Kinetics of Recovery of Naïve and Memory T Cells in Acute Leukemia Patients after Allogeneic Stem Cell Transplantation Depending on Different GVHD Prophylaxis Regimens.

Background: Memory T cells are a heterogeneous population of immune cells that provide adaptive immunity. Its full recovery seems essential for graft-versus-tumor reactions that provide an opportunity for biological cure in patients with acute leukemia. The use of mismatched or haploidentical donors has increased, which has become possible because of modifications in graft versus host disease (GVHD) prophylaxis. Materials and Methods: Sixty-five leukemia patients (acute myeloid leukemia - 40, acute lymphoblastic leukemia - 25), median age 33 (17-61) years, underwent allo-HSCT from 2016 to 2019 in the National Research Centre for Hematology. Patients were divided into three groups based on the impact of GVHD prophylaxis on T cell recovery: horse antithymocyte globulin (ATG)-based regimen (n=32), horse ATG combined with posttransplant cyclophosphamide (PT-Cy) (n=18), and ex vivo T cell depletion (n=15). Results: The early period after transplantation (before day +100) was characterized by significantly lower absolute numbers of T naïve, memory stem and T central memory cells in peripheral blood in patients after ATG+PT-Cy-regimen or ex vivo T cell depletion than after ATG-based prophylaxis (p<0.05). Moreover, strong depletion of naïve T and memory stem cells prevents the development of GVHD, and determining the absolute number of CD8+ naïve T and memory stem cells with a cutoff of 1.31 cells per microliter seems to be a perspective in assessing the risks of developing acute GVHD (p=0.008). The dynamics of T cell recovery showed the involvement of either circulating or bone marrow resident T effector cells shortly after allogeneic transplantation in all patients, but the use of manipulated grafts with ex vivo T cell depletion requires the involvement of naïve and memory stem cells. There was no significant effect of T cell recovery on leukemia relapse after allogeneic transplantation. Conclusion: These experimental outcomes contribute to providing the best understanding of immunological events that occur early after transplantation and help in the rational choice of GVHD prophylaxis in patients who will undergo allogeneic transplantation. Our study demonstrated the comparable immunological effects of posttransplant cyclophosphamide and ex vivo T cell depletion and immunological inefficiency of horse ATG for GVHD prevention.

Gene Expression Profile-Guided Personalized Intraperitoneal Chemotherapy for Gastric Cancer Peritoneal Carcinomatosis.

Background: Peritoneal carcinomatosis (PC) is one of the most unfavorable sites of metastasis for malignant tumors of various localizations, especially gastric cancer (GC). According to the literature, synchronous PC in GC is common in 15-52% of patients. The purpose of this study was to examine the long-term results using personalized systemic and intraperitoneal chemotherapy as part of the combined treatment of stomach cancer presenting with synchronous PC. Methods: Cytoreductive surgical treatment was performed for 70 patients at the first stage. The control group (n = 35) received standard postoperative chemotherapy according to the FOLFOX scheme. Personalized postoperative systemic and intraperitoneal chemotherapy was administered in the basic group (n = 35), based on the expression levels of the eight genes in the primary tumor, lymph node, and peritoneal metastases. Results: The median progression-free survival was 14.9 months in the basic group, and in the control group it was 11.2 months (P < 0.001). The median life expectancy in the basic group was 16.8 (13.7 - 18.8) months, in the control group it was 12.5 (11.3 - 13.1) months (P < 0.001). Conclusions: Developing algorithms of personalized systemic and intraperitoneal chemotherapy in patients with GC with synchronous carcinomatosis, based on the analysis of molecular genetic characteristics of the tumor and metastases, allows to improve the long-term results of combined treatment.

The Role of Intravesicular Proteins and the Protein Corona of Extracellular Vesicles in the Development of Drug-Induced Polyneuropathy.

Extracellular vesicles (EVs) as membrane structures of cellular origin participating in intercellular communication are involved in the molecular mechanisms of the development of various variants of polyneuropathy. Taking into account the increasing role of the protein corona of EVs and protein-protein interactions on the surface of EVs in the pathogenesis of various diseases, we focused our attention in this review on the role of intravesicular proteins and the protein corona of EVs in the development of chemotherapy-induced polyneuropathy (CIPN). It has been shown that EVs are effectively internalized by the mechanisms of endocytosis and macropinocytosis by neurocytes and glial cells, carry markers of insulin resistance, functionally active proteins (receptors, cytokines, enzymes), and may be involved in the pathogenesis of CIPN. The mechanisms of CIPN associated with the EVs protein corona can be related with the accumulation of heavy chains of circulating IgG in it. G-class immunoglobulins in EVs are likely to have myelin hydrolyzing, superoxide dismutase, and oxidoreductase enzymatic activities. Moreover, circulating IgG-loaded EVs are a place for complement activation that can lead to membrane attack complex deposition in neuroglia and neurons. The mechanisms of CIPN development that are not associated with IgG in the EVs protein corona are somehow related to the fact that many anticancer drugs induce apoptosis of tumor cells, neurons, and neuroglial cells by various mechanisms. This process may be accompanied by the secretion of EVs with modified cargo (HSPs, 20S proteasomes, miRNAs).

MORPHOLOGICAL AND FUNCTIONAL FEATURES OF THE MUCOUS MEMBRANE OF SMALL AND LARGE INTESTINE IN PATIENTS WITH COVID-19 AND IN POST-COVID-19 PERIOD.

OBJECTIVE: The aim: To reveal the morphological and functional features of the mucous membrane of small and large intestine in patients with COVID-19 and in post-COVID-19 period. PATIENTS AND METHODS: Materials and methods: In the present study, the authors used biopsy and autopsy material represented by the fragments of the mucous membrane of small and large intestine. All studied material was divided into 10 groups. Group 1 (comparison group) included autopsy material from the deceased who did not have COVID-19 during their lifetime. Groups 2-4 included autopsy material from the deceased who had COVID-19 of varying severity during their lifetime. Groups 5-7 included biopsy material from patients who had recovered from COVID-19 of varying severity, while the duration of the post-COVID period ranged from 1 to 50 days. Groups 8-10 included biopsy material from patients who had in anamnesis COVID-19 of varying severity (the duration of the post-COVID period lasted from 51 to 100 days). Histological, immunohistochemical, morphometric and statistical research methods were used. RESULTS: Results: The comparative analysis showed a more expressed deficiency of ACE2 in the mucous membrane of small and large intestine in patients with moderate and severe COVID-19 compared with patients in post-COVID-19 period of different duration. In patients who had moderate and severe COVID-19 in anamnesis, ACE2 deficiency decreases with increasing duration of post-COVID-19 period. In patients recovered from mild COVID-19, the ACE2 content increases with the duration of post-COVID-19 period from 1 to 50 days and corresponds to the norm with the duration of this period from 51 to 100 days. CONCLUSION: Conclusions: The comprehensive morphological study conducted by the authors made it possible, firstly, to clarify the morphological and functional features of the mucous membrane of small and large intestine in patients with COVID-19 of various degrees of severity; secondly, to obtain new data about the morpho-functional state of the mucous membrane of small and large intestine in patients, taking into account different duration of the post-COVID-19 period and the severity of the infection.

SEBORRHEIC KERATOSIS: CURRENT STATE OF THE PROBLEM.

OBJECTIVE: The aim is to identify age, gender, clinical and morphological features of seborrheic keratosis. PATIENTS AND METHODS: Material and methods: The study used biopsy material from 196 patients with a clinical diagnosis of "Seborrheic keratosis". In all cases, when studying directions for pathohistological examination, the authors analyzed gender and age characteristics, as well as localization of seborrheic keratosis. The resulting material was fixed in a 10% solution of neutral formalin (ph 7.4) for 24-48 hours. The procedure was carried out according to the generally accepted technique and the material was embedded in paraffin. Sections of 4-5×10-6m thick were made from paraffin blocks for staining with hematoxylin and eosin. The microspecimens were examined using an Olympus BX-41 microscope (Japan). RESULTS: Results: The study revealed a modern feature of seborrheic keratosis -"rejuvenation" of this tumor, as evidenced by its predominant development in patients aged 31 to 50 years. Seborrheic keratosis is more common in men, and is localized mainly on the skin of the face, scalp, neck and back. When diagnosing seborrheic keratosis, the author notes a significant discrepancy between clinical and morphological diagnoses, which actualizes the problem of improving the available clinical research methods and emphasizes the importance of morphological research. Hyperkeratotic and acanthotic histological variants of seborrheic keratosis are the most common. The case of seborrheic keratosis with malignancy and transformation into squamous cell carcinoma, identified by the author, should develop oncological alertness in doctors and patients. CONCLUSION: Conclusions: The study revealed age, gender, clinical and morphological features of seborrheic keratosis, which will contribute to a better understanding of this pathology by the doctors of various specialties, and improve the treatment and diagnostic process.

Bowel wall thickness is a strong predictor of steroid-refractory acute graft-versus-host disease with gut involvement after allo-HSCT.

OBJECTIVE: Acute graft-versus-host-disease (aGVHD) develops in 10-80% of allo-HSCT patients. More than half of all aGVHD cases are refractory to first-line therapy with steroids. We hypothesized that bowel wall thickness at the time of aGVHD diagnosis could be an early sign of steroid-refractory aGVHD with gut involvement. METHOD: Our prospective study included 85 patients with hematological malignancies who had undergone allo-HSCT. We used an inexpensive, widespread and simple method of transabdominal ultrasonography to examine bowel wall thickness in patients suspected to have gut aGVHD. RESULTS: Descending colon wall thickness was significantly greater in patients with gut aGVHD later found to be steroid-refractory than in patients with steroid-sensitive gut aGVHD, with AUC-0.73 (95% CI 0.58-0.87, p = 0.013). We showed that bowel wall thickness could predict the steroid-refractoriness of aGVHD. CONCLUSION: Transabdominal ultrasonography could be used as a marker of steroid-refractory aGVHD with gut involvement after allo-HSCT.

Explaining Consumer Intentions for Foods with Antioxidant Properties: Predictors of Choice and Purchase Barriers.

Today, food products not only should serve the source of main nourishment but also must minimize the risk of negative impact on the human body. Products enriched with antioxidants can be referred to such category. For food producers, the development of products "for health" is usually connected with significant investment, whereas the final success of innovative products does not always meet the expectations. The greatest part of such products is withdrawn from the market during the first year. It is important for manufacturers to learn consumer behavior in order to ensure sale growth and a stable market position. The purpose of this study was to study consumer reception of products containing antioxidants. (It is important to conduct market research to identify the needs of buyers in order to maintain a stable position in the market. In order to study this issue, we analyzed the consumer perception of foods with antioxidant properties.) We studied the consumer perception of products with antioxidant properties with regard to choice predictors and barriers for purchasing, which finally determine the success of the product on the market. For this purpose, we conducted a survey of 721 consumers of the South Urals. The results of the statistical analysis done with the help of SPSS proved that South Ural consumers in general are ready to purchase products containing antioxidants. Besides, producers must bring information concerning the real value of the product and win the consumer trust and confidence as far as these are the main predictors determining the choice for purchasing the products containing antioxidants. Misunderstanding of the role antioxidants play in the human body may lead to perception of risk concerning consumption of such products and rejection of the purchase.

MORPHOLOGICAL EFFICACY EVALUATION OF GEL WITH CARBON DIOXIDE EXTRACT OF HOPS IN CASE OF COMPLICATED WOUND INFECTION ACNE VULGARIS.

OBJECTIVE: The aim of the study is to reveal in the experiment the morphological features of the infected skin wounds healing, which are a manifestation of acne vulgaris severe and very severe forms, using a gel with carbon dioxide extract of hops. PATIENTS AND METHODS: Materials and methods: An experimental study was carried out on 80 male WAG rats of three months of age. The animals were divided into 9 groups. Group 1 consisted of intact animals (n=6). Group 2 was represented by animals (n=6), which had hair epilation on the dorsal surface of the body in an area of 1 сm2, followed by application of 2 ml of placebo gel to this area. Group 3 included animals (n=6), which were epilated on the dorsal surface of the body in an area of 1 сm2 and applied 2 ml of 1 % gel with carbon dioxide extract of hops. Group 4 included rats (n=6), which were epilated on the dorsal surface of the body in an area of 1 сm2 and simulated thermal damage. Group 5 was represented by 10 rats, who were epilated on the dorsal surface of the body in an area of 1 сm2, simulated thermal damage, followed by applying 2 ml of placebo gel to the wound surface. Group 6 included rats (n=10), who underwent measures similar to group 5, followed by application of 2 ml of 1 % gel with carbon dioxide extract of hops to the wound surface. In group 7, there were 6 rats, which were epilated on the dorsal surface of the body in an area of 1 сm2, thermal damage to the skin with underlying soft tissues was simulated, followed by application to the wound surface the reference strains of Staphylococcus aureus, Streptococcus pyogenes, Proteus vulgaris, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Malassezia slooffiae, Malassezia pachydermatis, Candida albicans, Candida parapsilosis. In groups 8 and 9, there were 15 rats each, which underwent measures similar to group 7, followed by applying 2 ml of placebo gel on its surface on the next day after infection of the wound in group 8, and in group 9 - 2 ml of 1 % gel with carbon dioxide extract hops. The material for the study was the skin with underlying soft tissues. It was used histological, histochemical, morphometric and statistical methods. RESULTS: Results: This experimentally created gel with carbon dioxide extract of hops activates separation processes of horny masses from the surface of the epidermis, cleaning the pores of the skin; stimulating the proliferative activity of the epidermis, which is located in the marginal sections of the wound or covers the surface of the regenerate; activating the processes of cleansing the wound from necrotic tissue; activating the growth and maturation of granulation tissue with its subsequent transformation into connective tissue. It has anti-inflammatory, bactericidal and antimycotic effects, normalizing skin microbiocenosis. CONCLUSION: Conclusions: The complex morphological study has showed that gel with carbon dioxide extract of hops is a highly effective drug in treatment of severe and very severe acne vulgaris, characterized by the development of deep and infected wound defects.

High expression of granzyme B in conventional CD4+ T cells is associated with increased relapses after allogeneic stem cells transplantation in patients with hematological malignancies.

Granzyme B is known to be a serine protease contained in granules of cytotoxic T cells. We have previously reported an influence of granzyme B expression in T regulatory cells (Tregs) on the risk of acute graft versus host disease (GVHD) onset. However, it is still unknown if conventional T cells (Tcon) use the granzyme B pathway as a mechanism of alloimmunity. We hypothesized that granzyme B in Tcon may affect recurrence within the first 6 months after allogeneic transplantation (allo-HSCT). A total of 65 patients with different hematological malignancies were included in this study. Blood samples were collected on day +30 after allo-HSCT. The percentage of granzyme B positive conventional T cells in patients who developed relapse in the first 6 months after allo-HSCT was 11.3 (4.5-35.3) compared to the others in continuous complete remission-1.3 (3.65-9.7), р = 0.011. The risk of relapse after allo-HSCT was in 3.9 times higher in patients with an increased percentage of granzyme B positive conventional T cells. The findings demonstrated that the percentage of granzyme B positive conventional T cells on day +30 after allo-HSCT could be a predictable marker of relapse within the first 6 months after allo-HSCT.

Study of anti-virus actions of metabolites of lactobacteria.

OBJECTIVE: The aim: of the work was to study the antiviral activity of the metabolites of the probiotic strain Lactobacillus rhamnosus GG (LGG or ATCC 53103) regarding clinical strains of enteroviruses (Coxsackie B-5, ECNO21) isolated from the feces of intestinal infections. PATIENTS AND METHODS: Materials and methods: The object of the study was substrate-dependent cell cultures of HeLa, Vero, Hep-2 lines. The titer of the virus was determined by the presence of a clear cytopathic action (CPA) in the monolayer infected cells of the virus. RESULTS: Results: Determination of the enteric virus infections activity in the culture fluid showed that in samples with the LGG metabolites, the infections activity of the clinical strains of enteroviruses decreased after 24 hours, at 1.5-1.7 (p <0.05) times, and after 96 hours in 3, 6 - 5,7 times (p <0,01). the processing of cell cultures by metabolites in the amount of 0.3 mg / ml contributed to a decrease in the titer of viruses by 2.77 ± 0.11 lg TCDD50 / cm3, 2.83 ± 0.11 lg TCD50 / cm3 and 2.94 ± 0.13 lg TCD50 / cm3 for Vero, HeLa and Hep-2 line cells in 24 hours. CONCLUSION: Conclusions: It has been experimentally determined that the maximum tolerated dose (MTD) of L. rhamnosus GG metabolites was 0.3 µg / ml for all cultures of cell lines. Determination of the antiviral activity of L. rhamnosus GG metabolites in clinical viruses of enteroviruses (Coxsackie B-5 and ECNO-21) showed a decrease in infection activity in 1.5-1.7 times, (p <0.05) of clinical trials in clinical trials enteroviruses.

Recovery of Donor Hematopoiesis after Graft Failure and Second Hematopoietic Stem Cell Transplantation with Intraosseous Administration of Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cells (MSCs) participate in the formation of bone marrow niches for hematopoietic stem cells. Donor MSCs can serve as a source of recovery for niches in patients with graft failure (GF) after allogeneic bone marrow (BM) transplantation. Since only few MSCs reach the BM after intravenous injection, MSCs were implanted into the iliac spine. For 8 patients with GF after allo-BMT, another hematopoietic stem cell transplantation with simultaneous implantation of MSCs from their respective donors into cancellous bone was performed. BM was aspirated from the iliac crest of these patients at 1-2, 4-5, and 9 months after the intraosseous injection of donor MSCs. Patients' MSCs were cultivated, and chimerism was determined. In 6 out of 8 patients, donor hematopoiesis was restored. Donor cells (9.4 ± 3.3%) were detected among MSCs. Thus, implanted MSCs remain localized at the site of administration and do not lose the ability to proliferate. These results suggest that MSCs could participate in the restoration of niches for donor hematopoietic cells or have an immunomodulatory effect, preventing repeated rejection of the graft. Perhaps, intraosseous implantation of MSCs contributes to the success of the second transplantation of hematopoietic stem cells and patient survival.

Development of the Composition and Manufacturing Technology of a New Combined Drug: Lavaflam.

OBJECTIVES: Treatment of diseaes of the biliary system is one of the urgent problems of modern medicine. MATERIALS AND METHODS: An original pharmaceutical drug "Lavaflam" in the form of combined tablets, which includes a composition of herbal components flamin (0.05 g) and lavender oil (0.02 g), was proposed for the complex treatment of diseases of the biliary system. Flamin is phytomedicine from the immortelle flowers [Helichrysum arenarium (L.) Moench, Asteraceae], which contains a complex of active substances from the flavonoids group (salipurposide, isosalipurposide, kaempferol, luteolin). It is applied as a choleretic and an anti-inflammatory agent in cholecystitis, cholangitis, and biliary dyskinesia. Tablets were prepared by pressing for separate granulation technology. RESULTS: The lavender oil granulate was prepared using the solid phase method, and ß-cyclodextrin was used as an excipient substance. The flamin granulate was prepared by mixing with the spherical-shaped filler mannitol PARTECK M 200. On the basis of previous studies, the excipients of the designed composition tablet "Lavaflam" were ß-cyclodextrin (0.27 g), mannitol PARTECK M 200 (0.20 g), croscarmellose sodium (0.03 g), potato starch (0.022 g), PEG 6000 (0.002 g), and magnesium stearate (0.006 g). The assay of the main components of lavender oil with the references linalol and linalyl acetate was performed using gas chromatography. The assay of the total flavonoids of flamin was performed using spectrophotometry with isosalipurposide as the reference. CONCLUSION: The new phytomedicine tablets "Lavaflam" meet European Pharmacopoeia requirements on the following parameters: appearance, geometric size, average weight, disintegration, friability, resistance of tablets to crushing, and quantification.

Level of Granzyme B-positive T-regulatory cells is a strong predictor biomarker of acute Graft-versus-host disease after day +30 after allo-HSCT.

Acute Graft-versus-host-disease (aGVHD), the major complication and one of the main causes of poor outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nowadays there are no widely accepted cell, plasma or another biomarker that can be used for aGVHD prediction. We hypothesized that a level of Granzyme B-positive T regulatory (GZMB-positive Treg) cells on day+30 after allo-HSCT could be the measure of immune response suppression and could predict aGVHD development after day +30. We applied a widespread and easy-to-perform method of multicolor flow cytometry to measure level of GZMB-positive Treg cells. Levels of GZMB-positive Tregs on day +30 after allo-HSCT were significantly higher in those patients who never developed aGVHD in comparison with the other group of patient with aGVHD after day +30 (p=0.0229). We conclude that the level of GZMB-positive Treg cells is a strong predictor of acute Graft-versus-host disease after day +30 after allo-HSCT.

Frequency of EGFR mutations in non-small cell lung cancer patients: screening data from West Siberia.

BACKGROUND: Incorporation of molecular analysis of the epidermal growth factor receptor (EGFR) gene into routine clinical practice has shown great promise to provide personalized therapy of the non-small cell lung cancer (NSCLC) in the developed world. However, the genetic testing of EGFR mutations has not yet become routine clinical practice in territories remote from the central regions of Russia. Therefore, we aimed to study the frequency of major types of activating mutations of the EGFR gene in NSCLC patients residing in West Siberia. MATERIALS AND METHODS: We examined EGFR mutations in exons 19 and 21 in 147 NSCLC patients (excluding squamous cell lung carcinomas) by real time polymerase chain reaction. RESULTS: EGFR mutations were detected in 28 of the 147 (19%) patients. There were 19 (13%) cases with mutations in exon 19 and 9 cases (6%) in exon 21. Mutations were more frequently observed in women (42%, p=0.000) than in men (1%). A significantly higher incidence of EGFR mutations was observed in bronchioloalveolar carcinomas (28%, p=0.019) and in adenocarcinomas (21%, p=0.024) than in large cell carcinomas, mixed adenocarcinomas, and NOS (4%). The EGFR mutation rate was much higher in never-smokers than in smokers: 38% vs. 3% (p=0.000). The frequency of EGFR mutations in the Kemerovo and Tomsk regions was 19%. CONCLUSIONS: The incorporation of molecular analysis of the EGFR gene into routine clinical practice will allow clinicians to provide personalised therapy, resulting in a significant increase in survival rates and improvement in life quality of advanced NSCLC patients.

NCBI's Database of Genotypes and Phenotypes: dbGaP.

The Database of Genotypes and Phenotypes (dbGap, http://www.ncbi.nlm.nih.gov/gap) is a National Institutes of Health-sponsored repository charged to archive, curate and distribute information produced by studies investigating the interaction of genotype and phenotype. Information in dbGaP is organized as a hierarchical structure and includes the accessioned objects, phenotypes (as variables and datasets), various molecular assay data (SNP and Expression Array data, Sequence and Epigenomic marks), analyses and documents. Publicly accessible metadata about submitted studies, summary level data, and documents related to studies can be accessed freely on the dbGaP website. Individual-level data are accessible via Controlled Access application to scientists across the globe.

Association of SNPs of CD40 gene with multiple sclerosis in Russians.

Multiple sclerosis (MS) is a serious, incurable neurological disease. In 2009, the ANZgene studies detected the suggestive association of located upstream of CD40 gene in chromosome 20q13 (p = 1.3×10(-7)). Identification of the causal variant(s) in the CD40 locus leads to a better understanding of the mechanism underlying the development of autoimmune pathologies. We determined the genotypes of rs6074022, rs1883832, rs1535045, and rs11086996 in patients with MS (n = 1684) and in the control group (n = 879). Two SNPs were significantly associated with MS: rs6074022 (additive model C allele OR = 1.27, 95% CI = [1.12-1.45], p = 3×10(-4)) and rs1883832 (additive model T allele OR = 1.20, 95% CI = [1.05-1.38], p = 7×10(-3)). In the meta-analysis of our results and the results of four previous studies, we obtain the association p-value of 2.34×10(-12), which confirmed the association between MS and rs6074022 at a genome-wide significant level. Next, we demonstrated that the model including rs6074022 only sufficiently described the association. From our analysis, we can speculate that the association between rs1883832 and MS was induced by LD, whereas rs6074022 was a marker in stronger LD with the functional variant or was the functional variant itself. Our results indicated that the functional variants were located in the upstream region of the gene CD40 and were in higher LD with rs6074022 than LD with rs1883832.

Interaction of Muc2 and Apc on Wnt signaling and in intestinal tumorigenesis: potential role of chronic inflammation.

Somatic mutations of the adenomatous polyposis coli (APC) gene are initiating events in the majority of sporadic colon cancers. A common characteristic of such tumors is reduction in the number of goblet cells that produce the mucin MUC2, the principal component of intestinal mucus. Consistent with these observations, we showed that Muc2 deficiency results in the spontaneous development of tumors along the entire gastrointestinal tract, independently of deregulated Wnt signaling. To dissect the complex interaction between Muc2 and Apc in intestinal tumorigenesis and to elucidate the mechanisms of tumor formation in Muc2(-/-) mice, we crossed the Muc2(-/-) mouse with two mouse models, Apc(1638N/+) and Apc(Min/+), each of which carries an inactivated Apc allele. The introduction of mutant Muc2 into Apc(1638N/+) and Apc(Min/+) mice greatly increased transformation induced by the Apc mutation and significantly shifted tumor development toward the colon as a function of Muc2 gene dosage. Furthermore, we showed that in compound double mutant mice, deregulation of Wnt signaling was the dominant mechanism of tumor formation. The increased tumor burden in the distal colon of Muc2/Apc double mutant mice was similar to the phenotype observed in Apc(Min/+) mice that are challenged to mount an inflammatory response, and consistent with this, gene expression profiles of epithelial cells from flat mucosa of Muc2-deficient mice suggested that Muc2 deficiency was associated with low levels of subclinical chronic inflammation. We hypothesize that Muc2(-/-) tumors develop through an inflammation-related pathway that is distinct from and can complement mechanisms of tumorigenesis in Apc(+/-) mice.

The NCBI dbGaP database of genotypes and phenotypes.

The National Center for Biotechnology Information has created the dbGaP public repository for individual-level phenotype, exposure, genotype and sequence data and the associations between them. dbGaP assigns stable, unique identifiers to studies and subsets of information from those studies, including documents, individual phenotypic variables, tables of trait data, sets of genotype data, computed phenotype-genotype associations, and groups of study subjects who have given similar consents for use of their data.

Bcl-2 protein in 518A2 melanoma cells in vivo and in vitro.

PURPOSE: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. EXPERIMENTAL DESIGN: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. RESULTS: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors. CONCLUSIONS: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.

Histone deacetylase 3 (HDAC3) and other class I HDACs regulate colon cell maturation and p21 expression and are deregulated in human colon cancer.

Inhibitors of histone deacetylases (HDACs) induce growth arrest, differentiation, and apoptosis of colon cancer cell lines in vitro and have demonstrated anti-cancer efficacy in clinical trials. Whereas a role for HDAC1 and -2 in mediating components of the HDAC inhibitor response has been reported, the role of HDAC3 is unknown. Here we demonstrate increased protein expression of HDAC3 in human colon tumors and in duodenal adenomas from Apc1638(N/+) mice. HDAC3 was also maximally expressed in proliferating crypt cells in normal intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 and other Class I HDACs inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. However, the magnitude of the effects elicited by silencing of individual Class I HDACs was significantly less than that induced by HDAC inhibitors. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression. These findings also demonstrate that multiple Class I HDACs are involved in repressing p21 and suggest that the growth-inhibitory and apoptotic effects induced by HDAC inhibitors are probably mediated through the inhibition of multiple HDACs.