Human effector CD8+ T cells with an activated and exhausted-like phenotype control tumour growth in vivo in a humanized tumour model.

BACKGROUND: Humanized tumour models could be particularly valuable for cancer immunotherapy research, as they may better reflect human-specific aspects of the interfaces between tumour and immune system of human cancer. However, endogenous antitumour immunity in humanized models is still largely undefined. METHODS: We established an autologous humanized mouse tumour model by using NSG mice reconstituted with human immune cells from hematopoietic progenitors and tumours generated from transformed autologous human B cells. We demonstrate growth of solid lymphoid tumours after subcutaneous implantation, infiltration by endogenous human immune cells and immunocompetence of the model. FINDINGS: We found human T cell subsets described in human cancer, including progenitor exhausted (Tpex), terminally exhausted (Tex-term) and tissue-resident (TRM) cells in tumour-bearing humanized mice with accumulation of Tex-term and TRM in the tumour. In addition, we identified tumour-reactive CD8+ T cells through expression of CD137. This subpopulation of de novo arising human CD137+ CD8+ T cells displayed a highly proliferative, fully activated effector and exhausted-like phenotype with enhanced expression of activation and exhaustion markers like PD-1, CD39, CD160, TIM-3, TIGIT and TOX, the senescence marker CD57 (B3GAT1) and cytolytic effector molecules such as PRF1, GZMH and NKG7. Moreover, these CD137+ CD8+ T cells exhibited tumour-specific clonal expansion and presented signature overlap with tumour-reactive CD8+ T cells described in human cancer. We demonstrate superior anticancer activity of this activated and exhausted-like human CD8+ T cell subset by adoptive transfer experiments using recipients bearing autologous human tumours. Mice adoptively transferred with CD137+ CD8+ T cells showed reduced tumour growth and higher CD8+ T cell tumour infiltration, correlating with control of human tumours. INTERPRETATION: We established an immunocompetent humanized tumour model, providing a tool for immunotherapy research and defined effective anticancer activity of human effector CD8+ T cells with an activated and exhausted-like phenotype, supporting clinical exploration of such cells in adoptive T cell therapies. FUNDING: Swiss Cancer Research foundation.

Molecular Characterization and Differentiation of Mesenchymal Progenitor Cells from Human Rheumatoid Arthritis Cartilage.

The presence of mesenchymal progenitor cells (MPCs) in rheumatoid arthritis (RA) articular cartilage is sparsely investigated largely owing to the persistent pathogenic disease condition and lack of specific biomarkers. Considering the recent advancements for potential cell-based therapies in immunomodulatory diseases, such as RA, this in vitro study was aimed at investigating the cellular, molecular, and differentiation characteristics of human RA cartilage-derived MPCs. Articular cartilage fragments from RA patients were obtained for the isolation of MPCs and characterization of their cellular and biological properties, cytogenetic stability, pluripotency, and plasticity. Established MPCs were phenotypically identified using a panel of markers, and their differentiation ability into mesenchymal lineages was assessed by cytochemical staining and the expression of molecular markers. MPCs displayed a heterogenous population of cells with characteristic features of multipotent stem cells. Cells had higher viability, proliferative rate, and colony-forming ability. Further, MPCs showed the expression of pluripotency markers, cytogenetic stability, and minimal replicative senescence. In addition, MPCs differentiated into osteocytes, adipocytes, and chondrocytes, and modulated the expression of each lineage-specific gene markers. The results demonstrated the availability of a viable pool of MPCs residing in RA cartilage, which could serve as an ideal cell source for reinstating native homotypic cartilage.

Clinical Features, Phenotypic Markers and Outcomes of Diffuse Large B-Cell Lymphoma between HIV-Infected and HIV-Uninfected Chinese Patients.

Background: The effect of HIV infection on the clinicopathological characteristics of diffuse large B-cell lymphoma (DLBCL) remains debatable. Methods: Fifty-three HIV-infected and ninety-three HIV-uninfected DLBCL patients were enrolled in the retrospective study by propensity score matching for sex, age, body mass index and international prognostic index (IPI) at a ratio of 1:2. The clinicopathological characteristics were compared between the two groups. Results: HIV-infected DLBCL patients had lower white blood cell counts [×109/L; 4.4 (3.4−5.6) vs. 6.1 (4.2−8.2), p < 0.001], platelet counts (×109/L; 184.7 ± 89.3 vs. 230.0 ± 113.9, p = 0.014) and serum albumin (g/L; 37.3 ± 6.9 vs. 41.3 ± 6.2, p < 0.001) but higher incidences of central nervous system (CNS) involvement (9.4% vs. 1.1%, p = 0.014), bone marrow involvement (24.5% vs. 11.5%, p = 0.044) and Epstein−Barr viremia (61.1% vs. 26.7%, p = 0.002) than HIV-uninfected patients. In terms of histopathology, HIV-infected patients had higher positivity of Epstein−Barr virus-encoded small RNA (EBER) (41.7% vs. 6.7%, p = 0.002), but lower CD20 (90.2% vs. 98.7%, p= 0.029) and CD79a (23.1% vs. 53.7%, p < 0.001) expression. The overall response rate (ORR) at the end of chemotherapy (70.2% vs. 87.8%, p= 0.012) and 1-year overall survival (OS) (61.7% vs. 84.2%, log-rank p = 0.006) in HIV-infected patients were significantly lower than those in HIV-uninfected patients. Multivariate analysis suggested IPI ≤2.0 [adjusted odds ratio (AOR) (95% confidence interval): 5.0 (1.2−21.2), p = 0.030] was associated with ORR, hypoalbuminemia [AOR: 3.3 (1.3−9.1), p = 0.018] and CNS involvement [AOR: 3.3 (1.0−10.5), p = 0.044] were associated with reduced 1-year OS in HIV-infected patients. Conclusion: HIV-infected DLBCL patients have unique blood profiles and phenotypic markers. Low ORR and 1-year OS were observed in HIV-infected DLBCL patients in our study, even in the HAART era.

Looking for a Simplified Diagnostic Model to Identify Potentially Lethal Cases of Prostate Cancer at Initial Diagnosis: An ImGO Pilot Study.

The recurrent genetic anomalies used to classify prostate cancer (PC) into distinct molecular subtypes have limited relevance for clinical practice. In consideration of WHO 2016 histological classification, which includes the introduction of Gleason Score 4 for patients with cribriform component and the definition of intraductal carcinoma as a new entity, a retrospective pilot study was conducted to investigate, by histological review, if there were any variations of Gleason Score and the incidence of intraductal carcinoma and cribriform pattern, intended as "phenotypic" markers of potentially lethal PC, among metastatic castration-sensitive PC (mCSPC) and metastatic castration-resistant PC (mCRPC) samples. Potentially predictive factors were also assessed. Among 125 cases, a variation in the Gleason Score was reported in 26% of cases. A cribriform (36%) or intraductal (2%) pattern was reported in a higher percentage. Of them, a primary Gleason pattern 4 was reported in 80% of cases. All patients with intraductal carcinoma present a BRCA2 mutation, also found in 80% of cases with a cribriform pattern. This pilot study documented some hypothesis-generating data, as the evaluation of de novo mCSPC and mCRPC as phenotypic/biologic model to be translated in clinical practice. A cribriform pattern/intraductal carcinoma might be a marker of potentially lethal PC. The high incidence of TP53 and BRCA2 mutations in de novo mCSPC may also have a therapeutic implication.

Recent insights into the characteristics and role of peritoneal macrophages from ascites of cirrhotic patients.

Macrophages are a diverse myeloid cell population involved in innate and adaptive immune responses, embryonic development, wound repair, and regulation of tissue homeostasis. These cells link the innate and adaptive immunities and are crucial in the development and sustainment of various inflammatory diseases. Macrophages are tissue-resident cells in steady-state conditions; however, they are also recruited from blood monocytes after local pathogen invasion or tissue injury. Peritoneal macrophages vary based on their cell complexity, phenotype, and functional capabilities. These cells regulate inflammation and control bacterial infections in the ascites of decompensated cirrhotic patients. Our recent work reported several phenotypic and functional characteristics of these cells under both healthy and pathological conditions. A direct association between cell size, CD14/CD16 expression, intracellular level of GATA-6, and expression of CD206 and HLA-DR activation/maturation markers, indicate that the large peritoneal macrophage CD14highCD16high subset constitutes the mature phenotype of human resident peritoneal macrophages during homeostasis. Moreover, elevated expression of CD14/CD16 is related to the phagocytic capacity. The novel large CD14highCD16high peritoneal subpopulation is increased in the ascites of cirrhotic patients and is highly sensitive to lipopolysaccharide (LPS)-induced activation, thereby exhibiting features of inflammatory priming. Thus, phosphorylation of ERK1/2, PKB/Akt, and c-Jun is remarkably increased in response to LPS in vitro, whereas that of p38 MAPK is reduced compared with the monocyte-derived macrophages from the blood of healthy controls. Furthermore, in vitro activated monocyte-derived macrophages from ascites of cirrhotic patients secreted significantly higher levels of IL-6, IL-10, and TNF-α and lower amounts of IL-1ß and IL-12 than the corresponding cells from healthy donor's blood. Based on these results, other authors have recently reported that the surface expression level of CD206 can be used to identify mature, resident, inflammatory peritoneal macrophages in patients with cirrhosis. Soluble CD206 is released from activated large peritoneal macrophages, and increased concentrations in patients with cirrhosis and spontaneous bacterial peritonitis (SBP) indicate reduced odds of survival for 90 d. Hence, the level of soluble CD206 in ascites might be used to identify patients with SBP at risk of death. In conclusion, peritoneal macrophages present in ascites of cirrhotic patients display multiple phenotypic modifications characterized by reduced ratio of cells expressing several membrane markers, together with an increase in the ratios of complex and intermediate subpopulations and a decrease in the classic-like subset. These modifications may lead to the identification of novel pharmaceutical targets for prevention and treatment of hepatic damage.

Validation of a Feed Protocol in a Mouse Model That Mimics Marasmic Malnutrition.

Host nutritional status directly interferes with immunity and/or susceptibility to infectious diseases. To understand the mechanisms behind this relationship, the use of animal models and feeding protocols is necessary. In the literature, studies reporting marasmic malnutrition in mice are not common. In this context, the objective of this study was to validate a feed methodology that mimics marasmic malnutrition, examining the nutritional, biochemical, and hematological status in BALB/c mice. Weaned BALB/c mice were or were not fed a Restricted diet (36.26% carbohydrate, 8.79% protein, 4.95% fat, and 7.62 kJ/100 g). Some malnourished mice underwent a refed process with a Control diet (65.93% carbohydrate, 24.18% protein, 9.89% fat, and 15.24 kJ/100 g). The nutritional status of the mice was evaluated through phenotypic markers and hematological and biochemical parameters. Our results showed that the Restricted diet was able to induce mild malnutrition in mice, resulting in mouse weight loss of 12%, which could be reversed after refeeding. Malnourished mice demonstrated slow body growth and low body mass index (BMI) values. Malnourished mice also showed physical and behavioral changes, a reduction of 47.5% in leukocyte counts and a 2-fold increase in cholesterol levels. In conclusion, our feeding protocol was able to generate mild malnutrition and cause changes in the nutritional status of mice that could be similar to those observed in marasmic malnutrition.

Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review.

The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-ß, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Situation of and phenotypic markers of susceptibility to helminth infection among sheep on farms in the Brazilian cerrado biome / Diagnóstico de situação e marcadores fenotípicos das parasitoses gastrintestinais em ovinos de fazendas localizadas no Distrito Federal: bioma cerrado brasileiro

Abstract This study aimed to determine the intensity, seasonality, and distribution by genera of, as well as to identify phenotypic markers of susceptibility to, gastrointestinal parasites among sheep on farms within the Brazilian savanna (cerrado) biome. We evaluated 1271 sheep, on seven farms, during the rainy season (in December 2017 and December 2018) and dry season (in July 2018 and July 2019). Parasitological evaluation was based on culture and EPG. We calculated hematocrit, as well as the body condition score and feces score. Of the sheep evaluated, 34.15% had moderate-to-severe parasitic infection. The factors of herds' phenotypic characterization about helminth infections were (p ≤ 0.05 for all): anemia (OR = 5.72); leanness (OR = 1.80); loose stools or diarrhea (OR = 1.54); breed other than Santa Inês (OR = 2.31); "weaned lamb" category (OR = 4.76); "lambing ewe" category (OR = 4.66); and dry season (OR = 2.37). Haemonchus, Trichostrongylus, Oesophagostomum, and Cooperia accounted for 76.40%, 20.23%, 2.89%, and 0.47%, respectively, of the helminth genera identified, with their proportional distributions being comparable between the rainy and dry seasons. Changes in health management, with regard to helminth infection control, are urgently needed in order to combat the disease more effectively and sustainably.

Resumo A presente pesquisa objetivou determinar a intensidade, sazonalidade, predominância de gêneros e marcadores fenotípicos às parasitoses gastrointestinais em fazendas de criação de ovinos do Distrito Federal, no bioma cerrado brasileiro. Foram avaliados 1.271 ovinos oriundos de sete propriedades, durante os períodos chuvoso (dezembro de 2017 e dezembro de 2018) e seco (julho de 2018 e julho de 2019). Procedeu-se a avaliação parasitológica por coprocultura e OPG, dosagem de hematócrito, escore de condição corporal e de fezes. Dos indivíduos avaliados, 34,15% deles apresentaram infecção parasitária moderada a grave. Os fatores de caracterização fenotípica dos rebanhos quanto às infecções helmínticas (p ≤ 0,05), foram: anemia (OR = 5,72),magreza (OR = 1,80), fezes pastosas/diarreicas (OR = 1,54), raças distintas à raça Santa Inês (OR = 2,31), categorias de produção animal "jovens" (OR = 4,76) e "fêmeas paridas" (OR = 4,66), e período seco (OR = 2,37). Haemonchus, Trichostrongylus, Oesophagostomum e Cooperia foram os gêneros de helmintos observados nas seguintes proporções: 76,40%, 20,23%, 2,89% e 0,47%, respectivamente, sem distinção em sua distribuição entre os períodos chuvoso e seco. Mudanças no manejo sanitário com relação às helmintoses são urgentemente necessárias, para um controle da doença de forma mais eficaz e sustentável.

The frequency of visceral and phenotypic markers in patients with the combination of undifferentiated connective tissue disease and gastroesophageal reflux disease.

OBJECTIVE: The aim: The aim of the research was to study the prevalence of visceral and phenotypic markers of UCTD syndrome in patients with GERD for the purpose of early diagnosis of this comorbidity. PATIENTS AND METHODS: Materials and methods: The study included 120 patients: 75 patients (Group II) - GERD was on the background of UCTD, 45 (Group I ) - the patients with GERD. The average age of the patients was 42.05 ± 6.5 years. Evaluations of UCTD's were performed accordingly to the criteria recommended by M. Moska et al., A. Doria et al., T. I. Kadurina, L. M. Abbakumova in the modification of T. Milkovskaya-Dimitrova, and the degree of their expression on the scale of T. Y. Smolnova. RESULTS: Results: Among the examined patients, the specific criteria for the certain connective tissue diseases were detected in the patients with UCTD from 2.7 and 20.0% more often. Bone, joint and skin phenotypic signs of dysplasia were observed in patients with GERD associated with UCTD by 4-4.5 times more often. Various abnormalities of internal organs development were detected in the majority of patients of Group II, namely in 88.0%, and only in 6.6% of the patients of Group I. According to the data on daily pH monitoring, esophagus AET constituted 4.6% of the total monitoring period in Group I and 5.48% in the patients of Group II. The number of refluxes with pH<4 recorded in the patients of Group I constituted 57±8 episodes, and 79±6 episodes in the patients of Group II. CONCLUSION: Conclusions: The obtained data indicated that the number of pathological GER was significantly higher in the setting of comorbidity. Our research also showed that the chances of diagnosing Reynaud's Syndrome, arthralgia, unmotivated body weight loss, dysphagia, skin rash, oral ulcers, proximal muscle weakness in the patients with GERD associated with UCTD are higher in comparison with the patients with GERD without comorbidity (р<0.05). This should necessarily be taken into account in the early diagnosis and when assigning a complex therapy in case of this pathology.

High-Parameter Immune Profiling with CyTOF.

Mass cytometry, or CyTOF, is a useful technology for high-parameter single-cell phenotyping, especially from suspension cells such as blood or PBMC. It is particularly appealing to monitor the systemic immune changes that could accompany cancer immunotherapy. Here we present a reference panel for identification of all major immune cell populations, with flexibility for addition of trial-specific markers. We also describe best-practice measures for minimizing and tracking batch variability. These include: sample barcoding, use of spiked-in reference cells, and lyophilization of the antibody cocktail. Our protocol assumes the use of cryopreserved PBMC, both for convenience of batching samples and for maximum comparability across patients and time points. Finally, we show an option for automated analysis using the Astrolabe platform (Astrolabe Diagnostics, Inc.).

Phenotypic and Molecular Analyses of Rhizoctonia spp. Associated with Rice and Other Hosts.

Forty-two Rhizoctonia isolates were collected from rice, mung bean, and grasses from Laguna, Philippines. Sixteen isolates were binucleate Rhizoctonia (BNR), while 26 were multinucleate Rhizoctonia (MNR). BNR isolates produced white to brown, small sclerotia (<1.0 mm) except for mung bean isolates. Twenty MNR isolates produced big (>1.0 mm), light to dark brown sclerotia, three produced salmon-colored masses in the medium, and three did not produce sclerotia. Twenty-three MNR isolates were identified as R. solani AG1-IA using specific primers. Deduced Internal Transcribed Spacer (ITS) sequences of BNR isolates D1FL, NVL, and ScNL shared 100, 97, and 100% identity with R. oryzae-sativae, respectively, while MNR isolates BMgL, IbMgL, and MaSL that produced salmon-colored masses shared 100, 90, and 100% identity with R. oryzae, respectively. Preliminary analysis of the DNA fingerprint patterns generated by repetitive-element PCR (rep-PCR) clustered the 42 isolates into three: R. solani, R. oryzae-sativae, and R. oryzae, together with Ceratobasidium sp. R. solani isolates were pathogenic on rice (TN1), barnyard grass, mungbean (Pagasa 3), and tomato (Athena), while R. oryzae and R. oryzae-sativae isolates were only pathogenic on rice, Echinochloa crus-galli, and tomato. R. solani and R. oryzae were found to be more virulent than R. oryzae-sativae.

Primer reporte de la variabilidad genética del gato (Felis catus), con marcadores fenotípicos en Coveñas, Sucre / First report of genetic variability in cats (Felis catus), with phenotypic markers, in Coveñas, Sucre / Primeiro repórter da variabilidade genética do gato (Felis catus), com marcadores fenotípicos em Coveñas, Sucre

Resumen El objetivo de esta investigación fue determinar la variabilidad genética de las poblaciones de gatos domésticos (Felis catus) utilizando genes que codifican la coloración, el diseño y la longitud del pelaje en Coveñas, Sucre, Colombia. Se realizaron muestreos aleatorios entre septiembre y diciembre de 2014, en 187 animales adultos presentes en cinco barrios de Coveñas, donde se caracterizó fenotípicamente cada animal. La nomenclatura utilizada está en concordancia con el Committee Standardized Genetic Nomenclature For Cats (1968), y atiende a los marcadores autosómicos de codificación morfológica: el locus ligado al sexo Orange (O) y los loci autosómicos non-agouti (a), tabby blotched (Tb), dilution (d), long hair (l) spotting white (s) y dominant white (W). Se calcularon los parámetros genéticos poblacionales: frecuencia alélica, diversidad genética, flujo génico, equilibrio Hardy-Weinberg y distancia genética, y se infirieron las relaciones filogenéticas entre las poblaciones de gatos. Se encontró que el marcador non-agouti fue el de mayor frecuencia, mientras los genes tabby blotched y dominant white presentaron los valores más bajos. La mayor parte de la diversidad genética se encontró dentro de las poblaciones (HS) y poca entre las poblaciones (D) y un elevado flujo génico. Se observó un exceso de heterocigotos en la población. No hubo equilibrio Hardy-Weinberg. Las poblaciones se encuentran muy relacionadas genéticamente. Además, se evidenció una posible selección natural y artificial del locus non-agouti.

Abstract This research aimed to determine genetic variability in domestic cat populations (Felis catus) using genes that codify the coloration, design and length of the coat in Coveñas, Sucre, Colombia. Random samples were collected between September and December 2014 from 187 adult animals in five neighborhoods of Coveñas, and each animal was characterized phenotypically. The nomenclature used in this research follows the Standardized Genetic Nomenclature for the Domestic Cat (1968), and examines the autosomal markers of morphological coding: the locus linked to sex Orange (O) and the autosomal loci Non-agouti (a), tabby blotched (Tb), dilution (d), long hair (l) spotting white (s) and dominant white (W). The genetic parameters of the population were calculated: allele frequency, genetic diversity, gene flow, Hardy-Weinberg equilibrium, and genetic distance; and phylogenetic relationships among cat populations were inferred. It was found that the Non-agouti marker was the most frequent, while the tabby blotched and dominant white genes had the lowest values. Most genetic diversity was found within the studied populations (HS), with little diversity between populations (D), and high gene flow was evidenced. An excess of heterozygotes was observed in the population. There was no Hardy-Weinberg equilibrium. Populations are genetically closely related. In addition, a possible natural and artificial selection of the Non-agouti locus was evidenced.

Resumo O objetivo desta pesquisa foi determinar a variabilidade genética das populações de gatos domésticos (Felis catus) utilizando genes que codificam a coloração, o desenho e a longitude da pelagem em Coveñas, Sucre, na Colômbia. Realizaram-se amostras aleatórias entre setembro e dezembro de 2014, em 187 animais adultos presentes em cinco bairros de Coveñas, onde se caracterizou fenotipicamente cada animal. A nomenclatura utilizada está em concordância com o Committee Standardized Genetic Nomenclature For Cats (1968), e atende a os marcadores autossômicos de codificação morfológica: o locus ligado a sexo Orange (O) e os loci autossômicos Non-agouti (a), tabby blotched (Tb), dilution (d), long hair (l) spotting white (s) e dominant white (W). Calcularam-se os parâmetros genéticos populacionais: frequência alélica, diversidade genética, fluxo génico, equilíbrio Hardy-Weinberg e distância genética, e se inferiram as relações filogenéticas entre as populações de gatos. Constatou-se que o marcador Non-agouti foi o de maior frequência, e enquanto que os genes tabby blotched e dominant white apresentaram os valores mais baixos. A maior parte da diversidade genética foi encontrada dentro das populações (HS) e pouca entre as populações (D) e um elevado fluxo génico. Observouse um excesso de heterozigotos na população. Não houve equilíbrio Hardy-Weinberg. As populações encontram-se muito relacionadas geneticamente. Além do mais, evidenciou-se uma possível seleção natural e artificial do locus Non-agouti.

Comparison of nucleus pulposus stem/progenitor cells isolated from degenerated intervertebral discs with umbilical cord derived mesenchymal stem cells.

Mesenchymal stem-cell based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. The development of these treatment strategies, however, has been hindered by the incomplete understanding of the origin, biological properties of nucleus pulposus (NP) derived stem/progenitor cells and their effects on the IVD degeneration. The goal of this study is to explore the biological properties of NP stem/progenitor cells isolated from degenerated IVD (D-NPMSCs) regarding immunotype, proliferative capacity, multi-lineage differentiation abilities, and the expression of NP specific cell surface markers compared to human umbilical cord mesenchymal stem cells (UCMSCs). Our results indicate that although D-NPMSCs shared the mesenchymal stromal cells (MSCs) characteristics with UCMSCs, significant differences exist in phenotype signatures and biological capacities between D-NPMSCs and UCMSCs. D-NPMSCs expressed lower expression levels of CD29 and CD105, the phenotype markers of MSCs, and exhibited reduced proliferation capability and differentiation potentials, which might account for the distinct NP microenvironment and the poor capacity for disc regeneration. This study will lay a foundation for further understanding the mechanism of stem cell-based therapy for IVD degeneration.

Expression and its clinical significance of gastric and intestinal phenotypic markers in 47 Siewert type Ⅱ and type Ⅲ early gastroesophageal junctional cancers / 中华消化杂志

Objective To investigate the expression of gastric and intestinal phenotypic markers in Siewert typeⅡand Ⅲ early gastroesophageal junction(GEJ) cancer, and to explore its correlation with clinic-pathological features.Methods From April 2010 to July 2015, 53 cases diagnosed as early GEJ cancer were enrolled.The gastric and intestinal phenotypic markers such as mucin5AC(MUC5AC),mucin6(MUC6),mucin2(MUC2),caudal related homeodomain transcription 2(CDX2) and cluster of differentiation 10(CD10) were detected, and then the patients were divided into gastric type, gastrointestinal type, intestinal type and non-classified type according to the results of immunohistochemical staining.Combined with Siewert classification the clinicopathological features were analyzed.Chi square test or Fisher′s exact test was performed for statistical analysis.Results In the cancer tissues of 47 patients with Siewert type Ⅱand Ⅲ early GEJ cancer, the case numbers of positive expression of MUC5AC,MUC6,MUC2, CDX2 and CD10 were 21(44.7%),19(40.4%),31(66.0%),27(57.4%) and 17(36.2%),respectively;the case numbers of gastric type, gastrointestinal type, intestinal type and non-classified type were 11(23.4%),14(29.8%),21(44.7%) and one(2.1%), respectively.The positive expression rates of MUC5AC and MUC6 in Siewert typeⅡwere 55.9%(19/34) and 50.0%(17/34),which were higher than those of Siewert typeⅢ(2/13), and the positive expression rate of MUC2 was 55.9%(19/34), which was lower than that of Siewert typeⅢ(12/13), and the differences were statistically significant (x2=6.240,4.679 and 4.053;all P<0.05).In Siewert typeⅡ, the proportion of intestinal type was 32.4%(11/34), which was lower than that of Siewert typeⅢ(10/13), and the differences were statistically significant (x2=7.142,P=0.010).In patients with Siewert typeⅡand Ⅲ early cancer, males predominated in intestinal type which were mostly well differentiated type with less submucosal carcinoma.The maximum diameter of tumor was less than those of gastric type and gastrointestinal type.In paracancerous mucosal tissues, the incidences of intestinal metaplasia in gastrointestinal type and intestinal type were 11/14 and 81.0%(17/21), which were higher than that of gastric type (3/11);the incidences of atrophy in gastrointestinal type and intestinal type were 12/14 and 85.7%(18/21),which were higher than that of gastric type (4/11),and the differences were statistically significant (Fisher′s exact test,all P<0.05).Conclusions Siewert typeⅡand Ⅲ early GEJ cancer can directly originated not only from gastric mucosa, but also from gastrointestinal and intestinal mucosa.Atrophy and intestinal metaplasia could exist before cancer genesis.

Nucleus pulposus phenotypic markers to determine stem cell differentiation: fact or fiction?

Progress in mesenchymal stem cell (MSC) based therapies for nucleus pulposus (NP) regeneration are hampered by a lack of understanding and consensus of the normal NP cell phenotype. Despite the recent consensus paper on NP markers, there is still a need to further validate proposed markers. This study aimed to determine whether an NP phenotypic profile could be identified within a large population of mature NP samples.qRT-PCR was conducted to assess mRNA expression of 13 genes within human non-degenerate articular chondrocytes (AC) (n=10) and NP cells extracted from patients across a spectrum of histological degeneration grades (n=71). qRT-PCR results were used to select NP marker candidates for protein expression analysis.Differential expression at mRNA between AC and non-degenerate NP cells was only observed for Paired Box Protein 1 (PAX1) and Forkhead box F1 (FOXF1). In contrast no other previously suggested markers displayed differential expression between non-degenerate NP and AC at mRNA level. PAX1 and FOXF1 protein expression was significantly higher in the NP compared to annulus fibrosus (AF), cartilaginous endplate (CEP) and AC. In contrast Laminin-5 (LAM-332), Keratin-19 (KRT-19) and Hypoxia Inducible Factor 1 alpha (HIF1α) showed no differential expression in NP cells compared with AC cells.A marker which exclusively differentiates NP cells from AF and AC cells remains to be identified, raising the question: is the NP a heterogeneous population of cells? Or does the natural biological variation during IVD development, degeneration state and even the life cycle of cells make finding one definitive marker impossible?

[The superficial markers of neoplastispecificity of c cell line MT-4 and perspectives of its application as a model for studying activity of immune modulating preparations.]

The article considers expression of markers of activation of neoplastic CD4+ T-lymphocytic transplantable cellular line M T-4, transformed by T-lymphotropic human virus type I. It is demonstrated that in cells are detected such external proteins as CD25+, CD28+, CD38+, CD62L+, CD69+, CD95+ and HLA-DR+. The maximal number of these components was detected in three days after transplantation of cells. These indices reached average level for markers CD25+, CD28+, CD38+, CD69+, CD95+ and HLA-DR+ - more than 90% and for CD62L+ - 48%. The obtained results and cultivation of cells indicate that cells MT-4 can be used as a convenient model for testing of activity of immune modulation preparations.

Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells.

AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC). METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 and human leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR. RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82% vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34), GFAP (1.12), Tau (1.08), MAP-1B (0.92), MAP-2 (1.14) and NSE (0.4) (P < 0.001 for all). CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.

Identification of phenotypic markers of B cells from patients with Chagas disease.

Chagas disease was discovered more than a hundred years ago, but its pathogenesis is still not completely understood. Autoimmunity is one of the mechanisms shown to contribute to its pathogenesis, which may indicate an important participation of B lymphocytes. Patients with Chagas disease have shown increased percentage of B cells producing IL-10. However, there are no reports of the phenotypic markers of B cells producing IL-10 in patients with Chagas disease. For the first time in the literature, we evaluated the phenotypic profile of distinct markers of B cells from peripheral blood of noninfected individuals and patients with Chagas disease. Our results showed that patients with Chagas disease had a higher expression of CD21 and CD24 on the surface of CD19+ B cells, while CD43 and CD23 were expressed equally in all groups. Moreover, the expression of MHC-II (HLA-DR), CD80, CD86, caspase-3, granzyme B and intracellular IL-10 and TGF-ß by CD19+ B cells was higher in patients with Chagas disease. The results of IL-10 production within CD19+ CD5+ CD1d+ B cells showed a higher percentage of this cytokine in patients with Chagas disease. Thus, our data bring a new knowledge about distinct markers of B cells in immune responses of Chagas disease.