Application of specific ELISAs for BMP15 and GDF9 to cumulus cell extracts from infertile women.

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific paracrine factors which regulate ovarian cumulus cell (CC) functions. This study aimed to investigate if BMP15 and GDF9 bound to CCs can be characterized, quantified, and show an association with IVF outcomes in infertile women. BMP15 and GDF9 ELISAs were validated and applied to discarded CC extracts. Pooled CCs from individual patients were collected from 120 (cohort 1; BMP15 only) and 81 infertility patients (cohort 2; BMP15 and GDF9) undergoing superovulation. BMP15 and GDF9 levels expressed per CC DNA were correlated with maternal age, clinical and embryology data. Total BMP15 and GDF9 were highly correlated with each other (r = 0.9, p < 0.001). The GDF9:BMP15 ratio was unrelated to oocyte number or age. BMP15/CC DNA and GDF9/CC DNA were unaffected by the type of superovulation and were not related to oocyte/embryo outcomes.

The impact of breast cancer surgery on functional status in older women - A systematic review of the literature.

Primary endocrine therapy as treatment of breast cancer is only recommended in older women with limited life expectancy. However, many older women opt for endocrine therapy due to concerns regarding frailty and potential decline in function after surgery. A decline in functional status after surgery is documented in some cancer types, such as colorectal, however, the full impact of breast cancer surgery is less understood. A systematic review was performed to examine the evidence for impact of breast cancer surgery on functional status in older women. PubMed and Embase databases were searched. Studies were eligible if performed within the last 10 years; included patients over the age of 65 years undergoing breast cancer surgery; included stratification of results by age; measured functional status pre-operatively and at least six months following surgery. A total of 11 studies including 12 030 women were appraised. Two studies represented level-II and nine level-IV evidence. Overall, physical activity level was negatively impacted by breast cancer surgery and this was compounded by the extent of surgery. Evidence for impact of breast cancer surgery on quality of life, fatigue and cognition, was conflicting. The possibility of decline in functional status after breast cancer surgery should be discussed in all older women considering surgery. A structured exercise program may improve the negative effects of surgery on physical activity. Further work is required in the areas of quality of life, fatigability and cognition.

Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Two unusual complications of neuroleptic malignant syndrome.

Neuroleptic malignant syndrome (NMS) is a rare but well described complication of the administration of antipsychotic agents. Compartment syndrome, with increased pressures within the confined space of fascial sheaths leading to compression damage of the contained tissue, similarly is well described. Brachial plexus injuries caused by patient malposition are also very rare but a few cases have been reported. We report a case where these three complications occurred together. This was attributable to the patient developing NMS whilst asleep in the prone position overnight.

Inhibin and premature ovarian failure.

BACKGROUND: Elucidation of the causes of premature ovarian failure (POF) is difficult due to the heterogeneity of the condition. Inhibin is a potential candidate gene for POF based on its dual actions on FSH secretion by the pituitary and gametogenesis in the gonads. A missense mutation in the inhibin alpha subunit gene (INHA G769A) is associated with POF in several populations. However, there is phenotypic heterogeneity in INHA G769A mutation carriers. METHODS: Relevant studies were identified by searching PubMed and mutational frequencies combined for meta-analysis. RESULTS: Meta-analysis of published studies revealed a risk difference of 0.04 (-0.030 to 0.11). The occurrence of asymptomatic carriers in populations suggests incomplete penetrance and/or a multi-genetic cause of POF. We propose that a decline in inhibin bioactivity caused by the mutation could increase FSH levels; and in a susceptible individual, the heightened sensitivity to gonadotrophins causes POF. Impaired paracrine effects of inhibin could impact folliculogenesis due to reduced antagonism of activin, bone morphogenetic protein 15 and growth differentiation factor 9. Functional studies of this mutation indicate normal production of dimeric inhibin A and B and impaired bioactivity of inhibin B. CONCLUSIONS: The identification of an autosomal mutation in the inhibin alpha subunit gene that is significantly linked to POF in certain ethnic populations highlights the role of inhibin in the regulation of ovarian biology and fertility. Although the reduction of inhibin B bioactivity by the INHA G769A mutation is clearly not the only cause, evidence suggests that this change may serve as a susceptibility factor, increasing the likelihood of POF.

Transglutaminase inhibitors induce hyperproliferation and parakeratosis in tissue-engineered skin.

BACKGROUND: The transglutaminase (TG) family consists of eight distinct isoforms. TG types 1, 3 and 5 play a major role in normal skin development, with TG2 also being elevated during dermal wounding. TG1, 3 and 5 are responsible for the cross-linking of keratin precursors and formation of the cornified envelope during keratinocyte differentiation. TG2 may play a role in keratinocyte basement membrane cross-linking. Abnormal TG expression has been demonstrated in Darier disease, Netherton syndrome, psoriasis and lamellar ichthyosis. During a recent investigation of skin contraction in tissue-engineered skin, transglutaminase inhibitors were found to produce hyperproliferation and parakeratosis. OBJECTIVES: Accordingly, this study was designed to study the effect of pan-transglutaminase inhibition on morphology of tissue-engineered skin and expression of keratinocyte differentiation and proliferation-associated antigens. METHODS: We used a tissue-engineered model of human skin, based on de-epidermized acellular human dermis, seeded with normal keratinocytes and dermal fibroblasts and cultured at an air-liquid interface. The pan-transglutaminase inhibitors putrescine, NTU283 (1-dimethyl,2-[(oxopropyl)thio]imidazolium) and NTU285 (N-benzyloxycarbonyl-l-glutaminyl-6-dimethylsulfonium-5-oxo-l-norleucine) were added to the culture medium. After 28 days, histology and immunohistochemistry for collagen IV, involucrin and cytokeratins 6, 10 and 16 were performed. RESULTS: Keratinocyte hyperproliferation and parakeratosis were seen in response to transglutaminase inhibition. Inhibition of transglutaminase also resulted in loss of basement membrane collagen IV. Involucrin and cytokeratins 6 and 16 were confined to the basal layers in control composites but expressed throughout the epidermis in response to transglutaminase inhibition. A distinct band of expression of cytokeratin 10 was seen in the upper stratum granulosum of control composites but only patchy expression was seen after transglutaminase expression. CONCLUSIONS: Pan-transglutaminase inhibition inhibits terminal differentiation of keratinocytes, leading to a hyperproliferative epidermis with parakeratosis and enhanced expression of involucrin and cytokeratins 6 and 16. Expression of the differentiation-associated cytokeratin, cytokeratin 10, is reduced. Basement membrane integrity is also lost as a result of transglutaminase inhibition.

Role of calcineurin in the regulation of human lung mast cell and basophil function by cyclosporine and FK506.

BACKGROUND AND PURPOSE: Cyclosporine and FK506 are thought to act by targeting the Ca2+-dependent protein phosphatase, calcineurin. The aim of the present study was to determine whether cyclosporine and FK506 stabilize mast cells and basophils by interacting with calcineurin. EXPERIMENTAL APPROACH: The effects of cyclosporine and FK506 on the IgE-mediated release of histamine from mast cells and basophils were evaluated. The presence of calcineurin in cells was determined by Western blotting. Ca2+-dependent protein phosphatase activities were assessed in cell extracts using a synthetic phosphorylated peptide that is known to serve as a substrate for calcineurin. KEY RESULTS: FK506 was about 100-fold more potent than cyclosporine as an inhibitor of IgE-dependent histamine release from mast cells and basophils. Immunoblotting of solubilized preparations of purified cells demonstrated the presence of calcineurin in mast cells and basophils. In enzyme assays, mast cells expressed approximately 7-fold higher Ca2+-dependent protein phosphatase activity than basophils. Whereas cyclosporine effectively inhibited Ca2+-dependent protein phosphatase activity in cell extracts, FK506 was considerably less effective. CONCLUSIONS AND IMPLICATIONS: FK506 and cyclosporine inhibit the stimulated release of histamine from mast cells and basophils. However, the ability of cyclosporine, but not FK506, to inhibit Ca2+-dependent protein phosphatase activity questions whether FK506 stabilizes mast cells and basophils by interacting with calcineurin.

Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model.

BACKGROUND: Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. OBJECTIVES: To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. METHODS: We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyte-conditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo[tumour necrosis factor (TNF)-alpha] or modify collagen biochemistry [putrescine, estrone, estradiol and beta-aminopropionitrile (beta-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte/fibroblast cocultures. RESULTS: Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-alpha and beta-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. CONCLUSIONS: Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.

Spindle cell haemangioendothelioma in an arteriovenous fistula of the ring finger after blunt trauma.

We present a case of traumatic arteriovenous fistula of the palm and ring finger, which posed management dilemmas and eventually necessitated ray amputation. Subsequent histology revealed a spindle cell haemangioendothelioma that had developed within the fistula. We report the clinical features and management of this patient.

Inhibin binding sites and proteins in pituitary, gonadal, adrenal and bone cells.

Activin signals via complexes of type I (50-55 kDa) and II (70-75 kDa) activin receptors, but the mechanism of inhibin action is unclear. Proposed models range from an anti-activin action at the type II activin receptor to independent actions involving putative inhibin receptors. Two membrane-embedded proteoglycans, betaglycan and p120, have recently been implicated in inhibin binding, but neither appears to be a signalling receptor. The present studies on primary cultures of rat pituitary and adrenal cells, and several murine and human cell lines were undertaken to characterise inhibin binding to its physiological targets. High affinity binding of inhibin to the primary cultures and several of the cell lines, like that previously described for ovine pituitary cells, was saturable and reversible. Scatchard analysis revealed two classes of binding sites (K(d) of 40-400 and 500-5000 pM, respectively). Affinity labelling identified [125I]inhibin binding proteins with apparent molecular weights of 41, 74, 114 and >170 kDa in all cell types that displayed high affinity, high capacity binding of inhibin. Additional labelling of a 124 kDa species was evident in gonadal TM3 and TM4 cell lines. In several cases, activin (> or =20 nM) competed poorly or not at all for binding to these proteins. The 74, 114 and >170 kDa inhibin binding proteins in TM3 and TM4 cells were immunoprecipitated by an anti-betaglycan antiserum. These three proteins correspond in size to the activin receptor type II and the core protein and glycosylated forms of betaglycan, respectively, that have been proposed to mediate anti-activin actions of inhibin, but the identity of the 74 kDa species is yet to be confirmed. Studies of [125I]inhibin binding kinetics and competition for affinity labelling of individual binding proteins in several cell lines suggest these three species and the 41 and 124 kDa proteins form a high affinity inhibin binding complex. In summary, common patterns of inhibin binding and affinity labelling were observed in inhibin target cells. Novel inhibin binding proteins of around 41 and 124 kDa were implicated in the high affinity binding of inhibin to cells from several sources.

Identification of specific inhibin A-binding proteins on mouse Leydig (TM3) and sertoli (TM4) cell lines.

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (K(d(1)) = 85 pM and 4,160 sites/cell; K(d(2)) = 520 pM and 12,500 sites/cell) and TM4 (K(d(1)) = 61 pM and 2,620 sites/cell; K(d(2)) = 520 pM and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed [(125)I]inhibin A binding (6-8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of [(125)I]inhibin A to both cell lines identified five [(125)I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [(125)I]inhibin A from each of these cross-linked species (ED(50) = 60-110 pM) closely resembled displacement from intact TM3 (ED(50) = 97 +/- 32 pM) and TM4 (ED(50) = 75 +/- 28 pM) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.

The U.S. study of work incapacity and reintegration.

In many countries, including the United States, the number of persons being awarded long-term or permanent disability benefits has risen dramatically in recent years. Government agencies, advocates for the disabled, and others are looking for ways to help persons with disabilities return to the labor force. The Work Incapacity and Reintegration (WIR) Study was developed to address that issue. The United States and five other countries--Germany, Denmark, Sweden, Israel, and the Netherlands--have participated in a cross-national study of work incapacity under the auspices of the International Social Security Association. The study had two objectives: to examine the factors that influence the pattern of work resumption among persons disabled by a back condition and to identify the medical and nonmedical interventions that are most effective in helping such persons reenter the labor force. Samples for the U.S. national study were drawn from four cohorts: Social Security Disability Insurance (DI) beneficiaries, Supplemental Security Income (SSI) beneficiaries, and recipients of temporary disability insurance (TDI) benefits from the states of California and New Jersey. Only the TDI recipients were included in the comparative study. This article discusses the study design and methodology and summarizes the findings of the U.S. national study. Findings from the U.S. study show significant differences between the two cohorts in terms of work resumption and other characteristics. The proportions of respondents from the TDI cohorts who were working at the third and final study contact ranged from 53 percent to 65 percent, compared with less than 5 percent of the DI and SSI respondents. Respondents from the DI and SSI cohorts were on average about 10 years older than the TDI respondents, were less well educated, and reported more physical demands in their usual work. They also reported lower levels of functional capacity, higher levels of pain, and a much greater tendency to have other chronic illnesses. The types of medical treatments provided were remarkably uniform across cohorts and, within cohorts, between those who did and did not resume working. Thus, no medical intervention was identified that showed a significantly higher success rate in terms of facilitating a return to work. However, changes made in the work environment by the employer were an important factor in work reintegration; about 80 percent of respondents who resumed working did so with the help of workplace accommodations. In addition, since respondents with fewer physical demands in their job were more likely to return to work, there appears to be some potential for job retraining as a means of promoting a return to work. The Social Security Administration should consider these findings in developing strategies to help disabled workers reenter the labor force.

Oxidation regulates the inflammatory properties of the murine S100 protein S100A8.

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.

Correlates of psychological distress following armed robbery.

Although mental health professionals have long been aware of the impact of traumatic events, it was not until 1980 that the term posttraumatic stress disorder (PTSD) was introduced into the DSM-III. Since then, one major goal of research has been to identify factors associated with distress following trauma; as yet, few reliable indicators have emerged. Within the population of armed robbery victims, this is particularly true. The purpose of this study was to investigate possible correlates of posttrauma distress in armed robbery victims, and to assess the overall level of distress within this group. A questionnaire was mailed out of 57 robbery victims, aged 15 to 65, who were recruited as study volunteers via community outreach. Severity of the trauma, vulnerability attributions, and avoidant coping were significantly related to distress level, and victims exhibited a high level of distress.

Family caregivers' criticism of patients with schizophrenia.

OBJECTIVE: Relationships were examined between patients' negative symptoms, family caregivers' knowledge of schizophrenia, caregivers' attributions about the cause of patients' symptoms, and caregivers' response to the symptoms. METHODS: A sample of 84 caregivers of patients with schizophrenia in Brisbane, Australia, were interviewed using a structured format and measures designed for the study. RESULTS: Results of regression analyses indicated that three variables significantly predicted caregivers' criticism of patients--a smaller proportion of negative symptoms in the patient's overall symptom pattern, the caregiver's low level of knowledge about the illness, and the caregiver's attributing the cause of negative symptoms to the patient's personality rather than to the illness. CONCLUSIONS: Overall, findings supported the utility of an attributional framework in enhancing conceptions about and research on schizophrenia and family caregiving.

Immunodetection of the murine chemotactic protein CP-10 in bleomycin-induced pulmonary injury.

The murine S-100 protein designated CP-10 is a potent chemotactic factor for phagocytic cells, exhibiting optimal activity in the picomolar range. We assessed the role of this cytokine in the inflammatory response to pulmonary injury following intratracheal administration of bleomycin to mice. In the lungs of normal animals, strong cytoplasmic immunostaining for CP-10 was demonstrable in all recognisable neutrophil leucocytes sequestered within alveolar capillaries. Following induction of pulmonary inflammation in susceptible C57BL/6 mice, numerous CP-10-positive neutrophils were observed, but many of the recruited neutrophils did not exhibit staining for CP-10. No other cells were immunoreactive. The concentration of CP-10 in bronchoalveolar lavage (BAL) fluids from normal mice and mice administered intratracheal saline was below the level of detection by enzyme immunoassay. In contrast, nanomolar levels of CP-10 were detected in unconcentrated BAL fluids from C57BL/6 mice after bleomycin-induced injury, and the presence of monomeric CP-10 was demonstrable by Western blotting. Elevation of CP-10 levels correlated with the influx of inflammatory cells in C57BL/6 mice, but was not demonstrable in BAL fluids from BALB/c mice, which are resistant to pulmonary injury by bleomycin. We conclude that CP-10 may contribute to the recruitment of inflammatory cells in bleomycin-induced lung damage.

Characterization of a mutant recombinant S100 protein using electrospray ionization mass spectrometry.

Two recombinant proteins derived by thrombin cleavage of a fusion protein between glutathione-S-transferase and CP10 (Chemotactic protein 10 kDa) were separated by C4 reversed-phase high-performance liquid chromatography (RP-HPLC). Both proteins were recognised by a polyclonal antibody to native CP10 following sodium dodecyl sulphate/polyacryamide gel electrophoresis (SDS/PAGE) and Western blotting. The major form (approximately 90%) had a mass of 10308 Da, by electrospray mass spectrometry (ESI-MS), which compared well with the theoretical mass of rCP10 (10307.6 Da) whereas the minor component (approximately 10%) had a mass of 11333 Da, 1025 mass units greater than expected. One sequence was obtained by N-terminal sequencing, suggesting that the N-terminus was not modified. The mass of peptides isolated after Asp-N digestion and C18 RP-HPLC were determined by ESI-MS and each assigned a probable sequence based on the expected peptide man of rCP10. The mutant protein produced one additional peak at 10.0 min with mass 1639 Da and the sequence DSHKEQQRGIPGNSS by Edman degradation. The first 5 amino acids corresponded to the last 5 C-terminal amino acids of rCP10. Analysis of the cDNA sequence of the expression vector used to produce rCP10 indicated that the 10 additional C-terminal amino acids were translated after the insertion of glutamine at the normal TAG stop codon. Another stop codon (TGA) located 27 base pairs downstream halts translation. The calculated mass of the mutant protein is 11332.7 Da, in good agreement with the experimental mass. Readthrough occurs in strains of E. coli (eg JPA101) with the amber mutation supE, and this allowed substitution of glutamine at TAG codons in approximately 5-10% of transcripts.

Induction of the S100 chemotactic protein, CP-10, in murine microvascular endothelial cells by proinflammatory stimuli.

Microvascular endothelial cells (EC) have multiple functions in inflammatory responses, including the production of chemoattractants that enhance leukocyte transmigration into tissues. Chemotactic protein, 10 kD (CP-10), is an S100 protein with potent chemotactic activity for myeloid cells in vitro and in vivo and is expressed in neutrophils and lipopolysaccharide (LPS)-activated macrophages. We show here that CP-10 is induced in murine endothelioma cell lines (bEnd-3, sEnd-1, and tEnd-1) after activation with LPS and interleukin-1 (IL-1) but not tumor necrosis factor alpha (TNFalpha) or interferon gamma (IFNgamma). Induction was not mediated by endogenous release of IL-1 or TNFalpha and was not directly upregulated by phorbol myristate acetate, calcium ionophore, or vitamin D3. EC were exquisitely sensitive to IL-1 activation (3.4 U/mL) and CP-10 mRNA induction with IL-1 occurred earlier (8 hours) than with LPS (12 hours). Furthermore, some microvessels and capillaries in delayed-type hypersensitivity lesions expressed cytoplasmic CP-10. Responses to LPS and not IL-1 in vitro were regulated by the degree of cell confluence and by TNFalpha costimulation. The related MRP-14 mRNA had a different induction pattern. Monomeric and homodimeric CP-10 upregulated by activation was predominantly cell-associated. EC-derived CP-10 may contribute to amplification of inflammatory processes by enhancing leukocyte shape changes and transmigration in the microcirculation.

Characterization of the mouse transforming growth factor alpha gene: its expression during eyelid development and in waved 1 tissues.

The spontaneous mouse waved 1 (wa1) mutation is allelic with the transforming growth factor alpha (TGF-alpha) gene and produces phenotypes similar to those of TGF-alpha knockout mice. Here, we show that TGF-alpha mRNA and protein levels are measurable in wa1 tissues but reduced 5- to 30-fold relative to wild type. Because the wa1-coding sequence is identical to that of the normal mRNA, wa1 is not a null mutation. Nuclear run-on analyses revealed decreased transcription of the TGF-alpha gene in wa1 tissues, but the sequence of a 3.2-kb 5' flanking fragment containing the promoter was unaltered. Moreover, pulsed field gel electrophoresis analysis did not reveal alterations within 750 kb upstream or 350 kb downstream of the gene, and chromosome 6 was karyotypically normal. Hence, we speculate that the wa1 mutation may be subtle and/or reside at a greater distance from the TGF-alpha gene. TGF-alpha deficiency elicits a spectrum of variably penetrant eye anomalies in wa1 and knockout mice that are associated with open eyes at birth. We found that late-gestation wa1 and TGF-alpha-null embryos display a significant delay in eyelid closure, although the eyes of most embryos fuse prior to birth. In situ hybridization localized TGF-alpha expression to the advancing margins of the eyelid epithelium and epidermal growth factor receptor expression throughout the eyelid and corneal epithelia. These results suggest that eye problems observed in TGF-alpha-deficient adult mice arise from premature exposure and trauma to open eyes during or following parturition.