Las trombopoyetinas en el tratamiento de la púrpura trombocitopénica inmunitaria y otras trombocitopenias / Thrombopoietines in the treatment of the thrombocytopenic purpura and other thrombocytopenics

La purpura trombocitopénica inmunitaria y las trombocitopenias secundarias representan condiciones patológicas graves cuyo tratamiento plantea diversos grados de dificultad. La aproximación terapéutica convencional ha sido la administración de esteroides, la esplenectomía y el uso de inmunoglobulina intravenosa u otros tipos de anticuerpos (e.g., anti-D). La mejor comprensión de la fisiología y fisiopatología de la trombopoyesis aunado a los avances en biología molecular ha permitido el desarrollo de una nueva aproximación terapéutica, la aplicación de las trombopoyetinas sintéticas o no inmunogénicas. Dentro de este grupo resaltan dos compuestos: el romiplostin (una proteína de fusión) y el eltrombopag (un compuesto sintético de bajo peso molecular). Ambas se encuentran disponibles comercialmente. Los estudios clínicos indican que estos medicamentos tienen un efecto satisfactorio en el tratamiento de las trombocitopenias, particularmente en los casos refractarios a los tratamientos convencionales.

Immune thrombocytopenic purpura and the secondary thrombocytopenias are conditions potentially severe with diverse degrees of treatment difficulties. Steroids administration, splenectomy and the use of intravenous immunoglobulin and other antibodies (e.g., anti-D) had been the conventional therapy. The better understanding of the thrombopoiesis physiology and physiopathology togetter with the biology advances have permitted the development of a new terapheutic approach: the use of synthetic or nonimmunogenic thrombopoietines. Among this group highlights composites: romiplostim (a fusion protein) and eltrombopag (a synthetic composite with low molecular wheigt). Both are already available and produce a satisfactory effect particularly in nonrespondent cases to the conventional treatment.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Protective and Anti-Pathology Effects of Sm Fructose-1,6-Bisphosphate Aldolase-Based DNA Vaccine against Schistosoma mansoni by Changing Route of Injection

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 microg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection.

Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10(9)TCID50/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-gamma and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.

Construction of a recombinant plasmid harbouring the glyceraldenyde-3-phosphate dehydrogenase gene of periodic Brugia malayi and observation on DNA immunity.

Purpose: Controlling and eliminating lymphatic filariasis will require further research of preventative measures and implementation. Parasite is dependent on glycolysis for ATP production. The glycolytic enzyme glyceraldenyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and therefore is either a potential target for anti-parasite drug development or a vaccine candidate. Therefore, we tried to investigate the DNA vaccine-elicited immune responses in BALB/c mice. Materials and Methods: We cloned a gene encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from periodic Brugia malayi into vector pcDNA3.1. Mice were injected at a dosage of 100 μg recombinant plasmid DNA with CpG intramuscular injection and immunized three times at 2-week intervals. pcDNA3.1 and normal saline were used as control. The tissue of muscles at the 4 weeks after the third injection was collected and target genes were detected using RT-PCR. The humoral responses elicited in mice by inoculation with the recombinant plasmid pcDNA3.1-BmGAPDH were detected using a standard ELISA. Two weeks after the third immunization, stimulation index (SI) was measured using the MTT method and the level of secreted IL-4 and INF-g were detected using ELISA. Results: Specific gene fragment coding GAPDH was amplified and the recombinant plasmid pcDNA3.1-BmGAPDH was constructed. Post-challenge sera from the mice immunized with the DNA vaccine had specific antibody titres of 1:1600 to 1:6400, and the highest titre was observed in the mice that were inoculated by pcDNA3.1-BmGAPDH/CpG at 6 weeks. At 4 weeks after immunization, the spleens of the mice were obviously enlarged. The proliferation of spleen T lymphocytes seen on the MTT assay was higher in the pcDNA3.1-BmGAPDH group than in the control group (P value <0.05). The levels of IL-4 and INF-g in serums from the immunized mice were significantly higher than that of the control (P value <0.05). Conclusions: We conclude that the recombinant eukaryotic plasmid pcDNA3.1-BmGAPDH could elicit humoral and cellular immune responses in mice.

Antibody response following Hepatitis B vaccination in peritoneal dialysis patients: does normalized urea clearance matter?

OBJECTIVES: Data on the factors that contribute to the antibody response to hepatitis B virus vaccination in peritoneal dialysis patients are scarce. The current study was conducted on a group of peritoneal dialysis patients to learn how the response to hepatitis B virus vaccination varies according to the patient's clearance of urea normalized to total body water (Kt/V). METHODS: A convenience sample of 33 peritoneal dialysis patients (13 women and 20 men, with a mean age of 49¡12 years) was administered double doses (20 μg IM in each deltoid muscle) of recombinant hepatitis B vaccine at 0, 1, 2, and 6 months. Response to immunization was measured at one to three months after the final dose of vaccine. The subjects were divided into groups according to the level of antibodies to hepatitis B surface antigen (anti-HBs), including non-responders ( < 10 IU/L), weak responders (10-100 IU/L), and good responders ( > 100 IU/L). RESULTS: Among non-responders, weak responders, and good responders, significant differences were found in age (54 ± 12 vs. 56 ± 9 vs. 45¡12 years, respectively; p = 0.049) and recombinant human erythropoietin use (20 vs. 29 vs. 76 percent, respectively; p = 0.016). No significant differences in weekly total Kt/V (p = 0.704), weekly peritoneal Kt/V (p = 0.064) and residual glomerular filtration rate (p = 0.355) were found across the three groups. CONCLUSIONS: Delivered clearance measured by weekly peritoneal Kt/V and total clearance measured by weekly total Kt/V did not predict the response to hepatitis B virus vaccination in patients on peritoneal dialysis.

Plasmodium vivax thrombospondin related adhesion protein: immunogenicity and protective efficacy in rodents and Aotus monkeys

The thrombospondin related adhesion protein (TRAP) is a malaria pre-erythrocytic antigen currently pursued as malaria vaccine candidate to Plasmodium falciparum. In this study, a long synthetic peptide (LSP) representing a P. vivax TRAP fragment involved in hepatocyte invasion was formulated in both Freund and Montanide ISA 720 adjutants and administered by IM and subcutaneous routes to BALB/c mice and Aotus monkeys. We measured specific humoral immune responses in both animal species and performed a sporozoite challenge in Aotus monkeys to assess the protective efficacy of the vaccine. After immunization both mice and Aotus seroconverted as shown by ELISA, and the specific anti-peptide antibodies cross reacted with the parasite in IFAT assays. Only two out of six immunized animals became infected after P. vivax sporozoite challenge as compared with four out of six animals from the control group. These results suggest that this TRAP fragment has protective potential against P. vivax malaria and deserves further studies as vaccine candidate.

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.

Protection against hepatitis B by the Butang® recombinant vaccine in newborn children in South Brazil

The prevention of hepatitis B by vaccination is one of the most efficient tools to avoid the transmission of the virus. This study evaluated the immunogenicity of the national vaccine Butang® in children born in Campo Mourão City, state of Paraná, Brazil, aged 7 to 12 months, by determining the anti-HBsAg antibodies levels after completion of the National Immunization Program Protocol for hepatitis B. All 70 children evaluated by the MEIA method (immune-enzymatic micro particles) showed seroconversion to the Butang® vaccine. Nine children (12.9 percent) presented a low response, with anti-HBs titers between 11 and 100 mUI/ml; 39 children (55.7 percent) showed a good response to the vaccine, with titers between 101 and 1000 mUI/ml; and 22 children (31.4 percent) showed antibodies titers higher than 1000 mUI/ml. The mean titer of the anti-HBs antibody titers was 1408.1 ± 2870.26 mUI/ml (15.7 to 19560.0 mUI/ml). The levels of antibodies produced by the prematurely-born children were not statistically different from those found in the newborns. Fifty-five children were also evaluated through the ELFA method (ELISA with a final detection in fluorescence), which presented similar results. The results obtained in our study corroborated the effectiveness of the national vaccine Butang® in newborn children of Campo Mourão City, Paraná, even if they were premature.

Imunização indireta de morcegos hematófagos Desmodus rotundus em cativeiro, com vacina anti-rabica V-RG, veiculada em pasta neutra / Indirect immunization of Demodus rotundus, in captivity, with anti-rabies V-RG vaccine throughh of neutral vehicle

Experimentos em cativeiro com o morcego Desmodus rotundus e o vírus rábico foram desenvolvidos em um novo modelo de gaiola de biosegurança. Os indicadores mostraram boa adaptação dos animais à gaiola. A suscetibilidade dos morcegos ao vírus rábico foi avaliada através de infecção experimental, via intramuscular, com variante de vírus rábico homologa. A dose de vírus rábico que desenvolveu doença em 60 por cento dos animais, foi usada como desafio em experimentos de vacinação dos morcegos. A vacina recombinante de vírus vaccinia e glicoproteína rábica (V-RG) foi testada por duas vias: esofágica e homogeneizada em pasta neutra aplicada no dorso de um ou dois morcegos vetores devolvidos à colônia, e mostrou-se segura e imunogênica para os morcegos, protegendo contra o desafio e produzindo altos títulos de /It was developed a new model of biosafety cage for experimental purposes with respect to hematophagous bat Desmodus rotundus and rabies virus. The index demonstrated the good adaptation of bats in this type of cage. In order to determine the susceptibility to rabies virus, bats were inoculated, by intramuscular route, with a rabies virus isolated from a naturally infected hematophagous bat. The dose that produced disease in 60 per cent of the animals of the experiment was used as challenge in experimental vaccination of bats. The bats were vaccinated orally by two routes: esophagal and applied in a neutral vehicle spread on the back of one or two vector bats comeback to the colony. The vaccine showed safety, immunogenic, produced high titers of neutralizing antibodies and a significant number of bats survive to challenge...

Antibody response to hepatitis B immunization in infants born to HIV-infected mothers.

OBJECTIVES: To determine the antibody response of hepatitis B immunization begun at birth in HIV-1 exposed infants. DESIGN: Prospective, clinical trial. SITE: King Chulalongkorn Memorial Hospital, Bangkok, Thailand. MATERIAL AND METHOD: Seventy six infants born to HIV-1 seropositive mothers, who were not hepatitis B carriers, received three 10 microgram doses of recombinant DNA hepatitis B vaccine (Engerix B, Smith Kline, Belgium) in a 0, 1 and 6 month schedule. The first dose was given at birth. Serum hepatitis B surface antibody (Anti -HBs) was measured at age 3, 9 and 12 months. Anti-HBs levels were determined by enzyme-linked immunoassay using the commercial kits (AUSAB EIA diagnostic kits, Abbott Laboratories, Chicago, USA) Antibody titer > or = 10 mIU/ml was defined as seroconversion. HIV infection was diagnosed by a positive test of HIV antibody at age > or = 18 months and/or by positive test of HIV polymerase chain reaction at age > or = 3 months. RESULTS: There were 14 HIV-1 infected (group 1) and 62 HIV-1 non infected (group 2) infants enrolled in this study. Anti-HBs titers of group 1 infants were significantly lower than those of groups 2 infants at both 3 and 6 months after the 3rd dose of vaccine, (Mann Whitney U test, p=0.019 and 0.001 respectively). Ten infants in group 1 and 57 infants in group 2 had anti-HBs titer > or = 10 mIU/ml. Their peak antibody titers were also noted at both 3 and 6 months after the 3rd dose of vaccine. Seroconversion rates were 71.4 per cent and 91.9 per cent in group 1 and 2 infants respectively, (p<0.05). Among the infants who had blood tests performed at age 12 months or 6 months after the 3rd dose of vaccine, anti-HBs titers declined in approximately 50 per cent of both groups of infants. There was a significantly higher percentage of seroconverters in group 1 who lost their protective titers than those in group 2, (p<0.001). CONCLUSION: The results in this study suggested that HIV-1 infected infants have poor antibody response to hepatitis B immunization and the protection was less durable. A fourth dose of vaccine at 6 months after the 3rd dose may be necessary.

Humoral immune response following hepatitis B vaccine booster dose in children with and without prior immunization.

One hundred and twenty-three children who had received no, incomplete and complete primary hepatitis B vaccination but had negative or very low anti-HBs titer were immunized with a single dose of recombinant hepatitis B vaccine. Blood tests for anti-HBs were obtained at 30 +/- 5 days after the booster immunization. Twelve of 18 (66.7%) children without prior immunization (group 1) seroconverted following the single dose Seroconversion rates in children who had undetectable anti-HBs with incomplete and complete primary immunization (group 2 and 3) were 83.34% and 94.5%, respectively. All children with complete 3- dose vaccination but who had low anti-HBs titer (group 4) also seroconverted. This study confirmed that immunological memory, allowing a protective anamnestic response, lasted at least 8 years in children who had received primary HB immunization with undetectable anti-HBs. Therefore, we conclude that the booster dose after complete vaccination is not necessary in healthy children.

Response to hepatitis B vaccination in high risk population.

Hepatitis B vaccine is well established as very efficacious, but immune response to the vaccine is highly individual specific. A study involving fifty vaccinees was undertaken at the Hepatitis Laboratory, National Institute of Communicable Disease, Delhi. One ml (20 microgram) of Engerix B vaccine (recombinant yeast derived vaccine) was administered in the standard three dose schedule (0, 1 and 6 months). The sero-conversion of the vaccinees was 24%, 66%, 76% and 78% at 1 month, 6 months, 7 months, and 12 months respectively. There was no seroconversion in 22% of the vaccinees. Sero-conversion was assessed using Macro ELISA test (Ausab, Abbott Labs) for Anti HBs reactivity.

Eficacia comparativa de las vías intradérmica e intramuscular en la inmunización activa contra la hepatitis B: resultados a largo plazo / Comparative efficacy of intradermally and intramuscular routes for active immunization against hepatitis B: long-term results

Se presentan los resultados a largo plazo de un estudio prospectivo, comparativo y randomizado con 119 voluntarios jóvenes sero-negativos, con riesgo aumentado de infección por el VHB, que evaluó la inmunogenicidad comparativa de una vacuna recombinante contra la hepatitis B, administrada por vía intramuscular (IM) o intradérmica (ID). La vacuna fue administrada en tres dosis consecutivas, en un esquema de inmunización de corta duración de 0,1 y 2 meses. Una dosis de refuerzo fue administrada al cabo de 1 año, por la misma vía utilizada en la inmunización original. La distribución por edad y sexo en ambos grupos fue similar. Los resultados obtenidos al cabo de las tres dosis mostraron tasas de seroconversión y seroprotección similares para ambos grupos (96 por ciento y 96 por ciento para la vía IM Vs. 98 por ciento y 93 por ciento para la vía ID, respectivamente). El promedio geométrico del título de anti-HBsAg en el suero de los individuos inmunizados por vía IM (155 UI/L) fue significativamente superior al obtenido en aquellos inmunizados por vía ID (71 UI/L). Por otro lado, la tasa de buenos respondedores (niveles de anti-HBsAg >100 UI/L ) fue igualmente más elevada en los vacunados por vía IM (67 por ciento Vs. 39 por ciento). El porcentaje de buenos respondedores en las mujeres fue consistentemente superior al de los varones para ambos grupos (75 por ciento Vs. 55 por ciento vía IM y 54 por ciento Vs. 13 por ciento vía ID, respectivamente). En 82 individuos estudiados 1 años más tarde, el promedio geométrico de los niveles de anti-HBsAg fue significativamente superior en los que recibieron la vía IM (278 UI/L) en comparación con los vacunados por vía ID (82,3 UI/L) y la tasa de seroprotección fue también superior (100 por ciento Vs. 84,8 por ciento respectivamente). De la misma manera, la respuesta de anti-HBsAg sérica a la dosis de refuerzo, fue mejor en los vacunados por vía IM (promedio geométrico 5.090 UI/L) con respecto a los que recibieron la vía ID.

Long-term immunogenicity and efficacy of a recombinant hepatitis B vaccine in Egyptian children

In 1992, Egypt adopted a hepatitis B vaccine schedule at 2, 4 and 6 months of age. We evaluated the long-term immunogenicity and efficacy of vaccination using this schedule in 180 children whose time lapse since last vaccination varied between 1 month and 5 years. None of the participants had clinical hepatitis, HBsAg was not detected in any participant and all but one had negative results for anti-HBc test. Although a high seroprotection rate [93.3%] was elicited 1 month after vaccination, there were low initial anti-HBs concentrations and both declined rapidly over time. Thus, the short interval [2 months] between the second and third doses of vaccine is less desirable in the long term. We recommend booster inoculations for all previously vaccinated children and a new vaccination schedule at 1, 2 and 9 months