SIV Infection Facilitates Mycobacterium tuberculosis Infection of Rhesus Macaques.

Tuberculosis (TB) is a common opportunistic infection and the leading cause of death for human immunodeficiency virus (HIV)-infected patients. Thus, it is necessary to understand the pathogenetic interactions between M.tb and HIV infection. In this study, we examined M.tb and/or simian immunodeficiency virus (SIV) infection of Chinese rhesus macaques. While there was little evidence that M.tb enhanced SIV infection of macaques, SIV could facilitate M.tb infection as demonstrated by X-rays, pathological and microbiological findings. Chest X-rays showed that co-infected animals had disseminated lesions in both left and right lungs, while M.tb mono-infected animals displayed the lesions only in right lungs. Necropsy of co-infected animals revealed a disseminated M.tb infection not only in the lungs but also in the extrapulmonary organs including spleen, pancreas, liver, kidney, and heart. The bacterial counts in the lungs, the bronchial lymph nodes, and the extrapulmonary organs of co-infected animals were significantly higher than those of M.tb mono-infected animals. The mechanistic studies demonstrated that two of three co-infected animals had lower levels of M.tb specific IFN-γ and IL-22 in PBMCs than M.tb mono-infected animals. These findings suggest that Chinese rhesus macaque is a suitable and alternative non-human primate model for SIV/M.tb coinfection studies. The impairment of the specific anti-TB immunity is likely to be a contributor of SIV-mediated enhancement M.tb infection.

High monocarboxylate transporter 4 protein expression in stromal cells predicts adverse survival in gastric cancer.

BACKGROUND: Increasing evidence suggests that stromal monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX) may play key roles in tumor development. However, their clinical value remains largely unexplored in gastric cancer (GC). The present study aimed to determine clinicopathological significance and prognostic values of stromal MCT4 and CA IX in GC. MATERIALS AND METHODS: Specimens from 143 GC patients were immunohistochemically stained using polyclonal anti-MCT4 and anti-CA IX antibodies. Expression was correlated with patient clinicopathologic characteristics and survival data. RESULTS: High stromal MCT4 expression was detected in 72 of 143 (50.3%) GCs and high CA IX in 74 (51.7%). Both high stromal MCT4 and CA IX were correlated with advanced TNM stage (p=0.000; p=0.000). High CA IX expression was positively related to depth of invasion (p=0.022) and positive lymph nodes (p=0.002) as well. Survival analysis indicated high expression of stromal MCT4 to be an independent factor in predicting poor overall survival (OS) (HR and 95%CI=1.962, 1.032-3.729, p=0.040) and disease free survival (DFS) (HR and 95%CI=2.081, 1.158-3.741, p=0.014) of GC patients. However, high CA IX expression exhibited no significant predictive value. CONCLUSIONS: These findings suggest that high expression of stromal MCT4 and CA IX proteins is significantly correlated with GC progression. High stromal MCT4 heralds worse outcome of GC patient, suggesting a novel candidate prognostic marker and therapeutic target.

Azithromycin inhibits neutrophil accumulation in airways by affecting interleukin-17 downstream signals.

BACKGROUND: Azithromycin can reduce neutrophil accumulation in neutrophilic pulmonary diseases. However, the precise mechanism behind this action remains unknown. Our experiment assessed whether azithromycin inhibits neutrophil accumulation in the airways by affecting interleukin-17 (IL-17) downstream signals. METHODS: Mice were pretreated with azithromycin before murine IL-17A (mIL-17) stimulation. After the mIL-17 stimulation, the levels of six neutrophil-mobilizing cytokines were determined by enzyme-linked immunosorbent assay (ELISA) tests in bronchoalveolar lavage (BAL) fluid; IL-6, CXC chemokine ligand-1 (CXCL-1), CXCL-5, macrophage inflammatory protein-2 (MIP-2), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). The number of neutrophils in BAL fluid were evaluated by cytospin preparations. RESULTS: (1) Azithromycin pretreatment significantly inhibited both the release of three neutrophil-mobilizing cytokines (MIP-2, CXCL-5 and GM-CSF) and the accumulation of neutrophils in airways caused by mIL-17 stimulation. (2) The levels of three neutrophil-mobilizing cytokines (IL-6, MIP-2 and GM-CSF) were positively correlated with the numbers of neutrophil in BAL fluid. CONCLUSIONS: Azithromycin can inhibit neutrophil accumulation in the airways by affecting IL-17 downstream signals. This finding suggests that macrolide antibiotic application might be useful in prevention of neutrophilic pulmonary diseases characterized by high levels of IL-17.

[Establishment and evaluation of a Chinese rhesus model of tuberculosis].

OBJECTIVE: To establish and evaluate the Chinese rhesus model of tuberculosis. METHODS: Twelve Chinese rhesus macaques, randomly divided into 3 groups, were inoculated with 2 different doses of Mycobacterium tuberculosis H(37) Rv strain via both bronchoscopic and intratracheal instillation into the lungs. Clinical observation and laboratory examinations were performed, including erythrocyte sedimentation rate, C-reactive protein, tuberculin skin test and X-ray examination. Histopathological assessments were performed in the 24th week postinfection. Statistical analysis was performed by ANOVA in the 3 groups. RESULTS: After infection all the animals manifested fever, weight lose, lack of appetite, coughing and other symptoms of tuberculosis. The temperature gradually increased and reached a peak [(40.1 ± 0.2)°C] at the 8th week postinfection. The weight decreased significantly at 24th week postinfection (-5.5 ± 5.6)%. Erythrocyte sedimentation rate elevated significantly at the 6th to 8th week postinfection (36 ± 40) mm/1 h. C-reactive protein was significantly increased at the 6th to 24th week after infection (75.8 ± 49.8) mg/L. The positive rate of tuberculin skin test was 100%. In Group I (bronchoscopic instillation, 20 CFU) the disease developed slowly, and the main manifestation of chest X-ray was patchy shadows. In group II (bronchoscopic instillation, 100 CFU) and group III (intratracheal instillation, 100 CFU) the disease developed rapidly, and the main manifestation of chest X-ray was patchy and nodular lesions during the 4th to the 12th week postinfection, but became large patchy and consolidation lesions during the 12th to the 24th week postinfection. Tuberculosis granuloma and caseous necrosis, similar to the pathological changes of human tuberculosis, were found in the lungs, mediastinal lymph nodes, kidney and spleen. The results of acid-fast stain were positive. The most serious pathological manifestations were observed in group II, followed by group III and group I. The highest bacterial load of the right lung was seen in group II, followed by group I and group III. CONCLUSIONS: A chinese rhesus model of tuberculosis was successfully developed via both bronchoscopic and intratracheal instillation. Their clinical manifestations, disease progression and pathological changes were similar to human primary tuberculosis and hematogenous disseminated tuberculosis.

M. tuberculosis H37Rv infection of Chinese rhesus macaques.

Mycobacterium tuberculosis is the most common communicable infectious disease worldwide and the top killer of human immunodeficiency virus (HIV)-infected people. Because of common dual HIV and M. tuberculosis infections, the emergence of multidrug-resistant M. tuberculosis strains, the lack of effective vaccination, the morbidity, and the mortality of M. tuberculosis infection are increasing sharply. Therefore, there is an urgent need for vaccine and drug development against M. tuberculosis infection. These require appropriate animal models that closely resemble human disease. To this end, we infected Chinese rhesus macaques with the M. tuberculosis H37Rv strain. Bronchoscopy was used to inoculate nine monkeys with different doses of M. tuberculosis H37Rv strain. Regardless of the M. tuberculosis dose, all monkeys were infected successfully. This was shown by clinical, laboratory, and histopathology assessments. Among nine infected monkeys, six developed acute rapid progressive tuberculosis and the remaining animals mirrored early-stage chronic disease. These data, taken together, demonstrate that Chinese rhesus macaques are highly susceptible to M. tuberculosis infection and develop similar manifestations of disease that are seen in humans. This model affords new opportunities for studies of M. tuberculosis disease pathology and therapeutics.

[The effect of CD4+ CD25+ regulatory T cell inactivation combined with levofloxacin on murine tuberculosis].

OBJECTIVE: To investigate the effects of inactivation of CD(4)(+)CD(25)(+) regulatory T cells (Treg) combined with the administration of levofloxacin (LFX) on the cellular immune response of murine tuberculosis. METHODS: Inactivation of Treg was achieved by intraperitoneal injection of anti-CD(25), clone PC61. Female C57BL/6 mice were divided into 4 groups, PC61 alone, LFX alone, PC61 plus LFX, and the control, with 19 mice in each group. The LFX group and the control group were treated with rat-IgG isotope control. Mice were inoculated with H(37) Rv (1 x 10(6) CFU) via the tail vein 3 days later. From the 2nd day, the LFX group and the PC61 plus LFX group received intragastric administration of LFX at 200 mg x kg(-1) x d(-1) per mouse for 45 days. Blood and spleen Tregs were measured by flow cytometry. The cellular immune response and pulmonary histopathology at different time points were also evaluated after infection. RESULTS: At the 10th and 30th day, the ratio of CD(4)(+)CD(25)(+)/CD(4)(+)T cells in the spleen was (30 +/- 4)% and (17.3 +/- 1.6)% respectively in the control group, (21 +/- 4)% and (16.1 +/- 1.3)% respectively in the PC61 group, (44 +/- 6)% and (24.7 +/- 2.0)% respectively in the LFX group, (24 +/- 3)% and (10.4 +/- 1.0)% respectively in the PC61 plus LFX group. The differences were significant between groups (q = 3.62 - 5.56, P < 0.05), but the difference between the PC61 plus LFX group and the PC61 group at the 10th day. Same results were obtained from the peripheral blood. PC61 plus LFX therapy resulted in BCG specific cytokine response (IL-17, IFN-gamma, TNF-alpha) from murine spleen cells at the 10th and the 30th day, and also in milder pathologic changes and the lowest mortality. CONCLUSIONS: The cellular immune response was enhanced by Treg inactivation and LFX therapy, which decreased the pathologic changes and the mortality of murine tuberculosis.

[Expression and pathobiological implication of hypoxia-inducible factor-1alpha in human colorectal carcinoma].

OBJECTIVE: To investigate the transcription level and protein expression of HIF-1alpha and VEGF in SW480 cell line and colorectal adenocarcinoma, and to determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF. METHODS: HIF-1alpha mRNA expression was analyzed by in situ hybridization. HIF-1alpha and VEGF protein expressions were determined by immunochemical streptavidin/peroxidase (SP) in SW480 cells and colorectal carcinoma tissue samples and Western blot, using proteins extracted from SW480 cells. Tumor tissue microvessel density (MVD) was determined by CD34 immunostaining of colorectal carcinomas. RESULTS: The levels of HIF-1alpha mRNA changed significantly in response to different oxygen concentrations and an addition of genistein in SW480 cells. Immunocytochemistry revealed that the levels of HIF-1alpha, VEGF protein expression in SW480 cells were significantly higher under hypoxia than those in nomoxia (P < 0.01, P < 0.05 respectively). However, addition of genistein, an inhibitor of HIF-1alpha, suppressed such responses to hypoxia. Western blot analysis showed that SW480 cells exposed to hypoxia expressed a high level of HIF-1alpha protein, compared to a weak expression in nomoxia. The addition of genistein in hypoxia suppressed the over-expression of HIF-1alpha. The positive rates of HIF-1alpha mRNA by in situ hybridization in colorectal adenomas and adenocarcinomas were 38.9% (7/18) and 67.7% (42/62), respectively. The percentage of HIF-1alpha mRNA positive cells varied significantly from colorectal adenomas to adenocarcinomas at different Duke stages (P < 0.05), and HIF-1alpha mRNA was higher in adenocarcinomas than in adenomas (P < 0.01). The positive rates of HIF-1alpha and VEGF protein expression in adenocarcinomas were 43.5% (27/62) and 37.1% (23/62), respectively. The expression of VEGF elevated as the Duke tumor staging increased. The conformation rate of HIF-1alpha and VEGF was 74.2% (46/62). MVD was significantly higher in HIF-1alpha and/or VEGF positive tumors than those without (P < 0.01 and P < 0.05 respectively). Among the four groups, i.e. HIF-1alpha+/VEGF+, HIF-1alpha+/VEGF-, HIF-1alpha+/VEGF- and HIF-1alpha-/VEGF-, the difference of MVD was highly significant (P < 0.01). HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density. CONCLUSIONS: These findings suggest hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinoma. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinoma.

Expression and significance of PTEN, hypoxia-inducible factor-1 alpha in colorectal adenoma and adenocarcinoma.

AIM: To investigate the expression and significance of PTEN, hypoxia-inducible factor-1 alpha (HIF-1alpha), and targeting gene VEGF during colorectal carciogenesis. METHODS: Total 71 cases colorectal neoplasms (9 cases of colorectal adenoma and 62 colorectal adenocarcinoma) were formalin fixed and paraffin-embedded, and all specimens were evaluated for PTEN mRNA, HIF-1alpha mRNA and VEGF protein expression. PTEN mRNA, HIF-1alpha mRNA were detected by in situ hybridization. VEGF protein was identified by citrate-microwave SP immunohistochemical method. RESULTS: There were significant differences in PTEN, HIF-1alpha and VEGF expression between colorectal adenomas and colorectal adenocarcinoma (P<0.05). The level of PTEN expression decreased as the pathologic stage increased. Conversely, HIF-1alpha and VEGF expression increased with the Dukes stage as follows: stage A (0.1029+/-0.0457: 0.1207+/-0.0436), stage B (0.1656+/-0.0329: 0.1572+/-0.0514), and stage C+D (0.2335+/-0.0748: 0.2219+/-0.0803). For PTEN expression, there was a significant difference among Dukes stage A, B, and C+D, and the level of PTEN expression was found to be significant higher in Dukes stage A or B than that of Dukes stage C or D. For HIF-1alpha expression, there was a significant difference between Dukes stage A and B, and the level of HIF-1alpha expression was found to be significantly higher in Dukes stage C+D than that of Dukes stage A or B. The VEGF expression had similar results as HIF-1alpha expression. In colorectal adenocarcinoma, decreased levels of PTEN were significantly associated with increased expression of HIF-1alpha mRNA (r=-0.36, P<0.05) and VEGF protein (r=-0.48, P<0.05) respectively. The levels of HIF-1 were positively correlated with VEGF expression (r=0.71, P<0.01). CONCLUSION: Loss of PTEN expression and increased levels of HIF-1alpha and VEGF may play an important role in carcinogenesis and progression of colorectal adenocarcinoma.

[Expression of HIF-1 alpha and its relationship to apoptosis and proliferation in lung cancer].

BACKGROUND & OBJECTIVES: Hipoxia-inducible factor-1 is a transcriptive factor that regulates genes involved in metabolism, angiogenesis, proliferation, and apoptosis. This study was designed to investigate the expression of hypoxia inducible facter-1 alpha(HIF-1 alpha) and its relationship to bcl-2, Bax, PCNA in lung cancer. METHOD: Immunohistochemical streptavidin/peroxidase(SP) was used to examine the expression of HIF-1a, bcl-2, Bax, and PCNA in 60 cases of lung cancer. RESULTS: In 60 cases of lung cancer, positive rate for HIF-1a was 28.3% (17/60), specially the positive rate of small cell lung cancer(66.7%) was significantly higher than non-small cell lung cancer (21.6%). HIF-1a expression increased as clinical stage and metastasis increased(P < 0.01). The positive rate of bcl-2, Bax, and PCNA were 31.7% (19/60), 40.0% (24/60), 76.7% (46/60), respectively. Inverse relationship was found between the expression of HIF-1 alpha and bcl-2; while the correlation of HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and Bax was positive(P < 0.01). The relationship between HIF-1 alpha and PCNA was not observed(P > 0.05). CONCLUSION: HIF-1 alpha is correlated with apopotosis, but has no relationship with proliferation.

[A study on cyclooxygenase-2 protein expression and vasculature during experimental rat lung carcinogenesis].

BACKGROUND & OBJECTIVE: There is evidence that cyclooxygenase-2(COX-2) involved in the occurrence and development of neoplasms. Elevated expression of COX-2 in carcinomas and antitumor effects of nonsteroidal antiinflammatory drug(COX-2 inhibitors) have been reported, but COX-2 expression in precancerous lesions and its association with angiogenesis is not clearly defined. The aim of this study was to investigate the expression of COX-2 protein and its association with microvessel density (MVD) during the experimental rat lung carcinogenesis. METHODS: Eighty Wistar rats were intra-leftlobar-bronchial instilled with 3-methylcholanthrene and diethylinitrosamine to induce lung squamous cell carcinoma, and ten rats were instilled lipiodol as control group. To acquire every pathological phase during the carcinogenesis, rats were sacrificed at intervals from fifteen days to nine months. COX-2 expression and MVD count in the samples of every pathological phase during the carcinogenesis were examined by immunohistochemistry. The immunohistochemical score of COX-2 was calculated by combining an estimate of the percentage of immunoreactive cells with an estimate of the staining intensity. Inter tumor MVD was marked by anti-Von Willebrand factor monoantibody. RESULTS: One hundred and forty seven specimens of every pathological phase during the carcinogenesis were obtained which including fourteen hyperplasia, twenty five squamous metaplasia, thirty three dysplasia, twelve carcinoma in situ, fifty four infiltration carcinoma, nine metastasis. The expression of COX-2 and MVD count increased during rat lung carcinogenesis. COX-2 immunohistochemical score was significantly higher in dysplasia, carcinoma in situ and metastasis(P < 0.01, P < 0.05, P < 0.05 respectively), and significant increased MVD were found in carcinoma in situ, infiltration carcinoma and metastasis (P < 0.01). During carcinogenesis, there was a significant correlation between COX-2 expression and MVD(r = 0.9521, P < 0.001, b = 11.51). CONCLUSION: COX-2 may play an important role during the carcinogenesis in experimental rat lung squamous cell carcinoma as well as its metastasis, partly by stimulating angiogenesis.

[Expression and clinical significance of IGF-II mRNA and bcl-2 protein in colorectal adenocarcinomas].

BACKGROUND & OBJECTIVE: Insulin-like growth factor II (IGF-II) can stimulate cell proliferation; B cell lymphoma 2 (bcl-2) protein can strongly inhabit cell apoptosis. Recently, the overexpression of IGF-II and bcl-2 in colorectal cancer has been found, but the combined detection of them has not been reported. The objective of this study was to investigate the relationship between the expression of IGF-II, bcl-2 and the invasion, metastases of colorectal adenocarcinomas and to analyze the clinical significance of combined detection of these factors. METHODS: Forty-eight paraffin embedded samples from colorectal adenocarcinomas were selected. IGF-II mRNA was detected by using in situ hybridization. The expression of bcl-2 and PCNA protein were determined immunohistochemically, and TUNEL technique was used to detect apoptosis. Ten normal colorectal tissues were used as controls. The positive cell ratio of cancers was calculated by computer. The specimens with positive cell ratio < or = 30% were defined as negative. RESULTS: The expressions of IGF-II mRNA and bcl-2 protein were significantly higher in colorectal adenocarcinomas (38.70% +/- 7.80% and 30.97% +/- 7.40%) than in normal colorectal tissues(23.12% +/- 4.07% and 12.69% +/- 1.31%) (P < 0.01) and were related to Dukes' stage and lymph node metastases but were not associated with patient's age, gender, tumor site, tumor size and tumor differentiation. A negative correlation was observed between IGF-II mRNA and bcl-2 protein (P < 0.05). A positive correlation between IGF-II mRNA and PCNA, apoptosis as well as a negative correlation between bcl-2 and apoptosis were observed (P < 0.01). There was no correlation between bcl-2 and PCNA (P > 0.05). The patients with IGF-II mRNA (+) and bcl-2 (-) were regarded as the worst prognosis. CONCLUSION: The overexpression of IGF-II and bcl-2 in colorectal adenocarcinomas play an important role in the pathogenesis, progression, invasion, and metastases of the tumor. Determination of both IGF-II and bcl-2 expression would be helpful for decision making of adjuvant therapy.