Marco Borderi

HIV-1 triggers apoptosis in primary osteoblasts and HOBIT cells through TNFα activation

Several HIV‐1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV‐1‐mediated direct and/or indirect effects on osteoblasts/osteoclasts cross‐talk regulation. This study investigated the effects of HIV‐1IIIb and HIV‐1ADA strains on osteoblasts using the osteoblast‐derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV‐1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV‐1 DNA and supernatant HIV‐1 RNA were not detected by real time PCR or b‐DNA assays respectively. Under the experimental conditions, even heat‐inactivated HIV‐1 or cross‐linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV‐1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up‐regulation of TNFα, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti‐TNFα polyclonal antibody tackled effectively HIV‐1 related induction of cell apoptosis. As a whole, these results indicate that HIV‐1 may impair bone mass structure homeostasis by TNFα regulated osteoblast apoptosis. J. Med. Virol. 80:1507–1514, 2008.

Metabolic bone disease in HIV infection.

HIV mainly replicates in CD4þ T lymphocytes andmonocyte/macrophages causing severe immunologicalimpairment. In addition to the immune system, HIVinfection affects tissues and organs such as kidney, liver,the central nervous system, heart and bone showing acomplex pathogenesis [1].The advent and widespread use of highly activeantiretroviral therapy (HAART) in the last two decadeshasled toa markedimprovementinthe treatmentofHIVdisease even though viral infection cannot be eradicatedbecause HAART does not completely eliminate the viralreservoirs [2]. HAART has dramatically changed thecourseofHIVinfection froma fatalinfectiontoachronicand relatively manageable disease. The increased lifeexpectancyofHIVpatientsandtheeffectsofHAARThavechanged the management of HIV infection. Nowadaysmedical treatment is no longer focused solely on HIVinfection, opportunistic diseases and monitoring immunederangement, but also includes the control of metabolic,cardiovascular, liver, bone and kidney complications. Inparticular, bone alterations have been observed in thecourse of HIV disease representing a pivotal clinicalprobleminthemanagementofHIVpatientsespeciallyforapossible development of bone fractures [3]. The majorbonelesionsdetectableinHIVpatientsarerelatedtobonedemineralization (osteopenia/osteoporosis and osteoma-lacia) and osteonecrosis ([4] for a review).This report will discuss the pathogenesis, diagnosis andtreatment of major bone complications represented bybone demineralization diseases during HIV infection andHAART treatment.

Recommendations for Evaluation and Management of Bone Disease in HIV

Thirty-four human immunodeficiency virus (HIV) specialists from 16 countries contributed to this project, whose primary aim was to provide guidance on the screening, diagnosis, and monitoring of bone disease in HIV-infected patients. Four clinically important questions in bone disease management were identified, and recommendations, based on literature review and expert opinion, were agreed upon. Risk of fragility fracture should be assessed primarily using the Fracture Risk Assessment Tool (FRAX), without dual-energy X-ray absorptiometry (DXA), in all HIV-infected men aged 40-49 years and HIV-infected premenopausal women aged ≥40 years. DXA should be performed in men aged ≥50 years, postmenopausal women, patients with a history of fragility fracture, patients receiving chronic glucocorticoid treatment, and patients at high risk of falls. In resource-limited settings, FRAX without bone mineral density can be substituted for DXA. Guidelines for antiretroviral therapy should be followed; adjustment should avoid tenofovir disoproxil fumarate or boosted protease inhibitors in at-risk patients. Dietary and lifestyle management strategies for high-risk patients should be employed and antiosteoporosis treatment initiated.

Prevalence of Hypovitaminosis D and Factors Associated With Vitamin D Deficiency and Morbidity Among HIV-Infected Patients Enrolled in a Large Italian Cohort

Background: A high prevalence of hypovitaminosis D (hypD) in HIV-infected patients has been reported, but reasons are unclear. Methods: The 25 hydroxy vitamin D (vitD) concentration was measured in a sample of HIV-positive patients from Italy enrolled in the Icona Foundation Study. The change in absolute levels of vitD pre/post combination antiretroviral treatment was modelled by linear regression controlling for confounders and seasonality. Factors associated with hypD were identified using logistic regression analysis, and survival analysis was employed to evaluate the prognostic value of vitD concentration to predict severe diseases (diabetes, cardiovascular, renal), AIDS, and death. Results: We studied 810 patients contributing 1408 vitD measures. Median age was 36 years (range: 20-69). VitD insufficiency (30-75 nmol/L) and deficiency (<30 nmol/L) were found in 47% and 6% of the measures. Factors independently associated with vitD deficiency were African or Centre/South American nationality [odds ratio (OR): 4.16 vs. European, P = 0.04], the sample being collected in spring (OR: 11.27, P = 0.001) or in winter (OR: 4.22, P = 0.03) vs. summer, and a previous history of severe diseases (OR: 5.43, P = 0.03) or AIDS (OR: 2.44, P = 0.04). Over a median follow-up of 6.3 years, patients with vitD insufficiency were at higher risk of subsequent severe diseases than those with normal levels (relative hazard = 1.60, P = 0.05). Conclusions: Our analysis shows that despite the relatively young age of our HIV-infected population, the prevalence of hypD was high. Classic risk factors for hypD in the general population were confirmed in this setting. HypD seems to be moderately associated with the risk of severe disease, AIDS, and death.

Morphologic Alterations in HIV-Infected People with Lipodystrophy Are Associated with Good Adherence to HAART

OBJECTIVE To evaluate the association between adherence to drugs and morphologic alterations (MOA) in a cohort of HIV-infected patients on HAART. METHOD This was a cross-sectional multicenter cohort study in eight tertiary Clinical Centers of Northern and Central Italy. Consecutive outpatients taking HAART were enrolled from August 2000 to March 2001. They completed a self-administered questionnaire for the evaluation of signs of MOA and the self-reported adherence to drugs. Main outcome measures were MOA according to the Multicenter AIDS Cohort Study (MACS) definition and adherence to drugs. RESULTS One hundred seventy-five persons were enrolled into the study. Median CD4 cell count was 522 (interquartile range [IQR] 306-720); 35% of people had undetectable HIV RNA. Patients had been taking HAART for a median of 53 months (IQR 33-62). Among enrolled patients, 83 (47%) had a diagnosis of self-reported MOA; 57 of them reported body changes of more than 12 months duration. Forty persons (23%) self-reported nonadherence in the previous week. Mean time on HAART was 48.7 months (SD = 19.7) for people with MOA and 42.1 months (SD = 21.8) for those without MOA (p =.043). The odds of adherence for people with MOA was 2.36 times (95% CI 1.11-5.00) higher than for people without MOA. On multivariate analysis, being older and female, having an undetectable HIV RNA, longer duration on HAART, and self-reported adherence were independently associated with the presence of MOA. In people with MOA, adherence seems to decrease over time. CONCLUSION Longer time on HAART and self-reported adherence were correlated to MOA. MOA was also associated with older age and female gender.

Development of Acute Psychotic Disorders and HIV-1 Infection

Objective: To gain more understanding about the relationship between human immunodeficiency virus type 1 (HIV-1) infection and new-onset psychosis, we compared clinical and immunological findings, psychiatric symptoms, global cognitive performance and, when available, computerized tomography (CT) findings between HIV-1-seropositive patients with new-onset psychosis and well-matched nonpsychotic HIV-1-seropositives. Methods: Two groups of subjects: HIV-1-seropositives with new-onset psychosis (n = 12) and HIV-1-seropositives without psychosis (n = 15) were recruited through outpatient departments. Organic Delusional Syndrome and Organic Hallucinosis were clinically diagnosed using DSM-III-R diagnostic criteria. Of the baseline participants, twenty-two participated in the two-year follow-up examination. Results: The prevalence of new-onset psychosis in HIV-1-infected subjects was 3.7 per 100 (95% C.I. = 1.6–5.7). HIV-1-seropositive persons with new-onset psychosis had more frequently a positive past psychiatric history, no antiretroviral therapy, and a lower global cognitive performance than did the nonpsychotic HIV-1-seropositives. CT was positive, showing generalized brain atrophy, in three out of nine patients. Remission of psychotic symptoms was observed only in two HIV-1-seropositive persons with new-onset psychosis. Death occurred in two psychotic HIV-1-seropositives with simple loosely held delusions. Autopsy results showed that cortical sulci and ventricle size were graded as with moderate/severe enlargement. Conclusions: New-onset psychosis in HIV infected patients could raise considerable problems in deciding whether a presentation is organic or functional. An interaction of the disease or of psychologically “having” the disease with the presence of a psychotic reaction should also be considered. Interestingly, a protective effect of antiretroviral therapy for new-onset psychosis is suggested.

Prospective evaluation of bone markers, parathormone and 1,25-(OH)2 vitamin D in HIV-positive patients after the initiation of tenofovir/emtricitabine with atazanavir/ritonavir or efavirenz

BackgroundIncreased risk of fractures and osteoporosis have been associated with the use of antiretroviral drugs. There is a paucity of prospective evaluations of bone markers after the initiation of drugs currently recommended to treat HIV infection and results on the evolution of these markers are conflicting. Lastly, the effect of tenofovir on 1,25-(OH)2 vitamin D is uncertain.MethodsWe performed a prospective study on the evolution of bone markers, parathormone and 1,25-(OH)2 vitamin D before and after standard antiretroviral regimens. This was a sub-study of a trial conducted in antiretroviral-naïve patients randomized to tenofovir + emtricitabine in combination with either atazanavir/ritonavir (ATV/r) or efavirenz (EFV). Follow-up lasted 48 weeks. The following bone markers were analyzed: C-terminal cross-laps (CTx), osteocalcin (OC), osteoprotegerin (OPG), and receptor activator of nuclear factor κB ligand (RANKL). Mixed-factorial analysis of variance with random-coefficient general linear model was used to compare their trends over time and linear multivariable regression was performed with a backward selection method to assess predictors of their variations from baseline to week 48. Trends of parathormone and 1,25-(OH)2 vitamin D were also evaluated.ResultsSeventy-five patients were studied: 33 received EFV and 42 ATV/r. Significant increases were found for all markers except for RANKL. There was a significant direct association between CTx and OC increases. Multivariable analysis showed that higher glomerular filtration rate (estimated through cystatin C clearance) predicted greater OPG increase, while older age, higher HIV RNA at baseline and use of ATV/r predicted greater CTx increase. A significant increase of parathormone accompanied the evolution of the study markers. 1,25-(OH)2 vitamin D remained stable, though a seasonality variation was demonstrated.ConclusionsThese data demonstrate CTx increase (bone resorption marker) corresponding to OC increase (bone formation marker) early upon HAART initiation. Moreover, predictors of bone marker increases have been suggested, possibly indicating that a stricter monitoring of bone health and pro-active interventions are needed in older patients, those with higher HIV RNA, prescribed ATV/r rather than EFV, and with decreased renal function at baseline. Further studies are needed to clarify the mechanisms responsible for up-regulation of bone turnover markers, as well as to understand if and what markers are best correlated or predictive of pathological fractures.

Statin therapy decreases serum levels of high-sensitivity C-reactive protein and tumor necrosis factor-α in HIV-infected patients treated with ritonavir-boosted protease inhibitors.

Abstract Background: Statins are lipid-lowering drugs that exhibit anti-inflammatory and immune-modulatory properties, leading to a reduction of serum levels of C-reactive protein (CRP) in the general population. Objective: Because very limited data are available today, our objective was to assess the lipid-lowering effects of statins and their capacity to decrease selected soluble markers of inflammation in HIV-infected patients. Methods: Retrospective cohort study of HIV-infected adult patients with hypercholesterolemia who were receiving a stable antiretroviral regimen including a ritonavir-boosted protease inhibitor and who started a lipid-lowering therapy with rosuvastatin (10 mg daily), atorvastatin (10 mg daily), or pravastatin (40 mg daily) and were followed-up for at least 12 months. One hundred and fifty-one patients were enrolled in the study: 51 in the rosuvastatin group, 47 in the atorvastatin group, and 53 in the pravastatin group. The primary observation was change in plasma lipid levels and serum markers of inflammation (high-sensitivity C-reactive protein [hsCRP], interleukin-6 [IL-6], and tumor necrosis factor-α [TNF- α]), while secondary observations include immunovirological parameters and safety profile of statins. Results: One year after starting the statin therapy, patients treated with rosuvastatin had significantly greater decreases in total cholesterol and LDL cholesterol than subjects on atorvastatin or pravastatin. All statins led to a similar, significant reduction in serum levels of hsCRP and TNF-α, without correlation between biomarkers and lipid values, and toxicity rates were similar for all 3 statins. Conclusion: Our findings suggest that rosuvastatin has a significantly greater lipid-lowering effect than atorvastatin or pravastatin, but all 3 statins exert a similar effect in lowering markers of inflammation as hsCRP and TNF-α.

HIV-1 DNA load analysis in peripheral blood lymphocytes and monocytes from naïve and HAART-treated individuals

OBJECTIVE To evaluate HIV-1 DNA load in PBLs and monocytes from both long-term HAART-treated and antiretroviral naïve HIV-1 infected patients. METHODS Cross-sectional quantitative analysis of HIV-1 DNA load was performed in PBLs and monocytes, purified from 34 long-term HAART-treated and 34 naïve HIV-1 infected patients, and compared to RNA viral load and CD4+ cell count. RESULTS HAART-treated patients showed significantly lower levels of viral DNA both in PBLs and monocytes in comparison with naïve individuals. Variable levels of HIV-1 DNA amount in monocytes were detected in all naïve patients but only in 12 of 34 HAART-treated individuals. PBLs HIV-1 DNA load was inversely correlated to CD4+ cell count in naïve and HAART-treated patients whereas no association was detected in monocytes. CONCLUSIONS Long-term HAART decreased HIV-1 DNA load in PBLs and monocytes demonstrating a valuable inhibitor effect, especially in short-lived reservoirs. In addition, the positive correlation of DNA burden between PBLs and monocytes may suggest a dynamic relation between these reservoirs in the course of disease. HIV-1 DNA load quantitative analysis in PBLs and monocytes may be considered an important approach to study the HIV-1 reservoir and the effectiveness of HAART therapy in HIV-1 seropositive patients.

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation.

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.