Search

Serpins represent one of the most diverse families of serine protease inhibitors. Despite their complexity, they are virtually found in all organisms and play an important role in homeostasis processes such as blood coagulation, inflammation, fibrinolysis, immune responses, chromatin condensation, tumor suppression, and apoptosis. There has recently been particular interest in studying serpin functions in infection and inflammation, especially since more serpins from parasites have been identified and characterized. Among helminths, Trichinella spiralis is one of the few parasites with an extremely strong ability to induce host immune suppression. Previous studies show that serpins are present in Trichinella and are expressed differentially at different parasite stages. More interesting, there is evidence of a recombinant serpin from Trichinella pseudospiralis that alters macrophage polarization in vitro. This finding could be relevant to comprehend the modulation process of the immune response. We expressed Tsp_01570, a putative serpin gene from Trichinella spiralis, in the eukaryotic system Pichia pastoris SMD1168H and evaluated its presence at different parasite stages, finding the serine protease inhibitor in the crude extract of adult worms. The effect of recombinant serpin on THP-1 cells was tested by quantification of IL-12p40, TNF-α, IL-4, and IL-10 cytokines released by ELISA. We also evaluated the expression of the M1 markers, CCR7 and CD86, and the M2 markers, CD163 and CD206, by immunofluorescence staining. This study represents the first insight in elucidating the importance of serpin Tsp_01570 as a potential molecular modulator., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Pichia pastoris has been widely used to produce antigenic proteins aimed to integrate subunit vaccines. Moreover, increasing interest in large-scale vaccine production at the lowest cost is rapidly focusing in the development of yeast surface display (YSD) systems for delivery of antigens. In this scenario, the safety of live yeast administration must be warranted, however, such information is very scarce. Here, we assess the intravenous administration (i.v.) of live P. pastoris cells in order to trace dissemination in BALB/c mice and to evaluate the immune response raised against the yeast compared to the well-defined pathogen Candida albicans. Our results demonstrate dissemination of P. pastoris to the heart, kidney, and spleen, but it is quickly eliminated during the first 48 h postinfection (hpi), with persistence in the liver along with mild mononuclear (MN) and polymorphonuclear (PMN) infiltrate, which was resolved at 144 hpi. In vivo delayed-type hypersensitivity test (DTH) or in vitro antigenic stimulation of mice splenocytes demonstrate that transient infection of P. pastoris did not induce a cell-mediated immune response nor increase the level of circulating IgG or IgM. These results demonstrate the innocuous profile of P. pastoris and support its use as a safe delivery system for vaccine development., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

We previously reported that the Trichinella nematode showed higher parasite loads in one gender than another, but also the parasite molting rate decreased when it was cultivated in the presence of progesterone. In this study we explored the hypothesis that the direct effect of progesterone on Trichinella spiralis could be mediated by a steroid-binding parasite protein. We sequenced, cloned and amplified the Cyt-domain of the progesterone receptor membrane component-2 of Trichinella spiralis (PGRMC2-Ts). Furthermore, we expressed the protein and developed an antibody to perform confocal microscopy and flow cytometry. The expression of the PGRMC2-Ts protein was exclusively detected at the oocyte and the parasite's cuticle in cross-sections of the parasite, and this expression was confirmed by western blot and flow cytometry. Molecular modeling studies and computer docking for the PGRMC2-Ts protein showed that it is potentially able to bind to progesterone, estradiol, testosterone, and dihydrotestosterone with different affinities. Furthermore, phylogenetic analysis demonstrated that T. spiralis PGRMC2 is related to a steroid-binding protein of another platyhelminth. Progesterone probably acts upon Trichinella spiralis oocytes by binding to PGRMC2-Ts. Our data showed that the PGRMC2-Ts protein is present in the parasite's oocytes, a development step that is crucial for the life cycle of the parasite. Indeed, this research might have implications in the field of host-parasite co-evolution and the sex-associated susceptibility to this infection. In a more practical matter, these results may contribute to the design of new drugs with anti-parasite effects., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: It has been established that the genomic background of Mycobacterium tuberculosis may influence disease progression, in particular for the Beijing family and the Latin American and Mediterranean (LAM)/RD Rio strains. The purpose of this study was to evaluate the prevalence of the LAM/RD Rio genotype in cases of tuberculosis from Mexico and their drug susceptibility profile., Methods: Two hundred eighteen M. tuberculosis isolates were screened by 43-spacer spoligotyping. The LAM/RD Rio genotype was confirmed by multiplex PCR, and the drug susceptibility testing was carried out in solid Löwenstein-Jensen media., Results: Among the LAM strains identified, 24 (63.1%) were confirmed as M. tuberculosis RD Rio . All RD Rio strains shared the RD174 deletion, that was associated with isoniazid resistance (p=0.0264)., Conclusions: We documented for the first time the isolation of the LAM/RD Rio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains., (Copyright © 2020 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation., Results: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10., Conclusion: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: It has been established that the genomic background of Mycobacterium tuberculosis may influence disease progression, in particular for the Beijing family and the Latin American and Mediterranean (LAM)/RD Rio strains. The purpose of this study was to evaluate the prevalence of the LAM/RD Rio genotype in cases of tuberculosis from Mexico and their drug susceptibility profile., Methods: Two hundred eighteen M. tuberculosis isolates were screened by 43-spacer spoligotyping. The LAM/RD Rio genotype was confirmed by multiplex PCR, and the drug susceptibility testing was carried out in solid Löwenstein-Jensen media., Results: Among the LAM strains identified, 24 (63.1%) were confirmed as M. tuberculosis RD Rio . All RD Rio strains shared the RD174 deletion, that was associated with isoniazid resistance (p=0.0264)., Conclusions: We documented for the first time the isolation of the LAM/RD Rio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains., (Copyright © 2020 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Publicado por Elsevier España, S.L.U. All rights reserved.)

Protease-activated receptors (PARs) have been described in a wide diversity of vertebrate cells, including human immune cells. Macrophages are pivotal cells in the host-pathogen interaction and their polarization in M1 or M2 cells has been described as a new central paradigm in the immune response to pathogens. In this context, we explored the involvement of PAR activation by serine proteases on M1/M2 macrophage differentiation and their impact on the Th1/Th2 cytokine profile in response to Mycobacterium tuberculosis antigen. Our results demonstrate that the serine proteases, thrombin and trypsin, induce interleukin (IL)-4 release from human monocytes, together with upregulation of the macrophage mannose receptor (CD206) in the same way that alternative M2a differentiated cells with M-CSF/IL-4. Protease stimulation of monocytes in the presence of PAR-1 (SCH-79797) or PAR-2 (FSLLRY-NH2) antagonists abolished IL-4 release from monocytes, whereas the use of the peptide agonist for PAR-1 (SFLLRNPNDKYEPF-NH2) or PAR-2 (SLIGKV-NH2) induced the secretion of IL-4 at a level comparable to thrombin or trypsin. When these protease-induced M2 macrophages from healthy human PPD + donors were co-cultured with autologous lymphocyte population in the presence of Mycobacterium tuberculosis antigen, we found a consistent inhibition of IFN-γ/IL-12 release together with persistent IL-4 expression, in contrast to the expected Th1 profile obtained with M2a macrophages. To our knowledge, this is the first observation that proteolytic activation of PAR1/2 receptors in monocytes induces M2-like macrophages with impaired plasticity and their implication in the driving of the Th1/Th2 cytokine profile., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Notwithstanding evidence that tuberculosis (TB) is declining, one of the greatest concerns to public health is the emergence and spread of multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB). MDR-TB are defined as strains which are resistant to at least isoniazid (INH) and rifampicin, the two most potent TB drugs, and their increasing incidence is a serious concern. Recently, notable efforts have been spent on research to pursue novel treatments against MDR-TB, especially on synergistic drug combinations as they have the potential to improve TB treatment. Our research group has previously reported promising synergistic antimicrobial effects between transition-metal compounds and antibiotics in Gram-negative and Gram-positive bacteria. In this work, we evaluated antimycobacterial activity of transition-metals/antibiotics combinatorial treatments against first-line drug resistant strains of Mycobacterium tuberculosis. Our data showed that INH/AgNO 3 combinatorial treatment had an additive effect (bactericidal activity) in an isoniazid-resistant clinical strain of Mycobacterium tuberculosis. Moreover, in vitro evaluation of cytotoxicity induced by both, the individual tratments of AgNO 3 and INH and the combinatorial treatment of INH/AgNO 3 in murine RAW 264.7 macrophages and human A549 lung cells; showed no toxic effects. Together, this data suggests that the INH/AgNO 3 combinatorial treatment could be used in the development of new strategies to treat resistant strains of Mycobacterium tuberculosis.

Trichosporon asahii is considered an opportunistic pathogen responsible for severe infections, mainly in immunocompromised patients. The aims of this study were to investigate the prevalent genotypes among 39 clinical isolates of this microorganism by sequencing the IGS1 region and to determine the in vitro production of DNAse, hemolysin, aspartyl proteinase, phospholipase and esterase, as well as the susceptibilities of the isolates to amphotericin B, anidulafungin, micafungin, caspofungin, voriconazole, posaconazole, fluconazole and 5-flucytosine. Our findings showed that genotype I was the most prevalent comprising 69.23% of the isolates. We confirmed the production of esterase for all our isolates, and report the production of DNAse and aspartyl proteinase in 84.62% and 23% of the isolates, respectively. Only one isolate of T. asahii produced hemolysin. None of the isolates showed phospholipase activity. Fifty-three percent of the T. asahii strains exhibited amphotericin B MICs ≥ 2 μg/ml. The three echinocandins evaluated yielded high MICs (≥2 μg/ml) in all isolates. Thirty-five percent of the isolates had high MICs for 5-flucytosine (≥32 μg/ml), and 97% of the isolates were susceptible to the evaluated triazoles., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To study the effect of the initiation time of posaconazole treatment from 1 to 3 days after systemic infection by Trichosporon asahii in mice., Methods: BALB/c mice, 4-5 weeks old, were intravenously infected with 1 × 10(7) cfu/mouse of T. asahii. The onset of treatment varied from 1 to 3 days after infection. Orally administered posaconazole at 0.5, 1, 2, 5 or 10 mg/kg body weight/day was compared with orally administered fluconazole (at 10 mg/kg/day) and intraperitoneally administered amphotericin B (at 1 mg/kg) on alternating days. Livers, kidneys and spleens of mice that died or survived to day 25 were removed to determine fungal tissue burdens., Results: When therapy began 1 day after challenge, posaconazole at ≥ 1 mg/kg significantly prolonged survival of mice compared with that of the control group and considerably reduced the fungal tissue burden over the control group. On the other hand, when treatment was started 3 days after infection, regimens of 5 and 10 mg/kg posaconazole significantly prolonged mice survival over that of the control group and appreciably diminished the fungal load compared with untreated mice. In this model, as the severity of trichosporonosis increased, higher doses of posaconazole were required to achieve equivalent activity levels. Fluconazole and amphotericin B were ineffective in preventing mice death and in significantly reducing fungal tissue burden. Posaconazole displayed potent in vivo activity against the strain tested., Conclusions: Posaconazole may be a suitable option in the treatment of disseminated T. asahii infection.

Objectives: To establish the species distribution and in vitro susceptibilities of 358 bloodstream fungal isolates from paediatric patients in Mexico., Methods: Isolates were collected during a 2 year surveillance programme in 14 medical centres in 10 Mexican states. A molecular approach was used to determine the Candida parapsilosis species complex. In vitro susceptibility to amphotericin B, fluconazole, voriconazole, itraconazole, posaconazole, caspofungin, anidulafungin and micafungin was determined according to CLSI procedures. Species-specific clinical breakpoints for fluconazole, voriconazole and echinocandins were applied., Results: Candida spp. accounted for 98.33% of fungaemias, including 127 Candida albicans isolates, 127 C. parapsilosis complex isolates (121 C. parapsilosis sensu stricto, 4 Candida orthopsilosis and 2 Candida metapsilosis strains) and 72 Candida tropicalis isolates. C. albicans and C. parapsilosis complex were the species predominant in neonates (48 cases each; 41.02%). C. parapsilosis complex was also the predominant species in patients 1 month to <2 years of age (P = 0.007). In contrast, C. albicans was the most frequent species in patients aged 2 to <12 years (P = 0.003). Antifungal resistance was rare among the subset of isolates. Candida glabrata showed the highest resistance rate to amphotericin B (1/9 isolates), fluconazole (1/9 isolates) and itraconazole (2/9 isolates)., Conclusions: The species distribution differed with the age of the patients, with C. albicans and C. parapsilosis complex being the most commonly isolated species. C. glabrata showed the highest resistance rate to amphotericin B, fluconazole and itraconazole. This is the first study of fungaemia episodes in Mexican children.

Systemic disease is the most severe clinical form of fusariosis, and the treatment involves a challenge due to the refractory response to antifungals. Treatment for murine Fusarium solani infection has been described in models that employ CFU quantitation in organs as a parameter of therapeutic efficacy. However, CFU counts do not precisely reproduce the amount of cells for filamentous fungi such as F. solani. In this study, we developed a murine model of disseminated fusariosis and compared the fungal burden with two methods: CFU and quantitative PCR. ICR and BALB/c mice received an intravenous injection of 1 × 10(7) conidia of F. solani per mouse. On days 2, 5, 7, and 9, mice from each mice strain were killed. The spleen and kidneys of each animal were removed and evaluated by qPCR and CFU determinations. Results from CFU assay indicated that the spleen and kidneys had almost the same fungal burden in both BALB/c and ICR mice during the days of the evaluation. In the qPCR assay, the spleen and kidney of each mouse strain had increased fungal burden in each determination throughout the entire experiment. The fungal load determined by the qPCR assay was significantly greater than that determined from CFU measurements of tissue. qPCR could be considered as a tool for quantitative evaluation of fungal burden in experimental disseminated F. solani infection.

The proliferation, directional migration to the vitreous and epithelial-mesenchymal transition (EMT) of quiescent, differentiated retinal pigment epithelium (RPE) cells is a major feature in the development of proliferative vitreoretinopathy (PVR) following exposure of the immuno-privileged eye niche to serum components, thrombin among them. We have previously documented thrombin induction of RPE cell proliferation and migration. We here analyzed the effect of thrombin on the E/N cadherin switch, a hallmark of EMT. Results show that thrombin induces the specific repression of epithelial E-cadherin gene transcription, alongside with the up-regulation of mesenchymal N-cadherin protein in RPE cells. We demonstrate, for the first time, that thrombin induces E-cadherin repression by stimulating snail-2 (SLUG) transcription factor expression, and the concomitant up-regulation of N-cadherin through the transcription-independent increase in protein translation promoted by PI3K/PKC-ζ/mTOR signaling. Our present findings suggest that the activation of protease-activated receptor-1 (PAR-1) by thrombin induces EMT of RPE cells, further supporting a central role for thrombin in PVR pathogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)

Currently, one third of the world's population is infected with Mycobacterium tuberculosis and 8.9-9.9 million new and relapse cases of tuberculosis are reported every year. The emergence of new cases, the increased incidence of multi-drug resistant strains of M. tuberculosis, and the adverse effects of first- and second-line antituberculosis drugs have led to renewed research interest in natural products in the hope of discovering new antitubercular leads. Interestingly, hundreds of natural products, possessing novel, uncommon, and known structural architectures, have been reported to exhibit activity towards non-resistant and multi-drug resistant strains of M. tuberculosis. The present review covers literature published during the last five years about those naturally occurring compounds with reported growth inhibitory activity in vitro towards sensitive and resistant M. tuberculosis strains. Compounds with antitubercular properties at minimal inhibitory concentrations (MICs) of less than 50 μg/mL or 60 μM were selected and grouped according to their source of origin (plants, bacteria, fungi, marine organisms, etc) and chemical type (terpenes, steroids, alkaloids, flavonoids, poliketides, peptides, etc). In some cases, the selection covers those structurally relevant natural products with low bioactivity (MICs of ≤128 μg/mL), and also those semisynthetic derivatives with remarkable antitubercular activity (MICs of ≤10 μg/mL). Additionally, this review includes a special section for those natural products that specifically target genes or enzymes of M. tuberculosis., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Tuberculosis (TB) is an infectious disease affecting people from all ages all over the world. It is estimated that one third of the world population lives infected with the causal agent: Mycobacterium tuberculosis. Despite availability and systematic administration of BCG vaccine in endemic areas, TB transmission remains elusive to control, partly because BGC efficacy has been shown to have wide variability (0-80%). Such variability in protection is attributed to factors including: the BCG strain used for immunization, pre-existing exposure to environmental saprophytic Mycobacterium species, and host genetic factors. In this context, efforts regarding to re-engineering BCG vaccines with the ability to prevent latent TB reactivation, providing long lasting protection, and devoid from collateral effects in immunosuppressed people are urgent. In this work we review the actual molecular «gene-by-gene» strategies aimed at generating BCG alternatives, and discuss the urgent necessity of high throughput technology methods for a rational design for a new TB vaccine., (Copyright © 2010 Elsevier España, S.L. All rights reserved.)

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial-mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose-dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR-2 and CCR-2 chemokine receptors. Whereas inhibition of CXCR2 by Sb-225002 and of CCR2 by Rs-504393 partially prevented hirudin-sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro-32-0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC-zeta pseudosubstrate and by the nuclear factor-kappa B (NF-kappaB) inhibitor BAY11-7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK-ERK-PI3K-mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC-zeta., (J. Cell. Biochem. (c) 2010 Wiley-Liss, Inc.)

The retinal pigment epithelium (RPE) plays an essential role in the maintenance and normal functioning of the neural retina. Alterations in RPE function are involved in several ocular pathologies involving the breakdown of the blood-retina barrier (BRB), which exposes RPE to serum components, thrombin among them. Our previous work has shown that thrombin stimulates the proliferation of RPE cells. We here analyzed the molecular pathways leading to this outcome, in order to support thrombin involvement in proliferative vitreoretinopathy (PVR), a major cause of retinal surgery failure. We demonstrated that thrombin activation of PAR-1 promotes cyclin D1 expression at the transcriptional level by stimulating c-Fos expression, mediated by PI3K, MAPK ERK1/2, and conventional PKC activity. Our results show that ERK activation is necessary but not sufficient for the induction of cyclin D1 expression and proliferation, since the inhibition of PI3K or cPKC prevents this outcome. Analysis of thrombin-activated PAR-1 downstream effectors demonstrated that c-Fos expression by the sustained activation of ERK and c-fos transcription triggers the expression and nuclear translocation of cyclin D1, a key regulator of cell cycle G1/S phase progression leading to proliferation. Evidence here provided contributes to the understanding of the mechanisms involved in proliferative eye diseases and enhances the possibility of controlling pathologies such as proliferative PVR, which eventually lead to blindness., ((c) 2009 Wiley-Liss, Inc)

Protection against tuberculosis (TB) is based on cell-mediated immune responses. TB is often characterized by immunological dysfunction of peripheral blood mononuclear cells, especially at chronic stages. Lipids from the Mycobacterium tuberculosis cell wall have been shown to produce various suppressive effects on cell-mediated immunity. The cell-surface lipid di-O-acyl-trehalose (DAT) is able to inhibit T-cell proliferation and cytokine secretion in cells from naïve mice. In the present study, we addressed the mechanisms involved in the suppressive effect caused by DAT. We found that DAT decreased the proliferation of spleen cells induced with PMA-ionomycin, suggesting that the suppressive mechanisms target intracellular functions just after phospholipase C-gamma activation. Addressing this possibility, the effect of DAT was found to involve down-modulation of the di-acyl glycerol-dependent activation of the MAPK-ERK1/2 pathway, one of the crucial signaling pathways leading to adaptive cell immune response against TB. Moreover, the inhibitory effect of DAT on proliferation was reproduced in antigen-stimulated T cells from M. tuberculosis-infected mice, involving the lowering of Th1-type cytokine transcription levels. The present findings thus reveal a new kind of bioactivity for a long-known M. tuberculosis cell wall lipid, DAT.

Mycobacterium tuberculosis is the single most deadly microorganism worldwide. A third of the world population is thought to have latent tuberculosis and approximately 2 million people die of the disease each year. Short and closely supervised treatment regimens are needed, but it is also essential to develop new strategies to ensure prompt diagnosis of the disease. In particular, cheap methods are needed to tackle tuberculosis from a population perspective. The present article reviews the advances in immunology and molecular strategies for epidemiological diagnosis and monitoring of tuberculosis patients.

cDNA encoding mature human placental variant growth hormone (HGH-V) was synthesized by retro-transcription polymerase chain reaction (RT-PCR) from total RNA recovered from human term-placenta and cloned in pBluescript plasmid (pBS) in Escherichia coli. cDNA was subcloned into pPIC9, fusing it to the flanking regulatory sequences of the Pichia pastoris alcohol oxidase 1 gene (AOX1) and finally introduced into the genome of this yeast by homologous recombination. The resulting new recombinant strain produced and secreted, towards the culture medium, mature HGH-V, whose activity was demonstrated in cell culture by the Nb2 proliferation assay.