Characterization of avirulent mutant Legionella pneumophila that survive but do not multiply within human monocytes.

Legionella pneumophila, the causative agent of Legionnaires' disease, is a Gram-negative bacterium and a facultative intracellular parasite that multiplies in human monocytes and alveolar macrophages. In this paper, mutants of L. pneumophila avirulent for human monocytes were obtained and extensively characterized. The mutants were obtained by serial passage of wild-type L. pneumophila on suboptimal artificial medium. None of 44 such mutant clones were capable of multiplying in monocytes or exerting a cytopathic effect on monocyte monolayers. Under the same conditions, wild-type L. pneumophila multiplied 2.5-4.5 logs, and destroyed the monocyte monolayers. The basis for the avirulent phenotype was an inability of the mutants to multiply intracellularly. Both mutant and wild-type bacteria bound to and were ingested by monocytes, and both entered by coiling phagocytosis. Thereafter, their intracellular destinies diverged. The wild-type formed a distinctive ribosome-lined replicative phagosome, inhibited phagosome-lysosome fusion, and multiplied intracellularly. The mutant did not form the distinctive phagosome nor inhibit phagosome-lysosome fusion. The mutant survived intracellularly but did not replicate in the phagolysosome. In all other respects studied, the mutant and wild-type bacteria were similar. They had similar ultrastructure and colony morphology; both formed colonies of compact and diffuse type. They had similar structural and secretory protein profiles and LPS profile by PAGE. Both the mutant and wild-type bacteria were completely resistant to human complement in the presence or absence of high titer anti-L. pneumophila antibody. The mutant L. pneumophila have tremendous potential for enhancing our understanding of the intracellular biology of L. pneumophila and other parasites that follow a similar pathway through the mononuclear phagocyte. Such mutants also show promise for enhancing our understanding of immunity to L. pneumophila, and they may serve as prototypes in the development of safe and effective vaccines against intracellular pathogens

Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors.

We have examined receptors mediating phagocytosis of the intracellular bacterial pathogen, Legionella pneumophila. Three mAbs against the type 3 complement receptor (CR3), which recognizes C3bi, inhibit adherence of L. pneumophila to monocytes by 64 +/- 8% to 74 +/- 11%. An mAb against the type 1 complement receptor (CR1), which recognizes C3b, inhibits adherence by 68 +/- 1%. mAbs against other monocyte surface antigens do not significantly influence adherence. Monocytes plated on substrates of L. pneumophila membranes modulate their CR1 and CR3 receptors but not Fc receptors; such monocytes bind 70% fewer C3b-coated erythrocytes and 53% fewer C3bi-coated erythrocytes than control monocytes. Adherence of L. pneumophila to monocytes in nonimmune sera is dependent on heat-labile serum opsonins; adherence is markedly reduced in heat-inactivated serum (84% reduction) or buffer alone (97% reduction) compared with fresh serum. mAbs against CR1 and CR3 receptors also inhibit L. pneumophila intracellular multiplication and protect monocyte monolayers from destruction by this bacterium. This study demonstrates that human monocyte complement receptors, CR1 and CR3, mediate phagocytosis of L. pneumophila. These receptors may play a general role in mediating phagocytosis of intracellular pathogens

Guinea pigs sublethally infected with aerosolized Legionella pneumophila develop humoral and cell-mediated immune responses and are protected against lethal aerosol challenge. A model for studying host defense against lung infections caused by intracellular pathogens.

We have employed the guinea pig model of L. pneumophila infection, which mimics Legionnaires' disease in humans both clinically and pathologically, to study humoral and cell-mediated immune responses to L. pneumophila and to examine protective immunity after aerosol exposure, the natural route of infection. Guinea pigs exposed to sublethal concentrations of L. pneumophila by aerosol developed strong humoral immune responses. By the indirect fluorescent antibody assay, exposed guinea pigs had a median serum antibody titer (expressed as the reciprocal of the highest positive dilution) of 32, whereas control guinea pigs had a median titer of less than 1. Sublethally infected (immunized) guinea pigs also developed strong cell-mediated immune responses. In response to L. pneumophila antigens, splenic lymphocytes from immunized but not control animals proliferated strongly in vitro, as measured by their capacity to incorporate [3H]thymidine. Moreover, immunized but not control guinea pigs developed strong cutaneous delayed-type hypersensitivity to intradermally injected L. pneumophila antigens. Sublethally infected (immunized) guinea pigs exhibited strong protective immunity to L. pneumophila. In two independent experiments, all 22 immunized guinea pigs survived aerosol challenge with one or three times the lethal dose of L. pneumophila whereas none of 16 sham-immunized control guinea pigs survived (p less than 0.0001 in each experiment). Immunized guinea pigs were not protected significantly from challenge with 10 times the lethal dose. Immunized but not control animals cleared the bacteria from their lungs. This study demonstrates that guinea pigs sublethally infected with L. pneumophila by the aerosol route develop strong humoral immune responses to this pathogen, develop strong cell-mediated immune responses and cutaneous delayed-type hypersensitivity to L. pneumophila antigens, are protected against subsequent lethal aerosol challenge, and are able to clear the bacteria from their lungs. The guinea pig model of L. pneumophila pulmonary infection is as an excellent one for studying general principles of host defense against pulmonary infections caused by intracellular pathogens

Interaction of the legionnaires' disease bacterium (legionella pneumophila) with human phagocytes. I. L. pneumophila resists killing by polymorphonuclearleukocytes, antibody, and complement

We have previously reported that virulent egg yolk-grown Legionella pneumophila, Philadelphia 1 strain, multiplies intracellularly in human blood monocytes. We now report on the interaction between virulent L. pneumophila and human polymorphonuclear leukocytes (PMN), antibody, and complement, in vitro, under antibiotic-free conditions. L. pneumophila in concentrations ranging from 10(3) to 10(6) colony forming units (CFU)/ml are completely resistant to the bactericidal effects of 0-50 percent fresh normal human serum, even in the presence of high concentrations of rabbit or human anti-L. pneumophila antibody. L. pneumophila bacteria fix the third component of complement (C3) to their surfaces, as measured by fluorescence microscopy using rhodamine- conjugated goat anti-human C3 IgG, only when the bacteria are incubated with both specific anti-L. pneumophila antibody and complement. Similarly, L. pneumophila adhere to PMN, as measured by fluorescence microscopy, only in the presence of both specific antibody and complement. Electron microscopy revealed that these opsonized bacteria are phagocytosed by the PMN. PMN require both antibody and complement to kill L. pneumophila; even then, PMN reduced CFU of L. pneumophila by only 0.5 log under conditions in which they reduce CFU of a serum-resistant encapsulated strain of Escherichia coli by 2.5 logs. Separation of PMN-associated and nonassociated CFU of L. pneumophila revealed that the major proportion of the surviving bacteria are PMN associated. Thus, the ineffective killing of opsonized L. pneumophila is a result of a failure of PMN to kill these bacteria after they become PMN- associated. With or without antibody, PMN do not support the growth of L. pneumophila. These findings suggest that PMN, even in conjunction with the humoral immune system, do not play a decisive role in defense against the Legionnaires' disease bacterium

Natural variation in immune responses to neonatal mycobacterium bovis bacillus calmette-guerin (BCG) vaccination in a cohort of Gambian infants

Background There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN- responses to BCG in this age group are poorly described. Characterisation of IFN- responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy. Methodology/Principal Findings 236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89-98% depending on the antigen) made IFN- responses and there was significant correlation between IFN- responses to the different mycobacterial antigens (Spearman’s coefficient ranged from 0.340 to 0.675, p=10-6-10-22). IL-13 and IL-5 responses were generally low and there were more non-responders (33-75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens Conclusions/Significance Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN- responses

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

In this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ‐chain fragment and the tailpiece from immunoglobulin μ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB‐PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N‐glycans linked to TB‐PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low‐affinity FcγRs. Immunization studies in mice indicated that TB‐PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen‐IgG γ‐chain molecules with inherent functional characteristics that are desirable in vaccines

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD

Mesoscopic organization reveals the constraints governing C. elegans nervous system

One of the biggest challenges in biology is to understand how activity at the cellular level of neurons, as a result of their mutual interactions, leads to the observed behavior of an organism responding to a variety of environmental stimuli. Investigating the intermediate or mesoscopic level of organization in the nervous system is a vital step towards understanding how the integration of micro-level dynamics results in macro-level functioning. In this paper, we have considered the somatic nervous system of the nematode Caenorhabditis elegans, for which the entire neuronal connectivity diagram is known. We focus on the organization of the system into modules, i.e., neuronal groups having relatively higher connection density compared to that of the overall network. We show that this mesoscopic feature cannot be explained exclusively in terms of considerations, such as optimizing for resource constraints (viz., total wiring cost) and communication efficiency (i.e., network path length). Comparison with other complex networks designed for efficient transport (of signals or resources) implies that neuronal networks form a distinct class. This suggests that the principal function of the network, viz., processing of sensory information resulting in appropriate motor response, may be playing a vital role in determining the connection topology. Using modular spectral analysis, we make explicit the intimate relation between function and structure in the nervous system. This is further brought out by identifying functionally critical neurons purely on the basis of patterns of intra- and inter-modular connections. Our study reveals how the design of the nervous system reflects several constraints, including its key functional role as a processor of information.Comment: Published version, Minor modifications, 16 pages, 9 figure