Anti-inflammatory effect of omega unsaturated fatty acids and dialysable leucocyte extracts on collagen-induced arthritis in DBA/1 mice.

Rheumatoid arthritis is a disabling autoimmune disease with a high global prevalence. Treatment with disease-modifying anti-arthritic drugs (DIMARDs) has been routinely used with beneficial effects but with adverse long-term consequences; novel targeted biologics and small-molecule inhibitors are promising options. In this study, we investigated whether purified omega unsaturated fatty acids (ω-UFAs) and dialysable leukocyte extracts (DLEs) prevented the development of arthritis in a model of collagen-induced arthritis (CIA) in mice. We also investigated whether the transcription factor NF-κB and the NLRP3 inflammasome were involved in the process and whether their activity was modulated by treatment. The development of arthritis was evaluated for 84 days following treatment with nothing, dexamethasone, DLEs, docosahexaenoic acid, arachidonic acid, and oleic acid. Progression of CIA was monitored by evaluating clinical manifestations, inflammatory changes, and histological alterations in the pads' articular tissues. Both DLEs and ω-UFAs led to an almost complete inhibition of the inflammatory histopathology of CIA and this was concomitant with the inhibition of NF-kB and the inhibition of the activation of NLRP3. These data suggest that ω-UFAs and DLEs might have NF-κB as a common target and that they might be used as ancillary medicines in the treatment of arthritis.

Induction and treatment of anergy in murine leprosy.

Leprosy is a disease consisting of a spectrum of clinical, bacteriological, histopathological and immunological manifestations. Tuberculoid leprosy is frequently recognized as the benign polar form of the disease, while lepromatous leprosy is regarded as the malignant form. The different forms of leprosy depend on the genetic and immunological characteristics of the patient and on the characteristics of the leprosy bacillus. The malignant manifestations of lepromatous leprosy result from the mycobacterial-specific anergy that develops in this form of the disease. Using murine leprosy as a model of anergy in this study, we first induced the development of anergy to Mycobacterium lepraemurium (MLM) in mice and then attempted to reverse it by the administration of dialysable leucocyte extracts (DLE) prepared from healthy (HLT), BCG-inoculated and MLM-inoculated mice. Mice inoculated with either MLM or BCG developed a robust cell-mediated immune response (CMI) that was temporary in the MLM-inoculated group and long-lasting in the BCG-inoculated group. DLE were prepared from the spleens of MLM- and BCG-inoculated mice at the peak of CMI. Independent MLM intradermally-inoculated groups were treated every other day with HLT-DLE, BCG-DLE or MLM-DLE, and the effect was documented for 98 days. DLE administered at a dose of 1.0 U (1 × 10(6) splenocytes) did not affect the evolution of leprosy, while DLE given at a dose of 0.1 U showed beneficial effects regardless of the DLE source. The dose but not the specificity of DLE was the determining factor for reversing anergy.

A test of the vulnerability-stress model with brooding and reflection to explain depressive symptoms in adolescence.

To date, few studies have attempted to test the effect of rumination and its components (brooding and reflection) on depression from a diathesis-stress approach, which involves an interaction between stressors and rumination. The purpose of this study was to assess whether rumination moderates the predictive association between stress and depressive symptoms in adolescents. The possible moderation effect of gender on the relationships between the two rumination components and depressive symptoms over time was also analyzed. It was hypothesized that brooding, both alone and in interaction with stressors, would predict an increase in depressive symptoms over time. In contrast, no main effects or similar interactions were expected for reflection. Finally, it was expected that the relationship between depressive symptoms and brooding would be higher in girls than in boys. A longitudinal study was carried out in three waves with a 6-month interval, in which a total of 998 adolescents (45 % female), aged between 13 and 17 years, completed measures of rumination, stressors, and depressive symptoms. The results showed that initial levels of stressors, brooding, and reflection predicted average levels of depressive symptoms over time. There was no significant interaction between rumination and stressors. Finally, brooding predicted depressive symptoms more strongly in girls than in boys. As a conclusion, these findings suggest that stressors and rumination components contribute separately to the development of depressive symptoms over time, and that brooding acts as a vulnerability factor for depression more strongly in girls than in boys.

ß7 integrins are required to give rise to intestinal mononuclear phagocytes with tolerogenic potential.

BACKGROUND AND OBJECTIVE: While pro-inflammatory monocyte trafficking to the intestine has been partially characterised, the molecules required for migration of tolerogenic mononuclear phagocytes (dendritic cells (DC) and macrophages) are unknown. We hypothesised that the gut-homing receptor integrin α4ß7 is required for this process. METHODS: We used a T cell-mediated colitis model to study the role of α4ß7 in the innate immune compartment. We then performed competitive bone marrow (BM) reconstitution experiments to assess the requirement of α4ß7 in the generation of intestinal retinoic acid (RA)-producing CD11c(hi) DC (ALDE(+)DC) and CD64 macrophages. Using mixed BM chimeras we also asked whether α4ß7 is required to give rise to tolerogenic mononuclear phagocytes. RESULTS: Lack of ß7 integrins in the innate immune compartment (ß7(-/-)RAG2(-/-) mice) markedly accelerated T cell-mediated colitis, which was correlated with lower numbers and frequencies of ALDE(+)DC in mesenteric lymph nodes. Consistent with a role of α4ß7 in the generation of intestinal mononuclear phagocytes, BM cells from ß7(-/-) mice poorly reconstituted small intestine ALDE(+)DC and Mφ when compared to their wild type counterparts. In addition, mice lacking ß7 integrins in the CD11c(hi) compartment showed decreased ability to induce Foxp3(+) T(REG) and IL-10-producing T cells. CONCLUSIONS: Mice lacking ß7 integrins in the innate immune compartment are more susceptible to intestinal inflammation, which is correlated with a requirement of ß7 integrins to reconstitute gut mononuclear phagocytes with tolerogenic potential.

Altered microRNA profiles in bronchoalveolar lavage fluid exosomes in asthmatic patients.

BACKGROUND: Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge. OBJECTIVE: To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure. METHODS: Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling. RESULTS: The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2) = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles. CONCLUSION: These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma.

A Phase I/IIa Prospective, Randomized, Open-Label Study on the Safety and Efficacy of Nebulized Liposomal Amphotericin for Invasive Pulmonary Aspergillosis.

Although nebulized liposomal amphotericin B (NLAB) is being used in invasive pulmonary aspergillosis (IPA) prophylaxis, no clinical trial has shown its efficacy as a therapeutic strategy. NAIFI is the inaugural randomized, controlled clinical trial designed to examine the safety and effectiveness of NLAB (dosage: 25 mg in 6 mL, three times per week for 6 weeks) against a placebo, in the auxiliary treatment of IPA. Throughout the three-year clinical trial, thirteen patients (six NLAB, seven placebo) were included, with 61% being onco-hematological with less than 100 neutrophils/µL. There were no significant differences noted in their pre- and post-nebulization results of forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), and oxygen saturation between the groups. Neither bronchospasm nor serum amphotericin B levels were reported in any patients given NLAB. 18F-Fluorodeoxyglucose positron emission tomography (FDG-PET-TC) was carried out at the baseline and after 6 weeks. A notable decrease in median SUV (standardized uptake value) was observed in NLAB patients after 6 weeks (-3.6 vs. -0.95, p: 0.039, one tail). Furthermore, a reduction in serum substance galactomannan and beta-D-Glucan was identified within NLAB recipients. NLAB is well tolerated and safe for patients with IPA. Encouraging indirect efficacy data have been derived from image monitoring or biomarkers. However, further studies involving more patients are necessary.

A critical view on the current use of daptomycin in Spain: The daptomise study.

BACKGROUND: The Study on the Clinical Use of DAPTOMycin in Spain (DAPTOMISE Study) is a national surveillance program of daptomycin use. The objectives of this study are to evaluate the current variability in daptomycin consumption across the different hospitals and the adequacy of therapy, specially focused on underdosing. METHODS: All adult and pediatric patients who received, at least, one dose of daptomycin in a single week in 98 institutions in Spain were included. The adequacy of daptomycin use was evaluated with respect to the indication, dosage, adjustments after microbiology results, switching to an oral agent and length of treatment. RESULTS: A total of 615 patients received daptomycin during the study week. The prevalence use was 2.3 patients / 100,000 inhabitants per week, 12.4 patients / 1000 admissions and 9.2 Days of Therapy (DOT) / 1000 hospital stays. These rates varied between hospitals: from 0 to 13.9 patients / 100,000 inhabitants, from 0 to 76.1 patients / 1000 admissions and from 0 to 49.4 DOT / 1000 hospital stays. The most frequent infections were bacteremia (31.6 %) and skin and soft tissue infections (17.9 %). Microbiological results were available in only 65.4 % of infections. The most frequent microorganisms were Staphylococcus aureus (192 isolates, of which 87 were resistant to methicillin) and coagulase-negative staphylococci (124 isolates). A total of 136 prescriptions (22.1 %) were underdosed. Dosages < 8 mg/kg were used for 35.6 % of endovascular infections and for 26.2 % of osteoarticular infections. Overall, 57.2 % of prescriptions were not optimal in, at least, one item. Clinical cure rate was 76.1% and mortality attributable to the infection 8.1%. CONCLUSION: This is the first registry that identifies the prevalence of use of daptomycin in Spain and shows a high variability in the consumption between the different hospitals. Daptomycin underdosing was present in more than 20 % of cases.

Necrosis, netosis, and apoptosis in pulmonary tuberculosis and type-2 diabetes mellitus. Clues from the patient's serum.

Pulmonary tuberculosis (PTB) and type 2 diabetes mellitus (T2DM) are two inflammatory diseases whose pathology involves neutrophils (NEU) as key participants. Countless inflammatory elements produced at the lesion sites leak into the blood and are distributed systemically. The study aimed to investigate the effect of the serum of patients with PTB, T2DM, and PTB + T2DM on the cellular and nuclear morphology of healthy NEU. Monolayers of NEU were prepared and incubated with sera from PTB (n꓿ 10), T2DM (n꓿10), PTB + T2DM (n꓿ 10) patients, or sera from healthy people (n = 10). Monolayers were stained for histones, elastase, and myeloperoxidase for NETosis, annexin V for apoptosis, and Iris fuchsia for necrosis. Hoechst stain (DNA) was used to identify the nuclear alterations. Necrosis was the predominant alteration. Sera from PTB + T2DM were the most potent change inducers. Normal sera did not induce cell alterations. The blood of TBP and T2DM patients carries a myriad of abnormal elements that induce necrosis of NEU in normal people, thus reflecting what might occur in the neutrophils of the patients themselves. These findings reinforce the participation of NEU in the pathology of these diseases. Necrosis is expected to be the most frequent neutrophil-induced alteration in tuberculosis and diabetes mellitus.

Functional state analysis of phagocytic cells of patients with type 2 diabetes and pulmonary tuberculosis.

BACKGROUND: The phagocytic function in pulmonary tuberculosis (PTB) and Type 2 diabetes (T2D) has been explored mainly in macrophages but not in polymorphonuclears (PMN). The purpose of this study was to determine the functional status of PMN leukocytes in patients with pulmonary tuberculosis (PTB), Type 2 diabetes (T2D), and in patients with both diseases. METHODS: An observational, prospective, and comparative study was carried out. 30 ambulatory patients with T2D, 10 with PTB undergoing treatment and 10 patients with PTB and T2D, and 44 healthy subjects were studied. PMN leukocytes were separated, the capacity of these cells to produce hydrogen peroxide and to reduce nitroblue tetrazolium (NBT) in response to stimulus with the phorbolic ester of myristic acid (PMA) was measured; and the capacity of PMN leukocytes to adhere to surfaces was determined. RESULTS: Concerning the test for adherence, on comparing healthy subjects with patients with T2D+PTB, we observed a clear decrease in cellular adherence in the group of patients with both diseases; it was statistically significant (p = 0.007).With regard to phagocytic function, we observed that in NBT reduction as well as in hydrogen peroxide production, statistically significant differences were not obtained on comparing healthy subjects with any of the three groups of patients. CONCLUSIONS: We observed a clear decrease in cellular adherence when both diseases co-exist. These results could indicate the need for the co-existence of T2D and TB to cause deterioration in the cells' adherence activity. The microtechniques employed permit the evaluation in a practical manner of certain phagocytic-activity expressions.

The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.

Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.

NeuroImmunoEndocrinology: A brief historic narrative.

Although no precise moment or unique event marks its birth, neuroimmunoendocrinology arguably shares a great deal of history with other medical and biologic disciplines. It originated from empirical observations and suppositions that failed to prevail upon the existing axioms. Despite the widespread resistance to embracing novel ideas, the seeming defeats inspired visionary researchers. Those pioneers managed to systematize the emerging knowledge and were able to contribute to science with real foundations. In consequence, new concepts and ideas arose in physiology, anatomy, endocrinology and early immunology. Together, they gave rise to a budding approach on the integration between the nervous, immune and endocrine systems. Then, neuroimmunoendocrinology emerged as a discipline integrating an intricate system with multidirectional functions and interactions that allow for responding to internal and external threats. Such response is mediated by cytokines, hormones and neurotransmitters, involved in different physiologic mechanisms of the organism homeostasis. Neuroimmunoendocrinology is no longer an area of scientific skepticism; on the contrary, it has cemented its position as a biomedical discipline worldwide for the past 70 years. Now, it offers a better understanding of pathologic processes.

A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis.

Background: Bovine tuberculosis (bTB) is still a prominent threat to animal health; lacking an efficient vaccine, other than BCG to get rid of tuberculosis, the most effective way for this is culling and slaughtering the infected animals. There are several cellular, serological, and molecular tests for the diagnosis of the disease but the most practical one at the field level is the double skin testing with bovine and aviary tuberculins. This is not a very specific test but is sensitive enough to identify most diseased animals; adjunct practical tests are desirable to strengthen the utility of skin tests. All lymphoid and myeloid cells participate, in diverse grades, in the immune response to tuberculosis with neutrophils playing an unintended pathologic role. The study aimed to investigate the response of neutrophils to agents present in the sera of tuberculous cows. Methods: We have developed a neutrophil-based test (N BT) to identify diseased cows within a herd suspected of having tuberculosis; a positive N BT correlates with a positive double skin test. In this test, healthy neutrophils are incubated with the sera of healthy or tuberculous cows for 3 and 6 h, and the nuclear morphologic changes are recorded and analyzed. Results: Sera from tuberculous but not from healthy cows induce nuclear alterations including pyknosis, swelling, apoptosis, and sometimes NETosis, in healthy neutrophils, and CFP 10 and ESAT 6 participate in the phenomenon. Conclusion: We propose the N BT as an auxiliary tool for substantiating the diagnosis of bTB reinforcing the PPD test outcome to help decide whether or not a cow should be sacrificed.

Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.

BACKGROUND: Exosomes are 30-100 nm membrane vesicles of endocytic origin produced by numerous cells. They can mediate diverse biological functions, including antigen presentation. Exosomes have recently been shown to contain functional RNA, which can be delivered to other cells. Exosomes may thus mediate biological functions either by surface-to-surface interactions with cells, or by the delivery of functional RNA to cells. Our aim was therefore to determine the presence of RNA in exosomes from human saliva, plasma and breast milk and whether these exosomes can be taken up by macrophages. METHOD: Exosomes were purified from human saliva, plasma and breast milk using ultracentrifugation and filtration steps. Exosomes were detected by electron microscopy and examined by flow cytometry. Flow cytometry was performed by capturing the exosomes on anti-MHC class II coated beads, and further stain with anti-CD9, anti-CD63 or anti-CD81. Breast milk exosomes were further analysed for the presence of Hsc70, CD81 and calnexin by Western blot. Total RNA was detected with a Bioanalyzer and mRNA was identified by the synthesis of cDNA using an oligo (dT) primer and analysed with a Bioanalyzer. The uptake of PKH67-labelled saliva and breast milk exosomes by macrophages was examined by measuring fluorescence using flow cytometry and fluorescence microscopy. RESULTS: RNA was detected in exosomes from all three body fluids. A portion of the detected RNA in plasma exosomes was characterised as mRNA. Our result extends the characterisation of exosomes in healthy humans and confirms the presence of RNA in human saliva and plasma exosomes and reports for the first time the presence of RNA in breast milk exosomes. Our results also show that the saliva and breast milk exosomes can be taken up by human macrophages. CONCLUSIONS: Exosomes in saliva, plasma and breast milk all contain RNA, confirming previous findings that exosomes from several sources contain RNA. Furthermore, exosomes are readily taken up by macrophages, supporting the notion that exosomal RNA can be shuttled between cells.

Sera from patients with tuberculosis increase the phagocytic-microbicidal activity of human neutrophils, and ESAT-6 is implicated in the phenomenon.

Background: It has been reported that sera from patients with active pulmonary tuberculosis (APT) induced nuclear changes in normal neutrophils that included pyknosis, swelling, apoptosis, and production of extracellular traps (NETs). Similar changes were observed with some sera from their household contacts but not with sera from healthy, unrelated individuals. It was suggested that those sera from household contacts that induced neutrophil nuclear changes might correspond to people with subclinical tuberculosis. Thus, our experimental approach might serve to identify individuals with early, ongoing disease. Methods: Nuclear changes in neutrophils were fully evident by 3 h of contact and beyond. Circulating mycobacterial antigens were the most likely candidates for this effect. We wanted to know whether the nuclear changes induced on neutrophils by the sera of APT patients would negatively affect the phagocytic/microbicidal ability of neutrophils exposed to APT sera for short periods. Results: We now provide evidence that short-term contact (30 min) with sera from patients with pulmonary tuberculosis increases several phagocytic parameters of normal neutrophils, including endocytosis, myeloperoxidase levels, production of free reactive oxygen species, phagolysosome fusion, and microbicidal activity on Staphylococcus aureus, with these effects not being observed with sera from healthy donors. We also give evidence that suggests that ESAT-6 and CFP-10 are involved in the phenomenon. Conclusion: We conclude that activation is a stage that precedes lethal nuclear changes in neutrophils and suggests that autologous neutrophils must circulate in an altered state in the APT patients, thus contributing to the pathology of the disease.

Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon γ (IFNγ)-producing T cells that cause inflammation and tissue damage in multiple organs, especially the lung. Exosomes are nano-sized immunomodulatory vesicles of endosomal origin released from a diverse range of cells and are also found in physiological fluids including bronchoalveolar lavage fluid (BALF) from healthy individuals. OBJECTIVE: To investigate whether exosomes are enriched in the lungs of patients with sarcoidosis compared with healthy individuals and whether they could contribute to pathogenesis. DESIGN: BALF exosomes from patients with sarcoidosis (n=36) and healthy controls (n=14) were compared by electron microscopy, flow cytometry, western blot analysis and mass spectrometry. BALF exosomes were incubated with autologous peripheral blood mononuclear cells (PBMCs) or the human bronchial epithelial cell line 16HBE14o-. Cytokines were measured by ELISPOT and ELISA. RESULTS: BALF from patients with sarcoidosis showed increased levels of exosomes compared with healthy individuals. Exosomes from patients showed significantly higher expression of MHC class I and II, tetraspanins CD9, CD63 and CD81 as well as neuregulin-1, known to be associated with cancer progression. Furthermore, BALF exosomes from patients induced significantly higher IFNγ and interleukin (IL)-13 production in autologous PBMCs compared with healthy individuals and could also stimulate IL-8 production from epithelial cells. CONCLUSION: The results indicate for the first time a role for exosomes in human lung disease with possible contributions to the initiation and progression of inflammation in sarcoidosis. This suggests that exosomes may be a new potential target for the clinical treatment of lung diseases.

Sleep characteristics of the parents of children admitted to a pediatric intensive care unit: risk factors and repercussion on their daily life activities.

OBJECTIVE: to analyze the sleep characteristics of the parents of children admitted to a pediatric intensive care unit (PICU), the possible risk factors and impact of sleep quality on their daily life activities. METHODS: Parents of children admitted to PICU for at least 48 h filled in a survey. Demographic data, sleep characteristics before and during admission and its impact on daily life activities measured by the FOSQ-10 questionnaire, were collected. RESULTS: 100 surveys from parents of 53 children admitted to the PICU were collected. Most children (74%) were cardiac patients. 55% of them had had previous PICU admissions. 45% of parents lived in a different city. They spent a median of 14 h a day (IQR 12-16) at the hospital and 89.2% did not attend work. Parents had significantly worse subjective sleep quality (p = 0.001), less sleeping hours/day (p = 0.001), more difficulty falling asleep (p = 0.001) and more night arousals (p = 0.001) during PICU admission than before. 77% of parents also had a bad FOSQ-10 score. Perceived sleep quality and FOSQ-10 score had a good correlation (p = 0.00, Kappa 0.43). Significant risk factors were living in a different city (p = 0.03), programmed admissions (p = 0.001), previous PICU admissions (p = 0.001), prolonged PICU length of stay (p = 0.03) and longer distance from home (p = 0.03). CONCLUSIONS: Three quarters of the parents of children admitted to PICU suffer from sleep disorders, which negatively affects their personal lives. Perceived sleep quality had a good correlation with FOSQ-10 score. Institutional support is needed to optimize parents' resting conditions during their child's hospitalization.

Effect of dialyzable leukocyte extract, sodium butyrate, and valproic acid in the development of anergy in murine leprosy.

Background: Murine leprosy is a chronic granulomatous disease caused by Mycobacterium lepraemurium (MLM) in mice and rats. The disease evolves with the development of cellular anergy that impedes the production of interferon gamma (IFNγ), tumor necrosis factor-alpha (TNFα), and nitric oxide (NO) required to kill the microorganism. In this study we investigated whether histone deacetylase inhibitors (HDACi) (valproic acid and sodium butyrate [NaB]) and the immunomodulator transfer factor in dialyzable leukocyte extracts (DLE) can prevent anergy in murine leprosy. Methods: Five groups of six Balb/c mice were intraperitoneally inoculated with 2 × 107 MLM. Thirty-days post inoculation, treatment was started; one group received no treatment, one was treated with rifampicin-clofazimine (R-C), one with sodium valproate (VPA), one with NaB, and one with DLE. The animals were monitored for the evidence of disease for 96 days. After euthanasia, their spleens were removed and processed for histologic, bacteriologic, and cytokine studies. Results: R-C completely controlled the ongoing disease. DLE and NaB significantly reduced the development of lesions, including granuloma size and the number of bacilli; VPA was less effective. DLE, NaB, and VPA reverted the anergic condition in diverse grades and allowed the expression of IFNγ, TNFα, and inducible NO synthase, also in diverse grades. Conclusion: Anergy in leprosy and murine leprosy allows disease progression. In this study, anergy was prevented, in significant degree, by DLE (an immunomodulator) and NaB (HDACi). VPA was less effective. These results suggest potential beneficial effects of DLE and NaB in the ancillary treatment of leprosy.

Etiological identification of viral agents in acute encephalitis in Guadalajara, México, 2011-2015

Introduction: Viral encephalitis is a well-known inflammatory process associated with neurological dysfunction that might derive into severe brain damage or a fatal outcome. In México there is no epidemiological data that describes the prevalence of viral agents responsible for acute encephalitis. Objective: To identify the main viral agents by real time PCR involved in acute encephalitis in Mexico. Materials and methods: We obtained cerebral spinal fluid (CSF) samples from all patients with suspected viral encephalitis admitted to the emergency service of the Hospital Civil de Guadalajara "Fray Antonio Alcalde". To identify pathogens, we performed nucleic acid extraction using real-time PCR and RT-PCR. Results: Sixty-six patients were diagnosed with acute encephalitis from 2011 to 2014. A definitive viral etiological diagnosis was established in 16 patients (24%); the main causative agents were enteroviruses in 50% of the 16 positive samples, followed by herpes simplex virus (37%) and cytomegaloviruses (12.5%). Patients with encephalitis were predominantly male (63.3%) and a seasonal predominance was observed during autumn (37.5%). The main clinical characteristics in the acute encephalitis phase were fever (48.45) and cephalea (36.3), followed by seizures, disorientation, and muscular weakness (30.3%). Kerning sign was present in two cases (3%) and other two cases presented Brudzinski's sign (3%). Conclusions: CSF PCR is a suitable diagnostic technique for the identification of viral encephalitis caused by viral infections that allows an appropriate antiviral therapeutic treatment.

Sera from patients with active pulmonary tuberculosis and their household contacts induce nuclear changes in neutrophils.

BACKGROUND: Resident alveolar macrophages, dendritic cells, and immigrating neutrophils (NEU) are the first cells to contact Mycobacterium tuberculosis in the lung. These cells, and additional lymphoid cells in the developing granuloma, release a series of components that may concentrate in the serum and affect disease progression. PURPOSE: The aim of this study was to investigate the effect of the serum from tuberculosis (TB) patients and their household contacts (HHC) on the nuclear morphology of NEU. MATERIALS AND METHODS: NEU from healthy (HLT) people were incubated with sera from patients with active pulmonary TB, their HHC, and unrelated people. Changes in the nuclear morphology of NEU were analyzed by light and electron microscopy. RESULTS: Sera from patients with TB induced changes in the nuclear morphology of NEU that included pyknosis, swelling, apoptosis, and netosis in some cases. Sera from some HHC induced similar changes, while sera from HLT people had no significant effects. Bacteria did not appear to participate in this phenomenon because bacteremia is not a recognized feature of nonmiliary TB, and because sera from patients that induced nuclear changes maintained their effect after filtration through 0.22 µm membranes. Neither anti-mycobacterial antibodies, TNFα, IL-6, IFNγ, or IL-8 participated in the phenomenon. In contrast, soluble mycobacterial antigens were likely candidates, as small quantities of soluble M. tuberculosis antigens added to the sera of HLT people led to the induction of nuclear changes in NEU in a dose-dependent manner. CONCLUSION: These results might help to detect subclinical TB within HHC, thus leading to a recommendation of prophylactic treatment.

Mycobacterium lepraemurium uses TLR-6 and MR, but not lipid rafts or DC-sign, to gain access into mouse macrophages.

OBJECTIVE/BACKGROUND: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages. MATERIALS AND METHODS: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy. RESULTS: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D). CONCLUSION: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.