Thesis (MPH)--University of Limpopo, 2010. / Introduction: Hepatitis B infection is a serious blood-borne disease caused by the hepatitis B virus (HBV) which attacks the liver, and is the leading cause of liver cancer and cirrhosis of the liver. HBV can be transmitted through exposure to infected blood and human secretions through needle stick / sharps injuries and splashes. Thus nurses are at high risk for HBV infection. The aim of the study: To investigate the knowledge, attitudes and practices (KAP) regarding the prevention of hepatitis B virus infections, in final year college student nurses in Gauteng province. Methods: A cross-sectional quantitative survey on 350 final year nursing students was conducted in three Gauteng province nursing colleges, using an anonymous self administered questionnaire with questions on knowledge, attitudes, and practices regarding HBV. The data were analysed using SPSS (statistical package for social science studies). Results: Of 350 questionnaires distributed, 312 student nurses returned completed forms (response rate: 89.14% [312/350]). The majority were females (86.8% [270/331]) and were below 31 years of age (30.1% [93/309]). The majority (87.6% [271/310]) had good knowledge of the causes and prevention of HBV. The unvaccinated respondents had fairly low positive attitudes, with a mean, mode and median score of 1 (possible score from -4 to +4). The majority (79% [244/310]) practiced good compliance with universal precautions of, and the majority (64.9% [202/311]) were vaccinated. College A displayed significantly higher knowledge (p<0.001), positive attitudes (p=0.001) and safer practices (p<0.001) than college B and C.

Thesis (D Phil. (Medical Virology))--2008. / Background and Objectives: South Africa is one of the countries highly affected by human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. Some drugs (e.g. lamivudine) used as part of combination antiretroviral regimens for HIV treatment have dual activity against HBV and HIV. Despite high infection rate with both viruses, routine screening for HBV before initiation of treatment for HIV is not yet a standard practice. This study undertook to investigate: (1) the burden of HBV co-infection in HIV-positive patients enrolling for highly active antiretroviral therapy (HAART) at Dr George Mukhari hospital, (2) the impact of anti-HBV containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, (3) the co-evolution of HBV and HIV drug-resistant strains, and (4) the correlation of HBV genotypes with response to anti-HBV containing HAART regimens. Study Population and Methods: To investigate the burden of HBV/HIV co-infections, a cohort of 192 HIV patients who were candidates for ARV treatment at Dr George Mukhari hospital were studied by screening for HBV serological markers (HBsAg, anti-HBs and anti- HBc) (Elecsys 2010, Roche Diagnostics) and HBV DNA with an in-house nested PCR assay targeting HBV polymerase gene. Quantitation of HBV DNA positive samples was performed with Roche Cobas Taqman HBV test 48 assay. To investigate the impact of lamivudine-containing HAART regimens on HBV during the management of HBV/HIV co-infected patients, as well as the coevolution of HBV and HIV drug-resistant strains, a total of 78 patients were studied. HBV virological response against lamivudine containing-HAART regimens [1a (lamivudine, stavudine and efaverenz); 1b (lamivudine, stavudine and neviripine)] was measured (Cobas Taqman HBV test 48, Roche diagnostics). HBV direct sequencing targeting HBV polymerase gene was performed on all baseline samples (n=78) and additional samples collected at various time points (n = 45). Direct sequencing was also performed on 30 HIV baseline samples targeting the HIV reverse transcriptase and protease genes (Spectru-Medix SCE 2410 Genetic Analysis System and ABI PRISM® 3100 Genetic Analyzer version 3.7). To explore the genetic diversity of HBV and HIV strains circulating in Pretoria and surrounding areas, as well as the correlation of HBV genotypes with response to lamivudine-containing-HAART regimens in co-infected patients, all baseline and follow-up HBV and HIV sequences were analysed, compared and correlated with treatment. Sequence alignments and phylogenetic studies for both HBV and vi HIV were conducted with MAFFT, Mega 4 and neighbour joining phylogenetic trees generated with the PHYLIP programme. Results: Three significant findings were observed in this study. Firstly, the majority of South African HIV patients enrolling for HAART were exposed to HBV infection and either had acute or chronic HBV infections. A total of 63.0% of patients were found to have one or more HBV markers, with 40.6% having detectable HBV DNA as an indication of replication. The study also detected 22.9% with positive HBsAg, and 23% of 77% HBsAg-negative patients having occult hepatitis B infection. Secondly, HBV/HIV co-infected patients do benefit during the management of HIV infections with lamivudine-containing HAART regimens. A total of 68.4% of patients responded to HAART, with undetectable HBV DNA during 18 to 24 months of follow-up. A total of 91.3% of HIV patients also responded to HAART with an undetectable HIV viral load during 6 to 12 months of follow-up. However, a total of 18% of patients had persistent HBV DNA, yielding various HBV virological responses against lamivudine containing-HAART regimens. This proportion of patients poses a question regarding the management of HBV and HIV coinfections, as guidelines on the use of HAART with anti-HBV activity from developed countries, may not necessarily be followed in developing countries. The results further showed that baseline drug-resistance was more frequent with HIV than HBV in this cohort of patients. The following HIV primary drug resistant mutants were observed: nine major NRT's primary mutants, M41L (1/30), E44A (1/30), V75M (1/30), F77L (1/30), V118I (1/30), M184V (1/30), L210S (1/30), T215Y (1/30) and V90I (1/30), and five major NNRT’s primary mutants were also detected, K103N (3/30), Y318CFSY (1/30), E138Q (1/30), P225H (1/30) and K238T. However, all followup samples had undetectable HIV viral load. In contrast to HIV, only one patient was detected with HBV mutant, M204I, at baseline. The mutant reversed to wild type during 6 months and other follow-up (12, 18 and 24 months). Finally, this study indicated that the HBV genotype A is still the most prevalent genotype circulating in South Africa. Of the 78 HBV sequences, 77 were genotype A and 1 sequence was genotype G. This is the first report from Africa of the detection of HBV genotype G. HIV subtype C remains the predominant prevailing subtype in South Africa. HBV genotype or HIV subtype C was not observed to influence any treatment outcome following treatment with vii lamivudine-containing HAART regimens. The study also indicated that patients on lamivudinecontaining HAART regimens do benefit not only by suppressing HIV and HBV viral load, but also improving immunity (i.e. CD4 cells count increases). Conclusion: Overall, the present study highlights the need for screening HBV before initiation of any HAART containing anti-HBV regimens in HBV/HIV co-infected patients. It necessitates the use of molecular assays for effective laboratory in diagnosis of occult HBV infections in HIVpositive patients, especially in developing countries where these assays are not widely available. While lamivudine-containing HAART regimens do benefit both HBV and HIV patients in co-infected individuals, however, whether HBV virological response is temporary or sustained is unknown at this stage. What is certain is that these patients require an effective monitoring programme as (1) a small percentage experience variable HBV virological responses (partial, reactivation, or no response), and (2) hepatitic flares are likely to develop if HAART is terminated (e.g. by patient), or the current HAART regimen is switched to another regimen without anti-HBV activity. HBV genotype A remains the dominant genotype in South Africa, but novel genotypes can be detected. HIV subtype C was found to be the prevalent subtype. HBV genotype or HIV subtype C were not seen to influence any treatment outcome following treatment with lamivudine-containing HAART regimens. Recommendations: HIV patients should be screened for HBV before initiation of anti-HBV containing HAART regimens. The screening of HBV in HIV patients is also important since some drugs included as part of HAART (e.g. nevirapine) may cause hepatotoxicity and exacerbate HBV infections leading to increased morbidity and mortality due to liver complications. Immunization and immune boosters of HIV patients with low (< 10IU/L) or no immunity against HBV should be done as this could be beneficial, although these patients may not respond optimally, or their immunity may wane faster due to immunocompromised status. Monitoring of both HBV and HIV resistant strains should be conducted for timely detection for the occurrence of multiple resistant mutations, which could limit future therapeutic option for both viruses.

Thesis (MPH)--University of Limpopo, 2010. / Introduction: Hepatitis B is a serious liver disease caused by the hepatitis B virus (HBV), with an estimated 360 million chronic infections worldwide, about a million of which die each year from chronic liver diseases. In South Africa (SA) over 50% of the population has been infected by HBV, and at least 3 million people are chronic HBV carriers. Chronic HBV carriers have the potential of transmitting HBV parenterally in the hospital setting, thus health care workers (HCWs) are at risk of contracting HBV, with the most likely exposure being via a needle stick injury (NSI). There is an effective vaccine against HBV which is recommended by the SA Department of Health, yet previous studies have shown that most HCWs are not vaccinated. Aim and objectives: The study aimed to investigate the knowledge, attitudes and practices regarding hepatitis B vaccination amongst HCWs in the Ekurhuleni Metro. Objectives were to determine: (1) the level of knowledge of HCWs about vaccination against HBV; (2) the attitudes of HCWs towards vaccination against HBV; (3) the practices of HCWs regarding HBV prevention and (4) the barriers to / predictors for effective HBV vaccination among HCWs at Ekurhuleni Metro Materials and Methods: This was a cross-sectional descriptive study which made use of a self-administered questionnaire that was sent to Ekurhuleni nurses and doctors who were working in 3 public hospitals, 7 district clinics, and 110 general practices. Results: Two hundred and fifteen questionnaires were distributed and 161 were returned giving an overall response rate of 74.9%. HCWs do not report their NSI; over a third [37.6% (41/81)] always reported the NSI; while 72% (116/161) of HCWs had been vaccinated, only 61.2% (71/116) of those vaccinated had received all 3 doses of the vaccine. For knowledge of HBV vaccination, 66.5% (107/161) scored poor; 31.7% (51/161) scored moderate; and 1.8% (3/161) scored high. For attitudes towards HBV vaccination, 0.6% (1/160) scored negative; 24.4% (39/160) scored neutral; and 74.5% (120/160) scored positive. A positive attitude score was a significant predictor for being vaccinated (OR=1.13, p=0.007) Conclusion: Guidelines should be put in place to increase vaccination uptake and reduce the risk of exposure to HBV infection by HCWs

No description available.

Reversible protein phosphorylation is an essential component of metazoan signaling and cardiovascular physiology. Protein kinase activity is required for regulation of cardiac ion channel and membrane receptor function, metabolism, and transcription, and aberrant kinase function is widely observed across disparate cardiac pathologies. In fact, multiple generations of cardiac therapies (eg. beta-adrenergic receptor blockers) have targeted cardiac kinase regulatory cascades. In contrast, essentially nothing is known regarding the mechanisms that regulate cardiac phosphatase activity at baseline or in cardiovascular disease. Protein phosphatase 2A (PP2A) is a key phosphatase with multiple roles in cardiac physiology. Here we demonstrate the surprisingly complex regulatory platforms that control PP2A holoenzyme activity in heart. We present the first full characterization of the expression and regulation of the PP2A family of polypeptides in heart. We identify the expression of seventeen different PP2A genes in human heart and define their differential expression and distribution across species and in different cardiac chambers. We show unique subcellular distributions of PP2A regulatory subunits in myocytes, strongly implicating the regulatory subunit in conferring PP2A target specificity in vivo. We report striking differential regulation of PP2A scaffolding, regulatory, and catalytic subunit expression in multiple models of cardiovascular disease as well as in human heart failure samples. Importantly, we demonstrate that PP2A regulation in disease extends far beyond expression and subcellular location, by identifying and describing differential post-translational modifications of the PP2A holoenzyme in human heart failure. Furthermore, we go to characterize a mechanism for this method of post-translational modification that may represent a pathway capable of being therapeutically manipulated in human heart failure. Lastly we provide evidence that dysregulation of phosphatase activity contributes to the cellular pathology associated with a previously described inheritable human arrhythmia syndrome, highlighting the importance of the PP2A in cardiovascular physiology and disease. Together, our findings provide new insight into the functional complexity of PP2A expression, activity, and regulation in heart and in human cardiovascular disease and identify potentially new and specific gene and subcellular targets for the treatment of human arrhythmia and heart failure.

In this work we wanted to investigate the role of NFATc1 in lymphocyte physiology and in pathological conditions (eg. psoriasis). NFATc1 is part of the signal transduction pathways that regulates B cells activation and function. NFATc1 has different isoforms that are due to different promoters (P1 and P2), polyadenylation and alternative splicing. Moreover, we tried to elucidate the points of interactions between the NFAT and the NF-κB pathways in activated B-cell fate. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA binding domain. We used mice which over-express a constitutive active version of NFATc1/α in their B cells with -or without- an ablated IRF4. IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt’s lymphoma cells and induces terminal differentiation toward plasma cells. Our experiments showed that a ‘double hit’ in factors affecting B cell activation (NFATc1 in this case) and late B cell Differentiation (IRF4 in this case) alter the development of the B cells, lead to increase in their numbers and increase in stimulation induced proliferation. Therefore, the overall picture indicates a link between these 2 genes and probable carcinogenic alterations that may occur in B cells. We also show that in splenic B cells, c-Rel (of the NF-κB canonical pathway) Support the induction of NFATc1/αA through BCR signals. We also found evidence that the lack of NFATc1 affects the expression of Rel-B (of the NF-κB non-canonical pathway). These data suggest a tight interplay between NFATc1 and NF-κB in B cells, influencing the competence of B cells and their functions in peripheral tissues. We also used IMQ-induced psoriasis-like inflammation on mice which either lack NFATc1 from B cell. Psoriasis is a systemic chronic immunological disease characterized primarily by abnormal accelerated proliferation of the skin keratinocytes. In psoriasis, the precipitating event leads to immune cell activation. Our experiments showed that NFATc1 is needed for the development of psoriasis. It also showed that IL-10 is the link that enables NFAT from altering the B cell compartment (eg Bregs) in order to affect inflammation. The important role of B cell in psoriasis is supported by the flared up psoriasis-like inflammation in mice that lack B cells. Bregs is a special type of B cells that regulate other B cells and T cells; tuning the immunological response through immunomodulatory cytokines. / Diese Arbeit befasst sich mit der Regulation und der Funktion des Transkriptionsfaktors NFATc1 (“nuclear factor of activated T-cells c1) in B-Lymphozyten. Hierzu wurde zum einen die transkriptionelle Kontrolle des Nfatc1-Gens in aktivierten B-Lymphozyten und zum anderen die Bedeutung dieses Faktors für die Wachstumskontrolle und Autoimmunität anhand verschiedener Modellsysteme analysiert. Sechs verschiedene NFATc1-Isoformen können in B-Lymphozyten durch die Nutzung zweier verschiedener Promotoren, zweier Polyadenylierungsstellen und eines alternativen Splicings generiert werden. Wir zeigen hier, dass insbesondere die NF-kB Faktoren c-Rel und p50 eine essentielle Bedeutung für die starke Induktion des Promoters P1 und damit der Expression der kurzen Isoform NFATc1A in B-Zell-Rezeptor-stimulierten B-Zellen spielen. Interessanterweise zeigen NFATc1-defiziente B-Lymphozyten eine geschwächte Aktivierung der NF-kB-Faktoren, was auf eine enge Verknüpfung dieser zwei Signalwege hindeutet. NFATc1-defiziente B-Lymphozyten weisen eine Aktivierungs- und Wachstumsschwäche auf (Bhattacharyya S., et.al.). Hier zeigen wir, dass die Überexpression von konstitutiv aktivem NFATc1A in B-Lymphozyten, insbesondere wenn dies im Kontext einer IRF4-Defizienz geschieht, zu einer verstärkten Expansion der B-Zellpopulation, insbesondere nach deren Aktivierung, führt. Dies belegt die kritische Bedeutung, die der wohldosierten Expressions- und Aktivierungskontrolle der NFATc1-Faktoren in B-Lymphozyten zukommt. Dies zeigt sich auch in einem Imiquimod-induziertem Psoriasis Mausmodell. Hier wird durch Applikation von Imiquimod auf die Haut eine der Schuppenflechte ähnelnde entzündliche Reaktion ausgelöst, die insbesondere durch eine stark verstärkte Proliferation der Keratinozyten gekennzeichnet ist. Wir können zeigen, dass die NFATc1-Faktoren in B-Lymphozyten kritisch an dieser Reaktion beteiligt sind. Fehlt den B-Lymphozyten das NFATc1-Gen, so produziert eine Subpopulation, die sogenannten regulatorischen B-Zellen, verstärkt das immunmodulatorischen Zytokins IL-10, wodurch die entzündliche Reaktion fast komplett unterdrückt wird. Dies ähnelt vorhergehenden Beobachtungen, in denen wir zeigen konnten, dass auch in einem Mausmodell der Multiplen Sklerose (EAE) die Immunreaktion durch den Verlust von NFATc1 in B-Zellen erheblich gelindert werden kann (Bhattacharyya S., et.al.).

Myocardial B-cell infiltration after LAD occlusion in mice is driven by CXCL13 After myocardial infarction, the immune system is activated and regulates wound healing and remodeling processes in the heart. While the role of T cells has been elucidated already, the function of B cells in myocardial infarction remained relatively unclear until now. It is, however, already known that B cells are of importance in healing processes in other tissues, for example in the skin. Our studies therefore addressed the role and function of B cells in healing and early remodeling processes in the myocardium after infarction. Under physiological conditions, only few B cells can be found in the heart. After myocardial infarction, however, which we modelled with a permanent ligation of the left anterior descending artery (LAD) in C57BL/6J mice, we could demonstrate that B lymphocytes accumulate in the early phase after tissue injury (days one to seven) in the myocardium. To detect B cells, we performed immunofluorescence stainings on cryosections of infarcted hearts using an anti-B220 antibody. Quantitative analysis of tissue infiltration revealed that B cells peaked at day seven. In flow cytometry, we further characterized the B cells infiltrating infarcted tissue. We found that most of them were mature B cells (IgM+, IgD+). Next, we wanted to outline a potential mechanism responsible for B-cell infiltration to the site of tissue injury. We therefore performed ELISA experiments revealing that CXCL13 was upregulated in scar tissue. Antibody-mediated neutralization of CXCL13 verifiably attenuated B-cell infiltration. Treated mice also showed – in the tendency – smaller infarct sizes and an improved survival. In conclusion, we could show that B lymphocytes infiltrate the myocardium after MI in mice following a local CXCL13 gradient and that it is, most likely, beneficial to inhibit this process. / Nach einem Herzinfarkt wird das Immunsystem aktiviert und bestimmt die Wundheilung sowie das Remodeling im Herzen. Während die Rolle von T-Zellen [dabei] bereits relativ gut untersucht ist, war die Funktion von B-Zellen beim Myokardinfarkt bis heute relativ unklar. Man weiß bisher allerdings, dass B-Zellen eine wichtige Rolle in Heilungsprozessen in anderen Geweben spielen, z.B. in der Haut. Unsere Untersuchungen beschäftigten sich daher mit der Rolle und Funktion von B-Zellen nach experimentellem Herzinfarkt im Mausmodell. Unter physiologischen Bedingungen finden sich nur wenige B-Zellen im Herzen. Nach Herzinfarkt, den wir mithilfe einer permanenten Ligatur der linken Herzkranzarterie in C57BL/6J-Mäusen induzierten, konnten wir jedoch zeigen, dass B-Zellen in der frühen Phase nach Gewebeschädigung (Tag eins bis sieben) im Myokard akkumulieren. Um B-Zellen zu detektieren, führten wir Immunfluoreszenz-Färbungen mit einem monoklonalen Anti-B220-Antikörper auf Gefrierschnitten des Herzens durch. Eine quantitative Auswertung der Gewebeinfiltration ergab, dass die Anzahl der B-Zellen an Tag sieben ihren Höhepunkt erreicht. In durchflusszytometrischen Untersuchungen charakterisierten wir [anschließend] die das infarzierte Gewebe infiltrierenden B-Zellen (weiter). Wir stellten dabei fest, dass es sich bei den meisten dieser Zellen um reife B-Zellen (IgM+, IgD+) handelte. Als nächsten Schritt wollten wir einen möglichen Mechanismus, der die B-Zell-Infiltration am Ort der Gewebeschädigung erklärt, umreißen. Wir führten daher ELISA-Messungen durch, die ergaben, dass CXCL13 im Narbengewebe hochreguliert war. Eine Antikörper-vermittelte Neutralisation von CXCL13 konnte nachweisbar die B-Zell-Infiltration hemmen. So behandelte Mäuse zeigten – in der Tendenz – kleinere Infarktgrößen und ein verbessertes Überleben. Zusammenfassend konnten wir somit zeigen, dass B-Lymphozyten nach Myokardinfarkt in der Maus das Myokard aufgrund eines lokalen CXCL13-Gradienten infiltrieren und dass es höchstwahrscheinlich vorteilhaft ist, diesen Prozess zu unterbinden.

No description available.

The transcription factor Pax5 is essential for commitment to the B lineage as the development of these cells is arrested at an early stage in the bone marrow of Pax5 deficient mice. Pax5 deficient pro-B cells display remarkable plasticity and are able to differentiate into other cell lineages both in vitro and in vivo. Several Pax5 target genes have been previously reported but none are able to explain the developmental block observed at the early to late pro-B cell stage. To determine the exact mechanisms by which Pax5 controls B cell development, I have undertaken a cDNA microarray screen with a custom generated B cell-specific cDNA library. By identifying genes that are differentially expressed between Pax5 deficient and wild type pro-B cells, I have identified a number of potential Pax5 target genes. The microarray screen identified lymphoid-restricted membrane protein (Lrmp or Jaw1) as a novel Pax5 activated transcript. Using RT-PCR and a Pax5-estrogen receptor inducible system, I confirmed that Jaw1 is a direct Pax5 target gene whose expression is confined, in resting cells, to the earliest stages of B and T cell development. The biological relevance of Jaw1 for cell fate specification has been tested by transducing bone marrow progenitor cells with murine retroviral vectors and reconstituting haemopoiesis in vivo. I have reported that over-expression of Jaw1 in these cells results in a decrease in the development of B and NK lymphocytes and a marked increase in the development of myeloid cells in the bone marrow of reconstituted mice. This result suggests that Jaw1 may play an important role in the early development of lymphocytes in the bone marrow. Whilst initial analysis of Jaw1 deficient mice has revealed no overt defects in lymphopoiesis, I postulate that Jaw1 is involved in IP3-induced calcium signaling downstream of the pre-BCR and BCR. This hypothesis has resulted from analysis of the function of IRAG, the only known Jaw1 homologue combined with data that Jaw1 is expressed in early B cells in the BM as well as in germinal centers in the spleen. Pax5 mutant mice usually die before weaning and the cause of death is currently unknown, suggesting that Pax5 is expressed in a previously unreported tissue. To investigate this hypothesis I produced a monoclonal antibody to Pax5 and screened for novel expression domains during embryonic development and also in neonate mice. These studies did not detect any new Pax5 expression domains, but did reveal that this antibody cross-reacts with Pax2 and/or Pax8 in the developing kidney and brain. Biochemical analysis of the serum from Pax5 deficient mice also did not reveal the likely cause of death in these animals, beyond the general signs of dehydration and starvation that are likely to be secondary to the underlying defect. / Doctor of Philosophy (PhD)

Although Australian epidemiology of HBV and HCV has been well described for populations groups at higher risk, but the information available for groups generally considered to be lower risk is much more limited. An understanding of the prevalence of these infections and their risk factors in antenatal women is important to guide testing policy and practice. A study was therefore conducted of the epidemiology of hepatitis B and hepatitis C infection in women. In addition, women were asked about their experience with antenatal testing. A total of 516 women participated in the survey, of these 479 (95%) women had been tested for HCV antibodies .The prevalence of HCV antibodies was 4% overall, and 2% among women who were unaware of their HCV status prior to their antenatal test. A history of injecting drug use and residing with a HCV positive person were significantly associated with HCV infection in multivariate analyses. HBV testing was conducted in 468 (99.6%) of women, and the overall prevalence was 2%. Risk factors identified were birthplace in countries of South East Asia. Women were asked about their perception of antenatal testing and pre-test information. Nearly a third (143, 30.5%) of women who had been tested for HCV infection either said that they did not know whether they had been tested, or said that they had declined testing. The corresponding proportion for HBV infection was 28.8% (135). Over 65% and 66% of women said that had not received any information about testing for HCV and HBV respectively. The finding that virtually all antenatal women were being tested for HCV was in contrast to government and non-government organisation policies of ???selective??? screening in place during the study period. Of concern was the substantial proportion of women who were tested despite reporting that they had declined their clinician???s offer to test for HCV and HBV, and the large number of women who reported an absence of pre-test information. Women who said they had received information reported the delivery and quality of information varied according to the antenatal clinician group, but perceived the overall quality as poor.