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ABSTRACTGallium ions have previously been shown to exhibit antibacterial and antibiofilm properties. In this study, we report differential bactericidal activities of two gallium complexes, gallium desferrioxamine B (Ga-DFOB) and gallium citrate (Ga-Cit). Modeling of gallium speciation in growth medium showed that DFOB and citrate both can prevent precipitation of Ga(OH)3, but some precipitation can occur above pH 7 with citrate. Despite this, Ga-Cit 90% inhibitory concentrations (IC90) were lower than those of Ga-DFOB for clinical isolates of Pseudomonas aeruginosaand several reference strains of other bacterial species. Treatment with Ga compounds mitigated damage inflicted on murine J774 macrophage-like cells infected with P. aeruginosaPAO1. Again, Ga-Cit showed more potent mitigation than did Ga-DFOB. Ga was also taken up more efficiently by P. aeruginosain the form of Ga-Cit than in the form of Ga-DFOB. Neither Ga-Cit nor Ga-DFOB was toxic to several human cell lines tested, and no proinflammatory activity was detected in human lung epithelial cells after exposure in vitro. Metabolomic analysis was used to delineate the effects of Ga-Cit on the bacterial cell. Exposure to Ga resulted in lower concentrations of glutamate, a key metabolite for P. aeruginosa, and of many amino acids, indicating that Ga affects various biosynthesis pathways. An altered protein expression profile in the presence of Ga-Cit suggested that some compensatory mechanisms were activated in the bacterium. Furthermore, the antibacterial effect of Ga was shown to vary depending on the carbon source, which has importance in the context of medical applications of gallium.

We investigated if a two-hit shock model, commonly referred to as generalized Shwartzman reaction (GSR), can prime for indirect acute respiratory distress syndrome (ARDS) in mice. The GSR was provoked in C57BL/6 mice by two consecutive i.p. injections of 100 μg lipopolysaccharide (LPS) at t = 0 and t = 20 h. These mice demonstrated a dramatic decrease in respiratory capacity and 80% mortality after the second injection. No such effect was observed when LPS was given as a single 200 μg dose at t = 0. Increased expression of proinflammatory cytokines in serum (interleukin-1β, interleukin-6 and interferon-γ), lung neutrophilia, and edema formation were observed in mice injected with one dose of LPS, but notably, mice exposed twice did not further increase their inflammatory response. Early treatment 1 h after the first LPS injection (t = 1 h) with either dexamethasone (10 mg/kg) or vitamin E (50 mg/kg) improved respiratory function and down-modulated the induction of proinflammatory cytokines in serum. In conclusion, mice with a generalized Shwartzman reaction exhibited features resembling some aspects of the pathophysiology in septic ARDS, i.e., neutrophilic inflammation, edema formation, impaired respiratory capacity, and mortality. Our data indicate that a systemic cytokine response and lung neutrophilia may prime for the GSR but that other mechanisms account for the rapid decline in lung function after the second challenge. We suggest that this model can be used for studies of pathogenesis and therapeutic prevention of acute respiratory failure.

ABSTRACTRecruitment of neutrophils to lung tissue and airspaces is a hallmark of inflammatory events following inhalation of endotoxins. We studied the role of different lymphocyte subsets in this inflammation, which is assumed to primarily involve the innate immune system. Inhalation of aerosolized Escherichia colilipopolysaccharide (LPS) in mice induced a dose-dependent increase in neutrophils in bronchoalveolar lavage fluid, reaching a maximum after 12 h at a low dose and after 24 h at a high dose. Profiles of gene expression in lung tissue indicated an early (2 h) and transient onset of proinflammatory cytokines and chemokines by a low dose of LPS, while a high dose caused more delayed and sustained (6 to 12 h) activation. Gamma interferon, interleukin-2 (IL-2), RANTES, and the α chain of the IL-2 receptor were not expressed at a low dose, whereas a high dose of LPS induced a strong expression of these genes, indicating a dose-dependent activation of T cells. A similar pattern was observed for IL-17, supporting a contribution of T cells to the neutrophilic inflammation only at high-dose exposure to LPS. The involvement of lymphocytes in the inflammatory response was further studied using mice with functional deficiencies in defined lymphocyte subsets. Both γδ T-cell- and B-cell-deficient mice displayed a response similar to that of the corresponding wild-type strains. Selective depletion of NK cells by in vivo administration of the pk136 antibody did not significantly affect the recruitment of neutrophils into airspaces. Thus, neither NK cells, B cells, nor γδ T cells appeared to participate in the host response, suggesting that among the lymphocyte subsets, αβ T cells are exclusively involved in endotoxin-induced airway inflammation.

Squalene is a cholesterol precursor, which stimulates the immune system nonspecifically. We demonstrate that one intradermal injection of this adjuvant lipid can induce joint-specific inflammation in arthritis-prone DA rats. Histopathological and immunohistochemical analyses revealed erosion of bone and cartilage, and that development of polyarthritis coincided with infiltration of αβ+T cells. Depletion of these cells with anti-αβ TcR monoclonal antibody (R73) resulted in complete recovery, whereas anti-CD8 and anti-γδ TcR injections were ineffective. The apparent dependence on CD4+T cells suggested a role for genes within the major histocompatibility complex (MHC), and this was concluded from comparative studies of MHC congenic rat strains, in which DA.1H rats were less susceptible than DA rats. Furthermore, LEW.1AV1 and PVG.1AV1 rats with MHC identical to DA rats were arthritis-resistant, demonstrating that non-MHC genes also determine susceptibility. Some of these genetic influences could be linked to previously described arthritis susceptibility loci in an F2 intercross between DA and LEW.1AV1 rats (ie, Cia3, Oia2and Cia5). Interestingly, some F2 hybrid rats developed chronic arthritis, a phenotype not apparent in the parental inbred strains. Our demonstration that an autoadjuvant can trigger chronic, immune-mediated joint-specific inflammation may give clues to the pathogenesis of rheumatoid arthritis, and it raises new questions concerning the role of endogenous molecules with adjuvant properties in chronic inflammatory diseases.

ABSTRACTFrancisella tularensisLVS is an effective live vaccine strain used for cutaneous vaccination against tularemia in man. In mice, injection of LVS causes invasive disease and subsequent development of immunity that is characterized by effective control of otherwise lethal doses of the organism. In the present investigation, it is shown that LVS-immune mice controlled an intradermal infection much more effectively than did naive mice; bacterial counts in skin samples were 1.5 to 2.0 log10lower 24 h after injection and 6 log10lower 72 h after injection in immune mice. Moreover, in contrast to naive mice, no bacteria were demonstrated in samples from livers and spleens of immune mice. By immunohistochemistry, skin samples from immune mice showed an intense staining for interleukin-12 (IL-12) and a moderate staining for tumor necrosis factor alpha (TNF-α) at 24 h postinoculation, after which staining for both cytokines faded. In naive mice, the staining for IL-12 was weak at all time points and no staining for TNF-α was observed. No staining for gamma interferon (IFN-γ) was observed in any group before 72 h. At that time point, skin samples from immune mice showed moderate staining and skin samples from naive mice showed weak staining. Reverse transcriptase PCR showed an induction of mRNA of the three cytokines in the skin within the first day after injection. A quantitative analysis demonstrated higher IFN-γ and TNF-α mRNA levels in immune mice at 24 h postinoculation. In conclusion, immunization with F. tularensisLVS conferred a capability to respond to cutaneous reinfection, with rapid local expression of IL-12, TNF-α, and IFN-γ, and this expression was paralleled by containment and mitigation of the infection. The cytokine response may be part of a local barrier function of the skin, important to host protection against tularemia.

The cytotoxic activity and T cell receptor (TCR) Vß repertoire in tumor-infiltrating lymphocytes (TIL) of three primary adrenal cell carcinomas were analyzed. Fresh, non-cultured TIL from two of the three tumors showed low but significant lysis of the autologous tumor, and for one of the patients this activity was strongly enhanced upon culture in interleukin-2. An allogeneic adrenal cell carcinoma line and the K562 or Daudi targets included as controls were not killed. Phenotypic analysis of freshly isolated TIL demonstrated that the cells from the two patients that demonstrated cytolytic capacity mainly consisted of CD45RO+ T cells. In vitro cultured TIL lines from these patients demonstrated a high percentage of CD8+ cells expressing either the Vß6 gene or the Vß8 gene product, as measured with a panel of mAb specific for TCR Va and Vß gene products. Analysis of the TCR Vß gene mRNA expression in freshly isolated non-cultured TIL, using a polymerase-chain-reaction-assisted cDNA-amplification assay, confirmed the strong expression of the genes coding for the TCR Vß6 or the Vß8. This assay also demonstrated a more restricted TCR Vß gene usage in the TIL as compared to peripheral blood lymphocytes from the same patient.

Three individual serine proteinases (I, II, III) originating from Antarctic krill (E. superba) were separated and highly purified using a combination of affinity and high resolution ion exchange chromatography. Each enzyme showed a single protein band (30 000 Daltons) in sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis indicating high purity and identical molecular weights. Moreover, each enzyme demonstrated one immunoprecipitate on crossed immunoelectrophoresis (two-dimensional agarose gel electrophoresis) using polyclonal rabbit antibodies which confirmed the high purity of the individual enzymes. A mixture of the three enzymes (I, II, III) revealed two immunoprecipitates, not one or three which should have been the case for identical or non-identical immunological properties. Double immunodiffusion test according to Ouchterlony exhibited immunological identity between enzyme II and III. Enzyme I showed only partial identity with II/III. These findings correlated well with biochemical data on the three serine proteinases. Enzyme I is able to liberate free amino acids from polypeptides in comparison with enzyme II and III (classical true endopeptidases), which do not. We suggest that these unique biochemical properties also have their immunological counterpart expressed as other antigenic determinants of the molecular structure.

Background: Sarcoidosis is a granulomatous disease characterized by the accumulation of activated T cells in the lungs. We previously showed that sarcoidosis patients expressing the HLA haplotype DR3(17), DQ2 had increased numbers of lung CD4+T cells using the T cell receptor (TCR) variable region (V) α2.3 gene segment product. In the present study, the composition of both the TCR α- and β-chains of the expanded CD4+lung T cells from four DR3(17), DQ2+sarcoidosis patients was examined. Materials and Methods: TCR α-chains were analyzed by cDNA cloning and nucleotide sequencing. TCR β-chains were analyzed for Vβusage by flow cytometry using TCR V-specific monoclonal antibodies or by the polymerase chain reaction (PCR) using Vβ- and Cβ-specific primers. Jβusage was analyzed by Southern blotting of PCR products and subsequent hybridization with radiolabeled Jβ-specific probes. Results: Evidence of biased Jαgene segment usage by the α-chains of Vα2.3+CD4+lung T cells was found in four out of four patients. Both different α-chain nucleotide sequences coding for identical amino acid sequences and a number of identically repeated α-chain sequences were identified. In contrast, the TCR β-chains of FACS-sorted Vα2.3+CD4+lung T cells were found, with one exception, to have a nonrestricted TCR Vβusage. Conclusions: The finding of Vα2.3+CD4+lung T cells with identical TCR α-chain amino acid sequences but with different nucleotide sequences strongly suggests that different T cell clones have been selected to interact with a specific sarcoidosis associated antigen(s). The identification of T cells with restricted TCR usage, which may play an important role in the development of sarcoidosis, and the possibility of selectively manipulating these cells should have important implications for the treatment of the disease.