Indirect Immunoflourescence Research Articles

Non-Nuclear and Rare Nuclear ANA Patterns in Indirect Immunoflourescence Testing and their Clinical Associations

Non-Nuclear and Rare Nuclear ANA Patterns in Indirect Immunoflourescence Testing and their Clinical Associations

SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30

The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

Prognostic value of connective tissue growth factor and c-Myb expression in IgA nephropathy and Henoch-Schönlein purpura—A pilot immunohistochemical study

AimAdverse and advanced prognostic signs in IgA nephropathy (IgAN) are interstitial fibrosis and tubular atrophy, but early predictors of bad outcome are still lacking. We investigated expression of connective tissue growth factor (CTGF) and c-Myb in renal biopsies of IgAN and Henoch-Schönlein purpura (HSP), because these gene products are indirectly included in fibrosis and epithelial-mesenchymal transition (EMT). MethodsThe sample included 23 patients and 8 controls who underwent nephrectomy due to renal cancer. The slides cut from the paraffin blocks were prepared for standard indirect immunoflourescence, using antibodies to CTGF and c-Myb. Ten high-power non-overlapping fields were photographed on Olympus IX51 microscope. Average percent of positive tubular cells, as well as number of positive cells per glomerulus were calculated. ResultsThe cytoplasmic tubular CTGF expression was higher in IgAN/HSP than in controls (P < 0.001), whereas no difference was found in glomeruli (P = 0.437). The nuclear c-Myb expresssion in glomeruli and tubules was higher in IgAN/HSP than in controls (P < 0.05). In the follow-up, decline in renal function correlated with glomerular and tubular c-Myb, as well as tubular CTGF expression (all P < 0.05). Conclusion: Our results proposed c-Myb and CTGF as novel, early and sensitive markers of chronic kidney disease and worse renal outcome, but larger series are needed.

Indirect Immunoflourescence assay for the screening of Zika antibodies in serum and cerebrospinal fluid (S40.004)

Indirect Immunoflourescence assay for the screening of Zika antibodies in serum and cerebrospinal fluid (S40.004)

Neutrophils Contribute to RBC Alloantibody Production

Background: Red blood cell (RBC) alloimmunization occurs in up to 10% of transfusion recipients (excluding RhD). Factors influencing alloimmunity are poorly defined; however, transfusion into an inflamed recipient significantly enhances alloimmunization rates in mouse models, and is associated with increased alloimmunization in some human studies. Our lab has recently reported that poly (I:C)-elicited inflammation leads to a significant increase in numbers of neutrophils in the spleen. Additionally, transfusion of syngeneic RBCs into an inflamed recipient leads to enhanced erythrophagocytosis by plasmacytoid dendritic cells (pDCs) and, unexpectedly, neutrophils. Although neutrophils typically eliminate and/or neutralize microorganisms, they can also secrete proinflammatory cytokines, present peptides to T cells through expression of MHC and costimulatory molecules, and elicit antibodies from B cells. Thus, given neutrophil participation in erythrophagocytosis, we hypothesized that neutrophils contribute to RBC-specific alloantibody production in an inflammatory setting.Methods: Recipient C57Bl/6 (B6) mice were treated with 0.5mg anti-Ly6G to deplete neutrophils (clone 1A8) - control mice received PBS. For long-term experiments, anti-Ly6G antibody was given every 48 hours to sustain neutrophil reduction. Post initial neutrophil reduction, recipient mice were given 200ug poly (I:C) to elicit inflammation followed by transfusion of 100uL of packed allogeneic HOD RBCs [HOD RBCs express a transgenic alloantigen (HOD), which is the only difference between syngeneic B6 and HOD RBCs]. For RBC consumption experiments, HOD RBCs were labeled with DiO, and erythrophagocytosis was analyzed (18-24 hours post transfusion) assessing DiO consumption by flow cytometry in different leukocyte subsets (visualized by staining with lineage specific antibodies). The presence of anti-HOD alloantibodies was evaluated in serum (14 days post-transfusion) via indirect immunoflourescence with both B6 and HOD RBC targets.Results: Anti-HOD alloantibodies (IgG2c and IgG3 subtypes) were significantly increased in serum of mice transfused with HOD RBCs in the context of poly (I:C)-elicited inflammation, compared to PBS controls. Neutrophil-depleted recipients that received a HOD RBC transfusion in the presence of poly (I:C) made significantly less anti-HOD alloantibodies (p<0.05), compared to their neutrophil-replete counterparts. In the absence of inflammation, red pulp macrophages (RPM) clear the bulk of RBCs. However, in an inflamed recipient, allogeneic HOD RBC consumption is significantly enhanced in pDCs and neutrophils (rates remain unchanged in RPM). Additionally, neutrophils upregulate MHC-II and costimulatory markers, compared to transfusion in the absence of inflammation. There was a significant increase in RPM numbers upon neutrophil depletion, but RBC consumption rates were unaltered; rather, neutrophil-depleted inflamed transfusion recipients had a significant decrease in overall RBC phagocytes (p<0.05), suggesting a lack of compensatory RBC clearance mechanism. However, neutrophil depletion also resulted in downregulation of MHC-II on RPMs and a slight, but statistically insignificant, increase in RBC consumption by pDCs.Conclusions: These data demonstrate that in an inflamed recipient, neutrophils consume transfused RBCs and express MHC-II and costimulatory molecules. Moreover, depletion of neutrophils significantly decreases alloantibody responses. Together, these findings suggest that consumption of transfused RBCs by neutrophils may play a previously unappreciated role in RBC alloimmunization. Neutrophils may play a role through direct antigen presentation; however, because alterations in other phagocytic populations were also observed with neutrophil depletion, alternate mechanisms cannot be ruled out. Together, these data indicate that while RPMs are the major consumer of transfused RBCs, they may not be the major antigen presenting cell. These findings strongly implicate neutrophils as having a functional contribution to RBC alloimmunization, either through serving directly as antigen presenting cells and/or by affecting other more traditional APC populations. DisclosuresZimring:BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties; Immucor Inc.: Research Funding.

HEp-2 cell classification using shape index histograms with donut-shaped spatial pooling.

We present a new method for automatic classification of indirect immunoflourescence images of HEp-2 cells into different staining pattern classes. Our method is based on a new texture measure called shape index histograms that captures second-order image structure at multiple scales. Moreover, we introduce a spatial decomposition scheme which is radially symmetric and suitable for cell images. The spatial decomposition is performed using donut-shaped pooling regions of varying sizes when gathering histogram contributions. We evaluate our method using both the ICIP 2013 and the ICPR 2012 competition datasets. Our results show that shape index histograms are superior to other popular texture descriptors for HEp-2 cell classification. Moreover, when comparing to other automated systems for HEp-2 cell classification we show that shape index histograms are very competitive; especially considering the relatively low complexity of the method.

Utility of serological assays in the diagnosis of paraneoplastic pemphigus

Background Paraneoplastic pemphigus (PNP) is a rare autoimmune mucocutaneous blistering condition which has a universal association with malignancy and a poor prognosis. PNP, which is characterised by the presence of autoantibodies against plakins in the skin epithelial layer, can be a challenging diagnosis to make, particularly in those without an existing history of malignancy, as tissue biopsy findings may resemble other disorders that have similar clinical features. Thus, serology may be helpful in the diagnosis. Historically, indirect immunoflourescence (IIF) has been the most widely used assay in diagnostic laboratories with reasonable, but not ideal, specificity. Western blotting for various anti-plakin antibodies is more specific but not routinely available. Recently many antiplakin antibody specific assays have been developed commercially. Aim The aim of our study was to compare the utility of three traditional assays, (IIF on rat bladder, monkey bladder and rat cardiac tissue) versus two newer plakin-based assays (envoplakin ELISA and HEK-transfected cell IIP) in patients presenting initially with oral ulceration alone. Method Fifty-nine samples [PNP n = 5, healthy controls n — 10, and disease controls n = 44, including pemphigus vulgaris (PV), mucous membrane pemphigoid (MMP), lichen planus (LP) and miscellaneous oral ulcers] were analysed on the five different assays and results were compared. Results The current assay employed by our laboratory, rat bladder IIF, is the most sensitive assay for our three confirmed PNP cases (3/3, 100%), and has high specificity among all controls (53/54, 98.1%). The Envoplakin ELISA was even more specific (54/54, 100%), although less sensitive (1/3, 33.3%). Monkey bladder IIF had equal sensitivity to rat bladder IIF, although poorer specificity (51/54, 94.4%). Rat cardiac and HEK-transfected cell IIF performed the most poorly with 78-88% specificity, with the latter also having 0% sensitivity. Conclusion Our findings show that the newer envoplakin ELISA compares favourably with more traditional IIF in its high negative predictive value for PNP in both disease and healthy controls. However, traditional IIF seems to have superior sensitivity in our limited cohort of PNP patients. Paraneoplastic pemphigus (PNP) is a rare autoimmune mucocutaneous blistering condition which has a universal association with malignancy and a poor prognosis. PNP, which is characterised by the presence of autoantibodies against plakins in the skin epithelial layer, can be a challenging diagnosis to make, particularly in those without an existing history of malignancy, as tissue biopsy findings may resemble other disorders that have similar clinical features. Thus, serology may be helpful in the diagnosis. Historically, indirect immunoflourescence (IIF) has been the most widely used assay in diagnostic laboratories with reasonable, but not ideal, specificity. Western blotting for various anti-plakin antibodies is more specific but not routinely available. Recently many antiplakin antibody specific assays have been developed commercially. The aim of our study was to compare the utility of three traditional assays, (IIF on rat bladder, monkey bladder and rat cardiac tissue) versus two newer plakin-based assays (envoplakin ELISA and HEK-transfected cell IIP) in patients presenting initially with oral ulceration alone. Fifty-nine samples [PNP n = 5, healthy controls n — 10, and disease controls n = 44, including pemphigus vulgaris (PV), mucous membrane pemphigoid (MMP), lichen planus (LP) and miscellaneous oral ulcers] were analysed on the five different assays and results were compared. The current assay employed by our laboratory, rat bladder IIF, is the most sensitive assay for our three confirmed PNP cases (3/3, 100%), and has high specificity among all controls (53/54, 98.1%). The Envoplakin ELISA was even more specific (54/54, 100%), although less sensitive (1/3, 33.3%). Monkey bladder IIF had equal sensitivity to rat bladder IIF, although poorer specificity (51/54, 94.4%). Rat cardiac and HEK-transfected cell IIF performed the most poorly with 78-88% specificity, with the latter also having 0% sensitivity. Our findings show that the newer envoplakin ELISA compares favourably with more traditional IIF in its high negative predictive value for PNP in both disease and healthy controls. However, traditional IIF seems to have superior sensitivity in our limited cohort of PNP patients.

A Comparative Study on Platelet Activation Markers Between Continuous and Intermittent Exercise Training Programs in Healthy Males

Background : Platelet plays an important role in the hemostatic system. The hyper-activation of platelet may lead to the development of thrombotic event in cardiovascular disease (CVD). Aerobic exercise is well known to reduce CVD risk and can reduce the platelet activation. The purpose of this study was to investigate the effect of the continuous and intermittent training programs on platelet activation. Methods : There were 24 healthy sedentary males mean aged 21.75 ± 3.73 years old participating in this study. They were divided into three groups, which are control group (n = 9), continuous group (n = 7) and intermittent group (n = 8). Continuous training group cycled at 60% VO 2 max for 45 minutes. For the intermittent training group, participants cycled at the 45% VO 2 max and had a rest before the second session at 75% VO 2 max, and the sessions were repeated twice. The exercise was performed non-stop until 45 minutes. Both exercise training groups performed the exercise three times per week for 12 weeks. Platelet activation was measured via detection of the P-selectin and glycoprotein IIb-IIIa using flow cytometry by indirect immunoflourescence. Results : This study showed that, intermittent type of training showed a significant difference of the gated percentage of CD62p and PAC-1 activation. There was also a significant difference of PAC-1 after training between two groups. Conclusions : As a conclusion, intermittent group pronounced more beneficial effects on platelet activation, whereas continuous group provided the beneficial changes on platelet activation. J Hematol. 2014;3(3):72-75 doi: http://dx.doi.org/10.14740/jh163w

Nm23-h1 binds to gelsolin and inactivates its actin-severing capacity to promote tumor cell motility and metastasis.

Nm23-H1 has been identified as a metastasis suppressor gene, but its protein interactions have yet to be understood with any mechanistic clarity. In this study, we evaluated the proteomic spectrum of interactions made by Nm23-H1 in 4T1 murine breast cancer cells derived from tissue culture, primary mammary tumors, and pulmonary metastases. By this approach, we identified the actin-severing protein Gelsolin as binding partner for Nm23-H1, verifying their interaction by coimmunoprecipitation in 4T1 cells as well as in human MCF7, MDA-MB-231T, and MDA-MB-435 breast cancer cells. In Gelsolin-transfected cells, coexpression of Nm23-H1 abrogated the actin-severing activity of Gelsolin. Conversely, actin severing by Gelsolin was abrogated by RNA interference-mediated silencing of endogenous Nm23-H1. Tumor cell motility was negatively affected in parallel with Gelsolin activity, suggesting that Nm23-H1 binding inactivated the actin-depolymerizing function of Gelsolin to inhibit cell motility. Using indirect immunoflourescence to monitor complexes formed by Gelsolin and Nm23-H1 in living cells, we observed their colocalization in a perinuclear cytoplasmic compartment that was associated with the presence of disrupted actin stress fibers. In vivo analyses revealed that Gelsolin overexpression increased the metastasis of orthotopically implanted 4T1 or tail vein-injected MDA-MB-231T cells (P = 0.001 and 0.04, respectively), along with the proportion of mice with diffuse liver metastases, an effect ablated by coexpression of Nm23-H1. We observed no variation in proliferation among lung metastases. Our findings suggest a new actin-based mechanism that can suppress tumor metastasis.

English

&nbsp; Human Adenoviruses (HAdVs) are human pathogens that normally are associated with acute respiratory disease (ARD). Here, in this study, we characterized human adenovirus isolated from respiratory specimens collected from 2003 to 2011. Positive adenovirus were confirmed by cell culture and indirect immunoflourescence (IF) techniques. The isolates and positive clinical samples were subjected to adenovirus polymerase chain reaction, followed by DNA sequencing. BLAST searches and phylogenetic analysis revealed that only 2 species of HAdV, species C at 64% (47/73) and species B at 36% (26/73) were found circulating amongst ARD cases in Malaysia. Four types of HAdV species C were detected namely: HAdV-2 (66%), HAdV-1(17%), HAdV-5(13%) and HAdV-6(4%). For HAdV species B, only 2 types were detected with HAdV-3 being the highest at 58% and HAdV-7 at 42%. HadV-7 was associated with severe infections. &nbsp; &nbsp; Key words:&nbsp;Human adenoviruses, acute respiratory disease, molecular characterization, gene sequences

Expression of Fas Protein ( Cd95 ) and Fas Ligand in Liver Tissue of Patients with Hcv-Induced Chronic Liver Disease and Its Correlation with the Disease Progression

This study evaluated hepatic expression of both Fas and Fas ligand (FasL) in patients with hepatitis c virus (HCV)-induced chronic liver disease and its correlation with the histopathological activity and laboratory parameters as an early predictor of advancement of the disease. The selected patients were (39) males and (21) females, their ages ranged from (20-67years) with a mean of 43.5 +/- 4.5 years, as well as (10) subjects (normal individuals) serving as a control group. They were (7) males and (3) females, their age ranged from (26-53 years) with a mean of 39.5 +/- 7.3 years. Patients were grouped as (1) Chronic hepatitis (CH) group including (30) patients with chronic viral hepatitis C. (2) Liver cirrhosis (LC) group including (30) patients with post hepatitis C cirrhosis. Liver biopsy was done for all subjects using an automated 18-gauge true cut needle. Sections were stained with Haematoxylin and Eosin for histopathological diagnosis and with Maisson and Trichrome for assessment of fibrosis. Unstained paraffin sections from each case were subjected for immuno-histochemical procedures using indirect immunoflourescence technique for detection of apoptotic hepatic and lymphocytic cells using monoclonal antibodies. Semiquantitative analysis of the pattern and distribution of the Fas antigen and Fas Ligand as indicators for hepatic apoptosis was studied and assessed.

RG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R

Background: Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy.Purpose: Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer.Methods: Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an in vivo antitumor activity experiment was tested in nude mice.Results: The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15–27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC50 value of 0.6–4 ng · mL−1. Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size.Conclusions and discussion: The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in E. coli and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.

Pro-inflammatory role of angiotensin II in mercuric chloride-induced nephropathy in rats

Mercuric chloride (HgCl2), which induces kidney toxicity, constitutes a potential threat to human health. In addition to direct toxic effects, kidney inflammatory events take place during the HgCl2-induced nephropathy. There is no information currently available about the role of angiotensin II (Ang II) in this inflammatory process. Accordingly, the aim of this study was to determine the expression of Ang II and Ang II-associated inflammatory molecules, i.e. intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and mono-cyte/macrophage infiltration (ED-1), in HgCl2-induced nephropathy. Three groups of Sprague Dawley rats that were to receive HgCl2 (2.5 mg HgCl2/kg BW, by gavage) were utilized: one had received Losartan at 30 mg/kg BW; one had received Enalapril at 30 mg/kg BW; and one had received distilled water, in each case daily for 3 days prior to the HgCl2 exposure. For these studies, an extra set of controls treated with saline solution in place of HgCl2 and water in place of the test drugs was employed. Renal biopsies were obtained 96 h after HgCl2 injection and the expressions of Ang II, ICAM-1, iNOS, and ED-1 were analyzed by indirect immunoflourescence while tubular damage was assessed via histopathology. An increased expression of Ang II, ICAM-1, iNOS, and ED-1 as well as increases in tubular necrosis were observed in all HgCl2-animals. Treatments with Losartan or Enalapril diminished the induced expressions as well as the extent of tubular damage. The data here suggest that Ang II is involved in the pro-inflammatory events during HgCl2-induced nephropathy, and that this is probably mediated, in part, by Ang II receptors Type 1 (AT-1).

13-gene signature to predict rapid development of brain metastases in patients with HER2-positive advanced breast cancer.

505 Background: Brain metastases (BM) of breast cancer constitute an important part of therapeutic failures and are associated with severe morbidity and mortality. The risk of BM is particularly high in HER2+ advanced breast cancer pts. We earlier developed in this group a 13-gene signature strongly predicting for rapid development of BM (J Clin Oncol 2008; 26: 45s). Now, we validated these results in an independent group of pts and on culture model system. Methods: Discovery group included 87 samples analyzed using cDNA synthesis, annealing, selection, extension, ligation and array hybridization (DASL). Independent validation group included 75 samples analyzed using quantitative reverse-transcriptase PCR. 3D culture validation model system used immortal, non-tumorigenic human MCF10A breast epithelial cells with and without ectopic expression of HER-2 and RAD51, a DNA double strand break repair gene (one of the three genes of this group overexpressed in 13-gene signature). The number and morphology of breast acini were scored using indirect immunoflourescence and confocal microscopy. Results: Median brain metastasis-free survival (BMFS) in the discovery group for ‘high’ vs. ‘low’ expression signature tumors was 36 months and 66 months, respectively (P=0.0068), and in the validation group 54 and 86 months, respectively (P=0.032). Short BMFS was also associated with ER-negativity; BMFS in the cohort of ‘high’ 13-gene signature and ER- tumors vs. other 3 groups was 31 months and 66 months in discovery group, and 41 and 77 months in validation group (P&lt;0.0001 and P=0.02, respectively). Overexpression of RAD51 in MCF-10A breast cells altered their three-dimensional acinar morphology and increased the percentage of invasive structures by 6.5 fold, both in the presence and absence of HER2 overexpression. Conclusions: 13-gene signature and ER-negativity predict rapid development of BM in HER+ advanced breast cancer pts. RAD51 may promote aggressiveness in breast epithelial cells. These data may be useful in the design of BM preventive trials and may prompt new treatment strategies.

Evaluation of the serum level of soluble E -cadherin and cell surface expression of α5β1 integrin in patients with acute leukemia whether lymphocytic or myeloid ,in order to extrapolate their prognostic value (127.3)

Abstract Background:E-cadherin is expressed on CD34+ stem cell,where it likely contributes to intracellular interactions during hematopoiesis. Also integrin such as α5β1 integrin represent an essential component of the bone marrow microenvironment that regulates cell survival .In leukemia, immature blasts are not retained within the marrow, suggesting a breakdown of adhesive mechanisms and cell migration. Objective: this study was done to evaluate serum soluble E -cadherin and cell surface expression of α5β1 integrin on PBMCs in patients with acute leukemia whether lymphocytic or myeloid Twenty patients (11 AML and 9 ALL)and ten age and sex matched normal individuals were studied. soluble E -cadherin were assayed using ELISA and cell surface expression of α5β1 integrin on PBMCs by indirect immunoflourescence . Results: Soluble E-cadherin was significantly lower in all leukemic and AML patients than control ( p=.0108,p=.0049 respectively).However no significant difference between AML and ALL. α5β1 integrin cell surface expression was significantly higher in both AML,ALL groups than control(p=.0001,0.0038 respectively). E-cadherin has negative significant correlation (r= -.573, p= .008) with bone marrow blast percentage after the induction of chemotherapy. Conclusion this suggests that a dual double way interaction between increased expression of α5β1 integrin and loss or decreased expression of E-cadherin that governs the process of tumour progression and metastasis.

Comparison of Immuno-Phenotypes of Stem Cells from Human Dental Pulp and Periodontal Ligament

It has been established that human dental pulp and periodontal ligament contain a population of mesenchymal stem cells (MSCs). However, the phenotypic analysis in terms of putative stem cell markers expressed by these stem cell populations is incomplete. It is relevant to understand whether stem cells derived from closely related tissues are programmed differently. The aim of the present study is to analyze whether these stem cells depict distinct characteristics by gaining insight into differences in their immunophenotype. Dental pulp and periodontal ligament tissue samples were obtained from extracted impacted wisdom teeth. Cell cultures were analyzed for surface and intracellular markers by indirect immunoflourescence. Detailed immunophenotype analysis was carried out by flow cytometry using relevant markers. The present study data shows dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) expressed embryonic stem (ES) cell markers Oct-4, Nanog and mesodermal marker Vimentin by indirect immunoflourescence. PDLSCs, however, had a weak expression of Nanog. Immunophenotyping revealed strong expression of MSC markers (CD73, CD90) in DPSCs and PDLSCs. Differences were observed in expression of stemness-related markers. DPSCs displayed increased percentages of SSEA4, CD13 and CD166 and decreased CD9 expression compared to PDLSCs. Both stem cells express common MSC markers, different levels of expression suggests there might be more than one stem cell population existing within these tissues which differ in their embryonic status, and DPSCs are a more primitive stem cell population in comparison to PDLSCs.

Abstract 2476: Acetylated Survivin functions in DNA damage repair

Abstract Survivin is an oncogenic protein that participates in cell division and inhibits apoptosis in cancer cells. These functions are dependent on its interaction with partner proteins within distinct subcellular compartments. Survivin is highly expressed in malignant tumors yet minimally expressed in normal tissues, making it an attractive target for molecular therapy. Inhibition of Survivin function enhances tumor cell sensitivity to radiation, the mechanism of which has been reported to result from a disruption of DNA-double-strand break repair processes. Recently, we demonstrated that Survivin is post-translationally modified by acetylation at numerous lysine residues, one of which is responsible for its subcellular transport. Acetylation at Lys-129 facilitates Survivin homodimerization which prevents its binding to the CRM1 nuclear export protein, inhibiting its translocation from the nucleus to the cytoplasm. The object of this study was to determine the potential relationship between Survivin acetylation and its function in nuclear repair processes. To elucidate the association between Survivin acetylation, nuclear localization and DNA double-strand break repair we subjected HeLa cells to 4Gy γXRT and examined Survivin acetylation at serial time points (20, 40, 60 min) after irradiation. An immediate deacetylation of Survivin was observed at 20 minutes, followed by an increase in both global acetylation and acetylation at Lys-129 at 40 minutes. The increase in Lys-129 acetylation correlated with its nuclear accumulation, as demonstrated by indirect immunofluorescence staining and immunoblotting after subcellular fractionation. Acetylated Survivin was found in endogenous immunoprecipitants with the DNA damage repair protein, Ku70, following γ-irradiation. Indirect immunoflourescence revealed co-localization of Ku70 and acetylated Survivin within the nucleus at similar time points after irradiation suggesting that Survivin acetylation may be required for Ku70 complex formation within the nucleus. These results implicate acetylated Survivin as a regulator of DNA damage repair through the Ku70 DNA repair pathway and further our understanding of Survivin function within the nuclear compartment of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2011-2476

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.

Successful treatment of recalcitrant oral pemphigus vulgaris with mycophenolate mofetil

Oral pemphigus vulgaris (OPV) is an intraepithelial autoimmune bullous disease with circulating autoantibodies against desmoglein 3. The disease is difficult to treat and in some cases may be fatal. Mycophenolate mofetil (MMF), a new immunosuppressive drug which selectively inhibits cytotoxic lymphocytes and antibody production by B-lymphocytes was originally used to treat acute graft rejections in renal transplanted patients. The objective of this trial was to assess the therapeutic efficacy of MMF on OPV. One 61 year-old male patient with oral pemphigus confirmed via histopathology, direct and indirect immunoflourescence, was treated with MMF. After the confirmation of the diagnosis, MMF was administered orally in a dose of 2 g daily. Routine laboratory studies and other recommended laboratory tests for MMF were performed at regular intervals. The patient responded very well to the therapy and all lesions healed completely within 4 months. Under a maintenance therapy of 200 mg MMF per day no relapse of the disease occurred for a follow-up period of 3 years. No side-effects were observed. MMF is a safe and effective therapy for oral OPV. More clinical double-blind studies should be performed to confirm our result.

The Quantitation of D Antigenic Sites in Partial D and Weak D Variants by Flowcytometry

Aim The aim of the study is to quantitate D antigenic sites on RBC in weak D and partial D variants in Indian population by flowcytometric method. Method Dantigenic sites were estimated in 48 cases of partial D, eight cases of weak D (identified by serological methods) and normal Rh phenotypes subjects by flowcytometric technique. The indirect immunoflourescence method was employed using thirty epitope specific monoclonal anti-D as primary antibody. The D antigenic sites were calculated using standard RBC with known D antigenic sites. Result The mean (±I S.D) D antigenic sites per RBC in weak D and partial D were 4503 ± 2067 and 6750 ± 3426 respectively. In R1R1 phenotype the range of D antigenic sites was 16500 ± 2500 and in R1r it was 10600 ± 2520. In four cases of weak D sites were in range of 1700 to 3500 and in other four it was 5000 to 7200 D sites/RBC. Among partial D variants DVI had minimum, and DVa had maximum number of D sites. Conclusion Flowcytometry is a very good tool for demonstrating minor differences in D antigen sites in partial D and weak D when serological methods are not conclusive.