Prevalence of active infection by herpes simplex virus type 2 in patients with high-risk human papillomavirus infection: A cross-sectional study.

The aim is to determine the prevalence of active infection by herpes simplex virus type 2 (HSV-2) among Mexican women with high-risk human papillomavirus (HR-HPV) cervical infection, recruited from public gynecology and colposcopy services. In a cross-sectional study, HSV-2 antibodies, HSV-2 DNA, and HR-HPV DNA were quantified. Significant differences in HSV-2 seroprevalence and HSV-2 active infection rates were found between negative and positive HR-HPV cases. HSV-2 seroprevalence was 28.15% and 16.1% (P = .0001), while HSV-2 active infection rates were 6.83% and 0.62% (P = .001) for positive and negative HR-HPV groups, respectively. The risk of HSV-2 seropositivity was 1.7 times greater for HR-HPV-positive cases (P = .02). Similarly, HR-HPV-positive cases were nine times more likely to have an HSV-2 active infection than HR-HPV-negative cases (P = .03). High HSV-2/h-HPV coinfection rates were observed among women recruited from public gynecology and colposcopy services. The main factors related to an HSV-2 active infection are a history of risky sexual behavior and HR-HPV infection. The prevalence of HSV-2 active infection among positive HR-HPV subjects indicate that these infections constitute an important group of STIs in Mexico.

Molecular mechanisms of cell death induced in glioblastoma by experimental and antineoplastic drugs: New and old drugs induce apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the development of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concentration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autophagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9, increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3, and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.

High risk HPV infection prevalence and associated cofactors: a population-based study in female ISSSTE beneficiaries attending the HPV screening and early detection of cervical cancer program.

BACKGROUND: Cervical cancer is the second cause leading of malignancy-related death among Mexican women. The present study determined the population-based prevalence of high risk Human Papillomavirus (HR-HPV) infection and associated cofactors in female beneficiaries of the Institute of Security and Social Services for State Workers (ISSSTE) attending the Program for HPV Screening and Early Detection of Cervical Cancer and registered in the Women's Cancer Detection System (SIDECAM). METHODS: In a cross-sectional study, cervical samples from 115,651 female users of the program for HPV screening and early detection of cervical cancer recruited in 23 ISSSTE care centers were analyzed for HR-HPV. Logistic regression analyses, adjusting for potential confounders, were performed to determine the association of HR-HPV infection with sexual health and behavior variables and with positivity to cervical premalignant lesions by cytology. RESULTS: The overall prevalence of HR-HPV infection among female ISSSTE beneficiaries in the 2013-2015 period was 13%. A bivariate analysis of relevant variables for HR-HPV infection showed a statistically significant association for age, number of sexual partners, use of hormonal contraceptives and smoking. A statistical association was found between infection by HR-HPV with the use of hormonal contraceptives, number of sexual partners and smoking and association of HPV 16 and other non-16/18 HR-HPV infection with number of lifetime sexual partners and tobacco use adjusted for age, history of hormonal contraception, number of sexual partners and tobacco use with the exception of exposition variable itself. Similarly, an association was found between HR-HPV infection, regardless of the virus genotype, with positivity to cervical premalignant lesions adjusted for age, number of lifetime sexual partners, history of hormonal contraception and tobacco use. CONCLUSIONS: HR-HPV prevalence in female ISSSTE Women's Cancer Program users is similar to the population-based prevalence previously reported in Mexican women without cervical alterations. The ISSSTE robust screening and early detection program, based on cytology studies and HPV co-testing, allows us to know the prevalence of HR-HPV infection among female users of the service.

A prospective cohort study to evaluate immunosuppressive cytokines as predictors of viral persistence and progression to pre-malignant lesion in the cervix in women infected with HR-HPV: study protocol.

BACKGROUND: Cervical cancer (CC) is caused by a persistent infection of high-risk human papillomavirus (HR-HPV). While most HPV infections are transient, persistent HPV infections are a significant health problem in Mexico. With an estimated HPV prevalence of 10% among women in reproductive age, approximately 25% of these women present at least a positive result in triage test, which according to previous studies is expected to be confirmed as positive CIN-2/3. The immune system has a key role in the natural history of HPV infection; alterations in the cellular immune response are responsible for the failure to eliminate HPV. The objective of this project is to assess the prognostic value of detecting immune markers (IL-10, IL-4, TGFß1, IFNγ, IL-6, and TNFα), the expression of HPV-HR E6/E7 proteins, and the viral load at the cervical level with respect to the persistence or clearance of HR-HPV infection, and the regression or progression of a cervical premalignant lesion. METHODS: A dynamic cohort study is being conducted in women with colposcopic, cytological, and histopathological results negative for squamous intraepithelial lesion (SIL) in the cervix and a positive HPV test; the subjects will be followed-up for 5 years, period from which 3 years have already elapsed, with yearly studies (colposcopy, cytology, and histopathology diagnosis, along with molecular HPV test, quantification of viral load and of IL-10, IL-4, TGFß1, INFγ, IL-6, and TNFα levels, along with the expression of the HR-HPV E6/E7 proteins in the cervix as a viral marker. The outcome will be categorized as viral persistence or clearance; and as SIL persistence, progression, or regression. Binomial and/or multinomial regression models adjusted for potential confounders will be used, associating the relative risk of the outcome with the immune and viral markers evaluated. DISCUSSION: This research will generate knowledge about immune markers with predictive value for the persistence and clearance of HPV, which will improve the triage of positive HPV women and thus reduce the economic burden for the Mexican health system imposed by the management of high-grade SIL and CC cases, which are still detected in late stages.

Risk allelic load in Th2 and Th3 cytokines genes as biomarker of susceptibility to HPV-16 positive cervical cancer: a case control study.

BACKGROUND: Alterations in the host cellular immune response allow persistent infections with High-Risk Human Papillomavirus (HR-HPV) and development of premalignant cervical lesions and cervical cancer (CC). Variations of immunosuppressive cytokine levels in cervix are associated with the natural history of CC. To assess the potential role of genetic host immunity and cytokines serum levels in the risk of developing CC, we conducted a case-control study paired by age. METHODS: Peripheral blood samples from patients with CC (n = 200) and hospital controls (n = 200), were used to evaluate nine biallelic SNPs of six cytokine genes of the adaptive immune system by allelic discrimination and cytokines serum levels by ELISA. RESULTS: After analyzing the SNP association by multivariate logistic regression adjusted by age, CC history and smoking history, three Th2 cytokines (IL-4, IL-6 and IL-10) and one Th3 (TGFB1) cytokine were significantly associated with CC. Individuals with at least one copy of the following risk alleles: T of SNP (-590C > T IL-4), C of SNP (-573G > C IL-6), A of SNP (-592C > A IL-10), T of SNP (-819C > T IL-10) and T of SNP (-509C > T TGFB1), had an adjusted odds ratio (OR) of 2.08 (95 % CI 1.475-2.934, p = 0.0001), an OR of 1.70 (95 % CI 1.208-2.404, p = 0.002), an OR of 1.87 (95 % CI 1.332-2.630, p = 0.0001), an OR of 1.67 (95 % CI 1.192-2.353, p = 0.003) and an OR of 1.91 (95 % CI 1.354-2.701, p = 0.0001), respectively, for CC. The burden of carrying two or more of these risk alleles was found to have an additive effect on the risk of CC (p trend = 0.0001). Finally, the serum levels of Th2 and Th3 cytokines were higher in CC cases than the controls; whereas IFNG levels, a Th1 cytokine, were higher in controls than CC cases. CONCLUSION: The significant associations of five SNPs with CC indicate that these polymorphisms are potential candidates for predicting the risk of development of CC, representing a risk allelic load for CC and can be used as a biomarker of susceptibility to this disease.

Cervical cancer-associated promoter polymorphism affects akna expression levels.

Cervical cancer (CC) is responsible for >260,000 deaths worldwide each year. Efforts are being focused on identifying genetic susceptibility factors, especially in genes related to the immune response. Akna has been proposed to be one of them, but data regarding its functional role in the disease is scarce. Supporting the notion of akna as a CC susceptibility gene, we found two polymorphisms associated with squamous intraepithelial lesion (SIL) and CC; moreover, we identified an association between high akna expression levels and CC and SIL, but its direction differs in each disease stage. To show the potential existence of a cis-acting polymorphism, we assessed akna allelic expression imbalance for the alleles of the -1372C>A polymorphism. We found that, regardless of the study group, the number of transcripts derived from the A allele was significantly higher than those from the C allele. Our results support the hypothesis that akna is a CC susceptibility genetic factor and suggest that akna transcriptional regulation has a role in the disease. We anticipate our study to be a starting point for in vitro evaluation of akna transcriptional regulation and for the identification of transcription factors and cis-elements regulating AKNA function that are involved in carcinogenesis.

Inhibitory activity of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) on transformed cells by human papillomavirus.

In this study, we investigated the effects of the aqueous extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, obtained from three localities (China; and Morelos and Michoacan, Mexico) on cervical cells transformed by human papillomavirus (HeLa and SiHa) and C-33A cancer cells. The cells were plated in DMEM medium supplemented, and were incubated in the presence of different concentrations of G. lucidum for 24 h. Cell proliferation was determined by MTT colorimetric assay and viability by trypan blue assay. Inhibitory dose was determined (IC50) of the three different extracts of G. lucidum in the culture cell lines mentioned above. The apoptosis process was confirmed by nuclear DNA fragmentation and the cell cycle was determined by flow cytometry. The results showed that aqueous extracts G. lucidum obtained from three localities produced inhibition in the proliferation of VPH transformed cells; they also induced apoptosis and cell cycle arrest in HeLa, SiHa, and C-33A cancer cells. Therefore, it was found that aqueous extracts G. lucidum obtained from three different locations produced inhibitory effect on cancer cells and may have a potential therapeutic use for the prevention and treatment of this disease.

IRS1, TCF7L2, ADRB1, PPARG, and HHEX polymorphisms associated with atherogenic risk in Mexican population.

Objective. We aimed to explore the association between polymorphisms of IRS1 (rs1801278), TCF7L2 (rs7903146 and rs12255372), ADRB1 (rs1801253), PPARG (rs1801282), and HHEX (rs5015480) genes with atherogenic risk (AI = Total cholesterol/HDL) in MetS, T2D, and healthy populations from the Mexican Social Security Institute. Methodology and Results. Four hundred thirty-five MetS, 517 T2D, and 547 healthy individuals were selected. The association between the SNPs and the atherogenic index was evaluated by multiple linear regression and multinomial logistic regression models. The ADRB1 gene showed a statistically significant association with high-risk atherogenic index, OR = 2.94 (IC 95% 1.64-5.24; P < 0.0001) for the Arg/Gly variant, under the dominant model an OR = 2.96 (IC 95% 1.67-5.25; P < 0.0001), and under the Log additive model an OR = 2.52 (IC 95% 1.54-4.15; P < 0.0001). Conclusions. The Arg389Gly polymorphism of the ADRB1 gene may be a worthy biological marker to predict the risk of developing cardiovascular diseases given a high-risk atherogenic index.

Molecular Mechanism and Potential Targets for Blocking HPV-Induced Lesion Development.

Persistent infection with high-risk HPV is the etiologic agent associated with the development of cervical cancer (CC) development. However, environmental, social, epidemiological, genetic, and host factors may have a joint influence on the risk of disease progression. Cervical lesions caused by HPV infection can be removed naturally by the host immune response and only a small percentage may progress to cancer; thus, the immune response is essential for the control of precursor lesions and CC. We present a review of recent research on the molecular mechanisms that allow HPV-infected cells to evade immune surveillance and potential targets of molecular therapy to inhibit tumor immune escape.

IL-10 expression is regulated by HPV E2 protein in cervical cancer cells.

It has been found that certain cytokines (IL-4, IL-10 and TGF-ß1) are highly expressed locally in biopsies from patients with premalignant lesions and cervical cancer, and may induce a local immune-suppression state. In particular, IL-10 is highly expressed in tumor cells and its expression is directly proportional to the development of HPV-positive cervical cancer, suggesting an important role of HPV proteins in the expression of IL-10. In fact, we demonstrated that E6 and E7 HPV proteins regulate TGF-ß1 gene expression in cervical cancer cells. Here, we found by band shifting analysis that the HPV E2 protein binds to the regulatory region of the human IL-10 gene (-2054 nt) and induces high promoter activity in epithelial cells. Additionally, cervical cancer cells transfected to express the HPV E2 protein induce elevated levels of IL-10 mRNA in human papillomavirus-infected cells. The elevated expression of IL-10 may allow for virus persistency, the transformation of cervical epithelial cells, and consequently cancer development.

Modulation of apoptosis by early human papillomavirus proteins in cervical cancer.

Cervical cancer (CC) constitutes a major women health problem. Clinical, molecular, and epidemiological investigations have identified persistent infection with high risk human papillomavirus (HR-HPV) as the major cause of CC. HR-HPVs lead to development of cervical carcinoma, predominantly through the action of E5, E6 and E7 viral oncoproteins. After HR-HPV infection, viral proteins employ strategies to modulate apoptosis. The E2 viral protein induces apoptosis in both normal and HPV-transformed cells through activation of caspase-8. The E5 protein can impair CD95L- and TRAIL-mediated apoptosis, which suggests that it may prevent apoptosis at early stages of viral infection. E6 inhibits apoptosis through the proteolytic inactivation of pro-apoptotic proteins such as p53, FADD, or procaspase-8, employing the ubiquitin proteasome pathway, or through interactions with proteins that form the death-inducing signaling complex (DISC) such as TNF-R1. On the other hand, E7 oncoprotein expressing cells are usually predisposed to undergo apoptosis. Useful targets for therapeutic strategies would interfere with expression or function of HR-HPV proteins to eliminate cells that express viral oncoproteins. In this review, we summarize the available data on the interaction of early HPV proteins with cellular factors that promote cell death, and the functional consequences of these interactions on apoptosis.

HPV 16 E2 protein induces apoptosis in human and murine HPV 16 transformed epithelial cells and has antitumoral effects in vivo.

OBJECTIVE: Our aims were to examine the ability of the human papillomaviruse (HPV) 16 E2 protein to induce apoptosis in a murine HPV-transformed cell line, and to evaluate its antitumor properties on HPV-associated tumors in vivo in immunocompetent mice. METHODS: HPV-transformed murine BMK-16/myc cells and human SiHa cells were transfected with the HPV 16 E2 gene to examine the effects of the E2 protein on cell growth and on the E6 and E7 oncogenes as well as DNA fragmentation and activation of the extrinsic pathway of apoptosis. Finally, to test the antitumor effect of the E2 protein on an experimental mouse tumor model, we generated a recombinant adenovirus expressing the E2 protein. RESULTS: The E2 protein inhibited the growth of SiHa and BMK-16/myc cell lines, and repressed the E6 and E7 oncogenes. Moreover, the E2 protein induced DNA fragmentation and apoptosis through activation of caspases 8 and 3 in BMK-16/myc cells. On the other hand, E2 also showed antitumor effects in vivo. CONCLUSIONS: Our findings indicate that E2 exerts pro-apoptotic activity in a murine HPV-transformed cell line as well as an antitumor effect in vivo.

[Transforming growth factor beta-1: structure, function, and regulation mechanisms in cancer]. / Factor de crecimiento transformante beta-1: estructura, función y mecanismos de regulación en cáncer.

Transforming growth factor beta-1 (TGF-beta 1) is produced by several cell lineages such as lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these and other cells. In general, TGF-beta 1 has pleiotropic properties on the immune response during the development of infection diseases and cancer; however, the mechanisms of action and regulation of gene expression of this cytokine are poorly understood, in this review, the biological properties and the molecular mechanisms that regulate TGF-beta 1 gene expression are described, to understand the role of this cytokine in growth and cell differentiation. The knowledge of molecular mechanisms of gene expression of TGF-beta 1 may serve to develop new cancer therapies. The English version of this paper is available at: http://www.insp.mx/salud/index.html

Cellular immune response and Th1/Th2 cytokines in human neurocysticercosis: lack of immune suppression.

Some reports have suggested that human neurocysticercosis (NCC) induces immunosuppression. To test this hypothesis, we performed a study on active cases of NCC who had not received cestocidal or immunosuppressive treatments. We examined blood counts and specific T cell markers (CD3, CD4, and CD8) by flow cytometry and found no differences between patients with NCC and healthy individuals. Both groups responded to concanavalin A (Con A), and patients with NCC responded more to a parasite crude antigen than uninfected individuals. Peripheral blood mononuclear cells were examined for interleukin (IL)-2, interferon-gamma, IL-10, and IL-4 mRNA. Regardless of infection status, more than 60% of individuals synthesized IL-2 mRNA and, less frequently, the other cytokines. These data suggest that immunosuppression does not occur in NCC patients.

Regulation of CD40 and CD40 ligand by the AT-hook transcription factor AKNA.

Proteins containing AT hooks bind A/T-rich DNA through a nine-amino-acid motif and are thought to co-regulate transcription by modifying the architecture of DNA, thereby enhancing the accessibility of promoters to transcription factors. Here we describe AKNA, a human AT-hook protein that directly binds the A/T-rich regulatory elements of the promoters of CD40 and CD40 ligand (CD40L) and coordinately regulates their expression. Consistent with its function, AKNA is a nuclear protein that contains multiple PEST protein-cleavage motifs, which are common in regulatory proteins with high turnover rates. AKNA is mainly expressed by B and T lymphocytes, natural killer cells and dendritic cells. During B-lymphocyte differentiation, AKNA is mainly expressed by germinal centre B lymphocytes, a stage in which receptor and ligand interactions are crucial for B-lymphocyte maturation. Our findings show that an AT-hook molecule can coordinately regulate the expression of a key receptor and its ligand, and point towards a molecular mechanism that explains homotypic cell interactions.

CD43-mediated signals induce DNA binding activity of AP-1, NF-AT, and NFkappa B transcription factors in human T lymphocytes.

Although numerous reports document a role for CD43 in T cell signaling, the direct participation of this molecule in cell activation has been questioned. In this study we show that CD43 ligation on human normal peripheral T cells was sufficient to induce interleukin-2, CD69, and CD40-L gene expression, without requiring signals provided by additional receptor molecules. This response was partially inhibited by cyclosporin A and staurosporine, suggesting the participation of both the Ca(2+) and the protein kinase C pathways in CD43 signaling. Consistent with the transient CD43-dependent intracellular Ca(2+) peaks reported by others, signals generated through the CD43 molecule resulted in the induction of NF-AT DNA binding activity. CD43-dependent signals resulted also in AP-1 and NFkappaB activation, probably as a result of protein kinase C involvement. AP-1 complexes bound to the AP-1 sequence contained c-Jun, and those bound to the NF-AT-AP-1 composite site contained c-Jun and Fos. NFkappaB complexes containing p65 could be found as early as 1 h after CD43 cross-linking, suggesting that CD43 participates in early events of T cell activation. The induction of the interleukin-2, CD69, and CD-40L genes and the participation of AP-1, NF-AT, and NFkappaB in the CD43-mediated signaling cascade implicate an important role for this molecule in the regulation of gene expression and cell function.

The response of hepatic acute phase proteins during experimental pulmonary tuberculosis.

A mouse model of pulmonary tuberculosis induced by the intratracheal instillation of live and virulent mycobacteria strain H37-Rv was used to study the relationship of the histopathological changes with the kinetics of local production and circulating levels of interleukin 6 (IL-6) and the gene expression of acute phase proteins (APP) in the liver. The histopathological studies showed a mononuclear inflammatory infiltrate located in the perivascular, peribronchial, and interstitial areas, with granulomas which started to form 2 weeks after the infection. Numerous IL-6 immunostained activated macrophages were observed in the inflammatory infiltrate, particularly in the interstitial-intralveolar compartment and granulomas, coexisting with a high IL-6 mRNA concentration determined by reverse transcription polimerase chain reaction in lung homogenates, particularly at day 21 of infection. Two peaks of IL-6 demonstrated by ELISA in lung homogenates and sera were observed at day 3 and 21 of infection, being higher on the latter. The hepatic APP mRNA transcription (alpha1-acid glycoprotein, fibrinogen, complement factor 4) analyzed by Northern blot showed a rapid and high increase at day one postinfection, which rapidly decreased and showed another second peak at day 21, when granulomas reached full maturity and the maximal production of IL-6 was observed. At the same time the liver mRNA concentrations of the negative APP albumin showed a substantial decrease. From 1 to 4 months after M. tuberculosis intratracheal instillation, histopathological changes of more severity (pneumonia, necrosis) and chronicity (interstitial fibrosis) were seen, as well as small groups of IL-6 immunostained macrophages in the pneumonic areas, granulomas and perivascular compartments, in coexistence with low IL-6 expression. During this advanced stage of the disease a high mRNA concentration of alpha1-acid glycoprotein and fibrinogen associated with low expression of the albumin gene in the liver continued. Thus, it seems that the time course of hepatic APP genetic expression in experimental pulmonary tuberculosis is related to the production of IL-6 and relevant histopathological changes, particularly the formation of granuloma.

[Perspectives for the development of vaccines and immunotherapy against cervico-uterine cancer]. / Perspectivas para el desarrollo de vacunas e inmunoterapia contra cáncer cervicouterino.

Cervical cancer represents a severe public health problem and has been associated to the presence of human papillomavirus. Strategies are presently being tested which target the virus to attempt to control disease progress. Studies on the humoral and cell-mediated immunity of the papillomavirus infection have been useful in the development of a vaccine. Synthetic virus-like particles have been validated as vaccine against several animal papillomaviruses and used to map the seroepidemiology of the human papillomavirus infection, and define neutralizing antibodies. Induction of cell-mediated immunity to HPV early proteins is bound to become a therapeutic approach to HPV infections. Recent advances have centered on directing the immune response to prevent infection, to virus-infected cells and to virally transformed cells, with favourable results.

Pathogenesis of tuberculosis in mice exposed to low and high doses of an environmental mycobacterial saprophyte before infection.

Mycobacteria are ubiquitous in the environment, but they are not part of the normal human microbial flora. It has been suggested that variable contact with mycobacteria can influence susceptibility to mycobacterial pathogens and the efficacy of subsequent Mycobacterium bovis BCG vaccination. To test this, mice were immunized with high or low doses of an environmental saprophyte, M. vaccae, that is intensely immunogenic as an autoclaved preparation. Two months later, they received an intratracheal challenge with M. tuberculosis H37Rv. Recipients of a low Th1-inducing dose (10(7) organisms) were partially protected and maintained a high ratio of interleukin 2 (IL-2)-positive to IL-4-positive cells in the perivascular, peribronchial, and granulomatous areas of the lung, whereas in unimmunized controls the IL-4-positive cells increased markedly between days 21 and 28. In contrast, recipients of the high dose (10(9) organisms), which primes Th2 as well as Th1 cytokine production, died more rapidly than unimmunized controls and showed massive pneumonia from day 7. The ratio of IL-2-positive to IL-4-positive cells in all compartments of the lung rapidly fell to 1 by day 14 for these animals. These events correlated with cytokine mRNA profiles and with increases in the local toxicity of tumor necrosis factor alpha (TNF-alpha), demonstrable only when a major Th2 component was present. These data indicate that cross-reactive epitopes present in an environmental saprophyte can evoke either protective responses or responses that increase susceptibility to M. tuberculosis. The latter are associated with the presence of a Th2 component and increased sensitivity to TNF-alpha.

Analysis of the local kinetics and localization of interleukin-1 alpha, tumour necrosis factor-alpha and transforming growth factor-beta, during the course of experimental pulmonary tuberculosis.

A mouse model of pulmonary tuberculosis induced by the intratracheal instillation of live and virulent mycobacteria strain H37-Rv was used to examine the relationship of the histopathological findings with the local kinetics production and cellular distribution of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha) and transforming growth factor-beta (TGF-beta). The histopathological and immunological studies showed two phases of the disease: acute or early and chronic or advanced. The acute phase was characterized by inflammatory infiltrate in the alveolar-capillary interstitium, blood vessels and bronchial wall with formation of granulomas. During this acute phase, which lasted from 1 to 28 days, high percentages of TNF-alpha and IL-1 alpha immunostained activated macrophages were observed principally in the interstium-intralveolar inflammatory infiltrate and in granulomas. Electron microscopy studies of these cells, showed extensive rough endoplasmic reticulum, numerous lysosomes and occasional mycobacteria. Double labelling with colloid gold showed that TNF-alpha and IL-1 alpha were present in the same cells, but were confined to separate vacuoles near the Golgi area, and mixed in larger vacuoles near to cell membrane. The concentration of TNF-alpha and IL-1 alpha as well as their respective mRNAs were elevated in the early phase, particularly at day 3 when the bacillary count decreased. A second peak was seen at days 14 and 21-28 when granulomas appeared and evolved to full maturation. In contrast, TGF-beta production and numbers of immunoreactive cells were low in comparison with the advanced phase of the disease. The chronic phase was characterized by histopathological changes indicative of more severity (i.e. pneumonia, focal necrosis and extensive interstitial fibrosis) with a decrease in the TNF-alpha and IL-1 alpha production that coincided with the highest level of TGF-beta. The bacillary counts were highest as the macrophages became large, vacuolated foamy cells, and containing numerous bacilli with immunoreactivity to mycobacterial lipids and lipoarabinomannan (LAM). These macrophages displayed poor and scarce TNF-alpha and IL-1 alpha immunostaining but still strong immunoreactivity to TGF-beta. These cytokine production kinetics and the spatial relationship between immunostained cells and lung lesions corroborate the important role of TNF-alpha and IL-1 alpha in the constitution of granulomas and immune protection during the early phase of the infection, and also suggest an important if not primary role for TGF-beta in the immunopathogenesis of the advanced forms of pulmonary tuberculosis.