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.

Safety profile of intravenous administration of live Pichia pastoris cells in mice.

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.

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.

Las proteasas de serina bacterianas y su implicación en la fisiopatología de la infección / Implication of bacterial serine proteases in the physiopathology of the infection

Las proteasas de serina son enzimas ampliamente distribuidas en la naturaleza, responsables de múltiples e importantes procesos biológicos. Durante las infecciones bacterianas los patógenos secretan y usan sus proteasas de serina como factores de virulencia para combatir contra el huésped, a través de diversos efectos como la desorganización de tejidos, la proteólisis de efectores inmunológicos o la inactivación de componentes relevantes para la fisiología del huésped; sin embargo, desde hace algunos años se ha observado que las proteasas de serina podían modular procesos fisiológicos por un mecanismo altamente específico, a través de la activación de los receptores activados por proteasas. En este artículo resumimos el conocimiento reciente sobre las proteasas de serina bacteriana y su relevancia en la fisiopatología de la infección, y destacamos la oportunidad de nuevas intervenciones antimicrobianas basadas en la inhibición de la interacción receptores activados por proteasas-proteasa

Serine proteases are enzymes widely distributed in nature, and are responsible for multiple and important biological processes. During bacterial infection, pathogens secrete and use their serine proteases as virulent factors to combat against the host, through diverse mechanisms, such as tissue disruption, proteolysis of immunological effectors or inactivation of relevant components for the host physiology. However, some years ago it was observed that serine proteases could modulate physiological processes by a highly specific mechanism, through the activation of protease activated receptors (PARs). In this paper, we review recent knowledge about bacterial serine proteases and their relevance in the pathophysiology of infection. The opportunity for new antimicrobial interventions based on the inhibition of PAR-protease interaction, is also highlighted

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.

Experiencia con furosemida subcutánea en una unidad de hospitalización a domicilio / Experience with subcutaneous furosemide in a home hospitalization unit

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Toracocentesis paliativa domiciliaria / Home palliative thoracentesis

INTRODUCCIÓN: El derrame pleural maligno (DPM) es una complicación frecuente, que suele cursar con disnea que puede disminuir significativamente la calidad de vida del paciente. Ante un DPM se acepta como primer paso la realización de una toracocentesis evacuadora que, en ocasiones, irá seguida de técnicas más definitivas. OBJETIVO: La práctica de toracocentesis fuera del ámbito hospitalario no es frecuente. En nuestra Unidad, realizamos toracocentesis evacuadora de intención paliativa en el domicilio del paciente, y para evaluar nuestros resultados revisamos las llevadas a cabo en los 4 últimos años. PACIENTES Y MÉTODOS: Estudio retrospectivo de las sucesivas toracocentesis paliativas (TP) realizadas en el domicilio en un periodo de 4 años. La indicación de TP se basa en la existencia de disnea de reposo/mínimos esfuerzos en pacientes con DPM y pronóstico vital inferior a 3 meses. Resultados Realizamos 56 TP en 26 pacientes. A su ingreso, la mediana del índice de Karnofsky de nuestros pacientes era de 40. El número de TP por paciente osciló entre uno y 9. En el 91,1% de los casos no se produjo ninguna complicación y se obtuvo alivio de la disnea en el 69,6% de ellos, con una supervivencia media tras la primera TP de 21 días. CONCLUSIONES: La TP es un método seguro, bien tolerado y eficaz para aliviar la disnea en enfermos oncológicos avanzados en los que ya no son posibles procedimientos más invasivos. Su práctica en el domicilio no parece añadir riesgos al procedimiento

INTRODUCTION: Malignant pleural effusion (MPE) is a common complication that usually causes breathlessness that can significantly decrease the quality of life of the PATIENTS: Faced with a MPE, performing a thoracentesis is accepted as a first step that could occasionally be followed by more definitive techniques. OBJECTIVE: Thoracentesis practice outside the hospital setting is rare. In our Unit, we carried out thoracentesis in the patient's home. We reviewed the procedures performed over the last4 years in order to evaluate our results PATIENTS AND METHODS: A retrospective study was conducted on the palliative thoracentesis (PT)performed at home over a period of 4 years. The indication for PT is based on the existence of dyspnea at rest/minimal exertion in patients with MPE, and a life expectancy of less than3 months. RESULTS: We performed 56 PT on 26 PATIENTS: On admission, the median Karnofsky index of our patients was 40. The number of PT per patient ranged between one and 9. In 91.1% of cases there was no complication and dyspnea relief was obtained in 69.6% of them, with a median survival after the first PT of 21 days. CONCLUSIONS: PT is a safe, well tolerated and effective in relieving dyspnea in advanced cáncer patients when invasive procedures are no longer possible. Its practice at home does not appear to add risk to the procedure