Heat-stable Antigen Research Articles

Role of RIPK1 in SMAC mimetics-induced apoptosis in primary human HIV-infected macrophages

Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.

Nucleophosmin/B23 promotes endometrial cancer cell escape from macrophage phagocytosis by increasing CD24 expression.

Despite recent therapeutic breakthroughs, advanced and/or recurrent endometrial cancer still poses a significant health burden globally. While immunotherapy can theoretically lead to durable responses, the benefits to patients remain limited. In an effort to identify novel immunotherapeutic targets, we specifically focused on the potential role of nucleophosmin (NPM, also known as B23) - a nucleolar phosphoprotein involved in tumorigenesis - in cancer immune evasion. Expression profiling with oligonucleotide microarrays was conducted to identify differentially expressed genes in NPM/B23-silenced endometrial cancer cells. CD24 - a heat-stable antigen commonly overexpressed in solid tumors and a target for cancer immunotherapy - was identified as one of the key NPM/B23-regulated molecules. We found that NPM/B23 was capable of inducing CD24 expression, with the Sp1 binding site in the CD24 promoter being essential for NPM/B23-mediated transcriptional activation. Interestingly, NPM/B23 silencing in endometrial cancer cells enhanced phagocytic removal by macrophages through a decreased exposure of CD24 on the cell surface. Conversely, restoration of CD24 expression in NPM/B23-silenced endometrial cancer cells inhibited macrophage-mediated phagocytosis. These results indicate that NPM/B23-driven CD24 overexpression enables endometrial cancer cells to evade from phagocytosis. We further suggest that CD24 may serve as a novel target for endometrial cancer immunotherapy. KEY MESSAGES: NPM/B23 induced CD24 expression in endometrial tumorigenesis. Sp1 binding site in the CD24 promoter is essential for the activation. NPM/B23 silencing enhanced phagocytosis by macrophages through decrease of CD24 on cancer cells. Restoration of CD24 expression in NPM/B23-silenced cancer cells inhibited macrophage-mediated phagocytosis.

CD24 for Cardiovascular Researchers: A Key Molecule in Cardiac Immunology, Marker of Stem Cells and Target for Drug Development.

The study of the membrane protein, CD24, and its emerging role in major disease processes, has made a huge leap forward in the past two decades. It appears to have various key roles in oncogenesis, tumor progression and metastasis, stem cell maintenance and immune modulation. First described in the 1980s as the homologous human protein to the mouse HSA (Heat Stable Antigen), it was reported as a surface marker in developing hematopoietic cell lines. The later discovery of its overexpression in a large number of human neoplasms, lead cancer researchers to discover its various active roles in critical checkpoints during cancer development and progression. Targeting CD24 in directed drug development showed promising results in cancer treatment. More recently, the chimeric CD24-Fc protein has shown exciting results in clinical trials as a specific modulator of auto-inflammatory syndromes. This report is aimed to summarize the relevant literature on CD24 and tie it together with recent advancements in cardiovascular research. We hypothesize that CD24 is a promising focus of research in the understanding of cardiovascular disease processes and the development of novel biological therapies.

Mouse HSA+ immature cardiomyocytes persist in the adult heart and expand after ischemic injury.

The assessment of the regenerative capacity of the heart has been compromised by the lack of surface signatures to characterize cardiomyocytes (CMs). Here, combined multiparametric surface marker analysis with single-cell transcriptional profiling and in vivo transplantation identify the main mouse fetal cardiac populations and their progenitors (PRGs). We found that CMs at different stages of differentiation coexist during development. We identified a population of immature heat stable antigen (HSA)/ cluster of differentiation 24 (CD24)+ CMs that persists throughout life and that, unlike other CM subsets, actively proliferates up to 1 week of age and engrafts cardiac tissue upon transplantation. In the adult heart, a discrete population of HSA/CD24+ CMs appears as mononucleated cells that increase in frequency after infarction. Our work identified cell surface signatures that allow the prospective isolation of CMs at all developmental stages and the detection of a subset of immature CMs throughout life that, although at reduced frequencies, are poised for activation in response to ischemic stimuli. This work opens new perspectives in the understanding and treatment of heart pathologies.

Validity of Katex Test for Diagnosis of Visceral Leishmaniasis

Background: Newly developed KAtex test can be used as a non invasive tool for diagnosis of Kala-azar.Objectives: The aim of the present study was to validate the KAtex method to diagnose VL.Methodology: This was a cross-sectional study carried out in the Department of Microbiology at Dhaka Medical College, Dhaka, Bangladesh in collaboration with the department of Parasitology at Institute of Epidemiology, Disease control and Research (IEDCR), Dhaka, Bangladesh for a period of one year. Clinically suspected Kala- azar (VL) cases of different age and sex attending IEDCR from different Kala-azar endemic areas of Bangladesh were selected for this study. Microscopy and culture was performed with Bone marrow (BM). KAtex was performed with urine sample. Urine samples taken from cases were pretreated to inactivate heat labile materials which might cause a false positive reaction. Antigen which is detected by KAtex is heat stable carbohydrate antigen. Latex sensitized with antibodies raised against Leishmania donovani antigen was mixed with the urine sample on a glass slide. No agglutination indicates absence of antigen in urine.Result: Cases were 130. Among 130 clinically suspected VL cases, 70 (53.85%) cases were BM positive and 60 (46.15%) cases were BM negative. All the 70 BM positive cases were positive by KAtex. Among 60 BM negative cases, 15 were positive by KAtex. The sensitivity of KAtex is 100% and specificity is 75%. Highest percentage (52.86%) of bone marrow positive cases were below 10 years of age group.Conclusion: In conclusion, KAtex test is a good diagnostic tool for the detection of VL.Bangladesh Journal of Infectious Diseases, December 2017;4(2):45-47

Detection of Edwardsiella tarda From African Catfish (Clarias garipienus) by Agar Gel Precipitation (AGP) Method in jambi

For the past few years, Edwardsiella tarda has become major problem in African catfish culture in Jambi. Detection by biochemical characteristic can lead to inaccurate result and there is a necessity to develop more specific and accurate method, one of which is Agar Gel Precipitation (AGP) method. Six samples each were collected from two African catfish farm located in District Sungai Gelam and Telanai Pura in Jambi, which was showed clinical signs of E. tarda outbreak with more than fifty percent mortality rate. Heat stable soluble antigen was prepared from 2 groups of pure culture isolated from sample for AGP test. Antiserum for test wells was antiserum of E. tarda (ATCC 15947) that have been produced by inoculating whole-cell antigen (heat-stable) and flagellar antigen (formalin-killed) in rabbit. For control positive, soluble antigen prepared from E. tarda (ATCC 15947), and control negative from Aeromonas hydophila (ATCC 35654) and Edwardsiella ictaluri (NCIMB 13272). Both antiserums were able to show positive reaction visible by the formation of specific precipitin lines between antiserum and antigen wells, and there was no precipitin reaction for negative control. In conclusion AGP method is a one of reliable technique to identify E. tarda.

CCR7 Controls Thymus Recirculation, but Not Production and Emigration, of Foxp3(+) T Cells.

SummaryCurrent models of Foxp3+ regulatory T cell (Treg) development involve CCR7-mediated migration of thymocytes into the thymus medulla to enable essential interactions with medullary epithelium. However, increased Foxp3+ thymic Treg numbers in Ccr7−/− mice challenge this view, and the role of CCR7 in Treg development, emigration, and/or recirculation is unknown. Here, we have examined CCR7 and Rag2pGFP levels during Treg development and generated Rag2pGFPCcr7−/− mice to study its impact on the intrathymic Treg pool. We reveal surprising developmental heterogeneity in thymocytes described as Treg precursors, showing that they contain recirculating CCR6+CCR7−Rag2pGFP− T cells. Although CCR7 defines bona fide Rag2GFP+ Treg precursors, it is not required for Treg production and emigration. Rather, we show that lack of CCR7 renders the thymus more receptive to Treg thymus homing. Our study reveals a role for CCR7 in limiting Treg recirculation back to the thymus and enables separation of the mechanisms controlling Treg production and thymic recirculation.

Immunogenicity and protective efficacy of a recombinant adenoviral based vaccine expressing heat-stable enterotoxin (STa) and K99 adhesion antigen of enterotoxigenic Escherichia coli in mice.

The diarrheal disease of domestic animals or in humans caused by enterotoxigenic Escherichia coli (ETEC) infections remains a major issue for public health in developing countries. Unfortunately, there is no effective vaccine available for preventing from an ETEC infection. Therefore, the development of a safe and effective vaccine against ETEC is urgently needed. In the present study, A recombinant adenoviral vector Ad5-STa-K99 that capable of expressing a fusion protein of heat-stable enterotoxin (STa) and K99 adhesion antigen of ETEC was generated and its immunogenicity was evaluated in a murine model. The intestinal mucosal secretory IgA(sIgA), serum anti-STa-K99 antibody responses, antigen-specific CD4(+) and CD8(+) T cells frequencies, as well as T-cell proliferation of mice immunized with the viral vector were determined as immunological indexes. The results demonstrated that Ad5-STa-K99 was able to enhance humoral responses with a dramatically augmented antigen-specific serum IgG antibody, and an elevated production of intestinal sIgA in immunized mice, suggesting the elicitation of both of humoral and mucosal immune responses. In addition, this adenoviral vector could significantly promote splenic T cell proliferation and increase the frequencies of CD4(+) and CD8(+) T cell populations in mice, indicative of a capacity to activate T cell responses. More importantly, vaccination of the Ad5-STa-K99 showed a potential to evoke a protective effect from ETEC challenge in mice. These data indicate that the Ad5-STa-K99 is a highly immunogenic vector able to induce a broad range of antigen-specific immune responses in vivo, and evoke a protective immune response against ETEC infections, implying that it may be a novel vaccine candidate warranted for further investigation.

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice.

BackgroundHealthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates.AimTo examine the role of CD24 in the wound healing process.MethodsAn excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA -/-) compared to wild-type (WT) mice.ResultsLarge full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA +/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA -/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice.ConclusionsDelayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.

Dynamic regulation of CD24 expression and release of CD24-containing microvesicles in immature B cells in response to CD24 engagement.

The glycophosphatidylinositol-anchored cell surface receptor CD24 (also called heat-stable antigen) promotes the apoptosis of progenitor and precursor B-lymphocytes. However, the immediate proximal events that occur after engagement of CD24 in B cells are not precisely understood. Using a bioinformatics analysis of mouse (Mus musculus) gene expression data from the Immunological Genome Project, we found that known vesicle trafficking and cellular organization genes have similar expression patterns to CD24 during B-cell development in the bone marrow. We therefore hypothesized that CD24 regulates vesicle trafficking. We first validated that antibody-mediated engagement of CD24 induces apoptosis in the mouse WEHI-231 cell line and mouse primary bone marrow-derived B cells. We next found that CD24 surface protein expression is rapidly and dynamically regulated in both WEHI-231 cells and primary immature B cells in response to engagement of CD24. The change in surface expression was not mediated by classical endocytosis or exocytosis. However, we found that CD24-bearing plasma membrane-derived extracellular microvesicles were released in response to CD24 engagement. Furthermore, in response to CD24 engagement we observed a clear exchange of CD24 between different populations of B cells. Hence, we show that engagement of CD24 in immature B cells results in a dynamic regulation of surface CD24 protein and a redistribution of CD24 within the population.

CD24: from a Hematopoietic Differentiation Antigen to a Genetic Risk Factor for Multiple Autoimmune Diseases

The autoantibody is an essential characteristic of inflammatory disorders, including autoimmune diseases. Although the exact pathogenic mechanisms of these diseases remain elusive, accumulated evidence has implicated that genetic factors play important roles in autoimmune inflammation. Among these factors, CD24 was first identified as a heat-stable antigen in 1978 and first successfully cloned in 1990. Thereafter, its functional roles have been intensively investigated in various human diseases, especially autoimmune diseases and cancers. It is currently known that CD24 serves as a costimulatory factor of T cells that regulate their homeostasis and proliferation, while in B cells, CD24 is functionally involved in cell activation and differentiation. CD24 can enhance autoimmune diseases in terms of its protective role in the clonal deletion of autoreactive thymocytes. Furthermore, CD24 deficiency has been linked to mouse experimental autoimmune encephalomyelitis. Finally, CD24 genetic variants, including single-nucleotide polymorphisms and deletions, are etiologically relevant to autoimmune diseases, such as multiple sclerosis and systemic lupus erythematosus. Therefore, CD24 is a promising biomarker and novel therapeutic target for autoimmune diseases.

Role of caspase-8 in thymus function

The thymus is the primary organ responsible for de novo generation of immunocompetent T cells that have a diverse repertoire of antigen recognition. During the developmental process, 98% of thymocytes die by apoptosis. Thus apoptosis is a dominant process in the thymus and occurs through either death by neglect or negative selection or through induction by stress/aging. Caspase activation is an essential part of the general apoptosis mechanism, and data suggest that caspases may have a role in negative selection; however, it seems more probable that caspase-8 activation is involved in death by neglect, particularly in glucocorticoid-induced thymocyte apoptosis. Caspase-8 is active in double-positive (DP) thymocytes in vivo and can be activated in vitro in DP thymocytes by T-cell receptor (TCR) crosslinking to induce apoptosis. Caspase-8 is a proapoptotic member of the caspase family and is considered an initiator caspase, which is activated upon stimulation of a death receptor (e.g., Fas), recruitment of the adaptor molecule FADD, and recruitment and subsequent processing of procaspase-8. The main role of caspase-8 seems to be pro-apoptotic and, in this review, we will discuss about the involvement of caspase-8 in (1) TCR-triggered thymic apoptosis; (2) death receptor-mediated thymic apoptosis; and (3) glucocorticoid-induced thymic apoptosis. Regarding TCR triggering, caspase-8 is active in medullary, semi-mature heat-stable antigen(hi) (HAS(hi) SP) thymocytes as a consequence of strong TCR stimulation. The death receptors Fas, FADD, and FLIP are involved upstream of caspase-8 activation in apoptosis; whereas, Bid and HDAC7 are involved downstream of caspase-8. Finally, caspase-8 is involved in glucocortocoid-induced thymocyte apoptosis through an activation loop with the protein GILZ. GILZ activates caspase-8, promoting GILZ sumoylation and its protection from proteasomal degradation.

Stem cells of the mammary epithelium and their role in breast cancer

Breast carcinogenesis is a multistage process in which genetic mutations in oncogenes and tumor suppressor genes cooperate with epigenetic alterations. Mammary tumor tissue is composed of normal and tumor cells. The tumor cells themselves seem to be clonal in origin, but heterogeneous in appearance. This suggests the existence of cancer stem cells (CSCs). They can originate from transformed tissue stem cells or progenitor cells with acquired self-renewal capacity and are able to escape from conventional therapy. Normal mammary stem cells (MaSCs) are required for the generation and maintenance of the mammary epithelium during the cyclical developmental stages of the mammary gland. They can reconstitute a functional gland upon transplantation into a mammary fat pad cleared from endogenous epithelium. CSCs share features with their normal counterparts; they are able to initiate tumor growth upon transplantation and express stem cell associated surface proteins, which can be utilized for their enrichment. Oncogenic transformation of MaSCs and their orthotopic transplantation in mice provides a promising strategy to model the early stages of mammary tumors and to follow the progression and drug responsiveness of these tumors in vivo. Oncogenic pathways e.g. estrogen and progesterone receptor (ER and PR), and signal transducers and activators of transcription (Stat) 3 and 5 signaling play important roles in normal development and malignant transformation of mammary cells. We are investigating the crosstalk between Stat5, Stat3 and ER signaling in the initiation of luminal tumors in mouse tumor models and their roles in tumor progression, metastasis formation and drug responsiveness. 1. Mammary stem cells in normal development and tumor formation The mammary gland is a unique organ, distinguished by its largely postnatal development. The mouse mammary gland shares many structural and functional features with the human breast and represents a valuable model for the study of organogenesis and tumorigenesis. During puberty, under control of the ovarian hormones, small rudimentary ductal structures invade into stromal fat pad. The mammary ducts grow at the terminal end buds (TEBs) and branch under the influence of steroid hormones during pregnancy. They are composed of an inner layer of luminal epithelial and an outer layer of myoepithelial located on the basement membrane. The expression of surface markers like EpCAM (epithelial cell adhesion molecule), CD24 (heat stable antigen,

Bone Marrow B cell Precursor Number after Allogeneic Stem Cell Transplantation and GVHD Development

Patients without chronic graft-versus-host disease (cGVHD) have robust B cell reconstitution and are able to maintain B cell homeostasis after allogeneic hematopoietic stem cell transplantation (HSCT). To determine whether B lymphopoiesis differs before cGVHD develops, we examined bone marrow (BM) biopsies for terminal deoxynucleotidyl transferase (TdT) and PAX5 immunostaining early post-HSCT at day 30 when all patients have been shown to have high B cell activating factor (BAFF) levels. We found significantly greater numbers of BM B cell precursors in patients who did not develop cGVHD compared with those who developed cGVHD (median = 44 vs 2 cells/high powered field [hpf]; respectively; P < .001). Importantly, a significant increase in precursor B cells was maintained when patients receiving high-dose steroid therapy were excluded (median = 49 vs 20 cells/hpf; P = .017). Thus, we demonstrate the association of BM B cell production capacity in human GVHD development. Increased BM precursor B cell number may serve to predict good clinical outcome after HSCT.

Significance of CD44 and CD24 as Cancer Stem Cell Markers: An Enduring Ambiguity

Cancer stem cell population is a subset of cells capable of dictating invasion, metastasis, heterogeneity, and therapeutic resistance in tumours. Eradication of this rare population is a new insight in cancer treatment. However, prospective identification, characterization, and isolation of these CSCs have been a major challenge. Many studies were performed on surface markers for potential identification and isolation of CSCs. Lack of universal expression of surface markers limits their usage and no best combination of markers has yet been confirmed to identify CSCs capable of initiating and metastasizing tumours. CD44, a hyaluronic acid receptor, is one of the most commonly studied surface markers, which is expressed by almost every tumour cell. CD24, a heat stable antigen, is another surface marker expressed in many tumour types. However, their expression and prognostic value in isolating CSCs are still an enduring ambiguity. In this critical review, we assess the role of CD44 and CD24 in tumour initiation, development, and metastasis. We mainly focus on analysing the significance of CD44 and CD24 as CSC surface markers in combination or with other putative markers in different types of cancer.

Reactivity of heat-stable Leptospira antigenic preparation used in enzyme-linked immunosorbent assay for detection of antibodies in swine serum

Serology plays an important role in laboratory diagnosis of leptospirosis. Apart from the most often used microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA) seems to be useful especially in screenings of animal herds. The ELISA used for detection of antibodies against selected Leptospira serogroups in swine serum samples was investigated during the study. An essential element of this test is heat-stable antigenic preparation from cultures of Leptospira interrogans serovars Icterohaemorrhagiae, Pomona and L. borgpetersenii serovar Sejroe. The aim of the present study was to identify and analyze ELISA heat-stable antigen fractions playing a role in the reaction with leptospiral antibodies indicated in swine serum. Reactivity of the three-component antigenic preparation was compared in immunoblotting with reactivity of six heat-stable antigenic preparations made from the following single serovars: L. interrogans serovars Icterohaemorrhagiae, Pomona, Canicola, L. borgpetersenii serovars Sejroe, Tarassovi and L. kirshneri serovar Grippotyphosa. All antigenic preparations were submitted to SDS-PAGE and transferred to a nitrocellulose membrane using a semidry system. After the transfer, the membrane was incubated with diluted swine serum containing antibodies specific for one of the six above mentioned Leptospira serovars. For the three-component antigenic preparation and antigens prepared from single serovars the immunoblot revealed reaction of sera with fractions of the 20-26 kDa region and around the 14.5 kDa region. The investigated heat-stable Leptospira antigenic preparation contains fractions demonstrating serogroup- and species-specificity. Fraction 20-26 kDa showed serogroup-specific activity, whereas the fraction around 14.5 kDa showed species-specific activity.

Serogrouping and Randomly Amplified Polymorphic DNA Fingerprinting of &lt;i&gt;Campylobacter Jejuni&lt;/i&gt;

Background: Thermophilic campylobacters are of worldwide significance in human and animal diseases. Sources of human infection remain mainly undetermined, but contaminated food, like poultry and row milk, are widely regarded as important vehicles of infection. Accurate methods of strain identification, differentiation and typing are essential for diagnosing and epidemiological purposes. Aim: The aim of our study was to determine the serologic and genetic diversity among the strains and to assess the discriminatory power of both typing methods in epidemiological studies. Material and Methods: Within a period of six months were isolated 26 strains of C. jejuni from faecal samples of children with acute gastroenteritis. Heat-stable specific antigen of C. jejuni was used for serogrouping of the strains by the reaction of passive hemagglutination. Purified genomic DNA was obtained and used in RAPD-PCR reaction. Results: Twenty one C. jejuni strains belonged to the following Penner’s serogroups: 8 strains were group A, 13 strains were group O. The remaining 5 strains were non-typable. RAPD-PCR analysis of 26 strains of Campylobacter jejuni yielded multiple amplification products in all of them. None of the strains processed by this method was non-typable. According to the number and sizes of amplification bands, 4 different genotypes (a, b, c, d) of C. jejuni strains were distinguished within 26 investigated strains. Conclusion: In this study we found that RAPD-PCR analysis provide better discrimination of C. jejuni strains than serogrouping by Penner’s method. Each of the three Penner’s antigenic groups comprised different genotypes. RAPD-PCR analysis of C. jejuni resulted in the generation of highly specific and reproducible DNA fingerprints that enable discrimination even between isolates of a single bacterial serogroup.

LKB1 regulates TCR-mediated PLCγ1 activation and thymocyte positive selection

The serine/threonine kinase LKB1 is a tumour suppressor that regulates cell growth, polarity, and proliferation in many different cell types. We previously demonstrated that LKB1 controls thymocyte survival via regulation of AMPK activation. In this study, we show that LKB1 was also involved in thymocyte positive selection through regulation of T cell receptor (TCR) signalling. Both Lck-Cre- and CD4-Cre-mediated deletion of LKB1 impaired the generation of mature CD4 and CD8 single positive (SP) thymocytes that might have resulted from the attenuated tyrosine phosphorylation of phospholipase C-γ 1 (PLCγ1) in the absence of LKB1. We found that LKB1 was directly phosphorylated by Lck at tyrosine residues 36, 261, and 365 and predominately interacted with LAT and PLCγ1 following TCR stimulation. Loss of LKB1 led to impaired recruitment of PLCγ1 to the LAT signalosome. Correlatively, LKB1-deficient thymocytes failed to upregulate lineage-specifying factors, and to differentiate into SP thymocytes even if their impaired survival was rescued. These observations indicated that LKB1 is a critical component involved in TCR signalling, and our studies provide novel insights into the mechanisms of LKB1-mediated thymocyte development.

Detection of canine echinococcosis by coproantigen ELISA

Objective To study the canine echinococcosis by coproantigen ELISA method. Methods During the present investigation experimental infection was established using evaginated worms of Echinococcus granulosus (E. granulosus). To check cross reactivity two pups were infected with Taenia hydatigena (T. hydatigena). In order to detect the presence of antigen, hyperimmune sera were raised against excretory-secretory products of adult worms E. chinococcus granulosus. Faecal sample collected either from experimentally infected pups or from other sources were heated at 70 °C to detect heat stable soluble antigen. Results Pups harbouring less than 104 worms showed negative results. Samples collected from 14 days onwards from experimentally infected animals harbouring more than 104 worms showed positive value. The maximum positive samples were detected in samples collected from in and around slaughter house and the least number of samples were detected positive maintained by dog squad. Conclusions The affinity purified IgG exhibited promising results for detection of canine echinococcosis by indirect ELISA.

Ontogeny of CD24 in the human kidney

The heat-stable antigen, CD24, is a cell-surface sialoglycoprotein expressed on immature cells that disappears after they have reached their final stage of differentiation. In mice, CD24 expression is preferentially upregulated in the developing mouse metanephros as compared with the surrounding intermediate mesoderm, but its role and expression in the developing human kidney has not been well described. Here we found in normal human fetal kidneys (8 to 38 weeks of gestation) that CD24 expression was upregulated and restricted to the early epithelial aggregates of the metanephric blastema and to the committed proliferating tubular epithelia of the S-shape bodies. Individual cells expressing CD24 were identified in the interstitium of later gestation and postnatal kidneys. In freshly isolated cells, FACS analysis identified distinct CD24(+) and CD24(+)133(+) cell populations constituting up to 16 and 14 percent, respectively, of the total cells analyzed. Early fetal urinary tract obstruction resulted in upregulation of CD24 expression both in developing epithelial structures of early stages and in the cells of the injured tubular epithelium of the later gestation kidneys. Our results highlight the cell-specific expression of CD24 in the developing human kidney and its dysregulation during fetal urinary tract obstruction.