Heterogeneity of autosomal dominant osteopetrosis

A review of the radiographs of 26 patients with autosomal dominant osteopetrosis disclosed two distinct and strictly family-related radiographic types. Both types had universal osteosclerosis. In type 1 the most striking finding was pronounced sclerosis of the cranial vault while the spine was almost unaffected. In type 2 the sclerosis of the skull was most pronounced at the base, the vertebrae always had end-plate thickening, and in the pelvis the iliac wings contained convex arcs of sclerotic bone. Age and sex distribution did not differ between the types. Autosomal dominant osteopetrosis may be a heterogeneous group of inherited bone disorders.     

A circumscribed projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: anatomical evidence for somatostatin-28-immunoreactive interneurons subserving reflex control of esophageal motility

Axonal transport and immunohistochemical methods were used to investigate the anatomical and biochemical organization of projections from the nucleus of the solitary tract (NTS) to the rostral, esophageal, part of the nucleus ambiguus (NA) in the rat. Discrete iontophoretic deposits of a retrogradely transported tracer, fluorogold, placed in the rostral NA labeled a column of cells within the NTS, termed the central part of the NTS (after Ross et al., 1985), situated just medial to the solitary tract and extending from about 300 to 1000 microns rostral to the obex. Iontophoretic deposits of the anterogradely transported tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L), placed in the central part of the NTS gave rise to dense and topographically restricted projections to the rostral NA. More caudal and ventral aspects of the NA did not receive prominent inputs from the central part of the NTS, and deposits that spared the central part of the NTS gave rise to only sparse projections to the rostral NA. Antisera against somatostatin-28 (SS-28) stained cell bodies within the central part of the NTS. In addition, a double-labeling procedure, capable of colocalizing anterogradely transported PHA-L and endogenous peptides to individual fibers and/or terminals, demonstrated an appreciable number of SS-28-immunoreactive terminals within the rostral NA that arose from the NTS. Correspondingly, unilateral lesions that involved the central part of the NTS resulted in a marked depletion of SS-28 immunoreactivity in the ipsilateral rostral NA. These data provide evidence for a discrete, partly somatostatinergic, projection from the central part of the NTS to the rostral NA. Anatomical and physiological studies implicating the central part of the NTS and the rostral NA in esophageal function suggest this pathway to be involved in the reflex control of esophageal motility.     

Novel hypothalamic and preoptic sites of prepro-melanin-concentrating hormone messenger ribonucleic Acid and Peptide expression in lactating rats

The deduced structure of the rat melanin-concentrating hormone (MCH) precursor predicted the existence of at least two peptides that may be processed from it, one similar to teleost MCH and a second novel neuropeptide, NEI. Cellular localization studies confirmed that prepro-MCH (ppMCH) mRNA and the MCH and NEI peptides are expressed predominantly in cells in the zona incerta and caudal lateral hypothalamic area with minor contingents seen in the olfactory tubercle and pons. A moderate MCH-and NEI-immunoreactive axonal projection to the median eminence and, particularly, to oxytocin-rich regions of the posterior pituitary suggested some anatomical heterogeneity of ppMCH-expressing neurons in the hypothalamus, and an involvement in neuroendocrine function. In the present study, immunohistochemical and hybridization histochemical methods were used to follow MCH gene and peptide expression as a function of reproductive status in female rats. Nursing dams sacrificed after 8 to 21 days of lactation consistently displayed ppMCH mRNA and MCH and NEI immunoreactivity in a discrete and contiguous band, encompassing the ventral aspect of the medial part of the medial preoptic nucleus, the periventricular preoptic nucleus, and the most rostral aspects of the paraventricular nucleus of the hypothalamus (PVH). Combined immunohistochemical (for oxytocin) and hybridization histochemical (for ppMCH mRNA) staining failed to reveal a significant degree of congruence in the two chemically-specified cell populations in the PVH of lactating dams. The apparent induction of ppMCH-derived peptides and mRNA in the preoptic area and PVH was not apparent in animals sacrificed 4 to 8 days after weaning, during late pregnancy, or at any point in the estrous cycle. Moreover, no frank alterations in ppMCH mRNA were evident in the principal sites at which ppMCH is expressed at constitutively high levels, i.e. in the lateral hypothalamic area and zona incerta, as a function of reproductive status. The loci and apparent state-dependency of the induction of ppMCH mRNA and peptide expression suggests a role for these gene products in the control of lactation.     

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of “free” CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.     

Evidence for involvement of a limbic paraventricular hypothalamic inhibitory network in hypothalamic‐pituitary‐adrenal axis adaptations to repeated stress

Emotional stressors activate a stereotyped set of limbic forebrain cell groups implicated in constraining stress‐induced hypothalamic‐pituitary‐adrenal (HPA) axis activation by inhibiting hypophysiotropic neurons in the paraventricular hypothalamic nucleus (PVH). We previously identified a circumscribed, anterior part of the bed nuclei of the stria terminalis (aBST) that houses stress‐sensitive, PVH‐projecting, γ‐aminobutyric acid (GABA)‐ergic neurons as representing a site of convergence of stress‐inhibitory influences originating from medial prefrontal and hippocampal cortices. Here we investigate whether exaggerated HPA axis responses associated with chronic variable stress (CVS; daily exposure to different stressors at unpredictable times over 14 days, followed by restraint stress on day 15) and diminished HPA output seen following repeated (14 days) restraint‐stress exposure are associated with differential engagement of the limbic modulatory network. Relative to acutely restrained rats, animals subjected to CVS showed the expected increase (sensitization) in HPA responses and diminished levels of activation (Fos) of GABAergic neurons and glutamic acid decarboxylase (GAD) mRNA expression in the aBST. By contrast, repeated restraint stress produced habituation in HPA responses, maintained levels of activation of GABAergic neurons, and increased GAD expression in the aBST. aBST‐projecting neurons in limbic sites implicated in HPA axis inhibition tended to show diminished activational responses in both repeated‐stress paradigms, with the exception of the paraventricular thalamic nucleus, in which responsiveness was maintained in repeatedly restrained animals. The results are consistent with the view that differential engagement of HPA inhibitory mechanisms in the aBST may contribute to alterations in HPA axis responses to emotional stress in sensitization and habituation paradigms. J. Comp. Neurol. 523:2769–2787, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural analysis of murine megakaryocyte maturation in vitro: comparison of big-cell and heterogeneous megakaryocyte colonies

Two morphologically distinct types of murine megakaryocyte (MK) colonies are present after three to seven days in soft agar culture: (a) "big-cell" colonies composed of ten to 30 large, mature-appearing megakaryocytes and (b) "heterogeneous" colonies consisting of approximately 100 or more cells at various stages of differentiation. Cytochemical and immunocytochemical techniques were used to study MK maturation in colonies as well as normal mouse bone marrow. Acetylcholinesterase (AChE), a specific marker for murine platelets and MK, was found in the perinuclear cisterna, endoplasmic reticulum, and occasionally, Golgi cisternae of MK in three-day big-cell colonies and immature bone marrow MK. MK in seven-day big-cell colonies and mature bone marrow MK showed additional reaction sites in the demarcation membrane system and occasional granules. In seven-day heterogeneous colonies, small cells resembled immature bone marrow MK with respect to AChE localization, whereas large cells corresponded to mature bone marrow MK. With immunogold procedures at the ultrastructural level, polyclonal antibodies against human platelet membrane glycoprotein IIIa and antimouse platelet antiserum labeled bone marrow MK and all MK from colonies grown in soft agar cultures for three to seven days. Granulocytes and macrophages in both bone marrow and soft agar cultures were negative for AChE and these immunocytochemical markers. These data indicate that the pattern of expression of AChE during maturation of MK is similar in vivo and in vitro and demonstrate, when using this marker at the fine-structural level, that a greater range of MK maturational stages is present in heterogeneous colonies than is observed in MK in big-cell colonies. Furthermore, we have confirmed that small cells in heterogeneous colonies are MK and that these colonies are composed solely of MK and their precursors.     

Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation

Human herpesvirus 6 activity (HHV-6) was studied in 15 allogeneic and 11 autologous marrow transplantation patients. After transplantation, HHV-6 was isolated from the peripheral blood mononuclear cells of 12 of 26 patients (6 allogeneic and 6 autologous). All isolates were variant B. Eleven of 26 and 12 of 19 patients showed salivary shedding of HHV-6 DNA before and after transplantation, respectively. The antibody titer increased in 7 of 26 patients. Thus, 23 of 26 patients showed evidence of active HHV-6 infection either by virus isolation, salivary shedding, or increases in antibody titers. The fraction of saliva specimens positive in 19 patients was negatively associated with their antibody titers (P= .005). The proportion of cultures positive increased after transplantation (P = .007). Sinusitis was associated with HHV-6 isolation in autologous recipients (P= .002). In allogeneic patients, active human cytomegalovirus infection was associated with HHV-6 isolation (P = .04). No association was observed between HHV-6 infection and GVHD, pneumonia, delay in engraftment, or marrow suppression. Of the 120 clinical events analyzed in 26 patients, HHV-6 was defined as a probable cause of 16 events in 9 patients based on the propinquity of HHV-6 activity and the clinical event plus the absence of other identified causes of the event.     

Human granulocytes generated in continuous bone marrow culture are physiologically normal

A long-term bone marrow culture system has been derived for maintenance and proliferation of human hemopoietic stem cells and granulocytes in vitro for up to 20 wk. The granulocytes generated in these cultures at 8 wk were comparable to fresh human peripheral blood granulocytes in physiologic properties, including phagocytosis, degranulation, respiratory burst, and bacterial killing: individual granulocytes generated up to 20 wk in several cultures demonstrated normal superoxide-generating capacity by NBT dye reduction slide test. Thus, human granulocytes generated in continuous marrow culture retain many biologic functions associated with bacterocidal capacity in vivo and indicate that this system should be of value in studies of disorders of granulocyte differentiation.