Immunization with LytB protein of Streptococcus pneumoniae activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis.

The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.

Pharmacological immunomodulation enhances peripheral nerve regeneration

To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38µm±1.16 and COL/MDP=3.54µm±1.16) compared to NOR (6.19µm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

Para avaliar o efeito do NAcetilmuramil- L-Alanil-D-Isoglutamina administrado topicamente em neurônios periféricos em regeneração, doze camundongos C57BL/6J machos adultos foram igualmente separados em três grupos. Quatro animais sofreram transecção unilateral do nervo ciático que foi ancorado no interior de um tubo de polietileno, mantendo-se 4 mm de distância entre as extremidades dos nervos e receberam colágeno + PBS (COL) dentro do tubo. Outros quatro animais sofreram o mesmo procedimento cirúrgico, porém receberam colágeno + MDP (COL/MDP) no interior da prótese. Quatro animais não foram operados e serviram como controle de normalidade (NOR). Após quatro semanas, os cabos de regeneração foram coletados para determinação do número de axônios mielínicos e da mêdia do diâmetro das fibras mielínicas regeneradas. O gânglio da raiz dorsal L5 também foi coletado para contagem e mensuração dos neurônios sensitivos. Os resultados revelaram diferença significativa no número de axônios entre os grupos NOR (4355±32), COL (1869±289) e COL/MDP (2430±223). Houve redução significativa no diâmetro das fibras mielínicas nos grupos que receberam as próteses tubulares (COL=3,38µm±1,16 e COL/ MDP=3,54µm±1,16) quando comparados ao grupo NOR (6,19µm±2,45). O número de neurônios não diferiu entre os grupos experimentais (COL=564±51 e COL/MDP=514±56), os quais apresentaram menor número de neurônios sensitivos em relação ao grupo não operado (NOR=1097±142). Os dados obtidos indicam que a aplicação local do MDP estimula a regeneração de nervos em camundongos.

Streptococcus pneumoniae protein vaccine candidates: properties, activities and animal studies.

Streptococcus pneumoniae is a causative agent for community acquired pneumonia, bacteremia, acute otitis media, and meningitis. Recent emergence of multi-drug resistant clinical isolates prompts the need of effective vaccine for the prevention of disease. The licensed polysaccharide-based pneumococcal vaccines only elicit protective antibodies against the infection of serotypes that are included in the vaccine. To broaden the protection, the use of pneumococcal proteins will be a feasible and preferable alternative. This communication provides a review on the biochemical properties of these protein candidates, their immunization results in animal studies, and perspectives on the development of protein-based pneumococcal vaccine.

Contribution of autolysin to virulence of Streptococcus pneumoniae.

Insertion-duplication mutagenesis was used to construct an autolysin-negative derivative of Streptococcus pneumoniae. This derivative was obtained by first transforming the nonencapsulated strain Rx1 with a derivative of the vector pVA891 carrying a 375-base-pair TaqI DNA fragment from the middle of the autolysin structural gene. DNA was extracted from the resultant erythromycin-resistant, autolysin-negative rough pneumococcus and used to transform S. pneumoniae D39, a virulent type 2 strain. Several erythromycin-resistant transformants were obtained from two independent experiments, and none of these transformants produced autolysin. Southern blot analysis confirmed that the autolysin gene in these transformants had been interrupted by the plasmid-derived sequences. The autolysin-negative mutants showed markedly reduced virulence for mice compared with that of strain D39; intranasal and intraperitoneal 50% lethal doses were increased 10(2)- and 10(5)-fold, respectively. Autolysin production was reinstated in one of the mutants by back-transformation with the cloned autolysin gene, with the concomitant loss of erythromycin resistance; the virulence of this isolate for mice was indistinguishable from that of D39. The importance of autolysin in pathogenesis was confirmed by immunization-challenge studies. Mice immunized with purified autolysin survived significantly longer than did control mice after intranasal challenge with strain D39. This study provides direct evidence that the pneumococcal autolysin contributes to virulence and identifies it as a potential vaccine antigen.

[Comparative biological value of protein hydrolysates and their fortified forms]. / Sravnitel'naia biologicheskaia tsennost' belkovykh gidrolizatov i ikh obogashchennykh form.

Experiments on Wistar male rats were made to study the biological value (with the use of the "growth" method) of basic and protein- and crystalline amino acids-enriched protein hydrolysates. Autolysine and protein hydrolysates obtained from formed elements of the blood of slaughtered animals (the degree of degradation 30 and 70%) and a mixture of crystalline amino acids composed according to the FAO/WHO scale were studied. Casein was used as control. The differences in the biological value of basic and protein hydrolysates-enriched preparations point to the necessity of correcting basic hydrolysates.