Immune modulator adamantylamide dipeptide stimulates efficient major histocompatibility complex class I-restricted responses in mice.

Adamantylamide L-alanyl-D-isoglutamine (AdDP) is a synthetic adjuvant which belongs to the family of the desmuramyl peptides. AdDP exerts its adjuvant properties when it is administered either by the parenteral or by the mucosal route, leading to the elicitation of strong humoral responses at both the systemic and the mucosal levels. However, very little is known about the effect of AdDP on cellular immunity. Here we demonstrate that AdDP is able to stimulate cellular responses, which are characterized by the release of gamma interferon by CD8+ T cells when they are restimulated with a major histocompatibility complex class I-restricted peptide and strong in vivo lymphocyte-mediated cytotoxic activity. The capacity of AdDP to stimulate the elicitation of both cellular and humoral adaptive responses makes this adjuvant a promising tool for the development of mucosal vaccine formulations.

Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia.

Chronic lung infection by opportunistic pathogens, such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex, is a major cause of morbidity and mortality in patients with cystic fibrosis. Outer membrane proteins (OMPs) of gram-negative bacteria are promising vaccine antigen candidates. In this study, we evaluated the immunogenicity, protection, and cross-protection conferred by intranasal vaccination of mice with OMPs from B. multivorans plus the mucosal adjuvant adamantylamide dipeptide (AdDP). Robust mucosal and systemic immune responses were stimulated by vaccination of naive animals with OMPs from B. multivorans and B. cenocepacia plus AdDP. Using a mouse model of chronic pulmonary infection, we observed enhanced clearance of B. multivorans from the lungs of vaccinated animals, which correlated with OMP-specific secretory immunoglobulin A responses. Furthermore, OMP-immunized mice showed rapid resolution of the pulmonary infection with virtually no lung pathology after bacterial challenge with B. multivorans. In addition, we demonstrated that administration of B. multivorans OMP vaccine conferred protection against B. cenocepacia challenge in this mouse infection model, suggesting that OMPs provide cross-protection against the B. cepacia complex. Therefore, we concluded that mucosal immunity to B. multivorans elicited by intranasal vaccination with OMPs plus AdDP could prevent early steps of colonization and infection with B. multivorans and also ameliorate lung tissue damage, while eliciting cross-protection against B. cenocepacia. These results support the notion that therapies leading to increased mucosal immunity in the airways may help patients with cystic fibrosis.

Intranasal vaccination with recombinant outer membrane protein CD and adamantylamide dipeptide as the mucosal adjuvant enhances pulmonary clearance of Moraxella catarrhalis in an experimental murine model.

Moraxella catarrhalis causes acute otitis media in children and lower respiratory tract infections in adults and elderly. In children the presence of antibodies against the highly conserved outer membrane protein CD correlates with protection against infection, suggesting that this protein may be useful as a vaccine antigen. However, native CD is difficult to purify, and it is still unclear if recombinant CD (rCD) is a valid alternative. We performed a side-by-side comparison of the immunogenicities and efficacies of vaccine formulations containing native CD and rCD with adamantylamide dipeptide as the mucosal adjuvant. Intranasal vaccination of mice stimulated the production of high CD-specific antibody titers in sera and of secretory immunoglobulin A in mucosal lavages, which cross-recognized both antigens. While vaccination with native CD increased the number of interleukin-2 (IL-2)- and gamma interferon-producing cells, rCD mainly stimulated IL-4-secreting cells. Nevertheless, efficient bacterial clearance was observed in the lungs of challenged mice receiving native CD and in the lungs of challenged mice receiving rCD (96% and 99%, respectively). Thus, rCD is a promising candidate for incorporation in vaccine formulations for use against M. catarrhalis.

Immunolocalization of the Epstein-Barr nuclear antigen-1 in conjunctival squamous carcinomas and dysplasias.

Epstein-Barr virus (EBV) has been linked etiologically to infectious mononucleosis, some non-Hodgkin as well as Hodgkin lymphomas, and lymphoepithelioma-like carcinomas. Moreover, various EBV antigens have been identified by a variety of techniques in a number of visceral carcinomas including breast, prostate, colon and lung primaries. We have now demonstrated by immunohistochemistry the presence of EBV nuclear antigen-1 (EBNA-1) in 4 of 15 cases of conjuntival squamous carcinomas and related dysplasias. At present, there is no significant evidence linking etiologically EVB to this type of tumor and dysplasia. However, our findings merit further investigation given the growing evidence that EBV may enhance proliferation and aggressiveness of tumor systems as well as the immortalization of non-neoplastic cells.

Epstein-Barr virus in breast carcinoma in Argentina.

CONTEXT: Because the etiology and progression of breast carcinoma remain unclear, novel mechanisms of disease pathogenesis need to be considered. Recent interest has focused on Epstein-Barr virus (EBV), an oncogenic ubiquitous herpesvirus. Investigations of this association could not only broaden understanding of breast cancer etiology but also have implications regarding early detection, treatment, and prevention. OBJECTIVE: To assess EBV presence in breast carcinoma in an Argentine series. DESIGN: Breast biopsy specimens of 69 women with breast carcinoma and fresh tumor tissue of 39 of these women were collected. As controls, 17 biopsy specimens of fibroadenomas, 9 of benign epithelial proliferation, 4 of atypical ductal hyperplasia, and 10 of usual ductal hyperplasia and 8 normal breast tissues from women were studied. The EBV-infected cells were identified by means of immunohistochemical analysis, using a monoclonal antibody against Epstein-Barr virus-encoded nuclear antigen 1 (EBNA-1). Polymerase chain reaction (PCR) was used to amplify EBV DNA, with primers that cover the EBV encoded RNA (EBER) and BamHIW regions. RESULTS: Nuclear expression of EBNA-1 was observed in tumor epithelial cells in 24 (35%) of the 69 cases. We confirmed both positive and negative immunohistochemical results by PCR in those cases where good quality DNA was also available, detecting amplification fragments of 108 base pairs (bp) from the EBER region and 122 bp from the BamHIW region. Neither immunohistochemical analysis nor PCR detected any positive EBV results in the control samples. CONCLUSIONS: Our results demonstrate the presence and expression of EBV restricted to epithelial tumor cells in a subset of breast carcinomas studied. However, no significant association was observed between EBV expression and worse clinical and pathologic patient characteristics.

Intranasal vaccination with recombinant P6 protein and adamantylamide dipeptide as mucosal adjuvant confers efficient protection against otitis media and lung infection by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a leading etiologic agent of otitis media in children and recurrent respiratory infections in patients with chronic obstructive pulmonary disease. The highly conserved outer membrane protein P6 constitutes a promising vaccine candidate antigen. However, the small amount of P6 produced by this fastidious microorganism renders large-scale production difficult. Controversial data also exist concerning the suitability of recombinant P6 (rP6) as a vaccine antigen. Therefore, we performed a comparative evaluation of the immunogenicity and efficacy of native P6 and rP6 in mice intranasally vaccinated with adamantylamide dipeptide (AdDP) as an adjuvant. High titers of P6-specific serum antibodies were elicited in mice vaccinated with either native P6 or rP6, which cross-recognized both antigens. However, rP6 stimulated stronger mucosal responses. Mice vaccinated with rP6 were protected against both pulmonary and middle-ear infections (P<.01). This demonstrates that rP6 plus AdDP constitutes a promising vaccine formulation against the most relevant forms of disease caused by NTHi.

Hepatitis C virus infection in infants and children from Argentina.

Hepatitis C virus (HCV) infection is uncommon in children, and its natural history is still unknown. Our aim was to analyze exposure to HCV in 48 infants and children in Argentina and to evaluate consecutive samples in 26 of them to study the outcome of HCV infection in early stages. HCV viremia, as determined by reverse transcription-PCR (RT-PCR) from the 5' untranslated region, showed continuously positive, occasionally positive, and negative patterns during follow-up. Restriction fragment length polymorphism was performed on RT-PCR-positive samples to evaluate HCV genotype. Genotype 1 turned out to be predominant, and no patient displayed a genotype shift during the observation period. Perinatal HCV infection was predominantly observed in patients born to mothers coinfected with HCV and human immunodeficiency virus. HCV viral load was detected by means of the AMPLICOR MONITOR, version 2.0, kit. No correlation was observed between HCV viral load and alanine aminotransferase and aspartate aminotransferase levels, although we detected a trend towards higher levels among patients displaying consecutive positive HCV RT-PCR results. Our results demonstrate that pediatric HCV infection is characterized by high viral loads and diverse HCV viremia patterns, independent of both age and route of transmission in the population under study. Further research is necessary to determine whether the high rate of HCV replication is related to virus variability or to host immune response.

Hepatitis C virus isolates from Argentina disclose a novel genotype 1-associated restriction pattern.

Hepatitis C virus isolates which disclosed a novel genotype 1-associated restriction pattern by restriction fragment length polymorphism analysis were characterized. Except for a mother and child pair, the patients were unrelated. Sequence analysis showed a G-->A substitution leading to a new RsaI recognition site. Phylogenetic analysis revealed that these isolates constitute a novel genetic lineage within the main cluster of genotype 1 strains.

Demonstration of Epstein-Barr virus in carcinomas of various sites.

EBV is an etiological agent in infectious mononucleosis, is implicated in some malignant lymphomas and lymphoepithelioma-like carcinomas, and has been sporadically reported in carcinomas of the breast, lung, and other sites. We studied immunohistochemically benign and malignant tumors of the breast, lung, colon, and prostate and found EBV in some carcinomas of those sites. Also, EBV reactions were noted in hyperplasias and dysplasias, e.g., breast carcinomas in situ and prostatic intraepithelial neoplasia. Benign tumor counterparts were negative. PCR analysis of selected cases confirmed the presence of EBV. Our results suggest that EBV is not restricted to lymphoepithelioma-like carcinomas but may play an oncogenic role in frequent epithelial cancers and possibly also in hyperplasias and certain dysplasias preceding carcinomas.

Assessment of Epstein-Barr virus association with pediatric non-hodgkin lymphoma in immunocompetent and in immunocompromised patients in Argentina.

CONTEXT: Epstein-Barr virus (EBV) has been classically associated with 3 malignancies, Burkitt lymphoma, B-cell lymphoproliferative syndromes, and nasopharyngeal carcinoma, and more recently with Hodgkin disease, T-cell lymphomas, and gastric and breast carcinomas, as well as with leiomyosarcoma and leiomyoma associated with immunosuppression. OBJECTIVE: To compare EBV expression in Argentine tumor samples with those reported elsewhere, we analyzed EBV expression in an Argentine pediatric population with non-Hodgkin lymphoma and correlated these results with clinical course and outcome. METHODS: We studied EBV presence by latent membrane protein-1 protein labeling by immunohistochemistry, by in situ hybridization, and by polymerase chain reaction for Epstein-Barr-encoded RNAs (EBERs) in formalin-fixed and paraffin-embedded non-Hodgkin lymphoma tissue samples (collected retrospectively) from 32 pediatric patients at Ricardo Gutiérrez Children's Hospital from 1993 to 2000. RESULTS: Eight out of the 32 (25%) non-Hodgkin lymphoma cases showed latent membrane protein-1 and EBERs by in situ hybridization positive staining in tumor cells. Among EBERs and latent membrane protein-1-positive cases, there were 5 immunocompromised patients, with either human immunodeficiency virus infection or primary immunodeficiency. The EBERs in situ hybridization results were confirmed by EBERs polymerase chain reaction in good-quality DNA from 11 samples, with 3 proving positive and 8 negative. CONCLUSIONS: The association of EBV with non-Hodgkin lymphoma in the Argentine pediatric population was low (25%), and this figure rose to 100% when only the immunocompromised patients subgroup was considered, confirming that the virus is probably a cofactor in the lymphomagenesis of some but not all pediatric non-Hodgkin lymphoma. So far, no differences in clinical outcome are discernible between EBV-positive and EBV-negative non-Hodgkin lymphoma patients.

Caracterización genotípica de la infección por el virus de hepatitis C en niños / Genotype characterization of hepatitis C virus infection in children

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5'untranslated region (5'UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36 per cent) were always positive for HCV RNA, in 5 (20 per cent) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36 per cent) showed alternating RT-nested PCR results and in 2 (8 per cent) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the host's response in the initial stages of it.

Caracterización genotípica de la infección por el virus de hepatitis C en niños / Genotype characterization of hepatitis C virus infection in children

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5untranslated region (5UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36 per cent) were always positive for HCV RNA, in 5 (20 per cent) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36 per cent) showed alternating RT-nested PCR results and in 2 (8 per cent) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the hosts response in the initial stages of it. (Au)

Prevalencia de la infección por el virus de Epstein-Barr en pacientes pediátricos / Prevalence of Epstein-Barr virus infection in pediatric patients

El virus de Epstein-Barr (VEB) está ampliamente distribuído en el mundo; el 80-90 por ciento de la población adulta ha sido infectada y posee anticuerpos. En países desarrollados, la primoinfección se produce en la adolescencia y la juventud; y en países subdesarrollados, en los primeros años de vida. En este estudio, se analizó la serología de la infección por VEB en un grupo de pacientes pediátricos de Capital y Gran Buenos Aires. La prevalencia de anticuerpos anti-VCA-IgG en la población total es del 72 por ciento. En pacientes de entre 6 meses y 1 año, sólo un 26 por ciento son seropositivos para VCA-IgG. A partir de 1 año de edad, se produce un aumento significativo (p<0,00001) del porcentaje de seropositivos para VCA-IgG que se mantiene en porcentajes comparables durante la infancia hasta la adolescencia, sin diferencias en la infección entre los sexos. Los resultados de este trabajo indican que el contacto con VEB se produce muy tempranamente en la niñez; los posibles factores que influencian la infección temprana por VEB son falta de higiene, desconocimiento de las normas básicas de sanidad, alto número de niños que comparten una vivienda o habitación, etc. El perfil de seroprevalencia hallado en este estudio coincide con el descripto para países y poblaciones subdesarrolladas(AU)

Prevalencia de la infección por el virus de Epstein-Barr en pacientes pediátricos / Prevalence of Epstein-Barr virus infection in pediatric patients

El virus de Epstein-Barr (VEB) está ampliamente distribuído en el mundo; el 80-90 por ciento de la población adulta ha sido infectada y posee anticuerpos. En países desarrollados, la primoinfección se produce en la adolescencia y la juventud; y en países subdesarrollados, en los primeros años de vida. En este estudio, se analizó la serología de la infección por VEB en un grupo de pacientes pediátricos de Capital y Gran Buenos Aires. La prevalencia de anticuerpos anti-VCA-IgG en la población total es del 72 por ciento. En pacientes de entre 6 meses y 1 año, sólo un 26 por ciento son seropositivos para VCA-IgG. A partir de 1 año de edad, se produce un aumento significativo (p<0,00001) del porcentaje de seropositivos para VCA-IgG que se mantiene en porcentajes comparables durante la infancia hasta la adolescencia, sin diferencias en la infección entre los sexos. Los resultados de este trabajo indican que el contacto con VEB se produce muy tempranamente en la niñez; los posibles factores que influencian la infección temprana por VEB son falta de higiene, desconocimiento de las normas básicas de sanidad, alto número de niños que comparten una vivienda o habitación, etc. El perfil de seroprevalencia hallado en este estudio coincide con el descripto para países y poblaciones subdesarrolladas

Rubéola congénita: un problema aún no resuelto en Argentina / Congenital rubella: still a problem without solution in argentina

Introducción. En 1995 se registró un epidemia de rubéola en Buenos Aires, incrementando el riesgo de las mujeres susceptibles de contraer la infección durante la gestación y trasmitirla al feto. Objetivo. Describir las consecuencias posneonatales de una epidemia de rubéola. Población. Se incluyeron 12 lactantes con rubéola congénita diagnosticados en el Hospital de Niños "Dr.Ricardo Gutierrez" de Buenos Aires durante 1996. Criterios y Diagnosticos. IGM específica reactiva, serología materna reactiva y clínica compatible con infección intrauterina. Resultados. La edad media de la madre fue de 24 años. Un niño era adopatado. En 5/11 se registró antecedente de contacto con niños con erupción rubeoliforme, 7/11 madres presentaron exantema en primer trimestre de la gesta. Ninguna madre recibió vacunación antirrubiólica previo a la gesta ni realizó estudio serológico. De los 12 lactantes con rubeóla congénita la edad media al diagnóstico fue de 2,2 meses, 8/12 presentaron retardo intrauterino, 2/12 neonatos fueron asintomáticos al nacer. Los datos clínicos relevante fueron. Alteraciones oculares, compromiso neurológico, cardioapatías congénitas, hepatoesplenomegalia y o alteraciones hematológicas, nuemonitis e hipoacusia. Dos niños fallecieron durante el primer año de vida

Rubéola congénita: un problema aún no resuelto en Argentina / Congenital rubella: still a problem without solution in argentina

Introducción. En 1995 se registró un epidemia de rubéola en Buenos Aires, incrementando el riesgo de las mujeres susceptibles de contraer la infección durante la gestación y trasmitirla al feto. Objetivo. Describir las consecuencias posneonatales de una epidemia de rubéola. Población. Se incluyeron 12 lactantes con rubéola congénita diagnosticados en el Hospital de Niños "Dr.Ricardo Gutierrez" de Buenos Aires durante 1996. Criterios y Diagnosticos. IGM específica reactiva, serología materna reactiva y clínica compatible con infección intrauterina. Resultados. La edad media de la madre fue de 24 años. Un niño era adopatado. En 5/11 se registró antecedente de contacto con niños con erupción rubeoliforme, 7/11 madres presentaron exantema en primer trimestre de la gesta. Ninguna madre recibió vacunación antirrubiólica previo a la gesta ni realizó estudio serológico. De los 12 lactantes con rubeóla congénita la edad media al diagnóstico fue de 2,2 meses, 8/12 presentaron retardo intrauterino, 2/12 neonatos fueron asintomáticos al nacer. Los datos clínicos relevante fueron. Alteraciones oculares, compromiso neurológico, cardioapatías congénitas, hepatoesplenomegalia y o alteraciones hematológicas, nuemonitis e hipoacusia. Dos niños fallecieron durante el primer año de vida

Diarrea por rotavirus: impacto en un hospital de niños de Buenos Aires / Rotavirus diarrhea: impact in a pediatric hospital of Buenos Aires

Ante la disponibilidad de una vacuna contra rotavirus, se desarrolló el presente estudio destinado a conocer el impacto de este virus en un hospital pediátrico del país. Se estudiaron entre septiembre 1997 y agosto 1998, 648 niños < 3 años con diarrea acuosa aguda asistidos en el Consultorio Externo de Clínica - Sector Diarreas y hospitalizados en las Salas de Internación Clínica Nº 4,5 y 6 del Hospital Ricardo Gutiérrez de la Ciudad de Buenos Aires. Se encontró rotavirus asociado al 36 por ciento de las diarreas ambulatorias y al 45 por ciento de las hospitalizadas. En el año de estudio, estimamos que el Hospital asistió a 1674 diarreas por rotavirus en forma ambulatoria, 14 de las cuales requirieron hospitalización. El estudio describe un pico de diarreas por rotavirus entre marzo y junio, mientras que las diarreas no causadas por rotavirus presentaron un pico entre enero y marzo (muy probablemente de origen bacteriano). Rotavirus presentó una mayor frecuencia entre 6 a 23 meses de edad; de las 233 diarreas por rotavirus identificadas, sólo el 10 por ciento ocurrieron en mayores de 2 años y el 13 por ciento durante los primeros 6 meses de vida. Esta situación es de relevancia dado que la vacuna de rotavirus se aplica en 3 dosis orales a los 2, 4 y 6 meses de edad, y podría haber prevenido la mayoría de los casos observados.

Diarrea por rotavirus: impacto en un hospital de niños de Buenos Aires / Rotavirus diarrhea: impact in a pediatric hospital of Buenos Aires

Ante la disponibilidad de una vacuna contra rotavirus, se desarrolló el presente estudio destinado a conocer el impacto de este virus en un hospital pediátrico del país. Se estudiaron entre septiembre 1997 y agosto 1998, 648 niños < 3 años con diarrea acuosa aguda asistidos en el Consultorio Externo de Clínica - Sector Diarreas y hospitalizados en las Salas de Internación Clínica Nº 4,5 y 6 del Hospital Ricardo Gutiérrez de la Ciudad de Buenos Aires. Se encontró rotavirus asociado al 36 por ciento de las diarreas ambulatorias y al 45 por ciento de las hospitalizadas. En el año de estudio, estimamos que el Hospital asistió a 1674 dia

Seroepidemiología de los rotavirus humanos en una comunidad del partido de Avellaneda, Provincia de Buenos Aires / Seroepidemiology of human rotavirus in a community of the Avellaneda district, Province of Buenos Aires

El presente trabajo describe los resultados obtenidos durante un estudio prospectivo llevado a cabo en 49 familias del Partido de Avellaneda, tendiente a conocer la seroepidemiología de los rotavirus humanos en nuestro medio. Cada familia fue incorporada estado la madre embarazada y el recien nacido fue estudiado hasta los 2 años de vida. La mayoría de las infecciones observadas durante el primer año fueron primarias (0,64 infecciones por niño-año; el 91,3% en niños seronegativos; p < 0,005). Esto coindidió con el período de mayor suceptibilidad a la diarrea por rotavirus (0,25 casos por niño-año; p < 0,01). La incidencia de infecciones en toda la población fue 0,63 casos por persona-año, sin variaciones signficativas para cada grupo de edad. El 61,6% de ellas fueron reinfecciones y en su gran mayoría asintomáticas. Por último se demonstró una relación signficativa entre el nivel de IgG específica circulante y la protección contra la infección y la diarrea causada por los rotavirus, durante los períodos de 6 meses estudiados (p < 0,005 para la infección; p < 0,03 para la diarrea). Aunque se encontró un mayor porcentaje de personas con anticuerpos y mayores niveles a medida que aumentaba la edad (p < 0,005), la incidencia de infecciones por rotavirus no presentó variaciones significativas con la misma. De acuredo con la alta incidencia de reinfecciones encontrada y por haber hallado la total desaparición del nivel de anticuerpos en un 5% de las infecciones un año después, se postula que la protección asociada al nivel de anticuerpos circulantes disminuye rápidamente luego de los 6 meses (AU)