Development of ISB 1442, a CD38 and CD47 bispecific biparatopic antibody innate cell modulator for the treatment of multiple myeloma.

Antibody engineering can tailor the design and activities of therapeutic antibodies for better efficiency or other advantageous clinical properties. Here we report the development of ISB 1442, a fully human bispecific antibody designed to re-establish synthetic immunity in CD38+ hematological malignancies. ISB 1442 consists of two anti-CD38 arms targeting two distinct epitopes that preferentially drive binding to tumor cells and enable avidity-induced blocking of proximal CD47 receptors on the same cell while preventing on-target off-tumor binding on healthy cells. The Fc portion of ISB 1442 is engineered to enhance complement dependent cytotoxicity, antibody dependent cell cytotoxicity and antibody dependent cell phagocytosis. ISB 1442 thus represents a CD47-BsAb combining biparatopic targeting of a tumor associated antigen with engineered enhancement of antibody effector function to overcome potential resistance mechanisms that hamper treatment of myeloma with monospecific anti-CD38 antibodies. ISB 1442 is currently in a Phase I clinical trial in relapsed refractory multiple myeloma.

ERß- and prostaglandin E2-regulated pathways integrate cell proliferation via Ras-like and estrogen-regulated growth inhibitor in endometriosis.

In endometriosis, stromal and epithelial cells from the endometrium form extrauterine lesions and persist in response to estrogen (E2) and prostaglandin E2 (PGE2). Stromal cells produce excessive quantities of estrogen and PGE2 in a feed-forward manner. However, it is unknown how estrogen stimulates cell proliferation and survival for the establishment and persistence of disease. Previous studies suggest that estrogen receptor-ß (ERß) is strikingly overexpressed in endometriotic stromal cells. Thus, we integrated genome-wide ERß binding data from previously published studies in breast cells and gene expression profiles in human endometriosis and endometrial tissues (total sample number = 81) and identified Ras-like, estrogen-regulated, growth inhibitor (RERG) as an ERß target. Estradiol potently induced RERG mRNA and protein levels in primary endometriotic stromal cells. Chromatin immunoprecipitation demonstrated E2-induced enrichment of ERß at the RERG promoter region. PGE2 via protein kinase A phosphorylated RERG and enhanced the nuclear translocation of RERG. RERG induced the proliferation of primary endometriotic cells. Overall, we demonstrated that E2/ERß and PGE2 integrate at RERG, leading to increased endometriotic cell proliferation and represents a novel candidate for therapeutic intervention.

Targeting EGFR and PI3K pathways in ovarian cancer.

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed in ovarian cancer, but agents targeting this pathway have shown little effect as single agents. This may be due to the presence of alternative pathways, particularly activation of the PI3K/Akt/MTOR pathway. METHODS: We have therefore examined the effect of inhibitors of this pathway (ZSTK474 and sirolimus) in combination with the EGFR inhibitors erlotinib and gefitinib in ovarian cancer primary cell cultures. RESULTS: The single-agent EGFR inhibitors showed little activity, although some activity was seen with the single-agent PI3K inhibitor, ZSTK474. Combinations of ZSTK474 with EGFR inhibitors showed enhanced activity with some evidence of synergy, whereas sirolimus combinations were less active. The results were not explicable on the basis of PIK3CA mutation or amplification, or PTEN loss, although one tumour with a KRAS mutation showed resistance to EGFR inhibitors. However, there was correlation of the EGFR expression with sensitivity to EGFR and resistance to PI3K active agents, and inverse correlation in the sensitivity of individual tumours to agents active against these pathways, suggesting a mechanism of action for the combination. CONCLUSION: Phase I/II clinical trials with these agents should include further pharmacodynamic endpoints and molecular characterisation to identify patients most likely to benefit from this strategy.

Ligand-activated peroxisome proliferator-activated receptor ß/δ modulates human endometrial cancer cell survival.

Endometrial cancer is the fourth most common malignancy among women and is a major cause of morbidity contributing to approximately 8,200 annual deaths in the USA. Despite advances to the understanding of endometrial cancer, novel interventions for the disease are necessary given that many tumors become refractory to therapy. As a strategy to identify novel therapies for endometrial carcinoma, in this study, we examined the contribution of the peroxisome proliferator-activated receptor ß/δ (PPARß/δ) to endometrial cancer cell proliferation and apoptosis. We found that when activated with the highly selective PPARß/δ agonists, GW0742 and GW501516, PPARß/δ inhibited the proliferation and markedly induced the apoptosis of three endometrial cancer cell lines. The specificity of the PPARß/δ-induced effects on cell proliferation and apoptosis was demonstrated using PPARß/δ-selective antagonists and PPARß/δ small interfering RNA in combination with PPARß/δ-selective agonists. Furthermore, we showed that PPARß/δ activation increased phosphatase and tensin homolog expression, which led to protein kinase B (AKT) and glycogen synthase kinase-3ß (GSK3ß) dephosphorylation, and increased ß-catenin phosphorylation associated with its degradation. Overall, our data suggest that the antitumorigenic effect of PPARß/δ activation in endometrial cancer is mediated through the negative regulation of the AKT/GSK3ß/ß-catenin pathway. These findings warrant further investigation of PPARß/δ as a therapeutic target in endometrial cancer.

HER2 amplification status in breast cancer: a comparison between immunohistochemical staining and fluorescence in situ hybridisation using manual and automated quantitative image analysis scoring techniques.

AIMS: To compare the results of breast cancer sections with HercepTesttrade mark immunohistochemistry (IHC) scores ranging from 0 to 3+ with fluorescence in situ hybridisation (FISH) for HER2 amplification. The HER2 digital scoring application of the Micrometastasis Detection System (MDS) was used, together with manual scoring of FISH and HercepTest, to determine whether this system provides an accurate alternative. METHODS: Paraffin wax embedded sections were stained using HercepTest and analysed by eye and automated quantitative image analysis. FISH was performed using the PathVysion fluorescent probe and scored by eye and automated quantitative image analysis using MDS. RESULTS: Of 114 cases, 26% were amplified by FISH, whereas only 18% scored 3+; 32% of IHC 2+ cases were amplified by FISH, and one showed borderline amplification. Six percent of IHC negative cases (0 or 1+) were amplified by FISH, and one showed borderline amplification. Of IHC 3+ cases, 10% were non-amplified by FISH. Classification discrepancies were seen in 18% of HercepTest cases scored by eye and using the MDS system. MDS was consistent with visual FISH scoring and correctly differentiated most ambiguous visual IHC scores. CONCLUSIONS: FISH provides a more accurate and consistent scoring system for determining HER2 amplification than HercepTest. The MDS system provides a reliable, consistent alternative to visual IHC and FISH scoring. IHC is still a valuable technique to aid in identification of isolated or heterogeneous tumour populations for subsequent FISH analysis, and a combined FISH and HercepTest approach to all breast cancer cases may be the most efficient strategy.

Wound healing assessment using 20 MHz ultrasound and photography.

BACKGROUND/AIMS: To compare two non-invasive techniques of assessing wound healing, photography and high resolution ultrasound (HRUS) scanning, in experimentally induced full-thickness human skin wounds. METHODS: Punch biopsy wounds, 4 mm in diameter, were made aseptically through locally anaesthetised skin on the anterior (volar) surface of the non-dominant forearm, 3 cm below the base of the cubital fossa, of 20 human participants. The wounds were treated with a topical antibiotic and covered for 3 days with Mepore sterile dressings. Wound healing was assessed on post-operative days 3, 7, 14 and 21 from photographs and HRUS B-scans. All photographs were taken of the wound site and adjacent intact skin under standardised conditions. The prints obtained were examined visually and digitised. Digital HRUS B-scans were taken through the centre of the wound bed and the adjacent intact skin parallel to the epidermis. Using the scanner's calibrated linear measurement capability, the wound width was measured adjacent to the deep surface of the scab, at the base of the wound, and midway between these two levels. RESULTS: The wound margins were more clearly defined in the HRUS scans than in the photographs of the wounds; in some of the latter the scab masked the wound margins. Changes in the surface width of the wound were affected by the time of scab dehiscence, which varied between volunteers. There was less individual variation in the width of the base of the wound, as measured from the HRUS scans. CONCLUSIONS: In contrast to photography, which allows recording of changes in the superficial aspect of the wound only, HRUS scanning permits the quantitative assessment of structural changes deep within the wound. Temporal changes in the width of the base of the wound can be used as an indication of the progress of repair.

Growth hormone improves clinical status in prepubertal children with cystic fibrosis: results of a randomized controlled trial.

OBJECTIVES: We conducted a 1-year randomized controlled trial to test the hypothesis that growth hormone (GH) improves the clinical status of children with cystic fibrosis. STUDY DESIGN: Nineteen prepubertal children were randomized to control (NonTX, n = 9) or to daily injections of GH (0.3 mg/kg/wk) (GHTX, n = 10) for 1 year. Every 3 months height, weight, and lean tissue mass were measured. Caloric intake, resting energy expenditure, pulmonary function, and respiratory muscle strength were measured every 6 months, as were total number of hospitalizations and courses of outpatient intravenous antibiotics. RESULTS: The GHTX group had significantly greater height, height velocity (NonTX = 3.8 +/- 1.4 cm/y, GHTX = 8.1 +/- 2.4 cm/y; P =.002), weight, weight velocity (NonTX = 2.1 +/- 0.9 kg/y, GHTX = 4.5 +/- 1.1 kg/y; P =.004), and change in lean tissue mass (NonTX = 2.1 +/- 1.6 kg, GHTX = 4.7 +/- 1.7 kg; P =.01) analyzed by the Student t test. The GHTX group had significant improvement in delta forced vital capacity compared with the year before study, and respiratory muscle strength improved. The number of hospitalizations and outpatient intravenous antibiotic courses significantly decreased in the GHTX group but did not change in the NonTX group. No subject had development of cystic fibrosis-related diabetes. CONCLUSIONS: Results of the first randomized controlled trial of GH treatment in cystic fibrosis indicate that GH improves growth and clinical status.

Growth hormone decreases protein catabolism in children with cystic fibrosis.

Despite aggressive nutritional therapy, low body weight and protein catabolism are common problems in children with cystic fibrosis. Previous studies by our group and others have demonstrated improvement in both height and weight in children with cystic fibrosis who were treated with human recombinant GH, and our group has recently documented improved clinical status and lean tissue mass as well. The purpose of this report is to summarize our findings of the effect of GH on whole body protein kinetics in cystic fibrosis and to relate these findings to changes in TNF-alpha levels. We conducted a 1-yr study of 19 prepubertal children with cystic fibrosis (age 7-12 yr, all <94% of ideal body weight). Ten children were randomly assigned to take daily injections of GH (0.3 mg/kg.wk), and nine were randomly assigned to be controls. Baseline results from the subjects with cystic fibrosis were compared with results obtained from nine age- and gender-matched healthy children. Whole body protein turnover was measured at baseline and every 6 months using the stable isotope [1-(13)C]leucine and mass spectrometric analysis. Leucine rate of appearance, a measure of protein catabolism, was similar in both cystic fibrosis subgroups at baseline and was significantly higher than in the control children without cystic fibrosis. Treatment with GH resulted in a significantly lower leucine rate of appearance, as well as significantly lower leucine oxidation. The rate of protein synthesis, as calculated from these numbers, actually decreased in the cystic fibrosis subgroup. TNF-alpha levels were higher in both cystic fibrosis subgroups than in controls and correlated with leucine rate of appearance. The results of this study suggest that one reason GH improves body weight and lean tissue mass is due to improved whole body protein catabolism and improved efficiency of whole body protein kinetics.

Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.

Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

An extended conformation of the macrophage mannose receptor.

The macrophage mannose receptor mediates phagocytosis of pathogenic microorganisms and endocytosis of potentially harmful soluble glycoproteins by recognition of their defining carbohydrate structures. The mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of 8-10 domains related to C-type carbohydrate recognition domains (CRDs), a fibronectin type II repeat and an N-terminal cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the receptor is a monomeric, elongated and asymmetric molecule. Proteolysis experiments indicate the presence of close contacts between several pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for modeled receptor conformations are consistent with an extended conformation with close contacts between three pairs of CRDs. The N-terminal cysteine-rich domain and the fibronectin type II repeat appear to increase the rigidity of the molecule. The rigid, extended conformation of the receptor places domains with different functions at distinct positions with respect to the membrane.

The ovarian phenotype of the aromatase knockout (ArKO) mouse.

Targeted disruption of exon 9 of the cyp19 gene gives rise to a non-functional aromatase enzyme incapable of converting androgens to oestrogens. The aromatase knockout (ArKO) mouse is, thus, characterised by a dysfunctional pituitary-gonadal axis, which manifests in non-detectable levels of oestrogen in serum. These mice also exhibit elevated levels of circulating gonadotrophins (luteinising hormone (LH) and follicle stimulating hormone (FSH)) and testosterone. The ArKO mouse is infertile due to folliculogenic disruption and a failure to ovulate. The age-dependent ovarian phenotype revealed a block in follicular development at the antral stage and a complete absence of corpora lutea. By 21-23 weeks of age haemorrhagic cystic follicles were present and by 1 year there were abnormal follicles, an absence of secondary and antral follicles and atretic primary follicles. Interstitial tissue remodelling was extensive and exemplified by an increase in collagen deposition and an influx of macrophages, coincident with the loss of follicles. In mice, maintained on a soy-free and, thus, phytoestrogen-free diet, the ovarian phenotype was accelerated and exacerbated. In conclusion, the ovarian phenotype of the ArKO mouse can be attributed to the altered hormonal environment brought about by the absence of aromatase and the failure of androgens to be converted to oestrogens in the presence of elevated gonadotropins.

The roles of activins, inhibins and estrogen in early committed follicles.

The hypothesis that activin and inhibin are autocrine/paracrine mediators of ovarian folliculogenesis has a solid basis. In mouse and rat models, granulosa cells (GC) of committed follicles express mRNA and protein for the activin/inhibin subunits and mRNA for the activin receptors (type I and II). Dimeric inhibin-A and -B are produced by postnatal ovarian cell dispersates and (GC) in culture. Similar levels of inhibin-A and -B are produced by postnatal ovarian cells, but thereafter as the ovary develops, inhibin-A becomes the predominant form. Activin was more effective than transforming growth factor-beta (TGF-beta) in enhancing follicle stimulating hormone (FSH)-stimulated inhibin production by ovarian cells. Evidence for a local regulatory role of estrogen in the ovary is also accumulating. Murine models of estrogen receptor (ERalpha or ERbeta) disruption produce mice with abnormal ovarian phenotypes. Female mice, which lack the capacity to produce estrogen (ArKO mice), have arrested folliculogenesis, no corpora lutea, elevated levels of luteinising hormone (LH), FSH and testosterone and are infertile. These data are consistent with autocrine/paracrine actions of activin in the early growth of committed follicles and estrogen in follicular maturation.

An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19 (aromatase) gene.

With the development of a mouse model of estrogen insufficiency due to targeted disruption of the aromatase gene [the aromatase knockout (ArKO) mouse], a new opportunity exists to examine the role of estrogen in ovarian follicular development. Ovaries and serum were collected from wild-type, heterozygous, and ArKO mice at 10-12 and 21-23 weeks and 1 yr of age. The ovaries were assessed histologically and stereologically, with primary, secondary, and antral follicles and corpora lutea counted. The uteri were hypoestrogenic, and serum levels of LH and FSH in ArKO females were elevated above those in heterozygote and wild-type animals at all ages studied. Although estrogen was not a prerequisite for reinitiation of follicle growth, there was a block of follicular development, and no corpora lutea were present in ArKO ovaries. Thus, the ArKO mouse was infertile as a consequence of disrupted folliculogenesis and a failure to ovulate. Hemorrhagic cystic follicles were present by 21-23 weeks of age. The ovarian phenotype degenerated with age, such that by 1 yr there were no secondary or antral follicles, and the primary follicles present were atretic. Extensive interstitial tissue remodeling occurred, exemplified by an influx of macrophages and collagen deposition, coincident with the loss of follicles. In conclusion, the ovarian environment in ArKO mice does not allow the characteristic development of follicles that culminates in ovulation and demonstrates an in vivo requirement of estrogen for normal ovarian function in the mouse.

Laser modulation of angiogenic factor production by T-lymphocytes.

BACKGROUND AND OBJECTIVE: In previous investigations, small variations in the energy densities of low level light therapy (LLLT) were found to produce significant differences in the proliferation of resting T-lymphocytes in vitro. Pulsing these cells with mitogen in addition to laser therapy produced inhibitory effects regardless of the amplitude of the energy density used. In the current study, the effect of LLLT on the production of angiogenic factor(s) by T-lymphocytes was investigated in vitro. STUDY DESIGN/MATERIALS AND METHODS: Human T-cells isolated from peripheral blood were prepared in suspension either with or without addition of mitogen. Cell suspensions were irradiated with laser by using the following energy densities: 1.2, 3.6, 6.0, and 8.4 J/cm(2). Wavelength, pulsing frequency, and power output were kept constant at 820 nm, 5,000 Hz, and 50 mW, respectively. After either 3 or 5 days of incubation, lymphocyte supernatants were collected and added as conditioned media to cultured endothelial cells (ECs). The effect on the proliferation of these ECs was assessed over a 72-hour period by using a methylene blue assay. RESULTS: Endothelial cell proliferation increased significantly when incubated with conditioned media collected from resting T-cells exposed to 1.2 and 3.6 J/cm(2). Day 5 conditioned media produced similar patterns of EC proliferation to that of day 3 but at lower magnitude. Pulsing of T-lymphocytes with mitogen in addition to laser irradiation significantly lessened their angiogenic capability. Conditioned media from 3.6 J/cm(2) laser-treated T-cells induced the maximal EC proliferation in all groups studied. CONCLUSION: It would seem that laser therapy stimulates lymphocytes to produce factor(s) that can modulate EC proliferation in vitro; this effect on the lymphocytes is influenced by (1) the amplitude of energy density used for T-cell irradiation, (2) exposing T-cells to both mitogen and laser, and (3) the duration of T-cell incubation in culture.

Two distinct segments of the hepatitis B virus surface antigen contribute synergistically to its association with the viral core particles.

The long surface antigen polypeptide (L-HBsAg) of hepatitis B virus (HBV) is believed to mediate contact between the virus envelope and nucleocapsid protein (HBcAg). The N and C termini of L-HBsAg were shortened progressively in order to define the minimum contiguous sequence of amino acids that contains the residues necessary for association with HBcAg. The resulting mutants were expressed in rabbit reticulocyte lysates and their interaction with HBcAg was examined with an immunoprecipitation assay and an equilibrium binding assay in solution to give relative dissociation constants. Binding of HBcAg particles by L-HBsAg displayed two widely differing dissociation constants, indicating two distinct binding sites between the molecules. The two distinct sites, one located between residues 24 and 191 and the other between residues 191 and 322 of L-HBsAg, contribute synergistically to high-affinity binding to HBcAg, but disruption of either of these segments resulted in a much weaker interaction showing only one dissociation constant. Inhibition of the interaction by peptides that bind to the tips of the nucleocapsid spikes differentiated contacts in HBcAg for the two binding domains in L-HBsAg and implied that the amino-terminal binding domain contacts the tips of the HBcAg spikes. Analysis of specific single amino acid mutants of L-HBsAg showed that Arg92 played an important role in the interaction.

Peptides that block hepatitis B virus assembly: analysis by cryomicroscopy, mutagenesis and transfection.

Peptides selected to bind to hepatitis B virus (HBV) core protein block interaction with the long viral surface antigen (L-HBsAg) in vitro. High resolution electron cryomicroscopy showed that one such peptide binds at the tips of the spikes of the core protein shell. The peptides contain two basic residues; changing either of two acidic residues at the spike tip to an alanine greatly reduced the binding affinity. Transfection of hepatoma cells with a replication-competent HBV plasmid gave significantly reduced production of virus in the presence of peptide, in a dose-dependent manner. These experiments show that the interaction of L-HBsAg with core particles is critical for HBV assembly, and give proof of principle for its disruption in vivo by small molecules.

Stimulatory effect of 660 nm low level laser energy on hypertrophic scar-derived fibroblasts: possible mechanisms for increase in cell counts.

BACKGROUND AND OBJECTIVE: Varying effects of red light wavelengths on in vitro cells have been reported. Low level lasers (LLL) are employed to assist wound healing especially for indolent ulcers. On healing, burn wounds may become hypertrophic, resulting in excessive wound contraction, poor cosmesis, and functional impairment. This study enquired whether 660 nm LLL affected hypertrophic scar-derived fibroblasts. STUDY DESIGN/MATERIALS AND METHODS: The experiments investigated the effect of a 660 nm, 17 mW laser diode at dosages of 2.4 J/cm2 and 4 J/cm2 on cell counts of two human fibroblast cell lines, derived from hypertrophic scar tissue (HSF) and normal dermal (NDF) tissue explants, respectively. The protocol avoided transfer of postirradiated cells. Estimation of fibroblasts utilized the methylene blue bioassay. RESULTS/CONCLUSION: The post-660 nm-irradiated HSFs exhibited very significantly higher cell counts than controls P < 0.01 on days 1-4 (Mann-Whitney U-test), and P < 0.01 on days 1-3 for similarly irradiated NDFs.

The effects of ovarian hormone deficiency on wound contraction in a rat model.

OBJECTIVE: To demonstrate the effect of a deficiency of ovarian hormones on the process of wound contraction, using the oophorectomised rat model of the human menopause. DESIGN: A randomised controlled trial. POPULATION: Ninety-six adult Wistar rats were randomly allocated into either an oophorectomised group or a sham-oophorectomised control group. METHODS: Having confirmed a significant reduction in plasma oestradiol levels in the oophorectomised rats, full-thickness excised lesions were made in the flank skin of the adult rats at either two weeks or four months after oophorectomy, so that the effects of two different durations of hormone deficiency could be assessed and compared with the sham-oophorectomised controls. Following wounding, the rats were left for 3, 5, 10 or 22 days; wound contraction was assessed from photographs of the wounds taken at these intervals after injury. RESULTS: In the rats wounded four months after oophorectomy there was a slower rate of wound contraction, resulting in larger wounds at days 3, 5, 10 and 22, compared with control rats. No significant difference was observed in rats wounded two weeks after oophorectomy, indicating that the effects of ovarian hormone deficiency on this process are delayed. CONCLUSION: Due to the pivotal role of wound contraction in the process of wound healing these findings may be of clinical relevance and could have an important impact on the administration of hormone replacement therapy.

Pilot study using high-frequency diagnostic ultrasound to assess surgical wounds in renal transplant patients.

BACKGROUND: The majority of renal transplant patients will sustain at least one acute rejection episode during the first 4 months, following transplantation. Clinical rejection is rarely an all-or-nothing reaction, and the first episode seldom progresses to complete renal destruction. The functional changes induced by rejection appear to be in large part reversible; therefore the recognition and treatment of the rejection episode, before the development of severe renal damage, are of extreme importance. METHODS: This pilot study describes a technique of non-invasive wound assessment used to monitor 14 transplant patients, over a 3 week post-operative period. The technique involved the use of 20 MHz diagnostic ultrasound, in conjunction with fractal image analysis. Ultrasound scans of both wounded and adjacent uninjured tissue were taken 5, 7, 11, 14 and 21 days post-surgery. RESULTS: When patients were divided into those who experienced allograft rejection and those who did not, a distinct trend appeared. In those who did not reject, the fractal signature of the wound progressed towards that of normal tissue in a linear fashion. In patients that did reject, the fractal signature initially progressed towards normal until day 11, then changed direction moving away from normal, between days 11 and 14. The patients experiencing allograft rejection were clinically diagnosed around a mean time of 14 days post-surgery. It is therefore possible that this shift in fractal signature may correlate with graft rejection. CONCLUSION: The results of this pilot study indicate that high frequency diagnostic ultrasound, coupled with appropriate image analysis, may be a useful adjunct to renal function assessment.

Differential responses of post-natal rat ovarian cells to FSH and activin.

A role for activin in the acquisition of gonadotropin responsiveness by the post-natal rat ovary was investigated. The inhibin/activin subunits in terms of protein and mRNA, were localised in granulosa cells of the rat ovary at days 4, 8 and 12 after birth. A characteristic pattern of responses to FSH for inhibin and progesterone (P) production was established using a dispersed ovarian cell bioassay. P production by day 4, 8 and 12 cultures was stimulated by FSH, but only when iso-butyl-methyl-xanthine (MIX) was present. In contrast, a basal level of inhibin production was measured in day 4 cultures which was not responsive to FSH or MIX. In day 8 and 12 cultures, inhibin production was FSH-responsive, but only in the absence of MIX. The addition of activin to cultures of day 4, 8 and 12 ovarian cells induced FSH-responsive P production and stimulated both basal and FSH-stimulated inhibin production. These studies indicate a differential response of neonatal ovarian cells to FSH in terms of P and inhibin production. Activin may play a role in facilitating the effects of FSH on signal transduction pathways leading to inhibin and steroid production and therefore be part of the mechanism which determines responsiveness of granulosa cells to FSH.