Spontaneous Co-Assembly of Cellulose Nanocrystals and TiO2 Nanorods Followed by Calcination to Form Cholesteric Inorganic Nanostructures.

Chiral nanomaterials possess unique electronic, magnetic, and optical properties that are relevant to a wide range of applications including photocatalysis, chiral photonics, and biosensing. A simple, bottom-up method to create chiral, inorganic structures is introduced that involves the co-assembly of TiO2 nanorods with cellulose nanocrystals (CNCs) in water. To guide experimental efforts, a phase diagram was constructed to describe how phase behavior depends on the CNCs/TiO2/H2O composition. A lyotropic cholesteric mesophase was observed to extend over a wide composition range as high as 50 wt % TiO2 nanorods, far exceeding other examples of inorganic nanorods/CNCs co-assembly. Such a high loading enables the fabrication of inorganic, free-standing chiral films through removal of water and calcination. Distinct from the traditional templating method using CNCs, this new approach separates sol-gel synthesis from particle self-assembly using low-cost nanorods.

The gut microbiome: what the oncologist ought to know.

The gut microbiome (GM) has been implicated in a vast number of human pathologies and has become a focus of oncology research over the past 5 years. The normal gut microbiota imparts specific function in host nutrient metabolism, xenobiotic and drug metabolism, maintenance of structural integrity of the gut mucosal barrier, immunomodulation and protection against pathogens. Strong evidence is emerging to support the effects of the GM on the development of some malignancies but also on responses to cancer therapies, most notably, immune checkpoint inhibition. Tools for manipulating the GM including dietary modification, probiotics and faecal microbiota transfer (FMT) are in development. Current understandings of the many complex interrelationships between the GM, cancer, the immune system, nutrition and medication are ultimately based on a combination of short-term clinical trials and observational studies, paired with an ever-evolving understanding of cancer biology. The next generation of personalised cancer therapies focusses on molecular and phenotypic heterogeneity, tumour evolution and immune status; it is distinctly possible that the GM will become an increasingly central focus amongst them. The aim of this review is to provide clinicians with an overview of microbiome science and our current understanding of the role the GM plays in cancer.

An experimental therapeutics test of whether adding dissonance-induction activities improves the effectiveness of a selective obesity and eating disorder prevention program.

OBJECTIVE: Compare the Healthy Weight obesity and eating disorder prevention program, which promotes participant-driven gradual lifestyle changes to bring energy intake and expenditure into balance, to a new intervention, Project Health, which adds activities to create cognitive dissonance about unhealthy eating, a sedentary lifestyle, and excess body fat, and an obesity education video-control condition. METHOD: College students at risk for both outcomes because of weight concerns (N=364, 72% female) were randomized to condition, completing pretest, posttest, and 6, 12 and 24-month follow-up assessments. RESULTS: Project Health participants showed significantly smaller increases in measured body mass index (BMI) through 2-year follow-up than both Healthy Weight participants and controls (both d=-0.18), and significantly lower onset of overweight/obesity over 2-year follow-up than Healthy Weight participants and controls (13 vs 21% and 22%). Healthy Weight and Project Health participants showed significantly greater eating disorder symptom reductions than controls through 2-year follow-up. Healthy Weight and Project Health participants showed marginally lower eating disorder onset over follow-up than controls (3 and 3% vs 8% respectively). CONCLUSIONS: The reduced increases in BMI and future overweight/obesity onset for Project Health relative to both an active matched intervention and a minimal intervention control condition are noteworthy, especially given the short 6-h intervention duration. The reduction in eating disorder symptoms for Healthy Weight and Project Health relative to controls was also encouraging. Results suggest that adding dissonance-induction activities increased weight loss effects. Yet, effects for both were generally small and the eating disorder onset prevention effects were only marginal, potentially because intervention groups included both sexes, which reduced eating disorder incidence and sensitivity.

First-in-man phase I trial of two schedules of the novel synthetic tetrahydroisoquinoline alkaloid PM00104 (Zalypsis) in patients with advanced solid tumours.

BACKGROUND: PM00104 binds guanines at DNA minor grooves, impacting DNA replication and transcription. A phase I study was undertaken to investigate safety, dose-limiting toxicities (DLTs), recommended phase II dose (RP2D), pharmacokinetics (PKs) and preliminary antitumour activity of PM00104 as a 1- or 3-h infusion three-weekly. METHODS: Patients with advanced solid tumours received PM00104 in a dose escalation trial, as guided by toxicity and PK data. RESULTS: A total of 47 patients were treated; 27 patients on the 1-h schedule (0.23-3.6 mg m(-2)) and 20 patients on the 3-h schedule (1.8-3.5 mg m(-2)). Dose-limiting toxicities comprised reversible nausea, vomiting, fatigue, elevated transaminases and thrombocytopenia, establishing the 1-h schedule RP2D at 3.0 mg m(-2). With the 3-h schedule, DLTs of reversible hypotension and neutropenia established the RP2D at 2.8 mg m(-2). Common PM00104-related adverse events at the RP2D comprised grade 1-2 nausea, fatigue and myelosuppression. In both schedules, PKs increased linearly, but doses over the 1-h schedule RP2D resulted in higher than proportional increases in exposure. A patient with advanced urothelial carcinoma had RECIST shrinkage by 49%, and three patients had RECIST stable disease ≥6 months. CONCLUSION: PM00104 is well tolerated, with preliminary evidence of antitumour activity observed. The 1-h 3-weekly schedule is being assessed in phase II clinical trials.

Comparative effects of various commercially available mouthrinse formulations on oral malodor.

OBJECTIVES: The primary aim of this study was to compare a new mouthwash (SB12®) containing 0.025% chlorhexidine and 0.3% zinc for oral malodor reduction against four commercially available mouthwashes and negative control. A secondary aim was to compare the two methods for measuring volatile sulphur compounds (VSC) by halimetry and OralChroma. METHODS: Organoleptic scale, halimeter and the OralChroma were used to assess oral malodour and VSC. The effects of five test formulations and water (negative control) were assessed after 30, 60, 90 and 180 min, with 1 week between the treatments to avoid any cross-over effect. RESULTS: Reduction in H(2) S by halimetry and malodour levels by organoleptic assessment ranged from, slight (LacerFresh®) (P > 0.05), moderate (BreathRx®, SmartMouth® (P < 0.01) to marked effects (SB12®, Listerine®) (P < 0.001) at all time points compared with water. The largest differences were observed at 30 min and decreased with time. SB12® showed separation from Listerine® at 180 min, using ANOVA plus Bonferroni's Multiple Comparison post-test (P < 0.05). Relationships between organoleptic, halimeter and OralChroma were between R² = 0.795 and 0.926. CONCLUSION: SB12 shows a consistent and reproducible inhibitory effect on oral malodor parameters, which in turn correlate well with each other.

The insulin-like growth factor-I receptor inhibitor figitumumab (CP-751,871) in combination with docetaxel in patients with advanced solid tumours: results of a phase Ib dose-escalation, open-label study.

BACKGROUND: This phase Ib trial assessed safety, tolerability, and maximum tolerated dose (MTD) of figitumumab (CP-751,871), a fully human monoclonal antibody targeting the insulin-like growth factor type 1 receptor (IGF-IR), in combination with docetaxel. METHODS: Patients with advanced solid tumours were treated with escalating dose levels of figitumumab plus 75 mg m(-2) docetaxel every 21 days. Safety, efficacy, pharmacokinetics (PKs), and biomarker responses were evaluated. RESULTS: In 46 patients, no dose-limiting toxicities were attributable to the treatment combination. Grade 3 and 4 toxicities included neutropaenia (n=28), febrile neutropaenia (n=11), fatigue (n=10), leukopaenia (n=7), diarrhoea (n=5), hyperglycaemia, lymphopaenia, cellulitis, DVT, and pain (all n=1). The MTD was not reached. Four partial responses were observed; 12 patients had disease stabilisation of > or =6 months. Pharmacokinetic and biomarker analyses showed a dose-dependent increase in plasma exposure, and complete sIGF-IR downregulation at doses of >or =3 mg kg(-1). Pharmacokinetics of docetaxel in combination was similar to when given alone. Out of 18 castration-resistant prostate cancer patients, 10 (56%) had > or =5 circulating tumour cells (CTCs) per 7.5 ml of blood at baseline: 6 out of 10 (60%) had a decline from > or =5 to <5 CTCs and 9 out of 10 (90%) had a > or =30% decline in CTCs after therapy. CONCLUSIONS: Figitumumab and docetaxel in combination are well tolerated. Further evaluation is warranted.

Modulation of phase separation between spherical and rodlike molecules using geometric surfactancy.

A Dyad consisting of C(60) linked to a pi-conjugated oligomer by a propylene spacer was synthesized to explore its ability to modulate phase separation between OFTB and PCBM using differential scanning calorimetry, hot-stage polarizing optical microscopy, X-ray diffraction, and atomic force microscopy techniques. Upon thermal annealing at 10 degrees C above its T(g) for 12-48 h, the 1:1 blend of OFTB and PCBM resulted in a eutectic mixture. Thermal annealing of a OFTB:Dyad:PCBM film with a 9:2:9 mass ratio at 10 degrees C above its T(g) for 12 h resulted in an amorphous film. Its AFM phase image indicated phase separation into two interspersed 30 nm amorphous domains at approximately equal fractions. Geometric surfactancy is inferred from the formation of microemulsions in analogy to widely reported traditional oil-surfactant-water systems and ternary polymer blends. In contrast, thermal annealing of a 7:6:7 film under a similar condition resulted in an amorphous film with compositional uniformity.

Phase 2 pilot study of Pathfinders: a psychosocial intervention for cancer patients.

PURPOSE: Pathfinders is a multi-faceted psychosocial care program for cancer patients; it was developed in community oncology and adapted to the academic oncology setting. This prospective, single-arm, phase 2 pilot study examined the acceptability and feasibility of Pathfinders for women with metastatic breast cancer. METHODS: Over 3 months, participants completed patient-reported surveys including the Patient Care Monitor (PCM, review of systems), Functional Assessment of Chronic Illness Therapy-Breast Cancer (FACT-B), Self Efficacy, and a single-item survey asking patients whether the program was helpful to them. A technology-based data collection system was used to capture electronic patient-reported outcomes at point of care, report symptoms in real time to clinicians, and collect warehouse data to provide a detailed longitudinal picture of the patient experience when receiving Pathfinders. RESULTS: Participants (n = 50) were: mean age 51 (SD 11); 76% white, 20% black; 74% married; 50% college degree. Forty-two (n = 42) patients completed baseline and 3-month assessments. Statistically significant improvements (all P < 0.05) occurred in PCM subscales for Distress (mean [SE] = -3.42 [1.21]), Despair (-4.53 [1.56]), and Quality of Life (2.88 [0.97]), and the FACT-B Emotional Wellbeing subscale (2.07 [0.46]). Of the 29 participants asked if Pathfinders was helpful, 27 (93%) responded positively and two did not respond. Other instruments measuring symptoms, quality of life, and self-efficacy showed improvement. CONCLUSIONS: In a phase 2 pilot study, Pathfinders was helpful to patients and is feasible in an academic medical center. Follow-up data collected at the 3-month assessment suggest that the program impacts various psychological outcomes, notably distress and despair.

Simulation of Circular Dichroism by Chromophores Coupled with Selective Reflection by Cholesteric Stacks.

The Good-Karali theory is extended to simulate composite circular dichroism (CD) through a cholesteric stack by incorporating chromophore's selective absorption (SA) and cholesteric stack's selective reflection (SR). Based on the independently evaluated anisotropic refractive indices and absorption coefficients, helical sense and pitch length, and film thickness, the theory is capable of describing transmission, reflection, and absorption through spin-cast cholesteric glassy liquid crystal films of nonafluorene. The resulting composite CD spectra agree quite well with experimental observations. The theory informs that SA plays a dominant role over SR in the composite CD. Specifically, the right-handed stack of the chromophore with its absorption dipole aligned with the local director in the cholesteric stack preferentially absorbs the left-handed over the right-handed circularly polarized light. The algebraic sign of the predicted composite CD flips by reversing the cholesteric host film's handedness without altering other parameter values. The established theory and computation constitute a solid foundation for optimizing circular polarizers by exploring the readily accessible parameter space targeting various potential applications.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

Earth tides, global heat flow, and tectonics.

The power of a heat engine ignited by tidal energy can account for geologically reasonable rates of average magma production and sea floor spreading. These rates control similarity of heat flux over continents and oceans because of an inverse relationship between respective depth intervals for mass transfer and consequent distributions of radiogenic heat production.

Development of the human Achilles tendon enthesis organ.

The attachment of the Achilles tendon is part of an 'enthesis organ' that reduces stress concentration at the hard-soft tissue interface. The organ also includes opposing sesamoid and periosteal fibrocartilages, a bursa and Kager's fat pad. In addition, the deep crural and plantar fasciae contribute to Achilles stress dissipation and could also be regarded as components. Here we describe the sequence in which these various tissues differentiate. Serial sections of feet from spontaneously aborted foetuses (crown rump lengths 22-322 mm) were examined. All slides formed part of an existing collection of histologically sectioned embryological material, obtained under Spanish law and housed in the Universidad Complutense, Madrid. From the earliest stages, it was evident that the Achilles tendon and plantar fascia had a mutual attachment to the calcaneal perichondrium. The first components of the enthesis organ to appear (in the 45-mm foetus) were the retrocalcaneal bursa and the crural fascia. The former developed by cavitation within the mesenchyme that later gave rise to Kager's fat pad. The tip of the putative fat pad protruded into the developing bursa in the 110-mm foetus and fully differentiated adipocytes were apparent in the 17-mm foetus. All three fibrocartilages were first recognisable in the 332-mm foetus--at which time adipogenesis had commenced in the heel fat pad. The sequence in which the various elements became apparent suggests that bursal formation and the appearance of the crural fascia may be necessary to facilitate the foot movements that subsequently lead to fibrocartilage differentiation. The later commencement of adipogenesis in the heel than in Kager's pad probably reflects the non-weight environment in utero. The direct continuity between plantar fascia and Achilles tendon that is characteristic of the adult reflects the initial attachment of both structures to the calcaneal perichondrium rather than to the skeletal anlagen itself.

Follow-up schedules after treatment for malignant melanoma.

BACKGROUND: Existing follow-up guidelines after treatment for melanoma are based largely on dated literature and historical precedent. This study aimed to calculate recurrence rates and establish prognostic factors for recurrence to help redesign a follow-up schedule. METHODS: Data were retrieved from the Sydney Melanoma Unit database for all patients with a single primary melanoma and American Joint Committee on Cancer (AJCC) stage I-II disease, who had received their first treatment between 1959 and 2002. Recurrence rates, timing and survival were recorded by substage, and predictive factors were analysed. RESULTS: Recurrence occurred in 18.9 per cent (895 of 4748) of patients overall, 5.2 per cent (95 of 1822) of those with stage IA disease, 18.4 per cent (264 of 1436) with IB, 28.7 per cent (215 of 750) with IIA, 40.6 per cent (213 of 524) with IIB and 44.3 per cent (86 of 194) with IIC disease. Overall, the median disease-free survival time was 2.6 years, but there were marked differences between AJCC subgroups. Primary tumour thickness, ulceration and tumour mitotic rate were important predictors of recurrence. CONCLUSION: A new follow-up schedule was proposed: stage I annually, stage IIA 6-monthly for 2 years and then annually, stage IIB-IIC 4-monthly for 2 years, 6-monthly in the third year and annually thereafter.

Update on the clinical utility of coronary computed tomographic angiography in stable angina pectoris.

Over the last decade, coronary computed tomographic angiography (CCTA) has emerged as a valuable non-invasive imaging modality with excellent diagnostic performance compared to invasive coronary angiography (ICA) for identifying patients with coronary artery disease (CAD). Beyond the diagnosis of CAD, CCTA also provides valuable prognostic information. While patients with normal CCTA have excellent long-term prognosis, among those with CAD, increasing CAD extent and severity is associated with increased cardiovascular event risk over both medium- and long-term follow-up in both men and women. The ability to image non-obstructive CAD is a particularly unique attribute of CCTA. Moreover, the ability to assess plaque features on CCTA has further enhanced our understanding of coronary plaque dynamics and the prediction of future cardiovascular events. The clinical impact of CCTA has been recently evaluated in two landmark prospective multicenter trials, which have provided insights into the influence of CCTA on the clinical management of symptomatic patients with suspected CAD. We review the value of CCTA in the evaluation of patients with stable chest pain including its diagnostic performance, prognostic utility and real-world clinical application.

Microscopic evaluation of the interface between glass-ionomer cements and tooth structures prepared using conventional instruments and the atraumatic restorative treatment (ART) technique.

OBJECTIVE: The purpose of this study was to evaluate the effect of cavity preparation using hand instruments and conventional rotary instruments on the bonding of glass-ionomer cements to formerly carious teeth. METHOD AND MATERIALS: In 2 experimental groups (12 teeth each with primary caries) caries was removed and cavities prepared using hand instruments according to the atraumatic restorative treatment (ART) technique or conventional rotary instruments. In the control group (12 caries-free teeth) Class 1 cavities were prepared using conventional instrumentation. Cavities in all teeth were restored with 1 of the commercial glass-ionomer cements designed for use with the ART technique, either Fuji IX (GC) or Ketac Molar (3M Espe). After 21 days of storage in physiologic saline at 37 degrees C, 3 400-microm-thick slices from each tooth were stained using the Mallory method and evaluated using a light transmitting microscope. RESULTS: In all samples, a region of interaction was observed between the cement and dentin and enamel. However, the interface in teeth from which caries had been removed was different from that in the control group. All were stained using Mallory staining, but only teeth which had been carious showed coloration. No differences were found in intensity of color or appearance between the cavity preparation techniques. CONCLUSION: The occurrence of caries in a tooth alters the bonding behavior of glass ionomers to that tooth. The method of caries removal (ART or conventional preparation) does not influence the quality of the interface between a glass ionomer and either dentin or enamel.

Mutation of the PTEN tumor suppressor gene in endometrial hyperplasias.

Mutation and deletion of the PTEN tumor suppressor gene occurs in about 40% of endometrial carcinomas. The purpose of this study was to determine whether PTEN mutations also are present in endometrial hyperplasias, which are premalignant precursors of invasive endometrial adenocarcinomas. Genomic DNA from 51 endometrial hyperplasias was extracted from paraffin blocks, and PCR was used to amplify the nine exons of the PTEN gene. These products were screened using single-strand conformation analysis, and variant bands were sequenced. Somatic mutations in the PTEN gene were seen in 10 of 51 cases (20%), and two mutations were found in one case. An identical 4-bp deletion in exon 8 was seen in three cases, and 8 of 11 PTEN mutations predicted truncated protein products. There was no higher frequency of PTEN mutations in endometrial hyperplasias with atypia (6 of 32; 19%) relative to those without atypia (4 of 19; 21%). These data suggest that inactivation of the PTEN tumor suppressor gene is an early event in the development of some endometrial cancers.

Ipilimumab and Bevacizumab in Glioblastoma.

The median survival in glioblastoma is just over a year, with no standard second-line therapy. Ipilimumab is an immune checkpoint inhibitor that activates the anti-tumour immune response by cytotoxic T-lymphocyte antigen-4 blockade. There is significant evidence supporting its role in the treatment of malignant melanoma, including in patients with brain metastases. The addition of the anti-angiogenesis agent, bevacizumab, seems to offer additional benefit and limit the immune-related side-effects of ipilimumab in melanoma. To date there have been no clinical trials investigating this combination in glioblastoma. In this single practice case series, 20 patients with glioblastoma were consented for and treated with ipilimumab and bevacizumab in combination. Safety, tolerability and the response to treatment were reviewed for all patients. Three patients were treated after palliative first-line radiotherapy, one patient after first-line chemoradiation and 16 patients were treated with recurrent disease. Sixty-five per cent of patients completed four cycles of 3 weekly ipilimumab therapy, administered with 2 weekly bevacizumab. Radiographic responses for patients with recurrent disease were evaluated by Response Assessment in Neuro-oncology (RANO) criteria; 31% of patients showed a partial response, 31% had stable disease and 38% had disease progression. The treatment combination was well tolerated, with treatment terminated before completion due to adverse events in two patients. Autoimmune toxicity was manageable with systemic corticosteroid therapy. Ipilimumab and bevacizumab in combination show promising activity with a predictable and manageable toxicity profile, warranting further clinical studies.

Long-term donepezil treatment in 565 patients with Alzheimer's disease (AD2000): randomised double-blind trial.

BACKGROUND: Cholinesterase inhibitors produce small improvements in cognitive and global assessments in Alzheimer's disease. We aimed to determine whether donepezil produces worthwhile improvements in disability, dependency, behavioural and psychological symptoms, carers' psychological wellbeing, or delay in institutionalisation. If so, which patients benefit, from what dose, and for how long? METHODS: 565 community-resident patients with mild to moderate Alzheimer's disease entered a 12-week run-in period in which they were randomly allocated donepezil (5 mg/day) or placebo. 486 who completed this period were rerandomised to either donepezil (5 or 10 mg/day) or placebo, with double-blind treatment continuing as long as judged appropriate. Primary endpoints were entry to institutional care and progression of disability, defined by loss of either two of four basic, or six of 11 instrumental, activities on the Bristol activities of daily living scale (BADLS). Outcome assessments were sought for all patients and analysed by logrank and multilevel models. FINDINGS: Cognition averaged 0.8 MMSE (mini-mental state examination) points better (95% CI 0.5-1.2; p<0.0001) and functionality 1.0 BADLS points better (0.5-1.6; p<0.0001) with donepezil over the first 2 years. No significant benefits were seen with donepezil compared with placebo in institutionalisation (42% vs 44% at 3 years; p=0.4) or progression of disability (58% vs 59% at 3 years; p=0.4). The relative risk of entering institutional care in the donepezil group compared with placebo was 0.97 (95% CI 0.72-1.30; p=0.8); the relative risk of progression of disability or entering institutional care was 0.96 (95% CI 0.74-1.24; p=0.7). Similarly, no significant differences were seen between donepezil and placebo in behavioural and psychological symptoms, carer psychopathology, formal care costs, unpaid caregiver time, adverse events or deaths, or between 5 mg and 10 mg donepezil. INTERPRETATION: Donepezil is not cost effective, with benefits below minimally relevant thresholds. More effective treatments than cholinesterase inhibitors are needed for Alzheimer's disease.

Developmental changes in luteinizing hormone/human chorionic gonadotropin steroidogenic responsiveness in marmoset granulosa cells: effects of follicle-stimulating hormone and androgens.

Factors regulating LH/hCG responsiveness in primate granulosa cells were examined in the marmoset monkey (Callithrix jacchus). Granulosa cells were isolated and pooled from small antral (0.5-1.0 mm) and large preovulatory (greater than or equal to 2 mm) follicles from mid- to late follicular phase ovaries of cyclic marmosets. The cells from small and large follicles were cultured in serum-free medium for 48 h in the absence or presence of increasing concentrations of hCG (0.1-100 ng/ml) with or without 0.1 microM androgen [testosterone or 5 alpha-dihydrotestosterone (DHT]). Granulosa cells from small follicles were also cultured in the absence or presence of a constant concentration of human FSH (30 ng/ml) with or without androgen for 48 h before exposure to hCG for an additional 48 h. Steroidogenic responsiveness was assessed by measuring progesterone accumulation in culture medium and aromatase activity in washed monolayers. Granulosa cells from large follicles showed dose-dependent increases in both progesterone accumulation and aromatase activity in response to treatment with hCG. In contrast, granulosa cells from small follicles were unresponsive to hCG. However, pretreatment of granulosa cells from small follicles for 48 h with FSH stimulated hCG responsiveness. The effects of both testosterone and DHT on hCG-stimulated aromatase activity and progesterone accumulation by granulosa cells from large preovulatory follicles were inhibitory. Testosterone and DHT also suppressed basal (no hCG) progesterone accumulation in these cells, but had no effect on basal aromatase activity. The effects of androgens on FSH-induced hCG responsiveness in immature granulosa cells were variable. The results show a development-related increase in marmoset granulosa cell responsiveness to LH/hCG and provide evidence that FSH and androgens interact to regulate the onset and expression of this critical event during preovulatory follicular development in the primate ovary.

Factors influencing follicle-stimulating hormone-responsive steroidogenesis in marmoset granulosa cells: effects of androgens and the stage of follicular maturity.

Preovulatory changes in the steroidogenic function of primate granulosa cells were studied using the cyclic marmoset (Callithrix jacchus) as a model. Antral follicles (greater than or equal to 0.5 mm diameter) were dissected from mid-late follicular phase ovaries (7 days after prostaglandin-induced luteolysis) and classified by diameter as small (0.5-1.0 mm), medium (1.1-1.9 mm) or large (greater than or equal to 2.0 mm). Granulosa cells from follicles in each size category were isolated and pooled to assess steroid biosynthesis. The aromatase activity of freshly isolated granulosa cells from large follicles was 200 times greater than that of small follicles, confirming their relatively advanced preovulatory status. Granulosa cells were cultured for 48 h in the presence and absence of human (h) FSH (0.1 ng/ml), with and without 0.1 microM androgen (testosterone or 5 alpha-dihydrotestosterone), to assess basal and hormone-responsive steroidogenesis (progesterone accumulation in culture medium and aromatase activity in washed granulosa cell monolayers). Basal granulosa cell steroidogenesis increased with follicular size, and there was a development-related pattern of response to hFSH and androgen. hFSH responsiveness (maximum fold-stimulation induced by hFSH) declined with follicular size, being 2-6 times greater for granulosa cells from small vs. large follicles. On the other hand, hFSH sensitivity increased with follicular size; the dose of hFSH giving 50% of the maximum response (ED50) for cells from large follicles being 10-20 times less than that of cells from small follicles. For granulosa cells from small follicles, treatment with 0.1 microM androgen in the presence of hFSH led to dramatic (up to 16-fold) enhancement of steroidogenic responses to hFSH. In contrast, for granulosa cells from large follicles, the presence of androgen substantially inhibited aromatase activity stimulated by hFSH and had weak inhibitory effects on progesterone accumulation. These results show that granulosa cell steroidogenesis becomes increasingly sensitive to hFSH during preovulatory follicular development in marmosets. The marked ability of androgen to directly augment hFSH-responsive steroidogenesis in vitro is lost during preovulatory development, such that androgen acts in mature granulosa cells to suppress hFSH-stimulated aromatase activity. These observations are evidence of development-dependent changes in granulosa cell responses to FSH and androgens which may contribute to the control of preovulatory follicular development in primates.