Absence Of Specific Immunity Research Articles

Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis

Replacement of the signal peptide (SP) of the bacteriocins enterocin P (EntP) and hiracin JM79 (HirJM79), produced by Enterococcus faecium P13 and Enterococcus hirae DCH5, respectively, by the signal peptide of Usp45 (SP(usp45)), the major Sec-dependent protein secreted by Lactococcus lactis, permits the production, secretion, and functional expression of EntP and HirJM79 by L. lactis. Chimeric genes encoding the SP(usp45) fused to either mature EntP (entP), with or without the immunity gene (entiP) or to mature HirJM79 (hirJM79), with or without the immunity gene (hiriJM79), were cloned into the expression vector pMG36c, carrying the P(32) constitutive promoter, and into pNZ8048 under control of the inducible PnisA promoter. The production of EntP and HirJM79 by most of the L. lactis recombinant strains was 1.5- to 3.7-fold higher and up to 3.6-fold higher than by the E. faecium P13 and E. hirae DCH5 control strains, respectively. However, the specific antimicrobial activity of the recombinant EntP was 1.1- to 6.2-fold higher than that produced by E. faecium P13, while that of the HirJM79 was a 40% to an 89% of that produced by E. hirae DCH5. Chimeras of SP(usp45) fused to mature EntP or HirJM79 drive the production and secretion of these bacteriocins in L. lactis in the absence of specific immunity and secretion proteins. The supernatants of the recombinant L. lactis NZ9000 strains, producers of EntP, showed a much higher antimicrobial activity against Listeria spp. than that of the recombinant L. lactis NZ9000 derivatives, producers of HirJM79.

TB or not TB? Fishing for Molecules Making Permissive Granulomas

Focal accumulations of mononuclear cells, called granulomas, are a hallmark of mycobacterial infections. A common misconception is that granulomas are uniformly protective. In transparent zebrafish larvae infected with Mycobacterium marinum, Volkman et al. demonstrate an interaction of mycobacteria with epithelial cells that helps recruit macrophages to the granuloma as "feeder cells."

T Cell-Mediated Control of HCMV Infection in Pediatric Patients Receiving Hematopoietic Stem Cell Transplantation.

We are studying the development of HCMV-specific CD4+ and CD8+ T cell response after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. A new technique was developed to simultaneously detect HCMV-specific CD4+ and CD8+ effector T cells using HCMV-infected autologous dendritic cells as stimulators and intracellular staining of IFN-γ production by T cells. This prospective study is based on monthly determination of both HCMV-specific T cell number and in vitro lymphoproliferative response to crude HCMV antigen. Patients are routinely monitored for HCMV infection/reactivation in blood (by determination of either antigenemia or quantitation of viral DNA) and treated according to a strategy of pre-emptive therapy. So far, of 41 patients receiving HSCT from an HLA-identical related donor (n=18), unrelated donor (n=15) or a T cell-depleted HSCT from a haploidentical relative (n=8), 25 patients have reached day +180, while 16 patients completed a follow-up of 90 days. Among the 28 HCMV-seropositive HSCT recipients, 25 developed HCMV-specific CD4+ and CD8+ T-cell response within the first 60 days after transplantation. In these patients, absolute CD4+ T cell count increased over time, but remained lower than that of healthy controls also at later time points. By contrast, CD8+ T cells reached and maintained absolute levels comparable to those of controls already from day +60. At this time, HCMV-specific CD4+ T cell count was comparable to that of controls, while HCMV-specific CD8+ T cell count was higher than that of controls, with no significant change thereafter. On the other hand, in vitro lymphoproliferative response to HCMV antigen was detectable only in about one half of these patients, even at day +180.HCMV infection was detected in blood of 22 of the 25 patients in whom HCMV-specific T cells were present. It was either self-limiting (n=14) or in 8 patients required shorter ganciclovir course (median 7 days, range 5-14) than in the 3 HCMV seropositive patients who developed HCMV infection in the absence of specific immunity (median 67 days, range 42–82, p<0.001). No patient developed HCMV disease or late viral infections. Conversely, HCMV-specific response was detected in only 3/13 HCMV seronegative recipients (none of whom developing detectable HCMV infection in blood). In these patients, both absolute and HCMV-specific T cell counts were lower than those of both controls and HCMV-seropositive HSCT recipients. Our data suggest that effective HCMV-specific T cell immunity can promptly develop after HSCT (regardless of donor type or T-cell depletion of the graft), particularly in seropositive recipients in whom latent virus may be a major antigenic drive for rapid reconstitution of T cell compartment, especially of CD8+ lymphocytes. On the other hand, transfer of memory T cell immunity from seropositive donors to seronegative recipients does not appear to be always sufficient to permit detection of virus-specific lymphocytes in patient's peripheral blood in the early period after the allograft, possibly also due to the lower chance of in vivo antigen stimulation. The frequent dissociation between IFN-γ production and lymphoproliferative response remains to be explained. Future studies could address modulation of antiviral intervention on the reconstitution of HCMV-specific T cell immune response.

Genetic control of experimental lyme arthritis in the absence of specific immunity.

Host genetics play an important role in determining resistance or susceptibility to experimental Lyme arthritis. While specific immunity appears to regulate disease resolution, innate immunity appears to regulate disease severity. Intradermal infection with Borrelia burgdorferi yields severe arthritis in C3H/He (C3H) mice but only minimal arthritis in BALB/c mice. Intradermal infection of immunodeficient C3H SCID mice also results in severe arthritis, but arthritis of only moderate severity in BALB/c SCID mice. In the present study, we examined immunodeficient recombinase-activating gene-knockout (RAG-1(-/-)) (RAG-) mice from resistant C57BL/6 (B6) and DBA/2 (DBA) mouse strains. B. burgdorferi-infected B6 RAG- and DBA RAG- mice had little or no ankle swelling, a low occurrence of inflammatory infiltrates in tibiotarsal joints, and low arthritis severity scores in comparison to RAG+ and RAG- BALB/c or C3H mice. Few differences in spirochete DNA levels in ankles of resistant and susceptible RAG- mice were seen. These data suggest that resistance to arthritis development following B. burgdorferi infection is not necessarily dependent on an acquired immune response and can occur despite the presence of high spirochete burden. Thus, genes expressed outside the specific immune response can be central regulators of experimental arthritis.

Genetic Control of Experimental Lyme Arthritis in the Absence of Specific Immunity

Genetic Control of Experimental Lyme Arthritis in the Absence of Specific Immunity

Liposomal Amikacin for Treatment of M. avium Infections in Clinically Relevant Experimental Settings

In an effort to optimize rational chemotherapy against M. avium infections in a clinically meaningful context, we tested whether liposome-encapsulated amikacin would effectively reduce the bacterial load in (i) intravenously infected immunodeficient SCID mice, (ii) immunocompetent mice in both early and late stages of intravenous infection, and (iii) immunocompetent mice with pulmonary M. avium infection. Although complete eradication of M. avium was never achieved following intravenous infection, mycobacterial CFUs decreased by 3 to 4 logs in the spleens and livers of mice treated for three weeks with twiceweekly intravenous injections of liposomal amikacin and continued to stay low in the liver, even in the absence of specific immunity. Mice treated in the chronic stage of infection equally benefited from therapy and showed signs of attenuated granulomatous inflammation in the liver. Even moribund mice responded to liposomal amikacin by significantly gaining weight and survived their infected untreated littermates by at least 4 months. In contrast, during pulmonary M. avium infection, treatment with liposome-encapsulated amikacin only resulted in a transient plateau of bacterial proliferation in the lungs, and the infection exacerbated immediately after cessation of therapy.

Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

Opsonin-independent phagocytosis of group B streptococci: role of complement receptor type three.

The role of complement receptor type 3 (CR3) in nonopsonic recognition of group B streptococci (GBS) by macrophages was investigated. Monoclonal anti-CR3 (anti-Mac-1) inhibited phagocytosis of GBS strains by as much as 50% in serum-free cultures of both mouse peritoneal macrophages and the macrophage cell line PU5-1.8. GBS uptake was unaffected by the presence of anti-C3 or salicylhydroxamate, an inhibitor of the covalent binding reaction of C3. Soluble antibodies to LFA-1 or to the common beta-chain (CD18) of the LFA-1/CR3/p150,95 family of cell adhesion molecules did not inhibit GBS uptake. Down-modulation of surface Mac-1 on macrophages following adherence to anti-Mac-1- or anti-CD18-coated surfaces also inhibited uptake of GBS. Further evidence for GBS interaction with CR3 was demonstrated by reduction of EC3bi rosette formation in macrophages adherent to GBS-coated plates. These studies suggest that GBS can interact with macrophage CR3, promoting phagocytosis in a C3-independent fashion. In the absence of specific immunity in neonates, this recognition mechanism may be a significant virulence determinant for GBS which poorly activate the alternate complement pathway.

Inhibition of Growth of Trichophyton rubrum by the Myeloperoxidase – Hydrogen Peroxide – Chloride System

The myeloperoxidase-hydrogen peroxide-chloride (MPO-H2O2-Cl) system is an antimicrobial system of polymorphonuclear leukocytes. We demonstrated that the MPO-H2O2-Cl system is fungicidal for Trichophyton rubrum. Fungal growth of a synchronous cell culture of T. rubrum germlings was assayed by measuring the uptake of tritiated N-acetyl-D-glucosamine, and the viability of the fungi was assayed by counting colony-forming units. Cytotoxins produced by the interaction of myeloperoxidase with hydrogen peroxide and chloride ion were fungicidal for T. rubrum. Growth inhibition was abolished in the presence of catalase or L-methionine. Polymorphonuclear leukocytes through the MPO-H2O2-Cl system may prevent invasion and sepsis by dermatophytes even in the absence of specific immunity.