TNF-Block Genotypes Influence Susceptibility to HIV-Associated Sensory Neuropathy in Indonesians and South Africans.

HIV-associated sensory neuropathy (HIV-SN) is a disabling complication of HIV disease and antiretroviral therapies (ART). Since stavudine was removed from recommended treatment schedules, the prevalence of HIV-SN has declined and associated risk factors have changed. With stavudine, rs1799964*C (TNF-1031) associated with HIV-SN in Caucasians and Indonesians but not in South Africans. Here, we investigate associations between HIV-SN and rs1799964*C and 12 other polymorphisms spanning TNF and seven neighboring genes (the TNF-block) in Indonesians (n = 202; 34/168 cases) and South Africans (n = 75; 29/75 cases) treated without stavudine. Haplotypes were derived using fastPHASE and haplotype networks built with PopART. There were no associations with rs1799964*C in either population. However, rs9281523*C in intron 10 of BAT1 (alternatively DDX39B) independently associated with HIV-SN in Indonesians after correcting for lower CD4 T-cell counts and >500 copies of HIV RNA/mL (model p = 0.0011, Pseudo R2 = 0.09). rs4947324*T (between NFKBIL1 and LTA) independently associated with reduced risk of HIV-SN and African haplotype 1 (containing no minor alleles) associated with increased risk of HIV-SN after correcting for greater body weight, a history of tuberculosis and nadir CD4 T-cell counts (model: p = 0.0003, Pseudo R2 = 0.23). These results confirm TNF-block genotypes influence susceptibility of HIV-SN. However, critical genotypes differ between ethnicities and with stavudine use.

Polymorphisms in CAMKK2 associate with susceptibility to sensory neuropathy in HIV patients treated without stavudine.

HIV-associated sensory neuropathy (HIV-SN) is a debilitating neurological complication of HIV infection potentiated by the antiretroviral drug stavudine. While stavudine is no longer used, HIV-SN now affects around 15% of HIV+ Indonesians. Here, we investigate whether polymorphisms within the P2X-block (P2X4R, P2X7R, CAMKK2) and/or ANAPC5 mark susceptibility to HIV-SN in this setting. As polymorphisms in these genes associated with HIV-SN in African HIV patients receiving stavudine, the comparison can identify mechanisms independent of stavudine. HIV patients who had never used stavudine (n = 202) attending clinics in Jakarta were screened for neuropathy using the AIDS Clinical Trials Group Brief Peripheral Neuropathy Screen. Open-array technology was used to type 48 polymorphisms spanning the four genes. Haplotypes were derived for each gene using fastPHASE. Haplogroups were constructed with median-joining methods. Multivariable models optimally predicting HIV-SN were based on factors achieving p < 0.2 in bivariate analyses. Minor alleles of three co-inherited polymorphisms in CAMKK2 (rs7975295*C, rs1560568*A, rs1132780*T) associated with a reduced prevalence of HIV-SN individually and after adjusting for lower CD4 T cell count and viremia (p = 0.0002, pseudo R2 = 0.11). The optimal model for haplotypes linked HIV-SN with viremia and lower current CD4 T cell count, plus CAMKK2 haplotypes 6 and 11 and P2X7R haplotypes 2 and 12 (p = 0.0002; pseudo R2 = 0.11). CAMKK2 haplogroup A (includes 16 haplotypes and all instances of rs7975295*C, rs1560568*A, rs1132780*T) associated with reduced rates of HIV-SN (p = 0.02, OR = 0.43 CI = 0.21-0.88). These findings support a protective role for these three alleles, suggesting a role in the pathogenesis of HIV-SN that is independent of stavudine.

Clinical diagnosis of sensory neuropathy in HIV patients treated with tenofovir: A 6-month follow-up study.

BACKGROUND: Sensory neuropathy (SN) is a common and often painful neurological condition associated with HIV-infection and its treatment. However, data on the incidence of SN in neuropathy-free individuals initiating combination antiretroviral therapies (cART) that do not contain the neurotoxic agent stavudine are lacking. AIMS: We investigated the 6-month incidence of SN in ART naïve individuals initiating tenofovir (TDF)-based cART, and the clinical factors associated with the development of SN. METHODS: 120 neuropathy-free and ART naïve individuals initiating cART at a single center in Johannesburg, South Africa were enrolled. Participants were screened for SN using clinical signs and symptoms at study enrolment and approximately every 2-months for a period of ~6-months. Diagnostic criteria for symptomatic SN was defined by the presence of at least one symptom (pain/burning, numbness, paraesthesias) and at least two clinical signs (reduced vibration sense, absent ankle reflexes or pin-prick hypoaesthesia). Diagnostic criteria for asymptomatic SN required at least two clinical signs only (as above). RESULTS: A total of 88% of the cohort completed three visits within the 6-month period. The 6-month cumulative incidence of neuropathy was 140 cases per 1000 patients (95% CI: 80-210) at an incidence rate of 0.37 (95% CI: 0.2-0.5) per person year. Height and active tuberculosis (TB) disease were independently associated with the risk of developing SN (P < .05). INTERPRETATION: We found that within the first 6 months of starting cART, incident SN persists in the post-stavudine era, with 11 (9%) of individuals developing asymptomatic SN, and 9 (8%) developing symptomatic SN.

Psychological Factors Associated With Painful Versus Non-Painful HIV-Associated Sensory Neuropathy.

HIV-associated sensory neuropathy (HIV-SN) is a common, and frequently painful complication of HIV, but factors that determine the presence of pain are unresolved. We investigated: (i) if psychological factors associated with painful (n = 125) versus non-painful HIV-SN (n = 72), and (ii) if pain and psychological factors affected quality of life (QoL). We assessed anxiety and depression using the Hopkins Symptoms Checklist-25. Pain catastrophizing and QoL were assessed using the Pain Catastrophizing Scale and Euroqol-5D, respectively. Presence of neuropathy was detected using the Brief Neuropathy Screening Tool, and pain was characterised using the Wisconsin Brief Pain Questionnaire. Overall, there was a high burden of pain, depression and anxiety in the cohort. None of the psychological variables associated with having painful HIV-SN. Greater depressive symptoms and presence of pain were independently associated with lower QoL. In those participants with painful HIV-SN, greater depressive symptom scores were associated with increased pain intensity. In conclusion, in a cohort with high background levels of psychological dysfunction, psychological factors do not predict the presence of pain, but both depression and presence of pain are associated with poor quality of life.

Emerging Role and Characterization of Immunometabolism: Relevance to HIV Pathogenesis, Serious Non-AIDS Events, and a Cure.

Immune cells cycle between a resting and an activated state. Their metabolism is tightly linked to their activation status and, consequently, functions. Ag recognition induces T lymphocyte activation and proliferation and acquisition of effector functions that require and depend on cellular metabolic reprogramming. Likewise, recognition of pathogen-associated molecular patterns by monocytes and macrophages induces changes in cellular metabolism. As obligate intracellular parasites, viruses manipulate the metabolism of infected cells to meet their structural and functional requirements. For example, HIV-induced changes in immune cell metabolism and redox state are associated with CD4(+) T cell depletion, immune activation, and inflammation. In this review, we highlight how HIV modifies immunometabolism with potential implications for cure research and pathogenesis of comorbidities observed in HIV-infected patients, including those with virologic suppression. In addition, we highlight recently described key methods that can be applied to study the metabolic dysregulation of immune cells in disease states.

Consumer engagement critical to success in an Australian research project: reflections from those involved.

This paper describes the people, activities and methods of consumer engagement in a complex research project, and reflects on the influence this had on the research and people involved, and enablers and challenges of engagement. The 2.5-year Integrating and Deriving Evidence Experiences and Preferences (IN-DEEP) study was conducted to develop online consumer summaries of multiple sclerosis (MS) treatment evidence in partnership with a three-member consumer advisory group. Engagement methods included 6-monthly face-to-face meetings and email contact. Advisory group members were active in planning, conduct and dissemination and translational phases of the research. Engaging consumers in this way improved the quality of the research process and outputs by: being more responsive to, and reflective of, the experiences of Australians with MS; expanding the research reach and depth; and improving the researchers' capacity to manage study challenges. Advisory group members found contributing their expertise to MS research satisfying and empowering, whereas researchers gained confidence in the research direction. Managing the unpredictability of MS was a substantive challenge; the key enabler was the 'brokering role' of the researcher based at an MS organisation. Meaningfully engaging consumers with a range of skills, experiences and networks can make important and unforeseen contributions to research success.

A haplotype spanning P2X7R, P2X4R and CAMKK2 may mark susceptibility to pulmonary non-tuberculous mycobacterial disease.

Despite widespread exposure to potentially pathogenic mycobacteria present in the soil and in domestic water supplies, it is not clear why only a small proportion of individuals contract pulmonary nontuberculous mycobacterial (NTM) infections. Here, we explore the impact of polymorphisms within three genes: P2X ligand gated ion channel 7 (P2X7R), P2X ligand gated ion channel 4 (P2X4R) and calcium/calmodulin-dependent protein kinase kinase 2 beta (CAMKK2) on susceptibility. Thirty single nucleotide polymorphisms (SNPs) were genotyped in NTM patients (n = 124) and healthy controls (n = 229). Weak associations were found between individual alleles in P2X7R and disease but were not significant in multivariate analyses adjusted to account for gender. Haplotypes spanning the three genes were derived using the fastPHASE algorithm. This yielded 27 haplotypes with frequencies >1% and accounting for 63.3% of the combined cohort. In univariate analyses, seven of these haplotypes displayed associations with NTM disease above our preliminary cut-off (p ≤ 0.20). When these were carried forward in a logistic regression model, gender and one haplotype (SH95) were independently associated with the disease (model p < 0.0001; R 2  = 0.05). Examination of individual alleles within these haplotypes implicated P2X7R and CAMKK2 in pathways affecting pulmonary NTM disease.

Genetics of HIV-associated sensory neuropathy and related pain in Africans.

Despite the use of safer antiretroviral medications, the rate of HIV-associated sensory neuropathy (HIV-SN), the most common neurological complication of HIV, remains high. This condition is often painful and has a negative effect on quality of life. Up to 90% of those with HIV-SN experience pain for which there is no effective analgesic treatment. Genetic factors are implicated, but there is a lack of a comprehensive body of research for African populations. This knowledge gap is even more pertinent as Africans are most affected by HIV. However, recent studies performed in Southern African populations have identified genes displaying potential as genetic markers for HIV-SN and HIV-SN-associated pain in Africans. Here, we review the published studies to describe current knowledge of genetic risk factors for this disease in Africa.

Polymorphisms in CAMKK2 may predict sensory neuropathy in African HIV patients.

HIV-associated sensory neuropathy (HIV-SN) is the most common neurological condition associated with HIV. HIV-SN has characteristics of an inflammatory pathology caused by the virus itself and/or by antiretroviral treatment (ART). Here, we assess the impact of single-nucleotide polymorphisms (SNPs) in a cluster of three genes that affect inflammation and neuronal repair: P2X7R, P2X4R and CAMKK2. HIV-SN status was assessed using the Brief Peripheral Neuropathy Screening tool, with SN defined by bilateral symptoms and signs. Forty-five SNPs in P2X7R, P2X4R and CAMKK2 were genotyped using TaqMan fluorescent probes, in DNA samples from 153 HIV(+) black Southern African patients exposed to stavudine. Haplotypes were derived using the fastPHASE algorithm, and SNP genotypes and haplotypes associated with HIV-SN were identified. Optimal logistic regression models included demographics (age and height), with SNPs (model p < 0.0001; R (2) = 0.19) or haplotypes (model p < 0.0001; R (2) = 0.18, n = 137 excluding patients carrying CAMKK2 haplotypes perfectly associated with SN). Overall, CAMKK2 exhibited the strongest associations with HIV-SN, with two SNPs and six haplotypes predicting SN status in black Southern Africans. This gene warrants further study.

Media discourses of low carbon housing: The marginalisation of social and behavioural dimensions within the British broadsheet press.

Decarbonising housing is a key UK government policy to mitigate climate change. Using discourse analysis, we assess how low carbon housing is portrayed within British broadsheet media. Three distinct storylines were identified. Dominating the discourse, Zero carbon housing promotes new-build, low carbon houses as offering high technology solutions to the climate problem. Retrofitting homes emphasises the need to reduce emissions within existing housing, tackling both climate change and rising fuel prices. A more marginal discourse, Sustainable living, frames low carbon houses as related to individual identities and 'off-grid' or greener lifestyles. Our analysis demonstrates that technical and economic paradigms dominate media discourse on low carbon housing, marginalising social and behavioural aspects.

A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA.

Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late 'point of no return' step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR's advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR's utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science.

ß-catenin represses expression of the tumour suppressor 15-prostaglandin dehydrogenase in the normal intestinal epithelium and colorectal tumour cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) overexpression in colorectal cancer increases levels of its pro-tumorigenic product prostaglandin E2 (PGE(2)). The recently identified colorectal tumour suppressor 15-prostaglandin dehydrogenase (15-PGDH) catalyses prostaglandin turnover and is downregulated at a very early stage in colorectal tumorigenesis; however, the mechanism responsible remains unclear. As Wnt/ß-catenin signalling is also deregulated early in colorectal neoplasia, a study was undertaken to determine whether ß-catenin represses 15-PGDH expression. METHODS: The effect of modulating Wnt/ß-catenin signalling (using ß-catenin siRNA, mutant TCF4, Wnt3A or GSK3 inhibition) on 15-PGDH mRNA, protein expression and promoter activity was determined in colorectal cell lines by immunoblotting, qRT-PCR and reporter assays. The effect of ß-catenin deletion in vivo was addressed by 15-PGDH immunostaining of ß-catenin(-/lox)-villin-creERT2 mouse tissue. 15-PGDH promoter occupancy was determined using chromatin immunoprecipitation and PGE(2) levels by ELISA. RESULTS: The study shows for the first time that ß-catenin knockdown upregulates 15-PGDH in colorectal adenoma and carcinoma cells without affecting COX-2 protein levels. A dominant negative mutant form of TCF4 (dnTCF4), unable to bind ß-catenin, also upregulated 15-PGDH; conversely, increasing ß-catenin activity using Wnt3A or GSK3 inhibition downregulated 15-PGDH. Importantly, inducible ß-catenin deletion in vivo also upregulated intestinal epithelial 15-PGDH. 15-PGDH regulation occurred at the protein, mRNA and promoter activity levels and chromatin immunoprecipitation indicated ß-catenin/TCF4 binding to the 15-PGDH promoter. ß-catenin knockdown decreased PGE(2) levels, and this was significantly rescued by 15-PGDH siRNA. CONCLUSION: These data suggest a novel role for ß-catenin in promoting colorectal tumorigenesis through very early 15-PGDH suppression leading to increased PGE(2) levels, possibly even before COX-2 upregulation.

Brain cell reservoirs of latent virus in presymptomatic HIV-infected individuals.

We detected HIV-1 DNA in pure populations of perivascular macrophages, parenchymal microglia, and astrocytes, isolated using laser microdissection from brain tissue of five untreated individuals who died in the presymptomatic stage of infection from non-HIV causes. HIV-1 DNA was detected in the three cell populations, most consistently in perivascular macrophages, without evidence of productive infection. The percentage of PCR reactions detecting HIV-1 DNA in perivascular macrophages correlated inversely with peripheral blood CD4 counts. These findings demonstrate that brain cell reservoirs of latent HIV-1 exist before pathological HIV encephalitis and suggest that perivascular macrophage trafficking of latent virus into the brain increases with immunosuppression.

HIV-associated sensory neuropathy: risk factors and genetics.

HIV-associated sensory neuropathy (HIV-SN) remains a common neurological complication of HIV infection despite the introduction of effective antiretroviral therapies. Exposure to neurotoxic antiretroviral drugs and increasing age have consistently been identified as risk factors for HIV-SN, while comorbid conditions with underlying predisposition to cause peripheral neuropathy (eg, diabetes mellitus, malnutrition, isoniazid exposure), ethnicity, and increasing height also have been implicated. Genetic association studies have identified genes affecting mitochondrial function and genes involved in the inflammatory response that modify the risk for HIV-SN among patients exposed to neurotoxic antiretrovirals. However, there is a lack of data on clinical, demographic, and genetic risk factors for HIV-SN in the modern era, with the rate of HIV-SN remaining unacceptably high despite the introduction of safer medications. Thus, more work is required to identify the principal factors that increase an individual's risk for HIV-SN so that effective preventative or therapeutic strategies can be implemented.

The role of CAMKK2 polymorphisms in HIV-associated sensory neuropathy in South Africans.

Human immunodeficiency virus-associated sensory neuropathy (HIV-SN) is a common neurological complication of HIV infection. It affected 57% of South African patients whose antiretroviral therapy (ART) included stavudine and was influenced by genotypes of the P2X-block (P2X7R, P2X4R and CAMKK2). We investigate associations between HIV-SN and P2X-block genotypes in patients who never received stavudine. An adjacent gene, ANAPC5, was included. 75 HIV+ individuals were assessed using the Brief Peripheral Neuropathy Screen before treatment and after 6-8 months on stavudine-free regimens. DNA was genotyped for 48 polymorphisms across the four genes using an OpenArray™ platform. Haplotypes were derived using fastPHASE. Associations with HIV-SN were assessed using bivariate and multivariate analyses. Nine individuals (12%) were diagnosed with HIV-SN prior to ART and a further 20 individuals (27%) developed HIV-SN within 6-8 months. Five polymorphisms, rs503720*G (OR = 133) in P2X7R, rs10849861*A (OR = 5.99), rs1653586*T (OR = 67.8) and rs11065504*C (OR = 0.02) in CAMKK2, and rs2089886*A (OR = 6.68) in ANAPC5, associated with HIV-SN after adjusting for body weight, nadir CD4 T-cell counts and prior tuberculosis (model p < 0.0001, n = 69, Pseudo R2 = 0.54). Three CAMKK2 haplotypes were associated with HIV-SN (OR = 2.82, 3.42 and 6.85) after adjusting for body weight, nadir CD4 T-cell counts and prior tuberculosis (model p < 0.0005, n = 71, Pseudo R2 = 0.26). The results support a role for CAMKK2 in HIV-SN, independent of mechanisms invoked by stavudine. SIGNIFICANCE STATEMENT: HIV-associated sensory neuropathy (HIV-SN) remains a clinically relevant complication of HIV infection and its treatment, affecting 38% of patients treated without neurotoxic stavudine. HIV-SN can impact an individual's ability to work and quality of life, with few effective therapeutic options, so an understanding of the underlying mechanisms would have clinical value. We confirm that CAMKK2 polymorphisms and haplotypes influence susceptibility to HIV-SN in South Africans treated without stavudine. This provides further evidence for a role for the protein encoded by CAMKK2 in the pathogenesis of HIV-SN, independent of mechanisms initiated by stavudine.

Brief Report: Demographic and Genetic Associations With Markers of Small and Large Fiber Sensory Neuropathy in HIV Patients Treated Without Stavudine.

Neurotoxic antiretroviral therapy (ART) such as stavudine has been now replaced with safer therapies, reducing the prevalence of neuropathy from 34% to 15% in HIV+ Indonesians. However, it is unclear whether the residual cases display damage to small or large nerve fibers and whether both are influenced by known risk factors, including alleles of CAMKK2 associated with neuropathy in HIV patients. The encoded protein influences the growth and repair of nerve fibers. HIV-positive adults on ART for >12 months without exposure to stavudine were screened for neuropathy using the AIDS Clinical Trials Group Brief Peripheral Neuropathy Screen (BPNS). Large fiber neuropathy was assessed by nerve conduction (NC) and small fiber neuropathy using stimulated skin wrinkling (SSW) applied to the fingers. CAMKK2 alleles were assessed by TaqMan OpenArray technology. Neuropathy diagnoses were more common with SSW than BPNS (49/173 vs 26/185, χ; P = 0.0009), with poor alignment between these outcomes (P = 0.60). NC and BPNS diagnosed neuropathy at similar frequencies (29/151 vs 26/185; P = 0.12) and were aligned (P < 0.0001). In bivariate analyses, all diagnoses were associated with patients' age and persistent HIV replication, with minor effects from CD4 T-cell counts and time on ART. CAMKK2 alleles associated with neuropathy diagnosed with BPNS and SSW but not NC. Multivariable analyses confirmed the importance of age and HIV replication, with distinct CAMKK2 polymorphisms affecting BPNS and SSW. Paradoxically, height was protective against skin wrinkling. Overall the data link CAMKK2 genotypes with small rather than large fiber damage. SSW may reflect pathology distinct from that identified using BPNS.

Measuring and monitoring apoptosis and drug toxicity in HIV patients by ligation-mediated polymerase chain reaction.

Apoptosis has a critical role in normal physiology while its dysregulation has causal links with certain pathologies. A biochemical hallmark of apoptosis, internucleosomal genomic DNA fragmentation, is detectable by ligation-mediated polymerase chain reaction (LM-PCR). Here we converted LM-PCR into a new apoptosis quantifier by dividing trace quantities of 600 bp apoptotic amplicons into those of a single copy house-keeping gene, generating the LM-PCR 'value'. Dynamic range was approximately 17-fold correlating with a approximately 200-fold difference in degree of apoptotic fragmentation. Inter- and intra-gel reliability were both excellent, supporting LM-PCR's utility with large sample sets. Validation experiments comprising cell exposure to staurosporine over time revealed LM-PCR is as sensitive as caspase-3/ELISA and more sensitive than terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling/flourescence-activated cell sorting (TUNEL/FACS) for distinguishing low degrees of apoptosis (the spectrum most relevant in vivo). The LM-PCR profile mirrored that of caspase-3/ELISA but not TUNEL/FACS. We then applied this molecular tool to clinical investigation. Increased apoptosis is implicated in lipoatrophy (subcutaneous fat wasting), a serious, persistent toxicity of some nucleoside analogue reverse transcriptase inhibitors (NRTIs) used in anti-HIV highly active antiretroviral therapy (HAART). We demonstrated in 105 peripheral blood mononuclear cell samples that elevated LM-PCR values are seen during therapy with stavudine (d4T), a particularly toxic NRTI (P< 0.0001 versus no HAART, unpaired t-test). Elevated values were also independently associated with clinical evidence of lipoatrophy (P= 0.007, multiple logistic regression modelling) but not with patient age, CD4 T-cell count nor HIV viral load (P> 0.8 for each). Together these data demonstrate that LM-PCR is a robust and reliable quantifier of apoptosis with potential for basic science and clinical investigation.

Evaluation of the Cavidi ExaVir Load assay (version 3) for plasma human immunodeficiency virus type 1 load monitoring.

We evaluated the new low-cost ExaVir Load (version 3) reverse transcriptase viral load assay against the Roche Cobas Amplicor assay. Results for samples tested using the reverse transcriptase assay correlated well with those obtained with the Roche assay (r = 0.85; n = 202). The version 3 reverse transcriptase assay shows improved sensitivity compared to the previous version.

In Vivo Model Systems for Hepatitis B Virus Research.

Hepatitis B virus (HBV) affects more than 257 million people globally, resulting in progressively worsening liver disease, manifesting as fibrosis, cirrhosis, and hepatocellular carcinoma. The exceptionally narrow species tropism of HBV restricts its natural hosts to humans and non-human primates, including chimpanzees, gorillas, gibbons, and orangutans. The unavailability of completely immunocompetent small-animal models has contributed to the lack of curative therapeutic interventions. Even though surrogates allow the study of closely related viruses, their host genetic backgrounds, immune responses, and molecular virology differ from those of HBV. Various different models, based on either pure murine or xenotransplantation systems, have been introduced over the past years, often making the choice of the optimal model for any given question challenging. Here, we offer a concise review of in vivo model systems employed to study HBV infection and steps in the HBV life cycle or pathogenesis.

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.