Notch-1 signaling activation sustains overexpression of interleukin 33 in the epithelium of nasal polyps.

BACKGROUND: Alterations in the nasal epithelial barrier homeostasis and increased interleukin 33 (IL-33) expression contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). AIMS: As Notch-1 signaling is crucial in repair processes of mucosa, the current study assessed Notch-1/Jagged-1 signaling and IL-33 in the epithelium of nasal polyps biopsies from allergic (A-CRSwNP; n = 9) and not allergic (NA-CRSwNP; n = 9) subjects by immunohistochemistry. We also assessed, in a model of nasal epithelial cells, the effects of stimulation of Notch-1 with Jagged-1 on the expression of IL-33 (by flow cytometry, immunofluorescence, and immunocytochemistry), Jagged-1 (by flow cytometry), and p-CREB transcription factor (by western blot analysis). RESULTS: Ex vivo (a) in normal epithelium, the expression of Notch-1 and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (b) in metaplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (c) in hyperplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in A-CRSwNP than in NA-CRSwNP; and (d) in basal epithelial cells, no differences were observed in the expression of Jagged-1, IL-33, and Notch-1. The expression of Notch-1 significantly correlated with the expression of IL-33. In vitro, stimulation of Notch-1 with Jagged-1 induced the expression of (a) Jagged-1; (b) IL-33; and (c) p-CREB transcription factor. The inhibitor of Notch-1, DAPT, reduced all the effects of Jagged-1 on nasal epithelial cells. CONCLUSIONS: The data herein provided support, for the first time, a putative role of Notch-1/Jagged-1 signaling in the overexpression of IL-33 in the epithelium of nasal polyps from patients with CRSwNP.

The role of transforming growth factor-ß1 in airway inflammation of childhood asthma.

TGF-beta-targeting structural and inflammatory cells has been implicated in the mechanisms leading to the inflammatory and restructuring processes in asthma, suggesting an impact of TGF-beta1 signaling on the development and persistency of this disease. We investigated the potential early involvement of TGF-beta1 activity in the immunological and molecular mechanisms underlying progression of inflammation in childhood asthma. We evaluated the levels of TGF-beta1 in induced sputum supernatants (ISSs) and the expression of small mother cell against decapentaplegic (Smad) 2 and Smad7 proteins in induced sputum cells (ISCs) from children with intermittent asthma (IA), moderate asthma (MA) and control subjects (C). Furthermore, we investigated the regulatory role of TGF-beta1 activity on eosinophil and neutrophil adhesion to epithelial cells using adhesion assay, and on the granulocyte expression of adhesion molecule CD11b/CD18 Macrophage-1 antigen (MAC-1), by flow cytometry. We found that the levels of TGF-beta1 are increased in ISSs of IA and MA in comparison to C, concomitantly to the activation of intracellular signaling TGFbeta/Smads pathway in ISCs. In MA, TGF-beta1 levels correlated with the number of sputum eosinophils and neutrophils. Furthermore, we showed the ability of sputum TGF-beta1 to promote eosinophil and neutrophil adhesion to epithelial cells, and to increase the expression of MAC-1 on the granulocyte surface. This study shows the activation of TGFbeta/Smad signaling pathway in the airways of children with IA and, despite the regular ICS treatment, in children with MA, and provides evidence for the contribution of TGF-beta1 in the regulation of granulocyte activation and trafficking.

Correlation between variable-number tandem-repeat-based genotypes and drug susceptibility in Mycobacterium avium isolates.

Little is known about the correlation between genotype and drug susceptibility in Mycobacterium avium (Mav) strains isolated from patients with Mav infections. To examine whether drug susceptibility profile of Mav is associated with genotype, we carried out variable-number tandem-repeat (VNTR) typing and drug susceptibility testing for Mav isolates from Japanese with nodular-bronchiectasis (NB)-type and cavitary disease (CA)-type diseases. We performed M. avium tandem repeat (MATR)-VNTR typing and drug susceptibility testing by the broth dilution method, using macrolides, rifamycins, ethambutol, isoniazid, aminoglycosides, and quinolones, for Mav isolates from patients with NB and CA-type diseases (NB-Mav and CA-Mav). Based on the VNTR genotyping, the Mav strains were grouped into three clusters. There was no difference with respect to the distribution of NB-Mav and CA-Mav among the clusters. We observed a strong association between VNTR genotype and susceptibility to quinolones (levofloxacin, moxifloxacin, gatifloxacin, sitafloxacin, and garenoxacin) and ethambutol. There was essentially no significant difference in drug susceptibility between NB- and CA-Mav strains, although NB-Mav was somewhat more resistant to fluoroquinolones, especially gatifloxacin, than CA-Mav. There was a significant association between VNTR genotype and susceptibility to quinolones and ethambutol in Mav isolates from Japanese patients.

Comparative study for the virulence of Mycobacterium avium isolates from patients with nodular-bronchiectasis- and cavitary-type diseases.

Mycobacterium avium (Mav) lung infections, called nodular-bronchiectasis (NB)-type M. avium complex (MAC) disease, are globally increasing. To elucidate whether there are unusual populations of Mav, causing NB-type disease rather than cavitary (CA)-type disease, we compared the virulence of Mav isolates from patients with NB-type (NB-Mav) and those from CA-type (CA-Mav) diseases, based on intracellular growth in various types of human cells. Five strains each of NB-Mav and CA-Mav were compared with each other for their invasiveness and ability to intracellularly replicate in various types of cultured cells of human origin. The two types of Mav isolates showed a similar ability, on average, to replicate in macrophages and lung epithelial cells. Moreover, they showed a similar ability to induce the production of reactive nitrogen intermediates and reactive oxygen intermediates by macrophages and susceptibility to antimicrobial molecules. Therefore, it appears that there is no essential difference in virulence in terms of infectivity to human macrophages and lung cells between Mav strains isolated from NB-MAC disease and those from CA-MAC disease. These findings indicate the importance of further studies to elucidate the mechanism for the establishment of NB-type MAC diseases based on host immunological conditions rather than the pathogenic nature of MAC organisms themselves.

Impaired activation of Notch-1 signaling hinders repair processes of bronchial epithelial cells exposed to cigarette smoke.

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.

Cigarette smoke extract modulates E-Cadherin, Claudin-1 and miR-21 and promotes cancer invasiveness in human colorectal adenocarcinoma cells.

BACKGROUND: Cigarette smoke is considered a risk factor for lung and colorectal cancer. A convincing link between epithelial-to-mesenchymal transition (EMT) with colorectal cancer progression and therapeutic resistance has emerged. Deregulated expression of E-Cadherin and Claudin-1 and increased miR-21 expression and invasiveness represent hallmarks of EMT. The effects of cigarette smoke exposure on EMT in colorectal adenocarcinoma cells are largely unknown. AIM: The aim of the study is to evaluate the effect of cigarette smoke extract (CSE) on miR-21, Claudin-1 and E-Cadherin, molecules associated to EMT in colorectal cancer cells. METHODS: A human colorectal adenocarcinoma cell line (Caco-2) was treated with CSE at different concentration (5% and 10%) and for different time points (3 h and 24 h). Metabolic activity (by MTS assay), cell necrosis/cell apoptosis (evaluating Propidium Iodide/Annexin V expression by flow cytometry), miR-21, Claudin-1 and E-Cadherin gene expression were evaluated by Real time PCR. Cell permeability, actin polymerization and cancer cell migration was assessed by Trans-Epitelial Electrical Resistance (TEER), Phalloidin expression and matrigel system, respectively. RESULTS: CSE at all the tested concentrations and at all time points reduced cell necrosis. CSE at 10% increased miR-21 and reduced the metabolic activity, cell necrosis, Claudin-1 and E-cadherin mRNA at 3 h. Cell permeability, actin polymerization and cancer cell migration were all increased upon CSE exposure. CONCLUSION: These results showed that CSE increasing miR-21, Claudin-1 and E-Cadherin and enhancing the aggressiveness of cancer cells, may concur to colorectal cancer progression.

A prospective study on primary gastric stump cancer following partial gastrectomy for benign gastroduodenal diseases.

A prospective study was made on 3827 Japanese patients who had undergone partial gastrectomy for benign gastroduodenal diseases to examine whether they are at a high risk of mortality from primary gastric stump cancer (PGSC) and whether the risk is determined by the surgical procedure. The patients were followed up from the time of surgery (from 1948 to 1970) to June 30, 1981. Of 3,701 patients (96.7%), the vital status at the end of observation was determined, the total person-years at risk being 62,286.33. The observed deaths were compared with the expected deaths calculated from the mortality rates of Japan. An elapsed time of 10 years from operation to death was set not only to exclude possible recurrent, remaining, or multiple cancers but also to allow a certain latency period for the development of PGSC. The observed and expected deaths from PGSC were 11 and 52.85, respectively, the ratio being 0.21 (p less than 0.01). The ratios were uniformly less than 1 for both sexes and across three operative groups: Billroth I, Billroth II with Braun's anastomosis; or Billroth II without Braun's anastomosis. No difference was observed between the death rates from PGSC by operation type. The possible role of the postoperative nonphysiological (pathological) environment or duodenogastric reflux in gastric stump carcinogenesis was not detected in the present study.

Time course of mycobacterial infection of dendritic cells in the lungs of intranasally infected mice.

SETTING: Dendritic cells (DC) could regulate between the protective and pathogenic immune responses following tuberculous infection. In this paper we investigated if their early infection in the lungs represents a plausible alternative to cross-priming with mycobacterial antigens acquired from infected macrophages. OBJECTIVE: To determine the extent and time course of infection of lung DCs following intranasal inoculation of BALB/c mice with green fluorescent protein (GFP) tagged Bacillus Calmette-Guerin (BCG). RESULTS: A fraction of GFP-BCG infected lung cells were classified as monocytic DCs with the CD11c+IA+33D1+CD8a- phenotype. These cells represented 5-18% of the total GFP+ cells, the bulk of which were macrophages. The infected DCs could be separated by cell size into two fractions with similar cell surface staining properties during the 2-72 h period after infection. An unexpected difference was observed for the time course of infection between DCs and macrophages: DC infection peaked at 48 h followed by decline at 72 h, while the proportion of infected macrophages remained steady during the same period. CONCLUSION: The presented results are direct evidence that monocytic DCs are recruited to the lungs and take up live bacilli within 48 h of intranasal infection with GFP-BCG. This finding is pertinent for the regulation of pulmonary and systemic immune responses and possibly for the dissemination of mycobacterial infection by DCs.

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mphis was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mphi anti-MAC functions. (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect. However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes (Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated. (3) ROI production by murine peritoneal mphis was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1-24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

Different role of human HLA-DR and -DQ molecules in xenogeneic transplantation using transgenic mice.

BACKGROUND: The role of T lymphocytes in graft rejection in xenotransplantation is still unclear. The ability of the human HLA class II molecules DR and DQ to function as xenoantigens was investigated in a murine model of skin grafting, using HLA-DR1 and -DQ6-transgenic mice. METHODS: Skin from HLA-DR1- or -DQ6-transgenic mice was transplanted in control littermates. Spleen cells from donors or recipients were tested in mixed lymphocyte reaction and cytotoxic assay. RESULTS: Skin from HLA-DR1-transgenic mice was rejected and spleen cells from rejecting mice were able to proliferate to donor cells, although no rejection was observed when the skin of HLA-DQ6-transgenic mice was engrafted in control littermates. No cytotoxicity was observed in any models. CONCLUSIONS: Taken all together these results clearly suggest a hierarchy in the xenogeneic potency of human HLA class II molecules, with the HLA-DR1 molecule functioning as a potent xenoantigen when compared with the HLA-DQ6 molecule.

Broad clonal heterogeneity of antigen-specific CD4+ T-cells localizing at the site of disease during tuberculosis.

The repertoire of CD4+ T-lymphocytes was investigated in six patients affected by tuberculosis, who had a negative PPD skin test at diagnosis. Polyclonal CD4+ T-cell lines from the peripheral blood failed to proliferate to PPD and to the 16- or 38-kDa proteins of Mycobacterium tuberculosis, while CD4+ T-cell lines from the site of disease responded to PPD, and to the 16- and 38-kDa proteins, and derived epitopes in vitro. The repertoire of CD4+ T-cells accumulating at the site of disease was found to be widely heterogeneous as demonstrated by the finding that at least seven different peptides from the 16- and 38-kDa proteins were recognized by every patient. These results indicate that CD4+ T-cells localized at the site of disease in tuberculosis recognize a vast array of M. tuberculosis epitopes.

Expression profiles of low molecular mass heat shock proteins by Mycobacterium avium and Mycobacterium intracellulare.

Closely related non-tuberculous mycobacterial species, Mycobacterium avium and Mycobacterium intracellulare, were compared for the profiles of their production of low molecular mass heat shock proteins at 45 degrees C, by performing polyacrylamide gel electrophoresis analysis of bacterial cell lysate proteins. All of the M. intracellulare but not M. avium strains potently increased the production of the 18-kDa heat shock protein, when cultured at 45 degrees C. Half of the M. intracellulare strains with high sensitivity to 45 degrees C produced not only the 18-kDa heat shock protein but also the 16-kDa heat shock protein at 45 degrees C. These findings indicate that M. avium and M. intracellulare differentially respond to 45 degrees C heat shock in terms of the production of low molecular mass heat shock proteins.

A new improved technique for placement of a pursestring suture on the edge of the distal part of the rectal stump using a skin stapler for low anterior resection.

Instead of a linear stapler or manual pursestring suture onto the lower part of the rectum, we placed a No. 2-0 Prolene suture on the edge of the rectal stump, using 12 to 16 clips and a disposable skin stapler. This technique is satisfactory for very low anterior resection.

Analyses of Gerstmann-Straussler syndrome with 102Leu219Lys using monoclonal antibodies that specifically detect human prion protein with 219Glu.

Two monoclonal antibodies that specifically detect human prion protein (PrP) were developed. The epitope of both antibodies was mapped using fusion proteins of glutathione-S-transferase and PrP peptides to the C-terminal region encompassing the polymorphic 219 residue. The antibodies recognized human PrP with 219Glu but not that with 219Lys. The unique property of the antibodies was utilized to determine the allelic origin of abnormal PrP deposited in the brain of a patient with Gerstmann-Straussler syndrome (GSS) with 102Leu/219Lys encoded by the same allele. Abnormal PrP was exclusively of mutant allelic origin, suggesting that 219Lys may be permissive to the formation of abnormal PrP in GSS. The antibodies may help to explore the relationship of 219Glu/Lys polymorphism to the pathogenesis of human prion diseases.

Effects of Yokuinin on the therapeutic efficacy of a new benzoxazinorifamycin KRM-1648 against Mycobacterium avium infection.

The Chinese traditional medicine, Yokuinin, which has anti-inflammatory effects and anti-human papilloma virus activity, was examined for its effects on the therapeutic efficacy of a benzoxazinorifamycin KRM-1648 (KRM) against Myobacterium avium infection in mice. Reverse transcription-PCR analysis revealed that Yokuinin increased the mRNA expression of all test cytokines in lung tissues of infected ice at week 8, in the order transforming growth factor-beta (TGF-beta) > IFN-gamma > TNF-alpha > IL-10. Mice given Yokuinin in combination with KRM had higher levels of TFG-beta mRNA expression than did mice given KRM alone, indicating that TGF-beta plays an important role in the expression of the anti-inflammatory effect of Yokuinin in vivo. Yokuinin reduced IL-10 production by M. avium-infected macrophages ph. (M phis) but did not affect M phi TFG-beta production. Although Yokuinin significantly modified cytokine expression in M. avium-infected mice, this drug did not influence the therapeutic efficacy of KRM against M. avium infection, suggesting that administration of Yokuinin in combination with KRM to the patients with M. avium infection does not cause severe disadvantages.

Thrombolytic therapy of synthetic graft occlusions before vascular reconstructive procedures.

The objective of this study was to evaluate the impact of thrombolysis of synthetic grafts before urgent vascular reconstruction. In 29 patients, 41 thrombosed synthetic grafts that underwent intraarterial thrombolysis were studied. The cases were divided into three groups: group I--complete thrombolysis followed by reconstruction; group II--complete thrombolysis alone; and group III--incomplete lysis requiring reconstruction or sympathectomy. Follow-up ranged from 1 to 556 days (mean: 149 days). Kaplan-Meier analysis was used to determine patency and limb salvage rates. One-year patency and limb salvage rates were 53% and 95%, 34% and 67%, and 38% and 48%, respectively, for groups I, II, and III. Eighteen complications occurred in 16 of the 41 (39%) episodes. One patient died of intracranial hemorrhage. The best results were achieved when complete lysis was followed by appropriate reconstruction. Patency was equally poor in complete thrombolysis alone and reconstructions required by incomplete thrombolysis. Limb salvage was better after complete thrombolysis, regardless of the appropriate reconstruction.

Anti-Mycobacterium tuberculosis activities of new fluoroquinolones in combination with other antituberculous drugs.

OBJECTIVES: Studies were undertaken in order to assess the anti- Mycobacterium tuberculosis (MTB) activities of newly developed fluoroquinolones in combination with other antituberculous drugs. METHODS: A new C-8-methoxyl fluoroquinolone, gatifloxacin (GFLX), and a new C-8-chloro fluoroquinolone, sitafloxacin (STFX), in combination with other drugs were examined for their activities against extracellular growing MTB organisms and those replicating in RAW264.7 macrophages (RAW-M phis s). RESULTS: STFX but not GFLX potentiated the activities of rifampin and rifalazil against extracellular MTB. Both GFLX and STFX exhibited combined activities against intramacrophage MTB, when used in combination with rifampin, rifalazil, isoniazid, pyrazinamide, ethambutol, streptomycin, or clofazimine. CONCLUSIONS: Although the observed combined effects varied to some extent from case to case depending on drug combinations, the present findings suggest the usefulness of these new fluoroquinolones in multi-drug regimens for tuberculosis patients.

Comparative roles of macrophages and NK cells in the host innate resistance of mice to Mycobacterium fortuitum infection.

OBJECTIVES: Profiles of host innate resistance to Mycobacterium fortuitum (MFT) infection in mice and the roles of macrophages (Mphis) and NK cells in host resistance to MFT infection were studied. METHODS: MFT-infected mice with or without the treatments to reduce Mphis and NK cells were examined for survival and the bacterial loads in the kidneys during the course of infection. RESULTS: A unique profile of strain difference was found in the innate resistance of mice to MFT. A/J, C3H/He and DBA/2 mice were susceptible, while BALB/c, B10A and C57BL/6 mice were resistant, in terms of survival after MFT infection. Such profiles of host resistance to MFT were essentially correlated with the ability of individual strain mice to prevent the bacterial growth in the early periods after infection. These profiles were different from the strain difference controlled by Bcg gene. Studies using carrageenan, anti-asialo GM1 antibody, and NK cell-deficient beige mice indicated the important roles of Mphis and NK cells in the host innate defense against MFT. CONCLUSIONS: These findings suggest that Bcg gene does not control the host resistance to MFT and that both Mphis and NK cells play crucial roles in the host innate resistance to MFT infection.

Changing patterns in colorectal carcinoma: a 25-year experience.

The pattern of colorectal carcinoma, especially with respect to stage and tumor location, has changed noticeably over the past 25 years. During that time period, 1,959 patients came to Hahnemann University Hospital with colorectal cancer, 1,584 of whom were reviewed in this study. There was a significant relationship between extent of disease and date of diagnosis, with the trend being toward decreasing stage at the time of diagnosis. In addition, there has been a demonstrable "rightward shift" in tumor location, especially over the past 15 years. This has been accompanied by a slight increase in the detection of rectal lesions as well. The trend toward earlier and more proximal lesions is likely due in large part to the increasingly widespread use of surveillance colonoscopy and sigmoidoscopy.

Revascularized diabetic limbs: positional changes in regional perfusion index.

We measured pre- and postoperative ankle:brachial index (ABI), regional perfusion index (RPI = foot/chest transcutaneous oxygen tension [TcpO2]), and variation in RPI with limb elevation in 22 ischemic lower extremities of 20 patients to compare ABI and RPI measurements for quantifying limb perfusion and analyze perioperative positional changes in RPI. Measurements were compared, using t tests, with all limbs grouped according to severity of clinical ischemia and, again, according to presence or absence of diabetes. Preoperative mean and mean post-revascularization increases in ABI values ranged from 0.27 to 0.48 and 0.40 to 0.54, respectively; corresponding RPI values ranged from 0.18 to 0.45 and 0.48 to 0.60, respectively. Pre- and postoperative decreases in RPI with elevation ranged from 0.07 to 0.11 and 0.11 to 0.23, respectively. ABI and RPI values were equally effective in assessing clinical ischemia preoperatively and increased perfusion postoperatively, regardless of degree of ischemia or diabetes. Upon elevation, all limbs exhibited larger decrements in blood flow to the skin postoperatively compared to preoperatively, as estimated by RPI. However, postoperative positional decrease in RPI was greater in diabetics compared with nondiabetics (0.23 +/- 0.12 vs 0.12 +/- 0.06; P < 0.05), suggesting postoperative elevation of diabetic limbs with ischemic skin lesions may be unadvisable.