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Objective: Education in microsurgery of the ear includes staged training to allow for mastering of the complex microsurgical procedures, particularly in the context of middle ear reconstruction and cochlear implantation. Traditional surgical training includes temporal bone preparations by cadaver dissection and supervised operating room practice. As these on-site trainings are limited, there is a need to broaden education facilities in an on-line format. Therefore, a first basic on-line training for otosurgery was developed., Materials and Methods: The system consists of an artificial temporal bone model together with a set of basic surgical instruments and implant dummies. As an essential part of the training kit, a high-resolution camera set is included that allows for connection to a video streaming platform and enables remote supervision of the trainees' surgical steps by experienced otological surgeons. In addition, a pre-learning platform covering temporal bone anatomy and instrumentation and pre-recorded lectures and instructional videos has been developed to allow trainees to review and reinforce their understanding before hands-on practice., Results: Over the three courses held to date, 28 participants with varying levels of prior surgical experience took part in this otological surgical training program. The immediate feedback of the participants was evaluated by means of a questionnaire. On this basis, the high value of the program became apparent and specific areas could by identified where further refinements could lead to an even more robust training experience., Conclusion: The presented program of an otosurgical online training allows for basal education in practical exercises on a remote system. In this way, trainees who have no direct access to on-site instruction facilities in ear surgery now have the chance to start their otosurgical training in an educational setting adapted to modern technologies., (© 2024. The Author(s).)

Objective: Education in microsurgery of the ear includes staged training to allow for mastering of the complex microsurgical procedures, particularly in the context of middle ear reconstruction and cochlear implantation. Traditional surgical training includes temporal bone preparations by cadaver dissection and supervised operating room practice. As these on-site trainings are limited, there is a need to broaden education facilities in an on-line format. Therefore, a first basic on-line training for otosurgery was developed., Materials and Methods: The system consists of an artificial temporal bone model together with a set of basic surgical instruments and implant dummies. As an essential part of the training kit, a high-resolution camera set is included that allows for connection to a video streaming platform and enables remote supervision of the trainees' surgical steps by experienced otological surgeons. In addition, a pre-learning platform covering temporal bone anatomy and instrumentation and pre-recorded lectures and instructional videos has been developed to allow trainees to review and reinforce their understanding before hands-on practice., Results: Over the three courses held to date, 28 participants with varying levels of prior surgical experience took part in this otological surgical training program. The immediate feedback of the participants was evaluated by means of a questionnaire. On this basis, the high value of the program became apparent and specific areas could by identified where further refinements could lead to an even more robust training experience., Conclusion: The presented program of an otosurgical online training allows for basal education in practical exercises on a remote system. In this way, trainees who have no direct access to on-site instruction facilities in ear surgery now have the chance to start their otosurgical training in an educational setting adapted to modern technologies., (© 2024. The Author(s).)

Although the regulatory influence of leptin on energy balance, glycemic control, immunity, reproduction, and cognition is well established, its clinical application to common obesity and its co-morbidities has been limited by impaired transport across the blood-brain barrier, and tendencies to induce adverse side effects. To circumvent these drawbacks, MA-[D-Leu-4]-OB3, a leptin-related synthetic peptide that mimics the metabolic and neurotrophic effects of leptin in mouse models of genetic and non-genetic obesity, diabetes, and cognitive dysfunction, has been developed. This report presents the results of our initial efforts to assess the safety of orally delivered MA-[D-Leu-4]-OB3. Two pre-clinical studies were done in male and female C57BL/6 mice: a short-term study with a high dose of MA-[D-Leu-4]-OB3 (50 mg/kg/100 μL/day) and a dose-response study with 3 increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/100 μL/day). Body weight, food and water intake, glucose tolerance, and episodic memory were evaluated. Once-daily cage-side clinical observations were conducted to detect any physical or behavioral indicators of toxicity. Our results indicate that all metabolic and neurologic endpoints tested were either unaffected or improved by MA-[D-Leu-4]-OB3, and no clinical indicators of toxicity were evident. Together with our previously reported efficacy data, these results provide additional evidence supporting further development of this novel synthetic peptide leptin mimetic as a first-in-class peptide drug candidate for the treatment of a number of metabolic and/or cognitive dysfunctions in humans., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Positioning systems are used in a wide range of applications which require determining the position of an object in space, such as locating and tracking assets, people and goods; assisting navigation systems; and mapping. Indoor Positioning Systems (IPSs) are used where satellite and other outdoor positioning technologies lack precision or fail. Ultra-WideBand (UWB) technology is especially suitable for an IPS, as it operates under high data transfer rates over short distances and at low power densities, although signals tend to be disrupted by various objects. This paper presents a comprehensive study of the precision , failure , and accuracy of 2D IPSs based on UWB technology and a pseudo-range multilateration algorithm using Time Difference of Arrival (TDoA) signals. As a case study, the positioning of a 4×4m2 area, four anchors (transceivers), and one tag (receiver) are considered using bitcraze's Loco Positioning System. A Cramér-Rao Lower Bound analysis identifies the convex hull of the anchors as the region with highest precision , taking into account the anisotropic radiation pattern of the anchors' antennas as opposed to ideal signal distributions, while bifurcation envelopes containing the anchors are defined to bound the regions in which the IPS is predicted to fail . This allows the formulation of a so-called flyable area , defined as the intersection between the convex hull and the region outside the bifurcation envelopes. Finally, the static bias is measured after applying a built-in Extended Kalman Filter (EKF) and mapped using a Radial Basis Function Network (RBFN). A debiasing filter is then developed to improve the accuracy . Findings and developments are experimentally validated, with the IPS observed to fail near the anchors, precision around ±3cm, and accuracy improved by about 15cm for static and 5cm for dynamic measurements, on average.

In this review, the current understanding of leptin's role in energy balance, glycemic regulation, and cognitive function is examined, and its involvement in maintaining the homeostatic "harmony" of these physiologies is explored. The effects of exercise on circulating leptin levels are summarized, and the results of clinical application of leptin to metabolic disease and neurologic dysfunction are reviewed. Finally, pre-clinical evidence is presented which suggests that synthetic peptide leptin mimetics may be useful in resolving not only the leptin resistance associated with common obesity and other elements of metabolic syndrome, but also the peripheral insulin resistance characterizing type 2 diabetes mellitus, and the central insulin resistance associated with certain neurologic deficits in humans., Competing Interests: PG was an unpaid scientific advisor to DIODEM Therapeutics, Inc., Albany, NY, United States, (Copyright © 2022 Grasso.)

Background: Extracellular beta-amyloid (Aβ), intra-neuronal hyper-phosphorylated tau protein, and chronic inflammation are neuropathological hallmarks of Alzheimer's Disease (AD). A link between AD, insulin dysfunction, and tumor necrosis factor-alpha (TNF-α) in promoting both tau and Aβ pathologies in vivo has been proposed., Methods: MA-[D-Leu-4]-OB3 was given, with or without insulin, to streptozotocin (STZ)-treated male Swiss Webster mice, and to male diet-induced obese (DIO) mice. Brains were excised, and coronal sections were imaged with fluoro jade-C (FJC), thioflavin-S, or hematoxylin and eosin (H&E). Serum TNF-α and IGF-1 were measured by ELISA. Histopathological changes in the cerebral cortex (CC) and hippocampus (HC) were correlated with changes in glycemic regulation, episodic memory, and serum levels of TNF-α and IGF-1., Results: In STZ-treated mice, blood glucose and serum TNF-α and IGF-1 were reduced by insulin alone, and normalized when MA-[D-Leu-4]-OB3 was given in combination with insulin. Improvement in episodic memory was inversely correlated with the number of FJC-positive cells in the CC and HC and serum TNF-α and IGF-1. FJC, thioflavin-S and H&E staining indicated no Aβ deposition. Similar results were observed in DIO mice treated with MA-[D-Leu-4]-OB3., Conclusions: The mechanism by which MA-[D-Leu-4]-OB3 improves episodic memory in mouse models of TIDM and T2DM appears to be related to improved insulin sensitivity and reduced TNF-α-induced neurodegeneration., General Significance: MA-[D-Leu-4]-OB3 may have application to human pre-clinical and clinical AD and AD-like dementia by interrupting the cascade of insulin resistance, neuro-inflammation, and neurodegeneration, that characterizes these diseases., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the exponential increase of studies on plastic debris in recent years, there are still few works focusing on the problem as it relates to inland waters: little is known about the accumulation and dispersion dynamics on lake shores, and there are no standardized sampling methods for monitoring purposes. The accumulation of plastic litter in natural habitats also threatens the resident organisms. In this paper, we investigated the abundance and accumulation of plastic particles, ranging in size from 1 to 50 mm, from the beach sediment of three volcanic lakes in Central Italy: Albano, Bracciano, and Vico. The collection was designed to define the most important variables that one must consider in order to obtain a representative sample of plastic litter in a lake environment. In view of the high heterogeneity of sampling protocols used, comparison among the obtained results is limited and sometimes impossible. By using one of the proposed sampling methodologies, and critically analyzing the results, we aimed to highlight a possible monitoring criterion and to identify specific elements that can be meaningful and representative. The samples were collected in May and September 2017. For each lake, we sampled plastic items and sediments from two beaches. Albano contained the largest amount of plastic (in weight), while Bracciano had the largest number of particles. Our observations lead us to infer that the number of particles is the parameter most sensitive to environmental variations, as well as the more suitable for monitoring with greater definition the differences between sites. Moreover, sampling should be taken in different seasons, following a sampling pattern that includes at least two beaches placed in strategic positions with respect to wind and waves.In order to identify new indicators to evaluate the entry points of plastic into the food web, we collected, from the same sites analyzed, some specimens of the Talitrid Amphipod Cryptorchestia garbinii, a detritivorous species having a critical role in debris turnover of these environments. To investigate the microplastic (MP) ingestion in natural conditions, we analyzed their digestive tracts with both Nile red staining method and micro-FTIR spectroscopy. The analyses confirmed that C. garbinii was able to ingest plastics in natural conditions. Therefore, it can signify one of the entry points for microplastics (MPs) in the trophic chain. This observation constitutes the first evidence of MP ingestion in this species.

The obesity-regulated gene, leptin, is essential for diet. Leptin resistance causes obesity and related diseases. Certain types of diet are able to decrease leptin resistance. However, leptin has been shown to be correlated with inflammation and stimulate proliferation of various cancers. Two synthetic leptin derivatives (mimetics), OB3 and [D-Leu-4]-OB3, show more effective than leptin in reducing obesity and diabetes in mouse models. OB3 inhibits leptin-induced proliferation in ovarian cancer cells. However, effects of these mimetics in hepatocellular carcinoma (HCC) have not been investigated. In the present study, we examined the effects of OB3 and [D-Leu-4]-OB3 on cell proliferation and gene expressions in human HCC cell cultures. In contrast to what was reported for leptin, OB3 and [D-Leu-4]-OB3 reduced cell proliferation in hepatomas. Both OB3 and [D-Leu-4]-OB3 stimulated expression of pro-apoptotic genes. Both compounds also inhibited expressions of pro-inflammatory, proliferative and metastatic genes and PD-L1 expression. In combination with leptin, OB3 inhibited leptin-induced cell proliferation and expressions of pro-inflammation-, and proliferation-related genes. Furthermore, the OB3 peptide inhibited phosphoinositide 3-kinase (PI3K) activation which is essential for leptin-induced proliferation in HCC. These results indicate that OB3 and [D-Leu-4]-OB3 may have the potential to reduce leptin-related inflammation and proliferation in HCC cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We have previously shown that following oral delivery in dodecyl maltoside (DDM), [D-Leu-4]-OB3 and its myristic acid conjugate, MA-[D-Leu-4]-OB3, improved energy balance and glucose homeostasis in genetically obese/diabetic mouse models. More recently, we have provided immunohistochemical evidence indicating that these synthetic peptide leptin mimetics cross the blood-brain barrier and concentrate in the area of the arcuate nucleus of the hypothalamus in normal C57BL/6J and Swiss Webster mice, in genetically obese ob/ob mice, and in diet-induced obese (DIO) mice. In the present study, we describe the effects of oral delivery of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control in diet-induced (DIO) mice, a non-genetic rodent model of obesity and its associated insulin resistance, which more closely recapitulates common obesity and diabetes in humans. Male C57BL/6J and DIO mice, 17, 20, and 28 weeks of age, were maintained on a low-fat or high-fat diet and given vehicle (DDM) alone or [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in DDM by oral gavage for 12 or 14 days. Body weight gain, food and water intake, fasting blood glucose, oral glucose tolerance, and serum insulin levels were measured. Our data indicate that (1) [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 restore glucose tolerance in male DIO mice maintained on a high-fat diet to levels comparable to those of non-obese C57BL/6J wild-type mice of the same age and sex maintained on a low-fat diet; and (2) the influence of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on glycemic control appears to be independent of their effects on energy balance. These results suggest that [D-Leu-4]-OB3 and/or MA-[D-Leu-4]-OB3 may have application to the management of the majority of cases of common obesity in humans, a state characterized at least in part, by leptin resistance resulting from a defect in leptin transport across the blood-brain barrier. They further suggest that these small molecule synthetic peptide leptin mimetics, through their influence on glycemic control, may prevent the pre-diabetic state associated with most cases of common obesity from escalating into overt type 2 diabetes mellitus., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study describes the localization of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics, in the hypothalamus of Swiss Webster and C57BL/6J wild-type mice, leptin-deficient ob/ob mice, and leptin-resistant diet-induced obese (DIO) mice. The mice were given [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in 0.3% dodecyl maltoside by oral gavage. Once peak serum concentrations were reached, the mice received a lethal dose of pentobarbital and were subjected to intracardiac perfusion fixation. The brains were excised, post-fixed in paraformaldehyde, and cryo-protected in sucrose. Free-floating frozen coronal sections were cut at 25-µm and processed for imaging by immunofluorescence microscopy. In all four strains of mice, dense staining was concentrated in the area of the median eminence, at the base and/or along the inner wall of the third ventricle, and in the brain parenchyma at the level of the arcuate nucleus. These results indicate that [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 cross the blood-brain barrier and concentrate in an area of the hypothalamus known to regulate energy balance and glucose homeostasis. Most noteworthy is the localization of [D-Leu-4]-OB3 immunoreactivity within the hypothalamus of DIO mice via a conduit that is closed to leptin in this rodent model, and in most cases of human obesity. Together with our previous studies describing the effects of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on energy balance, glucose regulation, and signal transduction pathway activation, these findings are consistent with a central mechanism of action for these synthetic peptide leptin mimetics, and suggest their potential usefulness in the management of leptin-resistant obesity and type 2 diabetes in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Obesity results in increased secretion of cytokines from adipose tissue and is a risk factor for various cancers. Leptin is largely produced by adipose tissue and cancer cells. It induces cell proliferation and may serve to induce various cancers. OB3-leptin peptide (OB3) is a new class of functional leptin peptide. However, its mitogenic effect has not been determined. In the present study, because of a close link between leptin and the hypothalamic-pituitary-thyroid axis, OB3 was compared with leptin in different thyroid cancer cells for gene expression, proliferation and invasion. Neither agent stimulated cell proliferation. Leptin stimulated cell invasion, but reduced adhesion in anaplastic thyroid cancer cells. Activated ERK1/2 and STAT3 contributed to leptin-induced invasion. In contrast, OB3 did not affect expression of genes involved in proliferation and invasion. In vivo studies in the mouse showed that leptin, but not OB3, significantly increased circulating levels of thyrotropin (TSH), a growth factor for thyroid cancer. In summary, OB3 is a derivative of leptin that importantly lacks the mitogenic effects of leptin on thyroid cancer cells., Competing Interests: The authors declare that they have no conflicts of interest.

We have previously described the pharmacokinetics of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, following delivery by subcutaneous (SC), intraperitoneal (IP), and intramuscular (IM) injection, and by oral gavage and intranasal instillation. These profiles suggested that the observed efficacy of [D-Leu-4]-OB3 on energy balance, glycemic control, and bone turnover in ob/ob and db/db mice might be improved by efforts directed toward improving its bioavailability, i.e., increasing maximum uptake (Cmax), extending serum half-life (t½), and reducing plasma clearance (CL). To address these issues, myristic (tetradecanoic) acid was conjugated to the N-terminal of [D-Leu-4]-OB3 (designated MA-[D-Leu-4]-OB3), and the pharmacokinetics of MA-[D-Leu-4]-OB3 in male Swiss Webster mice following SC, IP, and IM injection in PBS, and by oral and intranasal delivery in dodecyl maltoside (DDM, trade name Intravail®), a transmucosal absorption enhancing agent, were compared to those of [D-Leu-4]-OB. At a dose of MA-[D-Leu-4]-OB3 10-fold lower than that used previously for [D-Leu-4]-OB3 (0.1 mg vs.1.0 mg, respectively), Cmax of MA-[D-Leu-4]-OB3 was 11.1-, 7.5-, 1.9-, and 1.7-fold higher, t1/2 was 3.5-, 5.0-, 9.1-, and 86.7-fold longer, and CL was 17.0-, 11.6-, 5.7-, and 5.0-fold slower than [D-Leu-4]-OB3 following SC, IP, IM, and oral delivery, respectively. Furthermore, in leptin-resistant obese male db/db mice, oral delivery of MA-[D-Leu-4]-OB3 in DDM at concentrations up to 10-fold lower than those used with [D-Leu-4]-OB3 reduced fasting blood glucose levels in a dose-related manner.

The effects of leptin-related synthetic peptides [d-Leu-4]-OB3 and OB3 on energy balance and glucose homeostasis in ob/ob and db/db mice have been confirmed. The molecular basis of these effects, however, remains unclear. In the present study, we examined the ability of these peptides to activate signal transduction pathways known to be involved in transduction of the leptin signal. In a specific and concentration-dependent manner, [d-Leu-4]-OB3 induced phosphorylation of ERK1/2, PI-3K, Ser-727 STAT3, and Tyr-705 of STAT3. OB3 also induced activation of STAT3 via phosphorylation of ERK1/2, STAT3 Ser-727, STAT3 Tyr-705 and PI-3K p85, but to a lesser degree. Using PD98059 and LY294002, specific inhibitors of MEK and PI-3K, respectively, we were able to identify the signal transduction pathways involved in peptide-induced STAT3 activation. [d-Leu-4]-OB3 induced serine phosphorylation of STAT3 primarily through activation of ERK1/2. Tyrosine phosphorylation of STAT3, however, was induced primarily through activation of PI-3K. Our data suggest that in db/db mice, [d-Leu-4]-OB3 binding to short isoforms of the leptin receptor induces intracellular signaling cascades which do not require OB-Rb activation. These signals may ultimately result in peptide effects on transcriptional and translational events associated with energy balance and glycemic regulation. In summary, we have shown for the first time that, similar to leptin, bioactive leptin-related synthetic peptide analogs activate STAT3 via phosphorylation of serine and tyrosine residues by multiple signal transduction pathways., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The effects of oral delivery of exenatide or pramlintide acetate in dodecyl maltoside (DDM) on energy balance and glycemic control in insulin-resistant obese db/db mice are enhanced when given in combination with [D-Leu-4]-OB3. To examine the anti-hyperglycemic influence of [D-Leu-4]-OB3 in a non-obese insulin-deficient animal model, we compared the effects of metformin (200mg/kg) and [D-Leu-4]-OB3 (40 mg/kg) on energy balance and glycemic control in streptozotocin (STZ)-induced diabetic male Swiss Webster (SW) mice. Diabetic mice were given insulin (Levemir(®), sc) alone, or in combination with metformin or [D-Leu-4]-OB3 orally in DDM, for 14 days. Body weight and food and water intake were measured daily. Fasting blood glucose levels were determined every other day. Serum C-peptide was measured by ELISA. Diabetic mice receiving insulin alone for 14 days gained significantly more weight than DDM-treated control mice, or mice given insulin in combination with metformin or [D-Leu-4]-OB3. The weight gain seen in mice given insulin alone was accompanied by significant increases in both food and water intake. Mice treated with insulin in combination with metformin or [D-Leu-4]-OB3, consumed significantly less food and water. Blood glucose levels in STZ-treated mice receiving insulin alone were reduced to 65.3% of initial levels, while mice receiving insulin with metformin or [D-Leu-4]-OB3 were reduced to 44.5% and 38.9%, respectively. Our results indicate that [D-Leu-4]-OB3 is as effective as metformin in preventing the body weight gain associated with insulin therapy, and on a molar basis, that the efficacy of [D-Leu-4]-OB3 as an insulin sensitizer may equal or surpass that of metformin., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The escalation predicted for the incidence of both type 2 diabetes mellitus and obesity has prompted investigators to search for additional pharmacotherapeutic approaches to their treatment. Two of these approaches, combination pharmacotherapy and utilization of leptin-related bioactive synthetic peptides as anti-diabetes/anti-obesity agents, were used in the present study. Exenatide or pramlintide acetate was reconstituted in dodecyl maltoside (DDM) in the absence or presence of [D-Leu-4]-OB3, and delivered orally by gavage to insulin-resistant male C57BLK/6-m db/db mice twice daily for 14 days. Body weight gain, food and water intake, blood glucose, and serum insulin levels were measured. Mice given DDM alone for 14 days were 19.7% heavier than they were at the beginning of the study, while oral delivery of exenatide or [D-Leu-4]-OB3 in DDM reduced body weight gain to only 13.9% and 11.5%, respectively, of initial body weight. Mice receiving exenatide and [D-Leu-4]-OB3 were 4.2% lighter than they were at the beginning of the study. In another study, Intravail® treated control mice gained 38.2% of their initial body weight, while mice receiving pramlintide acetate or [D-Leu-4]-OB3 were only 26.8% and 25.4% heavier, respectively, at the end of the study, Co-administration of pramlintide acetate and [D-Leu-4]-OB3 did not further enhance the effect of pramlintide acetate on body weight gain. Food intake was reduced by exenatide, pramlintide acetate, and [D-Leu-4]-OB3 alone, and co-delivery with [D-Leu-4]-OB3 did not induce a further decrease. Water intake was not affected by exenatide, pramlintide acetate, or [D-Leu-4]-OB3 alone, but co-delivery of exenatide or pramlintide acetate with [D-Leu-4]-OB3 resulted in a significant reduction in water intake. Oral delivery of exenatide or pramlintide acetate in DDM significantly lowered blood glucose levels by 20.4% and 30.2%, respectively. Co-delivery with [D-Leu-4]-OB3 further reduced blood glucose by 38.3% and 50.5%, respectively. A concentration-dependent increase in serum insulin was observed in response to increasing concentrations of exenatide, and [D-Leu-4]-OB3 slightly reduced the insulin response to exenatide at all concentrations tested. Increasing concentrations of pramlintide acetate alone did not elevate serum insulin, and when given in combination with [D-Leu-4]-OB3, serum insulin levels fell below those of DDM-treated control mice. Our data indicate that (1) exenatide and pramlintide acetate, currently administered by subcutaneous injection, can be given orally in DDM; (2) the bioactivity of exenatide and pramlintide acetate is retained following oral delivery in DDM; and (3) the effects of exenatide and pramlintide acetate on energy balance and glycemic control can be enhanced by co-administration with [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Leptin, the protein product of the ob gene, is primarily an adipocyte-secreted hormone, whose functional significance is rapidly expanding. Although early research efforts were focused on defining leptin's role in reversing obesity in rodents, there is now substantial evidence indicating that its influence extends to a number of hypothalamic-pituitaryendocrine axes, including adrenal, gonadal, growth hormone, pancreatic islets, and thyroid. The pleiotropic nature of leptin has been confirmed by demonstration of a role for leptin in hematopoiesis, angiogenesis, immune function, osteogenesis, reproduction, and wound healing. Unfortunately, the results of the majority of clinical trials with recombinant human leptin indicated that its effectiveness in restoring energy balance and correcting obesity-related endocrinopathies in genetically obese rodent models extended only to the management of those rare forms of human obesity caused by mutation in the ob gene. Failure of leptin in the clinic, and withdrawal of phentermine from Europe, and fenfluramine and sibutrimine from clinical use in the United States, have stimulated new approaches in the development of anti-obesity and anti-diabetes pharmacophores. These efforts are focused on utilizing leptin-related synthetic peptides as leptin receptor antagonists or leptin-related synthetic peptide analogs or mimetics. This review summarizes patents on leptin-related peptide analogs, antagonists and mimetics.

The most effective option for the medical treatment of patients with acromegaly is the use of somatostatin analogs. Octreotide acetate is a synthetic analog of somatostatin, with similar effects but a prolonged duration of action. Octreotide acetate is routinely given by subcutaneous (s.c.) or intramuscular injection. In the present study, we examined the feasibility of oral delivery of octreotide acetate reconstituted with increasing concentrations (0.5%, 1.5% and 3.0%) of Intravail®, a patented alkylsaccharide transmucosal absorption enhancing agent. The pharmacokinetics of orally delivered (by gavage) octreotide acetate in Intravail® were compared to those of octreotide acetate administered subcutaneously in sodium acetate buffer to male Swiss Webster mice. Oral delivery of octreotide acetate in 0.5% Intravail® significantly enhanced total uptake (1254.08ng/ml/min vs. 311.63ng/ml/min, respectively), serum half-life (52.1min vs. 1.3min, respectively), and relative bioavailability (4.0 vs. 1.0, respectively) when compared to delivery by s.c. injection. Higher concentrations of Intravail ®did not further enhance uptake, serum half-life, or bioavailability. The results of this study indicate that oral delivery of octreotide acetate in Intravail®is feasible, and is an effective method of administration which significantly improves uptake, bioavailability and half-life when compared to s.c. injection. Thus, oral delivery of octreotide acetate in Intravail® may have significant potential as a novel, non-invasive approach to the treatment of acromegaly and octreotide-mediated symptoms of carcinoid and VIP-secreting tumors., (2011 Elsevier B.V. All rights reserved.)

Background: We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail(®) to male Swiss Webster mice resulted in significantly higher uptake and bioavailability when compared with commonly used injection methods of delivery., Aim and Methods: In this study, we examined the effects of intranasal delivery of mouse [D-Leu-4]-OB3 in Intravail(®) on energy balance, glucose regulation, insulin secretion and serum levels of osteocalcin, a specific and sensitive marker of bone formation. Genetically obese C57BLK/6-m db/db mice were allowed food and water ad libitum and given either Intravail(®) alone or mouse [D-Leu-4]-OB3 in Intravail(®) for 14 days by intranasal instillation., Results: Mouse [D-Leu-4]-OB3 reduced body weight gain, daily food intake, daily water intake and serum glucose by 11.5, 2.2, 4.0 and 61.9%, respectively. Serum insulin levels in db/db mice given mouse [D-Leu-4]-OB3 were approximately threefold lower than those in mice receiving Intravail(®) alone. Mouse [D-Leu-4]-OB3 elevated serum osteocalcin in db/db mice by 28.7% over Intravail(®) treated control mice., Conclusions: The results of our study indicate that intranasal delivery of biologically active mouse [D-Leu-4]-OB3 in Intravail(®) is feasible and has significant effects on regulating body weight gain, food and water intake, serum glucose, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice., (© 2010 Blackwell Publishing Ltd.)

Background: We have recently shown that intranasal administration of mouse [d-Leu-4]-OB3 reconstituted in Intravail to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injections methods of delivery. The absorption profile associated with intranasal delivery of mouse [d-Leu-4]-OB3 showed an early peak representing absorption across the nasal mucosa, and a later peak suggesting a gastrointestinal site of uptake., Aim and Methods: In the present study, we examined the effects of orally administered (by gavage) mouse [d-Leu-4]-OB3 on energy balance, glycaemic control and serum osteocalcin levels in male C57BL/6J wild-type and ob/ob mice allowed food and water ad libitum or calorie restricted by 40% of normal intake., Results: In wild-type mice fed ad libitum, oral delivery of mouse [d-Leu-4]-OB3 reduced body weight gain, food intake and serum glucose, by 4.4, 6.8 and 28.2% respectively. Serum osteocalcin levels and water intake were essentially the same in control and treated wild-type mice. In ob/ob mice fed ad libitum, mouse [d-Leu-4]-OB3 reduced body weight gain, food intake, water intake and serum glucose by 11.6, 16.5, 22.4 and 24.4% respectively. Serum osteocalcin in ob/ob mice treated with mouse [d-Leu-4]-OB3 was elevated by 62% over controls. Calorie restriction alone caused significant weight loss in both wild-type (9.0%) and ob/ob (4.8%) mice, and mouse [d-Leu-4]-OB3 did not further enhance this weight loss. As expected, serum glucose levels in wild-type and ob/ob mice were significantly reduced by calorie restriction alone. Mouse [d-Leu-4]-OB3 further reduced serum glucose in wild-type mice and normalized levels in ob/ob mice. Calorie restriction alone reduced serum osteocalcin levels by 44.2% in wild-type mice and by 19.1% in ob/ob mice. Mouse [d-Leu-4]-OB3 prevented this decrease in groups of mice., Conclusions: The results of this study suggest that oral delivery of mouse [d-Leu-4]-OB3 in Intravail is possible and may have potential not only as an alternative therapy in the treatment of human obesity and some of its associated metabolic dysfunctions, but also may help to prevent and/or reverse at least some of the bone loss which accompanies osteoporosis, anorexia nervosa and other wasting diseases.

We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injection methods of delivery. The absorption profile associated with intranasal delivery of mouse [D-Leu-4]-OB3 showed an early peak representing rapid uptake across the nasal mucosa, and a later peak suggesting a gastrointestinal site of absorption. In the present study, we show that gastrointestinal absorption of mouse [D-Leu-4]-OB3 does occur, and that reformulation of mouse [D-Leu-4-OB3 with Intravail significantly enhances its uptake. The pharmacokinetics of orally delivered (by gavage) mouse [D-Leu-4]-OB3 in the absence or presence of Intravail were examined, and compared to previously reported pharmacokinetic parameters of mouse [D-Leu-4]-OB3 following intraperitoneal (ip), subcutaneous (sc), intramuscular (im), and intranasal administration. When compared to oral delivery in PBS, Intravai significantly enhanced the total uptake (552,710 ng/ml/min vs.137,585 ng/ml/min) and relative bioavailability (4.0 vs. 1.0) of mouse [D-Leu-4-OB3. The relative oral bioavailabilities of mouse [D-Leu-4]-OB3 when compared to ip, sc, im, and intranasal delivery were 52.2%, 47.3%, 37.8% and 12.9%, respectively. The results of this study indicate that oral delivery of mouse [D-Leu-4]-OB3 in Intravail is an effective method of administration achieving relatively high serum levels of the bioactive peptide when compared to commonly used methods of injection. In addition to intranasal administration, oral delivery of mouse [D-Leu-4]-OB3 in Intravail may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans., (Copyright 2009 Elsevier B.V. All rights reserved.)

Using a synthetic peptide strategy, we localized an active domain in mouse leptin to a sequence between amino acids 106 and 140. Intraperitoneal (ip) administration of a number of synthetic peptide amides encompassed by this domain reduced body weight gain, food and water intake, blood glucose levels, and increased insulin sensitivity in genetically obese mice. In the present study, we examined the pharmacokinetics of mouse [D-Leu-4]OB3, our most potent peptide, in male Swiss Webster mice following ip, subcutaneous (sc), and intramuscular (im) injection, and after intranasal administration with Intravail, a new class of patented transmucosal delivery enhancement agents. Total uptake (1,072,270, 1,182,498; 1,481,060; ng/ml/min), serum half-life (48.8; 34.0; 30.0 min) and relative bioavailability (1.0, 1.1; 1.4;) of mouse [D-Leu-4]OB3 were similar when the peptide was given by ip, sc, or im injection, respectively. Total uptake and relative bioavailability were enhanced following intranasal delivery (4,336,963 ng/ml/min and 4.0, respectively), and serum half-life was 41.1 min. These results indicate that intranasal delivery of mouse [D-Leu-4]OB3 with Intravail is a more effective method of peptide administration than injection methods, and suggest that it may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.

We have previously shown that the activity of a synthetic peptide amide corresponding to amino acid residues 116-130 of mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116 and 122 (Ser-Cys-Ser-Leu-Pro-Glu-Thr). This peptide was named mouse OB3. The potency of OB3 was improved by replacing the L-leucine residue at position four with its D-isomer. Intraperitoneal administration (i.p.) of mouse OB3 or [D-Leu-4]OB3 to ob/ob and db/db mice reduces food intake, body weight gain and serum glucose levels, and enhances insulin sensitivity. These effects of OB3 and [D-Leu-4]OB3 are very pronounced in young mice and diminish with age. In the present study, we measured uptake, serum half-life, and bioavailability of mouse [D-Leu-4]OB3 in mice of different ages. Groups of male C57BL/6J mice, six and 25 weeks of age, were given a single i.p. injection of 1 mg mouse [D-Leu-4]OB3 in PBS. Five, 10, 20, 40, 60, 120, or 180 min after injection, the mice were anesthetized and exsanguinated. Serum samples were prepared and assayed for mouse [D-Leu-4]OB3 content by competitive ELISA. In six week-old mice, the maximum concentration of mouse [D-Leu-4]OB3 was reached in 10 min, and the serum half-life was approximately 52.5 min. In 25 week-old mice, however, mouse [D-Leu-4]OB3 peaked in 5 min, and the serum half-life was approximately 30.6 min. The relative bioavailability of mouse [D-Leu-4]OB3 in six and 25 week-old mice was determined by measuring the area under the uptake curves. Bioavailability of mouse [D-Leu-4]OB3 was approximately 20% greater in six week-old mice than in 25 week-old mice. The results of this study indicate that at least some pharmacokinetic parameters of peptide uptake change as mice age. They also suggest that differences in uptake, serum half-life, and relative bioavailability of mouse [D-Leu-4]OB3 may contribute, at least in part, to the reduced efficacy of bioactive leptin-related peptides we have consistently observed in ob/ob and db/db mice as they age.

Mitochondrial uncoupling protein 3 (UCP(3))-mediated uncoupling has been postulated to depend on several factors, including superoxides, free fatty acids (FFAs), and fatty acid hydroperoxides and/or their derivatives. We investigated whether there is an interrelation between endogenous mitochondrial superoxides and fatty acids in inducing skeletal muscle mitochondrial uncoupling, and we speculated on the possible involvement of UCP(3) in this process. In the absence of FFAs, no differences in proton-leak kinetic were detected between succinate-energized mitochondria respiring in the absence or presence of rotenone, despite a large difference in complex I superoxide production. The addition of either arachidic acid or arachidonic acid induced an increase in proton-leak kinetic, with arachidonic acid having the more marked effect. The uncoupling effect of arachidic acid was independent of the presence of GDP, rotenone and vitamin E, while that of arachidonic acid was dependent on these factors. These data demonstrate that FFA and O(2-) play interrelated roles in inducing mitochondrial uncoupling, and we hypothesize that a likely formation of mitochondrial fatty acid hydroperoxides is a key event in the arachidonic acid-induced GDP-dependent inhibition of mitochondrial uncoupling.

We investigated the mechanism by which 3,5-diiodo-l-thyronine (T2) affects skeletal muscle mitochondrial bioenergetic parameters following its acute administration to hypothyroid rats. One hour after injection, T2 increased both coupled and uncoupled respiration rates by +27% and +42%, respectively. Top-down elasticity analysis revealed that these effects were the result of increases in the substrate oxidation and mitochondrial uncoupling. Discriminating between proton-leak and redox-slip processes, we identified an increased mitochondrial proton conductance as the "pathway" underlying the effect of T2 on mitochondrial uncoupling. As a whole, these results may provide a mechanism by which T2 rapidly affects energy metabolism in hypothyroid rats.

Hypothermia is a medical emergency in patients with a body temperature lower than 35 degrees C (95 degrees F) due to prolonged exposure to ambient cold temperatures without appropriate protection. This condition has a 5-fold increased risk of death in the elderly. Usually, diagnosis is suggested by warning signs and symptoms like lethargy, weakness and loss of coordination, confusion and reduced respiratory or heart rate. We report the case of a 76-year-old woman who was referred to our center for symptomatic sinus bradycardia and with typical electrocardiographic abnormalities (Osborn wave) that suggested the diagnosis of severe hypothermia.

We describe the case of a patient who presented with narrow QRS tachycardia at a rate of 100 b/min. The R-R intervals were constant, and negative P waves were evident in the inferior leads, midway between two ventricular complexes. A few minutes later, the tachycardia rate suddenly increased to 190 b/min, and the electrocardiographic pattern became typical of atrioventricular nodal reentrant tachycardia. Intravenous verapamil succeeded in restoring sinus rhythm. These data suggested an atrioventricular nodal reentrant tachycardia that at the beginning was associated with a 2:1 anterograde block.

Persistence of left superior vena cava is a rare finding. The combination of persistent left superior vena cava and absent right superior vena cava with no other congenital cardiovascular abnormalities has been exceptionally reported. We describe a case of persistent left superior vena cava and absent right superior vena cava observed during pacemaker implantation and confirmed by transthoracic contrast echocardiography.

Background: To evaluate oxidative stress and myocardial damage after aortic crossclamping release (ACCR) during cardio pulmonary bypass (CPB) in children two parameters were investigated: total glutathione (GSH) and its oxidoreductive reactions (GSH/GSSG) as expression of oxidative stress, and plasmatic turnover of myocardial taurine (TAU) as expression of cell damage., Methods: The study was divided in two periods: 1) first period: analysis of oxidative stress and myocardial damage in 18 children. 2) Second period: evaluation of myocardial cell protection by controlled anterograde low oxygen warm reperfusion (ALOWR) before declamping. Twenty-one children were divided in two groups: not receiving (Group 1, 9 patients) and receiving (Group 2, 12 patients) ALOWR., Results: In the first period GSH values increased significantly after onset of mechanical ventilation (MV) in vein, after CPB start in artery and after ACCR in coronary sinus. Moreover TAU turnover in aortic and coronary sinus blood increased significantly after ACCR. In the second period, Group 2 showed a lower oxidative stress after ACCR, while no differences were observed in TAU turnover., Conclusions: 1) Assessment of TAU and GSH levels can be considered a good method to clinically evaluate myocardial injury during cardiac surgery. 2) MV and CPB can induce oxidative stress before aortic clamping and can decrease the physiologic scavengers. Therefore, to prevent that depletion, the strategy of these techniques must be adapted to the patient and to his cardiac disease. 3) Intramyocardial TAU turnover is not significantly modified by the reperfusion technique. 4) ALOWR can reduce myocardial oxidative stress and can improve heart recovery after the cardioplegic arrest.

Two concurrent surveys were conducted in four California counties to compare the prenatal HIV counseling and voluntary testing (C&VT) experiences of women with the self-reported practices of prenatal care providers. Participants were 850 women currently or recently receiving prenatal care and 254 providers. Although 79.9% of women reported being offered an HIV test during a prenatal visit, only 56.2% said they were told about the risks and benefits of taking an HIV test. Almost all providers (98.4%) indicated they offer an HIV test, and 76.8% reported offering counseling, to every patient. One third of the women (65.9%) knew that treatment exists for reducing the chance of prenatal transmission of HIV, and 78.7% of women said they were more likely to be tested given knowledge of such therapy. Women may have underreported prenatal C&VT because providers spend insufficient time discussing related issues or because C&VT information is not presented in a way that is relevant to all patients.

Leptin, the protein product of the ob gene, is primarily an adipocyte-secreted hormone, whose functional significance is rapidly expanding. Although early research efforts were focused on defining leptin's role in reversing obesity in rodents, there is now substantial evidence indicating that its influence extends to several hypothalamic-pituitary-endocrine axes, including gonadal, adrenal, thyroid, growth hormone, and pancreatic islets. A role for leptin in hematopoiesis, angiogenesis, immune function, osteogenesis, and wound healing has also been documented. The results of recent clinical trials with recombinant human leptin indicated that its effectiveness in restoring energy balance and correcting obesity-related endocrinopathies in genetically obese rodent models extended only partially to the management of human obesity. New efforts in drug development have focused on leptin-related synthetic peptide agonists as potential anti-obesity pharmacophores.

Introduction: The main purpose of this contribution is to set today's situation of technology assessment and point to some organizing and integrative ways with the evolution concern ASL (Local Health Firm)., Patients and Methods: We outline main critical processes that fix correct management of technological resource thought data available in national and international literature. We single out more common methods to quantify, evaluate, control and manage biomedical technologies., Results: We expound decisional process that lead Viterbo ASL (Local Health Firm) to a progressive controll of tecnological resource and its management after aggregation of the five USL (Local Health Units) that constituted Viterbo ASL and describe procedures assumed., Conclusions: We underline the importance of full and complete information in business and decisional process. Inside the complexity of italian view, we outline an organizative solution with a low economic impact and with a high technical content.

We have recently shown that the activity of a synthetic peptide corresponding to amino acid residues 116-130 of secreted mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116-122 (Ser-Cys-Ser-Leu-Pro-Gln-Thr, OB3). Substitution of the Leu residue at position 4 of OB3 with its D-isomer ([D-Leu-4]-OB3) enhanced the ability of OB3 (1 mg/day, ip, 7 days) to reduce body weight gain, food and water intake, and serum glucose in female C57BL/6J ob/ob mice. In the present study, we have utilized a pair-feeding approach to demonstrate that the antihyperglycemic action of [D-Leu-4]-OB3 is not solely due to its effects on caloric intake. One group of female C57BL/6J ob/ob mice (n=6) was fed ad libitum, and two additional groups (n=6 per group) were allowed 3.0 g food/mouse daily, an amount previously determined to satisfy [D-Leu-4]-OB3-treated mice. At the end of the 7-day test period, vehicle-injected mice fed ad libitum were approximately 10% heavier than their initial body weights, while pair-fed mice injected with vehicle and [D-Leu-4]-OB3-treated mice lost 5% of their initial body weights. After 1 day of treatment, blood glucose was reduced by 20% in pair-fed vehicle-injected mice, and by 40% in mice given [D-Leu-4]-OB3. Food restriction reduced blood glucose throughout the 7-day study, but not to levels seen in wild-type nonobese C57BL/6J mice of the same sex and age. After 2 days of treatment with [D-Leu-4]-OB3, however, blood glucose was reduced to levels comparable to those seen in wild-type nonobese mice. [D-Leu-4]-OB3 also lowered serum insulin levels by 53% when compared to mice fed ad libitum. Neither pair-feeding nor [D-Leu-4]-OB3 treatment had any apparent effect on thermogenesis. These results suggest that [D-Leu-4]-OB3 exerts its effects on serum glucose not only by suppressing caloric intake, but also through a separate effect on glucose metabolism which may involve increased tissue sensitivity to insulin.

We have previously reported that a synthetic peptide amide corresponding to amino acid residues 116-130 of mouse leptin, LEP-(116-130), reduces body weight gain, food intake, and blood glucose levels in ob/ob and db/db mice. In the present study we show that the activity of LEP-(116-130) resides in a restricted sequence between amino acid residues 116-122. A synthetic peptide corresponding to this sequence (Ser-Cys-Ser-Leu-Pro-Gln-Thr) has been named OB3. Single point D-amino acid substitution was used to study the structure-function relationship of each residue in OB3. D-Amino acid analogs of OB3 were synthesized by the solid phase method, purified to 98+%, and administered (1 mg/day, ip) for 7 days to female C57BL/6J ob/ob mice. The effects of the peptides on body weight gain, food and water intake, glucose homeostasis, and thermoregulation were assessed. In most cases, the efficacy of OB3 on all parameters tested was reduced by substitution of an L-amino acid with its corresponding D-isoform. A statistically significant increase (2.6-fold) in the weight-reducing effect of OB3, however, was observed by inversion of the configuration of the leucine residue at position 4 (Leu-4) of OB3 by substitution with its D-amino acid isoform [D-Leu-4]. Compared with OB3, mice treated with [D-Leu-4]-OB3 consumed 7.9% less food and 16.5% less water. Blood glucose was normalized to levels comparable to those in wild-type control mice within 2 days after initiation of [D-Leu-4]-OB3 treatment. Unlike native leptin, however, neither OB3 nor any of its D-amino acid-substituted analogs had any apparent effect on thermogenesis. Our results indicate that synthetic peptide strategies may be useful in the development of potent and stabile pharmacophores with potential therapeutic significance in the treatment of human obesity and its related metabolic dysfunctions.

We have recently reported that intraperitoneal (i.p.) administration of synthetic peptide amides corresponding to amino acids 106-140 of mouse leptin significantly reduced food intake and body weight gain in female C57BL/6J ob/ob mice. These results suggested that leptin activity was localized in domains toward its C-terminus between residues 106-140. In the present study, 14 overlapping peptides encompassing the complete sequence of secreted mouse leptin were synthesized, and their effects on body weight and food intake in female C57BL/6 J ob/ob mice were assessed. When given as seven daily 1-mg i.p. injections, only peptides corresponding to amino acids 106-120, 116-130 and 126-140 caused significant reductions in body weight and food intake. These results confirmed our earlier study and suggest that in contrast to the domain encompassed by amino acids 106-140, the N-terminal of mouse leptin between amino acids 21-105 may not contain functional epitopes that can be utilized as lead compounds in the development of peripherally administered bioactive peptide analogs or nonpeptide mimetics of leptin, which may have potential usefulness in treatment of the energy imbalance associated with obesity.

We recently reported that intraperitoneal administration of leptin-related synthetic peptide 116-130 [LEP-(116-130)] resulted in reduced food intake and significant weight loss in homozygous female C57BL/6J ob/ob mice. In this study, we used two in vitro bioassays to show that the interaction of LEP-(116-130) with the long form of the leptin receptor (OB-Rb), the receptor isoform that is predominantly expressed in the hypothalamus, is not required for the observed in vivo effects of the peptide on energy balance. LEP-(116-130) was unable to compete the binding of alkaline phosphatase-leptin fusion protein to OB-R. Moreover, LEP-(116-130) was unable to activate signal transduction by OB-Rb in vitro. In homozygous female C57BLKS/J-m db/db mice that do not express OB-Rb, intraperitoneal administration of LEP-(116-130) reduced body weight gain and blood glucose levels but not food intake, which further supports a mechanism of action that does not require peptide interaction with OB-Rb.

A back-propagation neural network to predict the carcinogenicity of aromatic nitrogen compounds was developed. The inputs were molecular descriptors of different types: electrostatic, topological, quantum-chemical, physicochemical, etc. For the output the index TD50 as introduced by Gold and colleagues was used, giving a continuous numerical parameter expressing carcinogenicity. From the tens of descriptors calculated, principal component analysis enabled us to restrict the number of parameters to be used for the artificial neural network (ANN). We used 104 molecules for the study. An Rcv2 = 0.69 was obtained. After removal of 12 outliers, a new ANN gave an Rcv2 of 0.82.

We have recently reported that a synthetic peptide corresponding to amino acid residues 81-95, a receptor-binding region of the human FSH-beta-subunit, and a subdomain within this region, hFSH-beta-(90-95) (DSTDCT), prolonged vaginal estrus when administered intraperitoneally (ip) to normally cycling Swiss Webster mice. These results were similar to those we reported for a synthetic peptide corresponding to hFSH-beta-(34-37) [TRDL, a subdomain within receptor-binding region hFSH-beta-(33-53)] in the same model system. TRDL also accelerated the onset of puberty in immature mice. We now report the effects of hFSH-beta-(90-95) in prepubertal female mice. In two separate experiments, a single ip injection of 200 microg/g body weight (BW) hFSH-beta-(90-95) in phosphate buffered saline (PBS, vehicle) administered to mice on day 28 delayed first vaginal estrus by 3 days in 50% (4/8) and 62.5% (6/8) when compared to mice given vehicle alone on day 28. Vaginal opening was also delayed in mice receiving hFSH-beta-(90-95) when compared to mice injected with vehicle alone. Serum estradiol levels of vehicle-injected control mice in first vaginal estrus were three-fold higher than in mice treated with hFSH-beta-(90-95), whose vaginal smears showed no evidence of first estrus. No significant differences in ovarian or uterine weights, or serum progesterone levels, were observed between vehicle-injected control mice achieving first vaginal estrus and mice receiving hFSH-beta-(90-95) in whom first estrus was delayed. The contrasting effects on the onset of puberty of hFSH-beta-(90-95) (delay) and hFSH-beta-(34-37) (acceleration) may reflect synthetic peptide binding to different domains of the FSH receptor, resulting in variable post-binding effects. These results are consistent with our earlier study suggesting that FSH-beta-subunit-related synthetic peptides can induce significant in vivo effects on the onset of puberty in female mice.

We have previously reported that synthetic peptide amides corresponding to subdomains of the human FSH 3-subunit, hFSH-beta-(33--53) and hFSH-beta-(81--95), interact with the external domain of the FSH receptor in two in vitro model systems. Consistent with these in vitro observations, we found that intraperitoneal (i.p.) administration of each of these peptides prolonged vaginal estrus in normally cycling mice in vivo. Both hFSH-beta-(33--53) and hFSH-beta-(81--95) contain cysteine (Cys) residues with free sulfhydryl groups of potential significance in receptor interactions. To assess the possible involvement of these groups in the in vivo effects of hFSH-beta-(33--53) and hFSH-beta-(81--95), synthetic peptide analogs were prepared in which all Cys residues were replaced with serine (Ser). In the present study, we demonstrate that the in vivo effect of hFSH-beta-(33--53) on the mouse estrous cycle, extension of vaginal estrus, was not changed by substitution of Cys-51 with Ser. In contrast, mice receiving the Ser-substituted analog of hFSH-beta-(81--95) had normal estrus stages, but were arrested in diestrus. hFSH-beta-(33--53)-(81--95), a linear peptide encompassing both domains, also prolonged vaginal estrus. The Ser-substituted analog of this peptide, however, prolonged vaginal estrus in some of the mice tested and induced cycle arrest at diestrus in others. hFSH-beta-(90--95), the active subdomain at the C-terminus of hFSH-beta-(81--95), extended vaginal estrus, but diestrus stages were of normal duration. Its Ser-substituted analog, however, prolonged the estrus stage of the majority of mice treated, but induced diestrus arrest in some. The differing responses to these peptides are presumably due to interactions of the synthetic peptides with different regions of the FSH receptor. This further suggests that one consequence of ligand interaction with multiple receptor binding domains may be variable effects on ovarian function, and that Cys to Ser analogs may have value in the design of a novel class of synthetic peptides capable of fertility regulation and control.

We used the duplicate portion method to measure the daily dietary intake of total and congener-specific polychlorinated biphenyls (PCB) and to assess their potential toxicity in a group of 20 subjects consuming a typical Italian diet. The mean +/- SD intake of total PCB, measured by GC-MS, was 3.72 +/- 1.51 micrograms/person/day, comparable to values reported in similar studies world-wide, with individual intakes varying within one order of magnitude, from 0.97 to 10.59 micrograms/person/day. The di-ortho congeners 153, 18 and 138 were the PCB found in the highest concentrations (respectively 13.8%, 11.4% and 10.9% of the total) while the non-ortho coplanar congeners (77, 126 and 169) amounted to 0.5% of the total. The corresponding levels of toxicity (TCDD-like TEQ values ascribable to PCB) ranged from 4.6 up to 119 pg/person/day of TCDD-equivalents in 18 subjects, i.e. presumed no-risk levels, but with peaks of 2109 and 4553 pg/person/day in two subjects with significant intakes of the congener 126. Principal components analysis and redundancy analysis showed dairy products, meat and fish were the principal sources of PCB, and vegetables those with the highest toxicity index in the Italian diet.

No carcinogenic activity was observed when snuff was inserted into the cheek pouch of the hamster or spread over the oral mucosa. This negative result was obtained in a number of experiments whether snuff was applied once only and left in place for several months or inserted repeatedly for up to 2 years. In the rat, a few tumours were observed when snuff was inserted into the artificial lip canal. The insertion appeared to cause a considerable reaction in the surrounding tissue so it is plausible to assume that trauma plays an important role in the development of these tumours. An extract of snuff applied to the oral mucosa of the rat did not produce any tumours, but an extract enriched by the addition of 10 times the naturally-occurring amounts of NNN and NNK produced a few benign tumours at the site of application. A higher incidence of tumours was produced when an equivalent amount of an aqueous solution of these two nitrosamines was applied directly to the oral mucosa, suggesting, according to the authors, that snuff inhibits the carcinogenic activity of TSNAs. Initiation/promotion studies were carried out on snuff in the rat in order to explore further its carcinogenic potential. The results were consistent with the conclusion that snuff does not possess any promotional activity. No increase in tumour incidence was observed in mice when snuff was given in the diet at concentrations of 25% gradually decreasing to 5% in a 14 month study. A negative result was also obtained in the rat given snuff at a concentration of 5% for 18 months. In hamsters given snuff at a concentration of 20% for 2 years, forestomach tumours occurred. A comparable incidence of this type of tumour occurred in animals given 20% cellulose. The result of this study does not provide valid evidence of carcinogenicity. HSV and snuff applied orally in the hamster produced a high incidence of squamous cell carcinomas. The sustained high level of squamous cell hyperplasia generated by the experimental design could account for the development of these tumours. Despite the defects in some of the earlier studies, the sum total of this experimental work suggests that snuff is not carcinogenic to the oral mucosa of the hamster or the rat. It is also unlikely to cause tumours in other tissues in these species. These results give some degree of reassurance that snuff is not likely to be carcinogenic to the human oral mucosa. The interaction of snuff and HSV viruses is, at the moment, questionable and requires further investigation.

It is essential to know whether drinking water contains any pesticides up to concentrations close to the European Union limit of 0.1 microgram/l, in order to protect the population. Mass spectrometry (MS) using a suitable internal standard (I.S.) should improve the quantitative analysis of pesticides, the I.S. circumventing loss of compounds during the analytical procedure and correcting the analytical variability. In this study we verified this assumption in GC-MS, synthesising specific internal standards for four organophosphorus pesticides with poor stability, comparing the performances with and without the I.S.

Objective: We surveyed the attitudes of Italian obstetricians toward the application of HIV infection control guidelines during labour and delivery proposed by the Italian National Committee for HIV Infection., Study Design: We identified 66 obstetric centres affiliated to the AOGOI (Association of Italian Gynecologists and Obstetricians). A postal questionnaire was sent to 752 physicians in charge in the centres., Results: A total of 419 clinicians (55.7%) completed and returned the form to the coordinating centre. Obstetricians were directly asked about the need for routine adoption of the Italian guidelines for delivery of women with positive or unknown HIV status (indicating the routine use during delivery of protective glasses, impermeable garments, mask and sterile latex gloves, the washing of hands with detergent solution after using gloves and collection of needles and sharp instruments in suitable containers). A total of 319 (76.1%) clinicians agreed that all these procedures should be adopted. In clinical practice, however, obstetricians declared that the use of latex gloves and collecting needles in suitable containers were always adopted, 'washing hands after using gloves' less frequently, and other procedures such as protective impermeable garments, glasses and mask were infrequently applied.

We have previously reported that a synthetic peptide corresponding to amino acid residues 81-95 of the human (h) FSH-beta subunit inhibited binding of [125I]hFSH to bovine calf testis membranes and stimulated estradiol biosynthesis in primary cultures of rat Sertoli cells. We have now obtained several lines of evidence demonstrating in vivo effects of hFSH-beta-(81-95) on the mouse estrous cycle. 1) A single i.p. injection of 200 micrograms/g BW hFSH-beta-(81-95) significantly (p < 0.001) prolonged vaginal estrus in comparison to that in vehicle-injected control mice. 2) Vaginal smears taken at estrus from mice given hFSH-beta-(81-95) were characterized by the complete absence of epithelial casts, a hallmark of spontaneous ovulation in mice. 3) Mice receiving hFSH-beta-(81-95) had significantly (p < 0.001) lower serum estradiol at proestrus and serum progesterone at diestrus than vehicle-injected control mice. 4) The proestrous effects of estrogen on uterine ballooning and weight gain, clearly evident in vehicle-injected control mice, were not observed in mice treated with hFSH-beta-(81-95). A synthetic peptide corresponding to the carboxy-terminal region of hFSH-beta-(81-95), hFSH-beta-(90-95), inhibited binding of [125I]hFSH to bovine calf testis membranes, antagonized FSH-stimulated estradiol biosynthesis by primary cultures of rat Sertoli cells, and prolonged vaginal estrus in normally cycling mice. A synthetic peptide corresponding to the amino-terminal domain, hFSH-beta-(81-86), was inactive in vitro and had no effect on the mouse estrous cycle. The results of the present study provide additional evidence for in vivo effects of FSH-related synthetic peptides.

Clusters of spontaneous abortion among video display terminal (VDT) users in North America and Canada in the late 1970s aroused suspicion about the potential risk of an association between VDT. exposure and pregnancy outcome. This case-control study considered the association between VDT use and the risk of miscarriage. Cases were 508 women admitted for spontaneous abortion to the Clinica Luigi Mangiagalli and a network of obstetric departments in the Milan area. Controls were 1,148 women who gave birth at term to healthy infants on randomly selected days at the same hospitals where cases were identified. No association emerged between VDT exposure and spontaneous abortion, the estimated odds ratio being 1.0 (95% CI: 0.8-1.2). This evidence agrees with studies conducted in different countries by various authors.

We have previously reported that a synthetic peptide amide corresponding to residues 34-37 (TRDL, threonine, arginine-aspartic acid-leucine) of the beta-subunit of human FSH induced prolonged vaginal estrus in normally cycling female mice (see Ref. 15). These results represented the first demonstration of an in vivo effect of a gonadotropin-related synthetic peptide on reproductive processes. We have extended these studies to examine possible effects of TRDL on the onset of puberty in female mice. In two replicated experiments, vehicle-injected control mice attained first vaginal estrus by day 39. An ip injection of 200 ug TRDL/g BW to 28-day-old prepubertal female mice, however, accelerated the onset of first vaginal estrus by 7 days in 11 of 12 (11/12) (Exp 1) and 7/9 (Exp 2) mice. Serum estradiol levels were significantly (P = 0.017) elevated in TRDL-treated mice, whereas progesterone was unchanged. Uteri of TRDL-treated mice were significantly (P = 0.003) heavier than uteri of vehicle-injected control animals of the same age and body weight. Intraluminal fluid accumulation (ballooning) at proestrus was absent in 20/21 TRDL-treated females, as were oviductal ova and ovarian corpora lutea. These phenomena are characteristic of the first estrous cycles of female mice isolated from males. To obtain further evidence for in vivo effects of TRDL, we assessed the ability of TRDL to accelerate the onset of puberty in male mice. When given as five consecutive daily ip injections of 200 ug/g BW to 28-day-old prepubertal male mice, TRDL significantly increased testis weight, when compared with vehicle-injected control mice of the same age and BW (171.3 +/- 3.8 mg vs. 151.6 +/- 4.3 mg, P = 0.001) and induced a 6.5-fold increase in serum testosterone levels. These studies confirm the previously reported in vivo activity of a synthetic peptide corresponding to human FSH-beta subunit 34-37 (TRDL) in adult female mice and extend its effects to the acceleration of the onset of puberty in immature male and female mice.

This paper describes changes in the livers of rats fed diets containing butylated hydroxytoluene (BHT) over two generations in two separate studies. BHT did not produce tumours when tested for carcinogenicity in several studies by the conventional way. However, when BHT was given to rats in a two-generation carcinogenicity study, a high incidence of hepatic tumours was found in males but not in female rats of the F1 generation. A sequential study has been carried out to gain an insight into this unexpected finding, paying particular attention to the perinatal period. In the dose-ranging study designed to assess the tolerance of rats to BHT, groups of male and female rats (F0 generation) were fed diets calculated to deliver 0, 500, 750 and 1000 mg/kg body weight/day. Following a loading period of 5 wk the rats were mated. The BHT content of the diet was not adjusted during pregnancy and lactation. Owing to the normal increase in food consumption during lactation, intakes peaked at double the nominal value by 21 days after the birth of pups. At this time the pups (F1) were weaned onto control diet and maintained on it for 4 wk. At birth, the body weights of pups from the BHT-treated dams were comparable to those of the controls but at weaning the body weights of the pups from all three dose levels were less than those of the controls. At the termination of the experiment (4 wk after weaning), the pups from BHT-treated dams still weighed less than those from untreated controls. In the main experiment the F0 generation were fed 0, 25, 100 and 500 mg/kg body weight/day. Their offspring (F1 generation) were weaned on diets containing the same amount of BHT as the respective parents, apart from the group given the highest dose level (500 mg/kg body weight/day). This dose level was reduced to 250 mg/kg body weight/day at weaning in order to conform with previously published findings. The pups from the dams given the highest dose level were maintained on a dietary concentration of 250 mg/kg body weight/day for the entire study. A group of age-matched non-pregnant females was also studied and the results obtained compared with those from pregnant dams. Pups from all groups were examined at day 20 of gestation, at weaning (21 days after birth), and at 4 and 22 wk post-weaning. There were no effects on fertility and no increase in foetal abnormalities at any dose of BHT. Dams receiving BHT at a nominal dose of 500 mg/kg body weight/day showed liver enlargement accompained by induction of pentoxyresorufin O-depentylase and glutathione S-transferase, and proliferation of the endoplasmic reticulum. Pups from these dams were of the same weight at birth as controls but lost weight during the lactation period. This deficit was not recovered by the time the experiment was terminated. Hence, in two independent studies, the only significant finding in rats treated with BHT in utero and during lactation was that the weight gain of pups during lactation was less than expected when dams received at least 500 mg BHT/kg body weight/day. The body weight of pups did not return to normal following a return to a control diet for 4 wk. It is postulated that the retardation in weight gain of the pups could be due to inadequate milk production.