Managing a nosocomial outbreak of carbapenem-resistant Klebsiella pneumoniae: an early Australian hospital experience.

BACKGROUND: Carbapenems are traditionally reserved as the last line of defence for treatment of serious infections with multiresistant Gram-negative bacilli. Reports of Klebsiella pneumoniae carbapenemase (KPC)-producing organisms have been emerging globally, but rare in Australasia to date. We describe an outbreak of KPC-2 producing K. pneumoniae at an Australian hospital. METHODS: After initial detection in October 2012, a retrospective review of patients with meropenem-resistant K. pneumoniae to June 2012, and ongoing prospective surveillance, was undertaken. Included patients were admitted to the hospital after June 2012 and had meropenem-resistant K. pneumoniae isolated from any site. Available isolates underwent detection of the KPC-2 gene by polymerase chain reaction and molecular typing was performed to determine genetic relatedness between isolates. Point-prevalence screening was performed on selected wards to detect asymptomatic carriage. Infection control procedures were implemented to contain the outbreak. RESULTS: Ten cases were identified in the initial cluster. Eight were localised to a single inpatient ward. Point-prevalence screening revealed one extra case. After temporary containment, re-emergence of KPC-producing isolates was observed post October 2013 with 18 further cases identified. Four K. pneumoniae isolates in the 2012 cluster and 16 from the 2013-2014 cluster were referred for further testing. All carried the KPC-2 beta-lactamase gene. The 2012 isolates were genetically similar to the 2014 isolates. CONCLUSION: KPC-2 mediated resistance is an emerging threat in Australia. The re-emergence of KPC despite initial containment emphasises the need for constant vigilance in the microbiology laboratory and ongoing maintenance of infection control and antimicrobial stewardship activity.

Rothia aeria mitral valve endocarditis complicated by multiple mycotic aneurysms: laboratory identification expedited using MALDI-TOF MS.

Rothia aeria has only rarely been described as a human pathogen. We describe a case of Rothia aeria causing mitral valve endocarditis and multiple mycotic aneurysms, including cerebral mycotic aneurysms. In the case described, early identification of Rothia aeria was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Growth hormone (GH), brain development and neural stem cells.

A range of observations support a role for GH in development and function of the brain. These include altered brain structure in GH receptor null mice, and impaired cognition in GH deficient rodents and in a subgroup of GH receptor defective patients (Laron dwarfs). GH has been shown to alter neurogenesis, myelin synthesis and dendritic branching, and both the GH receptor and GH itself are expressed widely in the brain. We have found a population of neural stem cells which are activated by GH infusion, and which give rise to neurons in mice. These stem cells are activated by voluntary exercise in a GH-dependent manner. Given the findings that local synthesis of GH occurs in the hippocampus in response to a memory task, and that GH replacement improves memory and cognition in rodents and humans, these new observations warrant a reappraisal of the clinical importance of GH replacement in GH deficient states.

Substrate-Controlled Magnetism: Fe Nanowires on Vicinal Cu Surfaces.

Here we present a novel approach to controlling magnetic interactions between atomic-scale nanowires. Our ab initio calculations demonstrate the possibility to tune magnetic properties of Fe nanowires formed on vicinal Cu surfaces. Both intrawire and interwire magnetic exchange parameters are extracted from density functional theory (DFT) calculations. This study suggests that the effective interwire magnetic exchange parameters exhibit Ruderman-Kittel-Kasuya-Yosida-like (RKKY) oscillations as a function of Fe interwire separation. The choice of the vicinal Cu surface offers possibilities for controlling the magnetic coupling. Furthermore, an anisotropic Heisenberg model was used in Monte Carlo simulations to examine the stability of these magnetic configurations at finite temperatures. The predicted critical temperatures of the Fe nanowires on Cu(422) and Cu(533) surfaces are well above room temperature.

20-kDa placental hGH-V has diminished diabetogenic and lactogenic activities compared with 22-kDa hGH-N while retaining antilipogenic activity.

Placental human growth hormone-variant (hGH-V) and pituitary human growth hormone-N (hGH-N) are of identical size (22 kDa) but differ in 13 residues scattered throughout the protein. Several isoforms of GH are produced by the hGH-N and hGH-V genes including a 20-kDa hGH-V resulting from a 45-bp deletion caused by the use of an alternative acceptor site within exon 3. To date, the biological properties of the 20-kDa GH-V have not been characterized in vivo. Using young male Wistar rats fed either chow or a high-fat (HF) diet for 4 wk postweaning, we investigated the effect of 7 days treatment with either 22-kDa hGH-N, 20-kDa hGH-V (5 ug x g(-1) x day(-1) sc), or vehicle on body composition and endocrine and metabolic profiles. Total body growth (absolute weight gain and linear growth trajectory) in the 20-kDa hGH-V-treated animals was intermediary between that of control and hGH-N-treated animals. Both 22-kDa hGH-N and 20-kDa hGH-V significantly reduced total body fat mass compared with control animals, and there were no differences between the GH isoforms in anti-lipogenic activity in animals fed the HF diet. Fasting plasma insulin and C peptide were significantly increased in animals on the HF diet and further increased by hGH-N but were unchanged in 20-kDa hGH-V-treated animals compared with saline-treated controls. Plasma volume as assessed by hematocrit was increased in hGH-N-treated animals but was unchanged in 20-kDa hGH-V-treated animals compared with controls. Furthermore, 20-kDa hGH-V had reduced lactogenic (prolactin receptor mediated) activity characteristic of hGH-N as tested in vitro compared with the 20-kDa hGH-N and 22-kDa hGH-N variants. In summary, placental 20-kDa hGH-V retains some of the growth-promoting and all antilipogenic activities of pituitary 22-kDa hGH-N but has diminished diabetogenic and lactogenic properties compared with the native 22-kDa hGH-N.

New insights into growth hormone receptor function and clinical implications.

Although used as a therapeutic for 50 years, it is only recently that the application of molecular techniques has provided a basis for understanding growth hormone's (GH) clinical actions. This article reviews progress in our current knowledge of the molecular mechanism of growth hormone (GH) receptor activation based on a number of physicochemical techniques, and documents insights gained into the means used by the activated GH receptor to control the expression of genes regulating growth and metabolism. These findings are related to disorders of short stature, and the therapeutic consequences are summarized.

A growth hormone receptor SNP promotes lung cancer by impairment of SOCS2-mediated degradation.

Both humans and mice lacking functional growth hormone (GH) receptors are known to be resistant to cancer. Further, autocrine GH has been reported to act as a cancer promoter. Here we present the first example of a variant of the GH receptor (GHR) associated with cancer promotion, in this case lung cancer. We show that the GHRP495T variant located in the receptor intracellular domain is able to prolong the GH signal in vitro using stably expressing mouse pro-B-cell and human lung cell lines. This is relevant because GH secretion is pulsatile, and extending the signal duration makes it resemble autocrine GH action. Signal duration for the activated GHR is primarily controlled by suppressor of cytokine signalling 2 (SOCS2), the substrate recognition component of the E3 protein ligase responsible for ubiquitinylation and degradation of the GHR. SOCS2 is induced by a GH pulse and we show that SOCS2 binding to the GHR is impaired by a threonine substitution at Pro 495. This results in decreased internalisation and degradation of the receptor evident in TIRF microscopy and by measurement of mature (surface) receptor expression. Mutational analysis showed that the residue at position 495 impairs SOCS2 binding only when a threonine is present, consistent with interference with the adjacent Thr494. The latter is key for SOCS2 binding, together with nearby Tyr487, which must be phosphorylated for SOCS2 binding. We also undertook nuclear magnetic resonance spectroscopy approach for structural comparison of the SOCS2 binding scaffold Ile455-Ser588, and concluded that this single substitution has altered the structure of the SOCS2 binding site. Importantly, we find that lung BEAS-2B cells expressing GHRP495T display increased expression of transcripts associated with tumour proliferation, epithelial-mesenchymal transition and metastases (TWIST1, SNAI2, EGFR, MYC and CCND1) at 2 h after a GH pulse. This is consistent with prolonged GH signalling acting to promote cancer progression in lung cancer.

Gene expression profiling of cells involved in periodontal regeneration.

Understanding the molecular mechanisms involved in periodontal regeneration is important for the development of more predictable clinical techniques. This study aimed to identify these mechanisms by comparing the gene expression profiles of cells derived from regenerating defects with patient-matched periodontal ligament cells. Gene profiling was carried out via Affymetrix U133A arrays containing probes for 22,000 genes. Robust differences in gene expression were obtained by identifying genes that consistently changed by a minimum of 2-fold. Analysis of molecular function as designated by gene ontology (GO) identified differentially regulated mechanisms including protein metabolism, tyrosine kinase activity, and skeletal development. The differentially expressed genes could be broadly divided into the categories of protein biosynthesis and turnover, structural constituents of the cytoskeleton and extracellular matrix, and signal transduction. The differential expression of 4 genes (EGR-1, elastin, osteoprotegerin, and IGFBP3) was confirmed via real-time polymerase chain reaction (PCR). Further, the expression of another 2 differentially expressed transcripts, decorin and biglycan, was immunohistochemically confirmed in a periodontal wound healing model and the protein expression was consistent with the pattern of gene expression. This study gives insight into the molecular processes involved in periodontal regeneration and identifies cell markers that are characteristic of regenerating periodontal tissues.

Mouse molar dentin size/shape is dependent on growth hormone status.

Growth hormone (GH) status affects dental development, but how GH influences tooth size/shape is unclear. Since GH affects dental epithelial proliferation, we hypothesized that GH influences the tooth crown and root dimensions. Dentin matrix dimensions were measured in longitudinal sections of decalcified first mandibular molars from 3 genetically modified mice: giant (GH-Excess) mice and dwarf (GH-Antagonist and GH-Receptor-Knockout) mice. GH status was found to influence crown width, root length, and dentin thickness. Analysis of these data suggests that GH influences both tooth crown and root development prior to dentinogenesis as well as during appositional growth of dentin. This is concordant with the expression of paracrine GH and GH receptors during tooth bud morphogenesis, and of GH receptors in the enamel organ, dental papilla, and Hertwig's epithelial root sheath during dentinogenesis. Based on prior studies, these GH morphogenetic actions may be mediated by the induction of both bone morphogenetic protein and insulin-like growth factor-1 expression.

Risk factors for KPC-producing Enterobacteriaceae acquisition and infection in a healthcare setting with possible local transmission: a case-control study.

BACKGROUND: Reports of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) in Australia were previously uncommon, with cases imported sporadically by travellers from higher prevalence countries. AIM: The study institution reported the first outbreak of KPC-Kp in Australia. The aim of this study was to identify risk factors for KPC-Kp colonization and infection using a matched case-control study. METHODS: The study included all hospitalized patients with KPC-Kp colonization or infection from January 2012 to September 2015. FINDINGS: Thirty-four cases of KPC-producing Enterobacteriaceae (including 31 KPC-Kp cases) were matched with 136 controls. Variables associated with KPC-Kp acquisition included: length of hospital stay >28 days in the past 12 months, prior vancomycin-resistant enterococci (VRE) colonization, central venous catheter (CVC), gastrointestinal disease and invasive procedures. Exposure to broad-spectrum antibiotics was also found to be a significant risk factor. In the multi-variate analysis, three factors independently associated with KPC-Kp acquisition were length of hospital stay >28 days in the past 12 months [odds ratio (OR) 23.6, 95% confidence interval (CI) 4.9-113.3], presence of a CVC (OR 15.4, 95% CI 2.7-86.9), and prior VRE colonization (OR 6.0, 95% CI 1.6-23.2). Very few patients had a history of overseas travel. CONCLUSION: This study demonstrates that patients with prolonged hospital exposure are more likely to acquire KPC-Kp in the setting of a local outbreak, and suggests that risk factors for KPC-Kp acquisition may be shared with those for VRE colonization. Local screening strategies targeting overseas travellers would likely miss many cases. The results of this study will help to inform screening policies for carbapenemase-producing Enterobacteriaceae.

New insights into growth hormone action.

It has been 75 years since Evans and Long identified a somatic growth-promoting substance in pituitary extracts, yet it is only in the last 20 years that the molecular basis for this action has been established. Three key elements in this elucidation were the cloning of the GH receptor, the identification of Janus kinase (JAK) 2 as the receptor-associated tyrosine kinase, and the delineation of signal transduction and activators of transcription (STAT) 5a/b as the key transcription factor(s) activated by JAK2. The interaction between these three elements results in enhanced postnatal growth and is the subject of this review. We describe a new model for GH receptor activation based on subunit rotation within a constitutive dimer, together with the phenotype and hepatic transcript profile of mice with targeted knockins to the receptor cytoplasmic domain. These support a central role for STAT5a/b in postnatal growth.

A GH receptor antisense oligonucleotide inhibits hepatic GH receptor expression, IGF-I production and body weight gain in normal mice.

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2'-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

Early responses of trans-activating factors to growth hormone in preadipocytes: differential regulation of CCAAT enhancer-binding protein-beta (C/EBP beta) and C/EBP delta.

Using the 3T3-F442A preadipocyte line as a model of GH-dependent differentiation, early changes in the DNA-binding affinity of transcription factors in response to GH addition were investigated. Addition of 50 ng/ml human GH to cells in chemically defined medium led to a rapid increase in binding activity of activator protein 1 (AP-1) and CCAAT enhancer-binding protein (C/EBP), which was significant at 30 min and reached maximal induction by 2 h (3-fold for AP-1, 2.5-fold for C/EBP). Induction in AP-1 DNA binding correlates with a concomitant GH trans-activation of c-jun and c-fos genes described previously. Using specific antibodies in electrophoretic mobility shift assays and Western blots, it was shown that the increase in activity of C/EBP is the result of an increase in synthesis of two alternatively translated forms of C/EBP beta: 40-C/EBP beta and 23-C/EBP beta. This increase in protein was not accompanied by alteration in mRNA level and could be blocked by a Janus kinase 2 tyrosine kinase inhibitor and a C kinase inhibitor at concentrations shown to inhibit GH-dependent activation of microtubule-associated protein (MAP) kinases. Concomitant with the translationally activated increase in C/EBP beta, a GH-dependent increase was observed in C/EBP delta transcription. This was accompanied by an increase in mRNA for C/EBP delta, which was superinduced by cycloheximide and, unlike the increase in C/EBP beta protein, was not observed with insulin. Thus GH exerts its effects on C/EBP isoforms at two levels: transcriptional activation of C/EBP delta and translational activation of C/EBP beta. It is proposed that GH-dependent phosphorylation results in the efficient translation of 40-C/EBP beta and 23-C/EBP beta (the mouse homolog of the inhibitor liver-enriched inhibitory protein), and that together with the induction of C/EBP delta, these may be involved in initiating the adipocyte differentiation program.

Ternary complex factors Elk-1 and Sap-1a mediate growth hormone-induced transcription of egr-1 (early growth response factor-1) in 3T3-F442A preadipocytes.

In our search for transcription factors induced by GH, we have analyzed immediate early gene activation in a model of GH-dependent differentiation. Here we describe the activation of early growth response factor-1 (egr-1) in GH-stimulated 3T3-F442A preadipocytes and the transcription factors responsible for its transactivation. Binding activity of egr-1 in electrophoretic mobility shift assay (EMSA) increased transiently 1 h after GH stimulation, accompanied by a concomitant increase in egr-1 mRNA. egr-1 induction appeared not to be related to proliferation since it was amplified in quiescent preadipocytes at a time when cells were refractive to GH-stimulated DNA synthesis. Truncations of the proximal 1 kb of the egr-1 promoter revealed that a 374-bp region (-624 to -250) contributes about 80% of GH inducibility in 3T3-F442A cells and approximately 90% inducibility in CHO-K1 cells. This region contains three juxtaposed SRE (serum response element)/Ets site pairs known to be important for egr-1 activity in response to exogenous stimuli. Site-specific mutations of individual SRE and Ets sites within this region each reduced GH inducibility of the promoter. Use of these site-specific mutations in EMSA showed that disruption of either Ets or SRE sites abrogated ternary complex formation at the composite sites. DNA binding of ternary complexes, but not binary complexes, in EMSA was rapidly and transiently increased by GH. EMSA supershifts indicated these ternary complexes contained serum response factor (SRF) and the Ets factors Elk-1 and Sap-1a. Coexpression of Sap-1a and Elk-1 resulted in a marked increase in GH induction of egr-1 promoter activity, although transfection with expression vectors for either Ets factor alone did not significantly enhance the GH response. We conclude that GH stimulates transcription of egr-1 primarily through activation of these Ets factors at multiple sites on the promoter and that stabilization of ternary complexes with SRF at these sites maximizes this response.

Effect of Deletion of Ghrelin-O-Acyltransferase on the Pulsatile Release of Growth Hormone in Mice.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium.

The hypothesis that growth hormone (GH) up-regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days. The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP-2 and -4), bone morphogenetic protein type IA receptor (BMPR-IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11). The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP-2 and -4, BMPR-IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH. Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP-2 and -4 and their receptor by GH compliments the role of GH-induced insulin-like growth factor 1 (IGF-1) in promoting bone and tooth root formation.

Growth hormone receptor expression in the rat gastrointestinal tract.

We have used immunohistochemistry to define the cellular distribution of GH receptors in the gastrointestinal tract (GIT) and its derivatives. Immunohistochemistry was performed in the adult rat GIT with a panel of characterized monoclonal antibodies to the GH receptor. The most intense and heterogeneous immunoreactivity was observed in epithelial cell subpopulations of GIT mucosa. Mesenchymal elements of the GIT were homogenously and moderately immunoreactive. Intense immunoreactivity was observed in the ductal epithelium of the sublingual gland, scattered basal epidermal cells of the esophageal mucosa, zymogen cells of the gastric glands, scattered surface epithelial cells of the stomach, and scattered peripheral pancreatic acinar groups. Scattered enteroendocrine cells and parietal cells, crypt and villous columnar cells of the small intestine, surface columnar cells of the cecum/colon, crypt base columnar cells of the colon, and contiguous peripheral cords of pancreatic islet cells displayed strong immunoreactivity. No immunoreactivity was detectable in the mucous and serous acini of the sublingual and submandibular gland, respectively, mucous-secreting cells of the base of the cardiac and pyloric glands, surface epithelial cells of the fundus, paneth cells, goblet cells of cecum/colon, or mucous cells at the base of the cecal crypt. Other elements of the GIT were moderately or weakly immunoreactive. In support of our localization we can detect high affinity binding (Ka = 3 x 10(9] of [125I]human GH with ovine GH as displacing ligand to crude homogenates of adult rat stomach and intestine. We conclude that discrete epithelial cell subpopulations of the GIT and its derivatives are directly responsive to GH action. GH may, therefore, act independently of or synergistically with hepatic insulin-like growth factor-I in executing its physiological and/or growth-promoting role in the GIT.

Immunological and physicochemical evidence for tissue specific prolactin receptors in the rabbit.

Late pregnant and midlactating rabbit tissues (adrenal gland, kidney cortex, liver, mammary gland, and ovary) were examined for evidence of tissue specific differences in PRL receptors by several criteria. These included analysis of association constants, gel filtration, analytical isoelectric focusing, and reactivity with specific anti-PRL receptor antibodies in both the direct competition and immunoprecipitation assays, using membrane bound and Triton X-100 solubilized receptors. Association constants of membrane bound PRL receptors were not significantly different (adrenal, 1.50 +/- 0.26 X 10(10) M-1; kidney cortex, 0.90 +/- 0.22 X 10(10) M-1; liver, 1.31 +/- 0.50 X 10(10) M-1; mammary, 0.95 +/- 0.36 X 10(10) M-1) except the ovarian receptor, which had a significantly higher affinity (2.05 +/- 0.04 X 10(10) M-1) than other tissue receptors. This difference did not result from a difference in tracer degradation. Higher affinity for the ovarian receptor may be a consequence of phospholipid modulation of receptor affinity, since Triton X-100 solubilization abolished this difference in affinity (ovary, 3.63 +/- 0.15 X 10(10) M-1; adrenal, 3.48 +/- 0.44 X 10(10) M-1; mammary, 3.61 +/- 0.41 X 10(10) M-1). However, both the solubilized kidney cortex receptor (2.68 +/- 0.52 X 10(10) M-1) and the solubilized liver receptor (2.40 +/- 0.38 X 10(10) M-1) were significantly different from PRL receptors in the other tissues, implying a possible structural distinction. In addition, clear differences in isoelectric point and immunoreactivity were evident between the kidney cortex receptor and PRL receptors in other tissues. Immunologic evidence of differences between mammary gland, adrenal, ovary, and liver PRL receptors was also obtained, although the degree of difference in the liver receptor was obscured by binding of 125I-ovine PRL to the GH receptor. No differences were seen in elution position of solubilized receptors by gel filtration on Sepharose CL6B. These differences support the notion of structurally distinct PRL isoreceptors in different tissues, although it is also possible that they result from differing proportions of two receptor forms (e.g. plasma membrane and golgi complex forms). It is suggested that the kidney cortex receptor is derived from a more primitive form of the PRL receptor concerned with the regulation of electrolyte balance, whereas PRL receptors in other tissues arose at later times to subserve other specialized functions.

The ontogeny of growth hormone receptors in the rabbit tibia.

To address the question of the mode of action of GH in stimulating longitudinal bone growth, we have used a panel of anti-GH receptor monoclonal antibodies to demonstrate GH receptors in the rabbit tibia and have studied the ontogeny of these receptors. In the neonate, receptors were localized in the hypertrophic zone between the cartilage canals, a region that develops into a secondary ossification center. In support of this finding, receptors were also localized on monolayer cultures of human infant costal chondrocytes. In 20- and 50-day-old rabbits, receptors were localized on reserve and proliferative chondrocytes in the growth plate. In 50- and 130-day-old rabbits receptors were localized on proliferative chondrocytes in the condylar cartilage. In older (180-day-old) rabbits with closed growth plates, GH receptors could not be detected, even in condylar cartilage. These results support the case for revision of the somatomedin hypothesis to accommodate a direct interaction between GH and receptors on epiphyseal chondrocytes.

Cellular localization of the growth hormone receptor/binding protein in the male and female reproductive systems.

We have used immunohistochemistry to localize GH receptor/binding protein (BP) in the male and female reproductive systems of adult rats. Testes and ovaries from neonatal animals were also examined to determine if GH receptor/BP expression in these tissues is developmentally regulated. Two monoclonal antibodies (MAb 43 and 263) were immunoreactive in identical locations whereas no immunoreactivity was evident when control monoclonal antibodies 7 and 50.8 were used. Localization of the receptor/BP was observed in both the nucleus and cytoplasm of immunopositive cells confirming our recent report of a nuclear GH receptor. Intense GH receptor/BP immunoreactivity in the male reproductive system was evident in the epithelium of the vas deferens and coagulating gland, the prostatic epithelium during the secretory phase, and the ductular epithelium of the coagulating and bulbourethral glands, respectively. Strong immunoreactivity was detectable in the Leydig and Sertoli cells, the epithelium of the ductus epididymis and seminal vesicles and smooth muscle of the tunica muscularis of the vas deferens, septae of the seminal vesicles, and in prostatic fibromuscular stroma. Cells of the seminiferous tubules (spermatogonia, primary and secondary spermatocytes, and spermatids) were moderately immunoreactive. No immunoreactivity was detectable in spermatozoa in the ductus epididymis or vas deferens, in scattered epithelial cells of the ductus epididymis, the prostatic epithelium in the nonsecretory phase, and mucous secreting cells of the bulbourethral glands. Leydig cells of 10-day postnatal rat testis were intensely immunoreactive whereas seminiferous tubular cells displayed homogenous immunoreactivity from moderate to strong. Intense GH receptor/BP immunoreactivity in the female reproductive system was evident in the germinal epithelium, the vascular endothelium of the myometrium, the epithelial lining of the fimbriae and oviduct, the endometrial epithelium and scattered endometrial glands, the mesothelium of the perimetrium, and the vascular endothelium of the endometrium. Strong immunoreactivity was exhibited by scattered oocytes, lutein cells of the corpus luteum, scattered endometrial glands, and the vascular endothelium of the endometrium. Moderate immunoreactivity was evident in scattered oocytes, granulosa cells, theca interna and externa, smooth muscle of the oviduct and myometrium, scattered endometrial glands, and luminally placed endometrial stroma cells. Ovarian granulosa cells from 10-day postnatal rats displayed strong immunoreactivity in contrast to moderate immunoreactivity in adult granulosa cells. In conclusion, we report a widespread distribution of the GH receptor/BP in the reproductive system of the rat by which GH may exert a direct action on reproductive function. The distribution is concordant with a role for GH in epithelial function and/or maintenance and also with a possible role for GH in the integrity of the endometrial vasculature.