Reciprocal Effects of Antiretroviral Drugs Used To Treat HIV Infection on the Fibroblast Growth Factor 21/ß-Klotho System.

Following antiretroviral therapy, HIV-infected patients show increased circulating levels of the antidiabetic hormone fibroblast growth factor 21 (FGF21). In contrast, the expression of the FGF21-obligatory coreceptor ß-Klotho (KLB) is reduced in target tissues. This situation is comparable to the FGF21 resistance status observed in obesity and type 2 diabetes. Here, we performed the first systematic study of the effects of distinct members of different antiretroviral drug classes on the FGF21/KLB system in human hepatic, adipose, and skeletal muscle cells. Most protease inhibitors and the nonnucleoside reverse transcriptase inhibitor efavirenz induced FGF21 gene expression. Neither nucleoside reverse transcriptase inhibitors nor the viral entry inhibitor maraviroc had any effect. Among the integrase inhibitors, elvitegravir significantly induced FGF21 expression, whereas raltegravir had minor effects only in adipose cells. In human hepatocytes and adipocytes, known target cells of FGF21 action, efavirenz, elvitegravir, and the lopinavir-ritonavir combination exerted inhibitory effects on KLB gene expression. Drug treatments that elicited FGF21 induction/KLB repression were those found to induce endoplasmic reticulum (ER) stress and oxidative stress. Notably, the pharmacological agents thapsigargin and tunicamycin, which induce these stress pathways, mimicked the effects of drug treatments. Moreover, pharmacological inhibitors of either ER or oxidative stress significantly impaired lopinavir-ritonavir-induced regulation of FGF21, but not KLB. In conclusion, the present in vitro screen study identifies the antiretroviral drugs that affect FGF21/KLB expression in human cells. The present results could have important implications for the management of comorbidities resulting from side effects of specific antiretroviral drugs for the treatment of HIV-infected patients.

Circulating fibroblast growth factor 23 (FGF23) levels are associated with metabolic disturbances and fat distribution but not cardiovascular risk in HIV-infected patients.

OBJECTIVES: Dyslipidaemia, insulin resistance, metabolic syndrome and HIV/HAART-associated lipodystrophy syndrome (HALS) are common comorbidities in HIV-1-infected patients, which may increase cardiovascular risk. Fibroblast growth factor 23 (FGF23) is a bone-derived hormone with effects on metabolism and phosphate homeostasis. The aim of this study was to determine the relationship between FGF23 levels, metabolic alterations, fat distribution and cardiovascular risk. METHODS: This was a cross-sectional study. Serum FGF23 levels were analysed in 152 patients and 34 healthy control individuals. Patients belonged to three groups: HIV-1-infected, antiretroviral-treated patients who have developed HALS (n = 60); HIV-1-infected, antiretroviral-treated patients without HALS (n = 43); and untreated (naive) HIV-1-infected patients (n = 49). Serum FGF23 levels were compared with lipid and glucose homeostasis parameters, fat distribution and cardiovascular risk. RESULTS: Serum FGF23 levels were increased in HIV-1-infected patients, but the increase was most marked in those with HALS. FGF23 levels showed a strong positive correlation with age, indicators of dyslipidaemia (LDL cholesterol, polyunsaturated fatty acids and monounsaturated fatty acids), HALS parameters (trunk/appendicular fat ratio), insulin resistance (fasting insulin and homeostasis model assessment of insulin resistance) and C-reactive protein. FGF23 levels correlated with cardiovascular risk but correlation was lost after age adjustment. CONCLUSIONS: FGF23 levels are increased in HIV-1-infected patients, especially in those with HALS, and this increase is associated with dyslipidaemia, insulin resistance, metabolic syndrome, fat distribution and parameters of inflammation. FGF23 is not associated with cardiovascular risk when age is taken into account.

Increased Circulating Levels of Growth Differentiation Factor 15 in Association with Metabolic Disorders in People Living with HIV Receiving Combined Antiretroviral Therapy.

OBJECTIVE: People living with HIV (PLWH) have an increased cardiovascular risk (CVR) owing to dyslipidemia, insulin resistance, metabolic syndrome, and HIV/combination antiretroviral therapy (cART)-associated lipodystrophy (HALS). Atherosclerosis and inflammation are related to growth differentiation factor-15 (GDF15). The relationship between metabolic disturbances, HALS, and CVR with GDF15 in PLWH is not known. RESEARCH DESIGN AND METHODS: Circulating GDF15 levels in 152 PLWH (with HALS = 60, without HALS = 43, cART-naïve = 49) and 34 healthy controls were assessed in a cross-sectional study. Correlations with lipids, glucose homeostasis, fat distribution, and CVR were explored. RESULTS: PLWH had increased circulating GDF15 levels relative to controls. The increase was the largest in cART-treated PLWH. Age, homeostatic model assessment of insulin resistance 1 (HOMA1-IR), HALS, dyslipidemia, C-reactive protein, and CVR estimated with the Framingham score correlated with GDF15 levels. The GDF15-Framingham correlation was lost after age adjustment. No correlation was found between GDF15 and the D:A:D Data Collection on Adverse Effects of Anti-HIV Drugs (D:A:D) score estimated CVR. CVR independent predictors were patient group (naïve, HALS-, and HALS+) and cumulated protease inhibitor or nucleoside reverse transcriptase inhibitor exposure. CONCLUSIONS: PLWH, especially when cART-treated, has increased GDF15 levels-this increase is associated with dyslipidemia, insulin resistance, metabolic syndrome, HALS, and inflammation-related parameters. GDF15 is unassociated with CVR when age-adjusted.

Association of thymidylate synthase gene polymorphisms with stavudine triphosphate intracellular levels and lipodystrophy.

The antiviral activity and toxicity of stavudine (d4T) depend on its triphosphate metabolite, stavudine triphosphate (d4T-TP). Therefore, modifications in intracellular levels of d4T-TP may change the toxicity profile of stavudine. d4T-TP intracellular levels in peripheral blood mononuclear cells were determined with a prominence liquid chromatograph connected to a triple-quadruple mass spectrometer. Polymorphisms in the thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), reduced folate carrier 1 (RFC1; SLC19A1), and cyclin D1 (CCND1) genes were determined by direct sequencing using an ABI Prism 3100 genetic analyzer or Fluidigm's Biomark system. The Mann-Whitney test, rank analysis of variance (with Bonferroni's adjusted post hoc comparisons), and logistic regression were used for the inferential analyses. Thirty-three stavudine-treated patients were enrolled in this cross-sectional study. d4T-TP intracellular levels were 11.50 fmol/10(6) cells (interquartile range [IQR] = 8.12 to 13.87 fmol/10(6) cells) in patients with a high-expression TS genotype (2/3G, 3C/3G, and 3G/3G), whereas in those with a low-expression TS genotype (2/2, 2/3C, and 3C/3C), they were 21.40 fmol/10(6) cells (IQR = 18.90 to 27.0 fmol/10(6) cells) (P < 0.0001). Polymorphisms in the MTHFR, DHFR, RFC1, and CCND1 genes did not influence the intracellular concentration of d4T-TP. d4T-TP levels were independently associated with the TS genotype (low versus high expression; odds ratio [OR] = 86.22; 95% confidence interval [CI] = 8.48 to nonestimable; P = 0.0023). The low-expression TS genotype was associated with the development of HIV/highly active antiretroviral therapy-associated lypodystrophy syndrome (HALS) (OR = 14.0; 95% CI = 2.09 to 108.0; P = 0.0032). Our preliminary data show that polymorphisms in the thymidylate synthase gene are strongly associated with d4T-TP intracellular levels and with development of HALS.

Relationship between HIV/Highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome and stavudine-triphosphate intracellular levels in patients with stavudine-based antiretroviral regimens.

BACKGROUND: The link between human immunodeficiency virus/highly active antiretroviral therapy (HAART)-associated lipodystrophy syndrome (HALS) and the use of thymidine analogues has been well established. However, to our knowledge, no relationship has been proven between intracellular levels of stavudine (d4T) and HALS. METHODS: We measured peripheral blood mononuclear cell intracellular levels of d4T-triphosphate (TP) in patients who were receiving d4T as part of their antiretroviral regimens. d4T-TP levels were determined by a validated liquid chromatography-tandem mass spectrometry assay method. The diagnosis of HALS was made in accordance with the criteria of a lipodystrophy severity grading scale. The Student t test, Pearson correlations, 1-way analysis of variance with Bonferroni correction, and stepwise logistic regression were used for statistic analyses. RESULTS: This was a cross-sectional study. There were 33 patients: 17 with HALS and 16 without HALS. The median concentration of d4T-TP for patients with HALS was 20.60 femtomoles (fmol)/1 x 10(6) cells (interquartile range [IQR], 14.90-26.92 fmol/1 x 10(6) cells) and for patients without HALS was 13.85 fmol/1 x 10(6) cells (IQR, 8.65-20.15 fmol/1 x 10(6) cells) (P=.013). The median d4T-TP intracellular level in patients who had developed an AIDS-defining condition was 22.50 fmol/1 x 10(6) cells (IQR, 15.80-27.37 fmol/1 x 10(6) cells) and in those who had not was 14.40 fmol/1 x 10(6) cells (IQR, 10.80-20.40 fmol/1 x 10(6) cells) (P=.037). There were no statistically significant differences in d4T-TP intracellular levels with respect to the presence of metabolic syndrome, the clinical form of HALS (pure lipoatrophic vs mixed), the degree of facial lipoatrophy, the presence of hepatitis C virus infection, and the pair of nucleosides in HAART. d4T-TP levels correlated only with cumulative d4T exposure in time and dose. d4T-TP intracellular levels were independently associated with HALS (odds ratio, 1.58; 95% confidence interval, 1.08-2.32; P=.019). CONCLUSIONS: Intracellular levels of d4T-TP are strongly associated with the development of HALS.

Human dendritic cell activities are modulated by the omega-3 fatty acid, docosahexaenoic acid, mainly through PPAR(gamma):RXR heterodimers: comparison with other polyunsaturated fatty acids.

There is accumulating evidence that omega-3 fatty acids may modulate immune responses. When monocytes were differentiated to dendritic cells (DCs) in the presence of docosahexaenoic acid (DHA), the expression of costimulatory and antigen presentation markers was altered in a concentration-dependent way, positively or negatively, depending on the markers tested and the maturation stage of the DCs. Changes induced by eicosapentaenoic acid and linoleic acid were similar but less intense than those of DHA, whereas oleic acid had almost no effect. DHA-treated, mature DCs showed inhibition of IL-6 expression and IL-10 and IL-12 secretion, and their lymphoproliferative stimulation capacity was impaired. The phenotypic alterations of DCs induced by DHA were similar to those already reported for Rosiglitazone (Rosi), a peroxisome proliferator-activated receptor gamma (PPAR gamma) activator, and the retinoid 9-cis-retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPAR gamma target genes pyruvate dehydrogenase kinase-4 and aP-2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPAR gamma inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPAR gamma:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.

Triglyceride Form of Docosahexaenoic Acid Mediates Neuroprotection in Experimental Parkinsonism.

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. The main treatment of PD consists of medication with dopamine-based drugs, which palliate the symptoms but may produce adverse effects after chronic administration. Accordingly, there is a need to develop novel neuroprotective therapies. Several studies suggest that omega-3 polyunsaturated fatty acids (n-3 PUFA) might provide protection against brain damage. Here, we studied several experimental models of PD, using striatal neuronal cultures, striatal slices, and mice, to assess the neuroprotective effects of docosahexaenoic acid (DHA), the main n-3 PUFA in the brain, administered in its triglyceride form (TG-DHA). Hence, we determined the beneficial effects of TG-DHA on neural viability following 6-hydroxydopamine (6-OHDA)-induced neurotoxicity, a well-established PD model. We also implemented a novel mouse behavioral test, the beam walking test, to finely assess mouse motor skills following dopaminergic denervation. This test showed potential as a useful behavioral tool to assess novel PD treatments. Our results indicated that TG-DHA-mediated neuroprotection was independent of the net incorporation of PUFA into the striatum, thus suggesting a tight control of brain lipid homeostasis both in normal and pathological conditions.

Aging is associated with increased FGF21 levels but unaltered FGF21 responsiveness in adipose tissue.

Fibroblast growth factor 21 (FGF21) has been proposed to be an antiaging hormone on the basis of experimental studies in rodent models. However, circulating FGF21 levels are increased with aging in rodents and humans. Moreover, despite the metabolic health-promoting effects of FGF21, the levels of this hormone are increased under conditions such as obesity and diabetes, an apparent incongruity that has been attributed to altered tissue responsiveness to FGF21. Here, we investigated serum FGF21 levels and expression of genes encoding components of the FGF21-response molecular machinery in adipose tissue from healthy elderly individuals (≥70 years old) and young controls. Serum FGF21 levels were increased in elderly individuals and were positively correlated with insulinemia and HOMA-IR, indices of mildly deteriorated glucose homeostasis. Levels of ß-Klotho, the coreceptor required for cellular responsiveness to FGF21, were increased in subcutaneous adipose tissue from elderly individuals relative to those from young controls, whereas FGF receptor-1 levels were unaltered. Moreover, total ERK1/2 protein levels were decreased in elderly individuals in association with an increase in the ERK1/2 phosphorylation ratio relative to young controls. Adipose explants from aged and young mice respond similarly to FGF21 "ex vivo". Thus, in contrast to what is observed in obesity and diabetes, high levels of FGF21 in healthy aging are not associated with repressed FGF21-responsiveness machinery in adipose tissue. The lack of evidence for impaired FGF21 responsiveness in adipose tissue establishes a distinction between alterations in the FGF21 endocrine system in aging and chronic metabolic pathologies.

Altered expression of nucleoside transporter genes (SLC28 and SLC29) in adipose tissue from HIV-1-infected patients.

BACKGROUND: Nucleoside transporter proteins (NTs) encoded by members of the SLC28 and SLC29 gene families contribute to nucleoside and nucleobase recycling but also modulate extracellular adenosine levels and thus adenosine-regulated metabolic targets. METHODS: We have examined the expression pattern of NT-encoding genes in human adipose tissue and we have further analysed whether the mRNA related to these genes show changes in their amounts associated with either HIV-1 infection, highly active antiretroviral therapy (HAART) or development of HIV-1-associated lipodystrophy syndrome (HALS). RESULTS: Human adipocytes express SLC28A1, SLC28A2 and SLC28A3 (encoding hCNT1, hCNT2 and hCNT3, respectively) and SLC29A1 and SLC29A2 (encoding hENT1 and hENT2, respectively). HIV-1 infection, prior to HAART and HALS development, is associated with the upregulation of the mRNA levels of the genes encoding hCNT1, hCNT3 and hENT2. The increase in the mRNA amounts for the former two genes may be due to the action of tumour necrosis factor-alpha (TNF-alpha), a cytokine with enhanced expression in adipose tissue following HIV-1 infection, as the effect is also observed in human adipocytes in culture after treatment with TNF-alpha. HAART and HALS development are associated with the upregulation of the mRNA levels encoding hCNT2 and hENT1, and further enhancement of hCNT1, hCNT3 and hENT2 gene expression. CONCLUSIONS: These data suggest that selected genes of the SLC28 and SLC29 families are not only targets of HIV-1 infection, but might also contribute to the development of adipose tissue alterations leading to lipodystrophy.

Effect of TLR ligands co-encapsulated with multiepitopic antigen in nanoliposomes targeted to human DCs via Fc receptor for cancer vaccines.

Nanoliposomes (NLs) hold promise as new highly specific nanomedicine for anti-tumor vaccines, since they could be targeted to specific receptors on dendritic cell (DC) to induce maturation and activation and increase the anti-tumor immune response. Here we studied a NLs formulation targeted or not to FcR (the receptor for the IgG Fc fragment) for the treatment of androgen-responsive prostate cancer. Luteinizing-hormone-releasing hormone (LHRH) peptide (B- and T-cell epitopes), in tandem with a tetanus toxoid T-helper epitope (830-844 region) and several TLR (Toll-Like Receptor) ligands as adjuvants were co-encapsulated. Specific uptake in vitro of LHRH-TT liposomes targeted to the FcRs of human DCs was enhanced. DC maturation/activation, cytokine production and lymphocyte activation were consistently higher in targeted than non-targeted liposomes. Similar increase was observed as more adjuvants were administrated. Targeting to specific receptor and co-encapsulation of several TLR adjuvants are essential factors for the immune response in peptide based liposome vaccine.

High FGF21 levels are associated with altered bone homeostasis in HIV-1-infected patients.

BACKGROUND: Fibroblast growth factor-21 (FGF21) has emerged as an important regulator of glucose, lipid, and body weight homeostasis. However, recent experimental studies have reported that increased FGF21 levels may lead to bone loss. OBJECTIVE: To assess the relationship of serum FGF21 levels and altered bone homeostasis in HIV-1-infected patients. DESIGN: Cross-sectional study of 137 HIV-1-infected patients and 35 healthy controls conducted at the Hospital de la Santa Creu i Sant Pau, Barcelona. Among HIV-1-infected patients, 35 were untreated (naïve), 43 were treated with antiretrovirals (HIV-1/ART) with no lipodystrophy, and 59 patients were HIV-1/ART and experienced lipodystrophy. Bone mineral density (BMD) and content (BMC) were assessed using dual-energy X-ray absorptiometry. Serum levels of FGF21, receptor activator of nuclear factor (NF)-KB ligand (RANKL), and C-telopeptide of type-I collagen (CTX-1) were measured by enzyme-linked immunosorbent assays. Serum levels of osteocalcin, osteoprotegerin, leptin, tumor necrosis factor-α, interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 were determined using an antibody-linked, fluorescently labeled microsphere bead-based multiplex analysis system. RESULTS: Alterations in bone parameters and bone homeostasis marker levels were consistent with higher turnover and bone loss in HIV-1 infected patients. FGF21 correlated negatively with BMD and BMC. FGF21 correlated positively with serum levels of osteoprotegerin and CTX-1, as well as with the CTX-1/osteocalcin ratio. CONCLUSIONS: Elevated FGF21 levels are associated with poor bone homeostasis in HIV-1-infected patients. Increases in FGF21 serum level may be an indicator not only of metabolic derangement but it may also serve as a biomarker of altered bone homeostasis in HIV-1 infected patients.

Natural Docosahexaenoic Acid in the Triglyceride Form Attenuates In Vitro Microglial Activation and Ameliorates Autoimmune Encephalomyelitis in Mice.

Many neurodegenerative diseases are associated, at least in part, to an inflammatory process in which microglia plays a major role. The effect of the triglyceride form of the omega-3 polyunsaturated fatty acid docosahexaenoic acid (TG-DHA) was assayed in vitro and in vivo to assess the protective and anti-inflammatory activity of this compound. In the in vitro study, BV-2 microglia cells were previously treated with TG-DHA and then activated with Lipopolysaccharide (LPS) and Interferon-gamma (IFN-γ). TG-DHA treatment protected BV-2 microglia cells from oxidative stress toxicity attenuating NO production and suppressing the induction of inflammatory cytokines. When compared with DHA in the ethyl-ester form, a significant difference in the ability to inhibit NO production in favor of TG-DHA was observed. TG-DHA inhibited significantly splenocyte proliferation but isolated CD4+ lymphocyte proliferation was unaffected. In a mice model of autoimmune encephalomyelitis (EAE), 250 mg/kg/day oral TG-DHA treatment was associated with a significant amelioration of the course and severity of the disease as compared to untreated animals. TG-DHA-treated EAE mice showed a better weight profile, which is a symptom related to a better course of encephalomyelitis. TG-DHA may be a promising therapeutic agent in neuroinflammatory processes and merit to be more extensively studied in human neurodegenerative disorders.

HIV-1 infection alters gene expression in adipose tissue, which contributes to HIV- 1/HAART-associated lipodystrophy.

BACKGROUND: The aetiopathogenic bases of HIV-l-/highly active antiretroviral treatment (HAART)-associated lipodystrophy (HALS) are poorly known, but this syndrome indicates that adipose tissue is highly sensitive to either HIV-1 infection, antiretroviral drugs or their combination. METHODS: To assess the relative contribution of infection and drugs, we compared the expression of marker genes corresponding to mitochondrial function, adipocyte differentiation and metabolism, and adipokines in subcutaneous adipose tissue from healthy controls, untreated HIV-1-infected patients, and HIV-1-infected patients treated with HAART with or without HALS. RESULTS: Subcutaneous adipose tissue from HIV-1-infected patients contained lower concentrations of the mRNA of the mitochondrial DNA-encoded cytochrome c oxidase subunit II than that of controls. These concentrations decreased further in association with HAART. The expression of nuclear genes coding for mitochondrial proteins, peroxisome proliferator-activated receptor-y, and adipocyte-specific markers was reduced in HIV-1-infected patients, treated or not, with respect to the controls. In contrast, the mRNA concentrations of uncoupling protein-3 and preadipocyte factor-1 increased in lipody-strophic HAART-treated patients. The genes coding for adipokines were strongly affected: tumour necrosis factor-alpha was upregulated, whereas adiponectin and leptin were downregulated in HIV-1-infected patients, treated or not. Thus, substantial alterations of gene expression were already present when naive patients were compared with controls. Further changes were associated with HAART and with the diagnosis of HALS. CONCLUSIONS: Disturbances in adipose tissue gene expression are already present in untreated HIV-1-infected patients, thus indicating a role of HIV-1 infection itself in eliciting adipose tissue alterations that are worsened by HAART, which ultimately leads to HALS.

Impact of elvitegravir on human adipocytes: Alterations in differentiation, gene expression and release of adipokines and cytokines.

Elvitegravir is a recently developed integrase inhibitor used for antiretroviral treatment of HIV infection. Secondary effects, including disturbances in lipid metabolism and, ultimately, in adipose tissue distribution and function, are common concerns associated with antiretroviral treatments. Here, we provide the first study of the effects of elvitegravir (in comparison with efavirenz, a non-nucleoside analog inhibitor of reverse transcriptase; and raltegravir, another integrase inhibitor) on human adipocyte differentiation, gene expression and secretion of adipokines and cytokines. Elvitegravir impaired adipogenesis and adipocyte metabolism in human SGBS adipocytes in a concentration-dependent manner (delaying acquisition of adipocyte morphology and reducing the expression of adipogenesis marker genes such as PPARγ, glucose transporter GLUT4, lipoprotein lipase, and the adipokines adiponectin and leptin). Compared with efavirenz, the effects of elvitegravir were similar but tended to occur at higher concentrations than those elicited by efavirenz, or were somewhat less intense than those caused by efavirenz at similar concentration. Elvitegravir tended to cause a more moderate induction of pro-inflammatory cytokines than efavirenz. Efavirenz induced a marked concentration-dependent increase in interleukin-8 expression and release whereas elvitregravir had little effect. Raltegravir had totally neutral actions of adipogenesis, adipocyte metabolism-related gene expression and release of adipokines and cytokines. In conclusion, elvitegravir alters adipocyte differentiation and function and promotes induction of pro-inflammatory cytokines similarly to efavirenz, but several effects were less intense. Further assessment of lipid metabolism and adipose tissue function in patients administered elvitegravir-based regimes is advisable considering that totally neutral effects of elvitegravir on lipid homeostasis cannot be anticipated from the current study in vitro.

Reverse transcriptase inhibitors alter uncoupling protein-1 and mitochondrial biogenesis in brown adipocytes.

OBJECTIVE: Human adipose depots contain remnant brown adipocytes interspersed among white adipocytes, and disturbances of brown with respect to white adipocyte biology have been implicated in highly active antiretroviral therapy (HAART)-induced lipomatosis. Brown adipocytes express the uncoupling protein-1 (UCP1) and contain a large number of mitochondria, potential targets of HAART toxicity. The aim of this study was to evaluate the effects of reverse transcriptase inhibitors (RTIs) on primary brown adipocytes differentiated in culture. DESIGN AND METHODS: We analysed the effects of RTIs, nucleoside analogues (NRTIs: stavudine, zidovudine, didanosine and lamivudine) and non-nucleoside analogues (NNRTIs: nevirapine and efavirenz), on differentiation, mitochondrial biogenesis and gene expression in brown adipocytes. RESULTS: None of the NRTIs altered brown adipocyte differentiation whereas NNTRIs had differing effects. Efavirenz blocked lipid deposition and expression of adipose marker genes but nevirapine induced lipid accumulation and adipose gene expression, promoted mitochondrial biogenesis and increased UCP1. Stavudine, zidovudine and didanosine reduced mitochondrial DNA (mtDNA) content. However, mitochondrial genome expression was only impaired in didanosine-treated adipocytes. Stavudine, but not zidovudine, induced expression of the mitochondrial transcription factors and this may explain compensatory mechanisms for the depletion of mtDNA by up-regulating mtDNA transcription. Stavudine caused a specific induction of UCP1 gene expression through direct interaction with a retinoic acid-dependent pathway. CONCLUSIONS: Specific disturbances in brown adipocytes in adipose depots may contribute to HAART-induced lipomatosis. Mitochondrial depletion does not appear to be the only mechanism explaining adverse effects in brown adipocytes because there is evidence of compensatory mechanisms that maintain mtDNA expression, and the expression of the UCP1 gene is specifically altered.

Liposomes containing NY­ESO­1/tetanus toxoid and adjuvant peptides targeted to human dendritic cells via the Fc receptor for cancer vaccines.

AIM: To improve the immunological response against tumors, a vaccine based on nanoliposomes targeted to the Fcg-receptor was developed to enhance the immunogenicity of tumor-associated antigens (TAAs). MATERIALS & METHODS: Using human dendritic cells in vitro, a fragment of the TAA NY-ESO-1 combined with a T-helper peptide from the tetanus toxoid encapsulated in nanoliposomes was evaluated. In addition, peptides Palm-IL-1 and MAP-IFN-g were coadministered as adjuvants to enhance the immunological response. RESULTS: Coadministration of Palm-IL-1 or MAP-IFN-g peptide adjuvants and the hybrid NY-ESO-1-tetanus toxoid (soluble or encapsulated in nanoliposomes without targeting) increased immunogenicity. However, the most potent immunological response was obtained when the peptide adjuvants were encapsulated in liposomes targeted to human dendritic cells via the Fc receptor. CONCLUSION: This targeted vaccine strategy is a promising tool to activate and deliver antigens to dendritic cells, thus improving immunotherapeutic response in situations in which the immune system is frequently compromised, as in advanced cancers.

Hypertrophied facial fat in an HIV-1-infected patient after autologous transplantation from 'buffalo hump' retains a partial brown-fat-like molecular signature.

BACKGROUND: Facial lipoatrophy, a common alteration among HIV-1-infected, antiretroviral-treated patients, is often corrected using autologous transplantation. In some cases, especially when enlarged adipose tissue from the dorso-cervical area (that is, a 'buffalo hump') is used as a source of fat for transplantation, the transplanted fat develops progressive hypertrophy. To gain insight into the molecular basis of this phenomenon, we evaluated the cell morphology and gene expression in this hypertrophied facial fat. METHODS: Quantitative real-time PCR was used to examine the expression of various marker genes in a sample of facial fat that underwent hypertrophy after autologous transplantation. The results were compared with gene expression data from 'buffalo hump' fat and subcutaneous fat from healthy controls. Optical and electron microscopic analyses were used to determine cell morphology. RESULTS: The enlarged facial adipose tissue did not exhibit the overt microscopic morphology of brown adipose tissue but (similar to 'buffalo hump' fat) it contained adipocytes heterogeneous in size. The enlarged facial fat retained the partial molecular signature of a distorted brown-to-white adipocyte phenotype, including expression of uncoupling protein-1 (UCP1) transcript, and showed unaltered adipogenesis and inflammation that are characteristic of 'buffalo hump' fat. CONCLUSIONS: Despite being implanted in a former lipoatrophic area, facially grafted 'buffalo hump' tissue appears to retain the altered phenotype of dorso-cervical adipose cells, thus accounting for its progressive enlargement. These results argue that caution should be exercised when considering 'buffalo hump' fat depots as a fat source for autologous transplantation.

Differentially altered molecular signature of visceral adipose tissue in HIV-1-associated lipodystrophy.

OBJECTIVE: Lipodystrophy in HIV-1-infected antiretroviral-treated patients is often associated with opposite alterations in adipose tissue depots as follows: lipoatrophy of subcutaneous adipose tissue (SAT) versus lipohypertrophy of visceral adipose tissue (VAT). We determined the specific molecular alterations in VAT relative to SAT in patients. DESIGN: We analyzed the expression of marker genes of mitochondrial function, adipogenesis, and inflammation in a unique collection of 8 biopsies of omental VAT from HIV-1-infected antiretroviral-treated patients with lipodystrophy. For comparison, we analyzed SAT from 10 patients, and SAT and VAT from 10 noninfected individuals. METHODS: Quantitative real-time polymerase chain reaction of mitochondrial DNA and gene transcripts; immunoblot and multiplex for quantification of specific proteins. RESULTS: Similar mitochondrial DNA depletion and abnormal increases in mitochondrial protein levels were found in VAT and SAT from patients. Transcript levels of adipogenesis and metabolism marker genes were unaltered in VAT but were decreased in SAT. Tumor necrosis factor α and CD68 were similarly induced in both adipose depots from patients, but other markers of inflammation-related pathways showed distinct alterations as follows: interleukin 18 and interleukin 1 receptor antagonist were induced only in SAT, whereas interleukin 6, interleukin 8, and monocyte chemoattractant protein 1 expression was reduced in VAT but not in SAT. CONCLUSIONS: Mitochondrial alterations are similar in VAT and SAT from patients, whereas adipogenic gene expression is decreased in SAT but unaltered in VAT, highlighting the relevance of adipogenic processes in the differential alterations of fat depots. Specific disturbances in inflammatory status in VAT relative to SAT are present. Milder induction of proinflammatory signaling in VAT could be involved in preventing fat wasting in this depot.

Effects of nevirapine and efavirenz on human adipocyte differentiation, gene expression, and release of adipokines and cytokines.

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz are drugs of choice for initial antiretroviral treatment for HIV-1 infection. Although NNRTIs have not traditionally been associated with the appearance of adipose alterations, recent data suggest that efavirenz may contribute to adipose tissue alterations in antiretroviral-treated patients, consistent with its ability to impair differentiation of adipocytes in cell cultures. No such effects have been reported for nevirapine, the other most commonly used NNRTI. In this study, we determined the effects of nevirapine on differentiation, gene expression and release of regulatory proteins (adipokines and cytokines) in differentiating human adipocytes, and compared them with those of efavirenz. Efavirenz caused a dose-dependent repression of adipocyte differentiation that was associated with down-regulation of the master adipogenesis regulator genes SREBP-1, PPARγ and C/EBPα, and their target genes encoding lipoprotein lipase, leptin and adiponectin, which are key proteins in adipocyte function. In contrast, nevirapine does not affect adipogenesis and causes a modest but significant coordinate increase in the expression of SREBP-1, PPARγ and C/EBPα and their target genes only at a concentration of 20 µM. Whereas efavirenz caused a significant increase in the release of pro-inflammatory cytokines (interleukin [IL]-8, IL-6, monocyte chemoattractant protein-1), plasminogen activator inhibitor type-1 and hepatocyte growth factor (HGF), nevirapine either had no effect on these factors or decreased their release (IL-6 and HGF). Nevirapine significantly increased adiponectin release, whereas efavirenz strongly repressed it. Moreover, nevirapine inhibited preadipocyte endogenous reverse transcriptase activity, whereas efavirenz did not alter it. It is concluded that, in contrast with the profound anti-adipogenic and pro-inflammatory response elicited by efavirenz, nevirapine does not impair adipogenesis.

Differential effects of efavirenz and lopinavir/ritonavir on human adipocyte differentiation, gene expression and release of adipokines and pro-inflammatory cytokines.

In the present study, a comparative assessment of the effects of efavirenz (EFV) and lopinavir/ritonavir (LPV/r; 4:1) on human adipocytes in culture has been performed. Human pre-adipocytes were treated with EFV or LPV/r during or after adipogenic differentiation. Acquisition of adipocyte morphology, expression of gene markers of mitochondrial toxicity, adipogenesis and inflammation, and release of adipokines and cytokines to the medium were measured. Results indicated that EFV and LPV/r impaired adipocyte differentiation in association with a reduction in transcript levels for adipogenic differentiation genes (adiponectin, lipoprotein lipase, leptin) and master regulators of adipogenesis (PPAR, C/EBP). The effects were greater with EFV than LPV/r. Both LPV/r and EFV induced increases in monocytechemoattactant protein-1 (MCP-1) mRNA levels, but the effect was greater with EFV. Similarly, the release of proinflammatory cytokines and other inflammation-related molecules (interleukins 6 and 8, MCP-1, PAI-1) was enhanced to a much higher degree by EFV than by LPV/r. Adiponectin and leptin release by adipocytes was reduced by both drugs, although to a higher extent by EFV. Neither drug affected mitochondrial DNA levels, transcripts encoding mitochondrial proteins or lactate release by adipocytes. In previously differentiated adipocytes, EFV caused a significant reduction in PPARγ and adiponectin expression, whereas LPV/r did not. We conclude that both EFV and LPV/r impair human adipogenesis, reduce adipokine release and increase the expression and release of inflammation-related cytokines, but the overall effects are greater with EFV. These findings may have implications for the pathogenesis of HIV-1-associated lipodystrophy and the development of HIV-1 therapies.