Synbiotic therapy decreases microbial translocation and inflammation and improves immunological status in HIV-infected patients: a double-blind randomized controlled pilot trial

BACKGROUND: HIV-infection results in damage and dysfunction of the gastrointestinal system. HIV enteropathy includes pronounced CD4+ T-cell loss, increased intestinal permeability, and microbial translocation that promotes systemic immune activation, which is implicated in disease progression. A synbiotic is the combination of probiotics and prebiotics that could improve gut barrier function. Our study goal was to determine whether the use of a synbiotic, probiotics or a prebiotic can recover immunological parameters in HIV-infected subjects through of a reduction of microbial translocation and pro-inflammatory cytokine production. METHODS: A randomized, double-blind controlled study was performed; twenty Antiretroviral treatment-naïve HIV-infected subjects were subgrouped and assigned to receive a synbiotic, probiotics, a prebiotic, or a placebo throughout 16 weeks. RESULTS: We had no reports of serious adverse-events. From baseline to week 16, the synbiotic group showed a reduction in bacterial DNA concentrations in plasma (p = 0.048). Moreover, the probiotic and synbiotic groups demonstrated a decrease in total bacterial load in feces (p = 0.05). The probiotic group exhibited a significant increment of beneficial bacteria load (such as Bifidobacterium; p = 0.05) and a decrease in harmful bacteria load (such as Clostridium; p = 0.063). In the synbiotic group, the CD4+ T-cells count increased (median: +102 cells/μL; p = 0.05) and the level of Interleukin 6 cytokine decreased significantly (p = 0.016). CONCLUSIONS: Our study showed a significant increase in CD4+ T lymphocyte levels in the synbiotic group, which could delay the initiation of antiretroviral therapy and decrease costs in countries with limited resources

Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

<p>Abstract</p> <p>Background</p> <p>Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK) cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity.</p> <p>Results</p> <p>We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT). Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells.</p> <p>Conclusions</p> <p>Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.</p

MHC class I-related chain A and B ligands are differentially expressed in human cervical cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Natural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line.</p> <p>Results</p> <p>Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress.</p> <p>Conclusions</p> <p>This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells.</p

Apoptosis induction in Jurkat cells and sCD95 levels in women's sera are related with the risk of developing cervical cancer

<p>Abstract</p> <p>Background</p> <p>Currently, there is clear evidence that apoptosis plays an important role in the development and progression of tumors. One of the best characterized apoptosis triggering systems is the CD95/Fas/APO-1 pathway; previous reports have demonstrated high levels of soluble CD95 (sCD95) in serum of patients with some types of cancer. Cervical cancer is the second most common cancer among women worldwide. As a first step in an attempt to design a minimally invasive test to predict the risk of developing cervical cancer in patients with precancerous lesions, we used a simple assay based on the capacity of human serum to induce apoptosis in Jurkat cells. We evaluated the relationship between sCD95 levels and the ability to induce apoptosis in Jurkat cells in cervical cancer patients and controls.</p> <p>Methods</p> <p>Jurkat cells were exposed to serum from 63 women (20 healthy volunteers, 21 with cervical intraepithelial neoplasia grade I [CIN 1] and 22 with cervical-uterine carcinoma). The apoptotic rate was measured by flow cytometry using Annexin-V-Fluos and Propidium Iodide as markers. Serum levels of sCD95 and soluble CD95 ligand (sCD95L) were measured by ELISA kits.</p> <p>Results</p> <p>We found that serum from almost all healthy women induced apoptosis in Jurkat cells, while only fifty percent of the sera from women with CIN 1 induced cell death in Jurkat cells. Interestingly, only one serum sample from a patient with cervical-uterine cancer was able to induce apoptosis, the rest of the sera protected Jurkat cells from this killing. We were able to demonstrate that elimination of Jurkat cells was mediated by the CD95/Fas/Apo-1 apoptotic pathway. Furthermore, the serum levels of sCD95 measured by ELISA were significantly higher in women with cervical cancer.</p> <p>Conclusion</p> <p>Our results demonstrate that there is a strong correlation between low levels of sCD95 in serum of normal women and higher apoptosis induction in Jurkat cells. We suggest that an analysis of the apoptotic rate induced by serum in Jurkat cells and the levels of sCD95 in serum could be helpful during the prognosis and treatment of women detected with precancerous lesions or cervical cancer.</p

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

<p>Abstract</p> <p>Background</p> <p>WNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the <it>WNT7A </it>gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.</p> <p>Methods</p> <p>We analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative <it>WNT7A </it>expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.</p> <p>Results</p> <p>WNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (<it>p </it>≤0.001). By restoring <it>WNT7A </it>expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of <it>WNT7A </it>expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.</p> <p>Conclusions</p> <p>To our knowledge, this is the first report evidencing quantitatively decreased <it>WNT7A </it>levels in leukemia-derived cells and that <it>WNT7A </it>restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of <it>WNT7A </it>as a tumor suppressor gene as well as a therapeutic tool.</p

Augmented serum level of major histocompatibility complex class I-related chain A (MICA) protein and reduced NKG2D expression on NK and T cells in patients with cervical cancer and precursor lesions

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is the second most common cancer in women worldwide. NK and cytotoxic T cells play an important role in the elimination of virus-infected and tumor cells through NKG2D activating receptors, which can promote the lysis of target cells by binding to the major histocompatibility complex class I-related chain A (MICA) proteins. Increased serum levels of MICA have been found in patients with epithelial tumors. The aim of this study was to compare the levels of soluble MICA (sMICA) and NKG2D-expressing NK and T cells in blood samples from patients with cervical cancer or precursor lesions with those from healthy donors.</p> <p>Methods</p> <p>Peripheral blood with or without heparin was collected to obtain mononuclear cells or sera, respectively. Serum sMICA levels were measured by ELISA and NKG2D-expressing immune cells were analyzed by flow cytometry. Also, a correlation analysis was performed to associate sMICA levels with either NKG2D expression or with the stage of the lesion.</p> <p>Results</p> <p>Significant amounts of sMICA were detected in sera from nearly all patients. We found a decrease in the number of NKG2D-expressing NK and T cells in both cervical cancer and lesion groups when compared to healthy donors. Pearson analysis showed a negative correlation between sMICA and NKG2D-expressing T cells; however, we did not find a significant correlation when the analysis was applied to sMICA and NKG2D expression on NK cells.</p> <p>Conclusion</p> <p>Our results show for the first time that high sMICA levels are found in sera from patients with both cervical cancer and precursor lesions when compared with healthy donors. We also observed a diminution in the number of NKG2D-expressing NK and T cells in the patient samples; however, a significant negative correlation between sMICA and NKG2D expression was only seen in T cells.</p

Substantial increase in the frequency of circulating CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade 1

Abstract Background The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8+ cytotoxic T cells and is classically considered absent in CD4+ T cells. However, recent studies have identified a distinctive population of CD4+ T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4+ T cell-immunocompromised individuals suggests that CD4+ T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4+ T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4+NKG2D+ T cell expansion were also measured. Results Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4+ T cells and CD8+ T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4+ T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4+NKG2D+ T cells was seen in patients with CIN 1, despite the overall levels of CD4+ T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4+NKG2D+ T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. Conclusions Taken together, our results suggest that the significant increase within the CD4+NKG2D+ T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion

Substantial increase in the frequency of circulating CD4+

BACKGROUND: The NKG2D receptor confers important activating signals to NK cells via ligands expressed during cellular stress and viral infection. This receptor has generated great interest because not only is it expressed on NK cells, but it is also seen in virtually all CD8(+) cytotoxic T cells and is classically considered absent in CD4(+) T cells. However, recent studies have identified a distinctive population of CD4(+) T cells that do express NKG2D, which could represent a particular cytotoxic effector population involved in viral infections and chronic diseases. On the other hand, increased incidence of human papillomavirus-associated lesions in CD4(+) T cell-immunocompromised individuals suggests that CD4(+) T cells play a key role in controlling the viral infection. Therefore, this study was focused on identifying the frequency of NKG2D-expressing CD4(+) T cells in patients with cervical intraepithelial neoplasia (CIN) 1. Additionally, factors influencing CD4(+)NKG2D(+) T cell expansion were also measured. RESULTS: Close to 50% of patients with CIN 1 contained at least one of the 37 HPV types detected by our genotyping system. A tendency for increased CD4(+) T cells and CD8(+) T cells and decreased NK cells was found in CIN 1 patients. The percentage of circulating CD4(+) T cells co-expressing the NKG2D receptor significantly increased in women with CIN 1 versus control group. Interestingly, the increase of CD4(+)NKG2D(+) T cells was seen in patients with CIN 1, despite the overall levels of CD4(+) T cells did not significantly increase. We also found a significant increase of soluble MICB in CIN 1 patients; however, no correlation with the presence of CD4(+)NKG2D(+) T cells was seen. While TGF-beta was significantly decreased in the group of CIN 1 patients, both TNF-alpha and IL-15 showed a tendency to increase in this group. CONCLUSIONS: Taken together, our results suggest that the significant increase within the CD4(+)NKG2D(+) T cell population in CIN 1 patients might be the result of a chronic exposure to viral and/or pro-inflammatory factors, and concomitantly might also influence the clearance of CIN 1-type lesion

MG132 proteasome inhibitor modulates proinflammatory cytokines production and expression of their receptors in U937 cells: involvement of nuclear factor-κB and activator protein-1

In response to inflammatory stimuli, monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors, such as nuclear factor κB (NF-κB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex, which plays a vital role in several cellular functions, including the regulation of transcription factors like NF-κB. In this study, we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132, a proteasome inhibitor, on the release of TNF-α, IL-1β and IL-6 and on the expression of their membrane and soluble receptors TNF-R1, IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-κB and AP-1 and on the IκB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IκB degradation, resulting in the abolition of NF-κB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors