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PubMed2001

Renal cyclooxygenase-2: evidence for recruitment of thick ascending limb of henle cells in microdissected nephron segments.

Prostaglandins participate in the regulation of sodium and water renal excretion. They are synthesized by cyclooxygenases (COX): the constitutive isoform and the enzyme regulated by physiological stimuli (COX-2). Our previous immunohistochemical studies have demonstrated the presence of COX-2 in a subset of thick ascending limb (TAL) of Henle cells and its induction during the postnatal period and after adrenalectomy. Previous results suggested that this induction phenomenon proceeds by recruitment of TAL cells from the cortex to the outer medulla. The present work aimed to specifically address these preliminary observations by using immunohistochemical techniques in single microdissected nephron segments. Normal adult rats, adrenalectomized rats, adrenalectomized rats on dexamethasone and 5, 10, and 15 days postnatal age were used (Sprague-Dawley rats, n= 5 each group). Glomeruli and different segments of nephron were microdissected from collagenase-treated kidney tissue. Tubules were immunostained with specific antibodies against COX-2. We confirmed that COX-2 was localized exclusively in TAL segments; it was induced after adrenalectomy and during postnatal age, peaking at 15 days after birth. We provided morphological evidence that the induction of COX-2 along TAL proceeded in a defined pattern by recruitment of cells from the cortical portion close to the glomeruli toward the outer medulla. No COX-2 was observed in the post-macula densa portion of the segments. Our results provide the anatomical basis for the contribution of COX-2 in physiological mechanisms such as renin secretion, tubuloglomerular feedback, and the interaction with neuronal NO synthase at the juxtaglomerular apparatus.

Adrenalectomy↗
PubMed2003

BetaB1-crystallin: identification of a candidate ciliary body uveitis antigen.

PURPOSE: Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS: Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS: Proteomic analysis suggested that beta B1 (betaB1)-crystallin is the primary ciliary body antigen. The presence of betaB1-crystallin in the human ciliary body was confirmed by Western blot with a betaB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-betaB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of betaB1-crystallin RNA in the ciliary body tissues. CONCLUSIONS: This study identified betaB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of betaB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.

Amino Acid Sequence↗
PubMed1991

Microquantitative analysis of the intra-acinar profiles of glutamate dehydrogenase in rat liver.

In adult male and female rat liver, the activity of NAD(+)-and NADP(+)-dependent glutamate dehydrogenase (GDH) was microquantitatively measured in tissue samples of 50-150 ng, microdissected continuously along the sinusoidal length. Total activity of GDH with NAD+ as co-factor was found to be higher by a ratio of about 1:2.3 than with NADP+. All intra-acinar enzyme profiles, irrespective of sex, showed an increasing gradient of GDH activity from the periportal beginning to the perivenous end. These findings are at variance with the immunohistochemical localization of GDH in rat liver. The microquantitative GDH profiles with higher perivenous values could indicate a more pronounced glutamine synthesis in Zone 3 of the liver acinus.

Animals↗
PubMed2005

Molecular determinants of cetuximab efficacy.

PURPOSE: To investigate whether mRNA expression levels of cyclin D1 (CCND1), cyclooxygenase 2 (Cox-2), epidermal growth factor receptor (EGFR), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), all members of the EGFR signaling pathway, are associated with clinical outcome in patients with EGFR-expressing metastatic colorectal cancer (CRC) treated with cetuximab. PATIENTS AND METHODS: Thirty-nine patients with metastatic CRC, refractory to both irinotecan and oxaliplatin, were enrolled on IMCL-0144 and treated with single-agent cetuximab. The intratumoral mRNA levels of CCND1, Cox-2, EGFR, IL-8, and VEGF were assessed from paraffin-embedded tissue samples using laser-capture microdissection and quantitative real-time polymerase chain reaction. RESULTS: There were 21 women and 18 men with a median age of 64 years (range, 35 to 83 years). Higher gene expression levels of VEGF were associated with resistance to cetuximab (P = .038; Kruskal-Wallis test). The combination of low gene expression levels of Cox-2, EGFR, and IL-8 was significantly associated with overall survival (13.5 v 2.3 months; P = .028; log-rank test). Both findings were independent of skin toxicity that was itself significantly correlated to survival. Patients with a lower mRNA amount of EGFR had a longer overall survival compared with patients that had a higher mRNA amount (7.3 v 2.2 months; P = .09; log-rank test). Patients with lower expression of Cox-2 had a significantly higher rate of grade 2 to 3 skin reactions under cetuximab treatment. CONCLUSION: This pilot study suggests that gene expression levels of Cox-2, EGFR, IL-8, and VEGF in patients with metastatic CRC may be useful markers of clinical outcome in single-agent cetuximab treatment.

Adult↗
PubMed1985

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system.

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.

Androstenedione↗
PubMed1982

Presence of melatonin in various cat brainstem nuclei determined by radioimmunoassay.

Microdissected samples of juvenile cat brain tissue were assayed for melatonin content using a double antibody radioimmunoassay. Immunoreactive melatonin was consistently detected, albeit in variable amounts, in pineal, habenula, the region of the nucleus gracilis, gigantocellular reticular formation of the pons and medulla oblongata. Among the negative areas were raphe nuclei, substantia nigra and locus caeruleus. These findings suggest that melatonin may play a role in some structures of the central nervous system outside the pineal-hypothalamo-pituitary axis. This immunoreactive melatonin could reflect a local synthesis, or a tissular uptake of melatonin from blood or cerebrospinal fluid.

Animals↗
PubMed2006

Transition from preinvasive carcinoma in situ to seminoma is accompanied by a reduction of connexin 43 expression in Sertoli cells and germ cells.

Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated, that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

Adult↗
PubMed2001

Loss of heterozygosity and allelic imbalance in apocrine adenosis of the breast.

Recently, there have been studies suggesting that apocrine adenosis of the breast is a putative precancerous lesion, despite the generally held view that apocrine adenosis is benign. Because apocrine adenosis is almost always present as a small area or areas, it cannot be easily studied by conventional methods. In this study, areas of apocrine adenosis were microdissected from archival paraffin-embedded tissue to examine loss of heterozygosity and allelic imbalance compared with normal breast tissue epithelium from the same patients. Seventeen cases of apocrine adenosis, four associated with carcinoma, were analyzed using polymorphic microsatellite markers and polymerase chain reaction for loss of heterozygosity/allelic imbalance at eight loci that were reported to show allele loss or imbalance in invasive and in situ breast cancer. Loss of heterozygosity/allelic imbalance was detected in six of 17 cases of apocrine adenosis; three of 12 (25%) informative cases at 1p (MYCL1), two of seven (28.6%) at 11q (INT2), one of three (33.3%) at 13q (D13S267), two of 12 (16.7%) at 16q (D16S539), and two of 10 (20%) at 17q (D17S250). Neither loss of heterozygosity nor allelic imbalance has been identified at 1p (D1S252), 17p (TP53), or 17p (D17S513). In two of the four cases associated with carcinoma, loss of heterozygosity/allelic imbalance was seen in the same allele as in the synchronous carcinoma. These results suggest that molecular alterations, such as loss of heterozygosity and allelic imbalance, identified in apocrine adenosis may constitute an early event in the pathogenesis of breast cancer; reinforcing the possibility of apocrine adenosis being a putative precancerous lesion.

Adult↗
PubMedDate not supplied

Multiple variants of HPV16 E6 gene in cervical invasive squamous cell carcinoma.

BACKGROUND: HPV16 is the most commonly detected genotype in cervical squamous cell carcinoma. E6 of HPV16 is a viral oncogene and has frequent DNA sequence variations whose encoded proteins have been shown to have heterogeneity in biochemical and biological properties. This study tried to establish whether the E6 variants derive from the infection pool or from spontaneous mutation in the host. MATERIALS AND METHODS: We combined the use of microdissection of multiple areas of tumor tissue, PCR-sequencing of HPV16 E6 and E5 genes and allele-specific amplification of PCR to analyze the E6 variations in four cases of cervical cancer (M4, M12, M13 and M23). RESULTS: We isolated two common (350G and 350T) and three rare (310G, 374T and 459C) E6 variations corresponding to five different E6 variants. The common E6 variations were always co-segregated with specific E5 variations. Both common variants persisted in all the four cases. Of three cases each had one additional rare E6 variant. CONCLUSION: The common E6 variants would derive from the infection pool, whereas the rare E6 variants may evolve from the mutation of either of the common E6 variants. This finding might have implications for the future study of natural HPV evolution, the design of viral vaccine and the carcinogenesis of cervical cancer.

Adult↗
PubMed1991

Detection and distribution of aromatase in hypothalamus of the male rat.

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase. An in vitro assay for aromatase activity, which quantitates the production of 3H2O during the conversion of 1 beta-3H-androstenedione to estrone, was developed and validated in the present study. The distribution of aromatase activity in discrete hypothalamic nuclei was also explored. Individual hypothalamic nuclei were microdissected from 200-micron frozen sections. Tissues from 3 animals were pooled and assayed for aromatase activity. It was found that the rate of aromatase activity was linear with time up to 1 hour, and with protein concentrations up to 900 micrograms/ml tissue homogenate. A maximal rate was obtained with a substrate concentration of 0.3 microM. The aromatase activity was heterogeneously distributed within the rat brain. The greatest amount of aromatase activity was found in the preoptic area. The suprachiasmatic nucleus also contained a high level of aromatase activity. The aromatase activities in the anterior hypothalamus, ventromedial nucleus, arcuate nucleus and dorsomedial nucleus were less than half that found in the preoptic area.

Animals↗
PubMed1984

[Floating cell cultures of the fetal pancreas].

A simple method for preparing human and animal fetal pancreatic cell cultures has been developed. It is based on enzymatic treatment of the fetal pancreatic tissue with collalitine in combination with microdissection. As a result of subsequent cultivation there form floating cytotypic and organotypic cultures consisting mainly of B cells in different phases of the secretory cycle. The floating cultures prepared by the above-described method produce insulin and can be successfully used in experimental and clinical transplantology.

Culture Techniques↗
PubMed1995

Allelotyping of ductal carcinoma in situ of the breast: deletion of loci on 8p, 13q, 16q, 17p and 17q.

In order to determine which tumor suppressor loci are involved in preinvasive breast cancer, we have assayed for loss of heterozygosity (LOH) in ductal carcinoma in situ (DCIS). Areas of DCIS were microdissected from archival paraffin-embedded tissue. DNA was extracted, and LOH was determined by PCR of microsatellite markers that map to 39 autosomal arms. Either uninvolved lymph node or white cell DNA was used as normal control. A total of 61 samples of DCIS were assayed. The average number of informative tumors examined for each marker was 19 (range, 8-48). The median fractional allelic loss was 0.037. The highest percentage of LOH was shown for loci on 8p (18.7%), 13q (18%), 16q (28.6%), 17p (37.5%), and 17q (15.9%). LOH on 18q was found in 10.7% of informative tumors. Fractional allelic loss was associated with LOH on 17p, with high nuclear grade and with the comedo subtype of DCIS. LOH on 17p correlated with LOH on 17q and on 13q. Additional markers were used for 16q and 17p to determine the smallest common region of deletion. These data provide evidence that tumor suppressor loci that map to these regions are involved in the oncogenesis of breast cancer before progression to the invasive phenotype. Our findings provide additional support that multiple loci on 17p and 16q are involved in the development of breast cancer.

Breast Neoplasms↗
PubMed1996

Allelotyping of all chromosomal arms in invasive cervical cancer.

The best characterized factor in the development of cervical cancer is the integration, of human papillomavirus into cervical cell chromosomes. In addition to HPV integration, the neoplastic process probably requires the activation of cellular protooncogenes and loss of tumor suppressor gene function. Loss of heterozygosity analysis in a large sample is used to identify regions which harbor putative tumor suppressor genes (TSG) since the deletion of normal alleles unmask mutated alleles. We evaluated tumor tissue from invasive cervical carcinomas, carefully microdissected to eliminate normal stroma and lymphocytes, for LOH at all 41 chromosomal arms with 50 polymorphic markers. We have evaluated tumor and normal DNA pairs from 48 invasive cervical cancers of which 85% of the tumors are confined to the cervix. The mean loss for all chromosomal arms was 12%. Three regions exhibited LOH two standard deviations above the mean: 3p14.1-12 (40%), 11q23.3 (36%), and 6p22-21.3 (32%). Three regions showed loss one standard deviation above the mean: 19q13.4 (30%), 6q21-23.33 (25%), and 2q33-37 (24%). Our results indicate that a significant number of invasive cervical cancers have lost specific chromsomal regions, thereby suggesting that genes involved in the cell cycle regulation or the suppression of tumor development are located in these regions.

Alleles↗
PubMed1998

Uneven regional distribution of nucleotide metabolism in human brain.

Adenine and uridine nucleotides and adenosine are proposed to act as neuromodulators and other nucleotides and nucleosides are also suggested to be involved in brain function. A following major step towards the verification of the functional role of nucleotides and nucleosides in the brain would be the examination of regional distribution of purines, pyrimidines and the enzymes involved in their metabolism. Using our recently developed chromatography-based assay for nucleosides from tissue homogenates, we analysed nucleosides in microdissected samples derived from various regions of human brain. Marked differences in the levels of nucleosides were measured in the cerebral cortex, cerebellar cortex, thalamus and white matter. The greatest levels of most nucleosides were found in the cerebral cortex, followed by the cerebellar cortex and the white matter while the smallest concentrations were found in the thalamus, although adenosine and xanthine showed a different distribution pattern in these brain areas. Within the cerebral cortex, the measured substances showed little variations except certain high levels in the cingulate and low levels in the frontal cortex. Even distribution of nucleosides was found in the thalamic nuclei while relative high values were measured in the medial geniculate body. Since a dramatic change in nucleoside concentrations occurs after death, the measured nucleoside concentrations are an interplay of original nucleotide and nucleoside concentrations and enzyme reactions following death. Thus our results suggest regional differences in nucleotide and nucleoside composition and nucleotide metabolising enzyme activities between brain areas.

Adenosine↗
PubMed2004

Laser microdissection: exploring host-bacterial encounters at the front lines.

The mucosal surfaces of tissues such as the stomach and intestines are in constant contact with indigenous bacterial populations and are major portals of entry for bacterial pathogens. Host responses to bacterial encounters at these surfaces frequently involve complex interactions between epithelial cells and immune cells, and are thus difficult to model in vitro. Laser microdissection is a technique in which pure populations of host cells are acquired from sections of complex tissue. When coupled with an expanding repertoire of techniques for molecular analysis of microdissected cells, laser microdissection allows host cellular responses to bacteria to be studied in their native tissue context. This approach has already yielded key insights into the nature of mucosal responses to commensal, as well as pathogenic bacteria, and promises to be an important addition to the cellular microbiologist's toolkit.

Animals↗
PubMed1983

Localization of tissue potassium pools in the amphibian kidney.

Techniques were developed to determine the location of exchangeable K pools that had been identified previously in kinetic studies of the intact perfused bullfrog kidney. Following perfusion of the kidneys with 42K, a washout of the isotope was begun and interrupted at various times; the kidneys were removed, frozen, dried at low pressure and temperature, and then microdissected. Glomerular capillary tufts, small segments of tissue containing early distal tubules (diluting segment), and other segments containing proximal tubular convolutions were removed and analyzed for total content of K and 42K. In the intact kidney 77% of tissue K exchanged in 60 min. The exchangeable K concentration was 95 mu eq/ml cell water. Correction for the K activity coefficient in Ringer solution yielded an activity of 72 mu eq/ml. Thirty-two percent of glomerular capillary K exchanged in 60 min; 7% exchanged with a half time of 3.5 min; and the remainder exchanged at a rate too slow to measure. The data from tissue containing proximal tubular segments were too scattered to permit analysis. In segments containing early distal tubules, 67% of tissue K was contained in two exchangeable pools: one pool exchanged at a rate 10-fold greater than did the other. The data for these two distal pools were analyzed in terms of a parallel model (two cell types?) and a nested model (cytoplasm and subcellular organelles?). Pool size and exchange rates were calculated for both models. Electron microscopic analysis revealed that early distal tubular segments contain only one cell type which has a large population of mitochondria. This suggests that the nested model is more plausible. The fast distal pool exchanged at the same rate as the fast-exchanging pool identified in kinetic studies of the intact functioning kidney and is considered to be the K secretory pool.

Animals↗
PubMed1992

Age-associated decrease in vasopressin-induced renal water transport: a role for adenylate cyclase and G protein malfunction.

Aging is associated with a decline in renal concentrating ability in response to dehydration-induced arginine vasopressin (AVP) release. To examine target tissue sensitivity, cortical collecting tubules (CCT) were individually microdissected from young (3 months), middle-aged (2-3 years) and old (4-5.5 years) rabbits and subjected to in vitro perfusion analysis and tissue culture of defined epithelial monolayers. Osmotic fluid transport was compared with adenylate cyclase activity and an age-associated decline in both parameters detected in response to increasing doses of AVP. [3H]-AVP binding was unchanged in CCT epithelia of different ages, excluding receptor alterations as the underlying mechanism. Also, hydraulic conductivity measurements of isolated young and old CCT were not significantly different, excluding post-cAMP events as a major mechanism of reduced sensitivity. Effects of cholera toxin and forskolin on hydraulic conductivity and adenylate cyclase activity showed a dramatic decrease in the ability of these compounds to elicit a response in CCT epithelia from old rabbits. It was concluded that alterations in Gs proteins and the catalytic unit of adenylate cyclase-were responsible for the age-associated decline in CCT response to AVP.

Adenylyl Cyclases↗
PubMed2003

In-depth analysis of molecular alterations within normal and tumour tissue from an entire bronchial tree.

Using laser capture microdissection (LCM), fluorescent microsatellite analysis and immunohistochemical analysis, we have constructed a detailed topographical molecular map of the entire bronchial tree surrounding a primary bronchial squamous carcinoma in order to establish the relationship between the molecular damage within the airway and that in the tumour itself. Allelic imbalance was analysed using markers on chromosomes 3, 9, 13 and 17. In addition, immunohistochemical analysis for p53 and cyclin D1 expression was performed. Analysis revealed allelic imbalance at several loci at the tumour site but also in 83% of the histologically normal airway specimens of the upper and lower lobes. The fractional allele loss (FAL) value was statistically higher (0.75+/-0.13) in the tumour site than in the distal site of the upper (0.42+/-0.09) and lower lobes (0.31+/-0.08). Immunohistochemical analysis revealed overexpression of p53 and cyclin D1 protein within histologically normal bronchial epithelium, thus confirming previous reports for their early involvement in lung tumour development. This is to date the largest in-depth study of allelic imbalance using LCM in a single individual. The patterns of allele-specific imbalance observed support a clonal or oligoclonal expansion model of outgrowths throughout the lung. The widespread incidence of genetic changes in the whole of lung most likely represents smoking-induced alterations and emphasize the complexity of the field cancerization concept. Our findings point to the need for in-depth studies of the whole bronchial tree tissue surrounding lung carcinomas, in order to identify the genetic changes that differentiate preneoplastic and neoplastic stages in lung carcinogenesis.

Aged↗