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PubMed2001

Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations.

Loss of heterozygosity (LOH) involving chromosomes 3p, 5q, 9p, or 17p and aberrant expression or mutation of p53 are reported previously in selected bronchial dysplasias and squamous cell cancers (SCCs). Yet, comprehensive analyses of LOH patterns at these chromosomal sites and of p53 alterations are not reported for histologically normal bronchial epithelium, high-grade bronchial dysplasia, and SCC present in the same pulmonary resections. Whether concordant or discordant genetic changes are detected in these bronchial tissues, especially when multiple high-grade dysplastic bronchial lesions are present, was studied. Genomic DNA was microdissected from eight pulmonary SCCs and high-grade dysplastic lesions that were associated with SCC. In four cases, two independent high-grade dysplastic bronchial lesions were identified. When available, histologically normal bronchial epithelium was microdissected. Germ-line genomic DNA was isolated from normal lymph nodes. LOH was assessed for 15 microsatellite markers on chromosomes 3p, 5q, 9p, or 17p, sites frequently deleted in lung cancers. Immunohistochemical p53 expression was studied and correlated with p53 DNA sequence analyses. Progressive LOH for these markers was found when SCCs were compared with high-grade dysplasia and histologically normal bronchial epithelium present in the same resections. Histologically normal bronchial specimens had LOH in up to 27% of informative markers. High-grade dysplastic lesions exhibited LOH for 18-45% and SCC had LOH for 18-73% of the markers. Common regions of LOH were found in some dysplasias compared with SCCs. In other dysplasias, discordance was found relative to SCCs, especially for p53 mutations. In cases with a single or second high-grade dysplasia associated with SCC, heterogeneity in LOH markers was detected. These concordant and discordant changes were consistent with convergent and divergent clonal selection pathways in pulmonary squamous cell carcinogenesis. Some histologically normal bronchial epithelial tissues had genetic changes more similar to those in the SCCs than in dysplastic lesions. DNA loss or mutations accumulate in SCC, but discordant genetic changes can exist in the same carcinogen-exposed bronchial tissues. These findings have implications for lung cancer prevention trials.

Bronchial Diseases↗
PubMed2005

Pulsed electron avalanche knife (PEAK-fc) for dissection of retinal tissue.

OBJECTIVE: To evaluate the effectiveness and precision of tractionless retinal tissue dissection by the advanced version of the pulsed electron avalanche knife for fine cutting (PEAK-fc; Carl Zeiss Meditec, Jena, Germany). METHODS: Porcine retina (in vivo) and human retina (in vitro) were incised with the PEAK-fc using various pulse parameters. The globes were then processed for light microscopy. Evaluation of all specimens focused on depth of the retinal cuts and on the degree of collateral damage. RESULTS: Retinal cuts performed both in vivo on porcine eyes and on human donor eyes showed very sharp edges with only little collateral damage. With probes of 600 mum in length, the optimal pulse parameters for precise and reproducible cutting of the retina were an amplitude of 350 to 380 V, a repetition rate of 300 Hz, and 30 "minipulses" per pulse of 100-microsecond duration. With increasing voltage, cuts also affected the retinal pigment epithelium and the choroid, followed by intravitreal bleeding during in vivo application. CONCLUSION: We demonstrated that PEAK-fc is capable of precisely cutting retinal tissue in vivo and in vitro using optimal pulse parameters. Further in vivo studies will be necessary to determine the efficacy of this new tractionless cutting device in vitreoretinal surgery.

Adult↗
PubMed2004

Mitochondrial DNA 4,977-bp deletion in paired oral cancer and precancerous lesions revealed by laser microdissection and real-time quantitative PCR.

Oral cancer is the fourth leading cause of cancer deaths among men in Taiwan and is closely associated with areca quid chewing habits. Recent studies showed that mitochondrial DNA (mtDNA) mutations occur in various tumors, including oral cancers, and that the accumulation of mtDNA deletions could be an important contributor to carcinogenesis. Using laser microdissection, we have analyzed mtDNA deletions by pairwise comparisons in oral cancer, precancerous cells, and their adjacent submucosal stoma tissues in 12 patients with areca quid chewing history. Real-time quantitative polymerase chain reaction (RTQPCR) was performed to detect and quantify mtDNA with the 4,977-bp deletion in the histologically defined specified cell groups. Quantitative analysis of 60 samples by RTQPCR revealed that the average proportions of 4,977-bp deleted mtDNA over total mtDNA were 0.137%, 0.367%, and 0.001% in cancer, precancer cells, and lymphocytes of lymph node biopsies, respectively. Pairwise analysis of the proportion of mtDNA deletion in cancer, precancer, and their stroma tissues revealed a consistent trend among these patients. All of the patients (12/12) presented a higher proportion of mtDNA with 4,977-bp deletion in the lesions than in the lymphocytes, with average increases of 198-fold in cancer and 546-fold in precancer cells. A decrease in the proportion of deleted mtDNA was observed in 8 of 12 patients when the disease progressed from precancer to cancer lesions. Interestingly, 7 of 12 cancer tissues and 8 of 12 precancer lesions exhibited an average of 6.3-fold and 17.4-fold increases in the proportion of 4,977-bp deleted mtDNA in the stromal cells than in the lesion cells, respectively. The observation that the proportion of 4,977-bp deleted mtDNA in all oral lesions was higher than normal and consistently decreased during cancer progression from precancer to primary cancer suggests that accumulation and subsequent cytoplasmic segregation of the mutant mtDNA during cell division may play an important role in oral carcinogenesis. This study also demonstrates that laser microdissection combined with RTQPCR is an efficient and sensitive tool to gain insight into the role that mtDNA mutation may play in carcinogenesis.

Adult↗
PubMed2005

Combined laser-assisted microdissection and short tandem repeat analysis for detection of in situ microchimerism after solid organ transplantation.

Following the transplantation of a solid organ leukocytes of donor origin migrate out of the organ, contributing to a chimeric blood cell population ("peripheral microchimerism"). At the same time, leukocytes and pluripotent precursor cells of the recipient migrate into the organ, creating an "in situ microchimerism." A method is described for the identification of cells with the recipient's genotype in the transplanted organ by combining laser-assisted microdissection and short tandem repeat analysis. The microdissection allows the contamination-free isolation of morphologically and immunohistochemically characterized cells or groups of cells from histological tissue sections. The subsequent analysis of highly polymorphic short tandem repeats enables unequivocal genotyping in nearly all donor-recipient instances. Employing this new methodological approach, we could identify in individual transplanted organs differentiated parenchymal cells of recipient's origin, which most probably are derived from circulating precursor cells from the bone marrow.

Humans↗
PubMed2002

Analysis of the gene-expression profile regarding the progression of human gastric carcinoma.

BACKGROUND: Tumor tissue consists of a variable mixture of tumor and host-cell populations. Recent developments in laser microdissection (LMD) and cDNA microarray analysis have encouraged us to study the differential gene expression profiles among normal cells, primary carcinoma cells, and metastatic carcinoma cells in cases of gastric carcinoma. METHODS: Total RNA was extracted from the cells obtained by means of LMD from the primary carcinoma, the corresponding gastric epithelium, and the lymph node metastasis in 5 cases of primary gastric carcinoma. RNA was amplified by the T7-based amplification system to be applied to a cDNA microarray. Thereafter, the differentially expressed genes among the 3 populations were evaluated. RESULTS: cDNA samples for microarray studies were successfully obtained from each cell population of 5 cases. The cDNA microarray demonstrated that several interesting genes, such as cell-cycle regulators and growth factors, were overexpressed in the metastatic cells compared with in the primary carcinoma cells. Oncogenes and cell-adhesion molecules were more overexpressed in the primary carcinoma cells than in the normal cells. On the other hand, caspase 8 and cadherin were more suppressed in the primary carcinoma cells than in the normal cells. Interestingly, among the matrix metalloproteinase family, only MMP7 was identified as a differentially overexpressed gene in both the primary carcinoma and the metastatic cells in comparison with the normal cells. CONCLUSIONS: This study demonstrated that the combined use of LMD, T7-based amplification, and a cDNA microarray enabled us to identify genes directly associated with each population of tumor tissue. The method will open up new possibilities for the precise gene analysis of tumor progression and metastasis.

Adenocarcinoma↗
PubMed2005

Histamine compartments of the Drosophila brain with an estimate of the quantum content at the photoreceptor synapse.

Reliable estimates of the quantum size in histaminergic neurons are not available. We have exploited two unusual opportunities in the fly's (Drosophila melanogaster) visual system to make such determinations for histaminergic photoreceptor synapses: 1) the possibility to microdissect successively from whole fly heads freeze-dried in acetone: the compound eyes; the first optic neuropils, or lamina; and the rest of the brain; and 2) the uniform sheaves of lamina synaptic terminals of photoreceptors R1-R6. We used this organization to count scrupulously the numbers of 30-nm synaptic vesicles from electron micrographs of R1-R6 profiles, and from microdissections we determined the regional contents of histamine in the compound eye, lamina, and central brain. Total head histamine averages 1.98 ng of which 9% was lost after freeze-drying in acetone and a further 28% after the brain was microdissected. Of the remainder, 71% was in the eye and lamina. Assuming that histamine loss from the tissue occurred mostly by diffusion evenly distributed among all regions, the overall lamina content of the head would be 0.1935 ng before dissection. From published values for the volumes of the brain's compartments, the computed regional concentrations of histamine are highest in the lamina (4.35 mM) because of the terminals of R1-R6. The concentration in the retina is approximately 13% that in the lamina, suggesting that most histamine is vesicular. There are approximately 43,500 +/- 7,400 (SD) synaptic vesicles per terminal and, if all histamine is allocated equally and exclusively among these, the vesicle contents would be 858 +/- 304 x 10(-21) moles or approximately 5,000 +/- 1,800 (SD) molecules at an approximate concentration of 670 mM. These values are compared with the vesicle contents at synapses using acetylcholine and catecholamines.

Analysis of Variance↗
PubMed2005

Frequent genomic alterations in epithelium measured by microsatellite instability following allogeneic hematopoietic cell transplantation in humans.

Although typically found in cancers, frameshift mutations in microsatellites have also been detected in chronically inflamed tissues. Allogeneic hematopoietic cell transplantation (HCT) may potentially produce chronic tissue stress through graft-versus-host reactions. We examined non-neoplastic epithelial tissues (colon, buccal) obtained 1 to 5061 days after human allogeneic HCT for the presence of genomic alterations at 3 tetranucleotide and 3 mononucleotide microsatellite loci. Novel bands indicative of microsatellite instability (MSI) at tetranucleotide repeats were detected in laser-microdissected colonic crypts and in buccal smears of 75% and 42% of patients who received an allograft, respectively. In contrast, no MSI was found in similar tissues from control subjects and from patients after intensive chemotherapy or in buccal cells from patients after autologous HCT. The MSI found in colon, which was often affected by graft-versus-host disease, was not due to loss of expression or nitrosylation of DNA repair proteins. MSI in clinically intact oral mucosa was more frequently found at later time points after HCT. MSI was also found in 3 posttransplant squamous cell cancers examined. Our data show that genomic alterations in epithelium regularly occur after allogeneic HCT and may be implicated in the evolution of posttransplantation diseases, including secondary cancer.

Adult↗
PubMed2005

Timing of critical genetic changes in human breast disease.

BACKGROUND: Breast cancer development has been characterized as a nonobligatory sequence of histological changes from normal epithelium through invasive malignancy. Although genetic alterations are thought to accumulate stochastically during tumorigenesis, little is known about the timing of critical mutations. This study examined allelic imbalance (AI) in tissue samples representing a continuum of breast cancer development to examine the evolution of genomic instability. METHODS: Laser-microdissected DNA samples were collected from histologically normal breast specimens (n = 25), atypical ductal hyperplasia (ADH, n = 16), ductal carcinoma-in-situ (DCIS, n = 37), and stage I to III invasive carcinomas (n = 72). Fifty-two microsatellite markers representing 26 chromosomal regions commonly deleted in breast cancer were used to assess patterns of AI. RESULTS: AI frequencies were <5% in histologically normal and ADH specimens, 20% in DCIS lesions, and approximately 25% in invasive tumors. Mann-Whitney tests showed (1) that levels of AI in ADH samples did not differ significantly from those in histologically normal tissues and (2) that AI frequencies in DCIS lesions were not significantly different from those in invasive carcinomas. ADH and DCIS samples, however, differed significantly (P < .0001). CONCLUSIONS: DCIS lesions contain levels of genomic instability that are characteristic of advanced invasive tumors, and this suggests that the biology of a developing carcinoma may already be predetermined by the in situ stage. Observations that levels of AI in ADH lesions are similar to those in disease-free tissues provide a genomic rationale for why prevention strategies at the ADH level are successful and why cases with ADH involving surgical margins do not require further resection.

Allelic Imbalance↗
PubMed1988

Renal cell culture.

Methods for the establishment and growth of renal cell types in culture are reviewed, with emphasis on current trends. General techniques available for the isolation and culture of glomerular cells have progressed from explant to enzyme dissociation and cloning techniques. The growth characteristics and properties of cultured glomerular endothelial, epithelial, mesangial, and bone-marrow-derived cells are discussed. Studies are described in which cultures of contractile mesangial cells have led to an elucidation of their role both in normally functioning glomeruli and in disease states. Renal tubule culture techniques also have progressed from mixed tissue explants and cell isolates to fractionation of enriched tubule populations and growth of specific, individually microdissected proximal convoluted, proximal straight, thick ascending limb of Henle's loop, and collecting tubules. The differentiated tubule epithelial-specific properties of such primary cultures are discussed in relation to those of permanently growing cell lines such as MDCK and LLC-PK1. Renal tubule cultures will be invaluable for the study of the role of hormones and extracellular matrix in epithelial growth and polarity of normal structure and function. In addition, in vitro models of cultured renal tubules have been established to study the effects of age, nephrotoxins, and anoxic injury.

Animals↗
PubMed2000

Immunostaining for cell picking and real-time mRNA quantitation.

Microdissection techniques allow a cell-type or even cell-specific mRNA analysis within complex tissues. Furthermore, valid mRNA quantitation can be performed by real-time reverse transcriptase-polymerase chain reaction from a few isolated cells obtained from cryosections. For a more precise access to many cell types, this technique has to be complemented by a cell-type-specific immunostaining. To evaluate its effect on mRNA quantitation, we analyzed alveolar macrophages (AMs) from control rat lungs and those undergoing stimulation with lipopolysaccharide and interferon-gamma nebulization. Whereas AMs from the left lung were directly harvested for mRNA extraction by bronchoalveolar lavage, tissue sections of the right lung were stained with an optimized immunofluorescence protocol detecting AMs. Fifteen AM profiles per sample were picked by laser-assisted sampling technique. Normalizing to a standard gene, nitric oxide synthase II (NOSII) and tumor necrosis factor (TNF)-alpha mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. In stimulated lungs, the percentage of picked samples positive for NOSII or TNF-alpha mRNA increased significantly. Moreover, a marked increase in the ratio of target gene mRNA to standard gene mRNA was noted for both NOSII and TNF-alpha in picked AMs from stimulated lungs, which matched very well the increase detected in the lavaged AMs undergoing direct RNA extraction. Thus, when using an optimized protocol for immunofluorescence, this approach may be reliably combined with laser-assisted cell picking and real-time mRNA quantitation in a few immunohistochemically characterized cell profiles within complex tissues.

Animals↗
PubMed2005

Expression of thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyl transferase in prostate cancer.

The enzymes thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT) are involved in the metabolism of the anticancer drug 5-fluorouracil. No reports have examined the expression of these enzymes in prostate cancer (CaP). A total of 25 previously untreated, hormone-sensitive CaP tissue samples and 11 benign prostatic hyperplasia (BPH) specimens were examined. Tissue of CaP and BPH tissue samples were obtained from formalin-fixed, paraffin-embedded sections by laser-captured microdissection, and then RNA was extracted. mRNA expression of TS, DPD, TP, and OPRT was analyzed by quantitative reverse transcriptase-polymerase chain reaction. TS and OPRT expression levels were significantly higher in CaP samples than in BPH. DPD expression level in poorly differentiated CaP was significantly lower than that in CaP with more favorable--well or moderately differentiated--histopathology.

Cell Differentiation↗
PubMed2001

Glicentin, an active enteroglucagon, has a significant trophic role on the small intestine but not on the colon in the rat.

BACKGROUND: Many experiments have indicated that the gut glucagons (enteroglucagons) are associated with cell proliferation in the small intestine. However, recent studies have failed to show trophic effects of glicentin (enteroglucagon) on the intestine. AIMS: To examine the effects of glicentin on intestinal proliferation in vivo in the rat. METHODS: Rats were established on total parenteral nutrition for 6 days. Four experimental groups were given daily doses of 1, 4, 20 and 80 microg/rat of glicentin via the jugular vein. Rats fed by total parenteral nutrition and rats fed chow ad libitum were used as controls. Tissues taken from the duodenum, jejunum, ileum and colon were fixed in Carnoy's fluid and microdissected to determine the metaphase arrest scores and crypt fission ratios. RESULTS: The mean metaphase arrest scores per crypt of the small intestine were significantly increased in the rats given 4, 20 and 80 microg of glicentin. These responses were dose-dependent, and were most prominent in the ileum. Crypt fission of the ileum was significantly decreased in the 20 and 80 microg glicentin groups. Glicentin had no effects on proliferation or fission in the colon. CONCLUSIONS: Glicentin is trophic to the rat small intestine, but not the colon.

Animals↗
PubMed2002

Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy.

PURPOSE: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy. METHODS: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis. RESULTS: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with non-dysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue. CONCLUSIONS: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.

Adolescent↗
PubMed2005

Nephrotic syndrome and renal failure after allogeneic stem cell transplantation: novel molecular diagnostic tools for a challenging differential diagnosis.

BACKGROUND: Sudden onset of nephrotic syndrome after allogeneic stem cell transplantation is rare and has been associated mostly with membranous glomerulonephritis related to chronic graft-versus-host disease (cGVHD). We report a case of nephrotic syndrome and rapidly progressive renal failure occurring in a young woman 3 years after allogeneic stem cell transplantation from her HLA-identical brother. In the renal biopsy, a diffuse mononuclear cell infiltrate was observed. Furthermore, histological analysis, immunofluorescence, and electron microscopy of the kidney specimen defined the diagnosis as minimal change disease, a T-cell-mediated glomerulopathy associated with lymphoproliferative disorders, but that has never been described as an isolated manifestation of cGVHD. METHODS: The differential diagnosis was performed by using immunohistochemistry and laser capture microdissection combined with Taq-Man quantitative polymerase chain reaction. RESULTS: Infiltrating mononuclear cells in renal tissue consisted of T cells expressing DNA levels of a Y chromosome-specific gene quantitatively similar to those observed in a male subject, showing that these cells derived from the transplant donor and definitely excluding leukemia relapse. However, the large number of infiltrating T cells allowed the possibility that in this patient, minimal change disease could be related to an atypical form of GVHD. CONCLUSION: This is the first study to use molecular techniques to show the differential diagnosis of nephrotic syndrome after allogeneic stem cell transplantation. This novel method approach might represent a key tool to characterize kidney infiltrate after allogeneic stem cell transplantation.

Acute Kidney Injury↗
PubMed2001

Molecular evidence for the independent origin of extra-ovarian papillary serous tumors of low malignant potential.

BACKGROUND: Molecular data suggest that peritoneal tumors in women with advanced-stage ovarian papillary serous adenocarcinoma are monoclonal in origin. Whether the same is true for ovarian tumors of low malignant potential is not known. We compared peritoneal and ovarian tumors from women with advanced-stage ovarian papillary serous tumors of low malignant potential to determine whether the peritoneal tumors arose from the same clone as the ovarian tumors. METHODS: We studied the clonality of 73 peritoneal and ovarian tumors from 18 women with advanced-stage ovarian papillary serous tumors of low malignant potential. Formalin-fixed, paraffin-embedded tumors and representative normal tissues were sectioned and stained with hematoxylin-eosin, representative sections from separate tumors were manually microdissected, genomic DNA was extracted from the microdissected tumors, and the polymerase chain reaction was used to amplify a CAG polymorphic site in the human androgen receptor locus on the X chromosome to determine the inactivation pattern of the X chromosome and the clonality of the tumors. RESULTS: The pattern of X-chromosome inactivation could be determined from the tumors of 13 of 18 patients. Of the 13 patients, seven (54%) had nonrandom inactivation of the X chromosome, and six of the seven had different inactivation patterns in the peritoneal and ovarian tumors. Three of these patients also had different patterns of nonrandom X-chromosome inactivation in tumors from each ovary. The remaining six patients had random patterns of X-chromosome inactivation in the peritoneal and ovarian tumors. CONCLUSIONS: Our data suggest that peritoneal and ovarian tumors of low malignant potential arise independently.

Adenocarcinoma, Papillary↗
PubMed2005

Leptin, cell proliferation and crypt fission in the gastrointestinal tract of intravenously fed rats.

Many peptides, hormones and growth factors have been implicated in the control of cell renewal in the gastrointestinal epithelium. Leptin is present in the stomach and salivary glands and leptin receptors are seen throughout the gut. Leptin can stimulate mitogen-activated protein kinase activity in vitro and short-term infusion has been reported to have a proliferative action on the colon in vivo, suggesting a biological link between obesity, physical activity and colon cancer. Food intake is one of the most important determinants of intestinal mucosal cell renewal, thus any direct effects of leptin on the gut may be hidden. This problem has been avoided experimentally by maintaining animals on total parenteral nutrition (TPN). Male Wistar rats were anaesthetized and cannulae were inserted into the jugular vein to deliver the TPN diet to which had been added 0, 0.5, 2.5, or 10 mg/kg of recombinant murine leptin. Orally fed rats were also studied. After 6 days of treatment, all animals were injected with vincristine and killed 2 h later. Tissue weight was recorded and crypt cell proliferation (arrested metaphases) and crypt fission were scored in 'microdissected' crypts. Leptin infusion led to a small decrease in body weight and in the weight of the caecum. Intestinal cell proliferation was significantly reduced by TPN when compared to the orally fed rats, but the addition of leptin had no effect on the small intestine or colon. Crypt fission was also significantly lowered in the TPN group. Fission was slightly but significantly increased in the proximal and mid-colon of the leptin-treated rats, but was decreased in the distal colon. Although leptin did not significantly alter cell proliferation, it had significant effects on the process of crypt fission in the colon, which varied according to the exact locality.

Animals↗
PubMed1995

Isolation of enteric ganglia from the myenteric plexus of adult rats.

Enteric neurons and glia cells were isolated from adult Sprague Dawley rats. A procedure is described using a combination of microdissection and mechanical dissociation after enzyme treatment which yields large numbers of cell clusters suitable for tissue culture and grafting into the injured spinal cord. Differentiated enteric ganglia remained viable for at least 5 days in vitro. Cultured neurons expressed histochemical reactivity for acetylcholinesterase and nicotinamide adenine dinucleotide phosphate diaphorase. Nestin positive glia, which represented a population of non-myelinating enteric Schwann cells, could also be identified in cultures maintained 5 days or longer in vitro. The myenteric plexus of adult rats can provide a readily available source of neurons and Schwann cells for grafting to the central nervous system.

Animals↗
PubMed2004

Spermine oxidation induced by Helicobacter pylori results in apoptosis and DNA damage: implications for gastric carcinogenesis.

Oxidative stress is linked to carcinogenesis due to its ability to damage DNA. The human gastric pathogen Helicobacter pylori exerts much of its pathogenicity by inducing apoptosis and DNA damage in host gastric epithelial cells. Polyamines are abundant in epithelial cells, and when oxidized by the inducible spermine oxidase SMO(PAOh1) H(2)O(2) is generated. Here, we report that H. pylori up-regulates mRNA expression, promoter activity, and enzyme activity of SMO(PAOh1) in human gastric epithelial cells, resulting in DNA damage and apoptosis. H. pylori-induced H(2)O(2) generation and apoptosis in these cells was equally attenuated by an inhibitor of SMO(PAOh1), by catalase, and by transient transfection with small interfering RNA targeting SMO(PAOh1). Conversely, SMO(PAOh1) overexpression induced apoptosis to the same levels as caused by H. pylori. Importantly, in H. pylori-infected tissues, there was increased expression of SMO(PAOh1) in both human and mouse gastritis. Laser capture microdissection of human gastric epithelial cells demonstrated expression of SMO(PAOh1) that was significantly attenuated by H. pylori eradication. These results identify a pathway for oxidative stress-induced epithelial cell apoptosis and DNA damage due to SMO(PAOh1) activation by H. pylori that may contribute to the pathogenesis of the infection and development of gastric cancer.

Apoptosis↗