[Pedicled vascularized iliac bone graft for treatment of osteonecrosis of the femoral head]. / Gefäßgestieltes Beckenkammtransplantat zur Behandlung der Femurkopfnekrose.

OBJECTIVE: Illustration of a nowadays only rarely performed operative procedure for the treatment of osteonecrosis of the femoral head to prevent or at least delay advanced arthrosis and the need for a total hip replacement. The pedicled vascularized iliac bone graft is raised without the need for special microsurgical techniques and has less vascular complications often seen in free vascularized grafts. INDICATIONS: Early stages of osteonecrosis of the femoral head stages II and III according to the Association Research Circulation Osseous (ARCO) up to the detection of fracture lines (crescent sign) but without mechanical insufficiency. CONTRAINDICATIONS: Osteonecrosis of the femoral head with collapse of the femoral head (ARCO stage ≥IIIB) and mechanical insufficiency. Patients who are noncompliant or a not able to take the weight off the operated leg. Patients who had radiotherapy or an operation on ipsilateral inguinal lymph nodes and patients who have vascular anomalies or severe arteriosclerosis. SURGICAL TECHNIQUE: Debridement of the femoral head osteonecrosis and implantation of a pedicled vascularized iliac bone graft. POSTOPERATIVE MANAGEMENT: Free movement of the hip joint 4 weeks after surgery. Outward rotation of the hip joint allowed after 3 months and restriction of weight load on the operated leg for at least 3-6 months postoperatively depending on the bony consolidation. RESULTS: Vascularized bone grafts for the treatment of femoral head necrosis show better clinical and radiological results than avascular bone grafts. Nevertheless, after 5 years follow-up approximately 25% of the operated hips formerly in stage II show further progression of radiological necrosis. In stage III all hips eventually show progress of femoral head collapse and the need of a total hip replacement. Concerning the outcome of a free vascularized bone graft (fibula flap) compared to the pedicled vascularized graft from the iliac crest for treatment, the anatomically demanding area and a higher complication rate should be considered even though the cancellous bone of the iliac crest is biologically ideal. Nowadays a free vascularized fibular graft is the most frequently used bone graft for treatment of femoral head necrosis.

Is end-organ surveillance necessary in patients with well-functioning metal-on-metal hip resurfacings? A cardiac MRI survey.

AIMS: Cardiac magnetic resonance (CMR) was used to assess whether cardiac function or tissue composition was affected in patients with well-functioning metal-on-metal hip resurfacing arthroplasties (MoMHRA) when compared with a group of controls, and to assess if metal ion levels correlated with any of the functional or structural parameters studied. PATIENTS AND METHODS: In all, 30 participants with no significant cardiac history were enrolled: 20 patients with well-functioning MoMHRA at mean follow-up of 8.3 years post-procedure (ten unilateral, ten bilateral; 17 men, three women) and a case-matched control group of ten non-MoM total hip arthroplasty patients (six men, four women). The mean age of the whole cohort (study group and controls) at the time of surgery was 50.6 years (41.0 to 64.0). Serum levels of cobalt and chromium were measured, and all patients underwent CMR imaging, including cine, T2* measurements, T1 and T2 mapping, late gadolinium enhancement, and strain measurements. RESULTS: None of the MoMHRA patients showed clinically significant cardiac functional abnormality. The MoMHRA patients had larger indexed right and left end diastolic volumes (left ventricular (LV): 74 ml/m2vs 67 ml/m2, p = 0.045; right ventricular: 80 ml/m2vs 71 ml/m2, p = 0.02). There was a small decrease in T2 time in the MoMHRA patients (median 49 ms vs 54 ms; p = 0.0003). Higher metal ion levels were associated with larger LV volumes and with shorter T2 time. CONCLUSION: Although cardiac function is not clinically adversely affected in patients with well-functioning MoMHRA, modern imaging is able to demonstrate subtle changes in structure and function of the heart. As these changes correlate with systemic ion measurements, they may be consequences of wear debris deposition. Longer, longitudinal studies are necessary to determine whether cardiac function will become affected. Cite this article: Bone Joint J 2019;101-B:540-546.

Induction chemotherapy (IC) followed by radiotherapy (RT) versus cetuximab plus IC and RT in advanced laryngeal/hypopharyngeal cancer resectable only by total laryngectomy-final results of the larynx organ preservation trial DeLOS-II.

Background: The German multicenter randomized phase II larynx organ preservation (LOP) trial DeLOS-II was carried out to prove the hypothesis that cetuximab (E) added to induction chemotherapy (IC) and radiotherapy improves laryngectomy-free survival (LFS; survival with preserved larynx) in locally advanced laryngeal/hypopharyngeal cancer (LHSCC). Patients and methods: Treatment-naïve patients with stage III/IV LHSCC amenable to total laryngectomy (TL) were randomized to three cycles IC with TPF [docetaxel (T) and cisplatin (P) 75 mg/m2/day 1, 5-FU (F) 750 mg/m2/day days 1-5] followed by radiotherapy (69.6 Gy) without (A) or with (B) standard dose cetuximab for 16 weeks throughout IC and radiotherapy (TPFE). Response to first IC-cycle (IC-1) with ≥30% endoscopically estimated tumor surface shrinkage (ETSS) was used to define early responders; early salvage TL was recommended to non-responders. The primary objective was 24 months LFS above 35% in arm B. Results: Of 180 patients randomized (July 2007 to September 2012), 173 fulfilled eligibility criteria (A/B: larynx 44/42, hypopharynx 41/46). Because of 4 therapy-related deaths among the first 64 randomized patients, 5-FU was omitted from IC in the subsequent 112 patients reducing further fatal toxicities. Thus, IC was TPF in 61 patients and TP in 112 patients, respectively. The primary objective (24 months LFS above 35%) was equally met by arms A (40/85, 47.1%) as well as B (41/88, 46.6%). One hundred and twenty-three early responders completed IC+RT; their overall response rates (TPF/TP) were 94.7%/87.2% in A versus 80%/86.0% in B. The 24 months overall survival (OS) rates were 68.2% and 69.3%. Conclusions: Despite being accompanied by an elevated frequency in adverse events, the IC with TPF/TP plus cetuximab was feasible but showed no superiority to IC with TPF/TP regarding LFS and OS at 24 months. Both early response and 24 months LFS compare very well to previous LOP trials and recommend effective treatment selection and stratification by ETSS. Clinical trial information: NCT00508664.

[Traumatic lesions of the brachial plexus : Clinical symptoms, diagnostics and treatment]. / Traumatische Läsionen des Plexus brachialis : Klinische Symptome, Diagnostik und Therapie.

Brachial plexus lesions mostly occur in young patients as a result of high-speed accidents. They are often diagnosed and treated after a delay. This has been shown to worsen the prognosis of surgical reconstructions evidently. In 70-80% of traumatic lesions functional reinnervation can be achieved by various surgical procedures. An early sufficient diagnosis and the subsequent referral of the patient to an appropriate competence center for consultation and, if necessary, surgery are therefore essential.

Toxicologic/transport properties of NCS-382, a γ-hydroxybutyrate (GHB) receptor ligand, in neuronal and epithelial cells: Therapeutic implications for SSADH deficiency, a GABA metabolic disorder.

We report the in vitro assessment of pharmacotoxicity for the high-affinity GHB receptor ligand, NCS-382, using neuronal stem cells derived from mice with a targeted deletion of the aldehyde dehydrogenase 5a1 gene (succinic semialdehyde dehydrogenase(SSADH)-deficient mice). These animals represent a phenocopy of the human disorder of GABA metabolism, SSADH deficiency, that metabolically features accumulation of both GABA and the GABA-analog γ-hydroxybutyric acid in conjunction with a nonspecific neurological phenotype. We demonstrate for the first time using MDCK cells that NCS-382 is actively transported and capable of inhibiting GHB transport. Following these in vitro assays with in vivo studies in aldh5a1-/- mice, we found the ratio of brain/liver GHB to be unaffected by chronic NCS-382 administration (300mg/kg; 7 consecutive days). Employing a variety of cellular parameters (reactive oxygen and superoxide species, ATP production and decay, mitochondrial and lysosomal number, cellular viability and necrosis), we demonstrate that up to 1mM NCS-382 shows minimal evidence of pharmacotoxicity. As well, studies at the molecular level indicate that the effects of NCS-382 at 0.5mM are minimally toxic as evaluated using gene expression assay. The cumulative data provides increasing confidence that NCS-382 could eventually be considered in the therapeutic armament for heritable SSADH deficiency.

In vitro toxicological evaluation of NCS-382, a high-affinity antagonist of γ-hydroxybutyrate (GHB) binding.

γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.

Therapeutic relevance of mTOR inhibition in murine succinate semialdehyde dehydrogenase deficiency (SSADHD), a disorder of GABA metabolism.

Aldehyde dehydrogenase 5a1-deficient (aldh5a1-/-) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1-/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1-/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1-/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1-/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.

Relationship between estimated glomerular filtration rate and biological half-life of 131I. Retrospective analysis in patients with differentiated thyroid carcinoma.

AIM: This retrospective study sought to investigate the relationship between biological half-life (t1/2 biol) of 131I and estimated glomerular filtration rate (eGFR) in patients with thyroid carcinoma. PATIENTS, METHODS: 96 patients with differentiated thyroid carcinoma (69 women, 27 men, mean age 64.0 ± 13.6 years) and diagnostic and therapeutic administration of 131I were considered. Patients with pronounced specific iodine storage were not included in the study. The eGFR was estimated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula, the t1/2 biol via dosimetry. Patients were subdivided in groups with normal clearance (NC) (n = 37, 38.5%), medium clearance (MC) (n = 48, 50.0%), and low clearance (LC) (n = 11, 11.5%) (eGFR ≥ 90; 60-89; 15-59 ml/min per 1.73 m2, respectively). The relationship between eGFR and t1/2 biol of 131I was modeled using a power function. RESULTS: The groups significantly differed in terms of age (NC 53.8, MC 68.6, and 78.0 years, respectively), serum creatinine levels (NC: 0.71; MC: 0.85; LC: 1.18 mg/dl), and t1/2 biol (NC: 0.53; MC: 0.71; LC: 1.01 days). The t1/2 biol was significantly influenced only by eGFR, and not by age, gender, or body weight. The relationship between t1/2 biol of 131I and eGFR was described by the formula t1/2 biol = 20.3 · eGFR-0.782. CONCLUSIONS: The calculated relationship between renal function and t1/2 biol of 131I can be used in principle to estimate a dose reduction for patients with renal insufficiency. The model, however, gives erroneous results in individual cases and therefore a routine utilization cannot be recommended. Prospective studies are necessary, based on larger patient numbers and more accurate methods for dose rate measurement and GFR.

Myelodysplasia in 2 pig-tailed macaques (Macaca nemestrina) associated with retroviral vector-mediated insertional mutagenesis and overexpression of HOXB4.

Gammaretroviral vectors are an efficient means to effect gene therapy. However, genotoxicity from insertion at nonrandom sites can confer a competitive advantage to transduced cells, resulting in clonal proliferation or neoplasia. Six pig-tailed macaques (Macaca nemestrina) underwent total body irradiation and reconstitution with autologous stem cells genetically modified by a gammaretroviral vector overexpressing HOXB4. Two animals were euthanized owing to irradiation- or transplantation-associated toxicity, whereas the other 4 had successful reconstitution. Of the 4 macaques with successful reconstitution, 1 has no long-term follow-up information; 1 was euthanized owing to infection with simian varicella virus infection 18 months post-total body irradiation; and the 2 others are described herein as case Nos. 1 and 2. After being stable for 3 years, case No. 1 developed pancytopenia and petechiation, and after 2 years of stability case No. 2 developed anemia and thrombocytopenia. Despite therapy, the animals deteriorated and were euthanized. Gross findings included emaciation; case No. 1 also had hemorrhage, peritonitis, and cholecystitis. Histologically, bone marrow was hypercellular with predominately blast cells of all hematopoietic lineages, though with myeloid predominance, and with maturation arrest and blast cell dysplasia (myelodysplasia). Myelodysplasia was likely from a combination of insertional mutagenesis by the retroviral vector and overexpression of HOXB4. Consequences of myelodysplasia included the blood dyscrasias and, in case No. 1, hemorrhage, bacterial cholecystitis, hepatitis, and peritonitis.

In vitro gas production as a surrogate measure of the fermentability of cellulosic biomass to ethanol.

Current methods for measuring ethanol yields from lignocellulosic biomass are relatively slow and are not well geared for analyzing large numbers of samples generated by feedstock management and breeding research. The objective of this study was to determine if an in vitro ruminal fermentation assay used in forage quality research was predictive of results obtained using a conventional biomass-to-ethanol conversion assay. In the conventional assay, herbaceous biomass samples were converted to ethanol by Saccharomyces cerevisiae cultures in the presence of cellulase enzymes. Cultures were grown in sealed serum bottles and gas production monitored by measuring increasing head space pressure. Gas accumulation as calculated from the pressure measurements was highly correlated (r(2)>0.9) with ethanol production measured by gas chromatography at 24 h or 7 days. The same feedstocks were also analyzed by in vitro ruminal digestion, as also measured by gas accumulation. Good correlations (r(2) approximately 0.63-0.82) were observed between ethanol production during simultaneous saccharification and fermentation and gas accumulation in parallel in vitro ruminal fermentations. Because the in vitro ruminal fermentation assay can be performed without sterilization of the medium and does not require aseptic conditions, this assay may be useful for biomass feedstock agronomic and breeding research.

Tumor vascularization in experimental melanomas: correlation between unenhanced and contrast enhanced power Doppler imaging and histological grading.

The purpose of this study was to evaluate the reliability of unenhanced and enhanced power Doppler sonography in visualization of intratumoral angioneogenesis. Thirty-seven malignant melanomas, which had been implanted intra- or subcutaneously in 22 mice, were examined. Various B-mode criteria, power Doppler criteria and spectral Doppler parameters were evaluated before and after IV-application of the d-galactose-based contrast agent Levovist. After sonographic examination, all tumors were analyzed histologically with semiquantitative grading of tumoral vascularization. Unenhanced, in 70% of the tumors, no intratumoral vessels were visible using power Doppler, but only in 11% of the intracutaneous and in 0% of the subcutaneous after injection of the contrast agent. The enhanced mode was definitely superior to unenhanced Doppler in showing the intratumoral vascularity. The intratumoral vascular structure could be sufficiently analyzed in 30% of all tumors by unenhanced Doppler, but in 92% enhanced. The mean percentage vessel area increased about 433% after application of Levovist (intracutaneous: 485%, subcutaneous: 280%). Despite the missing direct correlation between the sonographically and histologically determined grade of tumor vascularization (Pearson's correlation unenhanced 0,356, p <.05/enhanced 0.395, p <.05), the correlation between the percentage vessel area and the histologic grade of vascularization was improved after application of the contrast agent (Pearson's correlation unenhanced 0.347, p <.05/enhanced 0.686, p <.01). We did not find a significant direct correlation between histologically and sonographically determined degree of vascularization. However, the correlation was improved using a d-galactose-based signal-enhancing agent in power Doppler sonography.

Neurofibromin negatively regulates neurotrophin signaling through p21ras in embryonic sensory neurons.

Embryonic sensory and sympathetic neurons that lack neurofibromin, the protein product of the neurofibromatosis type 1 (Nfl) gene, survive and extend neurites in the absence of neurotrophins. To determine whether neurofibromin negatively regulates neurotrophin signaling through its interaction with p21ras, we used Fab antibody fragments to block Ras function in DRG, trigeminal, nodose, and SCG neurons isolated from Nfl(-/-) and wild-type mouse embryos. We show that introduction of anti-Ras Fab fragments significantly reduces the ability of neurofibromin-deficient neurons to survive in the absence of neurotrophins. Moreover, addition of H-ras protein enhances the survival of Nfl(-/-), but not wild-type, DRG neurons. Our results are consistent with a major role for neurofibromin in modulating Trk signaling through p21ras during neuronal development.

An in vivo model for load-modulated remodeling in the rabbit flexor tendon.

This study tested the hypothesis that eliminating in vivo compression to the wrap-around, fibrocartilage-rich zone of the flexor digitorum profundus tendon results in rapid depletion of fibrocartilage and changes in its mechanical properties, microstructure, extracellular matrix composition, and cellularity. The right flexor digitorum profundus tendons of 2.5-3-year-old rabbits were translocated anteriorly to eliminate in vivo compression and shear to the fibrocartilage zone and, at 4 weeks after surgery, were compared with tendons that had sham surgery and with untreated tendons. The translocated tissue showed a significant increase in equilibrium strain under a compressive creep load (p < 0.05). The thickness and area of the fibrocartilage zone also decreased significantly (p < 0.05). The nuclear density decreased by 40% in the fibrocartilage zone (p < 0.005); however, nuclear shape and orientation were not significantly altered. Glycosaminoglycan content in the fibrocartilage zone was also depleted by 40% (p < 0.02). The tightly woven basket weave-like mesh of collagen fibers in the zone appeared more loosely organized, suggesting matrix reorganization due to translocation. Moreover, immunoreactive type-II collagen and link protein in the fibrocartilage zone also decreased. With use of this unique in vivo model, this research clearly elucidates how changing tissue function (by removing compressive forces) rapidly alters tissue form.

Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin.

The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.

SPIO-MR imaging versus double-phase spiral CT in detecting malignant lesions of the liver.

PURPOSE: To assess the diagnostic performance of superparamagnetic iron oxide MR (SPIO-MR) imaging compared with double-phase spiral CT in detecting liver metastases and hepatocellular carcinoma. MATERIAL AND METHODS: Thirty-eight patients with a total of 144 malignant lesions of the liver were examined by CT and SPIO-MR. After definition of a gold standard by a panel of experts, the patient images were randomized and presented to a blinded jury of 5 independent observers whose task was to identify as many lesions as possible. The results were tested for statistical significance using multifactorial analysis of variance (alpha=5%). RESULTS: SPIO-MR produced the highest detection rate and was significantly superior (p<0.05) to unenhanced MR imaging and double spiral-phase contrast-enhanced CT (DPS-CECT). Maximum performance in DPS-CECT was obtained during the portal venous contrast phase but was significantly inferior to SPIO-MR imaging. The scores for unenhanced CT and unenhanced MR were significantly lower than for the corresponding enhanced procedures. SPIO-MR imaging produced a higher incidence of false-positive findings (n=39). CONCLUSION: SPIO-MR produced a significantly better detection rate for malignant focal liver lesions compared with double-phase spiral DPS-CECT but was associated with a higher rate of false-positive findings.

Mouse tumor model for neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by increased incidence of benign and malignant tumors of neural crest origin. Mutations that activate the protooncogene ras, such as loss of Nf1, cooperate with inactivating mutations at the p53 tumor suppressor gene during malignant transformation. One hundred percent of mice harboring null Nf1 and p53 alleles in cis synergize to develop soft tissue sarcomas between 3 and 7 months of age. These sarcomas exhibit loss of heterozygosity at both gene loci and express phenotypic traits characteristic of neural crest derivatives and human NF1 malignancies.

SNAP-23 and SNAP-25 are palmitoylated in vivo.

The neuronal presynaptic membrane t-SNARE complex consists of the transmembrane protein syntaxin with the palmitoylated protein SNAP-25. In non-neuronal tissues, SNAP-23 replaces SNAP-25 in the t-SNARE complex, although the mechanism of membrane anchoring of SNAP-23 has not been determined. We now report that like SNAP-25, SNAP-23 is palmitoylated in vivo on one or more cysteine residues present in a central "palmitoylation domain." Interestingly, SNAP-23 is palmitoylated less well than SNAP-25, and in vivo binding studies indicate a correlation between the extent of palmitoylation and the ability of SNAP-23 or SNAP-25 to bind to syntaxin in vivo.

Leptin antagonizes the insulin-like growth factor-I augmentation of steroidogenesis in granulosa and theca cells of the human ovary.

There is increasing evidence that leptin is a physiological link between obesity and infertility. Although leptin receptors have been demonstrated in human ovaries, there is no information regarding the effects of leptin on cells from developing ovarian follicles. To test the direct effects of leptin on human ovarian cells, granulosa cells (GC) and theca cells were isolated from the ovaries of regularly cycling women. Serum was obtained at the time of surgery, and follicular fluid was aspirated from the follicles before isolation of the ovarian cells. Leptin concentrations were similar in follicular fluid and serum. RT-PCR analysis demonstrated that the long, signaling form of the leptin receptor was expressed in both theca and GC. In cultured GC, leptin had no effect on estradiol production, alone or in the presence of FSH, but caused a concentration-related inhibition of the insulin-like growth factor I (IGF-I) augmentation of FSH-stimulated estradiol production. The effect of leptin was specific, because there was no effect on progesterone production. In cultured theca cells, leptin did not alter androstenedione production, alone or in the presence of LH. Leptin caused a concentration-related inhibition of the IGF-I augmentation of LH-stimulated androstenedione production. These data demonstrate that leptin can directly inhibit IGF-I action in ovarian theca and GC at concentrations commonly present in obese women.

Oral immunization with enterocoated microbeads induces antigen-specific cytolytic T-cell responses.

A cytolytic T-lymphocyte (CTL) response has been generated to a protein antigen via oral administration in an experimental murine system. This was achieved by the formulation of the test antigen ovalbumin (OVA) in a pH-sensitive microsphere particle (microbead) with an acrylic-based coating to avoid its degradation by gastric enzymes. Comparative studies of orally administered enterocoated microbead-OVA particles, versus the more traditional formulation of OVA in adjuvant (DETOX-PC) given subcutaneously as immunogen, were conducted; both elicited comparable responses in the generation of antigen-specific lymphoproliferative and CTL responses. CTL lines generated via oral administration of antigen were shown to be CD3(+), CD4(-), and CD8(+). CTL lysis of OVA peptide-pulsed targets was shown to be inhibited by anti-CD8 antibody. Whereas oral administration of pH-sensitive enterocoated microbeads containing proteins has previously been shown to elicit antibody and lymphoproliferative T-cell responses to protein antigens, this is the first demonstration of the generation of an antigen-specific cytolytic T-cell response via oral administration of a protein immunogen formulated in such microbeads.

Sympathetic neuron survival and proliferation are prolonged by loss of p53 and neurofibromin.

The proteins encoded by the p53 and Nf1 tumor suppressor genes are involved in cell signaling and regulation of proliferation during normal development and differentiation, as well as during tumor progression. To characterize the roles of these genes in the proliferation and survival of embryonic neurons, we have used dissociated cultures of sympathetic superior cervical ganglia (SCG) isolated from p53 and Nf1 single and compound-mutant mouse embryos. We have defined a temporal window for p53 involvement in sympathetic neuron survival and proliferation. Moreover, our results indicate that cooperativity between mutations in Nf1 and p53 prolongs SCG neuron proliferation and increases the incidence of neural tube defects in compound-mutant embryos.