Accuracy of histone H3 messenger RNA in situ hybridization for the assessment of cell proliferation in human tissues

Histone H3 mRNA in situ hybridization was compared to a reference method, iododeoxyuridine (IdUrd) immunohistochemistry of tissues labeled in vivo, as a means for assessing the proportion of S-phase cells (labeling index, LI) in oral tumor and normal mucosa. Paraffin sections from 16 patients with oral squamous cell carcinoma were studied. Patients received an IdUrd infusion before the biopsy was taken. Tissue sections were coded before counting the percentages of S-phase cells. A high correlation was found between the results obtained by the two techniques. The average histone H3 and IdUrd LIs of the tumors were 28.5 +/- 2.4% and 29.2 +/- 2.7%, respectively (P = 0.85), with a Spearman correlation coefficient r = 0.95 (P < 0. 0001). The histone H3 LI of the basal layer of normal mucosa was 3.1 +/- 0.8%, whereas the IdUrd LI was 2.7 +/- 0.9% (P = 0.74), with r = 0.78 (P = 0.004). In the suprabasal layers, these parameters were 21. 3 +/- 2.3% and 23.9 +/- 3.2%, respectively (P = 0.56), with r = 0.93 (P < 0.0001). In sections stained for both histone H3 and IdUrd, most cells were double labeled, with very few cells containing only one of the labels. In some specimens, large areas of H3-stained cells did not contain IdUrd-labeled cells, suggesting that during the IdUrd infusion, the precursor did not reach these areas. Two specimens were histone H3 negative. They were also negative when hybridized with beta-actin probe, indicating degradation of mRNAs in these samples. The results of this study demonstrate that the histone H3 mRNA in situ hybridization performed in human formalin-fixed, paraffin-embedded tissues provides the same data as does labeling the tumors in vivo with halogenated pyrimidine.     

Expression of acetylcholinesterase messenger RNA in human brain: an in situ hybridization study

The distribution of messenger RNA coding for acetylcholinesterase was studied in human post mortem brain and rhesus monkey by in situ hybridization histochemistry and compared to the distribution of acetylcholinesterase activity. Acetylcholinesterase messenger RNA had--similar to acetylcholinesterase enzymatic activity--a widespread distribution in human bain. Acetylcholinesterase messenger RNA positive cells corresponded to perikarya rich in acetylcholinesterase activity in most but not all regions. Examples for mismatches included the inferior olive and human cerebellar cortex. The presence of hybridization signals in cerebral cortex and an enrichment in layer III and V of most isocortical areas confirmed that perikaryal acetylcholinesterase in cerebral cortex is of postsynaptic origin and not derived from cholinergic projections. In striatum the expression of high levels of acetylcholinesterase messenger RNA was restricted to a small population of large striatal neurons. In addition, low levels of expression were found in most medium sized striatal neurons. Cholinergic neurons tended to express high levels of acetylcholinesterase messenger RNA whereas in cholinoceptive neurons the levels were moderate to low. However, some noncholinergic neurons like dopaminergic cells in substantia nigra, noradrenergic cells in locus coeruleus, serotoninergic cells in raphé dorsalis, GABAergic cells in thalamic reticular nucleus, granular cells in cerebellar cortex and pontine relay neurons expressed levels comparable to cholinergic neurons in basal forebrain. It is suggested that neurons expressing high levels of acetylcholinesterase messenger RNA may synthesize acetylcholinesterase for axonal transport whereas neurons with an expression of acetylcholinesterase confined to somatodendritic regions tend to contain lower levels of acetylcholinesterase messenger RNA.     

Immunohistochemical and in situ hybridization studies of beta-microseminoprotein in the human gastric mucosa

beta-Microseminoprotein is a 10-kDa disulphide-rich protein with unknown function which is present in the mucus of the airways, gastrointestinal tract and urogenital tract. In this paper, an investigation of the distribution of beta-microseminoprotein in the human stomach is reported. Immunohistochemistry and in situ hybridization were used. beta-Microseminoprotein was found to be localized mainly in the antrum part of the stomach and in two types of cells. Cells of the most abundant type (designated M-cells) were the neutral mucin-containing cells in the bottom part of the gastric glands and the surface epithelium. Virtually all these cells contained both beta-microseminoprotein mRNA and protein product. Cells of the second type (designated E-cells) were found in a zone one-third up from the bottom of the gastric glands, where gastric endocrine cells are located. The E-cells were fewer than the M-cells and usually solitary. They seemed to have a high concentration of protein compared with their low mRNA level. The majority of the E-cells contained chromogranin A and gastrin. The observations made have implications for the understanding of the differentiation of the mucosal cells in the antrum of the stomach and form a basis for future studies of beta-microseminoprotein in gastric disease.     

Extra-hypothalamic vasopressin neurons coexpress galanin messenger RNA as shown by double in situ hybridization histochemistry

Vasopressin (VP) neurons in the bed nucleus of the stria terminalis (BNST) and medial amygdala (AMe) exhibit sexual dimorphism and steroid dependency. VP neurons in the supraoptic nucleus and paraventricular nucleus have been shown to coexpress other transmitters including galanin (GAL). However, little is known about what other neurotransmitters may be colocalized with VP in the BNST and AMe. Here, we have used radio-labeled and digoxigenin-labeled cRNA probes to perform double in situ hybridization histochemistry for VP and GAL in the BNST and AMe of intact, adult male rats. We provide evidence that in the basal state, the majority of VP-synthesizing cells in the BNST and AMe of the adult male rat also express galanin mRNA. Likewise, the majority of GAL-expressing neurons in these regions also contain VP mRNA. These findings give further evidence for the similarity of the BNST and AMe and provide a rationale for studies investigating the role of GAL in functions involving extrahypothalamic VP pathways.     

Transient expression of acetylcholinesterase messenger RNA and enzyme activity in developing rat thalamus studied by quantitative histochemistry and in situ hybridization

The molecular basis for transient expression of acetylcholinesterase in noncholinergic regions of the early postnatal rat brain was studied by in situ hybridization histochemistry. A 33P-labelled 63-mer DNA oligonucleotide was used to probe acetylcholinesterase messenger RNA in the brains of rat pups at one, two, six, nine, 12, 16 and 21 days of age (birth = day 0). Cryostat brain-sections were hybridized with probe and exposed to X-ray film or emulsion coatings. Acetylcholinesterase messenger RNA was quantitated by counting silver grains and by measuring X-ray film density with video imaging and computer-based densitometry. Adjacent sections were stained histochemically for acetylcholinesterase activity, also quantitated by video densitometry. Overall there was a significant correlation between apparent levels of acetylcholinesterase activity and acetylcholinesterase messenger RNA. Increases in message tended to accompany the surges of acetylcholinesterase activity that marked the maturation of thalamocortical sensory relay pathways. Acetylcholinesterase expression in the youngest rats was generally sparse but it increased markedly during the first postnatal week, especially in the sensory relay nuclei of the thalamus. Levels of message and enzyme activity in the medial and dorsolateral geniculate and the ventral posteromedial and ventral posterolateral nuclei rose to a peak, typically about day 9. Beyond this time there was a gradual decline. By day 21 the staining and in situ hybridization patterns resembled those in adult brains, whose thalamic relay nuclei are impoverished in acetylcholinesterase activity and messenger RNA. Thus, acetylcholinesterase expression is strongly modulated in certain thalamic systems as they undergo neural morphogenesis.     

Multiparametric in situ messenger RNA hybridization analysis to detect metastasis-related genes in surgical specimens of human colon carcinomas

We examined the expression of several genes that regulate different steps of metastasis in surgical specimens of human colon carcinomas. The expression of epidermal growth factor receptor (growth), basic fibroblast growth factor [(bFGF), angiogenesis], type IV collagenase (invasion), E-cadherin (adhesion), and multidrug-resistant (mdr)-1 (drug resistance) mRNA was examined using an in situ mRNA hybridization (ISH) technique and Northern blot analysis. Dukes' stage C and D tumors exhibited a higher level of expression (P <0.05) for bFGF, type IV collagenase, and mdr-1 mRNA than Dukes' stage B tumors. The expression level of epidermal growth factor receptor and E-cadherin did not correlate with the stage of the disease. The ISH technique revealed intertumoral heterogeneity for expression of several genes among Dukes' stage B neoplasms. In some Dukes' stage B tumors, we also found intratumoral heterogeneous staining for bFGF and type IV collagenase, with the highest expression level at their invasive edge. In Dukes' stage C and D tumors, the expression of these genes was more uniform. These results recommend the suitability of the multiparametric ISH analysis for metastasis-related genes to identify individual colon cancers with metastatic potential.     

In situ mRNA hybridization technique for analysis of human telomerase RNA in gastric precancerous and cancerous lesions

Telomerase, the ribonucleoprotein enzyme that elongates telomerase, is repressed in normal somatic cells but is reactivated during tumor progression. The purpose of this study was to investigate the localization of human telomerase RNA (hTR) expression in human gastric precancerous and cancerous lesions by using in situ mRNA hybridization (ISH) with avidin-biotin staining. We also examined telomerase activity in these lesions by using hybridization protection assay connected with a telomeric repeat amplification protocol (TRAP/HPA). Analyzed tissue samples were as follows; 132 cases of chronic atrophic gastritis without intestinal metaplasia, 115 incomplete-type intestinal metaplasias, 40 complete-type intestinal metaplasias, 23 hyperplastic polyps, 23 tubular adenomas and 26 adenocarcinomas. In ISH analysis, high levels of hTR expression were observed preferentially in the nuclei at the single-cell level. hTR-expressing cells in carcinomas and adenomas were significantly more frequent than those of the other lesions (P < 0.001). The expression pattern of hTR in carcinoma and adenoma tissues was heterogeneous and similar intratumor heterogeneity was detected in Ki-67 immunoreactivity. Infiltrating lymphocytes in tissue also exhibited high levels of hTR expression. In TRAP/HPA analysis, carcinomas had significantly more frequent positivity for telomerase activity and a higher level of telomerase activity than the other lesions (P < 0.05). However, the amount of telomerase activity did not parallel the expression level of hTR. Our data suggest that hTR expression increases in the early stages of stomach carcinogenesis and that sufficient synthesis of hTR is a prerequisite for telomerase reactivation in tumorigenesis.     

An in vitro messenger RNA binding assay as a tool for identifying hybridization-competent antisense oligonucleotides

Targeting, use of appliances, and standards of outcome for General Dental Service orthodontic cases collected between 1990 and 1991 were compared with a sample of cases from an earlier study, collected between 1987 and 1988, using the PAR index and IOTN. Comparisons are made generally and in relation to the changes in prior approval regulations for cases started since October 1987. More lower-need cases were treated, but there were no more "unnecessary' treatments and there has been a limited improvement in outcomes, as assessed by the indices, associated with increased use of fixed appliances since 1987. Prior approval appeared to give no tangible benefits in terms of levels of unnecessary treatment or quality of outcome.     

Localization and differential expression of adenylyl cyclase messenger ribonucleic acids in rat adrenal gland determined by in situ hybridization

The expression of adenylyl cyclases (ACs) in the adult rat adrenal gland was examined. In situ hybridization revealed specific patterns of AC messenger RNA (mRNA) distribution. AC1 was limited exclusively to the adrenal medulla. AC5 and AC6 were mainly expressed in the adrenal medulla, with a weak expression in the zona glomerulosa. AC9 was found in all the three regions of the adrenal cortex but not in the adrenal medulla. All these ACs were detected on postnatal day 1 (PN1), and their pattern of expression was unchanged on PN7, PN21, and PN90 (adult). We analyzed the response of these ACs to various physiological conditions known to affect the synthesis of aldosterone and corticosterone in the adrenal cortex. Our study demonstrates a specific increase of AC6 but not AC5 mRNA in the zona glomerulosa of rats given a low sodium diet. AC9 mRNA was increased in all the three cortical zones of rats treated with ACTH. We suggest that AC6 and AC9 play important roles in different pathways associated with the regulation of aldosterone and corticosteroid production.     

An increased number of follicles containing activated CD69+ helper T cells and proliferating CD71+ B cells are found in H. pylori-infected gastric mucosa

Multiple sclerosis is characterized by myelin destruction and oligodendrocyte loss. The neuropathological hallmark of the disease is the presence of demyelinated plaques in the central nervous system. We have recently found a gliotoxic factor in MS cerebrospinal fluid which induces programmed cell death in vitro, in glial cells. Here we show DNA fragmentation and glial cell death in biopsy samples, obtained from a patient who underwent surgery with suspicion of tumor, and whose disease record, including brain autopsy, demonstrated an active multiple sclerosis. We used the in situ TUNEL technique, a method which sensitively detects the DNA fragmentation accompanying programmed cell death in tissue sections, and compatible with classical fixation techniques. We found intense DNA fragmentation in nuclei of glial cells at-or very near-to the site of demyelination. A double labeling technique showed that glial fibrillary associated protein positive astrocytes may undergo programmed cell death in multiple sclerosis.     

The structural organization of nuclear messenger RNA precursor. I. Reassociation and hybridization properties of double-stranded hairpin-like loops in messenger RNA precursor

The hybridization and renaturation properties of double-stranded hairpin-like loops isolated from giant nuclear messenger RNA precursor of mouse liver or ascites carcinoma cells were studied. About half of the hairpins in messenger RNA precursor appear to contain similar sequences, as indicated by the very fast kinetics of renaturation of the denatured double-stranded RNA sequences. These sequences have no tissue specificity. About one third of the hairpin sequences can hybridize to messenger RNA. It is suggested that the long hairpins in messenger RNA precursor play the role of sequences separating messenger RNA sequences from non-informative sequences and that these hairpins are recognized by processing enzymes.     

Reduction in enkephalin and substance P messenger RNA in the striatum of early grade Huntington's disease: a detailed cellular in situ hybridization study

The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S] oligonucleotides. Macroscopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs were noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms.     

Astrocytic messenger RNA responses to striatal deafferentation in male rat

This investigation describes the schedule and regional distribution of astrocytic responses in striatum following deafferentation by unilateral frontal cortex ablation. In the ipsilateral deafferented striatum, glial fibrillary acidic protein and clusterin (sulfated glycoprotein-2) messengerRNA showed peak elevations by 10 days postlesioning (Northern blots). Vimentin messengerRNA responded faster, with a transient elevation by three days postlesioning. The messengerRNA for glial fibrillary acidic protein, clusterin and vimentin returned toward control levels by 27 days postlesioning. However, the neuronal marker growth-associated protein messengerRNA, was decreased at all postlesion times. By in situ hybridization, the increased glial fibrillary acidic protein messengerRNA and clusterin messengerRNA signals were localized mainly to the dorsal half of the ipsilateral deafferented striatum and followed the same schedule as found by Northern blots. Glial fibrillary acidic protein messengerRNA was widely diffused in the dorsal striatum and was excluded from fascicles of the internal capsule; a similar distribution was found for glial fibrillary acidic protein-immunopositive astrocytes. While clusterin messengerRNA signal showed a distinct clustering, its immunoreactivity appeared as deposits in the deafferented striatal neuropil; Western blots confirmed the immunocytochemical results. By in situ hybridization, vimentin messengerRNA was mostly localized to the cortical wound cavity dorsal to the deafferented striatum and overlapped the distribution of vimentin-immunopositive cells. These findings suggest a coordination of striatal astrocytic messengerRNA responses with the degeneration of corticostriatal afferents. We also compared these same parameters with those from published reports on the hippocampus after deafferenting lesions. Certain astrocyte molecular responses to deafferentation are detected about five days earlier in the hippocampus than in the striatum. This different schedule in response to decortication may pertain to differences in synaptic remodeling in the hippocampus vs striatum.     

3' non-coding region sequences in eukaryotic messenger RNA

The sequence A-A-U-A-A-A is present in six different purified messenger RNA molecules (specifically the alpha-and beta-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRNA of mouse (MOPC 21) and the ovalbumin mRNA of chicken) about 20 residues away from the 3'-terminal poly (A) sequence. In addition, a large selection of the 3' non-coding regions of rabbit and human globulin mRNAs (both the alpha and beta globin mRNAs) are 85% homologous, demonstrating that this region is significantly conserved in evolution.     

Detection of trisomy 3 in primary gastric B-cell lymphoma by using chromosome in situ hybridization on paraffin sections

Recently, new backscatter factors for low-energy x rays derived from Monte Carlo calculations have been recommended in the UK code of practice for kilovoltage dosimetry published by (IPEMB). As these data, presented as a function of half-value layer, do not take account of the variation of the x-ray spectra for a given HVL, we have undertaken an experimental study in order to determine BSG for the beam qualities provided by a Darpac 2000 therapy unit. A RTL detector such as Li2B4O7:Cu and parallel-plate ion chambers specially designed for low-energy x-ray dosimetry have been used. The results obtained show very good agreement between the TLD and the Monte Carlo calculations, confirming values obtained by other authors with lithium borate TLD. On the contrary, the results obtained with plane-parallel ion chambers show discrepancies up to 9% that are discussed.