Collagen gene expression in human cancer

Type I collagen is the predominant collagen within the stroma and plays an important role in the processes of tumour cell invasion and metastasis during which the collagens within the stroma is degraded. Total RNA was extracted from different stages of breast cancer and adjacent normal tissue for analysis of collagen gene expression by Northern blot hybridisation. Stage I breast tumours had increased α1(I) and α2(I) collagen mRNA, whereas stages II and III tumours had decreased mRNA levels when compared to the adjacent normal tissue. This stage-specific change in collagen gene expression was confirmed by non-radioactive in situ hybridisation and the results indicated that α1(I) and α2(I) collagen mRNA was produced by the stromal fibroblasts and not the tumour cells. To determine whether this altered collagen gene expression was manifested in other cancers, α1(I) and α2(I) collagen mRNA levels were analysed in colorectal carcinoma samples by in situ hybridisation. Colon cancer as in the case of breast cancer, also showed stage specific changes in collagen gene expression. Dukes C and D colon cancer samples had decreased collagen mRNA levels compared to Dukes A and B. Mutated Ras has been shown to affect collagen mRNA levels in vitro (Slack et al, 1992), therefore the colon samples were analysed for Ras mutations in an attempt to correlate Ras mutations with the decreased levels of α1(I) and α2(I) collagen mRNA. Colorectal DNA samples were screened for Ras mutations by SSCP and direct sequence analysis. No possible association was found between the presence of Ras mutations and the decreased collagen gene expression. To gain greater insight into exactly how tumour cells modulate the collagen produced by normal fibroblasts, primary breast fibroblasts (prepared from breast tissue) were cocultured with various breast tumour cell lines. The fibroblasts were also incubated with conditioned media prepared from the tumour cells. Collagen production was analysed using the collagenase assay and the results showed that co-cultured tumour cells, as well as growth in the presence of tumour cell conditioned media, resulted in decreased type I collagen production by the fibroblasts. Type III collagen is often produced in conjunction with type I collagen and we have found that the breast tumour cells modulated type III collagen in the same way as type I collagen. These results demonstrated that a factor(s) was secreted by the tumour cells which affected collagen production. This factor was further shown to stimulate the fibroblasts to produce type I collagenase as analysis of the medium from co-cultured fibroblasts and tumour cells indicated the presence of collagenases. The tumour cell conditioned media was subsequently shown by Western blot analysis to contain a protein of similar molecular weight to the tumour cell derived collagenase stimulatory factor (known as EMMPRIN or extracellular matrix metalloproteinase inducer) which stimulates fibroblasts to secrete collagenases and has been shown to play a crucial role in tumour invasion (Biswas 1982, 1984 and Biswas et al, 1995). In order to determine whether fibroblasts of different origins reacted similarity when cocultured with breast tumour cell lines, WI-38 lung fibroblasts and FGo skin fibroblasts were co-cultured with breast tumour cells. WI-38 fibroblasts responded in the same way as breast fibroblasts (having decreased collagen production), FGo fibroblasts had no effect or slightly elevated collagen production, depending on the tumour cell line. These results suggested that the response to tumour cells is tissue specific. The decrease in type I collagen produced by the fibroblasts when incubated with the tumour cell conditioned media was not due to a decrease in α1(I) and α2(I) collagen mRNA as shown by Northern hybridisation. Type III collagen mRNA was affected differently, the levels were either decreased or increased depending on the tumour cell line being used. We postulate that the fibroblasts and tumour cells required contact for type I collagen mRNA to be decreased. Northern hybridisation showed that types I and III collagen mRNA levels were decreased when tumour cells were co-cultured with the fibroblasts. To demonstrate that specific contact was in fact required, the tumour cells were separated from the fibroblasts by a diffusible membrane and the levels of collagen mRNA were not adversely affected. Tumour cells, therefore can modulate collagen production by normal fibroblasts in two ways I); cause the fibroblasts to secrete collagenases which will degrade the collagen and 2); decrease collagen mRNA. Both of these mechanisms would aid the tumour in invasion and metastasis

Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum

As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis

Reprogramming the Mycobacterium tuberculosis transcriptome during pathogenesis

Transcriptional profiling has revealed that Mycobacterium tuberculosis adapts both its metabolic and respiratory states during infection, utilising lipids as a carbon source and switching to alternative electron acceptors. These global gene expression datasets may be exploited to identify virulence determinants and to screen for new targets for rational drug design. Characterising the changing physiological predicament of distinct M. tb populations during infection will help expose the fundamental biology of M. tb highlighting mechanisms that influence tuberculosis pathogenicity

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0·0001), granuloma (7/25, P = 0·0102), structural disorganization (14/25, P < 0·0001), and atrophy of the lymphoid follicles (20/25, P = 0·0036) and of the marginal zone (15/25, P = 0·0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis

Pleural Tuberculosis in Patients with Early HIV Infection Is Associated with Increased TNF-Alpha Expression and Necrosis in Granulomas

Although granulomas may be an essential host response against persistent antigens, they are also associated with immunopathology. We investigated whether HIV co-infection affects histopathological appearance and cytokine profiles of pleural granulomas in patients with active pleural tuberculosis (TB). Granulomas were investigated in pleural biopsies from HIV positive and negative TB pleuritis patients. Granulomas were characterised as necrotic or non-necrotic, graded histologically and investigated for the mRNA expression of IL-12, IFN-γ, TNF-α and IL-4 by in situ hybridisation. In all TB patients a mixed Th1/Th2 profile was noted. Necrotic granulomas were more evident in HIV positive patients with a clear association between TNF-α and necrosis. This study demonstrates immune dysregulation which may include TNF-α-mediated immunopathology at the site of disease in HIV infected pleural TB patients

Increased production of IL-4 and IL-12p40 from bronchoalveolar lavage cells are biomarkers of Mycobacterium tuberculosis in the sputum

BACKGROUND: Tuberculosis (TB) causes 1.45 million deaths annually world wide, the majority of which occur in the developing world. Active TB disease represents immune failure to control latent infection from airborne spread. Acid-fast bacillus (AFB) seen on sputum smear is a biomarker for contagiousness. METHODS: We enrolled 73 tuberculosis patients with extensive infiltrates into a research study using bronchoalveolar lavage (BAL) to sample lung immune cells and assay BAL cell cytokine production. All patients had sputum culture demonstrating Mycobacterium tuberculosis and 59/73 (81%) had AFB identified by microscopy of the sputum. Compared with smear negative patients, smear positive patients at presentation had a higher proportion with smoking history, a higher proportion with temperature >38.5 0 C, higher BAL cells/ml, lower percent lymphocytes in BAL, higher IL-4 and IL-12p40 in BAL cell supernatants. There was no correlation between AFB smear and other BAL or serum cytokines. Increasing IL-4 was associated with BAL PMN and negatively associated with BAL lymphocytes. Each 10-fold increase in BAL IL-4 and IL-12p40 increased the odds of AFB smear positivity by 7.4 and 2.2-fold, respectively, in a multi-variable logistic model. CONCLUSION: Increasing IL-4 and IL-12p40 production by BAL cells are biomarkers for AFB in sputum of patients who present with radiographically advanced TB. They likely reflect less effective immune control of pathways for controlling TB, leading to patients with increased infectiousness

Nitrate Respiration Protects Hypoxic Mycobacterium tuberculosis Against Acid- and Reactive Nitrogen Species Stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection

Differential cell reaction upon Toll-like receptor 4 and 9 activation in human alveolar and lung interstitial macrophages

BACKGROUND: Investigations on pulmonary macrophages (M&#934;) mostly focus on alveolar M&#934; (AM) as a well-defined cell population. Characteristics of M&#934; in the interstitium, referred to as lung interstitial M&#934; (IM), are rather ill-defined. In this study we therefore aimed to elucidate differences between AM and IM obtained from human lung tissue. METHODS: Human AM and IM were isolated from human non-tumor lung tissue from patients undergoing lung resection. Cell morphology was visualized using either light, electron or confocal microscopy. Phagocytic activity was analyzed by flow cytometry as well as confocal microscopy. Surface marker expression was measured by flow cytometry. Toll-like receptor (TLR) expression patterns as well as cytokine expression upon TLR4 or TLR9 stimulation were assessed by real time RT-PCR and cytokine protein production was measured using a fluorescent bead-based immunoassay. RESULTS: IM were found to be smaller and morphologically more heterogeneous than AM, whereas phagocytic activity was similar in both cell types. HLA-DR expression was markedly higher in IM compared to AM. Although analysis of TLR expression profiles revealed no differences between the two cell populations, AM and IM clearly varied in cell reaction upon activation. Both M&#934; populations were markedly activated by LPS as well as DNA isolated from attenuated mycobacterial strains (M. bovis H37Ra and BCG). Whereas AM expressed higher amounts of inflammatory cytokines upon activation, IM were more efficient in producing immunoregulatory cytokines, such as IL10, IL1ra, and IL6.CONCLUSION: AM appear to be more effective as a non-specific first line of defence against inhaled pathogens, whereas IM show a more pronounced regulatory function. These dissimilarities should be taken into consideration in future studies on the role of human lung M&#934; in the inflammatory response

The Enduring Hypoxic Response of Mycobacterium tuberculosis

deletion mutant. mutant. for long-term bacteriostasis in the face of oxygen deprivation