Effect of stretching on inflammation in a subcutaneous carrageenan mouse model analyzed at single-cell resolution.

Understanding the factors that influence the biological response to inflammation is crucial, due to its involvement in physiological and pathological processes, including tissue repair/healing, cancer, infections, and autoimmune diseases. We have previously demonstrated that in vivo stretching can reduce inflammation and increase local pro-resolving lipid mediators in rats, suggesting a direct mechanical effect on inflammation resolution. Here we aimed to explore further the effects of stretching at the cellular/molecular level in a mouse subcutaneous carrageenan-inflammation model. Stretching for 10 min twice a day reduced inflammation, increased the production of pro-resolving mediator pathway intermediate 17-HDHA at 48 h postcarrageenan injection, and decreased both pro-resolving and pro-inflammatory mediators (e.g., PGE2 and PGD2 ) at 96 h. Single-cell RNA sequencing analysis of inflammatory lesions at 96 h showed that stretching increased the expression of both pro-inflammatory (Nos2) and pro-resolution (Arg1) genes in M1 and M2 macrophages at 96 h. An intercellular communication analysis predicted specific ligand-receptor interactions orchestrated by neutrophils and M2a macrophages, suggesting a continuous neutrophil presence recruiting immune cells such as activated macrophages to contain the antigen while promoting resolution and preserving tissue homeostasis.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection.

Stretching Impacts Inflammation Resolution in Connective Tissue.

Acute inflammation is accompanied from its outset by the release of specialized pro-resolving mediators (SPMs), including resolvins, that orchestrate the resolution of local inflammation. We showed earlier that, in rats with subcutaneous inflammation of the back induced by carrageenan, stretching for 10 min twice daily reduced inflammation and improved pain, 2 weeks after carrageenan injection. In this study, we hypothesized that stretching of connective tissue activates local pro-resolving mechanisms within the tissue in the acute phase of inflammation. In rats injected with carrageenan and randomized to stretch versus no stretch for 48 h, stretching reduced inflammatory lesion thickness and neutrophil count, and increased resolvin (RvD1) concentrations within lesions. Furthermore, subcutaneous resolvin injection mimicked the effect of stretching. In ex vivo experiments, stretching of connective tissue reduced the migration of neutrophils and increased tissue RvD1 concentration. These results demonstrate a direct mechanical impact of stretching on inflammation-regulation mechanisms within connective tissue.

Identification of Nef-HIV-1 domains involved in p22-phox interaction and superoxide production.

Nef -HIV-1 has been shown to be involved in NADPH complex interaction and superoxide production. The aim of this work was to study the domains involved in the interaction between Nef and p22-phox. Two approaches were used: 1) in silico modelling, to determine the potential binding motifs and design Nef truncated forms and 2) functional assays. The results showed that GFPVT 68-72, FPDW 121-124 and REVLE 179-183 on Nef are critical for p22-phox (RPQIG 142-146 and PGGP 181-184) docking. However, only the region containing FPDW 121-124 on Nef is able to induce superoxide production. Understanding the molecular mechanisms involved in generating oxidative stress during HIV infection, is critical for therapeutic intervention, in order to minimize viral replication and dissemination.

[Effects of occupational exposure to pesticides on semen quality of workers in an agricultural community of Merida state, Venezuela]. / Efectos de la exposición ocupacional a plaguicidas sobre la calidad del semen en trabajadores de una comunidad agrícola del estado Merida, Venezuela.

Numerous studies report adverse effects of pesticides on male reproductive health. The objectives of this study were to investigate whether there is a relationship between occupational exposure to pesticides and semen quality, and to determine whether chronic exposure to pesticides differentially affects semen quality in men of different ages. A comparative study of 64 farmers and 64 control men was performed. The farmers were interviewed to determine their occupational history and particularly, activities that may involve exposure to pesticides. Semen parameters were evaluated and a comparative analysis of semen variables between exposed and control groups, as well as between age groups: 18-29, 30-37 and 38-60 years was done. Significant alterations of some semen parameters in the exposed group were found, such as: decreases in sperm concentration, slow progressive motility and sperm membrane integrity; at the same time, increases in eosin Y positive and sperm DNA fragmentation index. The results obtained by age groups showed significant differences between exposed and control groups for the parameters of membrane integrity, eosin Y positive and sperm DNA fragmentation index, being the exposed group between 18-29 years that showed the highest altered cases of these parameters. Our results prove that occupational pesticide exposure is associated with alterations in sperm quality, creating a risk to farm workers in their reproductive capacity.

Programmed hepatocytes cell death associated with FLIP downregulation in response to extracellular preS1/2.

Chronic hepatitis B virus (HBV) infection involves liver damage resulting in continuous cell injury and death. During HBV infection, hepatocytes exhibit changes in death receptor expression and in their susceptibility to death. These changes are observed not only in infected cells but also in bystander cells. Because excess viral surface protein (HBsAg) is secreted in large amounts as soluble particles containing preS proteins, the role of soluble preS1/2 in hepatocyte (HepG2) death modulation is an important issue to be explored. An increase of cell death induced by preS1/2 was observed. Also, cell death was associated with the down-regulation of FLIP and activation of caspase 8, caspase 9, and BID. Additionally, hepatocytes exhibited a sensitization to death mediated by the Fas receptor. These results, may contribute to understanding the role of envelope proteins (preS1/2) in the pathogenesis of HBV infection.

A deficient translocation of CD3ζ, ZAP-70 and Grb2 to lipid raft, as a hallmark of defective adaptive immune response during chronic hepatitis B infection.

Hepatitis B is considered to be a worldwide public health problem. An immunosuppressor microenvironment has been proposed to contribute to viral persistence during chronic disease. Understanding the intracellular signaling cascade in T-cells from HBV-infected patients, will contribute to unravel the mechanisms that control the development of immune response during hepatitis B. We analyze lipid rafts formation and early activation signals in chronic HBV infected patients, compared to naturally immune subjects (NIS). Patients show: (1) diminished GM1 clustering, (2) A deficient lipid rafts recruitment of CD3ζ/ZAP-70/Grb2, and (3) these proteins do not merge with GM1 within the lipid rafts. Finally, immunoprecipitation assays proved that ZAP-70 does not associate to CD3ζ. These results show for the first time, defects regarding early key events in T-cell activation, in chronically infected HBV patients, which may contribute not only to understand HBV immune tolerance, but to reveal new potential therapeutic targets to control the infection.

Fas and FasL expression in leukocytes from chronic granulomatous disease patients.

Abstract. Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by defects in superoxide (O2-) production, which result from mutations in one of the four NADPH oxidase components, predisposing to bacterial and fungal infections. Besides the O2-defect, it has been described that neutrophils from CGD patients are resistant to cell death, a phenomenon that has been connected to chronic inflammation and predisposition to autoimmune diseases. A diminished expression of Fas and its counterpart FasL, molecules known to play a major role in cell death, has been described in lymphocytes depleted of O2-reactive oxygen species (ROS), suggesting an involvement of ROS in Fas/FasL expression. In this work, Fas and FasL expressions were analyzed in T cells and neutrophils from two CGD families, previously known to harbor two different molecular defects: absence of either p47-phox or p67-phox. We found that T lymphocytes from CGD patients express low levels of Fas and FasL, while a diminished FasL expression was observed on neutrophils from a CGD A470 patient. These defects may contribute to understand altered cell death in CGD patients.

[Analysis of p22-phox and p47-phox subcellular localization and distribution in neutrophils from human immunodeficiency virus (HIV) infected patients]. / Localización y distribución celular de p22-phox y p47-phox en neutrófilos humanos de pacientes infectados con VIH.

INTRODUCTION: During human immunodeficiency virus (HIV) infection a dysfunction of polymorphonuclear (PMN) cells has been described including a progressively altered superoxide production as disease progression. The NADPH oxidase has been described as a major source of superoxide. The neutrophil NADPH oxidase comprises a plasma membrane-bound cytochrome b558 (which is a heterodimer of one p22-phox and one gp91-phox subunit) and cytosolic subunits, namely p47-phox, p67-phox and p40-phox. During neutrophil activation in response to various agonists, the cytosolic subunits translocate to and associate with the cytochrome b558, a process that results in oxidase activation. Therefore, an altered superoxide production could be a consequence of abnormal distribution or translocation of NADPH oxidase components in response to HIV infection. MATERIAL AND METHODS: We used several strategies including: confocal microscopy, subcellular fractionation and sucrose gradients, to analyze the cellular distribution of two of the NADPH oxidase components (p22-phox and p47-phox). RESULTS: We observed that in resting cells, a substantial proportion of p22-phox from HIV positive patients is distributed in regions close to the cytoplasmic membrane, sediment in high density sucrose fractions and is located in the cytoplasmic insoluble fraction. Additionally, a diffuse cytosolic distribution of p47-phox was observed in neutrophils from HIV infected patients. The results demonstrate an inappropriate cell distribution of NADPH-complex in PMN from HIV positive patients.

HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex.

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.

[Immune tolerance at 50 years from the Nobel Prize in Medicine and Physiology: a perspective as a mechanism of immune response against pathogens]. / La tolerancia inmunológica a 50 años del Premio Nobel en Medicina y Fisiología: una perspectiva como mecanismo de respuesta inmune frente a patógenos.

The immune system has the capability of protection against infectious disease which is accomplished by an enormous repertoire of receptors specifically reactive to foreign antigens, but it is tolerant to self-antigens, establishing biological identity. The ability to discriminate between self and non-self is a central property of the immune system, by using complex network of cellular and molecular mechanisms in order to prevent autoimmunity; this function is called immune tolerance. Thus, the Interaction between immune system and antigens is required for the generation of tolerance and it is critical in different physiological and pathological conditions in order to limit the damage to self tissues. Since Medawar description, who showed that the tolerance is an acquired property playing a central role in the homeostasis, several mechanism has been proposed to explain it. It is accepted today that an important group of antigen specific cells called regulatory T cell, both natural and induced, are critical as a unifying mechanism to maintain self-tolerance. These suppressor lymphocyte subpopulations had shown to play an important role not only by controlling autoimmune disease but also in the pathogenesis of many chronic infectious diseases, either manipulated by the microorganism to escape from the immune system, or induced by the host to reduce inflammatory damage. This review has the intention of updating about modern concepts in immune tolerance mechanisms, with special emphasis played by Treg cell in the tolerance which is unquestionable induced during the course of chronic infections diseases.

[Evaluation of the quality of the human spermatozoon: comparison between spermatic DNA integrity and semen variables]. / Evaluación de la calidad del espermatozoide humano: comparación entre la integridad del ADN espermático y variables del semen.

Semen analysis does not have an absolute predictive value on fertility, however it is a reflection of male fertility potential, which is related to its spermatozoa quality and other semen variables. Great variability in human semen parameters has been demonstrated within a single individual, an observation that could explain why a male with low semen quality can successfully fertilize an egg. Although conventional semen analysis, such as sperm concentration, motility and morphology, provide important information about the clinical status of male fertility, new procedures to predict the sperm functional capability have been developed in the last decade, such as analysis of nuclear DNA integrity, which have improved considerably the clinical diagnosis of male infertility, and increased the knowledge about spermatozoa function. DNA fragmentation consist in interruptions, both in single and double DNA strains, that frequently occur in sperm samples from infertile patients. We have conducted a clinical study in semen samples from patients who have attended the Andrology laboratory of the University of Los Andes, between March 2007 and March 2009. The aim of this study was to compare sperm DNA integrity, analyzed by flow cytometry, with traditional semen parameters. Our results show remarkable correlations between conventional human semen variables and sperm chromatin integrity, contributing to asses an integral evaluation of sperm quality allowing the analysis of its fertilizing potential in clinical studies.

Establishment of a Novel Porcine Model to Study the Impact of Active Stretching on a Local Carrageenan-Induced Inflammation.

OBJECTIVE: Active stretching of the body is integral to complementary mind-body therapies such as yoga, as well as physical therapy, yet the biologic mechanisms underlying its therapeutic effects remain largely unknown. A previous study showed the impact of active stretching on inflammatory processes in rats. The present study tested the feasibility of using a porcine model, with a closer resemblance to human anatomy, to study the effects of active stretching in the resolution of localized inflammation. DESIGN: A total of 12 pigs were trained to stretch before subcutaneous bilateral Carrageenan injection in the back at the L3 vertebrae, 2 cm from the midline. Animals were randomized to no-stretch or stretch, twice a day for 5 mins over 48 hrs. Animals were euthanized for tissue collection 48 hrs postinjection. RESULTS: The procedure was well tolerated by the pigs. On average, lesion area was significantly smaller by 36% in the stretch group compared with the no-stretch group (P = 0.03). CONCLUSION: This porcine model shows promise for studying the impact of active stretching on inflammation-resolution mechanisms. These results are relevant to understanding the stretching-related therapeutic mechanisms of mind-body therapies. Future studies with larger samples are warranted.

Altered T cell costimulation during chronic hepatitis B infection.

T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

[Impaired activation and costimulation of T CD4+ lymphocytes during chronic hepatitis C infection]. / Déficit en señales de activación y coestimulación en linfocitos T CD4+ de pacientes infectados con el virus de la hepatitis C.

Hepatitis C chronic infection occurs in 80% of the cases and eventually leads to cirrhosis and hepatocellular carcinoma. A deficient adaptive immune response has been described during chronic infection which contributes to viral persistence. This altered T cell response could be associated to deficient costimulation signals during priming of T cells. We have conducted an in vitro study to explore the activation phenomenon of CD4+ T cells focusing on costimulation via the CD28 receptor, associated to stimulation with purified Hepatitis C (HCV) core antigen. Our study involved the induction of CD69, CD25 and CD40L activation receptors, along with detection of intracellular cytokines such as IFN-gamma, TGF-beta and IL-10. Analysis was performed in chronically HCV infected patients, intrafamilial members of HCV-infected patients and healthy individuals. HCV core antigen induced CD40L expression in CD4+ cells from intrafamilial members, in contrast to chronically infected patients and control individuals. Association of CD28 crosslinking increased CD69 and IFN-gamma expression in chronically infected patients, suggesting a detriment in this signaling pathway. Additionally, an increased TGF-beta expression was observed in CD4+ cells from HCV-infected patients, which was corrected by addition of CD28 crosslinking. Our results may contribute to understand the underlying mechanism of T cell tolerance against HCV during chronic infection, and to provide new targets for the designing of therapeutic strategies to control the infection and to offer protective immunity against the virus.

CD40/CD40L expression in leukocytes from chronic granulomatous disease patients.

Chronic granulomatous disease (CGD) is an inherited disorder caused by defects in the NADPH oxidase complex, which generates superoxide, the precursor of hydrogen peroxide (H(2)O(2)) and other reactive oxygen derivatives with microbicidal activity. Because CGD patients are at risk of chronic inflammatory manifestations, including inflammatory bowel disease and autoimmune diseases, and it is not clear whether these pathologies are exclusively secondary to altered superoxide production, or whether distinct immunologic defects are involved, we explored cell proliferation, lymphocyte cell counts, immunoglobulin levels, presence of autoimmune antibodies and expression of costimulatory molecules in leukocytes from CGD patients. We found that CGD patients have a diminished phytohemagglutinin-induced proliferation of blood mononuclear cells. Following stimulation with PMA plus ionomycin, a reduced percentage of CD40L expression in T lymphocytes and a diminished expression of CD40 molecules in neutrophils were observed on leukocytes from these patients. Our results suggest an altered interplay between elements of innate and adaptive immunity in CGD patients, which may be reflected in an increased susceptibility to opportunistic infections.

Stretching Reduces Skin Thickness and Improves Subcutaneous Tissue Mobility in a Murine Model of Systemic Sclerosis.

OBJECTIVE: Although physical therapy can help preserve mobility in patients with systemic sclerosis (SSc), stretching has not been used systematically as a treatment to prevent or reverse the disease process. We previously showed in rodent models that stretching promotes the resolution of connective tissue inflammation and reduces new collagen formation after injury. Here, we tested the hypothesis that stretching would impact scleroderma development using a mouse sclerodermatous graft-versus-host disease (sclGvHD) model. METHODS: The model consists in the adoptive transfer (allogeneic) of splenocytes from B10.D2 mice (graft) into Rag2-/- BALB/c hosts (sclGvHD), resulting in skin inflammation followed by fibrosis over 4 weeks. SclGvHD mice and controls were randomized to stretching in vivo for 10 min daily versus no stretching. RESULTS: Weekly ultrasound measurements of skin thickness and subcutaneous tissue mobility in the back (relative tissue displacement during passive trunk motion) successfully captured the different phases of the sclGvHD model. Stretching reduced skin thickness and increased subcutaneous tissue mobility compared to no stretching at week 3. Stretching also reduced the expression of CCL2 and ADAM8 in the skin at week 4, which are two genes known to be upregulated in both murine sclGvHD and the inflammatory subset of human SSc. However, there was no evidence that stretching attenuated inflammation at week 2. CONCLUSION: Daily stretching for 10 min can improve skin thickness and mobility in the absence of any other treatment in the sclGvHD murine model. These pre-clinical results suggest that a systematic investigation of stretching as a therapeutic modality is warranted in patients with SSc.

[Pattern of T cell activation in absence of protective immunity against hepatitis B virus. Review]. / Patrón de activación de linfocitos T en ausencia de respuesta protectora contra el virus dela hepatitis B. Revisión.

Hepatitis B is an important cause of morbidity and mortality around the world. One-third of the world's population has been estimated to be infected with hepatitis B virus (HBV). A significant amount of evidence suggests that both humoral and cellular immune responses are important to eliminate the virus and that, cellular immunity is involved in the pathogenesis of the disease. Vaccination with HBsAg is considered as the main strategy for effective control of the infection and viral transmission. However, approximately 5-10% of immunized individuals fail to elicit detectable specific antibodies and remain at risk for hepatitis B infection. In this work we have reviewed the current status in the pathogenesis of the disease and the mechanisms described to explain nonresponsiveness to the vaccine as well. Since nonresponders to the vaccine are at risk for the infection, a common mechanism to explain the absence or inappropriate immune response to virus components is proposed. Within the suggested model an impaired activation of T lymphocytes against viral antigens, both in nonresponders to vaccination and chronically infected patients, is described. These observations could be consistent with potential differences in the MHC/Ag presentation; therefore contributing to our understanding of the altered T helper response as an underlying mechanism for the lack of protective immunity against VHB.

High prevalence of secondary resistance mutations in Venezuelan HIV-1 isolates.

The genetic variability was studied in HIV-1 from Venezuelan patients with and without treatment, in order to evaluate the presence of polymorphisms and drug resistance mutations. Proviral DNA from peripheral blood mononuclear cells or viral RNA from plasma was extracted from the blood of 30 patients. Two regions from the polymerase gene, protease (Pr) and reverse transcriptase (RT) and one genomic fragment from the envelope (Env) gene were amplified and sequenced. All HIV-1 samples analyzed were classified as subtype B, without evidence of recombination. Although no primary protease mutations were detected, a high frequency of secondary mutations (86%, 19/22), associated to restoration of viral replicative fitness, was observed in strains circulating both in treated and non-treated patients. Resistance mutations to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI) were detected in 35% (6/17) and 12% (2/17) of the viruses circulating in treated patients, respectively. Resistance mutations were also present in the virus infecting one antiretroviral naive individual (7.7%), suggesting that local screening for resistant mutation in naive patient might be important to minimize therapy failure. Future studies are warranted to assess the role of secondary mutation in the success of viral infection.

[Role of human immunodeficiency virus in leukocytes apoptosis from infected patients]. / Papel del virus de la inmunodeficiencia humana en 1a apoptosis de leucocitos de pacientes infectados.

The hallmark of the immunodeficiency virus infection is a progressive detriment of the immune response which has been associated to a gradual loss of its responsible components, in particularly, CD4 positive T cells. Although this cell population is considered the main target of the virus, there is a recent deal of interest in studying other components that may not be targets of the virus, but are important elements to control infectious microorganisms and that have been demonstrated to be altered during HIV infection. Neutrophils (PMN) are innate immune components that play a fundamental role against HIV infection and these cells have been described as functionally altered during AIDS. It has been suggested that such a dysfunction could be attributed to an increased susceptibility of these cells to accelerated spontaneous apoptosis. However, the underlying mechanisms that induce programmed cell death of neutrophils remain unknown. In previous works we have explored some events involved during cell death of neutrophils from HIV infected patients. It is the purpose of this work to review the current knowledge of apoptosis signals in neutrophils and to discuss our own data about some mechanisms involved in spontaneous and Fas mediated apoptosis, which may contribute to understand neutrophils dysfunction during HIV infection.