FACS Calibur Research Articles

Immunomodulating Potential of Ekebergia capensis in Murine Schistosoma mansoni Juvenile and Adult Infection

Schistosomiasis is a neglected tropical disease occurring in sub-Saharan Africa and affecting almost 250 million people. The drug of choice for treatment for Schistosomiasis has been Praziquantel which has been used for many years and there is a need to develop new drugs. The immunomodulatory potential of Ekebergia capensis extract on both juvenile and adult Schistosoma mansoni infection in vivo was evaluated in this study. Swiss albino mice were infected individually with 90 S. mansoni cercariae and randomized into groups of five each for i) plant extract treated groups ii) positive control groups treated with conventional drugs PZQ or artemether iii) infected but untreated (negative control) groups. The mice were treated orally with aqueous extracts of E. capensis at doses of 200 and 400 mg/kg at 2 weeks (juvenile worms) and 7 weeks (adult worms) post-infection. Immune immune-enhancing potential of the medicinal plant was determined by analyzing the levels of cytokines in serum samples that were collected before and after treatment. A BD-Cytometric Bead Array (CBA) mouse Th1/Th2/Th17 kit was used to quantitate the levels of cytokines using a flow cytometer (FACS Calibur) and analysis of the data was done using FCAP software. Results indicated that the medicinal plant extract had an immunomodulatory effect. There was a significant increase (P<0.05) in Th1 cytokines (IL-2, IFN- γ and TNF-α), a decrease in Th2 cytokines (IL-4, IL-6 and IL-10) and an increase in Th17 (IL-17). These findings confirm the potential use of medicinal plants in the management of schistosomiasis.

THYROID DYSFUNCTION IN HUMAN IMMUNODEFICIENCY VIRUS INFECTED PATIENTS AND THEIR CORRELATION WITH CD4 COUNT - A NON-RANDOMIZED, CROSS-SECTIONAL, SINGLE-CENTER STUDY

Background: Thyroid dysfunction has been reported in human immunodeficiency virus (HIV)-infected individuals. Objective: Objectives of this non-randomized, cross-sectional, single-center study was to study thyroid function in HIV positive patients at various stages of disease and to correlate the results obtained with CD4+ counts. Materials and Methods: This single-center study was carried out at Al-Ameen Medical College Hospital and Government District Hospital Bijapur, Karnataka, India from November 2020 to December 2022. The final selected study population included newly diagnosed adult and adolescent (17-60 years) HIV+ patients was composed of 100 participants of either gender. Patients were interviewed &enrolled in the study after examining in detail according to the proforma and then by taking their written consent and explaining the purpose of the study. The Thyroid hormone assays (S.TSH, FT3 and FT4) were done by Chemiluminescencelmmuno Assay (CLIA) using ADVIA Centaur-equipment.The CD4+ T-cell count was completed using FACS Calibur, Beckton Dickinson, USA27. Results: Overall mean age was 36 years (range in years: 17 – 66 years) and 66 patients (66%) were males.Male:Female ratio of 1.94:1 was recorded. Among 50 cases having CD4+ count <200/pl there were 38(76%) males and 12(24%) females. Among 50 cases having CD4 count >200/pl there were 28(56%) males and 22(44%) females.The CD4+ count ranged from 5 to 773/pl with a mean of 223±190.9/pl. This study observed that abnormalities of thyroid function are more common among patients having CD4+ count <200/pl. Clinically evident hyperthyroidism was not observed in any case.Direct correlation was observed between CD4 count and FT3 and FT4 values and Inverse correlation was observed when CD4+ counts were compared with serum TSH levels. Conclusion: All individuals with CD4 count less than 200 should be screened for hypothyroidism.An inverse correlation was seen between TSH and CD4 count indicating trend for hypothyroidism as HIV disease progress.

Abstract 2161: Fasting fueled ketogenesis inhibits ovarian cancer and promotes anti-tumor T cell response

Abstract Epithelial Ovarian Cancer (EOC) is the most lethal gynecologic cancer with no predominant genetic alteration identified that can be therapeutically targeted. Low-cost non-toxic approaches such as dietary modulations alter the responses of host and the cancer cells. Limited scientific investigations have examined the link between nutrition and EOC. Here, we tested the outcome of intermittent fasting (IF) on EOC progression with focus on fasting driven anti-tumor immune responses. Female C57/B6 mice were injected intraperitoneally with 5x106 mouse epithelial ovarian cancer ID8 (p53-/-, ID8 p53-/-, PTEN-/- and ID8 p53-/-, BRCA-/-) cells and fed either ad libitum (RD) or subjected to IF, where they underwent 16 hour fasting and 8 hour feeding window for 5 days/week. Tumor progression was monitored by ascites formation and abdominal circumference. Immune profiling was performed by flow cytometry using BD FACS Calibur. Untargeted metabolomics was performed in plasma from RD and IF mice using LC-MS/MS. Enhancement of immunotherapy was tested by combining IF with anti-PD 1 (100 µg/mice, every 5th day, 3 times) in ID8 p53-/- tumor bearing mice and similar parameters were examined. IF reduced the growth of ID8 (p53-/-, ID8 p53-/-, PTEN-/- and ID8 p53-/-, BRCA-/-) tumors, increased overall survival when compared to RD mice. IF promoted anti-tumor T cell response as seen by increase in CD4+ and CD8+ cells with enhanced expression of IFNγ, granzyme B and perforin compared to RD mice bearing ID8 cells in the ascites and blood. A striking observation was that the CD4+ T cells from IF mice exhibited a strong Th1 phenotype (CD4+IFNγ+), while Th2 phenotype (CD4+IL4+) was significantly decreased. IF when combined with anti-PD-1 treatment significantly improved survival and potentiated the T cell mediated anti-tumor response. Metabolic analysis revealed an enrichment in ketone body biosynthetic pathway as reflected by 5fold increase in serum ketone bodies such as beta hydroxy butyrate (BHB) and acetoacetate, when compared to RD. Receptors that have been reported to response to BHB as a ligand, specifically GPR109A and GPR41, were highly expressed on T cells derived from IF mice. BHB treatment enhanced the CD8+ T cell effector function as reflected by increased levels of IFNγ, granzyme B and perforin compared to untreated CD8+ T cells. Our study shows that IF significantly restricts EOC growth, improve survival, potentiate chemotherapy and immunotherapy anti-tumor response by augmenting T cell mediated anti-tumor immune responses. We also provide evidence that IF generated bioactive metabolite BHB which can be a potential substitute for availing the ant-tumor benefits of IF. Thus, our data provides strong evidence for IF and its metabolic mediator BHB for ameliorating EOC progression and as a viable approach in maintaining and sustaining an effective anti-tumor T cell response alone and in combination with immunotherapy. Citation Format: Mary Priyanka Udumula, Laila Poisson, Lin Chun-Hui, Nivedita Tiwari, Harshit Singh, Giri Shailendra, Ramandeep Rattan. Fasting fueled ketogenesis inhibits ovarian cancer and promotes anti-tumor T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2161.

Effects of thymulin 5cH in granuloma evolution and B1 cell differentiation: an experimental model to understand its biological mechanisms

In previous studies, we found that thymulin (a thymic hormone), when prepared in homeopathic 5cH potency, had the property to improve the productive performance of broiler chickens infected with reovirus, as well as modulate the development of Ehrlich tumor and granuloma inflammatory lesions in mice by immune-mediated mechanisms. The aim of the present work was to study the immunomodulatory mechanisms of thymulin 5cH in a granuloma experimental model, by subcutaneous inoculation of BCG in mice, focusing the B-1 cells and zinc involvement in this process. Three groups of male Balb/c SPF mice (group A treated with thymulin 5cH, group B treated with thymulin 5cH incubated in Chelex ® - a zinc chelant - and group C, control, treated with vehicle) were inoculated with BCG in the left footpad and subcutaneous granuloma and spleen were harvested for histomorphometry analysis, after 7, 14 and 21 days. Ziehl-Neelsen, HE and Prussia Blue staining methods were used. Flow cytometry was also used in the same times to characterize and quantify peritoneal cells. Positive cells for CD11b (activated phagocytes, B-1 cells), CD19 (B-1 and B-2 cells), CD23 (negative B-1 cells, positive B2 cells) and CD5 (B-1a cells) were analyzed in a FACS Calibur (BD) device. Statistical analysis was performed using Kruskal - Wallis / Dunn for nonparametric evaluations and ANOVA / Tuckey-Krammer for the parametric ones. The X² method was used to evaluate the cell count in flow cytometry. P values ≤ 0.05 were considered statistically significant. Mice treated with thymulin 5cH presented higher macrophage activity and increase in the follicular area were seen in spleen after 7 days. Increase in gross lesion diameter and decrease in local BCG infection were seen after 21 days. At this time, the flow cytometry demonstrated the increase in peritoneal phagocytes derived from B-1 cells in thymulin 5cH treated mice, independently of Chelex ® incubation. The incubation of thymulin 5cH with Chelex ® blocked its effects only upon the number of B2 cells in the peritoneum and reduces Mn levels in the medicine solution. We conclude that thymulin 5CH modulates the BCG-induced granuloma through more than one mechanism, especially by peritoneal B1 cell differentiation into phagocytes.

The Direct Influence of Cytomegalovirus Lysate on the Natural Killer Cell Receptor Repertoire.

Natural killer (NK) cells are essential for controlling certain viral infections, including cytomegalovirus (CMV). In particular, the importance of NK cells in the context of CMV infection is underscored by the adaptive capabilities of these cells. Evidence suggests that some viruses can directly interfere with NK cell compartments and their activation and lead to shape-shifting the NK cell receptor repertoire. Still, it remains unknown whether the CMV can interact with NK cells without intermediaries. Here, we examined whether the direct effects of CMV lysate alter phenotypical properties of NK cells. To investigate this issue, NK cells were isolated from the blood of CMV seropositive healthy donors by negative magnetic separation. Isolated NK cells were cultured in the presence of CMV lysate and analyzed for the expression of NKG2A, NKG2C, and CD57 by FACS caliber. The results showed that NKG2C expression is significantly upregulated in the presence of CMV lysate compared to without stimulated group (mean increase, 6.65 %; 95% CI, 0.2582 to 13.02; p=0.043; R square: 0.38). Likewise, results have shown a significant decrease in the frequency of NKG2A+CD57- NK cell subsets (p=0.005; 95% CI, -13.49 to -3.151; R square: 0.5957) in the stimulated group compared to without stimulated ones. According to these results, CMV may drive a direct influence on NK cell receptor repertoire, including the expansion of NK cells expressing NKG2C receptor, which is needed for further studies.

Comparison of modulation interference microscopy, DNA spectrometry, DNA cytometry, and flow cytofluorimetry in the assessment of phytohemagglutinin-induced activity of human blood lymphocytes

Rationale: The study of the structural particulars and functional state of immune cells and primarily lymphocytes is of great importance for both fundamental and clinical medicine. It requires the development of simple and reliable analytic methods that would allow for fast and effective real-time assessment of cell activity.Aim: To evaluate the effectiveness of the interference microscopy compared to DNA spectrometry, DNA cytometry, and flow cytometry with an internalized fluorescent label CFSE (carboxyfluorescein succinimidyl ester) in the assessment of PHA-induced proliferation of human blood lymphocytes.Materials and methods: Phytohemagglutinin (PHA)-induced proliferative activity of blood lymphocytes from 10 healthy volunteers was studied with various methodological strategies. Blast transformation of lymphocytes was induced by their incubation for 5 days with PHA 5 μg/mL. The cell proliferative activity was assessed as follows: 1) by DNA spectrometry at 260/280 nm using Tecan Infinite 200 Pro with a specialized NanoQuant Plate™; 2) by cytophotometry followed by cell distribution analysis assessing deoxyribonucleic acid (DNA) content after staining with Felgen's dye with an imaging system based on an Olympus BX41 light microscope with a ProgRes CF camera; 3) by flow cytometry using an internalized fluorescent label CFSE; the analysis was performed with a BD FACS Calibur flow cytometer; 4) by measurement of the lymphocyte interference profile with a modulation interference microscope MIM-340 (Schwabe, Russia). The functional activity of the nucleus (FAN) was determined and used as a criterion for assessment of the lymphocyte functional state.Results: Incubation of lymphocytes with PHA led to an increase in the linear size by 22.2±2.8%, a decrease in phase height by 46.3±4.7% (p=0.019), and an increase in FAN by 75.9±9.4%, vs control (p=0.046). As measured by isolated DNA spectroscopy, PHA stimulation of lymphocytes was associated with an increase in the amount of DNA by 55% vs baseline (409.8±22.3 ng/μL and 264.3±25.0 ng/μL, respectively, p=0.049). Felgen's reaction revealed that the proportion of nuclei containing more than 2n DNA was 2% in the control cells and 14.8% in the PHA-activated lymphocytes, with a difference between the groups of 12.8%. CFSE staining with subsequent incubation and assessment by flow cytofluorimetry demonstrated an increase in the percentage of proliferating cells from 1.68±0.9% in the control to 55.56±5.6% (p=0.00068) in the mitogen-stimulated sample.Conclusion: Modulation interference microscopy does not require the sample preparation and demonstrated comparable and even higher effectiveness compared to conventional methods for assessment of lymphocyte activity. At the same time, it allows for evaluation of the lymphocyte functional state in real time in the process of cultivation. This opens ample opportunities for evaluation immune cells for research and diagnostic purposes.

PSX-A-7 Late-Breaking: Correlation of mitochondrial membrane potential and rough motility scores in cryopreserved bovine spermatozoa

Abstract Spermatozoon motility is an important factor in successful artificial reproductive technologies. Successful reproduction requires properly developed spermatozoa with adequate forward, progressive motility that allows for transport through the female reproductive tract. Motility is driven by production of ATP; however, cryopreservation is known to have damaging effects on spermatozoa. Mitochondria utilize oxidative phosphorylation to synthesize ATP through an electrochemical proton motive force that is composed primarily of mitochondrial membrane potential (Δψm). The mitochondrial membrane potential can be measured using a fluorescent, carbocyanine JC-1 dye (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide). The objective of this study was to examine correlations between the mitochondrial membrane potential and motility score. Cryopreserved semen samples from three bulls were donated to Tarleton State University for research purposes. Samples were thawed, placed under a phase contrast microscope, and analyzed by three individuals with varying levels of training for rough motility. These samples were then prepared with JC-1 dye according to manufacturer’s instructions. Two microliters of dye were added to a stock preparation of each sample. Samples were examined on a FACS Calibur flow cytometer at University of Texas Southwestern Core in Dallas, TX. Flow cytometry analysis was performed using FlowJo V10.7. Statistical analysis was performed using SAS v9.4. The Pearson correlation coefficient showed a strong, positive correlation (r = 0.90) between the mitochondrial membrane potential and motility (P = 0.28), thus indicating as the mitochondrial membrane potential increases, so does the rough motility score. These data represent a subset of a population that demonstrates the need of further research on the ability for spermatozoa to produce ATP and the correlation in forward, progressive motility. This research can provide a foundation in which future researchers may develop an assay that allows for testing of mitochondrial membrane potential by producers to select bulls with greater breeding potential.

LRP1 Expression in Sickle Cell Disorders: Insights into the Heterogeneity of Monocytic Response to Heme

Introduction: The importance of heme and its main scavenger hemopexin in the pathophysiology of sickle cell disease (SCD) has been demonstrated in animal models of SCD vaso-occlusion. Increase in monocyte-derived inflammatory cytokines in SCD can be attributed to heme-mediated activation of TLR4 signalling to produce TNF-alpha, and amplification of IL-6 production through a mechanism dependent on heme iron. This suggests that monocytes not only respond to heme but also have receptors to acquire it. We hypothesized that varying levels of heme and hemopexin in SCD could affect how circulating monocytes express LDL receptor related protein (LRP1), the receptor that targets the heme/hemopexin complex to degradation, which can be cleaved to produce a soluble form (s-LRP1).Methods: Peripheral venous blood samples from 119 subjects (39 healthy volunteers - AA, 44 homozygous sickle cell anemia patients - SS, 8 not on hydroxyurea treatment, and 36 hemoglobin SC disease patients - SC) were collected upon signed informed consent approved by the local ethics board. Automated complete blood counts and serum lactate dehydrogenase (LDH), bilirubin levels, and heme and hemopexin concentrations were determined. LRP1 expression was measured by mean fluorescence intensity through conventional flow cytometry (FACS Calibur and FACS DIVA software) in different monocyte subsets, classified according to their surface markers into classical (CD14++ CD16-), intermediate (CD14+ CD16+) and nonclassical (CD14dim CD16+) monocytes.Results and Discussion: As expected, reticulocyte count, LDH, bilirubin, and heme were significantly more elevated in SS than in SC patients when compared to AA subjects, and hemopexin was reduced in both patient groups (P <0.0001). Monocyte subsets differed significantly among the groups: SS and SC patients had more circulating intermediate and nonclassical monocytes than AA subjects (P <0.0001 and P=0.0002, respectively), which is compatible with the systemic inflammation that characterizes SCD. Noticeably, expression of membrane-bound LRP1 was significantly higher in monocytes from SS patients than in those from SC or AA. We also found that LRP1 expression was higher in intermediate and classical monocytes, which are characterized by higher CD14 expression, than in nonclassical CD14dim CD16+ cells. LRP1 expression correlated with hemopexin (rS=0.58, 95%CI 0.26-0.79, P=0.0012) and heme levels (rS=-0.39, 95%CI -0.58 to -0.16, P=0.0012) in SS, but not in SC patients. To address whether monocytic LRP1 expression was associated with circulating LRP1 levels, we measured s-LRP1 and found higher mean s-LRP1 levels in SS than in SC patients (5.52±2.32ug/ml vs. 4.23±2.08ug/mL, P=0.01), but AA subjects showed intermediate s-LRP1 levels (4.70±1.67ug/ml). In SS patients, s-LRP1 correlated with LDH (rS=0.45, 95% CI 0.22-0.64, P=0.0002), but not with all biomarkers of hemolysis, suggesting that s-LRP1 production may also be influenced by mechanisms other than hemolysis. Our data show that more severe hemolysis in SS patients (higher LDH, heme, and lower hemopexin levels) is associated with elevated expression of monocyte LRP1 and its circulating form s-LRP1. Our finding that LRP1 expression in intermediate monocytes correlates with heme and hemopexin levels, particularly in SS patients, suggests this cell type responds to excessive heme. Along with previous publications that this monocyte subtype upregulates heme oxygenase gene HMOX, this supports that monocytes equipped with LRP1 can capture heme/hemopexin complexes and detoxify heme. In addition, higher LRP1 expression in CD14+ circulating monocytes corroborates that these cells can respond simultaneously to free heme with TLR4/CD14-dependent TNF-alpha production and to hemopexin-bound heme captured by LRP1 to foster iron-mediated augmentation of IL-6 production (Dagur et al. 2017).Conclusions: This study highlights that some monocyte subtypes are more prone to capture heme/hemopexin complexes and may react to both free and hemopexin-bound heme, adding to the complexity of the pathophysiology of SCD. Modulation of monocyte differentiation towards subsets with lower responsiveness to heme may be further explored in SCD and other hemolytic diseases in the future.

Functional activity of animal bone marrow cells after their treatment with nanocomplexes

Background. Previously, the antitumor activity of nanocomplexes (NCs) containing nanoparticles of rare earth metal orthovanadates GdYEuVO4 and cholesterol has been approved when applied in 9:1 ratio (the cells-to-NCs), which can be considered as a conditionally therapeutic dose. Therefore, studying the potential risks of NCs exposure in terms of functional activity of hematopoietic progenitor cells is relevant.&#x0D; Рurpose – determining a toxic effect of NCs on functional activity of hematopoietic cells of bone marrow (BM).&#x0D; Materials and Methods. The study was performed in BM cells of CBA/H mice. Nanocomplexes were synthesized at Institute for Scintillation Materials of the National Academy of Sciences of Ukraine. BM cells with NCs were incubated in the ratios as follows: 9BM:1NCs; 1BM:1NCs; 1BM:9NCs, followed by assessing the number of apoptotic/necrotic cells in BM using FITC Annexin V Apoptosis Detection Kit I (BD, USA) by means of “FACS Calibur” flow cytometer (“BD”, USA). Hematopoietic progenitor cells of BM were functionally evaluated in vivo by determining the content of colony-forming units of the spleen (CFUs) and the number of myelokaryocytes in lethally irradiated recipients on day 8 after administering BM cells, pre-incubated with NCs. Survival of irradiated recipient mice after BM administration was recorded 12 days long.&#x0D; Results and discussion. The dose-dependent effect of functional potential in- hibition for BM hematopoietic progenitor cells under NCs influence has been established. Although, in vitro processing the BM cells with a conditionally therapeutic dose of NCs (9BM:1NCs) before administration to irradiated animal caused remodeling of cell membranes and contributed to apoptotic manifes- tations, but it did not lead to strong changes in their colony-forming potential and did not reduce the number of BM cells in animals if compared with the introduced BM cells without NCs treatment. Increasing the NCs concentration five- and tenfold significantly reduced the colony-forming potential of BM cells, caused BM hypoplasia and a crucial reduction in the survival of recipient animals, indicating possible toxic effects of this compound when administered at high concentrations.&#x0D; Conclusions. The toxic effect of NCs is detected only when certain concen- trations, significantly exceeding the conditionally therapeutic dose previously determined when treating the experimental oncology diseases, are used.

Seroprevalence Toxoplasma Infection among HIV/AIDS Patients in Kathmandu, Nepal

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii affecting about one third of the world’s population. It can be asymptomatic to fatal toxoplasmic encephalitis depending on the immune status of infected individuals. Among HIV/AIDS patients, it usually manifest as life-threatening condition. We, therefore, studied the seroprevalence of T. gondii infection among HIV/AIDS patients in Nepal and this report constitutes the first report from Nepal. A total of 45 HIV/AIDS patients were included in this study. The serum samples collected and stored at -20°C were tested for Toxoplasma IgG and IgM antibodies at National Public Health Laboratory, Teku, Kathmandu using Snibe Maglumi 1000 Fully Automated Immunoassay Analyzer and the results were expressed in AU/ml. The blood put into the EDTA tube was used for CD4 count using BD FACS Calibur Flow Cytometer. In this study, 33.3% (15/45) HIV infected patients were seropositive for anti-T. gondii IgG. However, none of them were positive to anti- T. gondii IgM. Most of the patients (36 out of 45 patients) had &lt;200/mm3 CD4 cell count. However, out of them 36.1% (13/36) were seropositive to anti-T. gondii IgG whereas 22.2% (2/9) patients with ≥200 CD4 cell counts had Toxoplasma antibodies (p &gt;0.05).

POS-361 CD4 AND CXCR5 IN PATIENTS WITH DIABETIC NEPHROPATHY

Diabetes is a metabolic condition characterized by hyperglycemia caused by defects in insulin secretion, insulin activity, or both. Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus especially type 2 diabetes. Blood CXCR5+CD4+ T cells represent three subsets: T follicular helper 1 (Tfh1), Tfh2 and Tfh17 cells. Increasing the numbers of circulating Tfh cells and also stimulation of Tfh cells have been linked with autoimmune diseases and may have a role in pathogenesis of diabetic nephropathy. This study was conducted on 20 diabetic patients without nephropathy (Group I), 20 diabetic nephropathy patients (Group II) and 20 apparently healthy control subjects (Group III). Complete history taking and full clinical examination were done. Assessment of CD4 and CXCR5 by flowcytometry (FACS caliber, BD, CA,USA). The present study revealed a statistically significant difference between diabetic patients (Group I), diabetic nephropathy patients (GroupII) and healthy control (Group III) regarding CD4+CXCR5+T follicular helper cells level. CD4+CXCR5+T follicular helper cells levels were significantly higher in diabetic nephropathy patients than in diabetic patients without nephropathy and control subjects. CD4+CXCR5+T follicular helper cells level correlated significantly with fasting blood glucose, postprandial blood glucose, hemoglobin A1c, creatinine, urea and urinary protein level, and negatively correlated with estimated GFR in diabetic nephropathy patients. This study showed that CD4+CXCR5+T follicular helper cells level might be a useful marker for prediction and diagnosis of diabetic nephropathy patients.

Distribution of subtypes and immunophenotypic characterization of 1379 cases of paediatric acute leukaemia.

Objectives:Acute leukaemia is the most common and highly curable childhood malignancy; subtyping and identification of antigens via immunophenotyping helps in treatment plan as well as minimal residual disease monitoring.Methods:This retrospective study was conducted at the Haematology section of the clinical laboratories of Ziauddin University Hospital and The Indus Hospital, Karachi conducted at January 1st, 2012 to December 31st, 2017. The study included 1379 cases of de novo acute leukemia from 2012 to 2017. Among these, 80% were diagnosed by using four color flowcytometry (FACS Calibur), 9% and 11% via immunohistochemistry on bone marrow trephine biopsy samples and morphological examination respectively.Results:The mean age of patients was 7.4 ± 4.3 years while male to female ratio was 1.75:1. Lymphoblastic leukaemia accounted for 77.2% and myeloid leukaemia 21.2%. Amongst lymphoblastic lineage, B-ALL was 80.4% while T-ALL was 19.6%. Among the phenotypic expression of B-ALL, CD79a (99.8%) had the highest positivity. In B-ALL, CD13 (29.8%) was the most common aberrant myeloid marker. Aberrant expression of CD79a observed in 11.1% of T-ALL cases. In non APL AML, aberrant expression of CD79a and CD19 was observed in 6.6% and 5.5% of cases respectively.Conclusion:Overall immunophenotypic profile, expression of aberrant phenotypes and subtype distribution in our patients was similar to international literature except for a relatively high frequency of T-ALL which was discordant from the western data.

Predictors of Anemia Among HIV-Infected Children on Antiretroviral Therapy in Wolaita Zone, South Ethiopia: A Facility-Based Cross-Sectional Study.

PurposeAnemia is a global public health problem, and the majority of human immunodeficiency virus (HIV)-positive people become anemic at some point in the course of the disease. We lack adequate evidence on the magnitude of anemia among children on highly active antiretroviral therapy in Ethiopia and particularly in South Ethiopia. Thus, this study aimed at determining the proportion and associated factors of anemia among children on highly active antiretroviral therapy in Wolaita zone, South Ethiopia.Patients and MethodsA facility-based cross-sectional study was conducted from November to December 2018 on 256 children from 6 months to 14 years of age who were on antiretroviral therapy. Data were collected through an interview with caregivers and review of medical records. CD4+ cell count was analyzed using FACS Calibur, and hemoglobin level was measured with a Hem cue 301 analyzer. Stool samples were examined for the presence of intestinal parasites by direct wet mount technique. Data analyzed with Stata version 14.0 were conveyed in mean and standard deviation of the mean, median and inter-quartile range. Multivariate analysis was carried out to identify independent predictors of the outcome variable. Adjusted odds ratio with 95% confidence interval was reported.ResultsThe proportion of anemia was found to be 38.8%. Co-trimoxazole prophylaxis (AOR=0.45; 95% CI: 0.21, 0.95), caregivers not receiving nutritional counseling (AOR=0.90; 95% CI: 0.01, 0.98) and presence of intestinal parasites (AOR=3.10; 95% CI: 1.39, 6.88) were associated with anemia.ConclusionThe proportion of anemia found in this study is a moderate public health problem. Health education programs in antiretroviral therapy clinics should be targeted at appropriate dietary practice, and appropriate hand washing and other hygienic practices to prevent intestinal parasitic infections. Co-trimoxazole prophylaxis should be given to all eligible children based on the recommendation.

Determinants of periodontitis according to the immunological and virological profiles of HIV-infected patients in Yaound\xe9, Cameroon

BackgroundHIV infection is associated to different oral manifestations (including periodontal diseases), which have decreased with the advent of antiretroviral therapy (ART). Yet, the occurrence of periodontitis is still consistent among patients with HIV living in sub Saharan-Africa, with limited evidence on the driven factors and mitigating measures in these settings. We aimed at evaluating the occurrence of periodontitis and its associated immunological and virological factors in patients with HIV living in Yaoundé, Cameroon.MethodsWe included 165 (44 ART-naïve and 121 ART-experienced) patients > 18 years old attending the Yaoundé Central Hospital and the Chantal BIYA International Reference Centre, from January-April 2018. The periodontal status was assessed by measuring the clinical attachment loss, periodontal pocket depth, plaques index and gingival bleeding index. CD4+/CD8+ cells and viremia were measured using the fluorescence-activated cell sorting method (FACS Calibur) and the Abbott m2000 RT HIV-1 RNA kit respectively. A standard-questionnaire concerning participants’ medical records and oral hygiene methods was filled. Data was analyzed and p < 0.05 considered statistically significant.ResultsThere was a significantly high prevalence of periodontitis in the ART-naïve (53.2%) compared to the ART-experienced group (37.3%), with a twofold increased risk of the ART-naïve population presenting with periodontitis than the ART-experienced population (OR 2.06, p = 0.03). More importantly, ART-naïve, patients with CD4 < 200 cells presented with higher risk of having periodontitis compared to those with higher CD4-values, with a threefold difference (OR 3.21). Worth noting, males presented with a higher risk of having clinical attachment loss (OR 6.07). There was no significant association between the occurrence of periodontitis and the CD8 (p = 0.45) or viremia (p = 0.10).ConclusionIn the Cameroonian context, a considerable number of adults infected with HIV suffer from periodontitis regardless of their treatment profile. Nonetheless, ART-naïve patients have a higher risk, indicating the protective role of ART. Interestingly, severely immune-compromised patients and men are vulnerable to periodontitis, thereby highlighting the need for clinicians to refer patients for regular periodontal screening especially male patients and those with low CD4. Such measures could greatly improve the quality of life of the population living with HIV in Cameroon.

EVALUATION OF NATURAL KILLER CELLS USING DIFFERENT TECHNIQUES

Natural killer (NK) cells mediate spontaneous cytotoxicity toward abnormal and tumor cells and rapidly secrete numerous cytokines and chemokine`s which promote subsequent adaptive immune responses. The presented study aimed to compare between different methods for NK evaluation and determination. Ten samples of healthy individuals were collected from Al-Zahraa University hospital and evaluated for phenotypic identification for NK cells. Isolation procedure of NK was performed in Clinical Pathology Department, Hematology Unit, Flow-cytometry lab, Al-Azhar University. Protocols started with isolation of peripheral blood mononuclear cells (PBMCs) from fresh human whole blood depending on Ficoll-Paque density gradient centrifugation. Other protocol depended on flow cytometry sorting technique. All experiments were carried out in accordance with the approved guidelines. Sorting procedure using FACS Calibur machine yielded 64.97 % purity before cells were cultured, while mixed Ficoll separation of whole blood showed purity 22.74%. Through our study, it is clear to us that the percentage of purity of NK cells using the flow-cytometry sorting technique is much higher than the percentage of purity of NK cells using the separation method dependent on Ficoll-Paque density technique, Therefore, through our study, we see that it is better to rely on the flow-cytometry sorting technique using the flow cytometry device and CD56 +CD16 and CD3 antibodies in order to obtain the best purity of the NK cells (CD56+ CD16 positive /CD3 negative population) for use in medical purposes and laboratory research.

Effects of Complement Regulators and Chemokine Receptors in Type 2 Diabetes

ABSTRACT CD55 and CD59 are complement regulatory proteins suggested to be related with progression of diabetes and its complications. The stromal cell-derived factor 1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR-4) are chemokine proteins. We aimed to investigate the relation of CD55 and CD59 expression levels and polymorphisms of SDF-1 and CXCR-4 with type 2 diabetes mellitus (T2DM) and its complications. Seventy-five T2DM patients and 73 controls were enrolled. Expression levels of CD55 and CD59 were measured by FACS Calibur; qRT-PCR was used to determine SDF-1 and CXCR-4 gene polymorphisms. CD55 and CD59 expressions in patients with nephropathy, retinopathy and cardiovascular disease were significantly lower than controls. Frequency of CXCR-4 T allele carrying was high in patients and created 1.6 fold risk for the disease (p = .07). CXCR-4 a allele carriers had decreased nephropathy; although there was no statistical significance in carrying CXCR-4 T allele, presence of nephropathy was approximately 2 times higher (p = .254). The nephropathy risk increased 10-fold in CXCR-4 TT genotype carriers (p = .02). All SDF-1 CC genotype carriers had retinopathy, so, it was considered that the CC genotype was effective in retinopathy development (p = .031). For the presence of cardiovascular disease, significant difference was observed for SDF-1 genotypes. Increased cardiovascular risk of 5- and 1.9-fold in SDF-1 T (p = .007) and CXCR-4 T (p = .216) allele carriers, respectively, was observed. We suggest that CD55 and CD59 protein levels and SDF-1 and CXCR-4 have predictive importance in process, complications and tendency of T2DM.

MO008B LYMPHOCYTE SUBSET CHANGES IN PRIMARY MEMBRANEOUS NEPHROPATHY

Abstract Background and Aims Primary membranous nephropathy (PMN) is the most common cause of nephrotic syndrome (NS) in adults. Rituximab a chimeric monoclonal anti-CD20 antibody has been supposed to eliminate autoantibody-producing B cells via direct signaling, complement-mediated cytotoxicity (CMC), and antibody-dependent cellular cytotoxicity (ADCC). According to the fact that a wide range of B lymphocytes may carry this marker, we aimed to identify which subset is more (or less) frequent in PMN patients and which one is more affected by rituximab administration. Method Three groups were enrolled in the present study. They included a healthy control group and two patient groups having the clinical, laboratory, and pathological diagnostic criteria of PMN, comprising either patients on standard treatment or patients on standard treatment plus rituximab. The latter group was studied just before receiving rituximab (pre-rituximab) and two months later (post rituximab). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-hypaque (inno-train, Germany) gradient. Afterward, cells were adjusted to a concentration of 1 _ 106 cells/mL and stained with mouse antihuman CD19-Cy5-conjugated antibody (Cytognos, Spain), mouse antihuman CD24-PE-conjugated antibody (Cytognos, Spain), and mouse antihuman CD38-FITC-conjugated antibody (Cytognos, Spain). Flow cytometry performed by FACS Calibur (BDFacs Calibur Becton Dickinson, USA). Results The total lymphocyte percentage was higher in PMN patients receiving either standard treatment or rituximab than in healthy controls (standard-treatment vs. control: P value = 0.001, pre-rituximab vs. control: P value =0.001). In post-rituximab analysis, CD19+ cell count showed notable reduction (P value = 0.003) B CD19+CD24+CD38- cells, representing the memory B cell population, did not show any significant difference between healthy controls and patients. Furthermore, the count of these cells did not decrease significantly two months after rituximab administration. The subset of CD19+CD24-CD38+ B lymphocytes, a class of naïve/mature lymphocytes with normal function, was significantly higher in the control group than in standard treatment patients (P value = 0.01). However, no statistically significant difference was found in CD19+CD24-CD38+B lymphocytes neither between the rituximab and control groups nor between pre-rituximab and post-rituximab patients. Conclusion The number of regulatory B cells decreased in both standard treatment and rituximab-receiving PMN patients and the proportion of naïve/mature B-lymphocytes was lower in the former group. Moreover, the memory B cells count did not reduce significantly two months after rituximab administration. Hence, it might be the best choice to target the memory B cell subset in immunosuppressive therapy while avoiding the Breg or naïve/mature B lymphocyte depletion to obtain more favorable sustained outcomes.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

The Prognostic Significance of Blast Aberrancies By Flow Cytometry in Low/Int Risk MDS

Background: The myelodysplastic syndromes (MDS) represent heterogeneous disorders with varied clinical courses. The major prognostic tool in MDS is the IPSS-R, which helps estimate survival outcome and estimate risk for AML transformation. In low or intermediate risk pts, the IPSS-R has shortcomings; in these pts, the development of transfusion dependent anemia is the major disease associated complication, and this is not addressed by IPSS-R stratification. Previous studies have indicated that aberrancies detected by flow cytometry can risk stratify pts with MDS. The purpose of this study was to determine whether detection of neoplastic-specific blast aberrancies in pts diagnosed with low or intermediate risk MDS can identify pts at higher risk for transfusion dependent anemia after MDS diagnosis. Methods: We performed a retrospective chart review on MDS patients initially diagnosed at our institution between 1/2010 and 12/31/2017. Patients with low/intermediate risk by IPSS-R were identified. Flow cytometry findings on initial diagnostic BM biopsies performed at our institution only were reviewed. Flow cytometry (4- and 8-color) was performed on bone marrow aspirates for the following antigens: CD3, CD7, CD11b, CD13, CD14, CD15, CD19, CD20, CD33, CD34, CD36, CD38, CD45, CD56, CD64, CD117, and HLA-DR using FACS Calibur or FACSCanto II flow cytometers. Myeloblasts were identified by cluster analysis, as previously described (Am J Clin Pathol. 2010 Nov; 134(5):749-61), and compared to 20 control cases. Blast aberrancies were defined as an immunophenotypic difference of &gt; ¼ log compared to the blasts in the controls. Neoplasia-specific blast aberrancies were defined as: expression of CD7, CD11b, CD15, and/or CD56 and/or under expression of CD38 and CD45. We estimated probability of transfusion dependent anemia using Kaplan Meier product limit method and compared survival curves using log-rank test. Analyses were performed using Stata v12.0. Results: A total of 63 patients were identified, with median age of 68 years (range 31-89 years). Median hemoglobin (Hg) at diagnosis was 9.8 (range 5.2-15.3). Cytogenetic risk categories were very good, good, intermediate and poor in 3%, 71% 16%, and 10% respectively. IPSS-R category was very low or low in 70% (44 pts), and intermediate in 30% (19 pts). The presence of blast aberrancies was similar in proportion among low risk patients (61%, n=27) compared to intermediate risk patients (68%, n=13). Overall, the presence of only one blast aberrancy, whether neoplasia-specific or not, did not significantly segregate patients at greater risk for transfusion dependence. However, the presence of 2 or more aberrancies statistically defined two populations. Those possessing 0-1 blast aberrancy did not reach a median time to transfusion dependence, whereas those possessing 2+ aberrancies had a median time to transfusion dependence of 1.2 years (p=0.02). Additionally, when looking at neoplasia-specific blast aberrancies, pts with 0-1 aberrancy had a median time to transfusion of 4.7 years, compared to 2+ aberrancies, at 0.8 years (p=0.02). Figure 1 illustrates this finding. Conclusion: The determination of blast aberrancies by flow at time of MDS diagnosis may provide prognostic information in low/intermediate risk MDS patient and could help predict risk for early red blood cell transfusion dependence. Upfront risk stratification would be valuable information to plan follow-up for these patients, as well as treatment decision making including early initiation of ESAs. Disclosures Michaelis: Novartis: Consultancy; Celgene: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; JAZZ: Other: Data Safety Monitoring Board, uncompensated, Research Funding; BMS: Research Funding; Bioline: Research Funding; ASTEX: Research Funding; Janssen: Research Funding; Millenium: Research Funding; Macrogeneics: Research Funding; Pfizer: Equity Ownership, Research Funding; Incyte: Consultancy, Research Funding. Runaas:Agios: Honoraria; Blueprint Medicine: Honoraria. Atallah:Takeda: Consultancy, Research Funding; Pfizer: Consultancy; Jazz: Consultancy; Helsinn: Consultancy; Jazz: Consultancy; Novartis: Consultancy; Helsinn: Consultancy. Abedin:Actinium Pharmaceuticals: Research Funding; Pfizer Inc: Research Funding; Helsinn Healthcare: Research Funding; Agios: Honoraria; Jazz Pharmaceuticals: Honoraria.

Modulation of host immune status by cryptococcus co-infection during HIV-1 pathogenesis and its impact on CD+4 cell and cytokines environment

BackgroundCryptococcus infection is the second most common opportunistic infection in HIV patients with an increased rate of morbidity and mortality. Altered immune system during HIV- Cryptococcus co-infection is yet to be explored by laboratory. This study evaluates pro- and the anti-inflammatory cytokines in HIV patients with Cryptococcus co-infection and correlate them with CD4+T cell counts as well as viral loads before the initiation of drug therapy. This information would enable to understand host immune modulation and cellular environment during co-infection and understand its impact on HIV pathogenesis. MethodologyThe study comprised four categories of patients with cryptococcosis, HIV, HIV-cryptococcosis co-infected and asymptomatic Healthy volunteers. All the patients and healthy individuals were subjected to CD4+T cells count by FACS using monoclonal antibody cocktail CD4+T cell count (counts per mm3) which was counted using multiSET software on FACS caliber. The viral loads were counted in terms of viral RNA copy numbers which was estimated by real-time PCR using by Artus HIV-1 RG. The sensitivity of kit was >70 IU/ml. ELISA was performed for IL-12 p70, IL-12, IL-4, IL-10, IL-6, TNF-α and IFN-Y using commercially kits (BD Biosciences, USA). Significant variations were assayed by Student's t-test and P values ≤ 0.05 were considered statistically significant. ResultsReduction in CD+4 cell counts was highly significant in HIV patients with or without cryptococcosis. CD4+T cell counts were inversely proportional to viral load. TNF-α levels were raised in cryptococcosis patients significantly higher than healthy individuals. TNF-α was more or less not dependent on viral load but it was more related to the cryptococcosis IL-12 levels were increased in patients with infection and was highest in the HIV infected group. Level of IL-4 was similar in healthy and patients with cryptococcosis but it was elevated in HIV-Crypto co-infected patients. HIV infected patients showed a significant increase in IL-4 level and it was elevated higher in co-infected patients. IL-10 and IL-6 were significantly higher in HIV patients. The fungal infection did not influence the levels of IL-10 in HIV group but IL-6 was low in fungal infected patients. ConclusionThere are very limited studies related to the immune modulation status of HIV co-infected with Cryptococcus before the initiation of any drug therapy. Such information might through in-depth light to understand the initial state of the immune environment which certainly would play a pivotal role in the outcome of the immune modulation.