Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury.

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Heritable disorders of pituitary development.

Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism. Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis. Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland. Pit-1 has a POU-specific and a POU-homeo DNA-binding domain. The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH. Pituitary glands are either small or normally sized. The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain. Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH. Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension. Pituitary degeneration may produce acquired deficiency of ACTH. Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread. The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding. Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia. Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism.

The cellular localization of vasoactive intestinal peptide (VIP) in the mouse median eminence by immuno-electron microscopy.

The aim of this study was to examine, by use of pre-embedding immunocytochemistry, the ultrastructural localization of vasoactive intestinal peptide (VIP) immunoreactivity in the mouse median eminence. VIP immunoreactivity was observed in axonal profiles. The VIP-immunoreactive axonal profiles were in close proximity to non-immunoreactive axonal profiles that contained dense granular vesicles and clear vesicles and also to processes of tanycytes. VIP-immunoreactive terminals were observed in the proximity of the perivascular space and in the neuropil. Our results suggest that VIP-immunoreactive axon terminals may possibly interact with other non-immunoreactive axon terminals containing peptide and/or other transmitters at the level of the median eminence or may be released to the portal vasculature thereby to effect anterior pituitary cells.

Median eminence-afferent vasoactive intestinal peptide (VIP) neurons in the hypothalamus: localization by simultaneous tract tracing and immunocytochemistry.

Retrograde tract tracing and immunocytochemistry were used to investigate the CNS source of the VIP that is present in high concentrations in the hypophysial portal blood and has been shown to have a stimulatory effect on pituitary prolactin secretion. Fluoro-gold (FG), which enters the CNS through areas devoid of the blood-brain barrier, such as median eminence, was injected peripherally. Brain sections from FG-treated animals were immunostained for VIP. A small population of VIP-containing cell bodies in the parvocellular and periventricular parts of the paraventricular nucleus (PVN) was also labeled with FG. Vasoactive intestinal peptide-immunoreactive perikarya not labeled with FG were also observed in the PVN, as well as FG-labeled cells that did not contain VIP. The results suggest that some VIP-producing neurons in the PVN project to the median eminence and are, therefore, functionally related to pituitary regulation; the function of other VIP neurons in the PVN is unknown.

Quantification of vasoactive intestinal peptide immunoreactivity in the anterior pituitary glands of intact male and female, ovariectomized, and estradiol benzoate-treated rats.

There are considerable data suggesting that vasoactive intestinal peptide (VIP) is involved in the regulation of PRL secretion; however, the role and cell of origin of anterior pituitary VIP remain to be determined. Immunocytochemical (ICC) studies have generally failed to detect VIP-immunoreactive (IR) cells in the pituitary of the untreated rat, although VIP-IR cells have been observed in the pituitaries of hypothyroid or estrogen-treated rats. This study was designed to examine the cellular distribution and tissue content of VIP in the anterior pituitary gland of rats under selected endocrine conditions known to alter the rates of PRL and VIP synthesis and secretion. To this end, anterior pituitary VIP and PRL content (ICC and RIA) and serum PRL levels were determined in ovariectomized (OVX) and OVX rats 3 days after treatment with 7 or 70 micrograms estradiol benzoate (EB). For comparison, pituitary VIP and PRL content (ICC and RIA) and serum PRL levels in untreated male and diestrous female rats were determined. Immunostaining for VIP was accomplished using a newly developed primary antiserum. Significant numbers of VIP-IR cells per 5-microns section were found in the anterior pituitary glands of all animals examined (275 +/- 33 in diestrous to 481 +/- 103 cells in male rats). VIP was not colocalized with PRL in any of the pituitaries regardless of steroid treatment or sex. Furthermore, the number of VIP-IR cells per pituitary gland was not significantly correlated with sex or EB treatment. Treatment with 70 micrograms, but not 7 micrograms, EB significantly increased the pituitary content of VIP and serum PRL levels compared to those after ovariectomy. However, both EB treatments resulted in a significant increase in pituitary PRL content compared to that in untreated OVX rats. Pituitaries from male rats had several-fold more VIP and less PRL content than pituitaries from diestrous rats. These data show that 1) in contrast to previous ICC studies, VIP-IR cells are readily detected in the anterior pituitary of intact male and female and OVX as well as EB-treated rats; 2) VIP is localized to cells other than lactotrophs, regardless of the steroid background; and 3) marked changes in anterior pituitary VIP content are not accompanied by changes in VIP-IR cell number.

Catecholamine reinnervation of supraoptic nucleus after deafferenting mechanical lesions and superior cervical ganglionectomy: histofluorescence and functional assessments in young adult and aged rats.

Catecholaminergic innervation of the hypothalamic vasopressin (VP)-secreting supraoptic nucleus (SON) was examined at selected intervals after deafferenting neurosurgical lesions, with respect to potential contribution of peripheral vascular sympathetic fibers. Young adult (3 months) and aged (20 months) male F344 rats were subjected to mechanical knife-cut lesion just caudal and medial to the SON, superior cervical ganglionectomy (SCGx), or both surgeries. SON and lesion sites were assessed at 4, 14, 30 or 45 days after surgery, by CA histofluorescence. Functional evaluation in rats subjected to chronic lesions consisted of monitoring water balance (water consumption and urine volumes, and urine osmolality and VP content) in individual rats for presurgical and postsurgical intervals. Histofluorescence evaluation showed that SCGx did not affect the overall SON fluorescence pattern, although a minor sympathetic contribution to that pattern was discerned by comparing SON in rats subjected to lesion alone vs SCGx + lesion. Morphological reinnervation of SON was accomplished at 30 days in young rats, and 45 days in aged rats, after both lesion and SCGx. In young rats, histofluorescence density 30 days after deafferentation was denser than the innervation pattern seen in intact (sham-lesioned) animals, while reinnervation at 45 days postsurgically in aged rats only approximated the presurgical pattern. Vasopressin excretion and corresponding water conservation measures were compromised by SON deafferentation at both ages; excreted VP levels and water balance did not rebound to presurgical values at chronic postsurgical intervals in either young or aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characterization of the Clostridium difficile toxin A gene.

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Effects of bromocriptine on prolactin cellular hypertrophy, proliferation and secretory activity in diethylstilbestrol-induced pituitary tumors.

Pituitary tumors induced by chronic diethylstilbestrol (DES) treatment in female F344 rats were treated subsequently with bromocriptine (BC). Effects of BC on separable subpopulations of lactotrophs were examined. Enzymatically dissociated cells from individual pituitaries were assessed regarding total number, relative lactotroph population, intracellular prolactin (PRL) content, PRL release in primary culture, and density alterations by separation in Ficoll-Hypaque or after sedimentation at unit gravity. In addition to the treatment and analysis of in situ tumors, the effects of BC treatment in vitro were assessed, using tumor cells which were first separated on Ficoll-Hypaque. Cell proliferation was assessed by cell cycle analysis, using DNA measurement by laser flow cytometry. BC treatment of tumors reversed the effects of DES on pituitary weight, PRL content and in vitro PRL release. Total cell recovery was not affected by BC, but cell separation showed that BC reduced the number of larger PRL-containing cells. Cell cycle analysis showed a decrease in numbers of cells in S and G2 cycle phases after BC in only one of four experiments. BC had an effect on proliferation in only the upper gradient fractions, containing the smallest cells. Culture of Ficoll-separated tumor cells revealed greater PRL release among lighter/smaller cells. BC treatment inhibited PRL release from both light and dense cells. The results establish that PRL cell hypertrophy, as well as hyperplasia, results from DES treatment. Bromocriptine treatment reverses this hypertrophy concomitant with inhibiting PRL synthesis and release. Reversal of proliferation in tumor cells is not a major effect of bromocriptine treatment.

Isolated deficiency of tyrosine hydroxylase immunoreactivity in tuberoinfundibular neurons in pituitary prolactin-deficient Snell dwarf mice.

Light microscopic morphology and relative numbers of tyrosine hydroxylase (TH)-immunoreactive neurons were assessed in hypothalamus of hypopituitary Snell dwarf mice, compared with normal mice of the same strain. Qualitatively, a specific deficit in staining of tuberoinfundibular neurons was noted in dwarf hypothalamus; TH-positive terminals were absent in dwarf median eminence, and morphology of axons in this region was aberrant. Qualitative observations were supported by quantitative assessment of hypothalamic dopaminergic neuron groups. Dorsal and intermediate TH-positive cells numbered 80% of those normal mouse brain; tuberoinfundibular (A12) TH-positive neurons in dwarf hypothalamus were 2% of those in normal mouse brain. The results imply that absence of pituitary hormone feedback impedes both structural and biochemical development of hypophysiotropic hypothalamic neurons.

Effect of chronic hyperprolactinemia on tuberoinfundibular dopaminergic neurons: histofluorescence in aged and in diethylstilbestrol-treated male rats.

Catecholamine histofluorescence was used to examine morphology of hypothalamic tuberoinfundibular dopaminergic (TIDA) median eminence-afferent neurons (areas A12 and A14) in male Fischer 344 rats bearing prolactin (PRL)-secreting tumors. These tumors were either spontaneous, discovered in rats aged 20-32 months, or were induced by chronic diethylstilbestrol (DES) treatment in younger animals. In the latter case, histofluorescence was examined in two groups of animals: (1) those which had been treated, beginning at 6 months of age, with 8 weeks of DES, and subsequently had had the treatment discontinued for 10 months, but which continued to exhibit hyperprolactinemia; (2) animals treated continuously with DES, for 50 or 70 days. Histofluorescence was induced using either the Falck-Hillarp, aluminum-formaldehyde ('ALFA') or the formaldehyde-glutaraldehyde ('FAGLU') technique and pituitaries were examined for PRL immunoreactivity, as permitted by the fixation method chosen for histofluorescence. For selected animals, pituitaries were enzymatically dissociated, maintained in primary culture, and were assessed for PRL cell quantification, intracellular PRL content, and in vitro release. Among animals treated chronically with DES, histofluorescence was assessed by the Falck-Hillarp technique, and counts of A12 and A14 perikarya were made in each hypothalamus. Median eminence zona externa fluorescence was diminished in aged rats with normal pituitaries, in aged rats bearing spontaneous tumors, and in rats treated continuously with DES, in comparison with fluorescence in younger rats with normal pituitaries or from which DES treatment had been with-drawn. Perikaryal fluorescence showed typical cell morphology and numbers in aged rats, aged rats bearing spontaneous tumors, and in rats from which DES had been withdrawn. In one animal, treated continuously for 70 days with DES, numbers of A12 and A14 perikarya decreased. In aged rats bearing spontaneous tumors, histofluorescence morphology indicated stimulatory effects on TIDA perikarya and terminals, including increased perikaryal and terminal fluorescence intensity. The morphological results support biochemical evidence of PRL stimulation of tuberoinfundibular dopaminergic neurons, and suggest that inhibitory effects of increased PRL secretion on these hypothalamic cells in DES-induced hyperprolactinemia reflect a direct and reversible effect of DES on these neurons.

Pituitary lactotroph sedimentation profiles and in vitro secretory activity after ablation of the medial basal hypothalamus.

Pituitary cells from adult male rats subjected to chronic (6 and 10 weeks) medial hypothalamic ablation (MHA) were analyzed by unit gravity sedimentation to assess distribution of size and density of lactotrophs, and for subsequent in vitro prolactin (PRL) release in primary culture. Tinctorial staining (Herlant's tetrachrome) showed that initial preparations of cells from MHA rats were small and relatively undifferentiated. MHA cells did not sediment as far into the gradient as did cells from intact control pituitaries. Intracellular PRL content was lower in all gradient fractions of MHA cells. At 6 weeks after surgery, peak recovery of PRL was also in the upper portions of the gradient. In the 10-weeks group, however, peak PRL recovery from MHA cells was in a population that sedimented further, but more restrictedly, in comparison with control cells. At both postsurgical intervals, the majority of tinctorially or immunocytochemically identified lactotrophs from lesioned rats were lower in the gradient, indicating enlarged and denser cells. Relative numbers of lactotrophs (per pituitary) were increased 10 weeks after MHA. In vitro PRL release, over a maximum of 21 days culture, was comparable for cells from MHA rats and intact controls, according to daily per cell secretion rates and "production index" (hormone released/initial hormone content). By comparison, luteinizing hormone (LH) release was suppressed in culture compared to intact controls, and LH was recovered from gradient fractions of smaller cells. The results indicate that chronic removal of hypothalamic influence results in gradual prolactin cell hypertrophy and decreased hormone retention and in relative increase in numbers.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical analysis of prolactin cells in the adult rat adenohypophysis: distribution and quantitation relative to sex and strain.

Prolactin immunocytochemistry was conducted 1) in serial sections of whole fixed pituitaries, for cell distribution analysis; and 2) on preparations of enzymatically dispersed anterior pituitary cells maintained for 1-4 days in vitro, for quantitation of the relative population of prolactin (PRL) cells. Both types of analysis were conducted on glands from young adult rats of both sexes in Long-Evans, Sprague-Dawley, and Fischer 344 strains. Cell quantitation results were compared with serum levels, intracellular content, and in vitro release of prolactin, for individual cell preparations. The results show that both PRL cell distribution and relative population numbers are similar between sexes in all three rat strains. Average percentages ranged from 25.9% to 32.1% in all young males and diestrous females. Prolactin-positive numbers of cells correlated positively with intracellular hormone content but not with serum or medium PRL levels. The prolactin cell population was significantly larger in aged Fischer 344 males but was not correlated with intracellular PRL levels. The prolactin cell population was heterogeneous in staining intensity and distribution in both sexes of all three strains.

Plasticity of catecholaminergic neurons in aged rat brain: reinnervation and functional recovery after axotomy.

Regenerative growth at the lesion site, reinnervation of a target nucleus and functional manifestations of recovery were studied in aged (20 and 30 months old) rats subjected to long-term transection of catecholaminergic (CA) fibers which contact and influence neurons of the supraoptic nucleus (SON). Small bilateral knife cuts were placed stereotaxically just caudal and medial to the SON. CA histofluorescence, induced by formaldehyde-glutaraldehyde (FAGLU) or aluminum-formaldehyde (ALFA) methods, was examined in hypothalamus at 2, 14, 21 and 60 days postsurgically. Water consumption, and urine volume and osmolality, were monitored presurgically, and through survival times. Subtotal CA denervation in the SON, and typical axonal transmitter "pile-up" at the lesion site, were evident two days after surgery. Among these degenerative profiles, which persisted for up to three weeks, fine-sized new fibers were apparent at the lesion, beginning between 2 and 14 days, and persisting throughout the period studied. At 21 days, and progressively thereafter, SON neurons were rimmed with fluorescent varicosites. Water consumption initially was depressed, but returned to presurgical mean levels by nine days. Urine volume returned to normal by 32 days. Urine osmolality showed a recovery by approximately three weeks. These functional parameters rebounded to levels higher than presurgical means among 20 month old, but not 30 month old, rats beyond 6 weeks survival, concurrent with a morphological hyperinnervation. The results reaffirm morphological regeneration, and support reinnervation and functional recovery, which extend considerably into the aging process.