Ganglioside GM3 deficiency enhances mast cell sensitivity.

Mast cells are a significant source of cytokines and chemokines that play a role in pathological processes. Gangliosides, which are complex lipids with a sugar chain, are present in all eukaryotic cell membranes and comprise lipid rafts. Ganglioside GM3, the first ganglioside in the synthetic pathway, is a common precursor of the specifying derivatives and is well known for its various functions in biosystems. Mast cells contain high levels of gangliosides; however, the involvement of GM3 in mast cell sensitivity is unclear. Therefore, in this study, we elucidated the role of ganglioside GM3 in mast cells and skin inflammation. GM3 synthase (GM3S)-deficient mast cells showed cytosolic granule topological changes and hyperactivation upon IgE-DNP stimulation without affecting proliferation and differentiation. Additionally, inflammatory cytokine levels increased in GM3S-deficient bone marrow-derived mast cells (BMMC). Furthermore, GM3S-KO mice and GM3S-KO BMMC transplantation showed increased skin allergic reactions. Besides mast cell hypersensitivity caused by GM3S deficiency, membrane integrity decreased and GM3 supplementation rescued this loss of membrane integrity. Additionally, GM3S deficiency increased the phosphorylation of p38 mitogen-activated protein kinase. These results suggest that GM3 increases membrane integrity, leading to the suppression of the p38 signalling pathway in BMMC and contributing to skin allergic reaction.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.

Functional comparison of the human epidermal growth factor receptor and telomerase reverse transcriptase promoters in canine tumor cells.

We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far. Here, we compared the activity of these promoters in canine mammary tumor and urothelial carcinoma cells. Our results showed that compared with the TERT promoter, the hEGFR promoter was more useful as a tumor-specific promoter to induce efficient transgene expression in canine tumor cells.

The membrane-type estrogen receptor G-protein-coupled estrogen receptor suppresses lipopolysaccharide-induced interleukin 6 via inhibition of nuclear factor-kappa B pathway in murine macrophage cells.

The female sex hormone estrogen exerts anti-inflammatory effects. The G-protein-coupled estrogen receptor (GPER) has been recently identified as a novel membrane-type estrogen receptor that can mediate non-genomic estrogenic effects on many cell types. We previously demonstrated that GPER inhibits tumor necrosis factor alpha-induced expression of interleukin 6 (IL-6) through repression of nuclear factor-kappa B (NF-κB) promoter activity using human breast cancer cells. Although several reports have indicated that GPER suppresses Toll-like receptor-induced inflammatory cytokine expression in macrophages, the molecular mechanisms of the inhibition of cytokine production via GPER remain poorly understood. In the present study, we examined GPER-mediated inhibition of IL-6 expression induced by lipopolysaccharide (LPS) stimulation in a mouse macrophage cell line. We found that the GPER agonist G-1 inhibited LPS-induced IL-6 expression in macrophage cells, and this inhibition was due to the repression of NF-κB promoter activity by GPER. G-1 treatment also decreased the phosphorylation of inhibitor of κB kinases. Among the mitogen-activated protein kinases, the phosphorylation of c-jun N-terminal kinase (JNK) was increased by G-1. These findings delineate the novel mechanism of the inhibition of LPS-induced IL-6 through GPER-activated JNK-mediated negative regulation of the NF-κB pathway in murine macrophage cells, which links anti-inflammatory effects to estrogen.

Expression of HIF-1α ODD domain fused canine caspase 3 by EGFR promoter-driven adenovirus vector induces cytotoxicity in canine breast tumor cells under hypoxia.

Adenovirus (Ad) vectors are widely used in cancer gene therapies. However, compared to human patients, relatively limited information is available on gene transduction efficiency or cell-specific cytotoxicity in canine tumor cells transduced with Ad vectors. Since epidermal growth factor receptor (EGFR) is highly expressed on canine breast tumor cells, we sought to develop an Ad vector based on the RGD fiber-mutant adenovirus vector (AdRGD) that expresses canine caspase 3 under the control of EGFR promoter. The aims of this study were to achieve high transduction efficiency with transgene expression restricted to canine breast tumor cells. Using EGFR promoter-driven AdRGD, we were able to restrict transgene expression to canine breast tumor cells with no evidence of expression in normal cells. Canine breast tumor cells transduced with EGFR promoter-driven AdRGD carrying canine caspase 3 gene showed cytotoxic activity. We constructed a second AdRGD vector that expressed oxygen-dependent degradation (ODD)-caspase 3 under the control of the EGFR promoter; the fusion protein contains a core part of the ODD domain of hypoxia inducible factor-1 alpha (HIF-1α) fused to caspase 3. Transduction of canine breast tumor cells with EGFR promoter-driven AdRGD expressing ODD-caspase 3 induced a higher rate of cell death under hypoxic conditions compared with under normoxia. The results indicate that the EGFR promoter-driven AdRGD vectors will be of value for tumor-specific transgene expression and safe cancer gene therapy in dogs.

Detection of Bcl-2 mRNA and its product in the glomerular podocytes of the normal rat kidney.

Podocyte apoptosis underlies podocytepenia leading to glomerulosclerosis. An apoptosis inhibitory protein Bcl-2 is expressed in the podocytes in the early stage of nephrogenesis and downregulated in the maturing stage of human fetal kidneys. Recent studies reported changed localization and expression of Bcl-2 in the renal glomeruli under the pathologic conditions. This study aimed to confirm in situ localization of Bcl-2 mRNA and its product in the glomeruli, and to demonstrate the local expression of Bcl-2 mRNA in normal rat glomeruli. Paraffin sections of the kidneys from normal male Wistar rats were immunostained by anti-Bcl-2 monoclonal antibody. The localization of Bcl-2 mRNA in the glomeruli was evaluated by in situ hybridization. The glomeruli were dissected from frozen sections of the kidneys with the laser microdissection (LMD) system. Total RNA extracted from 10, 100 or 200 dissected glomeruli was used for reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. Bcl-2 mRNA and its product were detected in the podocytes but barely in the mesangial cells. In RT-PCR, the specific-sized bands of Bcl-2 from 100 or 200 dissected glomeruli were clearly observed. Real-time PCR for Bcl-2 showed that cDNA from 100 or 200 dissected glomeruli became amplified at 36 or 33cycles, respectively. Bcl-2 is expressed in the glomerular podocytes of the normal rat kidney and quantitative analysis of Bcl-2 mRNA in the renal glomeruli is possible using the LMD technique.

First case report of histoplasmosis in a cat in Japan.

Cytological, histopathological and immunohistochemical examinations were carried out on a presumed 10-year-old Japanese cat showing vomiting and emaciation. On cytologic examination of the mass of the upper abdominal cavity, many yeast-like organisms were detected in the macrophages. At necropsy, the upper part of colon was markedly dilated with a thickened wall. The lung did not show significant changes. Histologically, severe necrotic and granulomatous lesions were observed in the colon. In the colonic lesion, the cytoplasm of the macrophages contained yeast-like organisms with irregularly shaped dots, and the cell walls of these organisms were stained black by Grocott-Gomori methenamine-silver stain. Immunohistochemically, they were found to be positive for anti-histoplasma yeast antibody. This is the first report of feline histoplasmosis in Japan.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-beta(1) at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 muM SB431542, an inhibitor of activin and TGF-beta type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-beta(1), whereas <0.5 microM SB431542 effectively reduced TGF-beta(1)-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-beta-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-beta pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-beta occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-beta signaling.

Requirement of Smad3 for mast cell growth.

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.

Involvement of p38 MAP kinase and Smad3 in TGF-beta-mediated mast cell functions.

Transforming growth factor-beta (TGF-beta) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-beta-mediated cell responses in BMMCs. Treating BMMCs with TGF-beta induced the phosphorylation of p38 within 2 h and persisted for 24 h. The involvement of p38 in TGF-beta-induced cell responses depended upon mast cell functions; it was necessary for up-regulation of mmcp-1 and migration, but not for up-regulation of mmcp-7 and inhibition of metabolic activity. New protein synthesis was required for the up-regulation of mmcp-1 but not mmcp-7 in response to TGF-beta treatment, and stabilization of mRNA was partially responsible for the increase in gene transcript of mmcp-1. The decrease in metabolic activity in response to TGF-beta treatment was smaller in Smad3-deficient BMMCs compared to wild-type BMMCs. Maximal migration was detected at a TGF-beta concentration of 40 fM in wild-type BMMCs, whereas TGF-beta-induced migration was absent in Smad3-deficient BMMCs. Thus, the roles of p38 and Smad3 are different among TGF-beta-mediated cell responses in BMMCs.

Transcriptional regulation of mouse mast cell protease-7 by TGF-beta.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt -126 to -122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-beta pathway. In addition, a region from nt -104 to -98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-beta signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-beta. Treatment with TGF-beta in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-beta stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-beta pathway.

Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor-beta, activin A and microphthalmia-associated transcription factor.

Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-beta1, and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-beta1 did not increase PAI-1 mRNA levels in mast cells from mi/mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-beta1-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-beta1, whereas transcriptional activation by TGF-beta1 was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-beta family, enhanced transcription induced by MITF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-beta1 and activin A treatment was also detected in B16 melanocytes; TGF-beta1 transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-beta1 and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner.

Responses during cell preparation for functional analyses in mouse bone marrow-derived cultured mast cells.

Murine bone marrow-derived cultured mast cells (BMMCs) are most widely used in in vitro experiments for evaluation of mast cell functions. The present study has shown that cell preparation procedure, i.e., cell collection by centrifugation and the subsequent adjustment and culture of cell density at the desired concentrations, transiently induced gene expression of plasminogen activator inhibitor-1 (PAI-1) and the AP-1 components (c-fos, c-jun, and junB). The level of PAI-1 gene transcript was closely related to the cell density and the gene expression was enhanced by pretreatment with okadaic acid, an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A). The cell preparation procedure also caused dephosphorylation of MAP kinases, i.e., ERK, p38, and JNK, resulting from PP1/PP2A activation. In view of the cell responses to the cell preparation procedure itself, care is needed in the interpretation of in vitro data using BMMCs.

Up-regulation of mouse mast cell protease-6 gene by transforming growth factor-beta and activin in mast cell progenitors.

Previous studies have revealed that members of the transforming growth factor-beta (TGF-beta) including TGF-beta1 and activin A modulate the function of mast cells. Here we show the up-regulation of mouse mast cell protease-6 (mMCP-6), which is expressed in differentiated mast cells, by TGF-beta1 and activin A in bone marrow-derived cultured mast cell progenitors (BMCMCs). Quantitative real time RT-PCR analyses revealed that the mRNA level of mMCP-6 was slightly but reproducibly increased by treatment with TGF-beta1 or activin A, which was regulated at the transcription level. Reporter assays showed that Smad3, a signal mediator of the TGF-beta/activin pathway, was responsible for the transcription. The TGF-beta response element is located at -153 bp relative to the transcription initiation site, CAGA. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, the heart and skeletal muscle, also stimulated the transcription of mMCP-6. The region at -166 bp, GACCTG, was responsible for MITF-induced transcription. Mutations of the CAGA motif and the MITF responsive site indicated that the MITF site of mMCP-6 promoter is indispensable for the transcriptional activation by a constitutively active TGF-beta receptor (ALK5-TD), whereas the CAGA motif is dispensable for transcription by MITF. Transcriptional activation of mMCP-6 by the TGF-beta pathway was differently interacted with that by MITF isoform; ALK5-TD further enhanced MITF-E-induced transcription, whereas MITF-M-induced transcription abolished responsiveness to ALK5-TD. The positive regulation of mMCP-6 by the TGF-beta/activin pathway and the differential regulation by the MITF isoform suggest a rigorous regulation of mast cell function as effector cells of immune response.

Expression of tocopherol-associated protein in mast cells.

Tocopherol-associated protein (TAP) was expressed in mouse mast cells. TAP was predominantly localized in the cytoplasm, and the subcellular localization was not changed by alpha-tocopherol. The results suggest that the physiological role of TAP in mast cells is not regulation of tocopherol function but an as-yet-unidentified activity.

Effect of PCB-126 on intracellular accumulation and transepithelial transport of vinblastine in LLC-PK1 and its transformant cells expressing human P-glycoprotein.

The effects of 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB-126), which is the most toxic congener of coplanar polychlorinated biphenyls (Co-PCBs), on intracellular accumulation and transepithelial transport of vinblastine were examined in porcine kidney cells, LLC-PK1, and its transformant cells expressing human P-glycoprotein (LLC-MDR1). The accumulation decreased less than one-tenth in LLC-MDR1 compared to LLC-PK1. In both cells, the accumulation increased with the addition of PCB-126 and cyclosporine A (CYA), which are P-glycoprotein modulators, though the magnitudes were different in these two cell groups as well as for these two chemicals. Thus, PCB-126 might inhibit extrusion of vinblastine through the drug extrusion system as does CYA. In both the cells, there might be an endogenous drug extrusion system other than P-glycoprotein that was inhibited by CYA or PCB-126. The net basal-to-apical transepithelial transport of vinblastine increased 1.7-fold more in LLC-MDR1 than in LLC-PK1. By adding PCB-126 on the apical side, the transport was greatly decreased by -76% in the monolayer of both cells. By adding PCB-126 and CYA on the basal side in LLC-MDR1 monolayer, the transports increased -1.7-fold, so that PCB-126 might inhibit the extrusion of vinblastine on both the apical and basal sides. One of the causes to be considered for the adverse effects of Co-PCBs, in addition to the binding with an aryl hydrocarbon receptor, might be the modification of drug transport by its interaction with the drug transport system.

$3,3',4,4',5$ -Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein.

The effects on the drug efflux of $3,3',4,4',5$ -pentachlorobiphenyl (PCB-126), the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs), were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine ( ${\text{KB3 - Phe}};{61} $ ). In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In ${\text{KB3 - Phe}};{61} $, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 $\mu$ M PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in ${\text{KB3 - Phe}};{61} $. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

Reliability of RT-PCR methods for measuring relative gene expression in mast cells.

Three methods to quantify gene transcript levels in mast cells, real-time RT-PCR, competitive RT-PCR and conventional RT-PCR analyses, were compared. Linear regression analysis on five gene transcripts revealed that the mRNA levels measured by real-time RT-PCR analysis were minimally correlated with those by conventional RT-PCR analysis. In addition, differences in the mRNA level between samples measured by conventional RT-PCR analysis were smaller than those by real-time RT-PCR analysis, suggesting that conventional RT-PCR analysis is less sensitive at measuring mRNA levels. Results from competitive RT-PCR analysis correlated closely with those from real-time RT-PCR analysis. When the differences in mRNA level between samples are relatively smaller, however, the correlation tended to be weaker. Real-time RT-PCR analysis has higher reliability, but is expensive. In contrast, competitive RT-PCR analysis is inexpensive, but is weaker at detecting smaller differences in gene transcript level between samples. Therefore, the most appropriate analytical method to measure mRNA levels should be chosen, depending on the experimental conditions.

Contribution of mast cells to bacterial clearance and their proliferation during experimental cystitis induced by type 1 fimbriated E. coli.

Bacterial infections of the urinary bladder are very common, and the role of mast cells in these infections is invariably thought of as a detrimental one. However, recent studies have shown that mast cells play a key role in host defense against various enterobacterial infections. In this manuscript, using mast cell-deficient (WBB6F1 - W/Wv) and mast cell-sufficient (WBB6F1 - +/+) mice we have investigated the protective role of mast cells in urinary bladder infections in vivo. Our findings show that (i) the mast cells are activated by FimH-expressing E. coli, and release large amount of histamine in the urinary bladder; (ii) the number of surviving bacteria in the urine is dependent on the presence of mast cells, and (iii) mast cell number in the bladder increases following uropathogenic infection in mice which is likely due to an increase in the mast cell growth-promoting cytokine IL-3 in bacteria-activated mast cells. Taken together, these observations suggest a beneficial role of mast cells in urinary bladder infections in mice.