LABRAD : Vol 40, Issue 2 - November 2014

Significance of HLA Typing in Transplant Medicine Role of Protocol Renal Biopsies in Transplant Patients Blood Product Utilization in Haematopoietic Stem Cell Transplant Recipients Use of Magic Marker “C4d” in the Diagnosis of Acute Antibody Mediated Rejection in Renal Transplant Patients Coagulopathy in Renal Transplantation Therapeutic Drug Monitoring of Cyclosporine Signifi cance of CMV antigenemia Assay In Renal Transplant Patients Pretransplant Serological Evaluation Importance of Monitoring Cytomegalovirus (CMV) and BKV (Polyomavirus) Infection in Renal Transplant Patientshttps://ecommons.aku.edu/labrad/1003/thumbnail.jp

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Occurrence of Hepatotrophic Viral Infections among patients in Tertiary Care Center.

INTRODUCTION : Hepatic abnormalities are quite common among HIV infected persons. Thousands of people with HIV are also infected, or at risk of being infected with one of the several hepatitis viruses. Some of these viruses can cause chronic infection which may result in cirrhosis, liver failure, and hepatocellular carcinoma in a proportion of patients. Apart from being more prevalent, the natural history of these hepatitis viruses may be modified in patients with HIV infection and hence their significance3. Also it has been claimed that certain hepatitis virus infections may exacerbate the course of HIV infection, with rapid progression to AIDS, although this observation has not yet been confirmed. Viral hepatitis is not so much an opportunistic infection in patients with HIV infection, as it is companion infection. Thus because some forms of hepatitis are acquired through sexual spread, or parenteral contact with blood or blood products in the same way that HIV is, hepatitis and HIV infections often coexist. The enteral forms of hepatitis (HAV and HEV) are not strongly associated with HIV infection, whereas HBV, HDV, HCV and HGV are strongly associated because of shared routes of transmission. So screening of these hepatitis viral infections becomes mandatory in HIV positive patients. AIMS AND OBJECTIVES : • To study the serological status of hepatotrophic viral infections like HBV and HCV among HIV positive and HIV negative patients attending Voluntary Confidential Counselling and Testing Centre (VCCTC), Government General Hospital, Chennai. • To compare the progression of Hepatitis B virus infection among HIV positive and HIV negative study groups. • To compare the levels of anti HBs, among HIV positive and HIV negative study groups with hepatitis B virus infection, during follow up. • To detect the prevalence of HGV RNA using the RT-PCR among HIV positives with coexistent HBsAg and/ or anti HCV. • To study the clinical stage and immunological status of patients with HIV infection at presentation, and their correlation with hepatotrophic viral infections. MATERIALS AND METHODS : This study was conducted in serologically confirmed HIV positive and negative patients attending VCCTC, Government General Hospital, Chennai. Pre and post test counselling were given to the patients attending VCCTC, and informed consent was got from them for testing. Strict confidentiality regarding the results were maintained. For ELISA, blood samples were collected from the patients. Serum was separated, and stored at -20°C in cold storage, until use. For all the ELISAs performed, the manufacturer’s testing protocol instructions were strictly followed and results were interpreted only when the validity criteria were satisfied. RESULTS : In the present study 500 HIV positive and 300 HIV negative patients attending VCCTC, Government General Hospital, Chennai were screened for the coexistence of hepatotrophic virus infections. Majority of the HIV positives were in the sexually active age group of 30 – 39 years ( 52%), with males being more than females in the ratio of 1.9: 1. Prevalence of Hepatitis B surface antigen was more both among HIV positives and negatives, when compared to anti HCV antibodies. The persistence of Hepatitis B surface antigen after 6 months was three times more among the HIV positives, when compared to the HIV negative study group. The presence of HBeAg which indicates active replication was also more among HIV positives when compared to negative study group. HIV negative patients had higher antibody levels, when compared to HIV positive study group. Among the 5 patients who had chronic HBV infection (Table III), 2 had coexistent anti-HBs in their serum. Heterosexual route was the major mode of transmission of HIV. The heterosexuals also included the spouse of infected men. Heterosexual route was the major mode of transmission of HBV. HCV was more prevalent among IVDUs. Among the total 16 IVDUs (Table II), 2 (12.5% ) were positive for both HBsAg and Anti HCV. Majority of the HIV positives without hepatotrophic viral infections were in Clinical Stage II Category B of HIV infection. Majority of the patients who were positive for hepatitis viral markers were in Clinical Stage II Category C of HIV infection CONCLUSION : HGV infection occurs with high frequency in patients with HIV and HCV combined infections (35%), signifying the common modes of transmission of HGV and HCV. Among the HIV positive study group, HBsAg positives with HGV co-infection had higher mean CD4 counts, when compared to HBsAg positives without HGV co-infection. There was no significant difference observed between the HCV positives with or without HGV co-infection. This implies that the presence of HGV RNA is associated with milder HIV status in individuals with HIV and HBV combined infection. Among the HIV positive study group, HGV positive patients with hepatitis B and / or hepatitis C co-infections had higher mean ALT levels, when compared to HGV negative individuals. This implies that, presence of HGV RNA was associated with severe hepatitis in patients with HIV and HBV or HCV combined infections. The present study throws light upon the co existence of HIV with Hepatotrophic viruses in a tertiary care center like ours. Only very few studies have been performed in India analyzing the coexistence of HGV with HIV. Our study can be considered as pioneer study in this context. The results aid the central HIV programme implementing agencies, especially in the field of ART to give importance to hepatitis G virus, along with hepatitis B and hepatitis C viral infections

Impaired response to HBV vaccination in HIV-1 infected children : immunopathological mechanisms

HBV vaccination prevents HBV infection and related liver cancer. Immunological dysfunctions of Tfh and B cells in HIV-1 infected individuals may affect the response to HBV vaccine. The general objective of this thesis was to elucidate HBV vaccine response in HIV-1 infected children receiving ART, to assess functional and phenotypic properties of pTfh cells in HBV vaccinated children and to study whether HBV vaccination may have a role in reducing the size of HIV-1 reservoirs. In paper I, we showed reduced frequencies of pTfh cells in HIV-1 infected children compared to healthy controls and of pTfh cells expressing the co-stimulatory molecules ICOS and PD1, important to mediate the interaction of Tfh cells with B cells. The frequency of IL-4 expressing pTfh cells and of resting memory B cells was also lower in infected children; on the contrary, an expansion of exhausted memory B cells was detected in this group. In paper II, all children who received HBV vaccination, except four, displayed a strong vaccine response at 1 month post-vaccination. Lower plasma levels of anti-HBs antibodies (Abs) were measured in HIV-1 infected children compared to controls at 1 month and 6 months post-vaccination. HIV-1 infected children had elevated plasma CXCL13 levels compared to controls at all time points; changes in plasma CXCL13 concentration were however not observed following vaccination. As the functional and phenotypic properties of pTfh cells were similar in both groups pre- and postvaccination, alterations in pTfh properties could not explain the reduced vaccine response in HIV-1 infected children. In a yet unpublished study, we showed an altered frequency of B cell subsets in HIV-1 infected children which correlated with anti-HBs Ab titers after vaccination. In paper III, the number of HIV-1 DNA copies in PBMCs was unchanged after vaccination with a combined HBV and HAV vaccine in HIV-1 infected children; however, 54% of these individuals showed a decline in the size of HIV-1 DNA reservoir after vaccination. The change was most likely related to vaccination since the children were on ART for a median of 7.2 years and had therefore likely reached a plateau phase for HIV-1 DNA decay after ART initiation. EM CD8+ T cells were the stronger predictors of the change in HIV-1 DNA copies using multivariate analysis. In conclusion, three doses of accelerated HBV vaccination induced high anti-HBs Abs in both HIV-1 infected and control children. A rapid decline of anti-HBs Abs in plasma after 6 months from vaccination suggests the need of an additional booster dose for HBV vaccine. The role of HBV vaccination in reducing HIV-1 DNA reservoirs should be investigated further

Prevention from transfusion transmissive diseases in the regional center for transfusion medicine in Stip, Republic of Macedonia for the period 2009-2010

Introduction: Blood transfusion is a transplantation of fluid tissue or an introduction of human biological material that needs to survive in the donor organism and to play important biological functions. During the blood and blood products transfusion, it is possible to transmit many transfusion transmissive diseases, which increases the need of securing safe blood transfusion. Objective: To present the procedures and measures taken in order to prevent the transmission of transfusion transmissive diseases in the blood and blood products donors at the Clinical Hospital in Stip. Materials and methods: Each blood unit was mandatory tested for HBSAG, anti- HCV, anti-HIV and Treponema pallidum antibodies at the Regional center for transfusion medicine. The testing was done with the ELISA technique by using the Dade Berhing BEP 200 instrument and the tests from Siemens and Ortho for anti-HCV. The confirmation tests were done at the Institute of Transfusion Medicine in the capital Skopje. Results: In total, 6067 blood samples were tested. The presence of HBAGS was detected in 81 sample (1.33%), anti-HCV in 19 (0.313%), anti-HIV in one (0,016%) and Treponema pallidum antibodies in 5 samples (0.082%). Discussion and conclusion: In order to achieve high level of security of the transfusion blood and blood products it is essential to use highly specific and sensitive tests, modern equipment, well trained health personnel and sufficient financial resources allocated specifically for that aim

Correlation of heavy metals and theirs impact to epidemiological survey in the miners blood donors and other human population

Introduction: Miners who are blood donors, and work in mines for lead-zinc ores are constantly exposed to heavy metals (lead, zinc and cadmium) and this aspect is expected to increase or decrease many hematological parameters. Aim of the Study: The concentration of lead, zinc and cadmium was studied in exposed blood donors and non-exposed blood donors (control group). Knowing the structure of various heavy metals, all of the analysis was carried out to examine the impact of these heavy metals on the occurrence and severity of certain epidemiological diseases and hematological parameters on the miners who are blood donors. Material and Methods: In this research 120 miners were included who were blood donors (mining for lead and zinc) from the Republic of Macedonia and a control group of 30 participants that included blood donors not directly exposed to heavy metals, while living in the immediate vicinity of the lead and zinc mine. In this research biochemical analysis (inductively coupled plasma spectrometry (ICP) one of the most sensitive analytical techniques for the determination of elements in biological materials was applied and the basic haematological parameters were determined. Results: The observation of increased blood lead level on blood donors in the exposed group (mean = 0.089 mg/l) and 20% on blood donors in the control group (mean = 0066), increased blood zinc level in the exposed (mean = 1391) and in the control group (mean = 1074), increased blood cadmium level in 62% of exposed (mean = 0007) and in 50% of the control group (mean = 0006); If the normal BLL (blood lead level) is 0.04–0.07 mg/l, we concluded that all male blood donors in the exposed group had above normal BLL. In the control group 20% of male blood donors had above normal BLL; if the normal BZL (blood zinc level) is 0.1 mg/l, we concluded that all male blood donors exposed in the control group had above normal BZL. If the normal BCL is 0.005 mg/l, we concluded that 62% of the male blood donors in the exposed group had above normal BCL. In the control group 50% of male blood donors had above normal BCL; The blood lead, zinc and cadmium level will rise during exposure at work. forty eight percent of miners (exposed group) had an exposure period of 20 years, 29% between 10 and 20 years and the remaining 23% an exposure period under 10 years. Results showed negative correlation between the number of red blood cells and hemoglobin and blood levels of heavy metals; positive correlation between the number of leukocytes and blood heavy metals levels. Epidemiological survey showed that nearly all workers complained of headache. While 25 of 70 miners who were blood donors (with long exposure) were found to be suffering from various diseases such as asthma, respiratory tract, irritation and watering of eyes. Conclusion: The research confirms that the increased content of heavy metals in blood donors affects the concept of professional risk that involves probability that as a result of exposure of workers to certain harmful agents in the work environment negative effects are manifested on their health. The change of some haematological parameters in the blood donors, results in the emergence of certain diseases with complex etiologies and risks to their health

NAFLD and HBV interplay - related mechanisms underlying liver disease progression

Non-alcoholic fatty liver disease (NAFLD) and Hepatitis B virus infection (HBV) constitute common chronic liver diseases with worldwide distribution. NAFLD burden is expected to grow in the coming decade, especially in western countries, considering the increased incidence of diabetes and obesity. Despite the organized HBV vaccinations and use of anti-viral therapies globally, HBV infection remains endemic and challenging public health issue. As both NAFLD and HBV have been associated with the development of progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC), the co-occurrence of both diseases has gained great research and clinical interest. The causative relationship between NAFLD and HBV infection has not been elucidated so far. Dysregulated fatty acid metabolism and lipotoxicity in NAFLD disease seems to initiate activation of signaling pathways that enhance pro-inflammatory responses and disrupt hepatocyte cell homeostasis, promoting progression of NAFLD disease to NASH, fibrosis and HCC and can affect HBV replication and immune encountering of HBV virus, which may further have impact on liver disease progression. Chronic HBV infection is suggested to have an influence on metabolic changes, which could lead to NAFLD development and the HBV-induced inflammatory responses and molecular pathways may constitute an aggravating factor in hepatic steatosis development. The observed altered immune homeostasis in both HBV infection and NAFLD could be associated with progression to HCC development. Elucidation of the possible mechanisms beyond HBV chronic infection and NAFLD diseases, which could lead to advanced liver disease or increase the risk for severe complications, in the case of HBV-NAFLD co-existence is of high clinical significance in the context of designing effective therapeutic targets

Effects of human immunodeficiency virus infection and treatment with antiretroviral therapy on immunological responses to childhood vaccines

Original published work submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctorate of Philosophy in Virology. Johannesburg 2017.Introduction: HIV-infected and HIV-exposed-uninfected children have a heightened susceptibility to some vaccine preventable disease. There is a paucity of data on immunogenicity of vaccines in these children, including HIV-infected children who are initiated on early antiretroviral therapy (ART). We evaluated the effect of maternal HIV-exposure and timing of ART in HIV-infected children on antibody responses to combined diphtheria-toxoid (DT) -tetanus-toxoid (TT)-whole cell pertussis (wP) and Haemophilus influenzae type b conjugate vaccine (HibCV); monovalent hepatitis B vaccine (HepB) and live-attenuated measles vaccine (MV). Methods: Samples obtained from children aged 6–12 weeks who had been enrolled into the CIPRA-SA study were analysed. Briefly, HIV-uninfected children born to HIV-uninfected (HIV-unexposed) and HIV-infected mothers (HEU). Additionally, we enrolled perinatally HIV-infected children with CD4+%≥25% randomized to deferred-ART (i.e. initiated when clinically or immunologically indicated per the then WHO recommended treatment criteria; ART-Def) or immediate-ART initiation (i.e. initiated on ART immediately upon confirmation of HIV-infection status at 4-10 weeks of age; ART-Immed). Children enrolled in the ART-Immed arm were further randomized to interrupt ART at one-year (ART/12m) or two-years of age (ART/24m). Additionally, a convenience sample of HIV-infected children with CD4+<25% initiated on immediate-ART was enrolled (ART-CD4+<25%). Children received a primary series of DTwP-HibCV/HepB at 6, 10 and 14 weeks of age; and MV at 40 weeks of age. Booster dose of DTwP and MV was given at 15-18 months of age. Sampling time-points were: prior to the first dose of vaccine, four weeks after the third dose (18 weeks age), 24 weeks after the third dose (39.3 weeks of age), at the time of the booster dose (15- 18 months age), two to four weeks after the booster dose and at 24 months of age. Samples were analysed for antibodies for DT, TT, PT, FHA, HepB measured by Luminex microbead-immunoassay; and MV antibodies were quantified by an indirect enzyme immunoassay. Results: Antibody kinetics and response to primary series of DTwP-HibCV/HepB: Pre-vaccination GMCs were higher in HIV-unexposed than HEU children for TT, but lower for HepB, DT and FHA. Post-vaccination, sero-conversion, sero-protection and GMCs were similar in HEU and HIV-unexposed children for all vaccines. Furthermore, GMCs were higher in HIV-unexposed for TT, DT, HepB and FHA than in ART-Immed children; and for TT, HepB and PT than in ART-Def children. Nevertheless, there was no difference in proportion of HIV-unexposed and HIV-infected children who developed sero-protective vaccine-specific antibody levels post-vaccination. The timing of ART initiation generally did not affect immune responses to vaccines between HIV-infected groups. Antibody kinetics and booster responses to DTwP-HibCV/HepB vaccines: Pre-booster GMCs were generally higher in HIV-unexposed than HIV-infected children for all vaccine epitopes. Post-booster and at 24 months of age the ART-Def group had lower GMCs (except to FHA), and were less likely to have sero-protective antibody levels compared to HIV-unexposed group. Also, post-booster and at 24 months of age, GMC were generally higher in HIV-unexposed than ART-Immed children, and a higher percentage of HIV-unexposed than ART-Immed children maintained antibody levels ≥1IU/ml to TT and DT at 24 months of age. The GMCs and percentage of children with sero-protective thresholds were similar pre-booster and at 24 months of age between HIV-unexposed and HEU children. Antibody kinetics and response to measles virus vaccine: At 7.3 weeks of age, the proportion with sero-protective titers was higher in HIV-unexposed (65.2%) compared to any HIV-infected group (range: 16.7% to 41.8%); but dropped to <17% in all Groups at age 19.6 weeks. Twenty-eight weeks following the first measles-vaccine, ART/12m were less likely to have sero-protective titers (79.3%) compared to HIV-unexposed (94.8%; p<0.001), ART-Def (95.7%; p=0.003) or ART/24m (92.1%; p=0.02). Although the proportion with sero-protective levels were similar between groups immediately post-booster dose, this was lower in HEU (79.6%; p=0.002) and ART/12m (80.3%; p=0.01) compared to HIV-unexposed (94.3%) 41-weeks later. Conclusion: Primary vaccination with DTwP-HibCV/HBV of HIV-infected children initiated on early-ART confers similar immunity compared to HIV-unexposed children. HIV-infected children had poor anamnestic responses, if ART was not initiated prior to primary vaccination. In contrast, the memory response and persistence of antibody to most vaccine epitopes were similar between HIV-unexposed and HEU children. Increased waning of vaccine induced immunity over a 24 month period in ART-Def, ART/12m and HEU children following MV booster-dose; indicating the need for further booster doses after two-years of age in these children. I recommend close monitoring of HEU children, as this group makes up most children born to HIV-infected mothers and what facets of the immune system have been impacted by maternal exposure to HIV.MT201

Liver Illness and Psoriatic Patients

Psoriasis is a chronic inflammatory disease of the skin affecting approximately 2% of the world's population. Systemic treatments, including methotrexate and cyclosporin, are associated with potential hepatotoxicity, due to either direct liver damage or immunosuppression or both immunomediated and a direct liver injury; therefore, treatment of patients with psoriasis poses a therapeutic challenge. The aim of this minireview is to help clinicians in the management of psoriatic patients who develop signs of liver dysfunction. To find relevant articles, a comprehensive search was performed on PubMed, EMBASE, and Cochrane with appropriate combinations of the following keywords being considered: Viral hepatitis, nonalcoholic fatty liver disease, psoriasis, hepatotoxicity, drug toxicity, cholestasis, and autoimmune liver diseases