Lipid levels,insulin resistance and cardiovascular risk over 96 weeks of antiretroviral therapy: a randomised controlled trial comparing low-dose stavudine and tenofovir

Background

HIV infection and antiretroviral treatment are associated with changes in lipid levels, insulin resistance and risk of cardiovascular disease (CVD). We investigated these changes in the first 96 weeks of treatment with low-dose stavudine or tenofovir regimens.

Methods

This is a secondary analysis of a double blind, randomised controlled trial performed in South-Africa, Uganda and India comparing low-dose stavudine (20 mg twice daily) with tenofovir in combination with efavirenz and lamivudine in antiretroviral-naïve adults (n?=?1067) (Clinicaltrials.gov, NCT02670772). Over 96 weeks, data were collected on fasting lipids, glucose and insulin. Insulin resistance was assessed with the HOMA-IR index and 10-year CVD risk with the Framingham risk score (FRS). A generalized linear mixed model was used to estimate trends over time.

Results

Participants were on average 35.3 years old, 57.6% female and 91.8% Black African. All lipid levels increased following treatment initiation, with the sharpest increase in the first 24 weeks of treatment. The increase in all lipid subcomponents over 96 weeks was higher among those in the stavudine than the tenofovir group. Insulin resistance increased steadily with no difference detected between study groups. FRS rose from 1.90% (1.84–1.98%) at baseline to 2.06 (1.98–2.15%) at week 96 for the total group, with no difference between treatment arms (p?=?0.144). Lipid changes were more marked in Indian than African participants.

Conclusion

Lipid levels increased in both groups, with low-dose stavudine resulting in a worse lipid profile compared to tenofovir. Insulin resistance increased, with no difference between regimens. CVD risk increased over time and tended to increase more in the group on stavudine. The low CVD risk across both arms argues against routine lipid and glucose monitoring in the absence of other CVD risk factors. In high risk patients, monitoring may only be appropriate at least a year after treatment initiation.

     

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman’s disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL- 6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.     

The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies.

Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia. The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life. X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic. We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5. In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies. Mutations in the G4.5 gene were found in nine of the patients analyzed. The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069). Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a "less severe" phenotype.     

Cyclic variations in cell-mediated immunity to skin allografts detected by the technetium-99m microcytotoxicity assay

The technetium-99m microcytotoxicity assay has been used to detect cell-mediated immunity in CS7BL/6 mice sensitized with A/J skin allografts. Our initial studies of the quantitative in vitro assessment of lymphocyte-mediated cytotoxicity in mice rejecting first-set skin allografts revealed a simple monophasic response peaking at 14 days postgrafting and declining to control levels by 21 days. Subsequent experiments in which the development and persistence of immunity was assessed at daily intervals from 9–21 days postgrafting revealed that the response was considerably more complex. A cyclic rise and fall in killer activity was evident. The first peak occurred 10–13 days after grafting and the second one 3–5 days later. A third peak of cytotoxic activity sometimes could be detected 16–19 days postgrafting. An attempt was made to characterize the phenomenon by studying the cytocidal effects resulting from the admixture of high- and low-responding lymphocyte populations. An intermediate effect generally was observed when lymphocytes with maximal killer activity were combined in equal numbers with those having decreased reactivity. Varying the ratio of high-and low-responding cells resulted in changes in the net killing effect which was consistent with dilution of more reactive lymphocytes with less reactive ones. Mixing lymphocytes from two peak periods produced a maximum killing effect at all effector to target cell multiplicities. Failure to demonstrate modulation of the reactivity of high-responding cells by low-responding ones suggests that these cyclic variations were not mediated by suppressor cells although a role for humoral factors cannot be excluded at the present time. Alternatively, the cyclic pattern may have been due to the specific depletion and subsequent regeneration of cytotoxic lymphocytes in the lymph nodes of sensitized animals.     

Probing the activity of a recombinant Zn2+‐transporting P‐type ATPase

P‐type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP‐driven ion transport across biological membranes. Characterization of single‐cycle dynamics by time‐resolved X‐ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo‐electron microscopy sample preparation. To pave way for such time‐resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time‐dependent Fourier‐Transform Infra‐Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn²⁺‐transporting Type‐I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.     

Proton paths in the sarcoplasmic reticulum Ca(2+) -ATPase

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.