Structural and functional integrity of endocrine pancreas post administration of Karwinskia humboldtiana fruit to Wistar rats: a possible therapeutic application for cancer of exocrine origin.

AIMS: Pancreatic adenocarcinoma represents a therapeutic challenge due to the high toxicity of antineoplastic treatments and secondary effects of pancreatectomy. T-514, a toxin isolated from Karwinskia humboldtiana (Kh) has shown antineoplastic activity on cell lines. In acute intoxication with Kh, we reported apoptosis on the exocrine portion of pancreas. One of the mechanisms of antineoplastic agents is the induction of apoptosis, therefore our main objective was to evidence structural and functional integrity of the islets of Langerhans after the administration of Kh fruit in Wistar rats. METHODS: TUNEL assay and immunolabelling against activated caspase-3 were used to detect apoptosis. Also, immunohistochemical tests were performed to search for glucagon and insulin. Serum amylase enzyme activity was also quantified as a molecular marker of pancreatic damage. RESULTS: Evidence of toxicity on the exocrine portion, by positivity in the TUNEL assay and activated caspase-3, was found. On the contrary, the endocrine portion remained structurally and functionally intact, without apoptosis, and presenting positivity in the identification of glucagon and insulin. CONCLUSIONS: These results demonstrated that Kh fruit induces selective toxicity on the exocrine portion and establish a precedent to evaluate T-514 as a potential treatment against pancreatic adenocarcinoma without affecting the islets of Langerhans.

Expression of Trichinella spiralis serpin Tsp_01570 in Pichia pastoris: a first insight of its biomodulatory activity.

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.

Molecular identification of a PGRMC-2 receptor in maturing oocytes of the zoonotic nematode parasite Trichinella spiralis.

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.

RDRioMycobacterium tuberculosis strains associated with isoniazid resistance in Northern Mexico.

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)/RDRio strains. The purpose of this study was to evaluate the prevalence of the LAM/RDRio 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/RDRio 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 RDRio. All RDRio 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/RDRio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains.

Expression of recombinant protease MarP from Mycobacterium tuberculosis in Pichia pastoris and its effect on human monocytes.

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.

RDRioMycobacterium tuberculosis strains associated with isoniazid resistance in Northern Mexico.

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)/RDRio strains. The purpose of this study was to evaluate the prevalence of the LAM/RDRio 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/RDRio 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 RDRio. All RDRio 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/RDRio genotype in pulmonary cases of tuberculosis in Mexico, and we found resistance to the first-line anti-tuberculosis drug isoniazid in these strains.

Triggering of protease-activated receptors (PARs) induces alternative M2 macrophage polarization with impaired plasticity.

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.

Thrombin induces slug-mediated E-cadherin transcriptional repression and the parallel up-regulation of N-cadherin by a transcription-independent mechanism in RPE cells.

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.

Recent advances in antitubercular natural products.

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.

[From genetics to genomics in the rational design of new Mycobacterium tuberculosis vaccines]. / De la genética a la genómica en el diseño de nuevas vacunas contra la tuberculosis.

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-gene¼ 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.

Thrombin stimulates RPE cell motility by PKC-zeta- and NF-kappaB-dependent gene expression of MCP-1 and CINC-1/GRO chemokines.

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.

Thrombin stimulates RPE cell proliferation by promoting c-Fos-mediated cyclin D1 expression.

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.

Mycobacterial di-O-acyl trehalose inhibits Th-1 cytokine gene expression in murine cells by down-modulation of MAPK signaling.

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.

[Innovative strategies to diagnose and monitor tuberculosis patients]. / Estrategias innovadoras para el diagnóstico y seguimiento de los pacientes tuberculosos.

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.

Production of recombinant human placental variant growth hormone in Pichia pastoris.

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.