The biological basis of the bone-muscle inter-relationship in the algorithm of fracture healing.

The biological cascade of fracture healing is intimately linked to the muscle envelope. It further depends on the preservation of stable, perpetual axial micromovements. The current study was designed to demonstrate that high molecular weight bioactive substances diffuse from the muscle envelope to initiate osteoinductive activity at experimental fracture sites. Forty-eight rats underwent an experimental fracture of the left tibia and stabilization with an intramedullary 20-gauge needle. The animals were divided into 4 groups (A-D) of 12 rats each according to the post-fracture treatment. In group A (control) no additional treatment was applied following fracture and intramedullary fixation. In groups B, C, and D, a nitrocellulose membrane of various sizes was wrapped around the fracture, separating the periosteum from the muscle envelope. The groups differed by the membrane pore size, allowing passage of the following molecular sizes: 50 kilodaltons (kDa), 12 to 14 kDa, and 3.5 kDa in groups B, C, and D, respectively. Four animals in each group were sacrificed 2, 5, and 10 weeks after the procedure for radiographic and histological evaluation of fracture healing. Radiographic evaluation revealed a decreased rate of bone synthesis that correlated with the nitrocellulose pore size. Morphological and functional analysis of the bone explants indicated poorly healed fractures in groups B, C, and D. Direct contact between fractured bone and its muscle envelope is essential for the biological sequence of new bone formation. The extent of obstruction between the fracture and its muscle envelope correlates with the delay in fracture healing.

A targeted DNA vaccine encoding fas ligand defines its dual role in the regulation of experimental autoimmune encephalomyelitis.

This study used naked DNA vaccination to induce breakdown of tolerance to self and thus elicit immunological memory to native, membrane-bound Fas ligand (FasL). Upon induction of experimental autoimmune encephalomyelitis (EAE), this memory was turned on to provide protective immunity. FasL-specific autoantibodies isolated from protected animals differentially downregulated the in vitro production of TNF-alpha, but not IFN-gamma, by cultured T cells. These autoantibodies were highly protective when they were administered to rats at the onset of EAE. In contrast, administration of these FasL-specific Ab's to EAE rats after the peak of the acute phase of disease prevented spontaneous recovery from disease. This extended illness is partially explained by inhibition of mononuclear cell apoptosis at the target organ, which resulted in increased accumulation of T cells and macrophages at the site of inflammation. Hence, FasL exerts two distinct, stage-specific regulatory functions in the control of this T-cell mediated autoimmune disease of the central nervous system.

C-C chemokine-encoding DNA vaccines enhance breakdown of tolerance to their gene products and treat ongoing adjuvant arthritis.

Depending on the method of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic polyadjuvant-induced arthritis (AA). We administered naked DNA vaccines encoding MIP-1 alpha, MCP-1, MIP-1 beta, and RANTES to Lewis rats and confirmed that each of these vaccines induced immunological memory to the corresponding gene product. Upon induction of disease, this memory effectively inhibited the development of the autoimmune condition. Self-specific Ab's developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of each vaccine. Repeated administration of the constructs encoding MCP-1, MIP-1 alpha, or RANTES inhibited the development and progression of AA, even when each vaccine was administered only after the onset of disease. This suggests a highly effective way by which the immune system could be re-educated to generate protective immunity against its own harmful activities.

Is the Bishop-score significant in predicting the success of labor induction in multiparous women?

OBJECTIVE: To determine whether the Bishop-score upon admission effects mode of delivery, maternal or neonatal outcomes of labor induction in multiparous women. STUDY DESIGN: A retrospective study including 600 multiparous women with a singleton pregnancy, 34 gestational weeks and above who underwent labor induction for maternal, fetal or combined indications. Induction was performed with one of three methods- oxytocin, a slow release vaginal prostaglandin E2 insert (10 mg dinoprostone) or a transcervical double balloon catheter. The women were divided into two groups-Bishop-score <6 and Bishop-score ⩾6. We evaluated labor course, maternal complications (postpartum hemorrhage, manual lysis, uterine revision, perineal tear grade 3-4, need for blood transfusions, relaparotomy, prolonged hospitalization) and neonatal outcomes (Apgar score, cord pH, hospitalization in the neonatal intensive care unit, prolonged hospitalization). RESULTS: Both groups had a high rate of vaginal deliveries-93.7% and 94.9%, respectively. There was no difference between the two groups in terms of maternal or neonatal outcomes. CONCLUSION: Labor induction in multiparous women is safe and successful regardless of the initial Bishop-score. In multiparous women the Bishop-score is not a good predictor for the success of labor induction, nor is it a predictor for maternal of neonatal adverse outcomes and complications.

Insulin production by human embryonic stem cells.

Type 1 diabetes generally results from autoimmune destruction of pancreatic islet beta-cells, with consequent absolute insulin deficiency and complete dependence on exogenous insulin treatment. The relative paucity of donations for pancreas or islet allograft transplantation has prompted the search for alternative sources for beta-cell replacement therapy. In the current study, we used pluripotent undifferentiated human embryonic stem (hES) cells as a model system for lineage-specific differentiation. Using hES cells in both adherent and suspension culture conditions, we observed spontaneous in vitro differentiation that included the generation of cells with characteristics of insulin-producing beta-cells. Immunohistochemical staining for insulin was observed in a surprisingly high percentage of cells. Secretion of insulin into the medium was observed in a differentiation-dependent manner and was associated with the appearance of other beta-cell markers. These findings validate the hES cell model system as a potential basis for enrichment of human beta-cells or their precursors, as a possible future source for cell replacement therapy in diabetes.

Immediate postpartum ultrasound evaluation for suspected retained placental tissue in patients undergoing manual removal of placenta.

OBJECTIVES: Approximately 1% of term deliveries are complicated by retained products of conception. Untreated, this condition may cause bleeding, infection and intrauterine adhesions. This study assessed whether performing routine bedside uterine ultrasound immediately after manual removal of the placenta reduced the occurrence of undiagnosed, retained products of conception and its associated complications. STUDY DESIGN: A retrospective study was conducted using the records of patients who delivered and underwent manual removal of placenta at a single obstetrics center over a 6-year period. The outcomes of patients who were assessed using immediate bedside ultrasound were compared to a similar group who were treated based on clinical evaluation alone. All patients underwent ultrasound examination prior to discharge. Outcome variables included the rate of additional interventions (medical or surgical), abnormal pre-discharge uterine ultrasound findings, postpartum hemorrhage rate, puerperal fever and length of hospital stay. RESULTS: A total of 399 charts were reviewed. Immediate post-procedural ultrasound was performed in 235 patients. The remaining 164 women did not undergo immediate post-procedural ultrasound. All patients underwent an ultrasound examination prior to discharge. Among the patients who had an immediate post-procedural ultrasound, 12 (5.1%) received immediate re-intervention (2 methergine, 6 curettage and 4 manual uterine revision) vs. no intervention in the second group (p<0.001). No statistically significant difference was found between the group of patients who had immediate post-procedural ultrasound and those who did not, in the rates of postpartum hemorrhage (3.1% vs. 0.7%, p=0.13), abnormal ultrasound findings prior to discharge (14.9% vs. 14.8%, p=0.96) or additional late intervention (7.2% vs. 7.9%, p=0.79), respectively. CONCLUSIONS: Our findings suggest that immediate, bedside uterine ultrasound examination after manual removal of placenta might not change patient outcomes. Furthermore, it might increase unnecessary interventions. Further studies are needed to prospectively assess the benefit of routine uterine ultrasound examination after manual removal of placenta.

Intra-vaginal prostaglandin E2 versus double-balloon catheter for labor induction in term oligohydramnios.

OBJECTIVE: Compare mechanical and pharmacological ripening for patients with oligohydramnios at term. STUDY DESIGN: Fifty-two patients with oligohydramnios ⩽ 5 cm and Bishop score ⩽ 6 were randomized for labor induction with a vaginal insert containing 10 mg timed-release dinoprostone (PGE2) or double-balloon catheter. The primary outcome was time from induction to active labor. Time to labor, neonatal outcomes and maternal satisfaction were also compared. RESULT: Baseline characteristics were similar. Time from induction to active labor (13 with PGE2 vs 19.5 h with double-balloon catheter; P = 0.243) was comparable, with no differences in cesarean rates (15.4 vs 7.7%; P = 0.668) or neonatal outcomes. The PGE2 group had higher incidence of early device removal (76.9 vs 26.9%; P = 0.0001), mostly because of active labor or non-reassuring fetal heart rate. Fewer PGE2 patients required oxytocin augmentation for labor induction (53.8 vs 84.6% P = 0.034). Time to delivery was significantly shorter with PGE2 (16 vs 20.5 h; P = 0. 045). CONCLUSION: Intravaginal PGE2 and double-balloon catheter are comparable methods for cervical ripening in term pregnancies with oligohydramnios.

The insulin-sensitive glucose transporter (GLUT4) is involved in early bone growth in control and diabetic mice, but is regulated through the insulin-like growth factor I receptor.

Children with uncontrolled type I (insulin-dependent) diabetes mellitus are characterized by a slow growth rate, which improves upon adequate therapy. While skeletal growth is an energy-consuming process involving high glucose utilization, the role of glucose transporters (GLUT) and their regulation in the bone formation process are not yet fully understood. Thus, we studied both in vivo and in vitro early endochondral bone formation in control and streptozotocin-induced young diabetic mice. Using in situ hybridization and immunohistochemistry techniques, we demonstrated the novel existence of the insulin-sensitive glucose transporter (GLUT4), as well as GLUT1, in juvenile-derived murine mandibular condyles and in the humeral growth plate-two models for endochondral bone formation. Insulin-like growth factor (IGF) I receptors (IGF-I-R), but not insulin receptors (IR), were shown to have cellular distribution similar to GLUT4, being more abundant in mature chondrocytes. Further, in the skeletal growth centers of streptozotocin-induced diabetic mice, GLUT4, IGF-I, and IGF-I and insulin receptor levels, but not GLUT1 were markedly reduced. The decrease in GLUT4 and in IGF-I and insulin receptors was associated with severe histological changes in the mandibular condyles and humeral growth plate. Insulin therapy restored IR levels to normalcy, whereas IGF-I-R and GLUT4 levels were only partially recovered. Thus, GLUT4 and IGF-I-R have a potential role in early bone growth in mice. Further, during early bone growth GLUT4 may be regulated through the IGF-I receptor rather than via the insulin receptor. We propose that skeletal growth retardation in type I diabetes may be associated with reduced expression of the GLUT4 and IGF-I receptor in the bone growth center.

Testosterone stimulates insulin-like growth factor-I and insulin-like growth factor-I-receptor gene expression in the mandibular condyle--a model of endochondral ossification.

Puberty is associated with an increase in the plasma concentration of sex steroids, GH, and insulin-like growth factor-I (IGF-I). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induce skeletal growth is still not fully understood. To better understand the direct effect sex steroids have on bone growth, we studied an isolated organ culture system of the mandibular condyle, derived from 3.5-5.5-week-old male and female mice. We found that testosterone 10(-6) M, but not estradiol, stimulated thymidine incorporation into the DNA of male-derived condyle. Three days of testosterone treatment doubled the condyle size and increased the chondroprogenitor zone, while maintaining the normal gradient of the developing chondrocytes. Immunohistochemistry and in situ hybridization techniques showed that testosterone stimulated IGF-I and IGF-I-R and their messenger RNAs (mRNAs) mainly in the mature chondrocyte layer. Immunoneutralization of IGF-I in the testosterone-treated condyle caused the disappearance of the chondroblast and young chondrocyte layers, though the progenitor cell layer remained almost unaffected. Overtreatment with testosterone (dose or duration) accelerated condylar ossification. In the presence of testosterone 10(-5) M (high dose), calcification "climbs" up to the chondroprogenitor zone, and most of the condylar chondrocytes are replaced by bone tissue. Similar changes occurred after 7 days of testosterone treatment (long duration) with 10(-6) M. In conclusion, testosterone stimulates growth and local production of IGF-I and IGF-I-R in chondrocyte cell layers of an isolated organ culture of mice mandibular condyle. Part of the effect testosterone has on condylar growth is mediated by IGF-I.

In vivo anabolic effects of parathyroid hormone (PTH) 28-48 and N-terminal fragments of PTH and PTH-related protein on neonatal mouse bones.

We developed a neonatal mouse model to investigate in vivo anabolic effects of intact PTH (1-84) and its two fragments PTH (1-34) and PTH (28-48) and of the N-terminal fragment of PTH-related peptide [PTHrP (1-34)]. Two-day-old mice were injected with low-dose (0.05 microg/g body weight) and high-dose (0.2 microg/g body weight) of each of these peptides daily for 6 or 16 consecutive days. Long bones (tibias and femurs) and mandibular condylar cartilages were harvested. Total DNA and protein were analyzed as parameters for anabolic effects. DNA was increased significantly in tibias only by low doses of PTH (1-84) and PTH (1-34), but by both doses of PTH (28-48). In the cartilages of the mandibular condyles, both doses of all three peptides increased DNA. Total protein was increased in the tibia by the low dose of the three peptides, whereas in the condylar cartilage high doses of PTH (1-34) and PTH (28-48) also caused a 2- to 4-fold increase. When the effects of PTH (1-34) and PTHrP (1-34) on the tibias were compared, it became apparent that PTH (1-34) was more effective than PTHrP (1-34) when injected in low doses, but the latter caused a severalfold increase in DNA and protein at both doses. The outstanding anabolic effect of PTH (28-48) was further investigated using [3H]thymidine autoradiography, analysis of insulin-like growth factor I (IGF-I) protein, and localization of IGF-I messenger RNA (mRNA) by in situ hybridization. PTH (28-48) increased by 3-fold the number of [3H]thymidine-labeled cells in the epiphyseal cartilage of tibias removed from 8-day-old injected mice, and in the proliferative zone of the epiphyseal growth plate of tibias removed from 18-day-old injected mice. Femurs from the latter showed a 20% increase in their IGF-I content. In parallel, only tibias from 18-day-old injected mice showed IGF-I mRNA localization in proliferating chondrocytes, whereas those from vehicle-injected control mice did not exhibit IGF-I mRNA. In summary, our study showed that the neonatal mouse is a sensitive model to examine anabolic effects of different PTH and PTHrP fragments. It also revealed that PTH (28-48) has strong anabolic effects on this model, and suggests that IGF-I might mediate the anabolic effects of PTH (28-48).

Growth hormone and insulin-like growth factor-I increase macrophage uptake and degradation of low density lipoprotein.

Hyperlipidemia has been reported in adults with hypopituitarism, and human (h) GH therapy has been shown to lower plasma cholesterol in patients with hypercholesterolemia. Macrophage cholesterol accumulation is an early event in atherosclerosis, and these cells have been shown to respond to GH and insulin-like growth factor (IGF-I). The present study was aimed at investigating the activity of GH and IGF-I in macrophages, and used murine macrophages as a model system to investigate the effects of GH and IGF-I on cellular uptake and metabolism of low density lipoprotein (LDL). The J-774 murine macrophage cell line was shown to bind hGH, to respond to hGH by an increase in cell IGF-I content, and to have specific high affinity binding sites for IGF-I. Mouse peritoneal macrophages and the J-774 macrophage cell line respond to hGH with a dose-dependent stimulation of cellular association and degradation of LDL as well as an enhanced cholesterol esterification rate. A similar response was observed after in vitro treatment of the cells with IGF-I. Preliminary results in human monocyte-derived macrophages showed similar results. The dependency of the effect of hGH on locally produced IGF-I was shown by abrogation of the hGH effect after adding anti-IGF-I antibody to the culture medium. It is concluded that murine macrophages possess the machinery to bind GH, produce IGF-I, and bind IGF-I. This machinery is used by macrophages, and apparently by other cells, to execute GH-dependent IGF-I-mediated stimulation of cellular uptake and metabolism of LDL. This may provide the explanation for both the elevated plasma LDL concentration in patients with GH deficiency and the effect of GH therapy to reduce plasma LDL levels.

Human growth hormone enhances chondrogenesis and osteogenesis in a tissue culture system of chondroprogenitor cells.

GH is a systemic promoter of skeletal growth which increases the synthesis of insulin-like growth factor I in parenchymal tissues as well as in peripheral tissues such as the liver and growing cartilage. The present study was designed to examine whether GH possesses a direct effect on cellular proliferation and differentiation in mouse condylar cartilage. Cartilage progenitor cells were cultured in the form of a tissue culture on top of collagen sponges in medium containing 10% fetal calf serum and were treated with 50 ng/ml GH for 6 days. Three-day incubation with GH caused a marked increase in DNA synthesis and in the size of the culture, which developed into a nodule of differentiated chondrocytes and a well developed perichondrial layer around it. The effect of GH was even more pronounced after 6 days in culture; at which time a distinct network of trabeculae was noted throughout the extracellular matrix. The trabeculae contained osteocyte-like cells and were in close contact with both osteoblast-like and osteoclast-like cells. Using antibodies against bone-specific antigens, i.e. osteocalcin and osteopontin, provided further support for the notion that the newly formed trabecular formation was comprised of bone matrical components. Untreated control cultures lacked such structures, but contained hyaline-like cartilage. It, therefore, seems reasonable to suggest that GH induces a strong stimulatory effect on progenitor cell proliferation, cartilage differentiation, and extracellular mineralization, which supports de novo bone formation in vitro.

Leptin reverses the inhibitory effect of caloric restriction on longitudinal growth.

Caloric imbalance, particularly in critical periods of growth and development, is often the underlying cause of growth abnormalities. Serum levels of leptin are elevated in obesity and are low in malnutrition and malabsorption. The aim of the present study was to determine whether leptin integrates energy levels and growth in vivo, as shown previously in our ex vivo experiments, even in the presence of caloric restriction. In the first part of the study, mice were divided into three groups. Two groups were fed ad libitum and received leptin or vehicle only, and the third group was pair-fed with the group injected with leptin to dissociate leptin's effect on growth from its effect on food consumption. Mice given leptin had a significantly greater tibial length than untreated pair-fed animals and a similar tibial length as control mice fed ad libitum despite their lower weight. In addition, leptin significantly increased the overall size of the epiphyseal growth plate by 11%. On immunohistochemistry and in situ hybridization studies, leptin stimulated both the proliferation and differentiation of tibial growth plate chondrocytes without affecting the overall organization of the plate. There was also a marked increase in the expression and level of IGF-IR. In the second part of the study, two groups of mice were fed only 60% of their normal chow; one was injected with leptin, and the other was injected with vehicle alone. Caloric deprivation by itself reduced serum levels of IGF-I by 70% and the length of the tibia by 5%. Leptin treatment corrected the fasting-induced growth deficiency, but further reduced the level of serum IGF-I. These results indicate that leptin stimulates growth even in the presence of caloric restriction independently of peripheral IGF-I.

Spontaneous differentiating primary chondrocytic tissue culture: a model for endochondral ossification.

Primary cartilage-derived cell cultures tend to undergo dedifferentiation, acquire fibroblastic features, and lose most of the characteristics of mature chondrocytes. This phenomenon is due mainly to the close matrix-cell interrelationship typical of cartilage tissue, which is vital for the preservation of the cartilaginous features. In this study we present a model for spontaneous redifferentiation of primary chondrocytic culture. Mandibular condyles excised from 3-day-old mice, thoroughly cleaned of all soft tissue, were digested with 0.1% collagenase. These mandibular condyle-derived chondrocytes (MCDC) were cultured under chondrogenesis-supporting conditions; that is, 5 x 10(5) cells/mL were incubated in Dulbecco's modified Eagle medium supplemented with 100 microg/mL ascorbic acid, 1 mmol/L calcium chloride, 10 mmol/L beta-glycerophosphate, 10% fetal calf serum, and antibiotics. Development and growth rates of these cartilage-derived cultures were determined by following morphological and functional changes. MCDC proliferated intensively during the first 24-48 h following plating, showing fibroblast-like (long spindle-shaped) morphology and producing mainly type I collagen. The proliferation rate gradually declined, and the cells developed polygonal shapes and started to produce type II collagen. In the 10-14-day-old cultures, cells began to aggregate in cartilaginous nodules and exhibited positive staining for acidic Alcian blue, type X collagen, and von Kossa. Expression of core-binding factor alpha(1) increased between 3 and 5 days and declined gradually thereafter. The condylar-derived tissue culture presented here depicts a spontaneous redifferentiation chondrocytic tissue culture that exhibits features of mature chondrocytes typically found in skeletal growth centers. The present study offers a model for primary chondrocytic tissue culture, which might serve as a model for in vitro endochondral ossification.

Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways.

We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue.

The early postnatal development of the murine mandibular condyle is regulated by endogenous insulin-like growth factor-I.

Skeletal growth during the early postnatal period is thought to be GH-independent, and is probably regulated by intrinsic growth factors. We studied the involvement of locally produced insulin like growth factor-I (IGF-I) in the growth of the neonatal mandibular condyle. Immunofluorescence studies revealed intense staining with antibodies to IGF-I in the mandibular condyle of 2-day-old ICR mice. We have also shown that these mandibular condyles contain specific high-affinity binding sites (Kd = 0.157 nmol/l) for IGF-I (427 fmol/mg). Autoradiographical studies of iodinated IGF-I revealed that the distribution of the receptors for IGF-I was parallel to that of IGF-I production, mainly in the younger zones of the condyle, namely the chondroprogenitor and the chondroblast cell layers. Immunoinhibition of IGF-I resulted in an almost complete inhibition (-91%) of thymidine incorporation into DNA, as well as in marked degenerative changes in the morphological appearance of the condyle. Our studies support the hypothesis that early postnatal growth is dependent on the paracrine activity of endogenous GH-independent IGF-I.

The aromatase inhibitor letrozole increases epiphyseal growth plate height and tibial length in peripubertal male mice.

Sex hormones may influence longitudinal growth, either indirectly, by affecting the growth-hormone-insulin-like growth factor I (IGF-I) axis, or directly, by affecting changes within the epiphyseal growth plate (EGP). The aim of the present study was to investigate the effects of letrozole, an aromatase inhibitor, on longitudinal growth and changes in the EGP in vivo. Eighteen peripubertal male mice were divided into three groups. The first group was killed at baseline, the second was injected with letrozole (Femara) s.c., 2 mg/kg body weight/day, for 10 days, and the third was injected with the vehicle alone. Serum testosterone levels were found to be significantly higher in the treated group than in the controls. Letrozole induced a significant increase in body weight, tail length and serum growth hormone level, but had no significant effect on the level of serum IGF-I. On histomorphometric study, there was a significant increase (12%) in EGP height in the treated animals compared with controls. Immunohistochemistry showed a 3.4-fold letrozole-induced increase in the proliferation of the EGP chondrocytes, as estimated by the number of proliferation cell nuclear antigen-stained cells, and a decrease in the differentiation of the EGP chondrocytes, as estimated by type X collagen staining. Letrozole did not interfere with type II collagen levels. The study group also showed a twofold increase in the number of IGF-I receptor-positive cells compared with controls. In conclusion, the aromatase inhibitor, letrozole, appears to increase the linear growth potential of the EGP in mice.

Prenatal stress selectively alters the reactivity of the hypothalamic-pituitary adrenal system in the female rat.

A study was made of the effects of prenatal stress on the reactivity of the hypothalamic-pituitary adrenal (HPA) axis in male and female offspring. Rat dams were subjected to noise and light stress on an unpredictable basis throughout pregnancy. At 28 days of age mRNA for POMC, proenkephalin and prodynorphin were measured in the hypothalamus of the offspring. A marked reduction was found in POMC mRNA in PS females (PSF) but not in males (PSM), but the other mRNA's did not differ from controls (C). At 60 days of age, PSF has 3 times higher resting levels of serum corticosterone (COR) and significantly lower dexamethasone (DEX)3H hippocampal binding sites than CF. Overnight adrenalectomy abolished the difference in DEX binding. After 10 min exposure to open field PS males and females voided more fecal pellets and made fewer center entries than C offspring, testifying to increased emotionality. Open field stress caused a 3-5-fold rise in circulating COR in all groups within 15 min, which returned to baseline by 90 min in all rats except PSF. These data show that prenatal stress can cause permanent alterations in the behavior of both sexes in stressful situations but appears to cause a selective effect on the HPA axis in the female rat.

Reduced folate carrier (RFC-1) gene expression in normal and psoriatic skin.

Methotrexate is widely used in the treatment of severe psoriasis. However, little is currently known about the mechanisms underlying its therapeutic activity in the skin. Methotrexate has been shown to be carried into cells through the reduced folate carrier (RFC-1). The recent cloning and characterization of the human gene encoding this transmembranal carrier enabled us to investigate RFC-1 gene expression in human skin. Biopsies were obtained from the skin of healthy and psoriatic volunteers. RNA extracted from these biopsies was analyzed by the reverse transcriptase-polymerase chain reaction technique. While RFC-1 gene expression was barely detectable in the uninvolved skin of psoriatic patients and in the skin of healthy volunteers, high levels of RFC-1 transcripts were found in biopsies obtained from psoriatic plaques. To further investigate this pattern of gene expression, we studied skin biopsies by in situ hybridization with a labeled antisense riboprobe specific for the RFC-1 gene. The RFC-1 gene was found to be weakly expressed in the epidermis, in biopsies obtained from the skin of healthy subjects as well as in those from the uninvolved skin of psoriatic patients. In contrast, in biopsies obtained from psoriatic plaques, high levels of RFC-1 gene transcripts were found mostly in the spinous layer of the epidermis. These results suggest the existence of a specific methotrexate carrier in the human epidermis, and may bear relevance to the cutaneous manifestations of methotrexate toxicity.