Péptido inmunomodulador CIGB-258 para el tratamiento de pacientes graves y críticos con la COVID-19 / An immunomodulatory peptide CIGB-258 for the treatment of critical and severe COVID-19 patients

Introducción: El CIGB-258 es un péptido inmunomodulador con propiedades antiinflamatorias. Objetivos: Establecer la frecuencia de dosis y el tiempo de tratamiento con el péptido CIGB-258, para pacientes críticos con COVID-19. Además, definir los criterios de uso y el esquema terapéutico del péptido, para pacientes graves con COVID-19. Métodos: Se incluyeron 9 pacientes críticos y 3 pacientes graves. Las evaluaciones clínicas, radiológicas y de laboratorio se registraron de acuerdo al protocolo establecido. Se obtuvieron muestras de suero antes y después del tratamiento con la CIGB-258, para la determinación de los biomarcadores de la inflamación. Resultados: Se estableció el protocolo de actuación con el péptido CIGB-258, el cual consiste en la administración intravenosa de 1 mg del péptido cada 12 horas a los pacientes críticos. La dosis debe aumentarse a 2 mg cada 12 horas, para los pacientes que no muestren mejoría clínica y radiológica en 24 horas. Después de la extubación, los pacientes deben recibir 1 mg de CIGB-258 al día, durante otros tres días. Los pacientes graves deben recibir 1 mg de CIGB-258 cada 12 horas, hasta que resuelvan su condición clínica. Conclusiones: CIGB-258 mostró un buen perfil de seguridad. El protocolo de actuación establecido contribuyó a que todos los pacientes críticos se recuperaran de la dificultad respiratoria y fueran extubados. Los pacientes graves mejoraron considerablemente. Los niveles de los biomarcadores asociados con hiperinflamación y las citocinas disminuyeron significativamente durante el tratamiento(AU)

Introduction: CIGB-258 is an immunomodulatory peptide with anti-inflammatory properties. Objectives: To establish the therapeutic schedule with CIGB-258 peptide for COVID-19 critically ill patients. In addition, to define the criteria for use and schedule of this peptide for COVID-19 seriously ill patients. Methods: 9 critically ill patients and 3 seriously ill patients were included in this study. Clinical, radiological and laboratory evaluations were recorded according to the established protocol. Serum samples were obtained before and after treatment with CIGB-258, for the determination of the inflammation biomarkers. Results: The therapeutic protocol was established with the CIGB-258 peptide, which consists of intravenous administration of 1 mg of peptide every 12 hours for critically ill patients. The dose should be increased to 2 mg every 12 hours, for patients who do not show clinical and radiological improvement in 24 hours. After extubation, patients should receive 1 mg of CIGB-258 daily, for another three days. Seriously ill patients should receive 1 mg of CIGB-258 every 12 hours, until their clinical condition resolves. Conclusions: CIGB-258 showed an excellent safety profile. The established therapeutic protocol contributed to all critically ill patients recovering from respiratory distress and being extubated. Seriously ill patients improved considerably. The levels of the biomarkers associated with hyperinflammation and cytokines decreased significantly during treatment(AU)

Nontuberculous mycobacteria in patients with suspected tuberculosis and the genetic diversity of Mycobacterium avium in the extreme south of Brazil / Micobactérias não tuberculosas em pacientes com suspeita de tuberculose e a diversidade genética de Mycobacterium avium no extremo sul do Brasil

ABSTRACT Objective: Nontuberculous mycobacteria (NTM) are a heterogeneous group of bacteria that are widely distributed in nature and associated with opportunistic infections in humans. The aims of this study were to identify NTM in patients with suspected tuberculosis who presented positive cultures and to evaluate the genetic diversity of strains identified as Mycobacterium avium. Methods: We studied pulmonary and extrapulmonary samples obtained from 1,248 patients. The samples that tested positive on culture and negative for the M. tuberculosis complex by molecular identification techniques were evaluated by detection of the hsp65 and rpoB genes and sequencing of conserved fragments of these genes. All strains identified as M. avium were genotyped using the eight-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat method. Results: We found that NTM accounted for 25 (7.5%) of the 332 mycobacteria isolated. Of those 25, 18 (72%) were M. avium, 5 (20%) were M. abscessus, 1 (4%) was M. gastri, and 1 (4%) was M. kansasii. The 18 M. avium strains showed high diversity, only two strains being genetically related. Conclusions: These results highlight the need to consider the investigation of NTM in patients with suspected active tuberculosis who present with positive cultures, as well as to evaluate the genetic diversity of M. avium strains.

RESUMO Objetivo: As micobactérias não tuberculosas (MNT) são um grupo heterogêneo de bactérias amplamente distribuídas na natureza e relacionadas com infecções oportunistas em seres humanos. Os objetivos deste estudo foram identificar MNT em pacientes com suspeita de tuberculose e culturas positivas e avaliar a diversidade genética de cepas identificadas como Mycobacterium avium. Métodos: Foram estudadas amostras pulmonares e extrapulmonares provenientes de 1.248 pacientes. As amostras que apresentaram resultado positivo em cultura e negativo para o complexo M. tuberculosis na identificação molecular foram avaliadas por meio da detecção dos genes hsp65 e rpoB e de sequenciamento de fragmentos conservados desses genes. Todas as cepas identificadas como M. avium foram genotipadas pelo método mycobacterial interspersed repetitive unit-variable-number tandem-repeat com oito loci. Resultados: Das 332 micobactérias isoladas, 25 (7,5%) eram MNT. Dessas 25, 18 (72%) eram M. avium, 5 (20%) eram M. abscessus, 1 (4%) era M. gastri e 1 (4%) era M. kansasii. As 18 cepas de M. avium apresentaram alta diversidade, e apenas duas eram geneticamente relacionadas. Conclusões: Esses resultados mostram a necessidade de considerar a investigação de MNT em pacientes com suspeita de tuberculose ativa e culturas positivas e de avaliar a diversidade genética de cepas de M. avium.

Ca2+/Calmodulin-dependent kinase II delta B is essential for cardiomyocyte hypertrophy and complement gene expression after LPS and HSP60 stimulation in vitro

Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Estudo da expressão dos genes de choque térmico hsp90, hsp60 e hsp10 do fungo aquático Blastocladiella emersonii / Study of expression of heat shock genes Hsp90, hsp60, hsp10 of aquatic fungi Blastocladiella emersonii

A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA imcompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submitidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüênciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 710 resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado...

Response of human lymphocytes to low gamma ray doses

Linfocitos humanos fueron irradiados en un campo de radiación gamma de baja intensidad para determinar la expresión de las proteínas de choque calórico en función de la dosis. Los linfocitos fueron obtenidos de individuos cuyo trabajo los identifica como ocupacionalmente expuesto y no ocupacionalmente expuestos. La identidad de las proteínas se realizó utilizando anticuerpos contra las proteínas Hsp25, Hsp60, Hsp70 y Hsp90. De éstas, solamente la proteína hsp70 fue detectada antes y después de la irradiación. Los linfocitos del personal ocupacionalmente expuesto y no ocupacionalmente expuesto expresaron, antes y después de la irradiación, solamente la proteína Hsp70. La cantidad de proteína resultó directamente proporcional al tiempo de irradiación. Después de una dosis gamma de 70.5 mGy, los linfocitos del individuo ocupacionalmente expuesto expresaron una mayor cantidad de proteína Hsp70 que la expresada por los linfocitos del personal no ocupacionalmente expuesto. Este hecho es indicio de que el individuo ocupacionalmente expuesto tiene una mayor tolerancia a los rayos gamma (gamma-tolerancia), inducida por un proceso de adaptación generada por su condición laboral

Immunity to the 60kD heat shock proteins

A brief review on the role of the heat shock proteins (hsp), their common properties and possible consequences for early pregnancy development is described. The 60kD hsp plays an important role as immunogenic antigen of many microbial pathogens and possibly in postinfectious autoimmunity. The immune responce to hsp may cause pregnancy failure. The consequences of previous sensitization to microbial hsp and the effects of human autoantibodies to hsp, are demonstrated in a mouse embryo culture model.

O papel das Heat Shock Proteins no entendimento das infeccoes ginecologicas / The hole of heat shock proteins regards to gynecological infections

Os autores enfocam a importância da biologia molecular no avanço do entendimento dos processos fisiopatogênicos das doenças infecciosas. A Heat Shock Proteínas, tidas como importante mecanismo de defesa celular quando estas estäo submetidas ao estresse, säo descritas em detalhes, classificando-as e dando suas principais funçöes. Os efeitos destas proteínas no organismo humano foram considerados, mostrando-se como hipoteticamente o aumento de sua produçäo, poderia ajudar o indivíduo em determinadas circunstâncias, favorecendo o combate de algumas infecçöes e eventualmente, potencializando a recuperaçäo celular. As situaçöes desfavoráveis também foram destacadas, uma vez que mais esporadicamente, em alguns casos, pode haver possíveis reaçöes imunogênicas cruzadas, levando ao aparecimento de doenças crônicas

The heat-shock response in Trypanosoma cruzi and Crithidia fasciculata

Molecular aspects of heat-shock response were investigated in monogenetic and digenetic members of the Trypanosomatidae and the data obtained compared. Trypanosoma cruzi and Crithidia fasciculata differ in the number of heat-shock proteins (HSPs) induced and in the range of supra-optimal temperature induction of these proteins. Whereas low molecular weight Hsps were induced by high temperature in Crithidia, this effect was only seen in T. cruzi after ethanol treatment. The 61 kDa peptide of T. cruzi, induced by heat, was characterized as a HSP60 family member by Western blot using a Mycobacterium polyclonal anti-HSP60 antibody. The HSP61 aa. sequence, deduced from the isolated HSP60 gene and its mRNA product were characterized. The predicted aa. sequence has shown the presence of a mitochondrial peptide leader and no large domains of aa. sequence conservation were found when compared to other known HSP60, in contrast to what is observed in HSP70. Furthermore, the HSP60 gene is apparently conserved in T. cruzi, C. fascilulata and Leishmania as suggested by genomic Southern blot analysis.

Heat shock proteins in Trypanosoma cruzi: identification and localization of HSP70 and HSP60 proteins and structure of HSP60 genes (brief report)

To identify the members of the HSP70 and HSP60 families of Trypanosoma cruzi, we analysed 35S methionine epimastigote cells by two dimensional Western blot. At 29 degrees C, an HSP70 monoclonal antibody (anti-D. melanogaster) recognized eight isotypes. At least five of these were heat-induced. Polyclonal antibody against the 65 KDa antigen (anti-M. tuberculosis) recognized three isotypes with identical molecular weights, but different microliters. Only one isoform was heat induced. The cellular distribution of HSP70 and HSP60 was studied by immunoelectron microscopy. Anti-HSP70 reactive protein was localized in the cytoplasm, mitochondria and nucleus, while anti-HSP60 protein was found in the mitochondrion and in close association with the kinetoplast. To characterize the HSP60 gene and its proteins, we isolated a genomic T. cruzi clone encoding the HSP60 gene. T. cruzi HSP60 genes could be shown to be organized in 2100 nt tandem arrays. RELP in the HSP60 genes revealed that at least three different types of HSP60 genes were encoded in the T cruzi genome. The predicted open reading frame measured exhibits about 50 percent identity to other HSP60 described. Expression of these HSP60 genes could not be induced by 2 hours heat shock at 37 degrees C. Post-transcriptional mechanisms may be responsible for HSP60 induction in T. cruzi