Os vírus da hepatite B (HBV), C (HCV) e Delta (HDV) causam grande impacto para a saúde pública mundial. Noventa e duas, oito e quatro amostras para HBsAg, anti-HD e anti-HCV, respectivamente, foram identificadas em indivíduos no Maranhão. Cinquenta amostras positivas para HBV DNA foram classificadas em subgenótipo D4 (42/86%) e A1 (8/14%). Para o HDV, apenas quatro foram classificadas como HDV-8. As amostras positivas para anti-HCV não apresentaram RNA detectável. O HBV-D4 parece ser o principal vírus representante na região estudada. O estudo filogenético sugere que houve a introdução de uma única cepa do subgenótipo D4 no Maranhão, enquanto que para o subgenótipo A1 existiu introdução de diferentes cepas. A confirmação do achado do HDV-8 em coinfecção com HBV- D4 suporta a hipótese de origem desses vírus na África. A ausência de infecção ativa pelo HCV é provavelmente devido uma introdução recente desse vírus e/ou menor frequência de meios de transmissão eficientes. Mais estudos são necessários em regiões onde é desconhecido o perfil de infecção desses vírus. / Hepatitis B (HBV), C (HCV) and Delta (HDV) viruses cause a great universal public health concern. Ninety-two, eight and four positive individuals for HBsAg, anti-HD and anti-HCV were identified, respectively. Fifty samples for HBV were classified in. subgenotype D4 (42/86%) and A1 (8/14%). Concerning HDV, four samples were identified as HDV-8 genotype. Anti-HCV positive samples were negative for RNA. HBV-D4 seems to be the main representative in the studied region. The phylogenetic tree topology suggests there was the introduction of a single strain of D4 subgenotype in Maranhao, whereas subgenotype A1 had several introductions of different strains. The finding of HDV-8 in coinfection with HBV D4 confirms the hypothesis of origin of these viruses in Africa. The low number of HCV infection in this region may be due to the recent introduction of this virus and / or lower frequency of efficient means of transmission. More studies are necessary in other regions where the infection profile of these viruses is indefinite.

The human hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family of viruses. Various other hepadnaviruses are used as models to study human HBV infections as all Hepadnaviridae family members have similar virus structure and replication strategies. The studies performed and described in this thesis were carried out using duck hepatitis B virus (DHBV) infection of Pekin ducks as a model system. Hepadnavirus infections can have either an acute or a chronic outcome. The factors that contribute to these outcomes include the immune response, the age of the host at the time of infection as well as size of viral inoculum. The overall aim of this project was to gain a detailed understanding of the mechanisms involved in clearance of virus and resolution of acute DHBV infections. As a first step, molecular and immunohistochemical detection methods for a range of cellular markers in ducks had to be developed as assays were not readily available. Quantitative reverse transcription PCR assays (qRT-PCR) were developed for the detection of mRNA of the duck T-lymphocyte markers, CD3, CD4, CD8, duck cytokines, IFN-α, IFN-γ, TNF-α and the duck housekeeping genes, β-actin and GAPDH. Immunohistochemistry was developed for the detection of duck CD4 + and CD8 + on T cells and for the detection of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation. These methods were then widely used throughout the project. The innate immune response during HBV infections is not well understood. Toll-like receptors (TLR) are a family of pattern recognition receptors that form part of the innate immune response and are involved in the recognition of bacterial, fungal and viral pathogens. The only TLR that have been reported to recognise viral pathogens are TLR- 2, TLR-3, TLR-4, TLR-7 and TLR-9. The possible role of TLR during hepadnavirus infections had not been well characterized to date. In this project cDNA sequences for duck TLR-2, TLR-4 and TLR-7 were identified and characterised and qRT-PCR assays were developed for their detection. Changes in duck TLR-2, TLR-4 and TLR-7 mRNA expression during hepadnavirus infection were identified following DHBV infection of primary duck hepatocytes (PDH) in vitro. The results showed increased levels of expression of duck TLR early during infection indicating an involvement of TLR and the innate immune response during DHBV infection. During the in vivo DHBV infection studies performed to date TLR mRNA expression remained unchanged. As previously mentioned hepadnavirus infection can have an acute or chronic outcome. We aimed to understand the mechanisms involved during the resolution of acute DHBV infection and to elucidate specific factors contributing to the successful resolution of infection. During acute infections immune markers were monitored by qRT-PCR and histological analysis of fixed liver sections was performed. Liver sections were analysed to detect liver inflammation, the number and size of Kupffer cells, hepatocyte apoptosis and changes in hepatocyte proliferation throughout the course of acute DHBV infection in 6-week-old ducks. By determining the percentage of DHBV-positive hepatocytes two patterns of clearance of acute DHBV infection were observed; early clearance of infected hepatocytes occurring before day 14 post infection (p.i.), and late clearance occurring after day 14, but before or on day 31 p.i. This viral clearance was seen to occur in a cell by cell pattern. Higher levels of hepatocyte proliferation and apoptotic hepatocytes were detected during the clearance phase (on day 14 p.i.) of the late clearance group. Periodic acid schiff-diastase (PAS-D) staining was used to show significant increases in both cell number and size of Kupffer cells. Levels of IFN-γ mRNA increased significantly over the uninfected age-matched control ducks on day 14 p.i. Levels of CD3, CD4 and CD8 mRNA expression also increased over the uninfected controls on days 14 and 31 p.i. In summary, we found that resolution of acute DHBV infection occurred on a cell by cell pattern of clearance, it was accompanied by increases in hepatocyte proliferation, apoptotic hepatocytes and activated Kupffer cells, indicating that T lymphocytes and cell death play important roles in the rapid clearance of DHBV infection. Following resolution of acute hepadnaviral infections residual viral DNA has been found to persist. Residual HBV DNA in humans can result in reactivation of HBV infection following liver transplantation or immunosuppressive drug treatment. This leads to possible pathogenic outcomes thus the need for further investigations. Previous studies performed in the duck model have shown that the major form of residual DNA is present as covalently closed circular DNA (cccDNA). We aimed to understand how this residual cccDNA was being maintained and if replication was involved in the process. Following resolution of infection in ducks, levels of residual DHBV DNA were monitored by quantitative PCR (qPCR). Ducks were treated with the Bristol-Myers Squibb nucleoside analogue Entecavir (ETV) in order to suppress any possible replication that might be maintaining levels of residual cccDNA. In DHBV-infected but non-ETV treated ducks, the levels of residual DHBV DNA decreased gradually when measured on days 60, 221 and 316 p.i. The observed decrease in residual DHBV DNA occurred in parallel with decreases in the rate of hepatocyte proliferation measured by PCNA staining. This finding suggests that levels of residual DHBV DNA and hepatocyte proliferation are linked and we hypothesise that hepatocyte turnover is involved in the clearance of residual DHBV DNA. ETV treatment did not have an effect on the levels of residual DHBV DNA which suggests that it is present in a subset of long-lived hepatocytes that do not support virus replication. Mathematical modelling was performed to predict the rate of hepatocyte proliferation required for the elimination of residual cccDNA. The mathematical modelling showed that the predicted rate of hepatocyte proliferation was consistent with the rate of hepatocyte proliferation measured by PCNA. Further mathematical modelling showed that residual cccDNA is most likely to survive mitosis and it decreases due to several rounds of hepatocyte proliferation required for its elimination. Altogether, this project has elucidated mechanisms involved during the resolution of acute DHBV infection and also possible mechanisms by which residual DHBV DNA is maintained following resolution of infection. Detailed understanding of the virological and immunological events that occur during the resolution of an acute hepadnavirus infection would assist in the development of new therapeutic treatments for the cure of chronic HBV infections. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345121 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1997. / Typescript. Bibliography: leaves 189-204.

published_or_final_version / Surgery / Master / Master of Philosophy

Hepatitis C virus (HCV) causes severe illness in millions of people worldwide, but the epidemic strains responsible for most infections arose within the past hundred years and represent only a small part of total HCV diversity. In this thesis I combine laboratory and computational methods to study HCV in Africa. I aim to characterize its current genetic diversity and its historical transmission prior to the global HCV epidemic. In Chapter 2 I begin by screening samples from the Democratic Republic of the Congo (DRC) for HCV and the related human pegivirus. I find high HCV sequence diversity, including a putative new subtype, and find significantly higher HCV prevalence in those born before 1950. Chapter 3 continues this screening, and combines the sequences obtained with those from online databases. Using molcular clock methods I estimate that genotype 4 originated in central Africa around 1733, and that multiple lineages, including subtype 4a which dominates the HCV epidemic in Egypt, have moved to north Africa since ~1850. In Chapter 4 I analyse sequences sampled from an elderly population in Kinshasa to estimate HCV’s transmission history there during the 20th century. The results indicate a rapid increase in HCV transmission between 1950 and 1970 in multiple independent lineages. Possible causes of this increase are discussed. This study population also exhibits high HCV genetic diversity, including the second genotype 7 sample discovered to date. Finally, Chapter 5 uses a range of sequencing techniques, including RNAseq, to characterise two putative HCV recombinants from Cameroon. I confirm that both sequences are recombinants, and generate a full genome sequence for one. I also develop new tools to distinguish between dual infection and recombination in next-generation sequencing data, and discuss how recombination might affect HCV diversity and treatment.

Hepatitis B virus is a member of the hepadnavirus family. The hepatitis B virus core gene codes for two proteins viz. core protein and pre-core protein. These proteins assemble to form particles viz. HBcAg and HBeAg respectively. The structure of the HBcAg has been widely studied but very little is known about the structure of HBeAg. Therefore, the aim of this study was to identify the disulfide bonding patterns in HBeAg. Recombinant HBeAg was isolated from E.coli and used for this study along with various mutants of HBeAg. There are four cysteines present in HBeAg each at position -7, 48, 61 and 107. From this study it can be inferred that the cysteine at 61 and 48 were found to be involved in inter-molecular disulfide bonds between the cysteine at 61 and 48 of other identical monomers. These di-mers were further inter-molecularly linked with cysteine at -7 to form chains. Moreover, the cysteine at -7 and cysteine at 107 were sometimes involved in intra-molecular disulfide bond formation. Thus, the HBeAg in a solution was found be particulate with a heterogeneous pattern of inter chain disulfide bonds.

Introduction and methods: Hepatocellular carcinoma (HCC) remains a difficult disease to study even after a decade of genomic analysis. Metabolic and cell-cycle perturbations are known, large changes in tumors that add little to our understanding of the development of tumors, but generate “noise” that obscures potentially important smaller scale expression changes in “driver genes”. Recently, some researchers have suggested that HCC shares pathways involving the master regulators of embryonic development. Here, we investigated the involvement and specificity of developmental genes in HCV-cirrhosis and HCV-HCC. We obtained microarray studies from 30 patients with HCV-cirrhosis and 49 patients with HCV-HCC and compared to 12 normal livers. Differential gene expression is specific to liver development genes: 86 of 202 (43%) genes specific to liver development had differential expression between normal and cirrhotic or HCC samples. Of 60 genes with paralogous function, which are specific to development of other organs and have known associations with other cancer types, none were expressed in either adult normal liver or tumor tissue. Developmental genes are widely differentially expressed in both cirrhosis and early HCC, but not late HCC: 69 liver development genes were differentially expressed in cirrhosis, and 58 of these (84%) were also dysregulated in early HCC. 19/58 (33%) had larger-magnitude changes in cirrhosis and 5 (9%) had larger-magnitude changes in early HCC. 16 (9%) genes were uniquely altered in early tumors, while only 2 genes were uniquely changed in late-stage (T3 and T4) HCC. Together, these results suggest that the involvement of the master regulators of liver development are active in the pre-cancerous cirrhotic liver and in cirrhotic livers with emerging tumors but play a limited role in the transition from early to late stage HCC. Common patterns of coordinated developmental gene expression include: (1) Dysregulation of BMP2 signaling in cirrhosis followed by overexpression of BMP inhibitors in HCC. BMP inhibitor GPC3 was overexpressed in nearly all tumors, while GREM1 was associated specifically with recurrence-free survival after ablation and transplant. (2) Cirrhosis tissues acquire a progenitor-like signature including high expression of Vimentin, EPCAM, and KRT19, and these markers remain over-expressed to a lesser extent in HCC. (3) Hepatocyte proliferation inhibitors (HPI) E-cadherin (CDH1), BMP2, and MST1 were highly expressed in cirrhosis and remained over-expressed in 16 HCC patients who were transplanted with excellent recurrence-free survival (94% survival after 2 years; mean recurrence-free survival = 5.6 yrs), while loss in early HCC was associated with early recurrence and (2 year). Loss of HPI overexpression was also correlated with overexpression of c-MET and loss of STAT3, LAMA2, FGFR2, CITED2, KIT, SMAD7, GATA6, ERBB2, and NOTCH2.

Infection with the hepatitis C virus (HCV) has a big impact on global health. It is estimated that approximately 3 % of the world’s population carry HCV, putting more than 200 million people at risk of developing severe liver disease, including chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The HCV encoded viroporin p7 forms ion channels that are crucial for the assembly and secretion of infectious viruses, making it a potential drug target. Its hydrophobic nature makes p7 notoriously difficult to investigate in an untagged native form. A previously determined 16 Å electron microscopy single-particle reconstruction in detergent showed a hexameric, flower-shaped p7 protein. In conjunction with one hexameric and several monomeric p7 solution state NMR structures published, this constitutes the currently available structural information framework. An E. coli expression system is introduced, which is especially adapted to express isotopically labeled p7. For the first time, suitable solution-state NMR conditions at physiological pH and temperature were identified that gave rise to high quality spectra suitable to interrogate iminosugar drug interactions with untagged isotopically labeled J4 p7 (C27S) solubilised in detergent. A novel secondary structure topology was observed and preliminary iminosugar binding sites were determined. Further, a DIB (droplet interface bilayer) system to analyse p7 ion channel function was established, which is suitable to elucidate how inhibitors act on p7 genotypes and how different lipids influence the ion channel function of p7. The p7 oligomeric state was further investigated using native gel analysis, showing that isolates representing HCV genotypes 1 - 6 form oligomeric complexes. An ion channel defective dibasic mutant implicated in severely compromising viral fitness is also shown for the first time to form an oligomer, implicating that it is not an assembly problem that leads to the abrogated function.

Complex patterns of HCV infection are increasingly reported, particularly in highly exposed individuals, with multiple and variable subtype profiles seen in many chronic patients. This study aims to address some of the questions arising from this increasingly diverse and dynamic picture, both within hosts and at a population level. In Chapter 2 I find evidence for a highly dynamic infection profile in acute HCV, both in terms of viral load and the dominant subtype. I extrapolate these observations from individual patients to formulate a model of HCV transmission across a high-risk population in order to predict the impact of current and anticipated interventions in Chapters 3 and 4. I show that antiviral therapy and a putative vaccination can still have a significant impact on HCV prevalence at the population level, even when the latter offers only partial protection and in the epidemiological background of ongoing exposure. Thus, in an epidemic with more than one circulating strain it will be crucial for any individual or combination of interventions to target all variants present. In Chapter 5 I demonstrate that early viral load kinetics of patients initiating treatment are indicative of treatment outcome. Strain differences are also evident in the virologic response to treatment with hard-to-treat genotype 1 exhibiting a slower rate of viral load decline than genotypes 2 and 3.

Flavonoide sind weitverbreitete sekundäre Pflanzeninhaltsstoffe. Ihr Beitrag zur Prävention von chronischen Erkrankungen wird zu großen Teilen auf immunmodulatorische und neuroprotektive Effekte zurückgeführt. Eine Voraussetzung für die Nutzung dieser Eigenschaften der Flavonoide stellt die Erfassung cytotoxischer Effekte dar. Mit Ausnahme von Xanthohumol und Quercetin ist für alle im Rahmen der vorliegenden Arbeit untersuchten Flavonoide, Hispidulin, Baicalein, Scutellarein, Hesperetin, Chrysin, Apigenin, Naringenin, Catechin, Pelargonidinchlorid und EMD 21388, sowohl in T-Zellen (Jurkat) als auch in neuronalen (SK-N-SH)-Zellen nach 24-stündiger Inkubation eine geringgradige Cytotoxizität festzuhalten. Für Xanthohumol bzw. Quercetin wird ein halbmaximaler Verlust der Zellvitalität je nach Modell in Konzentrationen von 33-45 µM bzw. 118-208 µM erreicht. Der weiterführenden Charakterisierung (zVAD, DNA-Laddering) ist zu entnehmen, dass die zellulären Veränderungen substanzabhängig differieren und sowohl nekrotische Mechanismen (Xanthohumol) als auch apoptotische Vorgänge (Quercetin) einschließen. Eine erhöhte Lipidperoxidation im oberen Dosisbereich lässt darüber hinaus auf eine Beteiligung von oxidativem Stress an den von Xanthohumol-induzierten nekrotischen Prozessen schließen. Eine positive Einflussnahme auf die Zellvitalität durch Antioxidantien wie GSH und NAC lässt des Weiteren vermuten, dass die erfassten Flavonoid-induzierten Prozesse jeweils sensitiv zum Redoxzustand der Zelle sind. Während die Effekte von Xanthohumol auch in anderen Zellmodellen (HL-60) nachweisbar bleiben, verhält sich Quercetin nicht durchgehend vitalitätsmindernd. Unterschiede zwischen den Testsubstanzen bestehen auch hinsichtlich antioxidativer Effekte. Das Eliminieren freier Radikale zählt zu den wichtigsten Mechanismen, die bei Flavonoid-vermittelter Neuroprotektion eine Rolle spielen. Insgesamt sind alle diesbezüglich untersuchten Substanzen als starke Superoxidanionen-Radikalfänger einzustufen. Im Co-Inkubationsversuch zeigt Scutellarein den stärksten Effekt, gefolgt von Quercetin, Hispidulin und Xanthohumol. Im Prä-Inkubations-Versuchsmodell liegen in der Reihenfolge ihrer Effektstärken Xanthohumol vor Quercetin, Hispidulin und schließlich Scutellarein. Die modellabhängigen Konstanten können, unter Beteiligung einer passiven Diffusion der hydrophoben Flavonoidaglykone, auf eine substanzgebundene Membranpermeabilität zurückzuführen sein. Das antioxidative Potential der Flavonoide resultiert u.a. aus einer komplexen Einflußnahme auf die Genexpression in der Zelle. In der vorliegenden Arbeit sind anhand von cDNA-Arrays für mehrere Vertreter übereinstimmend Wechselwirkungen mit Genen der zellulären Abwehr dargestellt. Demnach führen Scutellarein, Hispidulin, Quercetin und Xanthohumol zu einer deutlich reduzierten Expressionsstärke von STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 und SOD2. Unter den Flavonoid-induzierten Veränderungen ragen die Effekte auf ADAR1 heraus, dessen Genexpression von Scutellarein bis auf ein 0,1-faches der Referenzwerte reduziert wird. Gleichsinnige Auswirkungen von Scutellarein auf die Expression von ADAR1-Protein in Western Blots unterstreichen diese Interaktion und legen nahe, dass ADAR-vermittelte enzymatische Deaminierungen durch Flavonoide moduliert werden können. Diese Beobachtung wird ergänzt durch den nachgewiesenen Effekt von Flavonoiden auf die Expression einer Reihe weiterer Gene (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F und APOBEC3G), die analoge posttranskriptionale Mechanismen steuern und gleichermaßen in Immunabwehr und Neuroprotektion eingebunden sind. Zu den wichtigsten Substraten von ADAR zählen Glutamatrezeptoren. Erwartungsgemäß ist nach der Einwirkung von Scutellarein auf humane Zellen, die Glutamatrezeptoren exprimieren, ein Rückgang der Deaminierung im Bereich der Glutamatrezeptoruntereinheit GluR 2 zu verzeichnen (Q/R-Position). Dem entspricht in elektrophysiologischen Modellen eine gesteigerte Ca2+-Permeabilität der jeweiligen Ionenkanäle und eine veränderte neuronale Exzitabilität. Hieraus ergibt sich ein breites Spektrum zusätzlicher Optionen für die Induktion von gesundheitsrelevanten Flavonoidfunktionen in der Zelle. So spielt die Modulation von Deaminierungen zugleich eine entscheidende Rolle im Vermehrungszyklus viraler Erreger. Die Annahme einer möglichen antiviralen Qualität von Scutellarein wird durch ein HBV-Infektionsmodell anhand drei Parameter der Virusreplikation (Virus-DNA-Konzentration, HBs- bzw. HBe-Antigenproduktion) bestätigt. Offen bleibt auch nach ausführlicher Prüfung, ob der deutliche antivirale Effekt als das Produkt von Flavonoid-induzierten Veränderungen der Deaminierungsraten oder als Folge eines Effekts auf die virale Polymerase zu interpretieren ist. Die hier dargestellten Wirkmechanismen leisten einen Beitrag zum Verständnis der Bedeutung von Flavonoiden für neue Anwendungen in Neuroprotektion und Immunabwehr. / Flavonoids are common secondary plant metabolites that confer numerous nutritional health effects. Their role in preventing chronic diseases is attributed to immunmodulatory and neuroprotective effects among others. In order to fully exploit these properties the limitations imposed by the compounds cytotoxic profiles must be addressed. For the majority of compounds investigated, hispidulin, baicalein, scutellarein, hesperetin, chrysin, apigenin, naringenin, catechin, pelargonidinchloride and EMD 21388, the present study confirms minimal cytotoxicity in T-cells (Jurkat) and in neuronal cells (SK-N-SH). As for xanthohumol and quercetin a 50% decline in cell-vitality is observed at concentrations of 33-45 µM and 118-208 µM, respectively. Further characterization using zVAD and DNA-laddering indicate that cell-vitality may be compromised both by necrotic mechanisms (xanthohumol) and by apoptotic effects (quercetin). An increase in lipidperoxidation in the upper dose range suggests that oxidative stress may be involved in xanthohumol toxicity. As this is counteracted by antioxidants such as GSH and NAC, these flavonoids impact on cell-vitality is likely codetermined by the cells redox state. While the effects of xanthohumol extend to other cell models, quercetin toxicity in HL-60 cells is less pronounced. Test compounds are also found to differ with regard to antioxidative profiles. The elimination of free radicals is a key mechanism in flavonoid-induced neuroprotection and is shown to vary with different incubation protocols. In short incubation experiments (5 min; co-incubation) scutellarein is identified as the most powerful scavenger, followed by quercetin, hispidulin and xanthohumol. In prolonged incubations (24 hrs; prä-incubation) xanthohumol and quercetin are followed by hispidulin and scutellarein. Model-specific constants suggest that passive diffusion of the hydrophobic flavonoid-aglyca may occur across cell membranes, alongside with other modes of permeation. Flavonoids antioxidative potential is mediated by complex effects on gene expression. The present work uses data from cDNA-arrays to highlight interactions with genes involved in cellular defense. Specifically, scutellarein, hispidulin, quercetin and xanthohumol downregulate expression for STK4, CHD4, ARHGDIB, IL16, ISG20, PFN1 and SOD2. In addition, flavonoids consistently downregulate ADAR1-expression, which drops to 0,1-fold of reference values and is paralleled by scutellarein-effects on ADAR1-protein-expression. Together, these findings indicate, that ADAR-mediated enzymatic deamination may be modulated by flavonoids. Similar effects are noted on related genes (ADAR2, APOBEC3B, APOBEC3C, APOBEC3F and APOBEC3G), relevant to posttranscriptional processing underlying immune defense and neuroprotection. Glutamate receptors count among the most important neuronal substrates of ADAR. Following exposure to scutellarein a decrease in deamination rates is confirmed with respect to the glutamate receptor subunit GluR 2 (Q/R-site). As a result, an enhanced Ca2+-permeability of the respective ion channels is anticipated, and modified neuronal excitability. Overall, the regulation of enzymatic deamination by flavonoids offers opportunities for multilevel balancing of cell homeostasis. Thus deaminations may interfere with the replication cycle of viral pathogens. Using an ex-vivo HBV-infection model and three parameters of viral replication (viral load, HBs and HBe indices), antiviral properties of scutellarein are illustrated. Despite extensive investigation, it remains to be seen whether these effects can be ascribed to deaminations of viral DNA or to an interaction with other substrates, e.g. the viral polymerase. In summary, the present observations serve to foster our understanding of flavonoids roles in neuroprotection and immune defense.