Location of intra- and extracellular M. tuberculosis populations in lungs of mice and guinea pigs during disease progression and after drug treatment

The lengthy treatment regimen for tuberculosis is necessary to eradicate a small sub-population of M. tuberculosis that persists in certain host locations under drug pressure. Limited information is available on persisting bacilli and their location within the lung during disease progression and after drug treatment. Here we provide a comprehensive histopathological and microscopic evaluation to elucidate the location of bacterial populations in animal models for TB drug development.To detect bacilli in tissues, a new combination staining method was optimized using auramine O and rhodamine B for staining acid-fast bacilli, hematoxylin QS for staining tissue and DAPI for staining nuclei. Bacillary location was studied in three animal models used in-house for TB drug evaluations: C57BL/6 mice, immunocompromised GKO mice and guinea pigs. In both mouse models, the bacilli were found primarily intracellularly in inflammatory lesions at most stages of disease, except for late stage GKO mice, which showed significant necrosis and extracellular bacilli after 25 days of infection. This is also the time when hypoxia was initially visualized in GKO mice by 2-piminidazole. In guinea pigs, the majority of bacteria in lungs are extracellular organisms in necrotic lesions and only few, if any, were ever visualized in inflammatory lesions. Following drug treatment in mice a homogenous bacillary reduction across lung granulomas was observed, whereas in guinea pigs the remaining extracellular bacilli persisted in lesions with residual necrosis. In summary, differences in pathogenesis between animal models infected with M. tuberculosis result in various granulomatous lesion types, which affect the location, environment and state of bacilli. The majority of M. tuberculosis bacilli in an advanced disease state were found to be extracellular in necrotic lesions with an acellular rim of residual necrosis. Drug development should be designed to target this bacillary population and should evaluate drug regimens in the appropriate animal models.     

Molecular cloning and expression of the IL-10 gene from guinea pigs

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.     

Uptake and accumulation of oxidized low-density lipoprotein during Mycobacterium tuberculosis infection in guinea pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence.     

The cellular immune response to Mycobacterium tuberculosis infection in the guinea pig

Pulmonary tuberculosis in guinea pigs is an extremely useful model for drug and vaccine testing due to the fact that its pathological disease process is similar to that present in humans. Progress in this field has been hindered because the tools necessary to undertake a complete immunological analysis of the guinea pig cellular immune response against Mycobacterium tuberculosis have been lacking. In this study, we combined a new flow cytometric gating strategy with immunohistochemistry to track T cells, B cells, and the MIL4 Ab, which detects both guinea pig heterophils (neutrophils) and eosinophils, to provide the first documentation of the kinetics of influx and positioning of these cell populations. The results show that the responding T cells are mostly CD4 cells and that after day 30 of the infection numbers of these cells in the lungs drops dramatically. These appear to be replaced by a steady increase in B cells and granulocytes which was associated with worsening lung pathology. These data reveal new information about the cellular phenotypes which mediate protective immunity or host immunopathogenesis during M. tuberculosis infection in this key animal model.     

Effect of delta 9-tetrahydrocannabinol on herpes simplex virus type 2 vaginal infection in the guinea pig

The present investigation was undertaken to determine whether delta 9-tetrahydrocannabinol (delta 9-THC) decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. The guinea pig was selected as the host since it has been shown to express a spectrum of primary herpes genitalis which is similar to that in humans. Animals were administered delta 9-THC or vehicle intraperitoneally on Days 1-4, 8-11, and 15-18. Herpes simplex virus was introduced intravaginally on Day 2. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding, and animal mortalities. Virus-infected animals treated with drug at doses of 4 and 10 mg/kg exhibited significantly greater severity of genital disease during the 30-day period of study when compared to virus-inoculated vehicle controls. A direct relationship was noted between dose of delta 9-THC and cumulative mortalities on Day 14 following primary infection. These results indicate that delta 9-THC decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig.     

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Prophylactic effect of a therapeutic vaccine against TB based on fragments of Mycobacterium tuberculosis

The prophylactic capacity of the RUTI® vaccine, based on fragmented cells of Mycobacterium tuberculosis, has been evaluated in respect to aerosol challenge with virulent bacilli. Subcutaneous vaccination significantly reduced viable bacterial counts in both lungs and spleens of C57Bl mice, when challenged 4 weeks after vaccination. RUTI® protected the spleen less than BCG. Following a 9 month vaccination-challenge interval, protection was observed for the lungs, but not for the spleen. Survival of infected guinea pigs was prolonged by vaccination given 5 weeks before challenge. Inoculations of RUTI® shortly after infection significantly reduced the viable bacterial counts in the lungs, when compared with infected control mice. Thus, vaccination by RUTI® has potential for both the prophylaxis and immunotherapy of tuberculosis.     

CURRENT VIEWS ON GENITOURINARY TUBERCULOSIS

Tubercle bacilli are spread by the blood stream to the kidney in miliary fashion from the primary pulmonary lesion. Activation, followed by arrest, may delay development of the disease in the kidney for many years or “healing” may occur. Renal ulcerative lesions are the most frequent source of infection of other genitourinary organs.In pyelograms there is no particular characteristic of lesions of tuberculosis. Cellular elements in the urine of a patient with tuberculosis of other organs should lead to urine culture and guinea pig inoculation for mycobacterium tuberculosis.Treatment with streptomycin, isonicotinic acid and/or para-aminosalicylic acid should be started as soon as genitourinary tuberculosis is proved. Patients with advanced lesions usually receive great benefit from these medications; even though organisms may not be eliminated they are definitely diminished in activity. Excision of diseased organs or tissue may be necessary in a few cases.     

Experimental aerosol inoculation of Mycobacterium bovis in North American opossums (Didelphis virginiana)

The goal of this study was to evaluate the susceptibility of North American opossums (Didelphis virginiana) to aerosol inoculation of Mycobacterium bovis at two dose levels in order to gain information on disease pathogenesis, fecal shedding of the organism, and the potential role that opossums play in the spread of this disease in nature. Six opossums received high dose (1 x 10(7) colony forming units (cfu) by aerosol inoculation, six opossums received low dose (1 x 10(3) cfu inoculation, and six opossums were sham-inoculated with sterile water and served as controls. Lungs were the most frequently infected tissues, with nine of 12 inoculated opossums positive for M. bovis on culture. Gross lesions consisted of multifocal pneumonia and enlarged lymph nodes. Microscopically, granulomatous pneumonia and granulomatous lymphadenitis associated with acid-fast bacilli were present in eight of 12 inoculated opossums. Fecal shedding of M. bovis was uncommon at both inoculation doses. While opossums were highly susceptible to aerosol inoculation of M. bovis, they did not become emaciated or develop widely disseminated lesions. From this study, opossums may transmit tuberculosis by aerosol infection to other opossums in close contact and serve as a source of infection to carnivores that feed upon them, however, transmission of the disease to large herbivores by fecal shedding or direct contact may be less likely.     

Studies on neutral exopolysaccharides produced by the ectomycorrhiza Thelephora terrestris

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.     

Passive serum therapy with polyclonal antibodies against Mycobacterium tuberculosis protects against post-chemotherapy relapse of tuberculosis infection in SCID mice

We investigated the protective role of immune-sera against reactivation of Mycobacterium tuberculosis infection in SCID mice and found that passive immunization with sera obtained from mice treated with detoxified M. tuberculosis extracts (delivered in liposomes in a composition known as RUTI) exerted significant protection. Our SCID mouse model consisted of aerosol infection by M. tuberculosis, followed by 3 to 8weeks of chemotherapy with isoniazid+rifampicin (INH+RIF) (25 and 10mg/kg, respectively). After infection and antibiotic administration, two groups of mice were treated for up to 10weeks with intraperitoneal passive immunization using hyperimmune serum (HS) obtained from mice infected with M. tuberculosis, treated with chemotherapy (INH+RIF) for 8weeks and inoculated with RUTI (HS group) or with normal serum (CT group). Significant differences were found between HS and CT groups in the number of bacilli in the lungs (3.68+/-2.02 vs. 5.72+/-1.41log(10) c.f.u.), extent of pulmonary granulomatomous infiltration (10.33+/-0.67 vs. 31.2+/-1.77%), and percentage of animals without pulmonary abscesses (16.7% vs. 45.5%). These data strongly suggest a protective role of specific antibodies against lung dissemination of M. tuberculosis infection.     

Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.     

The guinea pig as a model of infectious diseases

The words 'guinea pig' are synonymous with scientific experimentation, but much less is known about this species than many other laboratory animals. This animal model has been used for approximately 200 y and was the first to be used in the study of infectious diseases such as tuberculosis and diphtheria. Today the guinea pig is used as a model for a number of infectious bacterial diseases, including pulmonary, sexually transmitted, ocular and aural, gastrointestinal, and other infections that threaten the lives of humans. Most studies on the immune response to these diseases, with potential therapies and vaccines, have been conducted in animal models (for example, mouse) that may have less similarity to humans because of the large number of immunologic reagents available for these other species. This review presents some of the diseases for which the guinea pig is regarded as the premier model to study infections because of its similarity to humans with regard to symptoms and immune response. Furthermore, for diseases in which guinea pigs share parallel pathogenesis of disease with humans, they are potentially the best animal model for designing treatments and vaccines. Future studies of immune regulation of these diseases, novel therapies, and preventative measures require the development of new immunologic reagents designed specifically for the guinea pig.     

Pathogenesis of serratial infection: activation of the Hageman factor-prekallikrein cascade by serratial protease

A serratial protease with an apparent molecular weight of 56,000 (56K protease), which had been purified from the culture supernatant of a strain of Serratia marcescens isolated from a corneal lesion of a human eye [Matsumoto, K. et al. (1984) J. Bacteriol. 157, 225-232], greatly enhanced vascular permeability when injected into guinea pig skin. The 56K protease, which requires zinc ion for activity, was found to possess plasma kallikrein-like properties in vitro as judged by (i) preferential amidolysis of carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide and Pro-Phe-Arg-4-methylcoumaryl-7-amide, which are known substrates for plasma kallikrein; (ii) release of kinin from high-molecular-weight kininogen; and (iii) prompt activation of Hageman factor followed by generation of kallikrein from plasma prekallikrein. These results suggest that the 56K protease enhances vascular permeability through activation of a Hageman factor-kallikrein-kinin pathway in vivo, and this molecular process appears to be a rational mechanism of enhancement of permeability and serratial pathogenesis.     

Induction of CD1-restricted immune responses in guinea pigs by immunization with mycobacterial lipid antigens

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitable animal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, we found that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In this study, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags from Mycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to total lipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cells pulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells in cytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presented M. tuberculosis glycolipid Ags to T cells raised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived from M. tuberculosis lipid Ag-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate that CD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide support for the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterial pathogens.     

Host-parasite relationships in experimental airborne tuberculosis. I. Preliminary studies in BCG-vaccinated and nonvaccinated animals

Smith, D. W. (University of Wisconsin, Madison), E. Wiegeshaus, R. Navalkar, and A. A. Grover. Host-parasite relationships in experimental airborne tuberculosis. I. Preliminary studies in BCG-vaccinated and nonvaccinated animals. J. Bacteriol. 91:718-724. 1966.-Previous studies from this laboratory on immunogenicity and allergenicity of defatted mycobacterial vaccines involved subcutaneous challenge of guinea pigs and killing of the animals 6 weeks later to evaluate the amount of disease. This type of experiment has discontinued in this laboratory in favor of an airborne challenge type of experiment, with the advantages that animals can be challenged with small numbers of bacilli by a natural route, and the number of primary lesions, the rate of spread from those lesions, and the rate of bacillary multiplication can be used to evaluate protection. Experiments to determine uniformity of infection showed that a fair degree of uniformity resulted when seven guinea pigs were exposed simultaneously, and were studied 3 weeks later to determine numbers of primary lesions and bacilli in the tissues. A less satisfactory degree of uniformity was obtained when more animals were exposed at one time. BCG-vaccinated and nonvaccinated animals were studied to determine the earliest time and the optimal time for killing the animals to detect the effects of vaccination. In guinea pigs, the degree of protection assessed by lesion counts is time-dependent, but the degree of protection assessed by viable counts of bacilli in the tissues was relatively constant 3 to 12 weeks after infection. Mice vaccinated subcutaneously with BCG were not protected against infection at any interval between 2 and 19 weeks. Guinea pigs vaccinated subcutaneously with the same lot of vaccine were protected as judged by counts of viable bacilli in the tissues 3 weeks after infection.     

Targeting proteins to the cell wall of sporulating Bacillus anthracis

Dormant spores of Bacillus anthracis germinate during host infection and their vegetative growth and dissemination precipitate anthrax disease. Upon host death, bacilli engage a developmental programme to generate infectious spores within carcasses. Hallmark of sporulation in Bacillus spp. is the formation of an asymmetric division septum between mother cell and forespore compartments. We show here that sortase C (SrtC) cleaves the LPNTA sorting signal of BasH and BasI, thereby targeting both polypeptides to the cell wall of sporulating bacilli. Sortase substrates are initially produced in different cell compartments and at different developmental stages but penultimately decorate the envelope of the maturing spore. srtC mutants appear to display no defect during the initial stages of infection and precipitate lethal anthrax disease in guinea pigs at a similar rate as wild-type B. anthracis strain Ames. Unlike wild-type bacilli, srtC mutants do not readily form spores in guinea pig tissue or sheep blood unless their vegetative forms are exposed to air.     

Prokaryotic Expression and In Vitro Functional Analysis of IL-1β and MCP-1 from Guinea Pig

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.     

Dose-Response Model for Lassa Virus

This article develops dose-response models for Lassa fever virus using data sets found in the open literature. Dose-response data were drawn from two studies in which guinea pigs were given subcutaneous and aerosol exposure to Lassa virus. In one study, six groups of inbred guinea pigs were inoculated subcutaneously with doses of Lassa virus and five groups of out-bred guinea pigs were similarly treated. We found that the out-bred subcutaneously exposed guinea pig did not exhibit a dose-dependent trend in response. The inbred guinea pigs data were best fit by an exponential dose-response model. In a second study, four groups of out-bred guinea pigs were exposed to doses of Lassa virus via the aerosol route. In that study, aerosol diameter was less than 4.5 μ m and both mortality and morbidity were used as endpoints. The log-probit dose-response model provided a somewhat better fit than the Beta-Poisson model for data with mortality as the endpoint, but the Beta-Poisson is considered the best fit model because it can be derived using biological considerations. Morbidity data were best fit with an exponential dose-response model.