Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist, Vibegron: Evaluation of Antimuscarinic Receptor Selectivity for Combination Therapy for Overactive Bladder.

Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of ß3-adrenergic receptors (ß3ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel ß3AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of ß3AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full ß3AR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a ß3AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel ß3AR agonist probe, [3H]MRL-037, that selectively labels ß3 receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of ß3AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder.

Determination of blood loss in bimaxillary surgery: does the formula and the time point affect results?

The amount of blood loss determined in orthognathic surgery differs greatly among studies. This can be attributed to the inhomogeneity in study cohorts analysed, but may also be a result of the varying methodologies used for blood loss determination. However, this has yet to be explored. Thus, the aim of this study was to investigate the extent to which the formula and time point used to measure blood loss affect the blood loss volume, determined in a homogeneous cohort undergoing bimaxillary surgery. Blood loss was calculated at 24 and 48 hours postoperatively using the haemoglobin balance method and the formula of Hurle et al. The estimated total blood volume was established based on the formulae of Nadler et al. and Choi et al. Differences in blood loss volume with respect to time point and formula were analysed and compared. Fifty-four patients were included in the final analysis. Statistically significant differences in blood loss were observed: a significant increase in the blood loss volume from 24 hours to 48 hours postoperatively was detected. When comparing the formulae used, blood loss differed significantly at 24 hours after surgery; however no such difference resulted at 48 hours postoperatively. These findings imply that the time point of measuring blood loss is highly relevant, whereas the formulae applied seem to have less of an impact on the blood loss volumes calculated.

The postpartum abdomen: psychology, surgery and quality of life.

PURPOSE: The postpartum abdomen presents significant challenges to the surgeon. It is anatomically complex, with often substantial symptomatic divarication of the rectus abdominis, affecting all anterior abdominal wall layers. This may lead to profound functional sequelae, and often, of more importance to patients, a significant physical deformity. The complex interplay of functional/physical symptoms can result in reduced quality of life (QoL) as well as negative body image/self-esteem. Postpartum women may seek abdominoplasty to address the whole scope of these concerns. Whilst techniques have evolved achieving such goals operatively, the impact of such surgery on QoL/mental health has yet to be established. METHODS: We perform a comprehensive review of potential options of validated patient-reported outcome measures (PROMs) for consideration of use in postpartum women seeking abdominoplasty; in addition to discussing current driving factors for seeking surgery and associated ethics. RESULTS: Pressure on postpartum women to return their abdominal wall contour to a pre-pregnant state is high. This poses important ethical considerations for surgeons. There are several well-established/validated PROMs used in body contouring in massive weight loss/bariatric population groups, including Body-Q and Body-QoL scales, but none yet specific to postpartum women. CONCLUSION: PROMs use to enable establishment of the true value of abdominoplasty in postpartum women, not just in terms of functional/physical restoration, but also in terms of delivering a positive impact on patients' mental health and QoL, are important. Further research is needed to determine if those already developed are appropriate or whether a postpartum-specific PROM would be beneficial.

Influence of different techniques of secondary cleft lip revision surgery on upper lip projection.

Patient dissatisfaction with labial appearance in the adult cleft lip is frequently linked to poor upper lip projection. Other areas of concern include asymmetry and impaired upper lip height. Different surgical techniques are available to address volumetric deficiencies, according to extent and localization. However, data comparing outcomes in these different areas are limited. The main aim of this study was to assess the relative gains in upper lip projection. An evaluation of upper vermilion height and symmetry was also performed. Thirty-seven consecutive patients treated by a single surgeon had their pre- and postoperative results measured using standardized photographs; these were analysed using subjective and objective outcome measures. Seven examiners evaluated anonymized pre- and postoperative side and front views for subjective evaluation. The objective analysis was performed using Adobe Photoshop. Fifteen lip revisions, four Abbe flaps, 12 dermal grafts, and six PermaLip implants were performed. In bilateral cleft lip and palate patients, Abbe flaps showed the most significant improvement in labial projection, followed by PermaLip implants and dermal grafts. In unilateral cleft lip and palate patients, PermaLip implants best addressed impaired lip projection, followed by dermal grafts. Overall, functional lip revisions showed excellent outcomes for upper lip symmetry; however, only minor changes in labial projection were found.

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

Craniofacial implants at a single centre 2005-2015: retrospective review of 451 implants.

Craniofacial endosseous implants are regularly used to support prostheses in the rehabilitation of complex defects, but reported success rates vary. To review our own clinical practice over 10 years, and particularly to examine the impact of radiotherapy and the timing of placement on the survival of implants, we retrospectively audited the records for all patients who had endosseous implants for prosthetic rehabilitation in our unit between 2005 and 2015. We reviewed 167 records, which gave 451 implants, of which, 222 (49%) were auricular, 98 (22%) nasal, and 131 (29%) orbital. Most were placed after ablative operations for cutaneous malignancy (n=103 patients, 62%). The failure rate of implants placed in bone that was irradiated either before or after placement was significantly higher than that of those placed in non-irradiated bone (univariate analysis: 11% compared with 2%, p<0.001: Kaplan-Meier survival analysis: p<0.001). The timing of placement in relation to radiotherapy (before compared with after) seemed to have no impact on success (p=0.96). Our findings are in keeping with previous reports, and the principal observation is that radiotherapy adversely affects success. We work closely with our maxillofacial prosthetists and place implants at the time of ablation. Our findings seem to support this practice regardless of whether or not the patient will later require adjuvant radiotherapy.

Phaeochromocytoma and Paraganglioma Excision Involving the Great Vessels.

OBJECTIVE/BACKGROUND: Phaeochromocytomas and paragangliomas are vascular neuroendocrine tumours distributed between the neck and the pelvis and may be associated with catecholamine secretion. The aim of the study was to describe the complex surgical management required to excise these tumours when in close proximity to the great vessels (aorta and vena cava). METHODS: This was a retrospective case series. Patients included those undergoing surgical excision of a phaeochromocytoma or paraganglioma involving the great vessels. Data on clinical presentation; genetic mutations; tumour location; catecholamine/metanephrine secretion; surgical strategy; pre-, intra-, and post-operative course were collated. RESULTS: Five patients (age range 16-60 years) were identified; three had thoracic paragangliomas located under the arch of the aorta, one had an abdominal paraganglioma invading the aorta, and one had a massive phaeochromocytoma invading the inferior vena cava via the adrenal vein. Three patients had predisposing germline mutations. All patients had adrenergic blockade prior to surgery. A diverse range of complex surgical techniques were employed to excise tumours, including cardiopulmonary bypass, aortic resection, grafting and venotomy of the vena cava. Early post-operative complications were limited. CONCLUSIONS: Excision of phaeochromocytomas and paragangliomas involving the great vessels is high risk surgery optimally undertaken within a multidisciplinary setting in a tertiary referral centre. Comprehensive radiological and biochemical assessment, meticulous pre-operative preparation and close intra- and post-operative monitoring are essential. Radiological imaging may be unable to resolve the tumour extent and anatomy pre-operatively and direct visualisation of the tumour may be the only way to clarify the surgical strategy. Pre-operative knowledge of the genetic predisposition may influence surgical management.

Linkage of protonation and anion binding to the folding of Sac7d.

The temperature, pH, and salt dependence of the folding of recombinant Sac7d from the hyperthermophile Sulfolobus acidocaldarius is mapped using multi-dimensional differential scanning calorimetry (DSC) and folding progress surfaces followed by circular dichroism. Linkage relations are derived to explain the observed dependencies, and it is shown that the data can be explained by the linkage of at least two protonation reactions and two anion binding sites to a two-state unfolding process. Circular dichroism spectra indicate that a native-like fold is stabilized at acid pH by anion binding. An apparent binding isotherm surface (folding progress versus pH and salt) is used to obtain intrinsic chloride binding constants as a function of pH for both sites. A saddle is predicted in the folding progress surface (progress versus temperature and pH) at low salt with a minimum near pH 2 and 20 degrees C with approximately 25% of the protein folded. The position of the saddle is sensitive to the intrinsic delta C degrees of unfolding and provides a third measure of delta C degrees independent of that obtained by a Kirchoff plot of DSC data and chemical denaturation. The observed enthalpy of unfolding approaches zero near the saddle making the unfolding largely invisible to DSC under these conditions. The linkage analysis demonstrates that the delta C degrees for unfolding obtained from a Kirchoff plot of DSC data should be distinguished from the intrinsic delta C degrees of unfolding. It is shown that the discrepancy between the free energy of unfolding for Sac7d obtained by DSC and that obtained by chemical denaturation may be explained by the linkage of protonation and anion binding to protein folding. The linkage analysis demonstrates the limitations of using the delta Hcal/ delta Hvh ratio an indication of two-state unfolding.

Hyperthermophile protein folding thermodynamics: differential scanning calorimetry and chemical denaturation of Sac7d.

Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidocaldarius is shown to be stable towards acid, thermal and chemical denaturation. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 degrees C as indicated by near and far UV circular dichroism spectra. Thermal unfolding followed by differential scanning calorimetry (DSC) occurs as a reversible, two-state transition from pH 0 to 10, with a maximal Tm of 90.7 degrees C between pH 5 and 9. At pH 0 the protein unfolds with a Tm of 63.3 degrees C. Plots of the enthalpy of unfolding as a function of Tm are linear and yield an anomalously low delta Cp of 497 (+/-20) cal deg-1 mol-1 using the Kirchhoff relation. Guanidine hydrochloride and urea-induced chemical denaturation of Sac7d occur reversibly and can be followed by circular dichroism. Global non-linear regression of the chemical denaturation data constrained by DSC determined values for delta Hm and Tm yields a delta Cp of unfolding of 858 (+/-21) cal deg-1 mol-1. The higher delta Cp is in good agreement with that predicted from the buried polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable size, indicating that the packing and change in solvent-accessible surface area on unfolding are not unusual. Similarly, guanidine hydrochloride and urea m-values are in good agreement with those expected for a protein of 66 residues. Possible explanations for the difference in delta Cp determined by application of the Kirchhoff relation to DSC data and that determined by the global fit are discussed. Protein stability curves defined by either delta Cp values are similar to those observed for small mesophile proteins. Although the protein is thermally stable, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (+/-0.1) kcal mol-1 at the growth temperature of 80 degrees C. The large number of potential ion pairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monomethylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins. Additional stabilization in vivo is expected from compatible osmolytes (polyamines) and DNA-binding.

Crystal structures of the chromosomal proteins Sso7d/Sac7d bound to DNA containing T-G mismatched base-pairs.

Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation.

Rapid prototyping and patient-specific pre-contoured reconstruction plate for comminuted fractures of the mandible.

The clinical use of rapid prototype, 3-dimensional models and pre-bent reconstruction plates is recognised for mandibular reconstruction after resection of cancer. We describe a new approach using similar techniques in the management of complicated mandibular fractures.

The acid-induced folded state of Sac7d is the native state.

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.

Calcium regulates the expression of insulin-like growth factor binding protein-3 by the human keratinocyte cell line HaCaT.

The insulin-like growth factor (IGF) system is essential for epidermal homeostasis. Insulin-like growth factor binding protein 3 (IGFBP-3), a modulator of IGF action that also exhibits IGF-independent activity, is localized to selected keratinocytes in the basal epidermal layer and may thus contribute to keratinocyte differentiation. We have utilized the human keratinocyte cell line, HaCaT, to examine the effect of calcium on the regulation of components of the IGF system. Western ligand and northern blot analyses revealed secreted IGFBP-3 and IGFBP-3 mRNA were reduced by an elevation in calcium levels in the culture medium. At 1.0 and 1.2 mM CaCl2 culture conditions IGFBP-3 abundance was reduced to 36% +/- 1.6% and 26% +/- 7.1%, respectively, of that from cells grown at 0.03 mM CaCl2. IGFBP-3 mRNA levels in 0.7 mM and 1.2 mM CaCl2 were reduced to 46% +/- 17.4% and 24% +/- 4.6%, respectively, compared with IGFBP-3 mRNA levels at 0.03 mM CaCl2. The observed reduction of IGFBP-3 was not associated with IGFBP-3 proteolysis. In contrast IGF-I receptor protein and mRNA levels remained unchanged. The IGF-I stimulated proliferative response of HaCaT keratinocytes showed that under low (0.03 mM) and high (1.2 mM) CaCl2 conditions IGF-I at 100 and 1000 ng per ml similarly increased cell number 2.4- and 2.7-fold, respectively, with similar dose-response curves. HaCaT keratinocytes grown under medium (0.7 mM) and high (1.2 mM), but not low (0.03 mM), CaCl2 conditions for 21 d revealed an induction of profilaggrin mRNA, a marker of keratinocyte differentiation. These studies indicate that the exposure of HaCaT keratinocytes to elevated calcium levels is associated with a decline in IGFBP-3 but not IGF-I receptor levels. These findings suggest a potential mechanism for the distribution of IGFBP-3 in the epidermis, which may be involved in the process of keratinocyte differentiation.

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in the psoriatic lesion.

Epidermal hyper-proliferation is a key feature of psoriasis, and a role for IGF-I in this process has previously been proposed. Herein we investigated the expression of IGF binding proteins in the psoriatic lesion and compared it with normal skin. With in situ hybridization, we found that IGFBP-3 mRNA was expressed in the basal layer of the epidermis in normal skin. IGFBP-3 was also detected immunohistochemically, exclusively in the basal layer. In the psoriatic lesion, IGFBP-3 mRNA was similarly limited to the basal layer despite the characteristic expansion of the basaloid keratinocyte compartment and was detected only in the suprapapillary epidermis, where IGFBP-3 mRNA was more abundant than in normal or uninvolved epidermis, and IGFBP-3 protein could be readily detected with specific antibody. As with IGFBP-3 mRNA, which represents the likely site of IGFBP-3 synthesis, IGFBP-3 was strictly limited to the basal keratinocytes of the suprapapillary epidermis. By using an antibody to the cell cycle antigen Ki67, we also showed that the suprapapillary epidermis, where IGFBP-3 expression was maximal, contained few keratinocytes undergoing mitosis, whereas the tips of the rete pegs, where IGFBP-3 expression was conspicuously absent, contained many keratinocytes undergoing mitosis. We suggest that IGFBP-3 is a growth inhibitor in basal keratinocytes and that absence of IGFBP-3 in the tips of rete pegs may contribute to epidermal hyper-proliferation in the psoriatic lesion.

Identification, localization, and regulation of insulin-like growth factor binding proteins and their messenger ribonucleic acids in the newborn rat olfactory bulb.

Insulin-like growth factor binding proteins (IGFBPs) have been identified in most tissues, including the central nervous system, where the major IGFBPs have been localized. The regulation and roles of IGFBPs in IGF action in the developing brain remain unclear. In this study we examined the expression and anatomical distribution of IGFBP messenger RNAs (mRNAs) in the newborn rat olfactory bulb (OB) during the first postnatal week. We used our recently developed newborn rat OB organ culture system, which emulates the first week of in vivo development, to identify and characterize expressed and secreted IGFBPs and to determine the role of the local growth factors IGF-I and basic fibroblast growth factor (bFGF) in their regulation. Postnatal day 1 rat OBs were cultured serum free for 6 days in the absence or presence of IGF-I (150 ng/ml) and bFGF (25 ng/ml), alone or in combination, as previously shown by us to maintain morphology and differentiation of neuronal and glial cells. Conditioned medium was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western ligand blotting using [125I]IGF-I, and IGFBPs were characterized by immunoprecipitation. Western ligand blotting of conditioned medium revealed two bands at 24 kilodaltons (kDa) and 30 kDa and a doublet at 38-42 kDa. All bands were enhanced by IGF-I treatment, whereas bFGF enhanced the 24-kDa and 30-kDa bands only. In combination, IGF-I and bFGF enhanced all four bands above that seen with either growth factor alone. Total RNA was extracted from fresh day 1, day 6, and cultured OBs for Northern blotting using complementary DNA probes for IGFBP-2, -3, -4, and -5. In fresh day 1 OBs, mRNA was detected for IGFBP-2, -4, and -5, but not for IGFBP-3. In fresh day 6 OBs IGFBP-2 mRNA was more abundant, whereas IGFBP-4 mRNA showed lower expression than at day 1, and IGFBP-5 mRNA was similarly expressed. When day 1 OBs were cultured for 6 days, mRNA was also readily detected for IGFBP-2, -4, and -5, but not for IGFBP-3. All detected mRNA species were enhanced by IGF-I. Basic FGF enhanced IGFBP-2 mRNA whether alone or in combination with IGF-I and enhanced only IGFBP-4 mRNA when given alone. IGFBP-5 mRNA was not affected by bFGF alone, but its enhancement by IGF-I was attenuated by bFGF. Sites of transcription of IGFBP and IGF-I mRNAs were located by in situ hybridization in both fresh and cultured bulbs.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of growth hormone receptor/binding protein messenger ribonucleic acid (mRNA) during rat fetal development: relationship to insulin-like growth factor-I mRNA.

Although GH plays a key role in postnatal growth, prenatal growth is thought to be GH independent. However, recent data has shown GH receptor/binding protein (GHR/BP) to be present in rat fetal tissues as early as fetal stage E12. The aim of the present study was to investigate tissue-specific production of the GHR/BP messenger RNA (mRNA) and its relationship to locally transcribed insulin-like growth factor-I (IGF-I) mRNA in the fetus. We have used in situ hybridization to localize GHR/BP and IGF-I mRNAs in 16.5-, 18.5-, and 20.5-day-old rat fetuses. Furthermore, because the two parameters of the IGF-I gene differentially respond to GH stimulation, we have also investigated the presence and localization of promoter-specific IGF-I mRNAs. We found the distribution of IGF-I and GHR/BP mRNAs to be widespread but distinct during the fetal stages examined. High levels of IGF-I mRNA were found in connective tissues or their precursors, including the dermis, perichondrium, and gut. In contrast, GHR/BP mRNA exhibited three distinct patterns of distribution. First, GHR/BP mRNA was found at epithelial sites adjacent to sites of IGF-I transcription. Second, GHR/BP and IGF-I mRNAs were found to colocalize in some connective tissues, but GHR/BP mRNA levels in these sites were often lower than at other sites (i.e. epithelial) of GHR/BP gene transcription. Third, GHR/BP mRNA was also found in regions remote from IGF-I mRNA, including the nerve ganglia and inner olfactory bulb. Using promoter-specific IGF-I RNA probes, we detected only promoter 1 transcripts in all fetal tissues examined. The only exception occurred in specialized epithelial cells of the cochlea where we detected high levels of both promoter 1- and 2-derived IGF-I transcripts. We have thus demonstrated a distinct distribution of GHR/BP and IGF-I mRNAs in the developing rat fetus with coordinate expression at some sites. These findings suggest a role of GH or a GH-like peptide, acting both directly and indirectly via IGF-I, in fetal growth and development.

Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth.

Melanocytes, highly differentiated neural crest-derived cells, are located in the basal layer of the epidermis, where they play a role in protecting against UV damage in the skin. Previous studies suggest that both growth hormone (GH) and the insulin-like growth factor I (GH/IGF-I) system may be important for melanocyte growth and function. We have therefore characterized the role of the GH/IGF system in melanocyte growth in vitro and its interaction with the local growth factor basic fibroblast growth factor (bFGF). Analysis of the effects of GH, IGF-I, and bFGF and combinations of these growth factors on melanocyte growth in vitro revealed that 1) GH stimulates the growth of melanocytes when combined with IGF-I, des(1-3)IGF-I [an analog of IGF-I that has a reduced binding affinity for IGF-binding proteins (IGFBPs)], or bFGF, either separately or in combination; 2) in contrast to the lack of effect of GH or bFGF alone, both IGF-I and des(1-3)IGF-I enhance melanocyte growth in a dose-dependent manner; and 3) IGF-I is more efficacious in eliciting a growth response at low concentrations compared to des(1-3)IGF-I. Using Western ligand blotting, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we show that cultured human melanocytes synthesize and secrete minimal amounts of IGFBP. IGFBP-4 is the major IGFBP produced by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1-3)IGF-I alone. In conclusion, these studies provide support for a role for both GH and IGF-I in the growth of human melanocytes in vitro, involving synergy with bFGF. Low levels of melanocyte-derived IGFBP-4 may play a role in enhancing the modulation of IGF action.

Localization of messenger ribonucleic acid for insulin-like growth factor-binding proteins in human skin by in situ hybridization.

The role of the insulin-like growth factors (IGFs) in human skin physiology has been increasingly recognized, although relatively little is known about the cell types involved or the cellular mechanisms that mediate these responses. Epidermal keratinocytes and dermal fibroblasts both possess IGF-I receptors and are responsive to IGF-I. IGF-binding proteins (IGFBPs), known modulators of IGF action, may also be responsible for targeting IGF-I to its receptors and are produced by both cultured keratinocytes and fibroblasts. To demonstrate sites of production of IGFBPs in human skin, we have used in situ hybridization to localize messenger ribonucleic acid (mRNA) for the six IGFBPs. Antisense and sense RNA probes for the IGFBPs (IGFBP-1 to -6) were produced, and 5-microns sections of normal adult human male chest skin were probed. The control probe used was keratin-5, which is known to hybridize to the basal keratinocytes of the epidermis. mRNAs for human IGFBP-2, -3, -4, and -5 were identified, with mRNAs for IGFBP-2 and IGFBP-4 localized in sebaceous glands and eccrine sweat glands (epidermal origin), IGFBP-3 mRNA in the basal layer of the epidermis and mRNAs for IGFBP-4, and IGFBP-5 found throughout the dermis. mRNAs for IGFBP-1 and -6 were not identified in human skin. These studies demonstrate specific localization of IGFBP mRNAs in adult human skin, suggesting that each IGFBP may play a specific role in targeting IGF-I to its receptor on responsive cells and, ultimately, in modulation of IGF-I action in skin.

Radioprotection in rat spinal cord with WR-2721 following cerebral lateral intraventricular injection.

The capacity of WR-2721 to provide radioprotection in central nervous system (CNS) tissue was assessed in F-344 rats irradiated with Cs-137 to the cervical spinal cord 45 min following injection of either 0.33 mg (0.60 X LD50) of WR-2721 or carrier solution in the right lateral cerebral ventricle. The radiation dose groups were 20, 26, 32, or 38 Gy; the dose rate was 1.48 Gy/min. Following irradiation, the time in weeks to forelimb and hindlimb paralysis was measured and statistical significance was assessed by means of the log rank sum test. The median times in weeks to forelimb paralysis in control vs. WR-2721-treated rats were, respectively, 20 vs. 22 at 38 Gy, 19 vs. 31 at 32 Gy (p less than 0.01), 23 vs. 28 at 26 Gy (p less than 0.01), and 49 vs. 60 at 20 Gy (p less than 0.01). The median times to hindlimb paralysis in control vs. WR-2721-treated rats were respectively, 20 vs. 29 at 38 Gy (p less than 0.001), 20 vs. 35 at 32 Gy (p less than 0.01), 23 vs. 34 at 26 Gy (p less than 0.001), and 58 vs. 65 at 20 Gy (p less than 0.01). From these results, we calculated the DMF for forelimb paralysis to be 1.3 and for hindlimb paralysis, 1.6. Histological studies from selected spinal cords from symptomatic killed rats showed petechial hemorrhages, rare microvascular thrombi, and scattered microinfarcts in both gray and white matter. In the white matter columns, there were scattered microfoci of demyelination. The histological findings did not differ between the control and WR-2721-treated groups, but were worse in the higher dose groups. These data indicate that WR-2721 has the capacity to be radioprotective in CNS tissues, when it is administered by a route that bypasses the blood-brain barrier.

Toxicity and biodistribution of the radioprotectors, WR2721, WR77913, and WR3689, in the CNS following intraventricular or intracisternal administration.

The radioprotective capacity of the phosphorothioate compounds, WR2721, WR77913, and WR3689, in the CNS is being evaluated following injection of the drugs into the lateral cerebral ventricle or the cisterna magna of F-344 rats. This approach circumvents the blood-brain barrier and permits an assessment of the CNS toxicity and regional distribution of these compounds. Following intraventricular injection in 150-200 gm female rats, the LD50 doses for WR2721, WR77913, and WR3689 were respectively 0.60 +/- 0.07 mg (S.E.), 2.36 +/- 0.13 mg, and 3.56 +/- 0.26 mg. Following intracisternal injection the LD50 doses were 0.71 +/- 0.18 mg, 4.12 +/- 1.09 mg and 3.03 +/- 0.68 mg, respectively. WR 2721 produced lethargy, unsteady gait, and dishevelment but these signs all resolved completely within 1-3 days in survivors. In addition to these signs, WR77913 and WR3689 produced severe convulsions. At high doses, following intraventricular administration, all three drugs were associated with cerebral and diencephalic periventricular necrosis and ipsilateral necrosis of the lateral hippocampus. Biodistribution studies were performed with [S-35]-labeled derivatives of the drugs and tissue sampling. The three drugs demonstrated similar patterns. Forty-five minutes following either the intraventricular or intracisternal route of drug delivery the highest drug concentrations were in the brainstem, cerebellum, and cervical cord. Additional studies with autoradiography revealed that intraventricular injection was associated with high drug uptake in the cerebral white matter, the periventricular diencephalon, and the periaqueductal mesencephalon. The biodistribution and toxicity data together suggest that the drugs can be ranked, WR3689 greater than WR77913 greater than WR2721, according to the level of drug thiol that can be achieved in the CNS tissues with intraventricular or intracisternal injection. Tissue levels achievable with WR2721 following these two routes of administration are as high as levels others have reported as radioprotective in rodent skin and gut.