Clinical significance of TLR7/IL-23/IL-17 signaling pathway in patients with acute respiratory distress syndrome.

OBJECTIVES: To analyze the clinical significance of Toll-Like Receptor 7/Interleukin-23/Interleukin-17 (TLR7/IL-23/IL-17) signaling pathway in patients with Acute Respiratory Distress Syndrome (ARDS). METHOD: The clinical data of 85 patients with ARDS were retrospectively analyzed and set as the ARDS group, and the clinical data of 85 healthy participants during the same period were set as the healthy control group. Univariate and multivariate logistic regression were used to analyze risk the factors affecting the prognosis of ARDS patients. RESULTS: TheTLR7 mRNA expression and IL-23 and IL-17 levels in peripheral blood mononuclear cells were higher in the ARDS group than in the control group (p < 0.05). TLR7 mRNA expression, IL-23, IL-17, Surfactant Protein-D (SP-D), and Clara Cell protein-16 (CC-16) levels were the highest in the severe group, followed by the moderate group, and the lowest in the mild group, while Oxygenation Index (OI) was the lowest in the severe group, followed by the moderate group, and the highest in the mild group (p < 0.05). Multivariate logistic regression analysis found that the disease grade (severe), TLR7 mRNA expression, IL-23 level, and IL-17 level were the risk factors affecting the 28-d survival status of ARDS patients (OR > 1, p < 0.05). CONCLUSIONS: In ARDS patients, the TLR7/IL-23/IL-17 signaling pathway is activated. The expression of this pathway is closely related to the severity of the disease and the levels of lung injury markers, and it is a risk factor that may have a direct impact on the prognosis of ARDS patients.

HIV-1 controllers exhibit an enhanced antiretroviral innate state characterised by overexpression of p21 and MCPIP1 and silencing of ERVK-6 RNA expression.

BACKGROUND: Human immunodeficiency virus (HIV)-1 infection can activate the expression of human endogenous retroviruses (HERVs), particularly HERV-K (HML-2). HIV controllers (HICs) are rare people living with HIV (PLWHs) who naturally control HIV-1 replication and overexpress some cellular restriction factors that negatively regulate the LTR-driven transcription of HIV-1 proviruses. OBJECTIVES: To understand the ability of HICs to control the expression of endogenous retroviruses. METHODS: We measured endogenous retrovirus type K6 (ERVK-6) RNA expression in peripheral blood mononuclear cells (PBMCs) of HICs (n = 23), antiretroviral (ART)-suppressed subjects (n = 8), and HIV-1-negative (NEG) individuals (n = 10) and correlated the transcript expression of ERVK-6 with multiple HIV-1 cellular restriction factors. FINDINGS: Our study revealed that ERVK-6 RNA expression in PBMCs from HICs was significantly downregulated compared with that in both the ART and NEG control groups. Moreover, we detected that ERVK-6 RNA levels in PBMCs across all groups were negatively correlated with the expression levels of p21 and MCPIP1, two cellular restriction factors that limit the activation of macrophages and T cells by downregulating the activity of NF-kB. MAIN CONCLUSIONS: These findings support the hypothesis that HICs activate innate antiviral mechanisms that may simultaneously downregulate the transcription of both exogenous (HIV-1) and endogenous (ERVK-6) retroviruses. Future studies with larger cohorts should be performed to confirm this hypothesis and to explore the role of p21 and MCPIP1 in regulating HERV-K expression in physiological and pathological conditions.

Vitamin D treatment distinctly modulates cytokine production by peripheral blood mononuclear cells among patients with chronic cardiac and indeterminate clinical forms of Chagas disease.

INTRODUCTION: Chagas disease is caused by the protozoan Trypanosoma cruzi and is clinically divided into acute and chronic phases. Chronic Chagas cardiomyopathy is the most studied manifestation of the disease. Vitamin D deficiency has been suggested as a risk factor for cardiovascular disease. No studies demonstrate the action of this hormone in the cells of patients with chronic Chagas heart disease. OBJECTIVE: To evaluate the in vitro immunomodulatory effect of vitamin D on peripheral blood mononuclear cells of patients with the different chronic clinical forms of Chagas disease. Evaluating vitamin D's in vitro effect on blood cells by producing cytokines. METHODS: Thirteen patients of the undetermined form (IND), 13 of the mild cardiac form (CARD1) and 14 of the severe cardiac form (CARD2) of Chagas disease, and 12 with idiopathic heart disease (CARDid) were included. The cells obtained from peripheral blood were treated in vitro with vitamin D (1 × 10-7 M) for 24 h and cytokines were dosed in the culture supernatant. RESULTS: Although it was not possible to demonstrate statistically significant differences between the groups studied, our data showed that the cells treated with vitamin D modify (p < .05) the production of interferon-γ (IFN-γ) (decrease in IND), tumor necrosis factor-α (TNF-α) (decreased in CARD1 and CARDid), interleukin (IL)-6 (increased in all groups), and IL-10 (decreased in CARD1, CARD2, and CARDid) when compared to untreated cells. CONCLUSION: In vitro treatment with vitamin D distinctly modulated the production of cytokines by mononuclear cells of peripheral blood among patients with chronic and indeterminate cardiac clinical forms of Chagas disease.

5-nitro-thiophene-thiosemicarbazone derivative induces cell death, cell cycle arrest, and phospho-kinase shutdown in pancreatic ductal adenocarcinoma cells.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with limited treatment options. This study explores the potential of novel 5-nitro-thiophene-thiosemicarbazone derivatives as therapeutic agents for PDAC. METHODS: We evaluated the cytotoxicity of seven derivatives in peripheral blood mononuclear cells (PBMCs) and PDAC cell lines. Promising candidates (PR12 and PR17) were further analyzed for their effects on colony formation, cell cycle progression, and reactive oxygen species (ROS) production. PR17, the most promising derivative, was subjected to additional investigation, including analysis of autophagy-related genes and protein kinase inhibition. RESULTS: Three derivatives (PR16, PR19, and PR20) displayed cytotoxicity towards PBMCs. PR12 reduced colony formation and G0/G1 cell cycle arrest in PDAC cells. Notably, PR17 exhibited potent activity in MIA PaCa-2 cells, inducing S-phase cell cycle arrest, downregulating autophagy genes, and inhibiting key protein kinases. CONCLUSION: PR17, a 5-nitro-thiophene-thiosemicarbazone derivative, demonstrates promising antineoplastic activity against PDAC cells by potentially modulating cell cycle progression, autophagy, and protein kinase signaling. Further studies are warranted to elucidate the detailed mechanism of action and explore its efficacy in vivo.

Involvement of Expression of miR33-5p and ABCA1 in Human Peripheral Blood Mononuclear Cells in Coronary Artery Disease.

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression post-transcriptionally and are crucial in lipid metabolism. ATP-binding cassette transporter A1 (ABCA1) is essential for cholesterol efflux from cells to high-density lipoprotein (HDL). Dysregulation of miRs targeting ABCA1 can affect cholesterol homeostasis and contribute to coronary artery disease (CAD). This study aimed to investigate the expression of miRs targeting ABCA1 in human monocytes, their role in cholesterol efflux, and their relationship with CAD. We included 50 control and 50 CAD patients. RT-qPCR examined the expression of miR-33a-5p, miR-26a-5p, and miR-144-3p in monocytes. Logistic regression analysis explored the association between these miRs and CAD. HDL's cholesterol acceptance was analyzed using the J774A.1 cell line. Results showed that miR-26a-5p (p = 0.027) and ABCA1 (p = 0.003) expression levels were higher in CAD patients, while miR-33a-5p (p < 0.001) levels were lower. Downregulation of miR-33a-5p and upregulation of ABCA1 were linked to a lower CAD risk. Atorvastatin upregulated ABCA1 mRNA, and metformin downregulated miR-26a-5p in CAD patients. Decreased cholesterol efflux correlated with higher CAD risk and inversely with miRs in controls. Reduced miR-33a-5p expression and increased ABCA1 expression are associated with decreased CAD risk. miR deregulation in monocytes may influence atherosclerotic plaque formation by regulating cholesterol efflux. Atorvastatin and metformin could offer protective effects by modulating miR-33a-5p, miR-26a-5p, and ABCA1, suggesting potential therapeutic strategies for CAD prognosis and treatment.

Gene profiling of Epstein-Barr Virus and human endogenous retrovirus in peripheral blood mononuclear cells of SLE patients: immune response implications.

Systemic lupus erythematosus (SLE) is a multifactorial disease characterized by the convergence of genetic, immunological, and viral elements resulting in a complex interaction of both internal and external factors. The role of the Epstein-Barr virus (EBV) and human endogenous retroviruses (HERV-E) as triggers and maintenance elements in the pathogenesis of SLE has been widely recognized. Previous studies have independently evaluated the effects of EBV and HERV-E in this disease. In this work, for the first time, these viral factors are jointly investigated in SLE patients. This study aimed at assessing the differential expression of immune regulatory genes and the incidence of specific viral pathogens (EBV and HERV-E), alongside the detailed characterization of surface markers in T- and B-lymphocytes in patients with SLE and control participants. A comparative analysis between patients with SLE and control participants was performed, evaluating the expression of phenotypic markers and genes involved in the immune response (TNF-α, IL-2, IL-6, IL-10, IFNG, TLR3), as well as HERV-E gag and EBV viral genes (LMP1 and BZLF1).A significant association between SLE and EBV was found in this study. A notable increase in EBV LMP1 gene expression was observed in patients with SLE . Also, a significant overexpression of HERV-E was observed, in addition to a considerable increase in the distribution of the cell surface marker CD27 + on T- and B-lymphocytes, observed in individuals with SLE compared to the control group. This study provides evidence regarding the role that EBV virus plays in lymphocytes in the context of SLE, highlighting how both the virus and the host gene expression may influence disease pathogenesis by altering immune regulatory pathways mediated by TNF-α, IFN-γ, and IL-10, as well as parallel overexpression of HERV-E gag. The decrease in TLR3 could indicate a compromised antiviral response, which could facilitate viral reactivation and contribute to disease activity.

Unexplained fever with consumptive syndrome in the elderly: two cases of VEXAS syndrome with inflammasome dysregulation.

The aim of this study is to investigate the inflammasome dysregulation in peripheral blood leukocytes of VEXAS patients. The constitutive and in vitro triggered activation of inflammasome in PBMC and neutrophils was analyzed in two Brazilian patients with typical UBA1 mutations, and compared with healthy donors. Our findings highlight the constitutive activation of caspase-1 in VEXAS leukocytes, accompanied by increased plasma levels of IL-18. Furthermore, upon stimulation of isolated peripheral blood mononuclear cells (PBMC) and neutrophils, we observed not only the exhaustion of NLRP3 and NLRP1/CARD8 pathways in VEXAS PBMC but also a significant increase in NLRP3-mediated NETs release in VEXAS neutrophils. These findings support previous studies on the contribution of the inflammasome to VEXAS pathogenesis, identifying at least two profoundly affected pathways (NLRP3 and NLRP1/CARD8) in VEXAS peripheral blood.

Assessment of the role of high-density lipoproteins and their immunomodulatory activity in systemic lupus erythematosus immunopathology.

OBJECTIVE: To explore the potential associations between high-density lipoprotein (HDL) levels and inflammasome components in the context of systemic lupus erythematosus (SLE). METHODS: A cross-sectional study was conducted. A group of 50 patients with SLE and 50 healthy controls matched by sex and similar age ranges were enrolled. Serum HDL cholesterol (HDL-C) and C reactive protein (CRP) levels were quantified. Serum cytokine levels, including IL-1ß and IL-6, were determined by ELISA. The gene expression of inflammasome-related genes in peripheral blood mononuclear cells was measured by quantitative real-time PCR. RESULTS: HDL-C levels were lower in the patients with SLE (p<0.05), and on segregation according to disease activity, those with active SLE had the lowest HDL-C levels. Patients with SLE presented higher concentrations of the serum inflammatory cytokines IL-1ß and IL-6 (p<0.0001) but similar levels of CRP to those in controls. A similar scenario was observed for the gene expression of inflammasome components, where all the evaluated markers were significantly upregulated in the SLE population. These results revealed significant negative correlations between HDL levels and disease activity, serum IL-6 and IL-1ß levels and the mRNA expression of NLRP3, IL-1ß and IL-18. In addition, significant positive correlations were found between disease activity and serum IL-1ß and between disease activity and the mRNA expression of IL-18, and interestingly, significant positive correlations were also observed between active SLE and serum IL-1ß and the mRNA expression of NLRP3. CONCLUSION: Our results suggest that HDL is essential for SLE beyond atherosclerosis and is related to inflammation regulation, possibly mediated by inflammasome immunomodulation.

Elevated NS1 serum levels reduce CD119 expression and CXCL-10 synthesis in patients with dengue hemorrhagic fever.

BACKGROUND: The intensity of dengue virus (DV) replication and circulating non-structural protein 1 (NS1) levels may promote changes in the human immune response and favor severe forms of infection. We investigated the correlations between NS1 with CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels, and IFN-γ receptor α chain (CD119) expression, and CXCL10 production by peripheral blood mononuclear cells (PBMCs) stimulated with recombinant IFN-γ in DV-infected patients with different clinical forms. METHODS: Dengue virus NS1, CXCL-8, CXCL-10, IFN-γ, and IL-12p40 serum levels were measured in 152 DV-infected patients with different clinical forms and 20 non-infected individuals (NI) using enzyme-linked immunosorbent assay (ELISA). In addition, we investigated the CXCL-10 production after in vitro IFN-γ stimulation of PBMCs from 48 DV-infected individuals (with different clinical forms of dengue fever) and 20 NI individuals using ELISA, and CD119 expression on CD14+ cells with flow cytometry. RESULTS: Patients with dengue hemorrhagic fever (DHF) had significantly higher NS1, CXCL-8, and CXCL-10 serum levels than those with classic dengue fever (DF). The response of PBMCs to IFN-γ stimulation was lower in patients with DHF than in those with DF or dengue with complications (DWC), with lower CD119 expression and reduced CXCL-10 synthesis. In addition, these alterations are associated with high NS1 serum levels. CONCLUSIONS: Patients with DHF reported high NS1 levels, low CD119 expression, and low CXCL-10 synthesis in PBMCs, which may be associated with infection progression and severity.

A new insight into the pathway behind spontaneous recurrent pregnancy loss: decreased CYR61 gene expression.

OBJECTIVE: Investigating the potential role of CYR61 in recurrent pregnancy loss is critical for developing diagnostic approaches and treatments for recurrent pregnancy loss. METHODS: In this prospective case-control study, we have investigated the expression patterns of CYR61 in blood samples from participants with recurrent pregnancy loss in their medical history and control group (n=20 vs n=10). Peripheral blood mononuclear cells from study and control groups were isolated and the expression patterns of the CYR61 gene were determined by real-time semi-quantitative reverse transcriptase PCR. RESULTS: A significant decrease in CYR61 gene expression was demonstrated in patients with two or more clinically recognized miscarriages compared with patients without miscarriages or with a history of miscarriage (p<0.01), which may make the CYR61 gene a potential candidate for predicting the risk of recurrent pregnancy loss. DISCUSSION: This study provides a basis for a detailed investigation of candidate biomarkers and molecular players involved in the development of recurrent pregnancy loss and for the development of potential treatment approaches to prevent recurrent pregnancy loss.

IL10 promoter variants are associated with gene expression but they are not markers of susceptibility to acute coronary syndrome.

Interleukin-10 (IL-10) is an immunomodulatory cytokine that plays a pivotal role in the pathogenesis of acute coronary syndromes (ACS). Here, we evaluated the role of IL10 promoter variants as markers for ACS susceptibility in Western Mexican patients as well as its association with IL10 mRNA and IL-10 plasma levels. Three promoter variants (- 1082 A > G, - 819 T > C and - 592 A > C) were analyzed in 300 ACS patients and 300 control group (CG) individuals. IL10 relative gene expression was evaluated in peripheral blood mononuclear cells (PBMC) and IL-10 levels were quantified in plasma. The allelic, genotypic and haplotypic frequencies did not show significant differences between groups. ACS patients had sevenfold higher mRNA IL10 level compared to CG (p = 0.0013). Homozygous C/C carriers in both - 819 T > C and - 592 A > C variants had 0.4-fold higher IL10 mRNA expression than heterozygous and polymorphic allele homozygous genotypes (p = 0.0357) in ACS group. There were significant differences in plasma IL-10 levels in CG and ACS group (1.001 vs 1.777 pg/mL, p = 0.0051). The variants were not markers of susceptibility to ACS in Western Mexican individuals. ACS patients showed higher IL10 expression than CG individuals which could be mediated by - 819 T > C and - 592 A > C variants and pharmacotherapy.

RecQ helicase expression in patients with telomeropathies.

BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.

Safety and efficacy indicators of guarana and Brazil nut extract carried in nanoparticles of coenzyme Q10: Evidence from human blood cells and red earthworm experimental model.

This study characterized a nanosupplement based on coenzyme Q10 containing guarana (Paullinia cupana) and Brazil nuts oil (Bertholetia excelsa) (G-Nut). Determined cytotoxic and oxi-immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs) and its effect on mortality of red Californian earthworms (Eisenia fetida) and on the immune efficiency of its coelomocytes immune by in vitro exposure to yeast dead microorganism. The cytotoxic and immunomodulatory effects of G-Nut and the GN-Free extract (0.25-3 mg/mL) were determined in PBMC cultures. Apoptotic, oxidative, and inflammatory markers were determined using biochemical, immunological, and molecular protocols. The effects of G-Nut and GN-Free extracts on mortality and immune efficiency were investigated in earthworms. G-Nut and GN-Free did not induce cytotoxic events in PBMCs, triggering the decrease in apoptotic (caspases 3 and 8) gene expression, lipid and protein oxidation levels, or pro-inflammatory cytokine levels. G-Nut and GN-Free did not trigger earthworm mortality and improved coelomocyte immune efficiency by increasing Eisenia neutrophil extracellular DNA traps and brown body formation when exposed to dead yeasts. The G-Nut nanoformulation is safe and can be used as a new form of food supplement by oral or transdermal delivery.

Anti-inflammatory action of geopropolis produced by stingless bees on human peripheral blood mononuclear cells.

Natural products have been used to treat inflammatory reactions and led to the discovery of new anti-inflammatory drugs. Geopropolis (GEO) is produced by stingless bees and has been used by indigenous people to improve the immune functions. Here, a possible synergism between GEO and dexamethasone (DEX) was assessed on human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS). PBMC viability was evaluated by the MTT, apoptosis/necrosis by flow cytometry, cytokine and eicosanoids production by ELISA, and intracellular pathways by polymerase chain reaction. GEO and DEX alone or in combination did not affect cell viability. GEO in combination with lower concentrations of DEX inhibited cytokine production (TNF-α, IL-1ß, and IL-10). No effects were seen on eicosanoids nor in intracellular pathways. Despite not always being more efficient than the isolated treatments, GEO + DEX seemed to be promising and allow the use of DEX in lower concentrations, reducing adverse effects.

Human T-cell activation with Toxoplasma gondii antigens loaded in maltodextrin nanoparticles.

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct of Toxoplasma gondii total antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+ and CD8+ cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 µg NP/0.3 µg TE) achieved IFN-γ-specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+ effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

Effect of epicatechin consumption on the inflammatory pathway and mitochondria morphology in PBMC from a R350P desminopathy patient: A case report.

Desminopathy R350P is a human myopathy that is characterized by the progressive loss of muscle fiber organization. This results in the loss of muscle size, mobility, and strength. In desminopathy, inflammation affects muscle homeostasis and repair, and contributes to progressive muscle deterioration. Mitochondria morphology was also suggested to affect desminopathy progression. Epicatechin (Epi)-a natural compound found in cacao-has been proposed to regulate inflammatory signaling and mitochondria morphology in human and animal models. Hence, we hypothesize chronic Epi consumption to improve inflammatory pathway and mitochondria morphology in the peripheral blood mononuclear cells (PBMCs) of a desminopathy R350P patient. We found that 12 weeks of Epi consumption partially restored TRL4 signaling, indicative of inflammatory signaling and mitochondria morphology in the desminopathy patient. Moreover, Epi consumption improved blood health parameters, including reduced HOMA-IR and IL-6 levels in the desminopathy patient. This indicates that Epi consumption could be a useful tool to slow disease progression in desminopathy patients.

Microalgae extracts modulates the immune response in Trypanosoma cruzi-infected human cells.

Chagas disease (CD) is caused by the hemoflagellate protozoan Trypanosoma cruzi. The control of the infection depends of the innate and acquired immune response of host. Moreover, CD plays a significant role in the immune response, and, in this context, microalgae can be an interesting alternative due to its immunomodulatory and trypanocidal effects. This study aimed to evaluate, in vitro, immunomodulatory potentials of the aqueous extracts of Chlorella vulgaris and Tetradesmus obliquus. Both microalgae extracts (ME) were obtained by sonication, and the selectivity index (SI) was determined by assays of inhibitory concentration (IC50) in T. cruzi trypomastigotes cells; as well as the cytotoxic concentrations (CC50) in human peripheral mononuclear cells (PBMC). The immune response was evaluated in T. cruzi-infected PBMC using the IC50 value. ME led to inhibition of T. cruzi trypomastigotes after 24 h of treatment, in which the IC50 values were 112.1 µg/ml to C. vulgaris and 15.8 µg ml-1 to T. obliquus. On the other hand, C. vulgaris did not affect the viability of PBMCs in concentrations up to 1000 µg ml-1, while T. obliquus was non-toxic to PBMCs in concentrations up to 253.44 µg ml-1. In addition, T. obliquus displayed a higher SI against T. cruzi (SI = 16.8), when compared with C. vulgaris (SI = 8.9). C. vulgaris decreased the levels of IFN, indicating a reduction of the inflammatory process; while T. obliquus displayed an interesting immunomodulatory effect, since discretely increased the levels of TNF and stimulated the production of the anti-inflammatory cytokine IL-10. This study confirms that ME are effective against T. cruzi trypomastigotes, and may able to control the parasitemia and preventing the progress of CD while regulating the inflammatory process.

Leukocyte dysfunction and reduced CTLA-4 expression are associated with perianal Crohn's disease.

Although perianal Crohn's disease (PCD) is highly associated with the exacerbated inflammation, the molecular basis and immunological signature that distinguish patients who present a history of perianal lesions are still unclear. This paper aims to define immunological characteristics related to PCD. In this cross-sectional observational study, we enrolled 20 healthy controls and 39 CD patients. Blood samples were obtained for the detection of plasma cytokines and lipopolysaccharides (LPS). Peripheral blood mononuclear cells (PBMCs) were phenotyped by flow cytometry. Leukocytes were stimulated with LPS or anti-CD3/anti-CD28 antibodies. Our results show that CD patients had augmented plasma interleukin (IL)-6 and LPS. However, their PBMC was characterized by decreased IL-6 production, while patients with a history of PCD produced higher IL-6, IL-8, and interferon-γ, along with decreased tumor necrosis factor alpha (TNF). CD patients had augmented FoxP3 and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) regulatory markers, though the PCD subjects presented a significant reduction in CTLA-4 expression. CTLA-4 as well as IL-6 and TNF responses were able to distinguish the PCD patients from those who did not present perianal complications. In conclusion, IL-6, TNF, and CTLA-4 exhibit a distinct expression pattern in CD patients with a history of PCD, regardless of disease activity. These findings clarify some mechanisms involved in the development of the perianal manifestations and may have a great impact on the disease management.

Steroid immune responsive gene regulation in Mycobacterium tuberculosis infection in vitro.

Tuberculosis (TB) is an infectious disease displaying a multifactorial pathology. The immunomodulatory role attributed to steroid hormones, such as vitamin D3 (VD3) and 17ß-estradiol (E2), highlighted the importance of these hormones against Mycobacterium tuberculosis (Mtb) infection. In order to understand their influence upon gene expression of immune and inflammatory responsive genes against Mtb we tested it in vitro using peripheral blood mononuclear cells (PBMCs). Cells were pretreated with VD3 (50 ng/mL) or E2 (100 nM/mL) and co-cultured with H37Rv Mtb or stimulated with lipopolysaccharide from Escherichia coli (LPS). After 24 h and 72 h of co-culture the Mtb viability in macrophages test was performed, as well the total RNA isolation for gene expression analysis by RT-qPCR of the following target genes: NLRP3, DC-SIGN, IL-1ß, and IL-10. We also measured IL-10, TNF, IFN-γ, IL-4, IL-6, and IL-2 supernatant levels. As the main results, we found that VD3 and E2 downregulated the expression of inflammatory genes NLRP3, IL-1ß, and IL-10 expression in Mtb co-cultured cells. Finally, VD3 treatment increased the release of the cytokine IFN-γ in Mtb-infected cells, while E2 treatment inhibited the release of IL-10, TNF, IFN-γ, and IL-6. Therefore, we report an immunogenetic influence of VD3 and E2 upon Mtb co-culture.

CD4+ Th1 and Th17 responses and multifunctional CD8 T lymphocytes associated with cure or disease worsening in human visceral leishmaniasis.

Introduction: In VL, a proinflammatory phenotype is typically associated with enhanced phagocytosis and a Th1 mediated immune response resulting in infection control. In contrast, an anti-inflammatory phenotype, associated with a predominant regulatory response, typically enables intracellular multiplication of Leishmania parasites and disease progression. Methods: To investigate the impact of chemotherapy on Th2 and Th17 immune responses in patients with visceral leishmaniasis (VL), we assessed all combinations of intracellular expression of IFN-γ, IL-10, IL-4 and IL-17 in the CD4+ and CD8+ T cell populations of peripheral blood mononuclear cell (PBMC) samples from patients, after antigenic stimulation with Leishmania lysate, throughout treatment and follow-up. As increases in spleen and liver sizes and decreases in hematocrit, hemogloblin, erythrocytes, monocytes, leukocytes and platelets levels are strongly related to the disease, we studied the correlations between the frequencies of T cells producing the afore mentioned cytokines, individually and in combination, and these variables, as markers of disease or cure. Results: We found that the frequency of IFN-γ-producingCD4+ T cells increased until the end of chemotherapy with Glucantime® or AmBisome ®, while IL-10, IL-4 and IL-17-producing CD4+ T cells peaked on day 7 following the start of treatment. Although the frequency of CD4+IL-17+ cells decreased during treatment an increase was observed after clinical cure. The frequency of CD4+ T cells producing only IFN-γ or IL-17 correlated with blood monocytes levels. Frequencies of double-producers of IFN-γ and IL-10 or IL-4 correlated positively with eosinophils and platelets levels. Together, this suggest that IFN-γ drives the immune response towards Th1 at cure. In contrast, and associated with disease or Th2 response, the frequency of CD4+ IL-10+ cells correlated positively with spleen sizes and negatively with circulating monocyte levels, while the frequency of CD4+ producing both IL-4 and IL-10 correlated negatively with platelets levels. The frequency of CD8+ single-producers of IFN-γ increased from day 21 to 90 while that of single-producers of IL-10 peaked on day 7, of IL-4 on day 30 and of IL-17, on day 180. IFN-γ expression in CD8+ single- and double-producers of cytokines was indicative of an immune response associated with cure. In contrast, frequencies of CD8+ double-producers of IL-4 and IL-10, IL-4 and IL-17 and IL-10 and IL-17 and producers of three and four cytokines, were associated with disease and were low after the cure. Frequencies of CD8+ T cells producing IFN-γ alone or with IL-17 were positively correlated with platelets levels. In contrast, as markers of disease: 1) frequencies of single producers of IL-10 correlated negatively with leukocytes levels, 2) frequencies of double producers of IL-4 and IL-10 correlated negatively with platelet, leukocyte, lymphocyte and circulating monocyte levels, 3) frequencies of triple-producers of IFN-γ, IL-4 and IL-10 correlated negatively with platelet, leukocyte and neutrophil levels and 4) frequencies of producers of IFN-γ, IL-4, IL-10 and IL-17 simultaneously correlated positively with spleen size, and negatively with leukocyte and neutrophil levels. Discussion: Our results confirmed that the clinical improvement of VL patients correlates with the decrease of an IL-4 and IL-10 CD4+Th2 response, the recovery of CD4+ Th1 and Th17 responses and the frequency of CD8+ single-producers of IFN-γ and double producers of IFN-γ and IL-17.