Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein / 第二军医大学学报

Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein..

Detection of Human Anti-Trypanosoma cruzi Antibody with Recombinant Fragmented Ribosomal P Protein

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and is endemic in many Latin American countries. Diagnosis is based on serologic testing and the WHO recommends two or more serological tests for confirmation. Acidic ribosomal P protein of T. cruzi showed strong reactivity against positive sera of patients, and we cloned the protein after fragmenting it to enhance its antigenicity and solubility. Twelve positive sera of Chagas disease patients were reacted with the fragmented ribosomal P protein using western blot. Detection rate and density for each fragment were determined. Fragments F1R1, F1R2, and F2R1 showed 100% rate of detection, and average density scoring of 2.00, 1.67, and 2.42 from a maximum of 3.0, respectively. Therefore, the F2R1 fragment of the ribosomal P protein of T. cruzi could be a promising antigen to use in the diagnosis of Chagas disease in endemic regions with high specificity and sensitivity.

Predictive Value of Gelatin Particle Agglutination Test (GPAT) in Leprosy Detection

Detection of Mycobacterium leprae infection prior to the onset of the clinical disease may be very important for epidemiological study of leprosy and its eradication. By adding diagnosis through early recognition, early treatment can be commenced, thus it would greatly help in the limitation of transmission of M. leprae and in reduction of the degree of deformities. The aims of our study was to evaluate the value of GPAT using semisynthetic trisaccharides antigen (NT-P-BSA, manufactured by FUJIREBIO-INC-Japan and provided by WHO) in early detection of leprosy. 1,030 apparently normal persons including 680 household contacts of patients of leprosy with average 3 years of contact and 350 people living in areas free of leprosy were tested with GPAT. Among 135 household contacts showing positive GPAT, 17 developed leprosy (12.8%) within 14-57 months of becoming positive while among 884 people with GPAT negative, none (0%) developed the disease during the same period of follow-up. In children, a high titer of antibodies constitutes a valuable indicator of high risk in developing the disease: 63.1% of them developed clinical leprosy while it was only 7.7% in adults. 100% leprosy children showing GPAT positive at serum dilution of 1:64 or over have developed leprosy. In conclusion, GPAT has shown that children with GPAT positive at high titer of 1:64 or over and whose mother/father being a leprosy patient can be considered as the highest risk group of eventually developing the disease. All household contacts with GPAT high positivity have been actively followed-up until now. In this sense, GPAT has proved to be an indicator for detection of sub-clinical leprosy infection with high chances of developing clinical disease.

Cepario autóctono de leptospiras en la prueba de micro - aglutinación / Micro - agglutination test of Autogenous leptospirosis

Introducción: leptospirosis. Zoonosis más frecuente en Cuba, como enfermedad infecto-contagiosa. Objetivos: identificar las cepas de leptospiras aisladas localmente, para ser empleadas en la prueba de microaglutinación. Apreciar su comportamiento serológico frente a las cepas de referencia. Método: estudio experimental, de sueros de pacientes presuntivos de padecer Leptospirosis, con 60 muestras, en el Instituto Carlos J. Finlay, de La Habana. Identificación de 40 sueros reactores a la hemaglutinación pasiva. Identificación de 34 cepas, de un total de 250 aislamientos, a partir de hemocultivos, de pacientes con antisueros policlonales a serogrupos y anticuerpos monoclonales a serovar. Se conformaron cinco cepasrepresentativas de los serovares: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi e Icterohaemorrhagiae Copenhageni, como las de mayor circulación en la región. Se realizó la prueba de microaglutinación con las cepas aisladas localmente, y las de referencia de forma paralela. Resultados: mayor reactividad con las cepas locales, y conformación de cinco cepas representantes de los serovares: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi e Icterohaemorrhagiae Copenhageni, como las de mayor circulación en la región. Se encontró una concordancia de 86,36% en la reactividad. Conclusiones: los resultados mostraron mayor reactividad con las cepas locales que con las de referencia, elevada concordancia e incremento de los títulos de anticuerpos de los sueros reactores usando cepas locales, y disminución del promedio de las reacciones cruzadas cuando se utilizaron las cepas locales. Se demostró su utilidad en la evaluación de estudios inmunológicos.

Introduction: leptospirosis, a contagious infectious disease considered the most common zoonosis in Cuba. Objectives: to identify locally isolated leptospirosis strains for micro- agglutination test. To evaluate their serological behavior against the reference strains. Method: an experimental study of 60 presumptive Leptospirosis patients' serum samples. Forty sera reactive to passive hemagglutination. To isolate 250 blood cultures of patients. To show 34 strains identified at the Carlos J. Finlay Institute, with polyclonal antisera to serogroups and monoclonal antibodies to serovar. Five strains represented by the serovars: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi and Icterohaemorrhagiae Copenhageni, were the most circulating strains in the region. The microagglutination test was performed in parallel form to locally isolated strains and reference strains Results: greater reactivity with the local strains. Five strains representing the serovars: Ballum Ballum, Canicola Canicola, Pomona Pomona, Hebdomadis Wolffi and Icterohaemorrhagiae copenhageni as the most circulating in the region. The reactivity showed 86.36% concordance. Conclusions: there was greater reactivity with the local strains than the reference strains. A higher concordance increased the titers of antibodies of the reactive sera, by the use of local strains. The average of cross reactions decreased when the local strains were used. The evaluation of immunological studies was demonstrated.

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay / 中华预防医学杂志

Objective@#To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.@*Methods@#HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.@*Results@#The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05).@*Conclusion@#We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Búsqueda de flavivirus en individuos con sospecha clínica de dengue y resultado negativo para el antígeno NS1 en Paraguay / Search of flavivirus in individuals with clinical suspicion of dengue and negative for the NS1 antigen in Paraguay

Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.

Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.

Serum fingerprint detection based on surface-enhanced Raman scattering / 军事医学

Objective To optimize the experiment conditions of surface-enhanced Raman spectroscopy detection of serum fingerprint spectra.Methods Normal human serum was used as the sample and Ag nanoparticles as the active substrate.The enhanced signals of different optimized experiments were obtained , including serum dose(2.5 to 500 μl), incubation time(10 to 30 minutes) temperature(4℃,room temperature and 37℃),and different treatment(extraction and protein removal).Results and Conclusion Serum doses should not exceed 50μl.The ratio should range from 1∶1 to 5∶1, the incubation time is from 10 to 30 minutes, and the incubation temperature from 4℃ to 37℃.The signals of samples directly mixed with an active substrate are stronger than those of samples which are extracted or protein removed .

Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

Rudolf Kraus em busca do "ouro da ciência": a diversidade tropical e a elaboração de novas terapêuticas, 1913-1923 / Rudolf Kraus in search of the "gold of science": tropical diversity and the development of new treatments, 1913-1923

Com base na trajetória de Rudolf Kraus, o artigo analisa a busca de curas para doenças infecciosas em regiões tropicais no início do século XX, dando especial atenção à elaboração por Kraus de novos terapêuticos biológicos como soros, vacinas e soluções proteicas. As regiões tropicais eram com frequência apresentadas como mais propícias à pesquisa devido à maior quantidade de organismos a identificar e também à concorrência supostamente menor entre pares. Os trópicos eram, assim, considerados um oásis para as pesquisas microbiológicas. Kraus dedicou-se à fabricação de diversos produtos de origem biológica, mas não teve o sucesso esperado com muitos deles.

Based on the career of Rudolf Kraus, the article analyzes the search for cures to infectious diseases in tropical regions in the early twentieth century, focusing especially on Kraus' development of new biological treatments like sera, vaccines, and protein solutions. At that time, the world's tropical regions were often portrayed as more propitious for research, given the larger number of organisms that could be identified in these realms and an allegedly lower level of peer competition as well. The tropics were thus seen as an oasis for microbiological research. Kraus dedicated himself to the production of various products of biological origin, but he failed to achieve his hoped-for success with many of them.

In vitro expression studies of non structural 1 protein of Canine Parvo virus 2 by polyclonal antiserum raised against CPV2-NS1 protein expressed in Escherichia coli as an antigen.

The canine Parvovirus 2, non-structural 1(NS1) is a novel candidate tumor suppressor gene. To confirm the expression of the NS1 in HeLa cells after transfection there was a need to raise antiserum against CPV2- NS1. Therefore, this study was carried out to express and purify the recombinant NS1(rNS1), and characterize the polyclonal serum. CPV2-NS1, complete coding sequence (CDS) was amplified, cloned in pET32a+ and expressed in BL21 (DE3) (pLysS). SDS–PAGE analysis revealed that the expression of the recombinant protein was maximum when induced with 1.5 mM IPTG. The 6 × His tagged fusion protein was purified on Ni-NTA resin under denaturing conditions and confirmed by western blot using CPV2 specific antiserum. The rabbits were immunized with the purified rNS1 to raise anti-NS1 polyclonal antiserum. The polyclonal serum was tested for specificity and used for confirming the expression of NS1 in HeLa transfected with pcDNA.cpv2.ns1 by indirect fluorescent antibody test (IFAT), flow cytometry and western blot. The polyclonal antiserum against NS1 could be very useful to establish functional in vitro assays to explore role of NS1 in cancer therapeutics.

Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.

In the present study recombinant VP3 (rVP3) was expressed in E.coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS–PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37ºC. The 6×His-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.

Detection of Enzootic Plague Foci in Peninsular India.

A Continuous serological and bacteriological surveillance in rodents was carried out in peninsular India i.e. Andhra Pradesh, Karnataka and Tamil Nadu to detect the role of different species of rodents in the maintenance of active enzootic plague foci. Live rodents were collected from wild and ruderal/peri-domestic situations by digging and trapping for sera and organ samples. During 1989 to 2007 serological evidence of plague was detected in different species of rodents in peninsular India. Plague antibodies were detected in 243 sera samples in three different rodent species. Sero-positivity (0.042 percent) amongst rodents tested were found in Tatera indica cuvieri (Hardwicke) followed by Rattus rattus and Bandicota bengalensis. Regular plague surveillance work enhanced the possibility of detecting and delimiting plague foci and helped in implementing necessary preventive anti plague measures to prevent the occurrence of human plague.

The screening of liver cancer specific binding peptide from serum / 中华肝胆外科杂志

Objective To find the liver cancer specific peptide for serological screen of liver cancer patients via screening phage-display peptide library. Methods Fifteen sera from liver cancer patients and physical examinates were collected for the four-round screening with Ph. D. 12TM phage display peptide kit. Highly specific phage monoclones were selected based on the ELISA results of the serological assay. The peptide labeled with FITC was synthesized according to the DNA sequencing of the optimal monoclone and tested with serum via fluorescent imagery. Results Nine highly specific monoclones were found among 50 selected ones after 4 rounds of screenings. The positive rate of the optimal monoclone,ZH-3, reached 46.7 %. The peptide sequence of ZH-3 was concluded by DNA sequencing as SAHGTSTGVPWP. Desirable specificity and affinity were also shown in the serum of liver cancer patients. Conclusion The peptide ZH-3 can be used as a diagnostic reagent for liver cancer.

Dengue NS1 antigen detection: A useful tool in early diagnosis of dengue virus infection.

Purpose: This study was undertaken to evaluate the efficacy of NS1 antigen (Ag) assay as an early marker for dengue virus (DV) infection. Materials and Methods: Group I evaluated the performance of NS1 antigen (Ag) assay in comparison to MAC-ELISA and their detection rate when performed together in a single sample. Six hundred acute/early convalescent sera were screened by both the assays. Group II evaluated the efficacy of a single assay in 30 acute phase sera of paediatric OPD patients screened only by NS1 Ag assay. Group III evaluated the specificity of NS1 assay in comparison to MAC-ELISA on 40 samples included as controls. Results: In Group I, 140 (23.3%) and 235 (39.1%) samples were positive by NS1 assay and MAC-ELISA respectively. The detection rate increased to 320 (53.3%) when both the assays were used together on a single sample. NS1 Ag positivity varied from 71.42% to 28.4% in acute and early convalescent sera, conversely IgM detection rate was 93.61% and 6.38% in early convalescent and acute phase sera respectively (P < 0.0001). In Group II, 66.66% (20) samples were positive by NS1 assay. All the samples in Group III were negative showing 100% specificity of both the assays. Conclusion: NS1 Ag assay holds promise in early diagnosis of dengue infection. When used in combination with MAC-ELISA on a single sample it significantly improves the diagnostic algorithm without the requirement of paired sera.

Experiment study on corneal moist chamber storage with aqueous removed and recipient serum tamponaded / 中国医师杂志

Objective To explore the new preservation method of cornea by evaluating the structure and function of rabbit's endothelia on the condition of whole-eye preservation with aqueous removed and recipient's serum tamponaded. Methods Forty New Zealand big white rabbits (80 eyes) were random divided into two groups, 40 eyes in group A (control group) and 40 eyes in group B (experimental group). The vitality of endothelial cell on the condition of tow corneal preservation methods which were the moist chamber preservation (group A) and the whole-eye preservation with aqueous removed and recipient's serum tamponaded (group B) was compared. At the 2nd day, the 5th day, the 7th day, the 10th day, the vitality of endothelial cell was appraised through ultra-microstructure by scanning electron microscope and the trpan biuealizarin red stain. The corneal thickness was measured, and corneal endothelial cells density was calculated, and cell size was observed by image analysis system. Result In group B, the corneas remained transparent for 7 days , and the rate of vitality was 90% for7 days and that was over 80% for 10 days. In group A, the corneas remained transparent for 2 days, and the endothelial cell losing and dying were found after 5 days. Cell vitality, cell density and cell size had no statistical difference between Group B for 7 days and group A for 2 days. (all P＞0. 05). In group A, at the 5th day, the 7th day ,the 10th day, the average corneal thickness were (0.64 ± 0.04) mm, (0. 79 ± 0. 03) mm , (1.06 ± 0. 03) mm. In the group B, at the 5th day, the 7th day ,the l0th day, the average corneal thickness were (0. 55 ±0.03)mm, (0.65 ±0. 02) mm , (0. 85 ± 0. 05) mm. The average corneal thickness had significant difference between group A and group B (all P ＜ 0. 05). Conclusion Recipient serum had the function with supporting the structure and function of rabbit's corneal endothelial cell, and it could prolong the storage time with the moist chamberstorage at the same time.

Screening, identification and significance of the phage-display random 7 amino acid peptide specific to the sera of patients with systemic lupus erythematosus / 中华微生物学和免疫学杂志

Objective To screen and identify the phage-display random 7 amino acid peptide specific to the systemic lupus erythematosus(SLE) and analyze its practical significance. Methods Using the phage random 7 peptide library screening, the SLE specific phage clones are obtained after binding with the mixture of sera from 30 SLE patients and 30 normal controls as ligand respectively. Then the Dot-ELISA is used to identify the SLE specific phage clones reactive to sera of the SLE patients and normal controls individually. Finally the identified phage-display random 7 amino acid peptides are sequenced and it's homology with the antigenic epitope of human being and other are also analyzed. Results Total 12 of the phage-display random 7 amino acid peptide are obtained by phage peptide library screening and the Dot-ELISA identification. Sequence analysis shows that the identified phage-display random 7 amino acid peptide epitope have homology with E. coli, Salmonella and human immunodeficiency virus, but not with that of human being. Conclusion SLE-specific peptides screened by phage random peptide library maybe used to diagnosis the SLE. Meanwhile, the antibodies in SLE patients which are combined with the Pathogen epitope, suggest that SLE maybe relate to pathogen infection.

Effects of Mesenchymal Stem Cells Feeder Layer, Culture Sera and Freeze-thaw Lysates on Expansion and Differentiation of Cord Blood CD34~+ Cells in Vitro / 中山大学学报(医学科学版)

[Objective]To investigate the effects of mesenchymal stem cells (MSC) feeder layer, culture sere and freeze-thaw lysates on expansion and differentiation of cord blood CD34~+ cells in vitro. [Methods] MSC were isolated from human bone marrow and cultured until the third passage. Sera were obtained from the cultured MSC, and freeze-thaw lysates were obtained by repeated freeze-thaw procedures. Cord blood CD34~+ cells were isolated by magnetic cell separation system, and were co-cultured with the MSC feeder layer, culture sera, freeze-thaw lysates and hematopeietic growth factors (HGFs), respectively. The nucleated cells, CD34~+ cells, CD34~+CD38~- cells, CD41~+ cells and CD3~+ cells in the above culture system were detected by flow cytometry on day 6 and day 12. [Results] ①MSC feeder layer had a strong effect on nucleated cells, CD34~+,CD34~+CD38~- cells expansion. The MSC sera and freeze-thaw lysates had similar effect on cell expansion, but the effect was weaker than that of feeder layer (P<0.05). ② Both MSC sera and feeder layer inhibited cord blood CD34~+ cells differentiation toward CD3~+ cells or CD19~+ cells, and no significant differences were found between these two groups (P>0.05). ③ Both MSC sera and feeder layer promoted cord blood CD34~+ cells differentiation toward CD41~+ cells, and the effect was stronger in the feeder layer than that of the sera (P<0.05). ④ Freeze-thaw lysates had no effect on cell expansion and differentiation, and were similar with that of HGFs (P>0.05). [Conclusions] The MSC sera have positive effects on expansion of cord blood CD34~+ and CD34~+CD38~- cells, moreover they have the ability of promoting cord blood CD34~+ cells differentiation toward CD41~+ cells.

Antiophidian sera sterility control: topics in perspective / Controle de esterilidade de soros antiofídicos: temas em perspectiva

The aim of this work is to review the most important topics about the antiophidic sera sterility, including obtaining methods, sterilization procedures and clean room control using Vital Brazil Institute (VBI) as an example. Bibliographical research was performed through Medline, Lilacs, PubMed, ISI and the Fundação Oswaldo Cruz - RJ and VBI Libraries, from 1960 to 2009. The antiophidic sera for human use are immunobiologic products produced in Brazil by three national laboratories, including VBI. Due to the parenteral use, these products should be sterile and pyrogen-free, which demands the microbiological control during the whole fabrication process. The sterility and pyrogen tests are important steps to ensure the quality and safety of these immunobiological products. Thus, these tests are target for continue evaluation and improvement. The most interfering aspects in the consistency and analytical patterns include the proper method selection, sampling, culture conditions and validation criteria. As the national and international legal requirements are cautious with the assays validation and approval of sterile parenteral products; the intrinsic limitations for established assays still require more investigation aiming the continue improvement of the microorganism and contaminants detection methods and optimization of the analysis extent.

O objetivo deste trabalho é revisar os tópicos mais relevantes para o controle da esterilidade de soros antiofídicos, abordando-se métodos de obtenção, procedimentos de esterilização e o controle de áreas limpas utilizando como exemplo os procedimentos adotados pelo Instituto Vital Brazil (IVB). Um levantamento bibliográfico foi realizado no Medline, ISI, Biblioteca da Fundação Oswaldo Cruz-RJ e IVB, no período de 1960 a 2009. Os soros antiofídicos para uso humano são produtos imunobiológicos fabricados no Brasil por três laboratórios nacionais, dentre eles o IVB. Por serem de administração parenteral, devem ser obrigatoriamente estéreis e apirogênicos, exigindo controle microbiológico durante todo o processo de fabricação. O teste de esterilidade e apirogenia são importantes instrumentos para garantir a qualidade e segurança microbiológica desses produtos, sendo alvo de avaliações constantes para seus aprimoramentos. Os aspectos que mais interferem na sua consistência e valor analítico incluem a escolha adequada do método, amostragem, condições de cultivo e critérios de validação. À medida que os requisitos legais nacionais e internacionais se mostram rigorosos na validação de ensaios e aprovação de produtos estéreis parenterais, limitações intrínsecas ao ensaio padronizado requisitam mais investigações, objetivando o aprimoramento contínuo nos métodos de detecção de microorganisms, contaminantes e otimização do tempo total de análise.