Amminikutty Jeevan

Alteration of the immune response to Mycobacterium bovis BCG in mice exposed chronically to low doses of UV radiation

BALB/c mice were exposed on shaved dorsal skin to 1 minimal erythemal dose (MED) of UVB radiation (2.25 kJ/m2) from a bank of six FS-40 sunlamps three times per week. The total number of irradiations ranged from 1 to 27. At regular intervals, groups of mice were injected in the left hind foot pad with 1 x 10(6) live mycobacteria (Mycobacterium bovis BCG) 3 days after the last UVB exposure. The mice were tested 21 and 42 days after infection for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli by injecting PPD into the right hind foot pad and measuring the foot pad swelling 24 hr later. The course of infection was followed by assessing the number of bacterial colony forming units in the lymph node draining the site of BCG infection and the spleen. Mice exposed from 1 to 15 times to 1 MED of UV radiation showed a significant suppression in their DTH response to PPD compared with the unirradiated mice. At the same time, the number of bacterial colony-forming units in the lymph node and spleen of the UV-irradiated mice was greater than in control mice. With continued exposure to UVB, however, the DTH response recovered to a normal level, and there was no longer an increase in the number of viable bacteria in the lymphoid organs. These results indicate that early in the course of chronic UV irradiation, mice were impaired in their ability to mount a DTH response to BCG and to clear these bacteria from their lymphoid organs; later the mice recovered from these effects of UV, with continued treatment. A dose-response study using single doses of UV radiation indicated that a dose of 2.7 kJ/m2 suppressed the DTH response by 50%. Thus, exposure of mice to a single or multiple low doses of UV radiation prior to infection can interfere with systemic immunity to mycobacteria.

Production and Characterization of Guinea Pig Recombinant Gamma Interferon and Its Effect on Macrophage Activation

ABSTRACT Gamma interferon (IFN-γ) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-γ (gpIFN-γ) and reported that BCG vaccination induced a significant increase in the IFN-γ mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-γ mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-γ with a histidine tag at the N terminus (His-tagged rgpIFN-γ) in Escherichia coli. rgpIFN-γ was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-γ. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-γ was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-γ did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-γ induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-γ has been successfully produced and characterized in our laboratory. The study of rgpIFN-γ will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

Differential Expression of Gamma Interferon mRNA Induced by Attenuated and Virulent Mycobacterium tuberculosis in Guinea Pig Cells after Mycobacterium bovis BCG Vaccination

ABSTRACT To determine whether Mycobacterium bovis BCG vaccination would alter gamma interferon (IFN-γ) mRNA expression in guinea pig cells exposed to Mycobacterium tuberculosis, we cloned a cDNA encoding guinea pig IFN-γ from a spleen cell cDNA library. The cDNA is composed of 1,110 bp, with an open reading frame encoding a 166-amino-acid protein which shows 56 and 41% amino acid sequence homology to human and mouse IFN-γ, respectively. Spleen or lymph node cells from naïve and BCG-vaccinated guinea pigs were stimulated with purified protein derivative (PPD) or M. tuberculosis H37Ra or H37Rv, and the total RNA was subjected to Northern blot analysis with a 32P-labeled probe derived from the cDNA clone. Compared to the IFN-γ mRNA expression in cells of naïve animals, that in spleen and lymph node cells exposed to various stimuli was enhanced after BCG vaccination. However, there was a significant reduction in IFN-γ mRNA levels when cells were stimulated with a multiplicity of infection of greater than 1 virulent M. tuberculosis bacterium per 10 cells. The enhanced IFN-γ mRNA response in BCG-vaccinated animals was associated with an increase in the proportions of CD4+ T cells in the spleens, as determined by fluorescence-activated cell sorter analysis. Furthermore, the nonadherent population in the spleens enriched either by panning with anti-guinea pig immunoglobulin G-coated plates or by purification on nylon wool columns produced more IFN-γ mRNA than whole spleen cells following stimulation with concanavalin A or PPD. This indicates that T cells are principally responsible for the upregulation of IFN-γ mRNA expression following BCG vaccination. The mechanism by which virulent mycobacteria suppress IFN-γ mRNA accumulation is currently under investigation.

Effect of Mycobacterium bovis BCG Vaccination on Interleukin-1β and RANTES mRNA Expression in Guinea Pig Cells Exposed to Attenuated and Virulent Mycobacteria

ABSTRACT The effect of Mycobacterium bovis BCG vaccination on interleukin-1β (IL-1β) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from naïve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1β or RANTES cDNA. Although IL-1β and RANTES mRNA could be detected in the spleen cells from naïve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1β expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1β mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1β and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.

Ultraviolet radiation reduces phagocytosis and intracellular killing of mycobacteria and inhibits nitric oxide production by macrophages in mice.

Exposure of mice to a single or multiple low doses of ultraviolet radiation (UVR) decreases the induction of the delayed‐type hypersensitivity (DTH) response to Mycobacterium bovis BCG and Mycobacterium lepraemurium (MLM) and impairs the clearance of bacteria from the lymphoid organs. This study is an attempt to address the mechanism by which UV radiation impairs the clearance of bacteria from the lymphoid organs by determining whether alterations in macrophage function such as ingestion and intracellular killing of mycobacteria or production of reactive nitrogen intermediates might be responsible for these effects. BALB/c or C3H/HeN mice were exposed to a single dose of UVB (280–320 nm) radiation ranging from 0.35 to 45 kJ/m2, and at regular intervals after irradiation, the peritoneal and splenic macrophages were collected, cultured, and infected with live BCG or MLM. Phagocytosis was assessed at 6 h by counting the number of acid‐fast bacteria per macrophage after Ziehl‐Neelsen staining. The rate of intracellular killing was assessed by lysing the macrophages at 6, 12, 24, and 48 h after BCG infection, plating the suspension on 7H11 agar, and counting the number of colony‐forming units 21 days later. Similarly, the nitric oxide production, as measured by nitrite, by macrophages obtained from UVB‐irradiated and nonirradiated mice in response to BCG was assessed. There was a significant reduction in the uptake of organisms by both peritoneal and splenic macrophages collected from UV‐irradiated mice. The intracellular killing of organisms was also significantly reduced, as was the production of nitric oxide by peritoneal macrophages infected with BCG in vitro. These results indicate that UVR affects macrophage functions and are consistent with our hypothesis that impaired clearance of bacteria in vivo results from an alteration in macrophage function. J. Leukoc. Biol. 57: 883–890; 1995.

Mechanism of UVB‐Induced Suppression of the Immune Response to Mycobacterium bovis Bacillus Calmette‐Guerin: Role of Cytokines on Macrophage Function

Previously we demonstrated that treatment of mice with either UVB radiation or supernatants derived from UVB‐irradiated PAM 212 keratinocytes decreased the induction of the delayed‐type hypersensitivity (DTH) response to Mycobacterium bovis bacillus Calmette‐Guerin (BCG), impaired the clearance of bacteria from their lymphoid organs and also altered macrophage functions. In order to characterize the cytokines involved in these phenomena, UV‐irradiated mice were injected with antibodies to interleukin‐10 (IL‐10), transforming growth factor‐β1 (TGF‐β1), or tumor necrosis factor‐α (TNF‐α). Injection of UVB‐irradiated mice with anti‐IL‐10 immediately after UV irradiation restored the DTH response and reversed the UV‐induced inhibition of bacterial clearance. Injection of UV‐irradiated mice with anti‐TGF‐β only partially restored the DTH response although it allowed a better clearance of BCG than injection of mice with the control antibody. In contrast, injection of anti‐TNF‐α did not affect the UVB‐induced suppression of DTH or impaired bacterial clearance. Similarly, the ability of macrophages to phagocytose BCG and kill the intracellular organisms was restored to almost normal levels after injecting UV‐irradiated mice with antibodies specific for IL‐10 or TGF‐β. Injection of mice with either recombinant IL‐10 or TGF‐β mimicked the effect of whole‐body UV irradiation on immune function. These results suggest that IL‐10 has a major role in UV‐induced suppression of both DTH to BCG and impairment in the clearance of bacteria and that TGF‐β has a more significant role in blocking bacterial clearance. Furthermore, these cytokines seem to modulate immune responses by altering macrophage functions in UVB‐irradiated mice.

Effects of ultraviolet radiation on the pathogenesis of Mycobacterium lepraemurium infection in mice

Abstract The purpose of this study was to determine whether exposing mice to ultraviolet radiation (UVR) would alter the pathogenesis of infection with Mycobacterium lepraemurium (MLM), which causes a chronic, progressive, lethal disease in susceptible mouse strains. BALB/c mice were irradiated on dorsal skin with various doses of UVR from FS40 sunlamps 3 days before infection with MLM in the hind footpad. The course of disease was followed by assessing the number of acid‐fast bacteria in the footpad, regional lymph node and spleen, and measuring the size of the lesion at the site of MLM infection at various times after infection. Mice were also tested periodically for a delayed‐type hypersensitivity (DTH) response by injecting MLM antigen into the uninfecled footpad and measuring footpad swelling 24 hours later. Mice treated with a single high dose of UVR (45 kJ/m2) had significantly more bacteria in the infected footpad, lymph node and spleen than unirradiated control animals. They also had larger lesions at the site of MLM infection and exhibited significant suppression of the DTH response at 3 and 6 months after infection. Injection of mice s.c. in the footpad with MLM 3 d after 45 kJ/m2 UVR reduced the median survival time from 391 to 305 d and after i.v. infection from 171 to 139 d. Dose‐response studies indicated that exposing mice to 2.3 kJ/m2 of UVR, which is approximately 1 minimal erythemal dose for this strain, suppressed the DTH response by 50% at 3 months alter infection. Significant increases in the number of bacteria in the footpad, spleen and lymph node were detected with doses of UVR≥5.6 kJ/m2. Mice exposed chronically to UV radiation also showed impaired responses to MLM. Thus, exposing mice to a single or multiple doses of UVR before infection increased the severity of a chronic mycobacterial infection and decreased the immune response to mycobacterial antigens.

A New Assay System for Guinea Pig Interferon Biological Activity

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Transporter associated with antigen-processing-1 (TAP1) alleles in gorilla gorilla: diversification of the locus postspeciation

The transporter associated with antigen-processing (TAP) proteins are required for the transport of cytosolic peptides into the endoplasmic reticulum for assembly with class I major histocompatibility molecules. In the rat, allelic variants of the TAP genes impart specificity to the process of peptide transport. However, differential transport has yet to be demonstrated with the human molecules. TAP genes from humans and rodents have been studied thus far; analysis of another species more closely related to humans is necessary for a clearer understanding of the evolution of TAP genes. Three TAP1 alleles from four gorilla cell lines were characterized in this study. There is limited genetic distance at the locus, either within the gorilla (0.2%) or between the two hominoid species (0.8%). Nucleotide substitution analysis demonstrates that TAP1 and TAP2 are evolving at comparable rates under similar selection pressures. This pattern is in marked contrast to that observed for MHC class I genes of hominoids or rodents. Although there is limited evidence for trans-species evolution of the gorilla locus, the bulk of the diversification occurred after speciation as evidenced by a lack of shared amino acid polymorphism with human homologues. An evolutionary scheme predicts that the ancestral hominoid TAP1 molecule most closely resembled the human TAP1*02011.

Molecular and Biochemical Characterization of Recombinant Guinea Pig Tumor Necrosis Factor-Alpha

Tumor necrosis factor alpha (TNF-α) is a cytokine which plays opposing roles in the context of infectious disease pathogenesis. TNF-α is essential for the development of a protective immune response to some pathogens, for example, Mycobacterium tuberculosis, by synergizing with other cytokines. However, exorbitant or uncontrolled TNF-α activity may also drive pathology and disease symptoms in many infectious diseases. In order to elucidate the beneficial and detrimental roles of TNF-α in tuberculosis (TB) and other diseases for which the guinea pig is the small animal model of choice, recombinant guinea pig (rgp)TNF-α has been produced using prokaryotic expression systems. However, it is unknown whether posttranslational modifications which cannot be made in the prokaryotic expression systems may be important for rgpTNF-α structure and function. Therefore, we carried out a comparative study by expressing rgpTNF-α in prokaryotic and eukaryotic expression systems and analyzed the eukaryotic-expressed rgpTNF-α for the presence of posttranslational modifications by subjecting it to NanoLC-MS/MS. We conclude that the eukaryotic-expressed rgpTNF-α lacks posttranslational modifications, and we found no significant difference in terms of the biological activity between prokaryotic- and eukaryotic-expressed rgpTNF-α. Taken together, results from our study show that a prokaryotic expression system can be used for generating large amounts of rgpTNF-α without concern for the biological integrity.