Transmitted drug resistance to NRTIs and risk of virological failure in naïve patients treated with integrase inhibitors.

OBJECTIVES: Nucleoside reverse transcriptase inhibitor (NRTI) transmitted drug resistance mutations (TDRMs) could increase the risk of virological failure (VF) of first-line integrase strand transfer inhibitor (InSTI)-based regimens. METHODS: Patients starting two NRTIs (lamivudine/emtricitabine plus abacavir/tenofovir) plus raltegravir or dolutegravir were selected from the EuResist cohort. The role of NRTI genotypic susceptibility score and of specific TDRMs in VF (i.e. two consecutive viral loads > 50 HIV-1 RNA copies/mL or a single viral load ≥ 200 copies/mL after 3 months from antiretroviral therapy start) was evaluated in the overall population and according to the InSTI employed. RESULTS: From 2008 to 2017, 1095 patients were eligible for the analysis (55.5% men, median age 39 years). In all, 207 VFs occurred over 1023 patient-years of follow-up. The genotypic susceptibility score (GSS) had no effect on the risk of VF in the overall population. However, the presence of M184V/I independently predicted VF of raltegravir- but not dolutegravir-based therapy when compared with a fully-active backbone [adjusted hazard ratio (aHR) = 3.09, P = 0.035], particularly when associated with other non-thymidine analogue mutations (aHR = 27.62, P = 0.004). Higher-zenith HIV-RNA and lower nadir CD4 counts independently predicted VF. CONCLUSIONS: NRTI backbone TDRMs increased the risk of VF with raltegravir-based but not dolutegravir-based regimens.

Efficacy of etravirine combined with darunavir or other ritonavir-boosted protease inhibitors in HIV-1-infected patients: an observational study using pooled European cohort data.

OBJECTIVES: This observational study in antiretroviral treatment-experienced, HIV-1-infected adults explored the efficacy of etravirine plus darunavir/ritonavir (DRV group; n = 999) vs. etravirine plus an alternative boosted protease inhibitor (other PI group; n = 116) using pooled European cohort data. METHODS: Two international (EuroSIDA; EUResist Network) and five national (France, Italy, Spain, Switzerland and UK) cohorts provided data (collected in 2007-2012). Stratum-adjusted (for confounding factors) Mantel-Haenszel differences in virological responses (viral load < 50 HIV-1 RNA copies/mL) and odds ratios (ORs) with 95% confidence intervals (CIs) were derived. RESULTS: Baseline characteristics were balanced between groups except for previous use of antiretrovirals (≥ 10: 63% in the DRV group vs. 49% in the other PI group), including previous use of at least three PIs (64% vs. 53%, respectively) and mean number of PI resistance mutations (2.3 vs. 1.9, respectively). Week 24 responses were 73% vs. 75% (observed) and 49% vs. 43% (missing = failure), respectively. Week 48 responses were 75% vs. 73% and 32% vs. 30%, respectively. All 95% CIs around unadjusted and adjusted differences encompassed 0 (difference in responses) or 1 (ORs). While ORs by cohort indicated heterogeneity in response, for pooled data the difference between unadjusted and adjusted for cohort ORs was small. CONCLUSIONS: These data do not indicate a difference in response between the DRV and other PI groups, although caution should be applied given the small size of the other PI group and the lack of randomization. This suggests that the efficacy and virology results from DUET can be extrapolated to a regimen of etravirine with a boosted PI other than darunavir/ritonavir.

Prediction of response to antiretroviral therapy by human experts and by the EuResist data-driven expert system (the EVE study).

OBJECTIVES: The EuResist expert system is a novel data-driven online system for computing the probability of 8-week success for any given pair of HIV-1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment. METHODS: The EuResist system was compared with 10 HIV-1 drug resistance experts for the ability to predict 8-week response to 25 treatment cases derived from the EuResist database validation data set. All current and past patient data were made available to simulate clinical practice. The experts were asked to provide a qualitative and quantitative estimate of the probability of treatment success. RESULTS: There were 15 treatment successes and 10 treatment failures. In the classification task, the number of mislabelled cases was six for EuResist and 6-13 for the human experts [mean±standard deviation (SD) 9.1±1.9]. The accuracy of EuResist was higher than the average for the experts (0.76 vs. 0.64, respectively). The quantitative estimates computed by EuResist were significantly correlated (Pearson r=0.695, P<0.0001) with the mean quantitative estimates provided by the experts. However, the agreement among experts was only moderate (for the classification task, inter-rater κ=0.355; for the quantitative estimation, mean±SD coefficient of variation=55.9±22.4%). CONCLUSIONS: With this limited data set, the EuResist engine performed comparably to or better than human experts. The system warrants further investigation as a treatment-decision support tool in clinical practice.

Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.

In this Phase I trial, patients' peripheral blood dendritic cells were pulsed with peptides eluted from the surface of autologous glioma cells. Three biweekly intradermal vaccinations of peptide-pulsed dendritic cells were administered to seven patients with glioblastoma multiforme and two patients with anaplastic astrocytoma. Dendritic cell vaccination elicited systemic cytotoxicity in four of seven tested patients. Robust intratumoral cytotoxic and memory T-cell infiltration was detected in two of four patients who underwent reoperation after vaccination. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed dendritic cell vaccine for patients with malignant glioma.

HIV-1 tat protein induces the production of interleukin-8 by human brain-derived endothelial cells.

This study focused on the role of the HIV-derived viral protein, tat, in activating central nervous system (CNS)-derived endothelial cells (EC) to produce interleukin-8 (IL-8), a stimulator and chemoattractant for neutrophils and lymphocytes. Human CNS-EC treated with tat (100 ng/ml) demonstrated a 2 to 3 fold upregulation in IL-8 mRNA and protein. Tumor necrosis factor-alpha (TNF) and tat were found to act additively in upregulating IL-8 production. In contrast, transforming growth factor beta (TGF beta), appeared to down modulate tat-induced IL-8 production. These data suggest that extracellular tat, especially in the presence of TNF, may be responsible for the local production of IL-8.

Endothelin-1 induces production of the neutrophil chemotactic factor interleukin-8 by human brain-derived endothelial cells.

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1-stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor-alpha (TNF) and IL-1beta functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-beta (TGF-beta), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

Thrombospondin, a negative modulator of megakaryocytopoiesis.

The effects of native thrombospondin (TSP), an 18 kd recombinant protein comprising residues 1-174 of TSP (TSP1-174) with heparin-binding domain and a fusion protein comprising residues 559-669 of TSP (TSP559-669) on murine hematopoiesis, were studied by using different in vitro culture systems. TSP by itself did not show an inhibitory effect on colony-forming unit-megakaryocyte (CFU-MK) growth in a serum-free agar system and on the growth of colony-forming unit-granulocyte and macrophage (CFU-GM) in a plasma clot system. It was, however, found that in the plasma clot culture system when using aplastic anemia serum as the source of thrombopoietin or C-Mpl ligand (TPO), TSP and TSP1-174, but not TSP559-669, were able to inhibit the growth of CFU-MK from unfractionated and lineage negative (Lin-) bone marrow cells in a dose-dependent manner. A statistically significant suppression was seen at 1 microg/ml of TSP and 5 microg/ml of TSP1-174. This inhibitory effect of TSP was further found in both the serum-free agar system and the plasma clot system without aplastic anemia serum, where megakaryocyte colony growth was stimulated by recombinant TPO, basic fibroblast growth factor (bFGF), or interleukin-3 (IL-3). In a methylcellulose system, where a combination of stem cell factor (SCF), IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) were used, TSP inhibited the growth of colony-forming unit-granulocyte-erythroblast, megakaryocyte, and macrophage (CFU-GEMM) but not CFU-GM and burst-forming unit-erythroblast (BFU-E). Interestingly, this inhibitory effect of TSP on megakaryocyte colony growth could be counteracted by Fraxiparin, a low-molecular-weight heparin. These results demonstrate that TSP is a negative modulator of megakaryocytopoiesis and suggest that its inhibitory effect is at least partially mediated by N-terminal heparin-binding domain.

Heparin-binding domain, type 1 and type 2 repeats of thrombospondin mediate its interaction with human breast cancer cells.

Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 micrograms/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 micrograms/ml) had little or no effect. Heparin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two beta-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity.

How much are homologous peptides homologous?

A statistical analysis designed to deal with the problem of identifying homologous pairs of "short sequences of amino-acids" (= peptides) belonging to different proteins is presented. The conceptual novelty of the searching strategy proposed here lies in the fact that both the degree of homology of the two peptides of the pair (measured by a suitably defined affinity score) and the level of statistical significance of its occurrence are taken into account on equal footing. They are combined in a sort of "biological indicator", characterising each pair. Pairs for which the value of the biological indicator is larger than an appropriate threshold are taken as statistically significant and (putatively) biologically relevant. The method is employed in various test cases and proves to be reliable and efficient. In particular we have studied the cases in which the known existence of an auto-immune response has lead to the identification of homologous peptide pairs between human and viral or bacterial proteins. The detection efficiency of the algorithm in these cases turns out to be especially good when the most naïve affinity table, the Identity matrix, is employed to measure the similarity of amino acidic pairs. In contrast, when the 250-PAM mutation matrix is used, the detection efficiency goes to zero.

Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species.

The aim of the present study was to investigate the reactivity of immunoreagents developed for clinical applications in humans in different animal species (hen, mouse, rat, rabbit, guinea-pig, dog, pig, sheep, baboon). Prothrombin fragment 1 + 2, thrombin-antithrombin III complex and fibrinopeptide A were tested for coagulation, platelet factor 4 and beta-thromboglobulin for platelet activation, glycoprotein IIb-IIIa, glycoprotein Ib and P-selectin for platelet membrane glycoproteins, D-dimers for fibrinolysis, thrombomodulin for activation of endothelial cells and thrombospondin and von Willebrand factor for adhesive proteins. Prothrombin fragment 1 + 2, platelet factor 4, beta-thromboglobulin and D-dimers were revealed only in baboons. Fibrinopeptide A was well detected in baboons but weakly in mice, dogs, pigs and sheep. Whereas glycoprotein IIb-IIIa was revealed on guinea-pig, dog and sheep platelets and glycoprotein Ib on rabbit and dog platelets, P-selectin and thrombomodulin were never detected. Thrombospondin was revealed in hens, mice, rats, guinea-pigs, pigs, sheep and baboons and von Willebrand factor in mice, rats, guinea-pigs, dogs, pigs, sheep and baboons. Interestingly, thrombin-antithrombin III complex (TAT) was detected in all species tested except the hen. A time- and dose-dependent increase in TAT was observed when rats, dogs or pigs were infused with thromboplastin (4.5-450 microliters/kg/h), while administration of hirudin (1 mg/kg) abolished this TAT generation. Thus, the TAT immunoassay could provide a tool for the screening of antithrombotic drugs in a number of animal species. However, the possibility of using a wider panel of human immunoreagents would appear to be restricted to baboons which display good species cross-reactivity.

Thrombospondin modulates human breast adenocarcinoma cell adhesion to human vascular endothelial cells.

Thrombospondin (TSP), a M(r) 450,000 cytoadhesive glycoprotein, has been shown to potentiate tumor cell metastasis in mice by a mechanism that involves the hemostatic system of the host. In this study, the potential involvement of TSP in the interaction of human mammary adenocarcinoma MCF-7 cells with human umbilical vein endothelial cells (HUVECs) in culture was investigated. Using an ELISA, preconfluent HUVECs synthesized 100-fold more TSP than did MCF-7 cells during 24 h of culture (20 versus 0.2 microgram/10(6) cells). Confocal microscopy localized TSP within intercellular junctions between aggregated MCF-7 cells in suspension. On adherent cells, TSP exhibited a patchy distribution both on the cell surface and in the cytosol. In HUVECs, TSP strongly stained the perinuclear space and was also found in association with cytoskeletal microfibrils. Flow cytometric analysis indicated the presence of a large number of unoccupied receptors for TSP on MCF-7 cells. Binding studies using [125I]TSP demonstrated the presence of 1.6 x 10(6) sites/cell with an apparent Kd of 28 nM. Attachment of radiolabeled MCF-7 cells to a TSP-coated substrate and to HUVEC monolayers was inhibited in the presence of a polyclonal antibody to TSP (10 micrograms/ml) or increasing concentrations (1-10 micrograms/ml) of soluble TSP. Neither nonimmune IgG nor the cell adhesion peptide Gly-Arg-Gly-Asp-Ser (100 micrograms/ml) inhibited these interactions. Inhibition was also observed with heparin (10 micrograms/ml), suggesting the participation of TSP heparin-binding domain(s) and heparin-like molecules. In the presence of an excess of soluble TSP or anti-TSP antibody, MCF-7 cells did not form aggregates in suspension and preformed aggregates were readily dissociated by the addition of soluble TSP. These results indicate that mammary adenocarcinoma cells use TSP to form aggregates and to attach to human endothelial cells. These interactions may have physiological implications during the hematogenous spread of tumor cells.

Involvement of thrombospondin in the adherence of human breast-adenocarcinoma cells: a possible role in the metastatic process.

The attachment of cancer cells to adhesive molecules, such as laminin (LN) and fibronectin (FN) in the extracellular matrix is a critical step in tumor invasion and metastasis. Recent data have suggested a potential role for thrombospondin (TSP), a 420-kDa cyto-adhesive glycoprotein, in the growth and spread of breast cancer. In this study, we have measured the ability of the human breast adenocarcinoma cell line, MDA-MB-231, to synthesize TSP and to use this molecule as an adhesion factor. The level of TSP in cells and secreted into the culture medium were determined by an enzyme-linked immunosorbent assay (ELISA). At pre-confluence, MDA-MB-231 cells were shown to produce a high level of TSP, most of which was retained within the cells. In comparison, FN was almost entirely secreted into the culture medium. An increased secretion of TSP was however measured at low cell density, suggesting that TSP might be required for cell/substratum or cell/cell interactions. As shown by flow cytometry, the cells expressed membrane-bound TSP as well as unoccupied TSP receptors. 125I-TSP bound saturably to 1.2 x 10(6) sites per cell with an apparent dissociation constant of 23 nM. The binding was inhibited by an excess of unlabeled TSP and by heparin, suggesting that the receptor could be a heparan-sulfate proteoglycan or a sulfatide. TSP promoted attachment but not spreading of MDA-MB-231 cells which attached and spread on FN and LN substrates. These results suggest that endogenously synthesized TSP may have a role in the cyto-adherence of tumor cells during the spread of breast cancer.