Gain of function in Mycobacterium bovis BCG Moreau due to loss of a transcriptional repressor.

The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1ß and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.

Gain of function in Mycobacterium bovis BCG Moreau due to loss of a transcriptional repressor

The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1β and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.

Tratamiento antiangiogénico del cáncer / Anatiangiogenic treatment of cancer

El proceso de formación de nuevos vasos a partir de los capilares preexistentes es lo que se denomina angiogénesis. La angiogénesis es un proceso complejo que involucra a distintas células, componentes solubles y factores de la matriz extracelular y tiene gran importancia en una gran variedad de procesos fisiológicos y patológicos del organismo. La regulación de la angiogénesis se lleva a cabo mediante un perfecto equilibrio entre la producción y la liberación de diversos factores estimulantes e inhibidores, que varían en función de las necesidades y el tipo de tejido. Un número elevado de enfermedades se caracteriza por alteraciones del proceso angiogénico, tanto por insuficiencia como por exceso de angiogénesis. Hace ya más de un siglo que se observó que la proliferación de los vasos sanguíneos era necesaria para el crecimiento tumoral. El tratamiento antiangiogénico realizaría una acción antitumoral de forma indirecta mediante la inhibición de la vascularización del tumor e impidiendo que se le aporten los nutrientes necesarios para su crecimiento y desarrollo (AU)

The process of formation of new vessels from pre-existing capillaries is called angiogenesis. Angiogenesis is a complex process which involves distinct cells, soluble components and factors related to the extra-cellular matrix and which is highly important in a large variety of physiological and pathological processes in the body. Angiogenesis regulation takes place through a perfect equilibrium between the production and release of different stimulatory and inhibitory factors which vary in relation to needs and tissue types. A large number of diseases are characterized by alterations in the angiogenic process, either by an insufficiency or by excessive angiogenesis. The requirement of blood vessel proliferation for tumor growth was observed more than a century ago. Angiogenic treatment would have an indirect antitumoral action, inhibiting tumor vascularization and impairing the supply of essential nutrients for tumoral growth and development (AU)

Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells.

Helicobacter pylori colonizes the human stomach and causes gastric disease. The resulting gastric damage is a multi-step process involving several molecular factors and different target cells. Th1 cytokines released by neutrophils and lymphoid cells that infiltrate gastric mucosa, nitric oxide production and inducible nitric oxide synthase (iNOS) are associated with immune activation and tissue injury. Many other molecular processes such as apoptosis, as well as angiogenic factors and integrins, are involved in H. pylori pathogenesis. We used cancer gastric cells AGS and MKN as experimental models to evaluate apoptotic rates, iNOS gene expression with and without the presence of interferon-gamma (IFN-gamma), placenta growth factor gene expression and alphav modulation. Our results show that AGS cells stimulated with H. pylori underwent apoptosis. Moreover, the addition of IFN-gamma caused a further increase in iNOS gene expression and in the apoptotic rates. We also found early modulation in PlGF and alphav expression, and noted that p53 and bax gene expression was involved in the apoptotic process. Taken together, these findings demonstrate that H. pylori employs a series of mechanisms to avoid the host defense and cause gastric mucosa damage. One H. pylori pathogenic mechanism for causing gastric damage is the induction of iNOS-dependent apoptosis that is strongly enhanced by IFN-gamma. Thus, data obtained indicate that Th1 cytokines such as IFN-gamma, via modulation of iNOS gene expression, may contribute to an increase in the pathogenicity of H. pylori infections.

Oxygen regulates invasiveness and virulence of group B streptococcus.

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (t(d)) of 1.8 h than at a slower t(d) of 11 h. Invasion was optimal when GBS was cultured at a t(d) of 1.8 h in the presence of >or=5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t(d) of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t(d) of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.

Bacillus Calmette-Guerin initiates intracellular signaling in a transitional carcinoma cell line by cross-linking alpha 5 beta 1 integrin.

PURPOSE: The adherence of bacillus Calmette-Guerin (BCG) to the surface of transitional carcinoma tumor cells initiates nuclear factor (NF)-kappa B signal transduction pathways that modulate the expression of proteins important in the antitumor response to BCG. We tested the hypothesis that BCG initiates NF-kappa B signaling as a consequence of cross-linking alpha 5 beta 1 integrin receptors present on the tumor cell surface. MATERIALS AND METHODS: The effect of alpha 5 beta 1 antibody mediated cross-linking on interleukin (IL)-6 mRNA expression, IL-6 promoter activation and activation of a specific NF-kappa B reporter construct was determined. A series of reporter constructs containing nonfunctional mutations in the AP-1, NF-IL-6 and NF-kappa B sites were used to determine the relative importance of these response elements in alpha 5 beta 1 cross-linking mediated activation of the IL-6 promoter. A final series of experiments assessed the role of alpha 5 beta 1 receptor occupancy by fibronectin (FN) in initiating antibody or BCG mediated signaling. RESULTS: Anti alpha 5 and anti beta 1 mediated cross-linking of alpha 5 beta 1 integrin initiated NF-kappa B signaling, IL-6 promoter activation and IL-6 mRNA expression. Deletion mutants demonstrated that alpha 5 beta 1 cross-link initiated, IL-6 promoter transactivation required intact NF-kappa B and AP-1 response elements. Receptor occupancy by FN was required for BCG but not for antibody initiated signaling. CONCLUSIONS: Cross-linking the alpha 5 beta 1 receptor present on the surface of human transitional carcinoma cells lines initiates signal transduction in a manner identical to that observed for BCG. We propose a model in which multiple FN binding sites present on BCG interact with alpha 5 beta 1 receptor bound FN molecules to cross-link alpha 5 beta 1 receptors and initiate intracellular signaling.

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific.

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.

Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1.

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8-1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.

Characterization of enteroaggregative Escherichia coli strains isolated from children with acute diarrhea, in Mendoza, Argentina.

Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4% sensitivity and 78.26% specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.

Characterization of enteroaggregative Escherichia coli strains isolated from children with acute diarrhea, in Mendoza, Argentina

Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4 sensitivity and 78.26 specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.(AU)

Genes of non-typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines.

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.

Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin-producing strain of Actinobacillus actinomycetemcomitans.

The periodontal pathogen Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT), a complex multicomponent toxin that arrests the growth of many types of eukaryotic cell. The kinetics of the effects of CDT-containing extracts, from an invasive strain of this bacterium, were examined on epithelial-like cells routinely used in invasion studies. Both KB and HEp-2 cells were exquisitely sensitive to the effects of the CDT with TD50 of 30 and 300 pg of total bacterial protein, respectively. Initial cell morphology changes were relatively rapid, occurring within the first 13 h of exposure. CDT-treated KB cells increased in size to 4-5 times the size of untreated controls. Cytotoxicity was irreversible when attached cells were incubated, for a minimum of 120 min, with nanogram quantities of CDT-containing extract. As cultures aged, the cells became more resistant to the effects of the CDT-containing extracts. These findings have important implications for understanding the ability of A. actinomycetemcomitans to invade and multiply in epithelial cells.

Optimal treatment of systemic bacillus Calmette-Guérin infection: investigations in an animal model.

PURPOSE: Hematogenous spread of bacillus Calmette-Guerin (BCG) after intravesical instillation for bladder cancer is rare but it may result in systemic infection and hypersensitivity reaction. We investigated fluoroquinolones and steroids in an animal model to improve the therapeutic options in local and systemic BCG infection. Furthermore, the antitumor effectiveness of intravesical BCG with simultaneous application of fluoroquinolones and/or steroids was tested. MATERIALS AND METHODS: Oral antimicrobial therapy with and without steroids was started immediately after intraperitoneal injection using fluoroquinolones or trimethoprim-sulfamethoxazole. To evaluate the therapeutic options against a hyperergic reaction after repeat systemic BCG infection re-challenge was performed with intraperitoneal BCG 7 days after primary infection and oral therapy was given with fluoroquinolones or trimethoprim-sulfamethoxazole with and without steroids. The influence of continuous oral fluoroquinolone therapy on the antitumor effect of BCG was also tested in the MB 49 orthotopic murine bladder tumor model. RESULTS: After primary systemic infection fluoroquinolone therapy alone led to significantly prolonged survival in mice (log rank test p = 0.041), whereas trimethoprim-sulfamethoxazole was ineffective. There was no additional effect of steroid administration. Steroids alone led to premature death (log rank test p = 0.022). After secondary BCG infection only steroid treated animals had prolonged survival (log rank test p = 0.032), whereas antimicrobials alone had no effect. The therapeutic efficacy of BCG in the orthotopic bladder tumor model was not affected by continuous oral fluoroquinolones in terms of survival (log rank test p = 0.001) or bladder weight (Wilcoxon test p = 0.001) compared with untreated controls. CONCLUSIONS: In a mouse model fluoroquinolones had a beneficial effect for primary systemic BCG infections, whereas the hyperergic reaction after repeat BCG infection was susceptible only to steroids. Administering fluoroquinolones during an intravesical treatment course does not affect the antitumor efficacy of BCG.

Type 1, P and S fimbriae, and afimbrial adhesin I are not essential for uropathogenic Escherichia coli to adhere to and invade bladder epithelial cells.

Fimbrial (type 1, P, and S) and afimbrial adhesins, the unique virulence traits of uropathogenic Escherichia coli (UPEC), are well recognized for their role in the initial step of uropathogenesis. In this study, we investigated whether these adhesins are dispensable for UPEC in adherence and invasion of uroepithelial cells by using E. coli isolates (n=40) from cystitis patients and T-24 cells, the bladder carcinoma cell line. We found all isolates adherent to T-24 cells within 15 min of infection. In invasion assay, all isolates could invade T-24 cells to a variable degree; 22.5% of them were found highly invasive. About 33% of isolates that do not have any recognized adhesins were as invasive as other isolates. The amplitude of invasiveness was also independent of the adhesins. In conclusion, this study demonstrates that type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesin I are not required for UPEC to adhere to and invade uroepithelial cells.

Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics.

Mycoplasmal contamination of cell culture systems continues to present major problems for basic research and for manufacturing of bioproducts. Previous work suggested that certain antibiotics have strong anti-mycoplasma properties and raised the prospect that the technically rather simple antibiotic treatment may be an appropriate means for mycoplasma eradication. We have developed and validated an effective strategy to eliminate mycoplasma from chronically infected cell cultures using antibiotics which have shown strong activity against these contaminants. Here, we describe our experience with the treatment of 123 consecutive mycoplasma-positive leukemia-lymphoma cell lines, comparing five different antibiotic regimens (in total 433 treatments). We optimized the antibiotic dose schedules and the duration of treatments. The various antibiotic treatments which were employed in parallel had a high efficacy, as 71% to 86% of the infected cultures were cleansed. Treatment failure may result from the resistance of the mycoplasmas to antibiotic therapy and the inability of the eukaryotic cells to survive the cytotoxic effects of the antibiotics. Resistance to mycoplasma eradication was observed in 3% to 20% of the cultures. Loss of the cell culture caused by cytotoxicity was seen in 3% to 11% of the treatments. With regard to the overall outcome, 96% of the cell lines were rendered mycoplasma-free with at least one of the antibiotic treatments and were permanently cured. In conclusion, antibiotic treatment represents the most practical and efficient option to cleanse mycoplasma-positive cell lines.

Detection of mycoplasma in leukemia-lymphoma cell lines using polymerase chain reaction.

The specific, sensitive and reliable detection of mycoplasma contamination in cell cultures is an important part of mycoplasma control. We have sought to develop and validate a method for mycoplasma detection which is sensitive and accurate, but also practical in the sense of time spent, costs, and applicability in the standard laboratory; finally, the method should be suitable for screening large numbers of test specimens. To that end, we adapted a previously developed polymerase chain reaction (PCR) method for daily routine application. This single-step PCR uses a mixture of primers annealing to gene sequences coding for evolutionarily conserved 16S rRNA of different mycoplasma species, including the ones most commonly found in cell cultures. An internal control was introduced to exclude any false-negative tests resulting from technical PCR problems. This mycoplasma detection by PCR has been validated prospectively on 201 consecutive leukemia-lymphoma cell lines received at the institute over a 3-year period and on 118 initially positive cell lines after anti-mycoplasma treatment with antibiotics. The sensitivity (detection of true positives) of this PCR detection assay was 96% and the specificity (detection of true negatives) was also 96%, with positive and negative predictive values (probability of correct result) of 86% and 99%, respectively. PCR defined the mycoplasma status with 96% accuracy (detection of true positives and true negatives). Besides the high sensitivity and specificity, further attractive features of the PCR approach are the ease and speed with which large numbers of specimens can be tested. PCR mycoplasma analysis provides a readily available, quick and reliable test system with which to manage the important issue of mycoplasma contamination of cell lines.

Effect of prolonged treatment with azithromycin, clarithromycin, or levofloxacin on Chlamydia pneumoniae in a continuous-infection Model.

Persistent infections with Chlamydia pneumoniae have been implicated in the development of chronic diseases, such as atherosclerosis and asthma. Although azithromycin, clarithromycin, and levofloxacin are frequently used for the treatment of respiratory C. pneumoniae infections, little is known about the dose and duration of therapy needed to treat a putative chronic C. pneumoniae infection. In this study, we investigated the effect of prolonged treatment with azithromycin, clarithromycin, or levofloxacin on the viability of C. pneumoniae and cytokine production in an in vitro model of continuous infection. We found that a 30-day treatment with azithromycin, clarithromycin, and levofloxacin at concentrations comparable to those achieved in the pulmonary epithelial lining fluid reduced but did not eliminate C. pneumoniae in continuously infected HEp-2 cells. All three antibiotics decreased levels of interleukin-6 (IL-6) and IL-8 in HEp-2 cells, but this effect appeared to be secondary to the antichlamydial activity, as the cytokine levels correlated with the concentrations of microorganisms. The levels of IL-1beta, IL-4, IL-10, tumor necrosis factor alpha, and gamma interferon were too low to assess the effect of antibiotics. These data suggest that the dosage and duration of antibiotic therapy currently being used may not be sufficient to eradicate a putative chronic C. pneumoniae infection.

Expression profiles of 10 members of Frizzled gene family in human gastric cancer.

Frizzled (FZD) genes encode seven-transmembrane type WNT receptors, which are implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, and FZD10. Here, we investigated expression profiles of all members of the FZD gene family in human gastric cancer. FZD mRNAs were detected by one-step cDNA-PCR. Specificity of cDNA-PCR was confirmed by nucleotide sequence analyses of cDNA-PCR products. Among seven gastric cancer cell lines, FZD7 was up-regulated in MKN7, which was consistent with a previous report. FZD5 was up-regulated in MKN45. FZD9 and FZD10 were up-regulated together in TMK1 and MKN74. FZD2 was up-regulated in TMK1, MKN7, MKN28, MKN45, MKN74 and KATO-III. Among 10 cases of primary gastric cancer, FZD9 was up-regulated in 2 cases, FZD2 and FZD8 were up-regulated in 4 cases. Effects of Helicobacter pylori (H. pylori) on expression of FZDs were further investigated, and it was revealed that FZDs were not up-regulated by H. pylori in MKN45 cells. These results indicate that FZD2, FZD8, and FZD9 might play key roles in human gastric cancer.

Up-regulation of WNT10A by tumor necrosis factor alpha and Helicobacter pylori in gastric cancer.

WNT signaling pathway is implicated in carcinogenesis and embryogenesis. We have previously cloned and characterized WNT10A and WNT6, which are clustered in human chromosome 2q35 region. In this study, we investigated expression of WNT10A and WNT6 in gastric cancer. The 3.0- and 2.4-kb WNT10A mRNAs were expressed in gastric cancer cell lines MKN7, MKN45 and MKN74. The 2.0-kb WNT6 mRNA was expressed in gastric cancer cell lines MKN28 and MKN74. WNT10A was up-regulated in 3 out of 6 cases of primary gastric cancer, while WNT6 was not up-regulated in primary gastric cancer. Effects of inflammatory cytokines and Helicobacter pylori (H. pylori) on expression of WNT10A and WNT6 were next investigated. Interferon gamma (IFNgamma) failed to induce up-regulation of WNT10A and WNT6. Tumor necrosis factor alpha (TNFalpha) induced up-regulation of WNT10A in MKN45 cells. Up-regulation of WNT10A reached maximum at 6 h after TNFalpha treatment. H. pylori also induced up-regulation of WNT10A in MKN45 cells. These results strongly suggest that up-regulation of WNT10A induced by TNFalpha and H. pylori might play key roles in human gastric cancer through activation of WNT--beta-catenin--TCF signaling pathway.

Mechanism of therapeutic effectiveness of cefixime against typhoid fever.

beta-Lactams have been considered ineffective against organisms growing inside mammalian cells because of their poor penetration into cells. However, cefixime has been shown to be clinically effective against typhoid fever. The probable mechanism of therapeutic effectiveness of cefixime against typhoid fever was investigated using Salmonella enterica serovar Typhimurium instead of S. enterica serovar Typhi both in a cellular and in a mouse infection model. Cefixime was able to inhibit the growth of serovar Typhimurium inhabiting monocyte-derived THP-1 cells. Elongation of serovar Typhimurium in THP-1 cells was observed microscopically. Apparent morphological changes of serovar Typhimurium in THP-1 cells were also observed by electron microscopy. The concentration of cefixime inside THP-1 cells was almost half (46 to 48%) of the concentration outside the cells when serovar Typhimurium coexisted in the solution. The length of time after oral dosing (8 mg/kg) that cefixime was present-calculated from levels in serum-at a concentration above the MIC at which 90% of the serovar Typhi organisms inside human cells were inhibited was presumed to be more than 12 h. Cefixime also showed excellent activity in the mouse systemic and oral infection models based on infections caused by serovar Typhimurium. It is concluded that a fair amount of cefixime can enter mammalian cells and inhibit the growth of bacteria inside cells when the bacteria are sensitive enough to cefixime, as are serovars Typhimurium and Typhi.