Extraction and Serological Properties of Mycobacterium Cell Surface and Excreted Proteins

© 2017, Springer Science+Business Media, LLC, part of Springer Nature. Modern medicine still faces the task of distinguishing active and latent tuberculosis cases at the early stage of the disease. Serological approaches have their advantages for their use in diagnostics. However, the progress of these approaches is ongoing but further progress is needed to meet the needs for this disease. Here, we extracted Mycobacterium tuberculosis H37Rv proteins from culture medium or from the cell surface and studied their reactivity with anti-M. tuberculosis serum in both ELISA and immunoblots. We found that M. tuberculosis surface proteins, extracted using dimethyl sulfoxide, with molecular weights in the range of 6.5–200 kDa, showed strong specific reactivity with anti-M. tuberculosis positive serum. While excreted proteins in the molecular weight range of 32–45 kDa had the highest reactivity. The latter was confirmed serologically when very weak signal was detected from the filtrate fractions compared to stronger activity from the Vivaspin 50 kDa MWCO retentates. Moreover, Mycobacterium bovis and tuberculosis proteins from the filter retentates had clear specific serum reactivity, which suggests that this approach can be used for differential diagnosis of two infections

The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

<p>Abstract</p> <p>Background</p> <p>The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.</p> <p>Results</p> <p>The 386 carbohydrate was not essential for folding of <it>wt </it>gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.</p> <p>Conclusion</p> <p>The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.</p

Statins Disrupt CCR5 and RANTES Expression Levels in CD4(+) T Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody

&lt;b&gt;BACKGROUND:&lt;/b&gt; In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo.&lt;p&gt;&lt;/p&gt; &lt;b&gt;RESULTS:&lt;/b&gt; Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134.&lt;p&gt;&lt;/p&gt; &lt;b&gt;CONCLUSIONS:&lt;/b&gt; The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.&lt;p&gt;&lt;/p&gt

ОЦЕНКА ЦИТОТОКСИЧНОСТИ ТРИХОТЕЦЕНА FUSARIUM SP. НА ЛИНИЮ РАКА МОЛОЧНОЙ ЖЕЛЕЗЫ IN VITRO

trichothecenes and their derivatives have recently attracted much attention of researchers with respect of their potential role in medicine, including for the treatment of different types of cancer. The purpose of the study was to investigate the cytotoxic effect of Fusarium trichothecene on human breast cancer cells,  human skin fibroblasts and embryonic kidney cells in vitro. Material and methods. Based on the Mtt assay, the cytotoxic effect of trichothecene on cell cultures was determined. Evaluation of morphological changes in cell cultures under the influence of trichothecene was performed by light microscopy. Results. Fusarium trichothecene was found to exhibit a dose-dependent toxic effect on cell lines in the range 1 nM to 1000 nM. the most pronounced cytotoxic effect of trichothecene was observed in human breast cancer cell line (IС50 94.72 ± 4.1 нМ). Lower doses of trichothecene led to a change in the size, shape of human breast cancer cells, human skin fibroblasts and embryonic kidney cells, and loss of contact between them and their isolation. the degradation of cell membranes, formation of unformed cell aggregates and fragments were observed in higher doses of trichothecene. Conclusion. Fusarium trichothecen is a biologically active compound and is less toxic to the normal than to the cancer cell lines, therefore, further studies of this agent are needed.В последнее время трихотеценовые соединения и их производные привлекают внимание исследователей в связи с их потенциальной возможностью применения в медицине, в том числе для лечения различных видов рака. Цель исследования – изучение цитотоксического действия трихотецена Fusarium sp. в отношении линий опухолевых клеток рака молочной железы, нормальных клеток фибробластов кожи и почек эмбриона человека in vitro. Материал и методы. С использованием общепринятого метода МТТ-теста проводилось определение цитотоксического действия трихотецена в отношении исследуемых культур клеток. Оценку изменения в морфологии клеток под воздействием трихотецена проводили методом световой микроскопии. Результаты. Было обнаружено, что трихотецен Fusarium sp. в диапазоне концентрации 1–1000 нM проявлял дозозависимое токсическое действие в отношении исследуемых линий клеток. Наиболее выраженное цитотоксическое действие трихотецена наблюдали при его действии на линию опухолевых клеток молочной железы (IС50 94,72 ± 4,1 нМ). Совместная инкубация трихотецена с линиями клеток рака молочной железы, клеток фибробластов кожи и почек эмбриона человека в более низких дозах приводила к изменению размеров, формы клеток, потере контактов между ними и их обособлению. При более высоких дозах трихотецена наблюдалась деградация мембран, образование неоформленных клеточных агрегатов и фрагментов (апоптозных тел). Заключение. Трихотецен Fusarium sp. обладает биологически активным потенциалом и является менее токсичным по отношению к нормальным клеткам человека по сравнению с опухолевыми, поэтому его целесообразно в дальнейшем исследовать как возможного противоопухолевого агента

Antiretroviral effect of lovastatin on HIV-1-infected individuals without highly active antiretroviral therapy (The LIVE study): a phase-II randomized clinical trial

<p>Abstract</p> <p>Background</p> <p>Highly active antiretroviral therapy produces a significant decrease in HIV-1 replication and allows an increase in the CD4 T-cell count, leading to a decrease in the incidence of opportunistic infections and mortality. However, the cost, side effects and complexity of antiretroviral regimens have underscored the immediate need for additional therapeutic approaches. Statins exert pleiotropic effects through a variety of mechanisms, among which there are several immunoregulatory effects, related and unrelated to their cholesterol-lowering activity that can be useful to control HIV-1 infection.</p> <p>Methods/design</p> <p>Randomized, double-blinded, placebo controlled, single-center, phase-II clinical trial. One hundred and ten chronically HIV-1-infected patients, older than 18 years and naïve for antirretroviral therapy (i.e., without prior or current management with antiretroviral drugs) will be enrolled at the outpatient services from the most important centres for health insurance care in Medellin-Colombia. The interventions will be lovastatin (40 mg/day, orally, for 12 months; 55 patients) or placebo (55 patients). Our primary aim will be to determine the effect of lovastatin on viral replication. The secondary aim will be to determine the effect of lovastatin on CD4+ T-cell count in peripheral blood. As tertiary aims we will explore differences in CD8+ T-cell count, expression of activation markers (CD38 and HLA-DR) on CD4 and CD8 T cells, cholesterol metabolism, LFA-1/ICAM-1 function, Rho GTPases function and clinical evolution between treated and not treated HIV-1-infected individuals.</p> <p>Discussion</p> <p>Preliminary descriptive studies have suggested that statins (lovastatin) may have anti HIV-1 activity and that their administration is safe, with the potential effect of controlling HIV-1 replication in chronically infected individuals who had not received antiretroviral medications. Considering that there is limited clinical data available on this topic, all these findings warrant further evaluation to determine if long-term administration of statins may benefit the virological and immunological evolution in HIV-1-infected individuals before the use of antiretroviral therapy is required.</p> <p>Trial registration</p> <p>Registration number NCT00721305.</p

HIV-2 interaction with cell coreceptors: amino acids within the V1/V2 region of viral envelope are determinant for CCR8, CCR5 and CXCR4 usage

© 2014 Santos-Costa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background: Human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2) use cellular receptors in distinct ways. Besides a more promiscuous usage of coreceptors by HIV-2 and a more frequent detection of CD4-independent HIV-2 isolates, we have previously identified two HIV-2 isolates (HIV-2MIC97 and HIV-2MJC97) that do not use the two major HIV coreceptors: CCR5 and CXCR4. All these features suggest that in HIV-2 the Env glycoprotein subunits may have a different structural organization enabling distinct - although probably less efficient - interactions with cellular receptors. Results: By infectivity assays using GHOST cell line expressing CD4 and CCR8 and blocking experiments using CCR8-specific ligand, I-309, we show that efficient replication of HIV-2MIC97 and HIV-2MJC97 requires the presence of CCR8 at plasma cell membrane. Additionally, we disclosed the determinants of chemokine receptor usage at the molecular level, and deciphered the amino acids involved in the usage of CCR8 (R8 phenotype) and in the switch from CCR8 to CCR5 or to CCR5/CXCR4 usage (R5 or R5X4 phenotype). The data obtained from site-directed mutagenesis clearly indicates that the main genetic determinants of coreceptor tropism are located within the V1/V2 region of Env surface glycoprotein of these two viruses. Conclusions: We conclude that a viral population able to use CCR8 and unable to infect CCR5 or CXCR4-positive cells, may exist in some HIV-2 infected individuals during an undefined time period, in the course of the asymptomatic stage of infection. This suggests that in vivo alternate molecules might contribute to HIV infection of natural target cells, at least under certain circumstances. Furthermore we provide direct and unequivocal evidence that the usage of CCR8 and the switch from R8 to R5 or R5X4 phenotype is determined by amino acids located in the base and tip of V1 and V2 loops of HIV-2 Env surface glycoprotein.This work was supported by grants from: Fundação para a Ciência e Tecnologia (FCT; PPCDT/SAU-IMI/55726/2004); Fundação para a Ciência e Tecnologia and Ministério da Saúde de Portugal (VIH/SAU/0006/2011); and from Gilead Sciences Portugal (Programa Gilead Génese).info:eu-repo/semantics/publishedVersio

Tracing the HIV-1 subtype B mobility in Europe: a phylogeographic approach

<p>Abstract</p> <p>Background</p> <p>The prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced.</p> <p>Results</p> <p>In the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred.</p> <p>Conclusion</p> <p>Subtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.</p

HIV interactions with monocytes and dendritic cells: viral latency and reservoirs

HIV is a devastating human pathogen that causes serious immunological diseases in humans around the world. The virus is able to remain latent in an infected host for many years, allowing for the long-term survival of the virus and inevitably prolonging the infection process. The location and mechanisms of HIV latency are under investigation and remain important topics in the study of viral pathogenesis. Given that HIV is a blood-borne pathogen, a number of cell types have been proposed to be the sites of latency, including resting memory CD4+ T cells, peripheral blood monocytes, dendritic cells and macrophages in the lymph nodes, and haematopoietic stem cells in the bone marrow. This review updates the latest advances in the study of HIV interactions with monocytes and dendritic cells, and highlights the potential role of these cells as viral reservoirs and the effects of the HIV-host-cell interactions on viral pathogenesis