Changes in circulating concentrations of testosterone and estrone sulfate after human chorionic gonadotropin administration and subsequent to castration of 2-year-old stallions.

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred.

Aortic stenosis in cardiovascular ochronosis.

Alkaptonuria (endogenous ochronosis) is a rare metabolic disorder caused by a deficiency of homogentisic acid oxidase, an enzyme responsible for the metabolic degradation of tyrosine. Patients with alkaptonuria commonly present with joint pain owing to degenerative arthritis. Other affected patients may present with pigmentation of the ear cartilage and sclera. This article reports a case of aortic stenosis associated with ochronosis in a 48-year-old man who presented with severe cardiac failure. He had no previous diagnosis of alkaptonuria, which was confirmed by mass spectrometry analysis of urine. The pathogenesis of cardiovascular ochronosis is unclear, but is probably related to the extensive extracellular deposits of ochronotic pigment in the cardiac tissue.

Matrix metalloproteinase-9 expression is associated with the presence of Chlamydia pneumoniae in human coronary atherosclerotic plaques.

OBJECTIVE: To investigate the association between Chlamydia pneumoniae and matrix metalloproteinase-9 (MMP-9) in atherosclerotic plaques. DESIGN: 31 coronary atherosclerotic plaque specimens were studied by immunohistochemistry, polymerase chain reaction (PCR), and reverse transcription PCR for the presence of C pneumoniae antigen and genomic DNA, and of MMP-9 protein and transcripts. RESULTS: Immunohistochemical analysis identified a strong association between the presence of C pneumoniae antigen and production of MMP-9 in coronary atherosclerotic plaques (p = 0.001). Furthermore, analysis of the intralesional amount of C pneumoniae and MMP-9 indicated an increased number of cells positive for MMP-9 in arterial sections that had increased C pneumoniae positivity (p < 0.05). CONCLUSIONS: This study provides evidence of an association between expression of MMP-9 and the intravascular presence of C pneumoniae and may suggest a potential pathological mechanism whereby C pneumoniae may contribute to the progression of coronary atherosclerosis.

The pathology of hypertrophic cardiomyopathy.

Sudden cardiac death (SCD) is devastating at any age, but even more so when the individual affected is young and asymptomatic, and the death is entirely unexpected. SCD is a catastrophic complication of hypertrophic cardiomyopathy (HCM) and may be the first manifestation of this disease. HCM is an inherited intrinsic disease of the myocardium characterized by left ventricular hypertrophy without chamber dilatation, in the absence of either a systemic or other cardiac disease, which may cause a similar magnitude of hypertrophy. HCM may be a clinically silent disease. Indeed, the pathologist may be the first to encounter a case of HCM at autopsy. HCM has wide-ranging implications for affected families, who will require cardiac screening and genetic counselling even if mutations are not known. Therefore, prompt and accurate diagnosis of HCM is vital. This review article will focus on the pathological diagnosis of HCM, recent advances in the genetics of this disease, and common pitfalls which may arise, leading to diagnostic uncertainty.

False-positive tuberculin skin tests in a state prison system.

OBJECTIVE: To assess the existence and extent of false-positive tuberculin skin test (TST) results in a regularly tested population subsequent to switching to a different skin testing product. METHOD: Over 9300 state prison inmates were tuberculin skin tested as part of a routine annual testing program. A shortage of Tubersol caused the prison system to purchase and test with Aplisol. Prison health services staff reported an apparent increase in skin test positivity using Aplisol compared to Tubersol. Record reviews were conducted in 34 prisons where inmates read as positive (> or = 5 mm) were retested with Tubersol. RESULTS: Among 368 inmates recorded as positive (> or = 5 mm) with Aplisol who were retested with Tubersol, 150 (40.8%) were read as negative (0-4 mm). CONCLUSION: The tuberculin skin test is a valuable tool in evaluating patients for TB infection. However, variations in planting and reading the test, and in the consistency between lots and manufacturers, can contribute to errors in determining an individual's infection status. The entire clinical and epidemiological picture for each patient must always be evaluated using the TST as a tool, and not an indisputable answer.

Pharmacokinetics and pharmacodynamics of 15-deoxyspergualin in a canine renal allograft model of local immunosuppression.

Local immunosuppression is based on the rationale that one can simultaneously prevent rejection and reduce systemic side effects by administering appropriately chosen immunosuppressive agents directly into the allograft. We utilized a mongrel canine renal transplant model with a programmable, implantable pump/catheter system to estimate the first-pass extraction of 15-deoxyspergualin (DSG) during renal artery infusion and to compare the efficacy and toxicity of continuous intraarterial (ia) versus intravenous (iv) DSG delivery. Six autotransplanted dogs were given DSG by both iv bolus (1 mg/kg) and ia infusion (1.0 mg/kg/d). DSG was administered to allograft recipients by continuous ia infusion at 0.5 (n = 11) and 0.75 (n = 8) mg/kg/day and by continuous iv infusion at 0.5 (n = 12) and 0.75 (n = 6) mg/kg/day. Mean +/- SD elimination half-life was 0.6 +/- 0.1 hr, and the transplanted kidney removed as much as 55-88% (mean 66%) of locally infused DSG. When compared with untreated controls [mean survival time (MST) = 8 days], low-dose (0. 5 mg/kg/day) DSG produced a significant antirejection effect when given ia (MST = 12 days; P = 0.04) but not iv (MST = 9 days; P = 0. 09), with equivalent overall mean drug levels during normal renal function. However, two of the four longest-surviving animals in the ia group died from severe systemic toxicity, manifested by anorexia, diarrhea, leukopenia, and sepsis. High-dose (0.75 mg/kg/day) DSG significantly prolonged survival via both local (MST = 12 days; P = 0.04) and systemic (MST = 11 days; P = 0.02) routes, but half of the iv-treated dogs died from, and four of the longer-surviving ia-treated animals manifested signs of, systemic toxicity, with significantly higher mean drug levels in the iv group. DSG significantly suppressed vascular rejection at both doses when administered locally and systemically, dose-dependently affected the severity of tubulointerstitial rejection and graft edema, and was not nephrotoxic. Our autotransplant pharmacokinetic data overestimated the allografted kidney's ability to extract DSG during local infusion of slightly lower, but immunosuppressive, doses, so that death from systemic toxicity was not prevented and a direct survival benefit of ia vs iv therapy was not realized. Local DSG administration might be combined with other immunosuppressants to therapeutic advantage.

Comparative pharmacokinetics and renal effects of cyclosporin A and cyclosporin G in renal allograft recipients.

Cyclosporin G (CSG) has produced less nephrotoxicity than cyclosporin A (CSA) at equivalent doses in animal models. Conflicting results have been reported concerning differences in the pharmacokinetics of CSA and CSG in preclinical studies, and no data exist regarding the effect of steady-state oral administration of CSG on renal function in transplant patients or CSG-induced release of endothelin and nitric oxide (NO) in vivo. The objective of the study was to examine steady-state pharmacokinetic profiles of adult renal allograft recipients receiving CSA and CSG in relation to concentrations of endothelin-1 and NO2/NO3 in urine and plasma, creatinine clearance (Clcr), and urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) 9 months after transplantation. Concentrations of CSA and CSG were measured in whole blood over a 12-hour dose interval by both a monoclonal and polyclonal fluorescence polarization radioimmunoassay for CSA. A metabolite fraction was defined as the numerical difference between the levels obtained at each time point by both assays. Patient groups were defined as follows: group 1: initial CSA (n = 6); group 2: initial CSG (n = 7); group 3: five of the seven patients in group 2 taking CSG subsequently undergoing conversion to CSA; group 4: the same five patients in group 3 restudied 1 month after 1:1 dosage conversion to CSA; and group 5: CSA groups 1 and 4 combined (n = 11). In group 1, the metabolite fraction accounted for 32% to 54% of the total measurable drug concentration at each time point, whereas in group 2, the metabolite fraction accounted for at most 10% to 15% of the total drug levels measurable by polyclonal fluorescence polarization radioimmunoassay. Although there were no significant differences in any of the mean pharmacokinetic parameters between groups using monoclonal fluorescence polarization radioimmunoassay, the normalized area under the concentration-time curve (NAUC) value was less in four of five patients after conversion from CSG to CSA, with a more variable and delayed time to reach peak concentration (tmax) but equivalent apparent oral clearance (Clpa) values. Clcr was found to change significantly with time in groups 1 and 5 but not in group 2, with CSA producing a more profound and sustained decrease than CSG. Endothelin-1 and NO2/NO3 levels in plasma and urine remained relatively constant after administration of both CSA and CSG, and there were no significant differences between groups 3 and 4 regarding mean endothelin-1 and NO2/NO3 concentrations in plasma, urinary release of endothelin-1 and NO2/NO3, and mean AUC of endothelin-1 and AUC of NO2/NO3. However, monoclonal NAUC correlated significantly with total urinary endothelin-1 within CSA groups 1 and 5 but not within CSG group 2. Metabolite NAUC correlated significantly with total urinary NAG within CSA group 1. Although limited by the small number of patients, this study suggests that 1) CSG may produce less of a reduction in Clcr over time after oral administration at steady state than does CSA, and 2) this beneficial effect of CSG may be in part due to decreased intrarenal release of endothelin-1, as urinary excretion of endothelin-1 seemed to correlate better with CSA than with CSG exposure.

Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues.

This report describes a systematic analysis of the expression of the fibroblast growth factor receptor (FGFR) multigene family (FGFR1, FGFR2, FGFR3, and FGFR4) in archival serial sections of normal human adult tissues representing the major organ systems, using immunohistochemical techniques. Polyclonal antisera specific for FGFR1, FGFR2, FGFR3, and FGFR4 and a three-stage immunoperoxidase technique were employed to determine the cellular distribution of these receptors at the protein level. The expression profiles for the tissue-specific cellular localization of the FGFR multigene family demonstrated wide-spread and striking differential patterns of expression of individual receptors in the epithelia and mesenchyme of multiple tissues (stomach, salivary glands, pancreas, thymus, ureter, and cornea) and co-expression of FGFR1-4 in the same cell types of other tissues. The wide-spread expression of FGFR1-4 in multiple organ systems suggests an important functional role in normal tissue homeostasis. Differences in the spatial patterns of FGFR gene expression may generate functional diversity in response to FGF-1 and FGF-2, both of which bind with equally high affinity to more than one receptor subtype. In vivo, this may lead to functional differences that are crucial for the regulation of normal physiological processes and are responsible for the pathological mechanisms that orchestrate various disease processes.

Testosterone, 11-ketotestosterone, and estradiol-17 beta modify baseline and stress-induced interrenal and corticotropic activity in trout.

Estradiol-17 beta (E), 11-ketotestosterone (KT), and testosterone (T) were administered to immature rainbow and brown trout by implantation of steroid-containing cocoa butter pellets. This procedure elevated the levels of these hormones in the blood of the treated fish and had significant effects on plasma ACTH and cortisol levels in both unstressed and stressed rainbow trout and in stressed brown trout. E treatment significantly elevated resting levels of ACTH and cortisol and KT significantly suppressed resting ACTH levels in rainbow trout, although no effect of KT was noted on baseline cortisol levels. One hour of confinement stress increased ACTH levels in rainbow trout, but less so in T- and KT-implanted fish than in sham-implanted fish. A similar pattern was observed in stress-induced plasma cortisol levels where T and KT treatment of rainbow trout resulted in a more than 50% attenuation of plasma cortisol levels while E implantation significantly increased stress-induced plasma cortisol levels. In brown trout subjected to confinement stress for 96 hr, within 1 hr of the onset of confinement the stress-induced increase in plasma ACTH and plasma cortisol was significantly lower in T- and KT-implanted fish than in sham-implanted controls. However, these differences were not sustained at subsequent sample points during the 96-hr period of continuous confinement. Nonetheless, overall mean ACTH levels for the entire confinement period were significantly enhanced in E-implanted brown trout and significantly reduced in KT-implanted fish. Overall mean cortisol levels were significantly lower in T- and KT-implanted fish. The enhancement of stress responsiveness observed in E-treated immature fish was not observed during confinement stress in untreated mature female trout, with naturally high plasma E levels. However, untreated mature male trout displayed a significantly reduced cortisol response to confinement. It is suggested that gonadal steroids are involved in the regulation of both baseline and stress-induced activity of the pituitary-interrenal axis in salmonid fish.

New immunosuppressive drugs in organ transplantation.

An examination of the mechanisms by which four new immunosuppressive drugs--rapamycin, mizoribine, mycophenolate mofetil, and 15-deoxyspergualin--exert their effects reveals three major categories. Rapamycin binds to an intracellular immunophilin. The resulting drug-immunophilin complex inhibits the action of cytokines, thus blocking the activation and proliferation of T cells. Mizoribine and mycophenolate mofetil are antimetabolites that inhibit a key enzyme in the de novo purine biosynthetic pathway. As a result, the proliferation of T cells and B cells is inhibited selectively compared with that of nonlymphoid cells because the salvage pathway is unavailable to lymphocytes. Finally, 15-deoxyspergualin has a unique mechanism of action, which includes suppressive effects on macrophage function, the induction of cytolytic T cells, B-cell reactivity, and antibody responses. The demonstrated synergism among certain immunosuppressants allows reduction in the dose of each agent to minimize the individual toxicities. As more of these new agents become available, it is likely that azathioprine will be replaced, and it may be possible to eliminate the need for long-term maintenance steroid therapy.

Localisation and differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal and atherosclerotic human arteries.

OBJECTIVE: Aberrant expression of FGF-1 and FGF-2 may be central to the atherosclerotic disease process, promoting both intimal hyperplasia and plaque neovascularisation. FGF-1 and FGF-2 mediate their biological effects by binding to a family of specific high-affinity cell surface receptors with protein tyrosine kinase activity. Four receptors have been identified in the human (FGFR1/flg gene product, FGFR2/bek gene product, FGFR3 and FGFR4), but little is known of their in vivo tissue distribution. Characterisation of the spatial distribution of the FGFR multigene family in both normal and atherosclerotic arteries is a prerequisite to further define the functional role of FGF-1 and FGF-2 in atherosclerosis. The objective of this study was to examine the cell-type-specific expression of the FGFR multigene family members in both normal and atherosclerotic human arteries. METHODS: FGFR expression was investigated immunocytochemically with polyclonal antisera to FGFR1-4 and by in situ hybridisation using FGFR1-4 riboprobes in archival material. Total cellular mRNA was analysed using poly d(T) and the levels correlated with the expression of FGFR1-4 mRNA. RESULTS: At the protein level, FGFR1-4 were expressed in the medial smooth muscle cells and adventitial vessels of normal arteries. In simple and advanced lesions, the expression profiles of FGFR1-4 showed variability between individual arteries, and cell-type-specific differential FGFR expression was apparent. Widespread co-expression of FGFR1 and FGFR2 was observed in intimal smooth muscle cells, foam cells and the plaque microvasculature of simple and advanced lesions. FGFR3 and FGFR4 exhibited more restricted patterns of distribution within the plaque. In situ hybridisation with poly d(T) confirmed high cellular transcriptional activity in archival atherosclerotic lesions. The high levels of total cellular mRNA and FGFR protein were not always reciprocated at the FGFR1-4 mRNA level, and only FGFR1 and FGFR2 mRNA transcripts were abundant in intimal lesions. CONCLUSION: These data provide evidence to suggest involvement of the FGF-FGFR multigene families in human atherogenesis. Differential FGFR expression in plaque subtypes may reflect distinct differences in receptor function which may be relevant to lesion progression during atherosclerosis.

Functional characterization of the spontaneously transformed human umbilical vein endothelial cell line ECV304: use in an in vitro model of angiogenesis.

To gain insight into the role of the endothelial cell during the pathophysiology of the angiogenic response, investigators have isolated micro- and macrovascular endothelial cells from a wide range of both animal and human vessels, including the umbilical vein. Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cords remain a readily available and popular source of endothelial cell. However, the isolation and culture of these cells have several disadvantages, including the risk of infection, exogenous growth factor requirement, and low proliferative capacity. The heterogeneity of endothelial cells from different vascular beds as well as the heterogeneity between HUVEC isolates from different cords can make the critical interpretation of results difficult. ECV304 is a unique spontaneously transformed human umbilical vein endothelial cell line. In this report, the novel use of ECV304 cells as an alternative to HUVECs in an in vitro model of angiogenesis using the reconstituted basement membrane extract (Matrigel) was investigated. ECV304 cells were characterized immunohistochemically and their angiogenic behavior on Matrigel was analyzed functionally by phase-contrast, electron, and time-lapse video microscopy. ECV304 cells had several practical advantages over HUVEC culture and in contrast to HUVECs, ECV304 cells exhibited an enhanced and highly reproducible capacity for in vitro angiogenesis. However, several differentiated functions were lost or reduced in the ECV304 cell line which also exhibited anomalous cytokeratin expression. ECV304 cells may provide novel insights into the mechanisms governing angiogenesis under both normal physiological and pathological conditions.

Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues.

BACKGROUND: The fibroblast growth factors (FGFs) and their receptors have a wide range of biologic actions in a variety of systems. The best characterized are the prototypical acidic and basic FGFs, both of which have the property of binding to the flg gene product, fibroblast growth factor receptor-1/FGFR-1. Although much is known at the molecular level, the available information about the in vivo distribution of FGFs and their receptors in human tissues is limited. Detailed characterization of the spatial distribution of ligands and receptors is a necessary prerequisite for understanding their function. EXPERIMENTAL DESIGN: Using immunohistochemical techniques, we have defined the tissue specific cellular location of both FGFR-1 and basic and acidic FGFs in serial sections of a broad range of normal human adult tissues. Immunodetection was performed using highly specific polyclonal antibodies to FGFR-1, acidic FGF, and basic FGF and the avidin-biotin immunoperoxidase method. RESULTS: Immunoreactive acidic and basic FGFs were present in almost all tissues examined. A distinct spatial pattern of distribution of these ligands was observed in the kidney, skin, liver, ureter, and the vasculature. FGFR-1 expression tended to be confined to the tissue microvasculature. Interestingly, marked numbers of FGFR-1 reactive vessels were observed in the stomach, salivary glands, lung, and appendix. Intense FGFR-1 positivity was observed in the basal epithelial cell layer of the cervix, palatine tonsil, and respiratory tract as well as breast myoepithelial cells, and the pitutary gland. CONCLUSIONS: We have been able to define the distribution of FGFR-1 and its ligands in normal human tissues. The results will be useful for determining the function of these growth factors and their receptors under both normal physiologic and pathologic conditions.

Expression of basic and acidic fibroblast growth factors and their receptor in normal and atherosclerotic human arteries.

OBJECTIVE: Acidic and basic fibroblast growth factors (FGF) are potent mitogens for vascular endothelial and smooth muscle cells and also promote angiogenesis. Given that atheroma is associated with the proliferation of a range of cell types, including smooth muscle cells, it has been proposed that these growth factors may play a part in the genesis and/or maintenance of atheromatous lesions. The aim of our study was to examine the distribution of acidic and basic FGF and their known receptor, fibroblast growth factor receptor 1 (FGFR-1), in normal and atherosclerotic arteries. METHODS: Formalin fixed was embedded archival material from a wide range of vessels was examined using a three stage avidin-biotin immunoperoxidase technique and specific polyclonal antibodies to acidic and basic FGF and FGFR-1. RESULTS: In normal arteries basic FGF immunoreactivity was found in medial smooth muscle cells and adventitial blood vessels, the luminal endothelium being non-reactive. Acidic FGF expression was observed predominantly in adventitial fibroblasts, while FGFR-1 expression was confined to the adventitial microvasculature. In early, simple, and advanced lesions acidic and basic FGF were expressed in macrophages and smooth muscle cells, the principal cell types involved in atherosclerotic lesion formation. Increased expression of basic FGF and FGFR-1 was particularly associated with neovascularisation of the atheromatous lesions. CONCLUSIONS: Acidic and basic FGF are expressed in early, simple and advanced atherosclerotic plaques. This suggests that in vivo these growth factors may have a role in the genesis of this disease. Basic FGF and FGFR-1 may potentially be involved in plaque neovascularisation. Such FGF driven angiogenic events may be central to the life threatening complications of atheroma and provide new options for therapeutic intervention.

Explorations of otic transplantation.

Embryonic rat inner ears were transplanted to the anterior chamber of the eyes of adult rats. While considerable development was evident, the structures present were limited to the vestibular division. We hypothesized that this selective survival could be due to the rate of vascularization. To test the effects of graft vascularization we made transplants in which the internal structures were exposed by removing the apex and base of the developing cochlea. The transplants were rapidly vascularized by the iris. Many of the soft labyrinthine structures of the cochlea from 1-day-old donors showed considerable development, including the spiral limbus, basilar membrane, and organ of Corti. To test the possibility that the cochlea requires inductive or trophic support beyond Embryonic Day 15 (E15), we cotransplanted the embryonic inner ear with developing brain stem. In these transplants, we observed improved development of the cochlea, with spiral ganglion cells and an organ of Corti possessing hair cells, Deiter's cells, and pillar cells. To further address the effect of developing CNS tissue on the development of grafted inner ear, we transplanted E15 inner ears to either the cortex or the brain stem of neonatal rats. In these experiments we have seen evidence of both vestibular and cochlear sensory surfaces. In the cochlea, an organ of Corti-like structure can be seen. The possibility of neural connections with the host brain has yet to be investigated.

Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the functional reconstruction of retinas lacking photoreceptors.

We have investigated the possibility of using transplantation of immature or mature rodent photoreceptors as well as mature human photoreceptors to reconstruct retinas in which photoreceptor degeneration is either inherited or environmentally induced. To this end, we have devised methods for isolating and transplanting the outer nuclear layer (ONL) (e.g., the photoreceptor layer) to the subretinal space of mature rodents. In addition we found that if portions of the inner retina are transplanted along with the intact photoreceptor sheet, photoreceptor organization is better maintained. In ultrastructural studies of the reconstructed retina an outer plexiform-like layer (OPL) is visible at the interface of the transplanted ONL and the host inner nuclear layer, with invaginating ribbon synapses characteristic of those formed by rod photoreceptors evident within this OPL. Ribbon synapses are found only rarely in unreconstructed retina. These results suggest that synaptic connections between transplanted photoreceptors and host cells may be made. Evidence for the potential recovery of function following photoreceptor transplantation is found in visually evoked cortical responses and behavioral responses (pupillary reflex) to light stimulation of the reconstructed eye. These findings suggest the possibility that neural transplantation can reconstruct a sensory end organ--in this case the retina--to restore evoked activity and an appropriate behavioral response to sensory stimulation.