El tratamiento con Jusvinza disminuye la hiperinflamación y la hipercoagulación en pacientes críticos con la COVID-19

RESUMEN Introducción: La infección con el SARS-CoV-2 induce un estado protrombótico en los pacientes, atribuible a la combinación de la respuesta hiperinflamatoria y la hipoxia. En Cuba, se usa el fármaco Jusvinza, basado en un péptido inmunomodulador, para el tratamiento de los pacientes con la COVID-19, que presenten signos y síntomas de hiperinflamación. Objetivos: Describir la evolución clínica y las variaciones de biomarcadores asociados con la inflamación y la coagulación, en un grupo de pacientes críticos con la COVID-19, tratados con Jusvinza, en comparación con un grupo de pacientes que no recibieron tratamiento con este péptido. Métodos: Se incluyeron 40 pacientes críticos con la COVID-19; se dividieron en 2 grupos: 20 pacientes tratados con Jusvinza y 20 no fueron tratados con dicho péptido (grupo control). Las características demográficas, comorbilidades, signos vitales, parámetros respiratorios, biomarcadores de la inflamación y de la coagulación se obtuvieron a partir de las historias clínicas de cada paciente. Resultados: El tratamiento con Jusvinza indujo una mejoría clínica en los pacientes, asociada con la disminución de varios biomarcadores de la inflamación y la coagulación. La sobrevida de los pacientes tratados con Jusvinza fue significativamente superior a la sobrevida de los pacientes no tratados con este péptido. Conclusiones: Jusvinza es capaz de controlar la hiperinflamación y la hipercoagulación en pacientes críticos con la COVID-19.

ABSTRACT Introduction: Infection with SARS-CoV-2 induces a prothrombotic state in patients, by the combination of hyperinflammatory response and hypoxia. In Cuba, the drug called Jusvinza, based on an immunomodulatory peptide, is used for the treatment of patients with COVID-19, who present signs and symptoms of hyperinflammation. Objectives: To describe the clinical course and behavior of various biomarkers associated with the inflammation and coagulation, in a group of critically ill patients with COVID-19 treated with Jusvinza, compared to a group of patients who did not receive treatment with this peptide. Methods: 40 critically ill patients with COVID-19 were included. The patients were divided into 2 groups: 20 patients were treated with Jusvinza and 20 were not treated with this peptide (control group). Demographic characteristics, comorbidities, vital signs, respiratory parameters and inflammation and coagulation biomarkers were obtained from the medical records of each patient. Results: Treatment with Jusvinza induced a clinical improvement in the patients, associated with the decrease of several inflammation and coagulation biomarkers. Patients treated with Jusvinza had a significantly higher survival than patients not treated with this peptide. Conclusions: Jusvinza is able to control hyperinflammation and hypercoagulation in critical ill patients with COVID-19.

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)

Immune Responses Induced by HSP60 DNA Vaccine against Toxoplasma gondii Infection in Kunming Mice

Toxoplasma gondii can infect all the vertebrates including human, and leads to serious toxoplasmosis and considerable veterinary problems. T. gondii heat shock protein 60 (HSP60) is associated with the activation of antigen presenting cells by inducing initial immune responses and releasing inflammatory cytokines. It might be a potential DNA vaccine candidate for this parasite. A pVAX-HSP60 DNA vaccine was constructed and immune responses was evaluated in Kunming mice in this study. Our data indicated that the innate and adaptive immune responses was elicited by successive immunizations with pVAX-HSP60 DNA, showing apparent increases of CD3e+CD4+ and CD3e+CD8a+ T cells in spleen tissues of the HSP60 DNA-immunized mice (24.70±1.23% and 10.90±0.89%, P < 0.05) and higher levels of specific antibodies in sera. Furthermore, the survival period of the immunized mice (10.53±4.78 day) were significantly prolonged during the acute T. gondii infection. Decrease of brain cysts was significant in the experimental group during the chronic infection (P < 0.01). Taken together, TgHSP60 DNA can be as a vaccine candidate to prevent the acute and chronic T. gondii infections.

Expression and significance of Neuritin and HSP60 in repair of acute liver injury / 实用医学杂志

Objective To explore the expression changes and possible molecular mechanisms of Neuritin and HSP60 during the repair of liver injury,and to provide an experimental basis for the study of the repair of liver injury. Methods Forty-eight Sprague-Dawley(SD)rats were randomly divided into control group without any treatment(n=6)and experimental group(n=42)underwent 70% hepatectomy to induce acute liver injury,and 6 h,12 h,24 h,48 h,3 d,7 d and 14 d after the operation,the left lobe resection of the residual liver was per-formed. Immunoblotting technique (Western blot) was used to detect the expression difference of Neuritin and HSP60 in liver tissue of the corresponding time points. Hematoxylin eosin staining(hematoxylin-eosin staining, HE)was used to observe the expression changes at each time point in liver pathology,and tail vein blood to detect ATL and AST changes. Results (1)Compared with those in the control group,the serum ALT and AST levels in the experimental group were increased at 6 h,12 h,24 h and 48 h after the operation,and reached the peak at 48 h postoperatively but those began to decrease 3 d postoperatively and was almost normal 7 d postoperatively. The difference was statistically significant(P < 0.05). Pathological findings:compared with that in the control group, the hepatic lobule structure in the experimental group was disorderly. The obvious balloon like change reached the peak 48 h postoperatively.(2)In the whole process of repair of liver injury,the expression of Neuritin and HSP60 showed differences in the opposite. There was a significantly negative correlation between Neuritin expression and the repair or the aggravation of the injury(P<0.001)and the lowest expression was observed 48 h postoperatively. There was a significantly positive correlation between HSP60 expression and the repair or the aggravation of the injury(P < 0.001)and the highest expression was observed 48 h postoperatively. Conclusions (1)With simple 70% left hepatic lobectomy,the repair of liver injury model of SD rats is successfully established and the heaviest injury is observed 48h postoperatively. With a high success rate(100%,42/42),simple and practical,the method provides a reliable and convenient animal model for the study of liver regeneration,liver injury and liver transplan-tation.(2)The expression of Neuritin decreases gradually after liver injury and reaches the lowest 48 h postopera-tively,while the expression of HSP60 increases gradually and reaches the highest 48 h postoperatively.(3)The change of expression of Neuritin and HSP60 is closely related to the process of liver injury repair(P < 0.001), showing a certain change rule. They may have some biological effects through interactions,and participate in and promote the regeneration and repair of liver injury.

Expression of heat shock protein 27, 60 and 90 in gastric cancer and its clinical value / 中国免疫学杂志

Objective: To investigate the expression of HSP-27,-60 and -90 in gastric cancer and its clinical significance. Methods:66 cases of gastric carcinoma was detected by immunohistochemistry HSP-27,60 and 90 of the expression and clinical significance of combined with clinical and pathological characteristics, tumor cell proliferation and survival analysis of three kinds of heat shock protein expression. Results: HSP-27,-60 and -90 were highly expressed in gastric cancer tissues. HSP-27 expression and tumor size (pT,P=0. 026),organ metastasis (pM,P=0. 046) and pathological staging (P=0. 041),HSP-27 staining intensity and lymph node status were significantly correlated ( pN, P=0. 042 ) . HSP-60 expression was associated with gender ( P=0. 011),and HSP-60 staining intensity was associated with age (P=0. 027) and tumor grade (P=0. 031). There was no correlation between HSP-90 expression and the clinical pathological parameters of this study; however, the intensity of HSP-90 staining was significantly correlated with tumor size (P=0. 020,pT). Single factor analysis showed that HSP-90 was significantly associated with longer survival (P=0. 033). Multivariate analysis demonstrated that HSP-90 was highly expressed as an independent prognostic factor for gastric cancer (P=0. 026). Conclusion: the HSP-27,-60 and -90 and some clinical pathological parameters. These parameters is very important for the treatment of patients with gastric cancer. The high expression of HSP-90 in patients with gastric cancer were inde-pendent prognostic indicators.

A potent multivalent vaccine for modulation of immune system in atherosclerosis: an in silico approach

PURPOSE: Atherosclerosis is classically defined as an immune-mediated disease characterized by accumulation of low-density lipoprotein cholesterol over intima in medium sized and large arteries. Recent studies have demonstrated that both innate and adaptive immune responses are involved in atherosclerosis. In addition, experimental and human models have recognized many autoantigens in pathophysiology of this disease. Oxidized low-density lipoproteins, beta2 glycoprotein I (beta-2-GPI), and heat shock protein 60 (HSP60) are the best studied of them which can represent promising approach to design worthwhile vaccines for modulation of atherosclerosis. MATERIALS AND METHODS: In silico approaches are the best tools for design and evaluation of the vaccines before initiating the experimental study. In this study, we identified immunogenic epitopes of HSP60, ApoB-100, and beta-2-GPI as major antigens to construct a chimeric protein through bioinformatics tools. Additionally, we have evaluated physico-chemical properties, structures, stability, MHC binding properties, humoral and cellular immune responses, and allergenicity of this chimeric protein by means of bioinformatics tools and servers. RESULTS: Validation results indicated that 89.1% residues locate in favorite or additional allowed region of Ramachandran plot. Also, based on Ramachandran plot analysis this protein could be classified as a stable fusion protein. In addition, the epitopes in the chimeric protein had strong potential to induce both the B-cell and T-cell mediated immune responses. CONCLUSION: Our results supported that this chimeric vaccine could be effectively utilized as a multivalent vaccine for prevention and modulation of atherosclerosis.

Serratia odorifera mediated enhancement in susceptibility of Aedes aegypti for chikungunya virus.

Background & objectives: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. Methods: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. Results: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. Interpretation & conclusions: tThe results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.

Seleção, caracterização e clonagem dos genes fljB e groEL agonistas dos receptores de reconhecimento de padrão do sistema imune inato das aves / Selection, characterization and cloning of the fljB and groEL genes agonists of the innate immune system pattern recognition receptors of birds

The recombinant production of innate immune system pattern recognition receptor agonists has provided a new tool for the production of immunostimulants for animals. The molecular pattern associated with the pathogen (PAMP), flagellin, coded by the fljB gene from Salmonella Typhimirium, and the molecular pattern associated to the damage (DAMP), HSP60, coded by the groEL gene from S. Typhimurium and S. Enteritidis, are recognized by pattern recognition receptors (PRRs) of the innate immune system of birds. In the present study, we performed the cloning of genetic fragments of the genes fljB, from S. Typhimurium, and groEL from S. Typhimurium and S. Enteritidis inserted in expression vector pET100/D-TOPO and transformed in E. coli TO10 cells. The clones were evaluated by colony PCR, plasmidial DNA PCR and genome sequencing in order to confirm the presence of these genes. In the colony PCR, we identified the presence of genes groEL (S. Enteritidis), groEL (S. Typhimurium) and fljB (S. Typhimurium) in 80%, 60% and 80% of the transformed colonies, respectively. The cloning system adopted allowed the production of HSP60 genetic fragment clones and flagellin of Salmonella strains, allowing the posterior use of these clones in gene expression trials, with the future potential of being used as non-specific immunostimulants for birds.

A produção recombinante de agonistas dos receptores do reconhecimento de padrão do sistema imune inato tem fornecido uma nova ferramenta para a produção de imunoestimulantes para animais. O padrão molecular associado ao patógeno (PAMP), flagelina, codificado pelo gene fljB de Salmonella Typhimurium e o padrão molecular associado ao dano (DAMP) HSP60, codificado pelo gene groEL da S. Typhimurium e S. Enteritidis, são reconhecidos por receptores de reconhecimento de padrões (RRPs) do sistema imune inato das aves. No presente estudo, foi feita a clonagem de fragmentos genéticos dos genes fljB de S. Typhimurium e groEL de S. Typhimurium e S. Enteritidis inseridos no vetor de expressão pET100/D-TOPO e transformados em células de E. coli TOP10. Os clones foram avaliados pela PCR de colônia, PCR de DNA plasmidial e sequenciamento genômico para a confirmação da presença desses genes. Na PCR de colônia, foram identificadas em 80%, 60% e 80% das colônias transformadas, a presença dos genes groEL (S. Enteritidis), groEL (S. Typhimurium) e fljB (S. Typhimurium) respectivamente. O sistema de clonagem adotado possibilitou a produção de clones dos fragmentos genéticos da HSP60 e flagelina das cepas de Salmonella, permitindo a utilização posterior desses clones em ensaios de expressão gênica, com potencial futuro de serem utilizados como imunoestimulante inespecífico das aves.

Expresión de HSP60 en infecciones murinas experimentales por Trypanosoma evansi / HSP60 expression in experimental murine infections by Trypanosoma evansi

La inducción de la síntesis de proteínas de choque térmico ha sido relacionada con la defensa del organismo frente a infecciones diversas. Este trabajo examina la acumulación de Hsp60 en Mus musculus infectados experimentalmente con un aislado equinos de Trypanosoma evansi, agente etiológico de la “derrengadera” en los Llanos venezolanos. Ratones NMRI (♀; 20 gr de peso corporal), normales e inmunosuprimidos (ciclofosfamida; 250 mg/kg) fueron inoculados intradermicamente con 1 tripomastigote/gr de peso corporal. La acumulación de Hsp60 se determinó mediante Western blot con el uso de un anticuerpo monoclonal en muestras de plasma obtenidas cada cuatro días post-inoculación. Los niveles de parasitemia también fueron registrados. Se observa acumulación diferencial y variable de Hsp60 que se correlaciona la parasitemia y la condición inmune del hospedador. Los resultados sugieren un control inmunológico en la regulación de la expresión de Hsp60, cuyos patrones de expresión dependen de la condición fisiológica del hospedador y los niveles de parasitemia.

Heat shock protein induction has been related to organism defense in diverse kinds of infection. This investigation examines Hsp60 accumulation in Mus musculus infected ex - perimentally with Trypanosoma evansi isolated from horse. This parasite is the causal agent of the “derrengadera” in Venezuelan planes. Normal and immunosupressed (cyclophosphamide( 250 mg/kg).) NMRI mice (♂; 20 gr body weight) were intra-dermal inoculated with 1 trypomastigote/ gr body weight; control groups were injected with distillated water in the same conditions. Hsp60 accumulation was analyzed by Western blot with a monoclonal antibody in plasma samples obtained each four days post-inoculation. Parasitemia levels were also registered. We observed a differential accumulation of Hsp60, which correlates with pa - rasitemia and immune conditions of the host. These results suggest an immunological control of Hsp60 expression that depends on the physiological condition of the host and parasitemia.

The Expression of Heat Shock Protein 60 kDa in Tissues and Cell Lines of Breast Cancer / 한국유방암학회지

PURPOSE: Breast cancer has been reported as the most common cancer of women in the United States, Western Europe and Korea and about 210,000 and 10,000 women in United States and Korea every year, respectively are diagnosed with it. Breast cancer is curable with an early diagnosis, and many researchers have made efforts to find a marker for this malady, heat shock protein (HSP) consists of 6 groups, it is highly preserved throughout both the prokaryotic and eukaryotic cells and it acts as a molecular chaperone that's involved in protein folding. HSPs have been recently reported to be related with breast cancer. In this study, we investigated the changes of expression of HSP60 in tissues and cell lines of breast cancer. METHODS: We obtained breast cancer tissues and normal tissues from breast cancer patients, and we purchased several cancer cell lines from American tissue culture correction. We treated the tissues and the cell lines of human breast cancer with heat shock protein. Proteins and mRNAs were isolated from the tissues and the cells and then we performed Western blotting, reverse transcriptase-Polymerase chain Reaction and fluorescence activated cell sorter analysis on them. RESULTS: On Western blot, HSP60 was more overexpressed in the tissues and the cell lines of breast cancer than in the normal breast tissues and cell lines. The expression of HSP60 showed 2 types of molecular weight differences in the tissues and cell lines of breast cancer, and specifically, low HSP60 was over-expressed in the cancer tissues. There was no difference between the breast cancer cell lines and the normal cell lines in the expressions of HSP60 mRNA, according to the treatment with heat shock. Also, there was no relationship between phosphorylation and the structural difference of HSP60 protein, according to HSP60 protein's molecular weight. The expression of HSP60 has been mostly reported at the mitochondria; however, in this study, it was more predominantly detected at the cytoplasm than at the mitochondria in the breast cancer cell lines. CONCLUSION: We conclude that HSP60 may be used as a diagnostic marker for breast cancer. Detailed investigation of the usefulness and significance of the HSP60 expression as a prognostic factor is required in further studies.

Production and characterization of cross-reactive anti-Porphyromonas gingivalis heat shock protein 60 monoclonal antibody / 대한치주과학회지

PURPOSE: Porphyromonas gingivalis (P. gingivalis) heat shock protein (HSP)60 may play a role in the immunopathogenesis of periodontitis as well as atherosclerosis by modulating autoimmune reaction due to its high level of sequence homology between bacteria and human counterpart. The purpose of this study was to identify immunodomiant epitope of P. gingivalis HSP60 that is reactive exclusively to the homologous bacteria without reacting with human HSP. MATERIALS AND METHODS: The present study was performed to identify the peptide specifically recognized by anti-P. gingivalis HSP60 monoclonal antibodies mono-reactive to P. gingivalis HSP60. RESULTS: Four different hybridomas were cloned producing monoclonal IgG antibodies exclusively to P. gingivalis HSP60. Thirty seven synthetic peptides (20-mer with 5-amino acid overlapping) were synthesized. All of these peptide were subject to SDS-PAGE for immunblot analysis. One peptide (TVPGGGTTYIRAIAALEGLK) and the other peptide (TLVVNRLRGSLKICAVKAPG) were recognized by all and one of the four monoclonal antibodies, respectively, that reacted solely with P. gingivalis HSP60. Immunohistochemistry to identify the localization of the HSP60 in the diseased gingival tissues revealed that all of the four monoclonal antibodies were highly reacted with the diseased gingival tissue than normal gingival tissue. CONCLUSION: The P. gingivalis HSP60 peptides (TVPGGGTTYIRAIAALEGLK and TLVVNRLRGSLKICAVKAPG, respectively) are positively involved in the immunopathologic process of periodontal disease. The peptide may potentially be developed as vaccine candidates. Further investigations are under way to identify more clones producing monoclonal antibodies reactive to P. gingivalis HSP and to other periodontopathogenic bacteria as well, while maintaining specificities to human counterpart.

Observation of HSP60 and Ras Protein in Gastric Mucosa of Atrophic Gastritis Induced by High-salt Hot Water in Rats Using Immune Fluorescence and Laser Scanning Confocal Microscope / 医学研究杂志

Objective To study the relationship between long-term high-salt water and the morbidity mechanism of chronic atrophic gastritis (CAG), The expression of HSP60 and ras protein were detected in the gastric mucosa of CAG rates induced by high-salt water.Methods The atrophic gastritis rat model was made by high-salt-hot water perfusion and the ultrastructure of gastric mucosa was observed by Laser Scanning ConfocaI Microscopy. Results There was no expression in gastric mucosa in normal group. The expression of HSP60 and ras was observed in the cell plasm of rats at 12 weeks. The higher expression was observed in the rats at 24, 32 and 65 weeks. Using laser scanning confocaI microscopy and immunofluorescence technique had observed coexistence of HSP60 and ras.Conclusion Long time high-salt-hot water can induce CAG and increasing the expression of HSP60 and ras. HSP60 and ras play an important role in the formation of atrophic gastritis.

CD4 and CD8 T cell response to the rHSP60 from Klebsiella pneumoniae in peripheral blood mononuclear cells from patients with ankylosing spondylitis / Respuesta de linfocitos T CD4 y CD8 contra la rHSP60 de Klebsiella pneumoniae en células mononucleares de sangre periférica de pacientes con espondilitis anquilosa

Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27 + AS patients and 15 B27 healthy controls was assessed by ³H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Objetivo.Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación. Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H-TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA-B27+y en 15 sujetos sanos HLA-B27-. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H-TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N-acetil-L-leucil-L-leucil-L-nor-leucinal (LLnL), y fijadas con p-formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo. Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno. Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.

Species Identification of Coagulase Negative Staphylococci by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism of Heat Shock Protein 60 Gene / 대한임상미생물학회지

BACKGROUND: An accurate and rapid method for specise identification of coagulase negative staphylococci(CNS) has been increasingly necessary for the clinical significance and planning the management of patients with staphylococcal infections. Recently, it has been reported that there is a highly conserved area on their 60KDa heat shock protein(HSP60) gene sequences between the interspecies of CNS and it can be amplified by a set of universal degenerate primer. This led us our attention to focus on whether the PCR-based RFLP method using Mse / restriction enzyme could be a useful tool for the species identification of CNS. METHODS: In the present study, we performed PCR-based RFLP analysis using a set of degenerate primers covering HSP60 and Mse / restriction enzyme on the reference strains and 25 clinical isolates(10 of S. epidermidis, 10 of S. haemolyticus, 4 of S. lugdunensis and 1 of S. warneri) which were previously identified by the API-STAPH, Vitek GPI card and/or with conventional biochemical test. RESULT: All the seven reference strains revealed that each strain has a distinct electrophoresed band patterns with combination of different number (up to 8) and size of fragments. And these distinct band patterns showed remarkable concordance with the seven reference strains and 25 clinical isolates. CONCLUSION: These result strongly suggest that the PCR-RFLP method using degenerate primers covering the HSP60 gene and Mse / digestion enzyme offer a convenient and accurate tool for species-specific identification of CNS.