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Abstract Background In the last years, plants are being used for the production of a wide variety of biopharmaceuticals, including cytokines, and have the potential to serve as vehicles for mucosal administration of these molecules. We had previously reported the expression of a cytokine, interleukin-12 (IL-12), in transgenic tomato plants and had demonstrated that it retained its biologic activity in vitro. Findings In this work, we administered crude extracts of IL-12-containing tomato fruits to mice through the intratracheal route, measuring endogenous IL-12 and determining biologic activity by quantification of interferon-gamma (IFN-γ) in lungs and by histological analysis. IFN-γ expression in lungs, as well as histological analysis, indicate that tomato-expressed IL-12 retains its biologic activity and, most importantly, its effects are restricted to the site of administration. Conclusion Our results indicate that the functional activity of tomato-expressed IL-12 is comparable to that of commercial recombinant IL-12 when given via the mucosal route. This opens the possibility of using crude extracts prepared from tomatoes expressing IL-12 for certain immunotherapies.

Non‐bilayer phospholipids arrangements (NPAs) are transient molecular associations different from lipid bilayers. When they become stable, they can trigger a disease in mice resembling human lupus, which is mainly characterized by the production of anti‐NPA IgG antibodies. NPAs are stabilized on liposomes or cell bilayers by the drugs procainamide or chlorpromazine, which produce drug‐induced lupus in humans. Here, we evaluated the participation of the TH2 response, through its hallmark cytokine IL‐4, on the development of the lupus‐like disease in mice. Wild‐type or IL‐4 knockout BALB/c mice received liposomes bearing drug‐induced NPAs, the drugs alone, or an anti‐NPA monoclonal antibody (H308) to induce the lupus‐like disease (the last two procedures stabilize NPAs on mice cells). IL‐4 KO mice showed minor disease manifestations, compared to wild‐type mice, with decreased production of anti‐NPA IgG antibodies, no anti‐cardiolipin, anti‐histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti‐NPA IgG antibodies. These findings indicate that IL‐4 has a central role in the development of the murine lupus‐like disease induced by NPA stabilization. [ABSTRACT FROM AUTHOR]

Abstract Background It has been established that hypothyroidism protects rats against renal ischemia and reperfusion (IR) oxidative damage. However, it is not clear if hypothyroidism is able to prevent protein tyrosine nitration, an index of nitrosative stress, induced by IR or if antioxidant enzymes have involved in this protective effect. In this work it was explored if hypothyroidism is able to prevent the increase in nitrosative and oxidative stress induced by IR. In addition the activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was studied. Control and thyroidectomized (HTX) rats were studied 24 h of reperfusion after 60 min ischemia. Methods Male Wistar rats weighing 380 ± 22 g were subjected to surgical thyroidectomy. Rats were studied 15 days after surgery. Euthyroid sham-operated rats were used as controls (CT). Both groups of rats underwent a right kidney nephrectomy and suffered a 60 min left renal ischemia with 24 h of reperfusion. Rats were divided in four groups: CT, HTX, IR and HTX+IR. Rats were sacrificed and samples of plasma and kidney were obtained. Blood urea nitrogen (BUN) and creatinine were measured in blood plasma. Kidney damage was evaluated by histological analysis. Oxidative stress was measured by immunohistochemical localization of protein carbonyls and 4-hydroxy-2-nonenal modified proteins. The protein carbonyl content was measured using antibodies against dinitrophenol (DNP)-modified proteins. Nitrosative stress was measured by immunohistochemical analysis of 3-nitrotyrosine modified proteins. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase was measured by spectrophotometric methods. Multiple comparisons were performed with ANOVA followed by Bonferroni t test. Results The histological damage and the rise in plasma creatinine and BUN induced by IR were significantly lower in HTX+IR group. The increase in protein carbonyls and in 3-nitrotyrosine and 4-hydroxy-2-nonenal modified proteins was prevented in HTX+IR group. IR-induced decrease in renal antioxidant enzymes was essentially not prevented by HTX in HTX+IR group. Conclusion Hypothyroidism was able to prevent not only oxidative but also nitrosative stress induced by IR. In addition, the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase seem not to play a protective role in this experimental model.

Abstract Background Potassium dichromate (K2Cr2O7)-induced nephrotoxicity is associated with oxidative and nitrosative stress. In this study we investigated the relation between the time course of the oxidative and nitrosative stress with kidney damage and alterations in the following antioxidant enzymes: Cu, Zn superoxide dismutase (Cu, Zn-SOD), Mn-SOD, glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT). Methods Nephrotoxicity was induced in rats by a single injection of K2Cr2O7. Groups of animals were sacrificed on days 1,2,3,4,6,8,10, and 12. Nephrotoxicity was evaluated by histological studies and by measuring creatinine clearance, serum creatinine, blood urea nitrogen (BUN), and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) and total protein. Oxidative and nitrosative stress were measured by immunohistochemical localization of protein carbonyls and 3-nitrotyrosine, respectively. Cu, Zn-SOD, Mn-SOD, and CAT were studied by immunohistochemical localization. The activity of total SOD, CAT, GPx, and GR was also measured as well as serum and kidney content of chromium and urinary excretion of NO2 -/NO3-. Data were compared by two-way analysis of variance followed by a post hoc test. Results Serum and kidney chromium content increased reaching the highest value on day 1. Nephrotoxicity was made evident by the decrease in creatinine clearance (days 1–4) and by the increase in serum creatinine (days 1–4), BUN (days 1–6), urinary excretion of NAG (days 1–4), and total protein (day 1–6) and by the structural damage to the proximal tubules (days 1–6). Oxidative and nitrosative stress were clearly evident on days 1–8. Urinary excretion of NO2-/NO3- decreased on days 2–6. Mn-SOD and Cu, Zn-SOD, estimated by immunohistochemistry, and total SOD activity remained unchanged. Activity of GPx decreased on days 3–12 and those of GR and CAT on days 2–10. Similar findings were observed by immunohistochemistry of CAT. Conclusion These data show the association between oxidative and nitrosative stress with functional and structural renal damage induced by K2Cr2O7. Renal antioxidant enzymes were regulated differentially and were not closely associated with oxidative or nitrosative stress or with kidney damage. In addition, the decrease in the urinary excretion of NO2-/NO3- was associated with the renal nitrosative stress suggesting that nitric oxide was derived to the formation of reactive nitrogen species involved in protein nitration.

Tuberculosis (TB) is one of the deadliest infectious diseases in humankind history. Although, drug sensible TB is slowly decreasing, at present the rise of TB cases produced by multidrug‐resistant (MDR) and extensively drug‐resistant strains is a big challenge. Thus, looking for new therapeutic options against these MDR strains is mandatory. In the present work, we studied, in BALB/c mice infected with MDR strain, the therapeutic effect of supra‐pharmacological doses of the conventional primary antibiotics rifampicin and isoniazid (administrated by gavage or intratracheal routes), in combination with recombinant human hepatocyte growth factor (HGF). This high dose of antibiotics administered for 3 months, overcome the resistant threshold of the MDR strain producing a significant reduction of pulmonary bacillary loads but induced liver damage, which was totally prevented by the administration of HGF. To address the long‐term efficiency of this combined treatment, groups of animals after 1 month of treatment termination were immunosuppressed by glucocorticoid administration and, after 1 month, mice were euthanized, and the bacillary load was determined in lungs. In comparison with animals treated only with a high dose of antibiotics, animals that received the combined treatment showed significantly lower bacterial burdens. Thus, treatment of MDR‐TB with very high doses of primary antibiotics particularly administrated by aerial route can produce a very good therapeutic effect, and its hepatic toxicity can be prevented by the administration of HGF, becoming in a new treatment modality for MDR‐TB. [ABSTRACT FROM AUTHOR]

Background During pregnancy, the Zika virus (ZIKV) replicates in the placenta and central nervous system (CNS) of infected fetuses; nevertheless, the ability of ZIKV to replicate in other fetal tissues has not been extensively characterized. Methods We researched whether dissemination of congenitally-acquired ZIKV outside the CNS exists by searching for the accumulation of the viral envelope protein, ZIKV ribonucleic acid (RNA), and infectious viral particles in different organs of a deceased newborn with Congenital Zika Syndrome. A real-time qualitative polymerase chain reaction (qPCR) was used to detect ZIKV RNA in the brain, thymus, lungs, kidneys, adrenal glands, spleen, liver, and small intestine. The same tissues were analyzed by indirect immunofluorescence and immunoperoxidase assays using the monoclonal antibody 4G2 to detect ZIKV envelope antigens. Isolation of infectious ZIKV in a cell culture was carried out using brain and kidney samples. Results A postmortem, virological analysis of multiple organs, such as the kidneys (epithelial cells in the renal tubules), lungs (bronchial epithelia), thymus (epithelial cells inside the Hassall's corpuscles), and brain (neurons, ependymal cells, and macrophages) revealed the presence of ZIKV RNA and envelope antigens. Other tissues of the deceased newborn tested positive by qPCR for Epstein-Barr virus and human herpesvirus 6, including the brain cortex (Epstein-Barr) and the thymus, kidneys, and adrenal glands (human herpesvirus 6). The kidneys were identified as a significant niche for viral replication, given that infectious particles were successfully isolated from renal tissues. Conclusions Our findings demonstrate the ability of congenitally-acquired ZIKV to produce disseminated infections and the viral tropism towards epithelial cells. [ABSTRACT FROM AUTHOR]

Previously, we described tracheal rat rings relaxation by several flavonoids, being 6‐hydroxyflavone (6‐HOF) the most active derivative of the series. Thus, its mechanism of action was determined in an ex vivo tracheal rat ring bioassay. The anti‐asthmatic effect was assayed in in vivo OVAlbumin (OVA)‐sensitized guinea pigs. Finally, the toxicological profile of 6‐HOF was studied based on Organization of Economic Cooperation and Development guidelines with modifications. 6‐HOF–induced relaxation appears to be related with receptor‐operated calcium channel and voltage‐operated calcium channel blockade as the main mechanism of action, and also through the production of relaxant second messengers NO and cGMP. Molecular docking supports that 6‐HOF acts as calcium channel blocker and by activation of nitric oxide synthase. In addition, the in vivo anti‐asthmatic experiments demonstrate the dose‐dependent significant anti‐allergic effect of 6‐HOF induced by OVA, with best activity at 50 /kg. Finally, toxicological studies determined a LD50 > 2,000 mg/kg and, after 28 day of treatment with 6‐HOF (50 mg/kg) by intragastric route, mice did not exhibit evidence of any significant toxicity. In conclusion, experiments showed that 6‐HOF exerts significant relaxant activity through calcium channel blockade, and possibly, by NO/cGMP‐system stimulation on rat trachea, which interferes with the contraction mechanism of smooth muscle cells in the airways. In addition, the flavonoid shows potential anti‐asthmatic properties in an anti‐allergic pathway. Furthermore, because the pharmacological and safety evidence, we propose this flavonoid as lead for the development of a novel therapeutic agent for the treatment of asthma and related respiratory diseases. [ABSTRACT FROM AUTHOR]

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success. [ABSTRACT FROM AUTHOR]

Introduction Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that mainly affects the lungs. Along the course of pulmonary TB there are remarkable changes in the production of cytokines that cause endocrine changes. So far, it is not known the physiological and histological changes in the male reproductive system during pulmonary TB. Objective To investigate whether pulmonary TB produces histological alterations of the BALB/c mice reproductive organs, as well as abnormalities in spermatogenesis, serum testosterone concentrations and expression of testicular cytokines. Methods BALB/c mice were infected intratracheally with high dose Mtb strain H37Rv. Groups of six non infected and infected animals were euthanized on days 1, 3, 7, 14, 21, 28, 60, 90 and 120 post-infection. Bacillary loads were determined by counting colony forming units (CFUs) in lungs, testes, prostate and seminal vesicles. Histological sections were obtained from the same organs. Spermatozoids number and quality were assessed by spermatobioscopy. Serum testosterone concentrations were determined by radioimmunoanalysis (RIA) in control and infected mice in each time of sacrifice. Results Mtb only grew in lung tissue. Serum androgens showed a trend to decrease in the infected mice compared to the healthy animals, the difference turn into statistically significance at post infection day 120. The weight of the testis was not modified throughout the study, and no histopathological changes were found. However, we detected a significant decrease in the weight of the seminal vesicles and prostate starting at 28 days post-infection. Atrophy of the seminal vesicles and prostate epithelia were significant, beginning after 60 days of infection. Spermatobioscopy revealed hypospermia in the later stages of the disease. We have observed in the testes a local significant disbalance on the cytokine profile (increase of IL-6 and decrease of IL-10 and TGF-b levels) together with a very significant reduction of the body weight during late pulmonary TB. Conclusion Pulmonary TB affects the histophysiology of the male reproductive system due to hormonal changes, an imbalance of pro-inflammatory cytokine profile, and a wasting syndrome during late disease. [ABSTRACT FROM AUTHOR]