Increased hypothalamic neuropeptide Y concentrations in diabetic rat.

Central and lateral hypothalamic concentrations of 10 regulatory peptides were measured by radioimmunoassay in streptozocin-induced diabetic (STZ-D) and matched control rats between 1 day and 14 wk after diabetes induction. After 2 wk, both central and lateral hypothalamic neuropeptide Y (NPY) concentrations in STZ-D rats were consistently higher than those found in control rats, with significant 30-50% increases at 4 wk in the central hypothalamus, and at 6 and 14 wk in both central and lateral hypothalamus. Immunocytochemical studies in 4- and 6-wk STZ-D animals showed the appearance of intensely NPY-positive swollen cell bodies in the supraoptic nucleus and a subjective increase in NPY staining of medial hypothalamic nerve fibers. Central hypothalamic concentrations of three other peptides were significantly greater in STZ-D animals than those in control animals at single points (neurotensin, 1 day; calcitonin gene-related peptide, 2 wk; neurokinin, 4 wk). Hypothalamic concentrations of the other six peptides examined (bombesin, galanin, neuromedin B, substance P, somatostatin, and vasoactive intestinal peptide) did not differ significantly between STZ-D and control groups at any time. However, galanin immunostaining in the supraoptic and magnocellular paraventricular nuclei was strikingly concentrated in a reduced number of distended cell bodies. Hypothalamic peptide changes in STZ-D could be related to metabolic disturbance, changes in energy and water balance, altered pituitary function, or other factors. Persistently elevated concentrations of NPY, a very potent central stimulant of eating and drinking, may mediate the hyperphagia and polydipsia characteristic of STZ-D.

Maternal origin of inflammatory leukocytes in preterm fetal membranes, shown by fluorescence in situ hybridisation.

The aim of this study was to determine the maternal or fetal origin of inflammatory leukocytes in fetal membranes from cases of chorioamnionitis. Fetal membranes were collected from male preterm infants and chorioamnionitis was diagnosed histologically. Fluorescence in situ hybridisation for X and Y chromosomes was used to determine the gender of infiltrating leukocytes in the chorion and amnion. Leukocytes, trophoblast and mesenchymal cells were identified using immunohistochemistry for CD45, cytokeratin-7 and vimentin, respectively. Leukocytes present in the chorion and amnion were labelled XX, indicating maternal origin, and these cells were immunoreactive for the leukocyte marker CD45 but not for vimentin or cytokeratin-7. All other cells in the chorion and amnion were labelled XY and of fetal origin. The results indicated that maternal leukocytes invade the amnion and chorion in chorioamnionitis and we suggest that this is part of the maternal inflammatory response to intrauterine infection.

Localization of 7B2, neuromedin B, and neuromedin U in specific cell types of rat, mouse, and human pituitary, in rat hypothalamus, and in 30 human pituitary and extrapituitary tumors.

The distributions of three novel peptides, 7B2, neuromedin B, and neuromedin U, in rat, mouse, and human pituitaries, rat hypothalamus, and 30 human pituitary tumors were investigated with immunocytochemistry. Immunoreactivity for 7B2 was present in rat, mouse, and human gonadotropes, in intermediate lobe cells and posterior lobe nerve fibers in rats and mice, in rat hypothalamus (particularly in the median eminence), and in eight human pituitary gonadotropinomas. In gonadectomized rats, larger, more numerous LH beta- and 7B2-immunoreactive gonadotropes were seen than in controls. Extractable 7B2-like immunoreactivity was elevated but not significantly so in gonadectomized rat pituitaries [males: castrated, 37.4 +/- 4.3 (mean +/- SE); controls, 26.9 +/- 4.3; females: ovariectomized, 27.2 +/- 2.7; controls, 19.1 +/- 2.2 pmol/gland]. Neuromedin B immunoreactivity was found in normal rat and mouse thyrotropes and weakly in "thyroidectomy" cells in hypothyroid rats, in which extractable pituitary neuromedin B was significantly depleted (thyroidectomized, 87.0 +/- 14.0; methimazole-treated, 82.0 +/- 11.4; control, 230.7 +/- 25.6 fmol/gland). Hyperthyroid rat pituitaries showed increased TSH beta and neuromedin B immunoreactivities and neuromedin B content (TRH-treated, 385.2 +/- 30.2; T4-treated, 352.6 +/- 20.2; control, 230.7 +/- 25.6 fmol/gland). Neuromedin U immunoreactivity occurred in corticotropes of all species, in rat and mouse intermediate lobe, and throughout the rat hypothalamus, with immunoreactive cell bodies in the arcuate nucleus. Neuromedin U-immunoreactive cells were present in six of six human pituitary and five of six human extrapituitary corticotropinomas. In adrenalectomized rats, corticotropes were larger and more numerous than in controls, but extractable anterior pituitary neuromedin U-like immunoreactivity was not raised (adrenalectomized, 3.30 +/- 0.45; control, 3.32 +/- 0.27 pmol/gland). Our findings suggest that 7B2, neuromedin B, and neuromedin U may be involved in pituitary function.

Identification of bombesin-immunoreactive cells in rat, human, and other mammalian pituitaries, their ontogeny and the effect of endocrine manipulations in the rat.

Bombesin and gastrin-releasing peptide are homologous peptides which have biological activity in mammals. The distribution of bombesin immunoreactivity in rat, guinea pig, cat, dog, pig, cow, monkey, and human pituitaries was investigated using immunocytochemistry with various different antisera. Polyclonal antisera identified bombesin-immunoreactive (IR) cells in the anterior pituitaries of all species except monkey and human, although positive nerves were present in the human posterior lobe. In contrast, a monoclonal antibody demonstrated bombesin-IR cells in anterior and intermediate lobe (or equivalent) of all species. Both types of antibodies identified the anterior pituitary cells as somatotrophs, which may be significant because bombesin and related peptides influence pituitary growth hormone secretion. Differences in bombesin immunoreactivity were seen in male and female rats, with males having more positive cells, and females showing more intense immunoreactivity in those cells which were positive. Ontogenetic studies in rats revealed that bombesin-IR cells were first seen at birth. The effect of estrogen on bombesin-IR cells was studied using ovariectomized and estrogen-treated female rats. Estrogen treatment decreased very significantly the number of bombesin-IR cells, compared with controls, whereas ovariectomy increased significantly the frequency of bombesin-IR cells, so that the staining pattern began to resemble that seen in normal male rats. No bombesin-IR cells were detected in pituitaries from thyroidectomized rats. These results suggest that immunoreactive bombesin/gastrin-releasing peptide in the pituitary is modulated by endocrine status and this peptide may be involved in paracrine interactions in this tissue.

Effect of endocrine manipulation on anterior pituitary galanin in the rat.

Galanin is widely distributed throughout the rat neural and endocrine system. The highest concentrations are found in the anterior pituitary, and it can influence classical pituitary hormone secretion. The effects of endocrine manipulation on pituitary galanin content, mRNA, and immunostaining have been investigated in the rat. In females, medical (39 +/- 4 fmol/gland), surgical (33 +/- 2), or combined (28 +/- 6) castration resulted in a highly significant decrease in galanin content (control, 223 +/- 14; P less than 0.0001). Estrogen in physiological and pharmacological doses produced a significant increase in galanin content (368 +/- 14 and 373 +/- 13, respectively; P less than 0.01) associated with an increase in galanin mRNA content. In the male, high dose dexamethasone and thyroidectomy caused a fall in galanin content, while galanin mRNA levels showed a rise and fall, respectively. Adrenalectomy caused a rise in galanin content, while adrenalectomy and castration produced a dramatic decrease in tissue galanin content. No change in galanin mRNA was observed in these groups. Galanin immunostaining paralleled the results of tissue content in all groups examined, except in the medically castrated group, in which there was some intragroup variation in staining patterns. In normal and high-dose estrogen-treated females, galanin expression was seen mainly in lactotrophs, with a small number of somatotrophs and thyrotrophs staining. In the male, galanin expression was confined to somatotrophs and thyrotrophs. Galanin mRNA was localized at the cellular level by in situ hybridization. In the normal pituitary only scattered lactotrophs contained message, while in high-dose estrogen-treated animals the number of positive cells, mostly lactotrophs, was vastly increased. Thus, the cellular localization of galanin immunostaining varies between the sexes. Galanin peptide and mRNA levels in the pituitary are powerfully influenced by endocrine status.

Localization of immunoreactivity for calcitonin gene-related peptide in the rat anterior pituitary during ontogeny and gonadal steroid manipulations and detection of its messenger ribonucleic acid.

Localization of calcitonin gene-related peptide (CGRP) expression in the rat anterior pituitary and its changes during ontogeny and after gonadal steroid manipulations were studied by immunocytochemistry, RIA, and in situ hybridization. Colocalization studies and the combined use of immunocytochemistry and in situ hybridization revealed that CGRP immunoreactivity is localized mainly in gonadotropes and alpha- and beta-CGRP messenger RNAs were detected in CGRP-immunoreactive cells. Immunoreactivity for CGRP also was detected in nerve fibers and colocalized with substance P immunoreactivity. Cells immunoreactive to CGRP antiserum were first detected in fetal rats at gestational day 18, and the incidence considerably increased between postnatal days 5 and 14. CGRP immunoreactivity was low in control adults of both sexes and in pregnant and ovariectomized females but increased in lactating, estrogen-supplemented ovariectomized and high-dose estrogen-treated females, and in high-dose estrogen-treated and castrated males. Testosterone supplement suppressed the effect of castration on CGRP immunoreactivity in males. Quantities of extractable immunoreactive CGRP under conditions of estrogen manipulation corresponded well to the immunocytochemical findings (females: controls, 96.4 +/- 13.1 fmol/gland; ovariectomized, 107.6 +/- 19.2; high-dose estrogen-treated, 212 +/- 23.0; estrogen-supplemented ovariectomized, 680 +/- 42.1). The present study suggests that pituitary CGRP is synthesized and stored in gonadotropes, is modulated by gonadal steroids, and may have a functional link with gonadotropins.

Novel peptide pancreastatin: its occurrence and codistribution with chromogranin A in the central nervous system of the pig.

The distribution of pancreastatin immunoreactivity was investigated in porcine brain, spinal cord, dorsal root ganglia, and pituitary. In the brain, immunoreactive cell bodies were present in many areas including the cortex, basal ganglia, hippocampus, thalamus, hypothalamus, mesencephalic reticular formation, cerebellum, and medulla oblongata. Immunoreactive fibres were most abundant in the globus pallidus, stria terminalis, entopeduncular nucleus, hippocampus, and in the substantia nigra. In the spinal cord, immunoreactive cells were found in laminae IV-IX. Immunoreactive fibres were concentrated in the dorsal horn. Pancreastatin immunoreactivity was localised to fibres and small cells (5-10% of the total) in the dorsal root ganglia. In the posterior pituitary, many immunoreactive fibres were present and in the anterior lobe subsets of gonadotrophs and thyrotrophs were pancreastatin-immunoreactive. The localisation of pancreastatin showed a parallel distribution with chromogranin A. Coexistence of pancreastatin with calcitonin gene-related peptide (CGRP) immunoreactivity in cell bodies in the spinal cord, including motoneurones, and with CGRP or galanin immunoreactivities in dorsal root ganglion cells was also noted. The differential pattern of pancreastatin immunostaining was reflected in the extractable levels of peptide with highest concentrations in the cortex (55.8 +/- 6.0 pmol/g wet weight, mean +/- S.E.M.), thalamus (60.0 +/- 5.0 pmol/g), hypothalamus (54.4 +/- 6.5 pmol/g), and anterior pituitary (2,714 +/- 380 pmol/g). Characterisation of pancreastatin immunoreactivity in the hypothalamus and pituitary by gel permeation and high-pressure liquid chromatography revealed multiple molecular forms, one of which was indistinguishable from natural porcine pancreastatin. The widespread distribution of pancreastatin immunoreactivity suggests this peptide may play a part in several neuroendocrine, autonomic, somatic, and sensory functions, and its colocalisation with chromogranin A is consistent with a precursor-product relationship.

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat.

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.

The anterior pituitary content of neuromedin U-like immunoreactivity is altered by thyrotrophin-releasing hormone and thyroid hormone status in the rat.

In this study we have examined the effects of TRH, thyroid hormones and dopamine on the rat anterior pituitary content of neuromedin U-like immunoreactivity. Oral administration of TRH (20 mg/100 g per day) to euthyroid animals evoked a fivefold increase in peptide content after 12 days of treatment. This effect was found to be dependent upon circulating levels of thyroid hormone, since administration of TRH to thyroidectomized animals failed to show a similar effect without simultaneous treatment with tri-iodothyronine. The possibility that the TRH-induced increase in anterior lobe neuromedin U content reflected alterations in prolactin secretion or synthetic rate was also examined. Treatment of euthyroid animals with a dopamine agonist and antagonist was, however, without effect. These results demonstrate a unique relationship between TRH and thyroid hormone levels in increasing the anterior lobe content of neuromedin U immunoreactivity.

Increased nitric oxide synthase immunoreactivity in rat dorsal root ganglia in a neuropathic pain model.

In rats, tight ligation of L5 and L6 spinal nerves produces symptoms of thermal hyperalgesia and mechanical allodynia, mimicking the symptoms which characterise painful peripheral neuropathies in humans. Immunoreactivity for nitric oxide synthase (NOS) was investigated in lumbar (L1, L4, L5 and L6) dorsal root ganglia from naive controls and from rats surviving for 3, 7, and 14 days after unilateral ligation of the L5 and L6 spinal nerves. Quantitative analysis revealed significant increases in the percentage of NOS-immunoreactive cell profiles in L5 and L6 ganglia on the operated side at all time points, with the number of labelled profiles increasing with time following ligation, but L1 and L4 ganglia were unaffected. These findings suggest that nitric oxide may have a role in the generation and/or maintenance of neuropathic pain.

Mycobacterium chelonei keratitis: a case report.

The case is reported of a patient who initially presented with a dendritiform corneal ulcer that ultimately failed to heal and in which Mycobacterium chelonei was repeatedly cultured. The organism was sensitive only to kanamycin and amikacin; however, topical administration of these antibiotics failed to achieve a complete cure. Penetrating keratoplasty was ultimately required to eradicate the organism from the cornea.

A robust method for isotopic riboprobe in situ hybridisation to localise mRNAs in routine pathology specimens.

In situ Hybridisation (ISH) to detect mRNA is widely applicable to studies of human pathology and experimental models including transgenic mice, where it can provide crucial information as to where a gene is expressed, that is not available in other ways. ISH was used to establish that relatively high levels of expression of E-cadherin mRNA were associated with long-term survival in colorectal cancer, before suitable antisera became available. There is increasing awareness that ISH can be used to help select between candidate disease genes found at a linked locus, and that ISH can help validate therapeutic targets. For instance, when several closely related receptor genes are expressed in a tissue, it may be possible to determine which combinations are expressed together in a single cell type. These diverse applications demand a robust method that works on a variety of clinical and experimental materials, and is easily interpreted. To date, the ICRF in situ Hybridisation Service has hybridised over 30,000 sections of principally formalin-fixed, paraffin-embedded tissues, with a high success rate. The practicalities of our preferred method are discussed and key steps for quality control highlighted.

Comparison of isotopic and non-isotopic labelling for in situ hybridisation of various mRNA targets with cRNA probes.

In situ hybridisation methods to localise messenger ribonucleic acid (mRNA) targets in tissue sections or cell preparations using riboprobes can be successful with either isotopic or non-isotopic labelling. Investigators often wish to decide which labelling method provides the maximum specificity, sensitivity and resolution, with minimum nonspecific background. In this study we compared isotopic (35S) and non-isotopic (digoxigenin) labelling, using a variety of probes and paraffin-embedded tissues. The targets were human beta-actin and von Willebrand Factor mRNAs in archival human tissues; and mRNAs for two closely related trefoil factor family (TFF) peptides, TFF2 and TFF3, in rat duodenum. Patterns of localisation with both isotopic and non-isotopic probes were broadly similar for each target. The 35S labelling provided good contrast and sensitive detection under darkfield illumination, but the cellular or subcellular resolution of the target was less precise than that obtained with the digoxigenin-labelled probes in transmitted light. Digoxigenin labelling in individual cells was more clearly demonstrated, but occasionally the contrast of positive staining with background was poor. The sensitivity of each method appeared to be similar for these high-abundance targets, therefore the choice between isotopic and non-isotopic labels is dependent upon the aim of the study and the cellular resolution required.

Making sense out of in situ PCR.

Given the limited sensitivity of existing in situ hybridization methods for detecting specific nucleic acid sequences, amplification in situ by means of the polymerase chain reaction (PCR) seems to be an attractive alternative. Recent studies using in situ PCR technology have not assessed the gain in signal strength that has been achieved, nor evaluated quantitatively the efficiency of amplification. An accompanying article in the current issue of the Journal examines the reproducibility and amplification efficiency of an RT-PCR in situ hybridization method that uses a sense probe, capable of detecting only amplified target sequences. The amplification procedure resulted in approximately 3-6-fold increased sensitivity that depended upon cell type and disease status.

Molecular approaches to neuroendocrine pathology.

Prognosis of many tumour types is influenced by the degree of neuroendocrine differentiation. Neuroendocrine tumours produce bioactive peptides and amines that can have major disruptive effects on physiology. In the past, investigation of neuroendocrine pathology has relied upon traditional histological staining methods and morphological analysis at light and electron microscopic level. While these methods are still invaluable, the use of immunocytochemical techniques has revolutionised the diagnosis and understanding of neuroendocrine tumours, allowing precise identification of tumour types by means of antibodies to general neuroendocrine markers and tumour-specific antigens. However, the histogenesis/oncogenesis of neuroendocrine neoplasia cannot be understood by characterising the tumour products alone. Molecular technology has made possible investigation of gene expression by in situ hybridisation, electrophoresis and Northern or Southern blotting, and highly specific and sensitive techniques such as the polymerase chain reaction. Where gene expression and gene product storage are poorly correlated, molecular pathology provides vital information to aid diagnosis. Understanding of genetic factors involved in the familial neuroendocrine syndromes such as multiple neuroendocrine neoplasia (MEN) has improved. Oncogenes, tumour-suppressor genes and transcription factors have been identified. The factors controlling cell proliferation, growth and progression of tumours can be investigated at molecular level. Expression of amidating enzymes along with bioactive products including growth factors raises the question of whether tumour growth can be controlled or prevented by inhibition of amidating enzymes that activate the growth factors.

Advantages of in situ hybridisation over direct or indirect in situ reverse transcriptase-polymerase chain reaction for localisation of galanin mRNA expression in rat small intestine and pituitary.

In situ hybridisation (ISH) and direct or indirect in situ reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect galanin mRNA in paraffin sections of rat intestine and pituitary. With conventional ISH, a subset of intestinal neuronal ganglion cells and anterior pituitary endocrine cells were labelled. Direct in situ RT-PCR also labelled some cells in pituitary but not in intestine. Negative controls were unlabelled, but sections with 3' primer alone for RT-PCR appeared positive. No signal was apparent using the indirect in situ RT-PCR method. Investigation of the specificity of solution phase RT-PCR using RNA extracts from pituitary or intestine revealed that additional PCR products were detected under some conditions. The sequences of these PCR products suggested that one was the result of mispriming and single primer PCR, which could also have occurred in situ. Alternative galanin primers gave only the predicted RT-PCR product in solution phase yet still gave artefacts in tissue sections using direct in situ RT-PCR. ISH with probes transcribed from the correct PCR product gave identical labelling to the original galanin riboprobe. In conclusion, direct in situ RT-PCR is unreliable and requires validation, while indirect in situ RT-PCR may fail even though sufficient target exists for detection with conventional sensitive riboprobe ISH.

Impaired mammary gland development in Cyl-1(-/-) mice during pregnancy and lactation is epithelial cell autonomous.

A specific defect of mice lacking cyclin D1 (Cyl-1(-/-)) is impaired development of the mammary gland during pregnancy. Here we show that when tissue from Cyl-1(-/-) mammary gland was transplanted into empty mammary fat pad of wild-type mice, the abnormal phenotype was maintained, indicating that it is epithelial cell autonomous. Nevertheless, in pregnancy the early proliferative response, which is characterized by extensive side branching, still occurs in the absence of cyclin D1. However, the response is atypical due to a marked reduction in the formation of accompanying alveoli. This reduction and delay in alveolar development persists throughout pregnancy. Moreover, although prolactin synthesis and release appear to be normal, lactogenesis is severely compromised. Consistent with the appearance of numerous side branches, progesterone receptor expression was readily detected in the mammary tissue of pregnant Cyl-1(-/-) mice, although there was a significant change in the ratio of the two (A and B) receptor isoforms. In Cyl-1(-/-) mammary glands during late pregnancy there was a decrease in the abundance of total and phosphorylated Stat5a, as well as delayed onset and substantial diminution of milk protein expression. The biochemical analysis suggests that there is a cumulative delay in growth and differentiation of the mammary gland during pregnancy that results in a severely compromised gland when, at parturition, further development is curtailed by the abrupt change in hormonal milieu.