Inhibition of Eph/ephrin interaction with the small molecule UniPR500 improves glucose tolerance in healthy and insulin-resistant mice.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

Expression and function of the stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in the swine ovarian follicle.

The most characterized stromal cell-derived factor-1 (SDF-1) variants are the isoform α, which is the predominant one but undergoes rapid proteolysis, and the ß isoform, which is more resistant. Through the interaction with a specific chemokine receptor called CXCR4, SDF-1 is able to regulate different physiological processes. The aim of this study was to verify the expression and potential functional role of SDF-1 and CXCR4 in the porcine ovary. Firstly, the expression of SDF-1 and its receptor in different ovarian districts was verified for the first time. Thereafter, the effect of SDF-1 ß isoform (51-72) fragment on functional parameters, such as proliferation, metabolic activity, redox status, nitric oxide production, and steroidogenic activity, was assessed on granulosa cells collected from follicles. In addition, the potential effect of this protein in vascular events was verified through investigations on porcine aortic (AOC) endothelial cells, such as the production of nitric oxide and viability tests. The proliferation and metabolic activity were not affected by treatment with the cytokine. As regard to steroidogenesis, the peptide stimulated both estrogen (P = 0.049) and progesterone production (P = 0.039). Redox status was affected by the examined substance since superoxide anion was inhibited (P = 0.001), while antioxidant power (P = 0.034), as well as nitric oxide generation, were stimulated (P = 0.034). Tests performed on AOCs showed significant stimulation of nitric oxide production (P = 0.004) by the examined peptide, while cell viability was unaffected. Therefore, the potential role of cytokine in the mechanisms involved in the regulation of follicular function can be hypothesized.

Orexin system in swine ovarian follicles.

Successful reproduction is strictly linked to metabolic cues. The orexins are a family of hypothalamic neurohormones, well known for their key role in the control of food intake and the involvement in several aspects of the reproductive process. The biological actions of both orexins are carried out through binding to the related Orexin 1 (OX1R) and Orexin 2 (OX2R) G-protein-coupled receptors. The purpose of this study was to investigate the presence of orexin system components in the porcine ovaries, to contribute to expand the knowledge about their pleiotropic role. First, we investigated the localization of orexin A (OXA) and its receptors by immunochemistry in different ovarian districts. Thereafter, we evaluated the expression of the prepro-orexin (PPO) gene and OXA effects on granulosa cell functions. Immunohistochemical study revealed the presence of orexinergic system components in porcine ovarian follicles. Moreover, our data show the expression of PPO messenger RNA in swine ovarian follicles >5 mm. In addition, OXA influences proliferation (P < 0.05), steroidogenic activity (P < 0.05), and redox status of granulosa cells (P < 0.05). Therefore, we hypothesize that OXA could exert a local physiological role in swine ovarian follicles even if further studies are required to deeply define the function of this pleiotropic system.

Orexin A in swine corpus luteum.

Orexin A (OXA) is a hypothalamic neuropeptide which acts on 2 known G-protein-coupled receptors. It has been demonstrated that OXA is a central molecular link between food intake and reproduction. More recently, its peripheral role has been investigated, and we demonstrated its involvement in regulating ovarian follicle function. The present study was undertaken to explore a potential physiological role of orexin system in swine corpus luteum, a transient ovarian endocrine organ. Our aim was, first, to analyze the localization and eventual colocalization of OXA and its 2 receptors within the different cell types composing the corpus luteum structure. Second, we wanted to explore the effects of OXA on isolated luteal cells, and finally to verify a potential involvement of OXA in angiogenesis, a crucial event in corpus luteum development. Our data demonstrate the local expression of OXA and its receptors in swine corpus luteum. Luteal cell functions were affected by treatment with OXA. In particular, progesterone production was inhibited (P < 0.05) and nonenzymatic scavenging activity was increased (P < 0.05). Moreover, OXA inhibited (P < 0.05) new vessel growth. Our results suggest that OXA could act locally to play a role in corpus luteum demise.

Lack of tissue-specificity of mucin markers in a lung-cancer model - biochemical approach.

Mucin-associated epitopes are recognized by monoclonal antibodies in the immunometric assays used for the diagnosis and monitoring of cancer. The recently developed new assays measure mucins as tumor markers, assuming that each mucin is associated with a particular tumor site, i.e. CA 15.3 and MCA with breast cancer, CA 125 with ovarian cancer, CA 19.9 and CA 195 with colon and pancreatic cancer. These associations are based on the frequency and the intensity of expression of the single markers for a certain organ. However, this theoretical organ specificity is not absolute, since the mucins are expressed also by tumors other than those mentioned above and they may also be present in inflammatory conditions and in normal tissues. These observations were confirmed by the present study, which used an experimental model consisting of a pool of 20 lung tissue samples (10 normal and 10 cancer). The tissue concentrations of the mucins MCA, CA 15.3, CA 125, CA 19.9, and of the glycoprotein CEA, were measured both in malignant tissue samples and in their normal counterparts. The marker levels were detected by immunometric assays in mucin fractions separated from the tissue extract by chromatographic methods. The comparison of the chromatographic profiles and the evaluation of the mucin levels in normal and malignant lung tissue specimens confirmed the absence of tissue specificity of these biochemical parameters. Recent developments in molecular biology and the discovery of genes coding for several apomucins may open new perspectives towards the understanding of the mechanisms regulating mucin pathways.

Chemotherapy vs supportive care in advanced non-small-cell lung cancer. Results of a meta-analysis of the literature.

STUDY OBJECTIVE: To contribute to the current debate about the relative merits of meta-analysis of the literature (MAL) and of individual patients data (MAP). DESIGN: Identification of published randomized trials and extraction of essential results directly from the published reports. SETTING: Chemotherapy vs supportive care in advanced non-small-cell lung cancer. MEASUREMENTS AND RESULTS: Survival probability at 6 months after randomization, as estimated from the published survival curves, has been considered as the end-point of interest. Quality scoring of the studies has also been performed. Specific methodologic issues concerning the estimation of relevant quantities necessary for the MAL have been addressed. The estimated pooled odds ratio of death was 0.44, with 95 percent confidence interval of 0.32 to 0.59, thus significantly favoring chemotherapy, and it corresponds to an estimated increase in median survival from 3.9 months for best supportive care to 6.7 for chemotherapy. CONCLUSIONS: The results of our MAL, favoring chemotherapy, are in line with those of a MAP recently published. However, they have to be considered in the light of their actual clinical relevance and of the balance between quality of life, toxicity, and costs of chemotherapy and best supportive care.

Single-agent chemotherapy versus combination chemotherapy in advanced non-small cell lung cancer: a quality and meta-analysis study.

STUDY OBJECTIVE: To estimate the quality of the studies and to compare single-agent with combination chemotherapy in advanced non-small cell lung cancer. DESIGN: Identification of published randomized trials and extraction of essential results directly from the published reports. MEASUREMENTS AND RESULTS: Survival probability at 1 year, as estimated from the published survival curves, has been considered as the end-point of interest. Quality scoring of the studies has also been performed. Arithmetical calculation, concerning the estimation of quantities necessary for the meta-analysis of the literature, has been addressed. The estimated pooled Odds Ratio of death was 0.8, with 95% confidence interval of 0.6-1.0, thus favoring combination chemotherapy. CONCLUSIONS: The results of our meta-analysis favor combination chemotherapy. They must, however, be considered in the light of their clinical relevance and of the balance between quality of life, toxicity and costs of chemotherapy.

Pattern of mucin gene expression in normal and neoplastic lung tissues.

This work evaluates the expression in lung cancer of the most well characterized mucin genes (MUC1, MUC2, MUC3) and of the recently described MUC4 in lung tissues, to check a correlation between the expression of any particular gene and this tumor. Hybridization with synthetic oligonucleotides obtained from a part of the sequences of MUC1, MUC2, MUC3 and MUC4, was performed on blotted RNA from 18 lung cancer tissue specimens and from 10 normal tissues samples taken, when possible, from the normal lung counterpart. By means of Northern blot analysis MUC1 revealed to be the most expressed mucin gene in lung cancer, followed by MUC4; by contrast, the expression of MUC2 and MUC3 was almost undetectable in all cancer specimens. The intensity of expression of MUC1 and MUC4 was always superior in cancer tissue than in the normal counterpart. As expected, the highest reactivity for MUC1 and MUC4 expression was observed mainly in the adenocarcinoma histotype which is mucin secreting. These findings represent a contribution to the study of mucin gene pattern in lung cancer, and, in particular, indicate that MUC4, in association with the MUC1 gene, seems to be strongly expressed in this neoplastic disease.

Radioimmunoassisted follow-up and surgery vs traditional examinations and surgery after radical excision of colorectal cancer.

The authors report their experience in the radioimmunodetection of recurrent colorectal cancer in comparison with traditional examinations. 485 patients were studied after radical surgery for colorectal cancer: 168 (Group 1) were studied with a radioimmunoassisted follow-up plan including: Immunoscintigraphy (IS), serum markers assays, Radioimmunoguided Endoscopy (RIGE), Intraoperative Radioimmuno-localization (IORIL) in case of reoperation; 317 (Group 2) underwent a protocol with traditional examinations. In 24 patients of Group 1 IS was performed even at the time of their admission for primary cancer. 7 patients underwent RIGE for primary cancer and 16 in the follow-up. IORIL was performed in 12 patients with primary cancer and in 16 in the course of reoperation. The radioimmunodiagnostic methods were performed after a single administration of the radiolabeled MAb (111In F(ab')2 a-CEA and 111In B72.3). The radioimmunoassisted follow-up plan detected a greater number of recurrences than traditional examinations (27% vs 13%). The rates of radical reoperation in Group 1 and 2 were 61% and 37% respectively. 33% of the patients of Group 2 were alive 24 months after radical reoperation vs 62.5% of the patients of Group 1. Immunoscintigraphy demonstrated a good sensitivity and specificity, mainly in the detection of pelvic recurrences (sens. 92%, spec. 84%). The radioimmunoassisted follow-up plan was well accepted by the patients. RIGE led to the detection of 3 periluminal recurrences of rectal cancer that traditional investigations failed to demonstrate and in 5 cases influenced the patients management. IORIL detected minimal tumor foci (2 mm.) where pre and intraoperative study were negative, while the histopathologic examination gave evidence of tumor. Considering that the costs of the radioimmunodiagnostic methods and of traditional examinations are very similar, we can conclude that the radioimmunoassisted follow-up plan has a favorable cost/benefit rate and a remarkable impact on the treatment of patients with colorectal cancer.

The response of pigs inoculated with a thymidine kinase-negative (TK-) pseudorabies virus to challenge infection with virulent virus.

12 Large-White-Landrace piglets were subdivided in four groups of 3 and housed in separate units. The piglets of three groups were inoculated with the 86/27V 6C2 thymidine kinase negative (TK-) mutant of pseudorabies virus (PRV), by different routes. A second inoculation with the same mutant was given to the pigs 21 days later. The animals of a fourth group were left as uninoculated controls. 21 days following the second inoculation with the TK- mutant all pigs were challenge infected with the virulent PRV. On post challenge day (PCD) 30 all pigs were killed and samples for virus detection and histology were taken from several organs. The inoculated TK- mutant of PRV did not induce any ill effects in the pigs except a transient febrile reaction in some animals. Virus was recovered from nasal swabbings from one pig 2 days after the first inoculation of the mutant. After challenge exposure with virulent PRV, the TK- mutant-inoculated pigs were apparently protected, whereas the control pigs all were severely affected and recovered very slowly over 3 weeks. Virus was isolated from the nasal swabbings from the TK- mutant-inoculated pigs on PCDs 2 and 4, whereas the nasal swabbings from the control piglets were all positive for virus from PCD 2 through PCD 10. DNA analysis of the virus recovered showed a pattern identical to that of the virulent PRV. Histologic lesions were found in the respiratory and the central nervous systems, however, the lesions in the TK- mutant-inoculated pigs were much milder compared to those registered for the control pigs. Virus was not isolated from any of the tissue samples that were tested, but viral DNA with sequences typical of PRV genome was detected by PCR in all samples of trigeminal ganglia from either the TK- mutant-inoculated pigs or from the controls.

Experimental infection of pigs with a thymidine kinase negative strain of pseudorabies virus.

Sixteen 20 day old pigs, devoid of neutralizing antibody to pseudorabies virus (PRV), were divided into two groups of eight, an the animals of each group were housed in a separate unit. In each group 6 pigs were inoculated intranasally with the thymidine kinase (TK-) mutant (Group 1) or the field strain of PRV (Group 2), each pig receiving an inoculum of 4 ml. The remaining 2 pigs in each group served as uninoculated controls. The only clinical sign observed in the pigs of Group 1 was a transient febrile reaction, in the case of six pigs inoculated with the TK- mutant of PRV, whereas no signs of disease were seen in the uninoculated controls. The virus was isolated from the 6 infected pigs of the group only on post infection day (PID) 2, whereas it was never isolated from the controls. By contrast, the pigs of Group 2, had a severe clinical response and one, among those that were inoculated with the field strain of the PRV, died on PID 9. Virus was consistently isolated from all pigs of Group 2, inoculated and control. On PID 30 all pigs, i.e. the 8 of Group 1 and 7 of the Group 2 which survived to the infection, were subjected to dexamethasone (DMS) treatment. After DMS treatment virus was never isolated from the nasal swabbings obtained from the pigs of Group 1, whereas it was consistently isolated from pigs of Group 2. After 30 d from the start of DMS treatment the pigs were killed and several tissues were collected from each pig for virus detection, by isolation in tissue culture and by PCR analysis. At necropsy no lesions were found in pigs of Group 1, whereas acute pneumonia and gliosis in the trigeminal ganglia were observed in pigs of Group 2. Virus was never isolated from any of the tissues taken from pigs of both, Group 1 and Group 2, nevertheless sequences of PRV were detected by PCR analysis in the trigeminal ganglia of the pigs of both Groups.

Study of the HLA-DP beta 1 locus by the polymerase chain reaction technique in patients with Hodgkin's disease.

BACKGROUND: A number of reports have studied associations between Hodgkin's disease and HLA. Some of them established correlation between several antigens and Hodgkin's disease, and others found no correlations. METHODS: The HLA DP locus was determined by the polymerase chain reaction method in 31 Hodgkin's disease patients and 58 healthy controls. RESULTS: No significant difference between patients and controls was noted. CONCLUSIONS: Further investigations are needed to confirm the hypothesis of a possible role of the HLA complex as one of the factors involved in Hodgkin's disease.

A model for phase noise generation in amplifiers.

In this paper, a model is presented for predicting the phase modulation (PM) and amplitude modulation (AM) noise in bipolar junction transistor (BJT) amplifiers. The model correctly predicts the dependence of phase noise on the signal frequency (at a particular carrier offset frequency), explains the noise shaping of the phase noise about the signal frequency, and shows the functional dependence on the transistor parameters and the circuit parameters. Experimental studies on common emitter (CE) amplifiers have been used to validate the PM noise model at carrier frequencies between 10 and 100 MHz.

[Clinical and cytogenetic variability in 12 Mexican families with Fanconi's anemia and its relationship with the complement gruoup to which they belong]. / Variabilidad clínica y citogenética en doce familias mexicanas con anemia de fanconi y su relación con el grupo de complementación al que pertenecen.

OBJECTIVE: To describe the clinical and cytogenetic features of Mexican patients with Fanconi anemia, while assessing whether the phenotypic variation is related to the complementation group. MATERIAL AND METHODS: The cytogenetic diagnosis was done using mitomycin C and diepoxybutane on peripheral blood lymphocytes. The severity of the anemia and each patient's clinical manifestations were classified using Alter's and Auerbach's clinical scores, respectively. Lymphoblastoid cell lines were established for eight patients and complementation group determined following cell fusion procedures in four propositi. RESULTS: Twenty-five Fanconi anemia patients from 12 families were studied. All patients had high, spontaneous and induced chromosomal breakages, no relationship was found between the clinical severity of the disease and the anemic state. Twelve patients were considered severely ill, while the remaining 13 were considered mild cases. Three individuals were anemia-free, while in 13 the anemia was mild, moderate in 7, and severe in 1. The mortality rate was 32% (8/25). No relationship was found between the clinical picture and degree of the anemia or mortality rate. Eleven patients were assigned to complementation group A with mild clinical findings and anemia. Their cytogenetic results showed variability. One patient assigned to group C was considered as a severe case with transfusion-dependent anemia and high sensitivity to mutagens. Thirteen patients were not classified. A lymphoblastoid cell line resistant to mitomycin C was obtained suggesting somatic mosaicism. CONCLUSIONS: The establishment of a complementation group does not necessarily explain variability. There are other important factors, such as somatic mosaicism, that modify the cellular phenotype.

Complete uterine prolapse without uterine mucosal eversion in a queen.

A five-year-old female cat weighing 3 kg was presented by the owner after noticing a large pink, bilobed mass protruding through the vulva during labour. The cat was in good condition, with appropriate lactation, and the newborn kittens were nursing normally. The uterus was not reverted or invaginated at examination, and there was rupture of the mesovarium, mesometrium and uterine-vaginal connection around the cervix. Manual reduction of the prolapsed uterus was not possible because of torn ligaments. A coeliotomy was performed to remove the ovaries, and the apex of the uterine horns was passed by the vaginal route. The remaining part of the mesometrium was disconnected, and the prolapsed uterus was removed. The queen and kittens were discharged from the hospital on the second day after surgery. An unusual feature of this case is that the prolapse was complete, without eversion of any part of the uterus through a vaginal tear.

Effects of intraarticular treatment with stanozolol on synovial membrane and cartilage in an ovine model of osteoarthritis.

Aim of the study was to verify the clinical and morphological effects of intra-articular stanozolol or placebo treatment, lasting 3 and 9 months, in sheep in which a femoro-tibial osteo-arthritis (OA) were surgically induced (medial bilateral meniscectomy). Twenty healthy sheep divided into four groups and two control animals group, after surgical medial bilateral meniscectomy, were weekly injected in femoral-tibial joint (FTJ) with stanozolol or placebo. Lameness evaluation was performed and synovial fluid was collected from all sheep at each treatment time. Necropsies were performed after 3 or 9 month as described in experimental design. Gross pathologies were described and specimen tissues collected from femoro-tibial articular joints were processed for routine histological examination. The gross anatomy of the FTJ was well-preserved in stanozolol-treated sheep; this also applied to the histological features of articular cartilage. Joint aseptic inflammation and fibrosis were observed in placebo-treated sheep, associated with a different degree of severity of condylar and tibial plate cartilage degeneration. Stanozolol intra-articular treatment reduces osteophytes formation and subchondral bone reaction and promotes articular cartilage regeneration.

D-dimer deposits in lungs and kidneys suggest its use as a marker in the clinical workup of dogs with heartworm (Dirofilaria immitis) disease.

It has been reported that dogs with heartworm disease (Dirofilaria immitis) show increased plasma levels of D-dimer, a fibrin degradation product present in the blood after a blood clot is degraded by fibrinolysis. In the present study the authors show that, in dogs with both experimental and natural infections with D. immitis, D-dimer deposits in lungs and kidneys are associated with pulmonary thromboembolism and microfilariemic status, as well as there was a clear association between increased plasma values of D-dimer and positive staining in immunohistochemistry. Results suggest that the monitoring of D-dimer levels in infected dogs could be useful in evaluating the presence of pulmonary thromboembolism in the lungs and that microfilariae may induce microthrombosis in kidneys, thus contributing to renal pathology.

Myocardial damage in dogs affected by heartworm disease (Dirofilaria immitis): immunohistochemical study of cardiac myoglobin and troponin I in naturally infected dogs.

It has recently been reported that dogs affected by canine heartworm disease (Dirofilaria immitis) can show an increase in plasma levels of myoglobin and cardiac troponin I, two markers of muscle/myocardial injury. In order to determine if this increase is due to myocardial damage, the right ventricle of 24 naturally infected dogs was examined by routine histology and immunohistochemistry with anti-myoglobin and anti-cardiac troponin I antibodies. Microscopic lesions included necrosis and myocyte vacuolization, and were associated with loss of staining for one or both proteins. Results confirm that increased levels of myoglobin and cardiac troponin I are indicative of myocardial damage in dogs affected by heartworm disease.