Omentum as non-strangled inguino scrotal hernia content in stallion: case report / [Omento como conteúdo de hérnia inguino-escrotal não estrangulada em garanhão: relato de caso]

Non-strangulated acquired hernias in stallions are rare, especially when the herniated content is not intestinal loops. Thus, the aim of the current study is to describe a case of acquired non-strangulated inguinoscrotal hernia in a stallion, whose herniated content was the omentum. The patient was a Criollo stallion with history of rhabdomyolysis, laminitis and bilateral scrotal volume increase observed in the left scrotal region. The animal presented pain in both thoracic limbs, abnormal blood test, especially hypoproteinemia, and mild pain during palpation in the left inguinal ring region. Hydrocele secondary to hypoproteinemia was suspected. After admission, the animal showed signs of acute abdomen, which were clinically reversed. With this, the animal was subjected to ultrasound examination of the scrotal region, whose findings suggested non-strangulated inguinal hernia, although the content could not be identified. Surgical treatment was chosen in order to identify the herniated content and remove the left testicle. Access to the affected scrotum was performed, in which the presence of fluid and a portion of the omentum was observed surrounding the testis and adhering to it. The animal was discharged after he recovered from the surgery and from laminitis. During the breeding season, the stallion remained with a herd of mares for natural mating. After 15 months of surgery, the animal was reassessed and showed no active signs of inflammatory and degenerative processes in the remaining testis. On this occasion, a pregnancy diagnosis was also performed, and all the mares were pregnant. It is concluded that the presence of omentum as a herniated content does not represent a surgical emergency but can make the reproductive prognosis reserved. In addition, removal of the affected testicle can benefit the spermatogenesis of the remaining testicle.(AU)

As hérnias adquiridas não estranguladas em garanhões são raras, principalmente quando o conteúdo herniado não é de alças intestinais. Assim, o objetivo deste trabalho foi descrever um caso de hérnia inguino escrotal adquirida e não estrangulada em garanhão, cujo conteúdo herniado era composto pelo omento. Foi atendido um garanhão da raça Crioula com histórico de rabdomiólise, laminite e aumento de volume escrotal bilateral, evidenciado na região escrotal esquerda. O animal apresentava dor nos membros torácicos, alterações na avaliação sanguínea, destacando-se a hipoproteinemia, e demonstrava dor leve à palpação na região do anel inguinal esquerdo. Suspeitou-se de hidrocele secundária a hipoproteinemia. Após a internação, o animal apresentou sinais de abdômen agudo, revertidos clinicamente. Com isso, realizou-se ultrassonografia da região escrotal, cujos achados sugeriram hérnia inguinal não estrangulada, sem que o conteúdo pudesse ser identificado. Optou-se pelo tratamento cirúrgico, com o intuito de identificação do conteúdo herniado e remoção do testículo esquerdo. Foi realizado acesso à bolsa escrotal afetada, na qual se observou presença de líquido e de uma porção do omento envolvendo o testículo e aderido a ele. O animal recebeu alta após restabelecimento da cirurgia e da laminite. Na propriedade, durante temporada reprodutiva, o garanhão permaneceu com uma manada de éguas para realização de monta natural. Passados 15 meses da cirurgia, o animal foi reavaliado e não demonstrou sinais ativos de processo inflamatório e degenerativos no testículo remanescente. Nessa ocasião, também foi realizado diagnóstico de gestação e todas as éguas encontravam-se prenhes. Conclui-se que a presença de omento como conteúdo herniado não representa uma emergência cirúrgica, mas pode tornar o prognóstico reprodutivo reservado. Ainda, a remoção do testículo afetado pode trazer benefícios à espermatogênese do remanescente.(AU)

Survey of dental diseases in Crioulo breed horses reared in an extensive farming system / Levantamento de afecções dentárias em equinos da raça Crioula mantidos em sistema de criação extensivo

The present study aimed to identify and quantify dental diseases in Crioulo horses reared exclusively under extensive farming conditions. In total, 254 Crioulo horses were evaluated, that were reared exclusively in native pastures with predominantly Eragrostis plana (Annoni grass). The animals were divided into three groups according to age: three to five years (G1), six to 12 years (G2), and 13 to 26 years (G3). In total, 46.1% of the animals in G1, 63.8% in G2, and 82.3% in G3 had two or more incisor disorders; 37.5% in G1, 52.5% in G2 and 45.5% in G3 had two or more disease findings in the canines; and 28.9% in G1, 35.4% in G2, and 64.7% in G3 had four or more disease findings in the second, third, and fourth premolars and molars. However, dental calculus was observed in 90.5% of G2 animals, whereas in G3, the occurrence rate of excessive enamel points and calculus was 82.4%. Although all of the horses studied were reared in an extensive farming system, dental changes were very common in the Crioulo breed reared under these management conditions.(AU)

O presente estudo teve por objetivo identificar e quantificar as afecções odontológicas de equinos da raça Crioula criados exclusivamente em regime extensivo. No total, foram avaliados 254 cavalos Crioulos, criados em pastagens nativas com predominância de Eragrostis plana (capim-annoni). Os animais foram divididos em três grupos, de acordo com a idade: de três a cinco anos (G1), seis a 12 anos (G2 ) e 13 a 26 anos (G3). No total, 46,1% dos animais do G1, 63,8% do G2 e 82,3% do G3 apresentaram dois ou mais transtornos incisivos; 37,5% do G1, 52,5% do G2 e 45,5% do G3 tiveram duas ou mais alterações nos caninos; 28,9% do G1, 35,4% do G2 e 64,7% do G3 mostraram quatro ou mais distúrbios no segundo, terceiro e quarto pré-molares e molares. Contudo, cálculo dentário foi observado em 90,5% dos animais do G2, enquanto no G3, a taxa de ocorrência de pontas excessivas de esmalte dentário e cálculo foi de 82,4%. Concluiu-se que, apesar de todos os equinos estudados serem mantidos em sistema extensivo, as afecções dentárias mostraram-se muito comuns na raça Crioula sob estas condições de manejo.(AU)

Biomechanical analysis of the masticatory movement before and after adjusting dental occlusion in equine / Análise biomecânica do movimento mastigatório antes e depois do ajuste oclusal dental em equinos

The aim of this study was to evaluate through three-dimensional kinematic analysis the influence of occlusal adjustment by tooth wear in masticatory biomechanics of horses. Seven clinically healthy thoroughbred Arabian horses with strong occlusal irregularities were used, of which seven castrated males and one female of between seven and nineteen years of age. Three digital video cameras and seven spherical reflective markers placed on the horses' face were employed. The animals were filmed twice in succession: while chewing hay before and after the occlusal adjustment by tooth wear. Following that, kinematic analysis was made of the movements obtained by means of the images which were synchronized, segmented and reconstructed three-dimensionally with the help of Dvideow program. Mathematical functions were applied in Matlab environment for obtaining the values of the amplitudes of movements. The results of the biomechanical analysis showed that the occlusal adjustment increases the range of mandibular movements in horses.(AU)

O objetivo deste estudo foi avaliar, através de análise cinemática tridimensional, a influência do ajuste oclusal por desgaste dentário na biomecânica da mastigação de equinos. Foram utilizados sete cavalos puro sangue árabe, clinicamente saudáveis, com irregularidades oclusais relevantes, sendo seis machos castrados e uma fêmea, de sete a dezenove anos de idade. Três câmeras de vídeo digitais e sete marcadores reflexivos esféricos foram colocados na face dos equinos. Os animais foram filmados duas vezes seguidas: durante a mastigação do feno antes e após o ajuste oclusal por desgaste dental. Na sequência, procedeu-se a cinemática dos movimentos obtidos por meio das imagens sincronizadas, segmentadas e reconstruídas em três dimensões, com a ajuda do programa Dvideow. Funções matemáticas foram aplicadas no ambiente Matlab para a obtenção dos valores de amplitudes dos movimentos. Os resultados da análise biomecânica mostraram que o ajuste oclusal aumenta a gama de movimentos mandibulares em equinos.(AU)

Survival of acral lentiginous melanoma in the National Cancer Institute of Colombia.

OBJECTIVE: To determine survival of a cohort of patients with acral lentiginous melanoma (ALM), treated at the National Cancer Institute of Colombia, the largest referral hospital of the country. METHODS: All patients diagnosed with an invasive ALM between 2003 and 2009 at the Colombian National Cancer Institute were included for analyses and followed for vital status and date of death. Using Kaplan-Meier methods, overall survival at 1, 3 and 5 years post diagnosis was determined, and Cox proportional hazards models were constructed for the variables showing a significant effect on survival in univariate analyses. RESULTS: Overall survival of this cohort of 90 patients was 77% at 1 year, 59% at 3 years and 54% at 5 years after diagnosis. Females had a better prognosis in univariate analyses but this advantage disappeared in multivariate models. Clinical stage was a strong predictor of survival, in univariate and multivariate models, particularly among elderly patients. CONCLUSIONS: Prognosis of ALM in Colombia is relatively poor, particularly for patients with higher clinical stage. The large proportions of ALM diagnosed in stage III and IV explain the relatively poor survival, and illustrate the importance of improving prognosis by lowering stage at diagnosis through better education and early detection programmes.

Influência do parasitismo intestinal sobre os parâmetros hematológicos e de líquido peritoneal em equinos de tração / Influence of intestinal parasitism on peritoneal fluid and hematological parameters in horses used for traction

O objetivo do presente estudo foi avaliar a influência do parasitismo intestinal crônico sobre parâmetros hematológicos e de líquido peritoneal por meio da comparação dessas características em equinos naturalmente parasitados e após administração de anti-helmíntico. Utilizaram-se 21 cavalos de tração urbana, entre dois e 19 anos, sem raça definida e com resultado de exame parasitológico superior a 300 ovos por grama de fezes. Foi realizada avaliação física e coleta de fezes, de líquido peritoneal e de sangue em dois momentos do experimento (D0 e D15), sendo efetuado tratamento antiparasitário no D0. No fluido peritoneal foram avaliadas características físicas, bioquímicas, bem como contagem de células nucleadas (CTCN) e diferenciação celular. No sangue foram determinados valores eritrocitários, leucocitários, proteínas plasmáticas totais, glicose e fibrinogênio plasmáticos, além de fosfatase alcalina (FA) sérica. A análise dos parâmetros avaliados não demonstrou diferença significativa entre animais parasitados e após administração de anti-helmíntico, exceto para valores de CTCN, contagem de neutrófilos segmentados e grau de turbidez do líquido peritoneal. As médias se mantiveram dentro dos intervalos de referência, com exceção da CTCN do líquido peritoneal no D0. No líquido peritoneal, houve predomínio de neutrófilos segmentados, seguidos por macrófagos, linfócitos e eosinófilos em ambos os momentos de avaliação. Observaram-se tendência do quadro eritrocitário em manter-se próximo aos limites inferiores e leve leucocitose no D0. A infecção parasitária nos animais estudados foi predominantemente moderada, o que oferece poucos riscos clínicos. Nessas condições, pode-se afirmar que a CTCN, a contagem absoluta de neutrófilos segmentados e o grau de turbidez do líquido peritoneal são influenciados e podem ser considerados ferramentas diagnósticas e prognósticas úteis nas parasitoses intestinais crônicas.(AU)

The aim of this study was to evaluate the influence of chronic intestinal parasitism on hematological parameters and peritoneal fluid. It was done by comparing these features in horses used for traction naturally parasitized and after the administration of anthelmintic. Twenty-one horses, between two and nineteen years of age, of mixed breed and with results of parasitological examination of more than 300 eggs per gram of feces were studied. Physical assessment and samples of feces were conducted, as well as blood and peritoneal fluid in the two phases of the experiment (D0 and D15). Antiparasitic treatment in D0 has also been done. The peritoneal fluid was evaluated for physical and biochemical features, and also total count of nucleated cells (TCNC) and cell differentiation. The blood was determined for erythrocyte, leukocyte, plasma total protein, glucose and plasma fibrinogen, and alkaline phosphatase (ALP) levels. The analysis of these parameters showed no significant difference between parasitized animals and after administration of anthelmintic except for TCNC values, segmented neutrophil count and degree of turbidity in peritoneal fluid. The averages remained within the reference ranges, except the TCNC in the peritoneal fluid in D0. In the peritoneal fluid there was a predominance of segmented neutrophils, followed by macrophages, lymphocytes and eosinophils in both time points. A trend was observed in erythrocyte frame to keep close to the lower limits and mild leukocytosis in D0. Parasitic infection of the animals studied was predominantly moderate, which offered minimal clinical risks. After that, it can be affirmed that the TCNC, absolute segmented neutrophil count and targeted degree of turbidity in peritoneal fluid are influenced and can be considered useful diagnostic and prognostic tools in chronic intestinal parasitism.(AU)

Caracterização ultrassonográfica das estruturas flexoras musculotendíneas e ligamentares da porção distal dos membros de novilhas cruzadas / Ultrasonographic characterization of flexor tendinous structure muscle and ligaments of the distal limb of crossbred heifers

Caracterizaram-se, por meio da ultrassonografia, as estruturas flexoras da porção distal dos membros de bovinos utilizando-se peças anatômicas da porção distal dos membros torácicos e pélvicos, provenientes de 20 novilhas mestiças da raça Nelore, com idades entre 24 e 36 meses. Para análise ultrassonográfica, foram estabelecidas cinco zonas de avaliação no plano transversal, denominadas, respectivamente, de zonas A, B, C, D e E, e duas em plano sagital, F-III e F-IV. Na face flexora, foram avaliados os tendões flexores digitais superficial e profundo, o músculo interósseo, o ligamento acessório do tendão flexor digital profundo e a manica flexoria, quanto à forma, limites, posição, ecogenicidade e mensurações das áreas transversais em cm². Sendo os resultados apresentados na forma descritiva e em tabelas, foi possível a caracterização das estruturas flexoras, identificando e determinando planos ultrassonográficos apropriados para a observação de imagens adequadas destes tecidos, além da obtenção de valores e parâmetros que possam ser utilizados como referência para esta espécie.

Anatomical specimens of the distal portion of the thoracic and pelvic limbs from 20 crossbred Nelore, aged between 24 and 36 months were studied. For ultrasound examination five areas of assessment in the transverse plane were established, respectively called zones A, B, C, D and E, and two in the sagittal plane, F-III and F-IV. On the face of the flexor tendons we assessed superficial and deep digital flexor, interosseous muscle, ligament tendon and deep digital flexor of shape, manica flexoria, boundaries, location, echogenicity and crosscutting areas in cm 2 , and the results were presented as descriptive and tables. This work was possible to characterize the structures flexor, identifying and determining appropriate plans for the sonographic observation of appropriate images of these tissues, in addition to obtaining values and parameters that can be used as reference for this species, increasing knowledge described in literature.

Injerto de tejido conjuntivo subepitelial. Consideraciones sobre la técnica / Subepithelial connective tissue graft. Details of the technique

En este estudio se evalúa, a través de una revisión bibliográfica, la utilización del injerto de tejido conjuntivo en el tratamiento de las recesiones gingivales, analizando su predictibilidad en el recubrimiento radicular, resultado estético final, complicaciones, malestar post-operatorio y aspectos histológicos de los tejidos neo-formados, además de discutir las variables de esta técnica. Inicialmente, el injerto de tejido conjuntivo tuvo como objetivo corregir deformidades en el reborde alveolar. Posteriormente, se modificó para intentar conseguir el recubrimiento radicular total en recesiones aisladas o múltiples. La cobertura radicular con injerto de tejido conjuntivo subepitelial es predecible en recesiones Clases I y II de Miller, obteniendo resultados estéticos satisfactorios. Si bien este procedimiento es una buena opción de tratamiento de cirugía plástica periodontal, la técnica quirúrgica no está completamente estandarizada (AU)

The main objective of this study search was to verify the utilization of the subepithelial connective tissue grafts in the treatment of the gingival recession, watching its foreseeing in the root coverage, final esthetic result, complications, post-surgery discomfort, and histological aspects. The connective tissue graft had at first, the purpose to correct the alveolar deformities. After, it was modified to try to obtain total root coverage. It was concluded that the root coverage with subepithelial connective tissue graft is predictable in the Miller’s Class I and II recessions; satisfactory results was obtained; there is not yet a standard technique, that is, the described procedure is a good option for the esthetical periodontal surgery (AU)

Aspectos morfométricos da cicatrização do cólon descendente de eqüinos submetidos a enterorrafias aposicionais com poliglactina 910 e com cianoacrilato / Morphometric aspects of the healing of descending colon of horses submitted to appositional suture techniques with polyglactin 910 and with cyanoacrilate

Foi realizado estudo morfométrico do cólon descendente de 15 eqüinos hígidos, nove fêmeas e seis machos, submetidos a enterorrafias aposicionais com fio de poliglactina 910 ou com cianoacrilato. Os animais foram distribuídos aleatoriamente em cinco grupos de três animais cada e sacrificados aos três, sete, 14, 35 e 70 dias de pós-operatório para colheita dos segmentos intestinais operados e posterior processamento histológico de rotina. A análise morfométrica foi feita em microscópio óptico adaptado a um sistema de análise de imagens, onde se quantificou o infiltrado inflamatório, mediante contagens de neutrófilos, eosinófilos, macrófagos, linfócitos e plasmócitos, não sendo observadas diferenças (P>0,05) entre as enterorrafias. Houve diferenças significativas entre os dias de pós-operatório para as contagens de neutrófilos, eosinófilos, macrófagos e linfócitos. Também se mensuraram as espessuras das regiões operadas (cicatriz) e das áreas contíguas não operadas (controle), registrando-se diferenças entre os dias de pós-operatório e as regiões (P<0,05), mas não entre as enterorrafias. O fio de poliglactina 910 e o cianoacrilato apresentam a mesma eficácia para serem usados em enterorrafias aposicionais do cólon descendente de eqüinos.

A morphometric study was carried out in descending colon of 15 healthy horses, nine females and six males, submitted to appositional suture techniques with polyglactin 910 or with cyanoacrilate. The animals were randomly distributed in five groups of three animals each and sacrificed on postoperative days 3, 7, 14, 35 or 70 in order to collect the operated intestinal segments. The morphometric evaluation was done with an optic microscope adapted to an image analysis system, where was counted the inflammatory infiltrated, through neutrophils, eosinophils, macrophages, lymphocytes and plasmacytes counting. No difference (P>0.05) was observed between the two suture techniques. There were significant differences between postoperative days related to the counting of neutrophils, eosinophils, macrophages and lymphocytes. It was also measured the thickness of operated regions (scar) and non operated nearby areas (control); it registered differences between the postoperative days and the regions (P<0.05), but not between the sutures. The polyglactin 910 and the cyanoacrilate showed the same efficacy to be used in appositional enterorrhaphies in equine descending colon.

[Acute renal failure secondary to paroxysmal cold hemoglobinuria]. / Insuficiencia renal aguda secundaria a hemoglobinuria paroxística al frío.

Paroxysmal cold hemoglobinuria is a type of hemolytic anemia that mainly affects children. Tubular renal injury induced by the heme pigment in intravascular hemolysis is a rare cause of renal failure. We describe the case of a 5-year-old boy who presented with dehydration and dark urine a few hours after exposure to cold. The child had had an upper respiratory infection the previous week. He developed anemia and acute renal failure. The direct antiglobulin test was positive with anti-C3c and C3d. The diagnosis of paroxysmal cold hemoglobinuria was confirmed by the presence of biphasic antibody in the Donath-Landsteiner test.

Insuficiencia renal aguda secundaria a hemoglobinuria paroxística al frío / Acute renal failure secondary to paroxysmal cold hemoglobinuria

La hemoglobinuria paroxística al frío es una forma de anemia hemolítica que afecta principalmente a niños. La toxicidad tubular renal del pigmento hemo en situaciones de hemólisis intravascular constituye una etiología rara de insuficiencia renal. Se presenta el caso de un paciente de 5 años que presenta deshidratación y orina oscura horas después de exponerse al frío. Una semana antes había un antecedente de infección de las vías aéreas superiores. En su evolución desarrolla anemia e insuficiencia renal aguda. El test de Coombs directo es positivo con anti-C3c y C3d. La presencia de anticuerpos bifásicos queda demostrada con la prueba de Donath-Landsteiner, lo que confirma el diagnóstico de hemoglobinuria paroxística al frío

Paroxysmal cold hemoglobinuria is a type of hemolytic anemia that mainly affects children. Tubular renal injury induced by the heme pigment in intravascular hemolysis is a rare cause of renal failure. We describe the case of a 5-year-old boy who presented with dehydration and dark urine a few hours after exposure to cold. The child had had an upper respiratory infection the previous week. He developed anemia and acute renal failure. The direct antiglobulin test was positive with anti-C3c and C3d. The diagnosis of paroxysmal cold hemoglobinuria was confirmed by the presence of biphasic antibody in the Donath-Landsteiner test

Impact of epitope permutations in the antibody response of mice to a multi-epitope polypeptide of the V3 loop of human immunodeficiency virus type 1.

Our group have produced in Escherichia coli and evaluated the immunogenicity of different multi-epitope polypeptides (MEPs) bearing one copy of V3 loop sequential B cell epitopes from several isolates of human immunodeficiency virus type 1 (HIV-1) gp120. One of these MEPs called TAB9 comprises the 15 central amino acids of the V3 loop from isolates LR150, JY1, RF, MN, BRVA and IIIB in this order. Antibodies against all V3 regions were elicited after immunization of rabbits, macaques and humans with TAB9. In contrast, mice immunized with this protein only developed antibodies against epitopes JY1, LR150 and MN in that order (JY1>LR150>MN>>>RF, BRVA, IIIB) resembling an immunodominant gradient from the N-terminus to the C-terminal portion of this construction. To assess what role the location of the V3 epitopes in TAB9 could play, we constructed the protein TAB16, by altering the position of V3 epitopes in TAB9 primary structure and compared the pattern of antibodies elicited by both MEPs in H-2(d) Balb/c mice. The MEP TAB16 elicited antibody titers comparable to that of the sera from mice immunized with TAB9. There were no statistical differences in antibody titers between both groups (P>0.05). JY1, LR150 and MN V3 epitopes were again immunodominant in mice immunized with TAB16 fusion protein. The highest antibody titers detected in both groups among V3 epitopes corresponded to JY1, now located at the C-terminus of the permuted chimera. Antibodies against V3 epitopes RF, BRVA and IIIB were again not detected. Additionally, the MN V3 epitope showed to be significantly more immunogenic in its new orientation in TAB16, possibly as a result of a higher degree of accessibility in the surface of the protein. The results of the present investigation strongly suggest that the sequential order or the intramolecular position of V3 epitopes inside the primary structure of TAB9 and TAB16 MEPs does not interfere with the global immunogenicity or with the hierarchy of immunodominance of these regions.

A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers.

A phase I clinical trial was performed to examine the safety and immunogenicity of a multi-epitope polypeptide comprising the central 15 amino acids of the V3 loop from six HIV-1 isolates. This protein called TAB9 was emulsified in Montanide ISA720 (Seppic, Paris) and administered intramuscularly at doses of 0, 0.2 and 1 mg to 24 healthy, HIV-1 seronegative adult males. Three immunisations were given at months 0, 1 and 6 in a randomised, double blind, placebo controlled clinical trial. The placebo was generally well tolerated. However, severe local reactions were observed in TAB9 vaccinated subjects after the second and third inoculations. Seven out of eight volunteers from the lower dose group showed moderate or severe local inflammation, while four out of eight subjects from the higher dose group developed granulomas and sterile abscesses. In general, the reactogenicity depended on the number of inoculations given and the dose of TAB9. Both doses were immunogenic, all immunised volunteers seroconverted and antibodies were broadly reactive against the V3 peptides included in the protein. All vaccine's sera reacted against gp120 in Western blot and 50% of them also neutralised at least one out of five laboratory isolates tested. No differences between doses were found. Anti TAB9 lymphoproliferative responses were observed, being more intense in the high dose group. Due to the strong local reactions that were found in this study, a change in the formulation will be required for further trials with this vaccine candidate in humans.

Isolates from four different HIV type 1 clades circulating in Cuba identified by DNA sequence of the C2-V3 region.

According to the epidemiology of human immunodeficiency virus infection in Cuba, the main sources of infection have been persons coming from foreign countries, mainly from Africa, and individuals who have had sexual contacts with foreigners in Cuba. However, the first Cuban HIV-1 isolates sequenced have been all classified as subtype B. In this note we report the sequence of the gp120 C2/V3 region from 11 HIV-1 isolates from Cuban patients. DNA was isolated either directly from blood PBMC or from primary isolates, PCR amplified and sequenced. Six isolates were classified as subtype B and three of them had the atypical sequences GRGR, GWGR, and TPGR on the tip of the V3 loop. Besides, two other sequences were classified as subtype A, two as subtype H, and one as subtype C. These results confirm that although subtype B seems to be predominant, HIV-1 isolates from various subtypes do circulate in Cuba.

A family of compact plasmid vectors for DNA immunization in humans.

DNA immunization technology is based on the availability of adequate vectors for cloning and expression of heterologous immunoactive proteins in mammalian cells. We have developed a family of DNA plasmid vectors suitable to manipulate antigen expression and location. Their in vitro and in vivo functionality and application are also reported. The developed immune response, the aspects considered for vector design, and the possible independent manipulation of both blocks for the generation of bicistronic constructs, make of the pAEC family of plasmid vectors a source for DNA vaccine candidate's development for further evaluation in human clinical trials, and for potential use in the gene therapy approach.

Efficiency of erythropoietin's signal peptide for HIV(MN)-1 gp 120 expression.

The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.

The V3 loop based multi-epitope polypeptide TAB9 adjuvated with montanide ISA720 is highly immunogenic in nonhuman primates and induces neutralizing antibodies against five HIV-1 isolates.

In a previous work we selected montanide ISA720 (M-ISA720) among different adjuvants for the vaccination with a V3 loop based multi-epitope polypeptide TAB9. In this paper we present the evaluation of the toxicity and immunogenicity of this formulation in non-human primates. TAB9 in M-ISA720 was highly immunogenic in macaques (Macaca fascicularis) inducing antibodies against TAB9 in all animals after one inoculation and a strong anamnestic response after booster injections. Furthermore 97% of the V3 peptides included were recognized by TAB9 sera. No differences between doses of 200 microg and 1 mg of TAB9 in M-ISA720 were observed after four immunizations. Neutralizing antibodies against five HIV-1 isolates were detected in most animals. Animals remain healthy throughout the study and did not show lesions at the inoculation site.

A prime-boost regime that combines Montanide ISA720 and Alhydrogel to induce antibodies against the HIV-1 derived multiepitope polypeptide TAB9.

A phase I clinical trial with the HIV-1-derived multi-epitope polypeptide (MEP) TAB9 in the oil adjuvant Montanide ISA720 (M-ISA720) was recently performed. Although severe local reactions were reported after the second and third injections of this vaccine candidate, the first inoculation was well tolerated. In this article we evaluated a prime-boost regime consisting of one inoculation of TAB9 in M-ISA720 followed by a booster with the same antigen in aluminum hydroxide. This combination of adjuvants elicited similar antibody levels in rabbits than the traditional two-dose schedule with M-ISA720. A control group injected three times with TAB9 in aluminum hydroxide developed markedly lower antibody titers. These results showed that although oil adjuvants are better than alum for priming the immune system for antibody production against TAB9, both kinds of adjuvants can be equally effective in booster immunizations. Therefore, by using the more reactogenic oil adjuvant only for priming, we should be able to eliminate the undesirable reactions characteristic of these compounds while achieving equivalent levels of specific antibodies.

An immunoassay with bovine serum albumin coupled peptides for the improved detection of anti V3 antibodies in HIV-1 positive human sera.

The V3 loop of the gp120 envelope glycoprotein contains the principal neutralizing determinant of HIV-1. Many serological studies have been carried out to assess the reactivity of HIV-1 infected individuals against V3 loop synthetic peptides from different HIV-1 subtypes. V3 directed serology has also been used to demonstrate the association between ELISA reactivity and progression to AIDS in HIV patients, and to study the reactivity against the V3 region in sera from vaccinated animals and human volunteers. The advantage of the use of bovine serum albumin (BSA) conjugated V3 peptides over free V3 peptides for ELISA is described. 15 meric V3 peptides representing several HIV-1 isolates were synthesized, chemically coupled to BSA, and used to coat ELISA microplates. Conjugated peptides were compared with free peptides for the detection of anti V3 antibodies in the sera from rabbits immunized with V3 containing chimeric proteins and from HIV-1 infected individuals. No differences in reactivity against free or BSA-peptide were found for most rabbit sera, however human plasma recognized preferentially the BSA conjugated peptides. Although technically more complex, ELISA with BSA coupled V3 peptides is more sensitive and appropriate for serological studies of HIV-1 infected persons.

Broadly reactive antibodies against a gp120 V3 loop multi-epitope polypeptide neutralize different isolates of human immunodeficiency virus type 1 (HIV-1).

A gene encoding for a novel multi-epitope polypeptide (TAB4) was synthesized and expressed in Escherichia coli. The protein was composed of 15 amino acid fragments derived from the V3 loop of HIV-1 isolates MN, IIIB, RF, JY1, BRVA and LR150, joined by five-amino-acid linkers. Immunogenicity of TAB4 in rabbits was studied, and the antibody response against individual peptides investigated. TAB4 was shown to be immunogenic in Complete Freund's Adjuvant in a dose-dependent manner, and was able to elicit a humoral response against all V3 epitopes included on the protein. Sera from some of the animals were able to neutralize the replication of viral strain MN, and in one case IIIB, with moderate titers. Some sera also neutralized several Cuban clinical strains, isolated in peripheral blood mononuclear cells, after one round of amplification in MT4 cells.