Lysosomal integral membrane protein 2 (LIMP-2) restricts the invasion of Trypanosoma cruzi extracellular amastigotes through the activity of the lysosomal enzyme ß-glucocerebrosidase.

Lysosomal integral membrane protein 2 (LIMP-2, SCARB2) is directly linked to ß-glucocerebrosidase enzyme (ßGC) and mediates the transport of this enzyme from the Golgi complex to lysosomes. Active ßGC cleaves the ß-glycosidic linkages of glucosylceramide, an intermediate in the metabolism of sphingoglycolipids, generating ceramide. In this study we used mouse embryonic fibroblasts (MEFs) deficient for LIMP-2 and observed that these cells were more susceptible to infection by extracellular amastigotes of the protozoan parasite Trypanosoma cruzi when compared to wild-type (WT) fibroblasts. The absence of LIMP-2 decreases the activity of ßGC measured in fibroblast extracts. Replacement of ßGC enzyme in LIMP-2 deficient fibroblasts restores the infectivity indices to those of WT cells in T. cruzi invasion assays. Considering the participation of ßGC in the production of host cell ceramide, we propose that T. cruzi extracellular amastigotes are more invasive to cells deficient in this membrane component. These results contribute to our understanding of the role of host cell lysosomal components in T. cruzi invasion.

Mucopolysaccharidosis in dog report of two cases / Mucopolissacaridose em cão Relato de dois casos

Mucopolissacaridoses são um grupo de afecções hereditárias do armazenamento lisossomal, causada por deficiência de hidrolases lisossomais, necessárias para a degradação das glicosaminoglicanas. Neste relato são escritos dois casos de mucopolissacaridose em cães: Um cão macho, Pitbull, de 60 dias, foi atendido devido à dificuldade de locomoção com os quatro membros e aumento de volume articular. No exame clínico constatou-se aumento de volume e deformidades em articulações e pectus cavinatum. Nas radiografias do sistema esquelético observou-se disostose multiplex. O outro paciente, uma canina fêmea, Rottweiler, de oito meses, foi atendida devido à dificuldade progressiva de locomoção. No exame clínico constatou-se tetraplegia, opacidade corneal, aumento de volume em língua e deformidade em crânio. Para triagem de mucopolissacaridose, realizou-se na urina dosagem qualitativa e quantitativa de glicosaminoglicanos e ensaios enzimáticos no plasma para dosar a atividade de enzimas lisossômicas. Apesar da confirmação de mucopolissacaridose com os testes de triagem, não foi possível diferenciar entre os tipos II ou VI.(AU)

Mucopolysaccharidosis are a group of inherited lysosomal storage disorders caused by deficiency of lysosomal hydrolases needed for the stepwise degradation of glycosaminoglycans. In this report we describe two cases of mucopolysaccharidosis in dogs. A two-month-old male Pitbull was referred with difficulty in locomotion in four limbs and swelling in joints. Clinical examination showed increasing volume of articulations, bad limb angulations with ambulation difficulties and pectus cavinatum. Radiographical exams of the skeletal system showed dysostosis multiplex. The other dog was an eight-month-old female Rottweiler that was referred due to progressive difficulty in walking. Clinical examination showed tetraplegia, corneal opacities, enlarged tongue and skull deformities. For screening mucopolysaccharidosis, a qualitative and quantitative measurement of urinary glycosaminoglycans and plasma enzymatic assays to evaluate the activity of lysosomal enzymes were made in both dogs. Despite the confirmation of mucopolysaccharidosis with the screening tests, the type between II and VI could not be distinguished.(AU)

Mucopolysaccharidosis in dog – report of two cases / Mucopolissacaridose em cão – Relato de dois casos

Mucopolissacaridoses são um grupo de afecções hereditárias do armazenamento lisossomal, causada por deficiência de hidrolases lisossomais, necessárias para a degradação das glicosaminoglicanas. Neste relato são escritos dois casos de mucopolissacaridose em cães: Um cão macho, Pitbull, de 60 dias, foi atendido devido à dificuldade de locomoção com os quatro membros e aumento de volume articular. No exame clínico constatou-se aumento de volume e deformidades em articulações e pectus cavinatum. Nas radiografias do sistema esquelético observou-se disostose multiplex. O outro paciente, uma canina fêmea, Rottweiler, de oito meses, foi atendida devido à dificuldade progressiva de locomoção. No exame clínico constatou-se tetraplegia, opacidade corneal, aumento de volume em língua e deformidade em crânio. Para triagem de mucopolissacaridose, realizou-se na urina dosagem qualitativa e quantitativa de glicosaminoglicanos e ensaios enzimáticos no plasma para dosar a atividade de enzimas lisossômicas. Apesar da confirmação de mucopolissacaridose com os testes de triagem, não foi possível diferenciar entre os tipos II ou VI.

Mucopolysaccharidosis are a group of inherited lysosomal storage disorders caused by deficiency of lysosomal hydrolases needed for the stepwise degradation of glycosaminoglycans. In this report we describe two cases of mucopolysaccharidosis in dogs. A two-month-old male Pitbull was referred with difficulty in locomotion in four limbs and swelling in joints. Clinical examination showed increasing volume of articulations, bad limb angulations with ambulation difficulties and pectus cavinatum. Radiographical exams of the skeletal system showed dysostosis multiplex. The other dog was an eight-month-old female Rottweiler that was referred due to progressive difficulty in walking. Clinical examination showed tetraplegia, corneal opacities, enlarged tongue and skull deformities. For screening mucopolysaccharidosis, a qualitative and quantitative measurement of urinary glycosaminoglycans and plasma enzymatic assays to evaluate the activity of lysosomal enzymes were made in both dogs. Despite the confirmation of mucopolysaccharidosis with the screening tests, the type between II and VI could not be distinguished.

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice.

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-γ- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis.

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of <49 and ≥20 KDa (M. hominis) and <102 and ≥58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado. Antí-genos citoplasmáticos endógenos, caracteristicamente codificados por vacinas de DNA plasmidial, são majoritariamente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I - MHC I. A via de apresentação MHC I é mais associada à resposta celular citotóxica e, frequentemente, não elicita uma resposta humoral satisfatória. Uma das principais estratégias para direcionar antígenos codificados pelas vacinas de DNA para o compartimento MHC II é expressar estes antígenos dentro da Proteína de Associação à Membrana Lisossomal (LAMP). A proteína do envelope dos flavivirus é reconhecidamente a principal proteína de superfície viral e o principal alvo para anticorpos neutralizantes. Diferentes grupos têm demonstrado que a co-expressão das proteínas de membrana e do envelope dos flavivirus em células de mamíferos, fusionada com a porção carboxi-terminal de LAMP, é capaz de induzir níveis satisfatórios de anticorpos neutralizantes. Neste trabalho revisamos a estratégia de co-expressão da proteína do envelope dos flavivírus, como quimeras de LAMP, com o objetivo de desenvolver vacinas de DNA contra a febre do Oeste do Nilo, dengue e febre amarela.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses.

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the development of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

The duration of TCR/pMHC interactions regulates CTL effector function and tumor-killing capacity.

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naïve CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.