former FAB classifications ALL-L1 and L2
● FAB L3 is now considered Burkitt lymphoma
WHO classification of acute lymphoblastic leukemia
=================================================================
Precursor B lymphoblastic leukemia / lymphoblastic lymphoma:
● ALL with t(9;22)(q34;q11.2); BCR-ABL (Philadelphia chromosome)
● ALL with t(v;11q23) (MLL rearranged)
● ALL with t(1;19)(q23;p13.3); TCF3-PBX1 (E2A-PBX1)
● ALL with t(12;21)(p13;q22); ETV6-RUNX1 (TEL-AML1)
● Hyperdiploid > 50
● Hypodiploid
● t(5;14)(q31;q32); IL3-IGH
General
=================================================================
De novo acute leukemia containing separate populations of blasts of more than one lineage (bilineal or bilineage), or a single population of blasts co-expressing antigens of more than one lineage (biphenotypic)Excludes:
● Acute myeloid leukemia (AML) with recurrent translocations t(8;21), t(15;17) or inv(16)
● Leukemias with FGFR1 mutations
● Chronic myelogenous leukemia (CML) in blast crisis
● Myelodysplastic syndrome (MDS)-related AML and therapy-related AML, even if they have MPAL immunophenotypeCriteria for biphenotypic leukemia:
● Score of 2 or more for each of two separate lineages:The European Group for the Immunological Classification of Leukemias (EGIL) scoring system2008 WHO classification of acute leukemias of ambiguous lineage
Poor, overall survival of 18 months
● Young age, normal karyotype and ALL induction therapy are associated with favorable survival
● Ph+ is a predictor for poor prognosis
● Bone marrow transplantation should be considered in first remission
Major Categories
MPAL with t(9;22)(q34;q11.2); BCR-ABL1
=================================================================
20% of all MPAL
● Blasts with t(9;22)(q34;q11.2) translocation or BCR-ABL1 rearrangement (Ph+) without history of CML
● Majority in adults
● High WBC counts● Most of the cases B/myeloid phenotype
● Rare T/myeloid, B and T lineage, or trilineage leukemiasMorphology:
● Many cases show a dimorphic blast population, one resembling myeloblasts and the other lymphoblastsCytogenetic abnormalities:
● Conventional karyotyping for t(9;22), FISH or PCR for BCR-ABL1 translocation
● Additional complex karyotypes
● Ph+ is a poor prognostic factor for MPAL, with a reported median survival of 8 months
● Worse than patients of all other types of MPAL
MPAL with t(v;11q23); MLL rearranged
=================================================================
Meeting the diagnostic criteria for MPAL with blasts bearing a translocation involving the 11q23 breakpoint (MLL gene)
● MPAL with MLL rearranged rare
● More often seen in children and relatively common in infancy
● High WBC counts
● Poor prognosis
● Dimorphic blast population, with one resembling monoblasts and the other resembling lymphoblasts
● Lymphoblast population often shows a CD19+, CD10- B precursor immunophenotype, frequently CD15+
● Expression of other B markers usually weak
● Translocations involving MLL gene include t(4;11)(q21;q23), t(11;19)(q23;p13), and t(9;11)(p22;q23)
● Cases with chromosome 11q23 deletion should not be classified in this category
B cell acute lymphoblastic leukemia (ALL) / lymphoblastic lymphoma (LBL)
Current 2008 WHO classification: B lymphoblastic leukemia / lymphoma, NOS or B lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities
See also lymphomas: B cell chapter
Also called B cell acute lymphoblastic leukemia / lymphoblastic lymphoma, pre B ALL / LBL
Usually children
B acute lymphoblastic leukemia presents with pancytopenia due to extensive marrow involvement, stormy onset of symptoms, bone pain due to marrow expansion, hepatosplenomegaly due to neoplastic infiltration, CNS symptoms due to meningeal spread and testicular involvement
B acute lymphoblastic lymphoma often presents with cutaneous nodules, bone or nodal involvement, < 25% lymphoblasts in bone marrow and peripheral blood; aleukemic cases are usually asymptomatic
Depending on specific leukemia, arises in either hematopoietic stem cell or B-cell progenitor
Tumors are derived from pre-germinal center naive B cells with unmutated VH region genes
Have multiple immunophenotyping aberrancies relative to normal B cell precursors (hematogones); at relapse, 73% show loss of 1+ aberrance and 60% show new aberrancies (Am J Clin Pathol 2007;127:39)
Favorable prognosis: age 1-10 years, female, white; preB phenotype, hyperdiploidy>50, t(12,21), WBC count at presentation <50×108/L, non-traumatic tap with no blasts in CNS, rapid response to chemotherapy < 5% blasts on morphology on day 15, remission status after induction <5% blasts on morphology and <0.01% blast on flow or PCR, CD10+
Intermediate prognosis: hyperdiploidy 47-50, diploid, 6q- and rearrangements of 8q24
Unfavorable prognosis: under age 1 (usually have 11q23 translocations) or over age 10; t(9;22) (but not if age 59+ years, Am J Clin Pathol 2002;117:716); male, > 50×108/L WBC at presentation, hypodiploidy, near tetraploidy, 17p- and MLL rearrangements t(v;11q23); CD10-; non-traumatic tap with > 5% blasts or traumatic tap (7%); also increased microvessel staining using CD105 in children (Leuk Res 2007;31:1741), MDR1 expression in children (Oncol Rep 2004;12:1201) and adults (Blood 2002;100:974), 25%+ blasts on morphology on day 15, remission status after induction ≥ 5% blasts on morphology and ≥ 0.1% blasts on flow or PCR
Bone marrow smears: small to intermediate blast-like cells with scant, variably basophilic cytoplasm, round / oval or convoluted nuclei, fine chromatin and indistinct nucleoli; frequent mitotic figures; may have “starry sky” appearance similar to Burkitt lymphoma; may have large lymphoblasts with 1-4 prominent nucleoli resembling myeloblasts; usually no sclerosis
Bone marrow biopsy: usually markedly hypercellular with reduction of trilinear maturation; cells have minimal cytoplasm, medium sized nuclei that are often convoluted, moderately dense chromatin and indistinct nucleoli, brisk mitotic activity
Other tissues: may have “starry sky” appearance similar to Burkitt lymphoma; collagen dissection, periadipocyte growth pattern and single cell linear filing
The World Health Organization (WHO) classification of the myeloid neoplasms James W. Vardiman, Nancy Lee Harris, and Richard D. Brunning
Blood 2002; 100(7) http://dx.doi.org/10.1182/blood-2002-04-1199
Lymphoma – Non B cell neoplasms
T/NK cell disorders/WHO classification (2008)
Principles of classification
=================================================================
Based on all available information (morphology, immunophenotype, genetics, clinical)
● No one antigenic marker is specific for any neoplasm (except ALK1)
● Immune profiling less helpful in subclassification of T cell lymphomas then B cell lymphomas
● Certain antigens commonly associated with specific disease entities but not entirely disease specific
● CD30: common in anaplastic large cell lymphoma but also classic Hodgkin lymphoma and other B and T cell lymphomas
● CD56: characteristic for nasal NK/T cell lymphoma, but also other T cell neoplasms and plasma cell disorders
● Variation of immunophenotype within a given disease (hepatosplenic T cell lymphoma: usually γδ but some are αβ)
● Recurrent genetic alterations have been identified for many B cell lymphomas but not for most T cell lymphomas
● No attempt to stratify lymphoid malignancies by grade
● Recognize the existence of grey zone lymphomas
● This multiparameter approach has been validated in international studies as highly reproducible
WHO 2008 classification of tumors of hematopoietic and lymphoid tissues (T/NK)
=================================================================
Precursor T-lymphoid neoplasms
● T lymphoblastic leukemia/lymphoma, 9837/3
Mature T cell and NK cell neoplasms
● T cell prolymphocytic leukemia, 9834/3
● T cell large granular lymphocytic leukemia, 9831/3
● Chronic lymphoproliferative disorder of NK cells, 9831/3
● Aggressive NK cell leukemia, 9948/3
● Systemic EBV-positive T cell lymphoproliferative disease of childhood, 9724/3
● Hydroa vacciniforme-like lymphoma, 9725/3
● Adult T cell leukemia/lymphoma, 9827/3
● Extranodal NK/T cell lymphoma, nasal type, 9719/3
● Enteropathy-associated T cell lymphoma, 9717/3
● Hepatosplenic T cell lymphoma, 9716/3
● Subcutaneous panniculitis-like T cell lymphoma, 9708/3
● Mycosis fungoides, 9700/3
● Sézary syndrome, 9701/3
● Primary cutaneous CD30-positive T cell lymphoproliferative disorders
● Lymphomatoid papulosis, 9718/1
● Primary cutaneous anaplastic large cell lymphoma, 9718/3
● Primary cutaneous gamma-delta T cell lymphoma, 9726/3
● Primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T cell lymphoma, 9709/3
● Primary cutaneous CD4-positive small/medium T cell lymphoma, 9709/3
● Peripheral T cell lymphoma, NOS, 9702/3
● Angioimmunoblastic T cell lymphoma, 9705/3
● Anaplastic large cell lymphoma, ALK-positive, 9714/3
● Anaplastic large cell lymphoma, ALK-negative, 9702/3
General considerations in the staging of chronic lymphocytic leukemia (CLL) and the revised Rai (United States) and Binet (Europe) staging systems for CLL are provided below.[1, 2, 3]
CLL and small lymphocytic lymphoma (SLL) are different manifestations of the same disease; SLL is diagnosed when the disease is mainly nodal, and CLL is diagnosed when the disease is seen in the blood and bone marrow
CLL is diagnosed by > 5000 monoclonal lymphocytes/mm3 for longer than 3mo; the bone marrow usually has more than 30% monoclonal lymphocytes and is either normocellular or hypercellular
Monoclonal B lymphocytosis is a precursor form of CLL that is defined by a monoclonal B cell lymphocytosis < 5000 monoclonal lymphocytes/mm3; all lymph nodes smaller than 1.5 cm; no anemia; and no thrombocytopenia
Revised Rai staging system (United States)
Low risk (formerly stage 0)[1] :
Lymphocytosis, lymphocytes in blood > 15000/mcL, and > 40% lymphocytes in the bone marrow
Intermediate risk (formerly stages I and II):
Lymphocytosis as in low risk with enlarged node(s) in any site, or splenomegaly or hepatomegaly or both
High risk (formerly stages III and IV):
Lymphocytosis as in low risk and intermediate risk with disease-related anemia (hemoglobin level < 11.0 g/dL or hematocrit < 33%) or platelets < 100,000/mcL
Binet staging system (Europe)
Stage A:
Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and < 3 enlarged areas
Stage B:
Hemoglobin ≥ 10 g/dL, platelets ≥ 100,000/mm3, and ≥ 3 enlarged areas
Stage C:
Hemoglobin < 10 g/dL, platelets < 100,000/mm3, and any number of enlarged areas
This discussion that completes and is an epicrisis (summary and critical evaluation) of the series of discussions that preceded it.
Innervation of Heart and Heart Rate
Action of hormones on the circulation
Allogeneic Transfusion Reactions
Graft-versus Host reaction
Unique problems of perinatal period
High altitude sickness
Deep water adaptation
Heart-Lung-and Kidney
Acute Lung Injury
The concept inherent in this series is that the genetic code is an imprint that is translated into a message. It is much the same as a blueprint, or a darkroom photographic image that has to be converted to a print. It is biologically an innovation of evolutionary nature because it establishes a simple and reproducible standard for the transcription of the message through the transcription of the message using strings of nucleotides (oligonucleotides) that systematically transfer the message through ribonucleotides that communicate in the cytoplasm with the cytoskeleton based endoplasmic reticulum (ER), composing a primary amino acid sequence. This process is a quite simple and convenient method of biological activity. However, the simplicity ends at this step. The metabolic components of the cell are organelles consisting of lipoprotein membranes and a cytosol which have particularly aligned active proteins, as in the inner membrane of the mitochondrion, or as in the liposome or phagosome, or the structure of the ER, each of which is critical for energy transduction and respiration, in particular, for the mitochondria, cellular remodeling or cell death, with respect to the phagosome, and construction of proteins with respect to the ER, and anaerobic glycolysis and the hexose monophosphate shunt in the cytoplasmic domain. All of this refers to structure and function, not to leave out the membrane assigned transport of inorganic, and organic ions (electrolytes and metabolites).
I have identified a specific role of the ER, the organelles, and cellular transactions within and between cells that is orchestrated. But what I have outlined is a somewhat limited and rigid model that does not reach into the dynamics of cellular transactions. The DNA has expression that may be old, no longer used messages, and this is perhaps only part of a significant portion of “dark matter”. There is also nuclear DNA that is enmeshed with protein, mRNA that is a copy of DNA, and mDNA is copied to ribosomal RNA (rRNA). There is also rDNA. The classic model is DNA to RNA to protein. However, there is also noncoding RNA, which plays an important role in regulation of transcription.
This has been discussed in other articles. But the important point is that proteins have secondary structure through disulfide bonds, which is determined by position of sulfur amino acids, and by van der Waal forces, attraction and repulsion. They have tertiary structure, which is critical for 3-D structure. When like subunits associate, or dissimilar oligomers, then you have heterodimers and oligomers. These constructs that have emerged over time interact with metabolites within the cell, and also have an important interaction with the extracellular environment.
When you take this into consideration then a more complete picture emerges. The primitive cell or the multicellular organism lives in an environment that has the following characteristics – air composition, water and salinity, natural habitat, temperature, exposure to radiation, availability of nutrients, and exposure to chemical toxins or to predators. In addition, there is a time dimension that proceeds from embryonic stage to birth in mammals, a rapid growth phase, a tapering, and a decline. The time span is determined by body size, fluidity of adaptation, and environmental factors. This is covered in great detail in this work. The last two pieces are in the writing stage that completes the series. Much content has already be presented in previous articles.
The function of the heart, kidneys and metabolism of stressful conditions have already been extensively covered in http://pharmaceuticalintelligence.com in the following and more:
The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I
2012pharmaceutical
Amandeep Kaur
Aashir Awan, Phd
Abhisar Anand
Adina Hazan
Alan F. Kaul, PharmD., MS, MBA, FCCP
alexcrystal6
anamikasarkar
apreconasia
aviralvatsa
David Orchard-Webb, PhD
danutdaagmailcom
Demet Sag, Ph.D., CRA, GCP
Dror Nir
dsmolyar
Ethan Coomber
evelinacohn
Gail S Thornton
Irina Robu
jayzmit48
jdpmd
jshertok
kellyperlman
Ed Kislauskis
larryhbern
Madison Davis
marzankhan
megbaker58
ofermar2020
Dr. Pati
pkandala
ritusaxena
Rick Mandahl
sjwilliamspa
Srinivas Sriram
stuartlpbi
Dr. Sudipta Saha
tildabarliya
vaishnavee24
zraviv06
zs22
