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Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX 1 R) may form complexes with the corticotropin releasing factor CRF 2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF 2 of OX 1 R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ 1 ) and 2 (σ 2 ) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ 1 receptors interact with CRF 2 receptors and that σ 2 receptors interact with OX 1 R. Moreover, we show that amphetamine effect on CRF 2 receptors was mediated by σ 1 R whereas the effect on OX 1 receptors was mediated by σ 2 R. Amphetamine did potentiate the negative cross-talk occurring within the CRF 2 -OX 1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF 2 R antagonist. These results show that amphetamine impacts on the OX 1 R-, CRF 2 R- and OX 1 R/CRF 2 R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: the incidence of obstetric sphincter tears has risen to 15-30% and the prevalence of anal incontinence (AI) symptoms after childbirth may be as high as 40%. The present study evaluates the correlation between obstetric injuries detected by endoanal ultrasound (3D-EUS) and AI symptoms, as well as their impact on the quality of life (QOL) of women after childbirth., Methods: a prospective observational study was performed of pregnant women evaluated before (baseline) and three months after childbirth to ensure the integrity of the anal sphincters and to evaluate possible injuries. The Fecal Incontinence Quality of Life (FIQL) questionnaire and the Cleveland Clinic Score of Incontinence (Wexner) were completed before and after childbirth. The questionnaire results were correlated with an assessment of sphincter defects performed by 3D-EUS., Results: a total of 56 females were included in the study. Overall, 48% developed symptoms of AI after childbirth, with a significant decrease in their FIQL compared to the initial evaluation, 3.9 (0.05) vs 3.4 (0.8), respectively (p = 0.000). In addition, 42% of the cohort presented with some kind of obstetric sphincter defect on the 3D-EUS. Instrumental assisted delivery and the sphincter defects were the only two significant factors identified via multivariate analysis that were associated with a decrease in QOL (0.4, 95% CI, 0.07-0.8)., Conclusions: AI after childbirth was associated with a huge impact on QOL, especially in patients with sphincter injuries. A complete clinical evaluation, including 3D-EUS, is recommended to prevent, manage or treat AI in primiparous females.

Objective: To evaluate the efficacy of respiratory muscular training in the weaning of mechanical ventilation and respiratory muscle strength in patients on mechanical ventilation of 48hours or more., Design: Randomized controlled trial of parallel groups, double-blind. Ambit: Intensive Care Unit of a IV level clinic in the city of Cali., Patients: 126 patients in mechanical ventilation for 48hours or more., Interventions: The experimental group received daily a respiratory muscle training program with treshold, adjusted to 50% of maximal inspiratory pressure, additional to standard care, conventional received standard care of respiratory physiotherapy. MAIN INTEREST VARIABLES: weaning of mechanical ventilation. Other variables evaluated: respiratory muscle strength, requirement of non-invasive mechanical ventilation and frequency of reintubation., Analysis: intention-to-treat analysis was performed with all variables evaluated and analysis stratified by sepsis condition., Results: There were no statistically significant differences in the median weaning time of the MV between the groups or in the probability of extubation between groups (HR: 0.82 95% CI: 0.55-1.20 P=.29). The maximum inspiratory pressure was increased in the experimental group on average 9.43 (17.48) cmsH20 and in the conventional 5.92 (11.90) cmsH20 (P=.48). The difference between the means of change in maximal inspiratory pressure was 0.46 (P=.83 95%CI -3.85 to -4.78)., Conclusions: respiratory muscle training did not demonstrate efficacy in the reduction of the weaning period of mechanical ventilation nor in the increase of respiratory muscle strength in the study population. Registered study at ClinicalTrials.gov (NCT02469064)., (Copyright © 2017 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.)

Objective: The CADIT-CAM study was designed to retrospectively analyze the clinical characteristics, treatments, and outcomes of patients with differentiated thyroid carcinoma (DTC) in Castilla La Mancha., Patients and Methods: A total of 1434 patients from 7 hospitals in Castilla La Mancha were enrolled into the study from 2001 to 2015., Results: Seventy-seven percent of patients were female, with a mean age at diagnosis of 48 years. Papillary thyroid carcinoma accounted for 93% of cases. Mean tumor size was significantly smaller at final follow-up (P<.05). Radioiodine ablation (RA) was performed in 84% of patients, and its use decreased during the study, especially in tumors with low recurrence risk. Recurrence occurred in 22% of patients and was associated to male gender, greater tumor size, multifocality, lymph node metastases, extrathyroid involvement, distant metastases and increasing thyroglobulin antibody titers. At the end of follow-up 76.2% of patients were alive and free of disease, 2.4% had died from DTC. Overall survival of the cohort was 95.1% at 15 years of follow-up., Conclusions: Characteristics of DTC in this Spanish cohort are similar to those reported in other studies in our country. Final results were excellent and use of treatment (RA) was consistent with risk-stratified recommendations., (Copyright © 2018 SEEN y SED. Publicado por Elsevier España, S.L.U. All rights reserved.)

Despite ancient knowledge on cocaine appetite-suppressant action, the molecular basis of such fact remains unknown. Addiction/eating disorders (e.g., binge eating, anorexia, bulimia) share a central control involving reward circuits. However, we here show that the sigma-1 receptor (σ 1 R) mediates cocaine anorectic effects by interacting in neurons with growth/hormone/secretagogue (ghrelin) receptors. Cocaine increases colocalization of σ 1 R and GHS-R1a at the cell surface. Moreover, in transfected HEK-293T and neuroblastoma SH-SY5Y cells, and in primary neuronal cultures, pretreatment with cocaine or a σ 1 R agonist inhibited ghrelin-mediated signaling, in a similar manner as the GHS-R1a antagonist YIL-781. Results were similar in G protein-dependent (cAMP accumulation and calcium release) and in partly dependent or independent (ERK1/2 phosphorylation and label-free) assays. We provide solid evidence for direct interaction between receptors and the functional consequences, as well as a reliable structural model of the macromolecular σ 1 R-GHS-R1a complex, which arises as a key piece in the puzzle of the events linking cocaine consumption and appetitive/consummatory behaviors.

Purpose: To analyse trends in hip fracture (HF) rates in patients with rheumatoid arthritis (RA) over an extended time period (17 years)., Methods: This observational retrospective survey was performed by reviewing data from the National Surveillance System for Hospital Data, which includes more than 98% of Spanish hospitals. All hospitalisations of patients with RA and HF that were reported from 1999 to 2015 were analysed. Codes were selected using the Ninth International Classification of Diseases, Clinical Modification: ICD-9-CM: RA 714.0 to 714.9 and HF 820.0 to 820.3. The crude and age-adjusted incidence rate of HF was calculated by age and sex strata over the last 17 years. General lineal models were used to analyse trends., Results: Between 1999 and 2015, 6656 HFs occurred in patients with RA of all ages (84.25% women, mean age 77.5 and 15.75% men, mean age 76.37). The age-adjusted osteoporotic HF rate was 221.85/100 000 RA persons/ year (women 227.97; men 179.06). The HF incidence rate increased yearly by 3.1% (95% CI 2.1 to 4.0) during the 1999-2015 period (p<0.001) and was more pronounced in men (3.5% (95% CI 2.1 to 4.9)) than in women (3.1% (95% CI 2.3 to 4.1)). The female to male ratio decreased from 1.54 in 1999 to 1.14 in 2015. The average length of hospital stays (ALHS) decreased (p<0.001) from 16.76 days (SD 15.3) in 1999 to 10.78 days (SD 7.72) in 2015. Age at the time of hospitalisation increased (p<0.001) from 75.3 years (SD 9.33) in 1999 to 79.92 years (SD 9.47) in 2015. There was a total of 326 (4.9%) deaths during admission, 247 (4.4%) in women and 79 (7.5%) in men (p<0.001)., Conclusion: In Spain, despite the advances that have taken place in controlling disease activity and in treating osteoporosis, the incidence rate of HF increased in both male and female patients with RA., Competing Interests: Competing interests: None declared.

Sigma σ 1 and σ 2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ 1 R interacting with dopamine D 1 and D 2 receptors, the potential regulation of dopaminergic transmission by σ 2 R is also unknown. We here demonstrate that σ 2 R may form heteroreceptor complexes with D 1 but not with D 2 receptors. Remarkably σ 1 , σ 2 , and D 1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ 2 R induces bias in signal transduction as σ 2 R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ 1 R, suggest that the D 1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ 1 , σ 2 , and D 1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit.

The mechanisms commanding the activity of dopaminergic neurons of the ventral tegmental area (VTA) and the location of these neurons are relevant for the coding and expression of motivated behavior associated to reward-related signals. Anatomical evidence shows that several brain regions modulate VTA dopaminergic neurons activity via multiple mechanisms. However, there is still scarce knowledge of how the lateral septum (LS) modulates VTA activity. We performed in-vivo dual-probe microdialysis to measure VTA dopamine, glutamate and GABA extracellular levels after LS stimulation in the presence or absence of GABAergic antagonists. Anterograde tracing and immunohistochemical analysis was used to reveal the anatomical relationship between LS and VTA. LS stimulation significantly increased dopamine and GABA, but not glutamate, VTA extracellular levels. Intra VTA infusion of bicuculline, GABA-A receptor antagonist, inhibited the increase of dopamine but not of GABA VTA levels induced by LS stimulation. Intra VTA infusion of indiplon, selective positive allosteric modulator of GABA-A receptors containing alpha1 subunit, significantly increases VTA dopamine extracellular levels induced by LS. Combined c-Fos and tyrosine hydroxylase immunohistochemistry, revealed that LS stimulation increases the activity of dopaminergic neurons in the antero-ventral region of the VTA. Consistently, anterograde tracing with biotinylated dextran amine revealed the existence of fibers arising from the LS to the antero-ventral region of the VTA. Taken together, our results suggest that LS modulates dopaminergic activity in the antero-ventral region of VTA by inhibiting GABAergic interneurons bearing GABA-A receptors containing alpha1 subunit., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Diabetes is the pandemic disease of the modern era, with 10% of these patients having type 1 diabetes mellitus. Despite the prevalence, morbidities, and associated financial burden, treatment options have not changed since the introduction of injectable insulin. To date, over 40,000 pancreas transplants have been performed globally. It remains the only known method for restoring glycemic control and thus curing type 1 diabetes mellitus. The aim of this review is to bring pancreatic transplantation out of the specialist realm, informing practitioners about this important procedure, so that they feel better equipped to refer suitable patients for transplantation and manage, counsel, and support when encountering them within their own specialty. This study was a narrative review conducted in October 2015, with OVID interface searching EMBASE and MEDLINE databases, using Timeframe: Inception to October 2015. Articles were assessed for clinical relevance and most up-to-date content, with articles written in English as the only inclusion criterion. Other sources used included conference proceedings/presentations and unpublished data from our institution (Oxford Transplant Centre). Pancreatic transplantation is growing and has quickly become the gold standard of care for patients with type 1 diabetes mellitus and renal failure. Significant improvements in quality of life and life expectancy make pancreatic transplant a viable and economically feasible intervention. It remains the most effective method of establishing and maintaining euglycemia, halting and potentially reversing complications associated with diabetes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cases of human visceral leishmaniasis (HVL) were not recorded until recently in the Chaco region of northwestern Argentina. Dogs were surveyed at the sites of infection of two HVL index cases in the Chaco region of Salta province. Canine cases (CanL) were diagnosed by two parasitological methods, two molecular methods targeting mini- and maxicircle DNA, and immunochromatographic dipstick. Among 77 dogs studied, 10 (13%) were found infected with Leishmania spp. In seven dogs and two humans, the infecting species was typed as Leishmania (Leishmania) infantum. The same genotype was detected in the human and two of the CanL. Although several diagnostic methods displayed weak or moderate agreement, the concordance values for serology versus maxicircle PCR were very good (Kappa index = 0.84). Sandflies captured in the area were identified as Lutzomyia migonei and Lu. cortelezzii/Lu. sallesi (cortelezzii complex). The focal appearance of leishmaniasis in dogs and humans in a sylvatic region and its relatively low prevalence of infection suggests that L. (L.) infantum transmission to dogs and humans may, in this region, stem from sylvatic reservoirs., (© The American Society of Tropical Medicine and Hygiene.)

Objective: Treatment of Cushing's disease poses interesting dilemmas in clinical practice. The aim of our study was to analyze the outcomes of the different treatments, the control and recurrence rates, and the complications derived from them., Material and Methods: Data were collected from the clinical records of 22 patients over 18 years of age (86.4% women). They had been diagnosed with Cushing's disease between 2000 and 2012, and were monitored at Complejo Hospitalario Universitario-Albacete, Hospital Virgen de la Salud-Toledo Hospital General Universitario de Ciudad Real, Hospital Virgen de la Luz-Cuenca, Hospital Nuestra Señora del Prado-Talavera de la Reina, and Complejo Hospitalario la Mancha Centro-Alcázar de San Juan., Results: Surgery was the treatment of choice in all patients. Biochemical cure was achieved in 72.2% of patients. Nine patients developed in the early postoperative period diabetes insipidus, which became in 2 patients only. Surprisingly, 3 patients with normal postoperative neurohypophyseal function later developed permanent diabetes insipidus. New hormone deficiencies occurred in 7 patients. Seventeen patients received ketoconazole before surgery (5 of them after surgery also), and 70% of them achieved normal urinary free cortisol levels. Three patients also received radiotherapy, and all of them were cured after a median follow-up of 85.5 months; they developed no tumors or other complications., Conclusions: Our study reports the outcomes of management of Cushing's disease in non-reference centers for this disease, possibly giving a realistic picture of standard clinical practice for the condition in Spain., (Copyright © 2014 SEEN. Published by Elsevier España, S.L.U. All rights reserved.)

Nanomedicine has focused on targeted neurotrophic gene delivery to the brain as a strategy to stop and reverse neurodegeneration in Parkinson's disease. Because of improved transfection ability, synthetic nanocarriers have become candidates for neurotrophic therapy. Neurotensin (NTS)-polyplex is a "Trojan horse" synthetic nanocarrier system that enters dopaminergic neurons through NTS receptor internalization to deliver a genetic cargo. The success of preclinical studies with different neurotrophic genes supports the possibility of using NTS-polyplex in nanomedicine. In this review, we describe the mechanism of NTS-polyplex transfection. We discuss the concept that an effective neurotrophic therapy requires a simultaneous effect on the axon terminals and soma of the remaining dopaminergic neurons. We also discuss the future of this strategy for the treatment of Parkinson's disease., From the Clinical Editor: This review paper focuses on nanomedicine-based treatment of Parkinson's disease, a neurodegenerative condition with existing symptomatic but no curative treatment. Neurotensin-polyplex is a synthetic nanocarrier system that enables delivery of genetic cargo to dopaminergic neurons via NTS receptor internalization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Serologic diagnosis of Trypanosoma cruzi infection is important due to the limited sensitivity of direct parasitologic methods for diagnosis in the indeterminate and chronic phases of disease. SAPA antigen has been used in several studies and has been shown to be a good marker for use in the diagnosis of T. cruzi infection. Chagas disease and leishmaniasis are endemic in northern Salta with overlapping zones of transmission, which frequently leads to T. cruzi-Leishmania spp. mixed infections. Diagnosis is complicated by the fact that there is significant cross-reactivity when non-specific antigens are used. We evaluated the reactivity of GST-SAPA antigen in the ELISA test (ELISA-SAPA) against sera from persons infected with T. cruzi (n = 154), leishmaniasis (n = 66), mixed infections (29), and healthy controls (n = 28) using commercial ELISA and IHA kits as reference tests. For ELISA-SAPA the sensitivity, specificity and kappa index were calculated for detection of T. cruzi infection. Among sera from patients infected with leishmaniasis, 30.5% of co-infections were detected. ELISA-SAPA sensitivity was 97.1% (confidence interval 95%: 94.5-99.9), specificity was 100% (confidence interval 95%: 99.4-100), and kappa index was 96% (confidence interval 95%: 93-99%), for detection of T. cruzi infection. Sensitivity, specificity and kappa indices have shown a high efficiency of ELISA-SAPA.

It is important to know whether the variability of species of Leishmania parasites circulating in a region affects the performance of the ELISA test for the diagnosis of leishmaniasis. Therefore, the aim of this study was to analyze the reactivity of the ELISA using homogenates of promastigotes of Leishmania (V.) braziliensis (ELISAb), Leishmania (L) amazonensis (ELISAa) and Leishmania (V.) guyanensis (ELISAg) against different sera groups. Samples from individuals with cutaneous leishmaniasis (n = 37), mucocutaneous leishmaniasis (n = 8), healthy controls (n = 52), persons infected with Trypanosoma cruzi (n = 11) and mixed infections (n = 14) were included in the study. We calculated sensitivities, specificities, cut offs, and predictive values for the three tests and compared them using ANOVA, kappa index, ROC curves comparison, and confidence intervals calculated by the bootstrap method. Significant differences were found when comparing the OD levels of sera from patients with cutaneous leishmaniasis against healthy controls, but there were no differences when comparing the different ELISAs. The sensitivities calculated for ELISAb and ELISAa were 84.6 and of 88.5% for ELISAg, while the value of specificity for the three tests was 96.2. The kappa index (0.87) and comparison of ROC curves showed similar performance for the three ELISAs (p = 0.225). The high reactivity obtained for these ELISAs in sera of patients with mucocutaneous leishmaniasis indicates this test as an important complement in the diagnosis of the disease.

The recent discovery of short neural precursors (SNPs) in the murine neocortical ventricular zone (VZ) challenges the widely held view that radial glial cells (RGCs) are the sole occupants of this germinal compartment and suggests that precursor variety is an important factor of brain development. Here, we use in utero electroporation and genetic fate mapping to show that SNPs and RGCs cohabit the VZ but display different cell cycle kinetics and generate phenotypically different progeny. In addition, we find that RGC progeny undergo additional rounds of cell division as intermediate progenitor cells (IPCs), whereas SNP progeny generally produce postmitotic neurons directly from the VZ. By clearly defining SNPs as bona fide VZ residents, separate from both RGCs and IPCs, and uncovering their unique proliferative and lineage properties, these results demonstrate how individual neural precursor groups in the embryonic rodent VZ create diversity in the overlying neocortex.

Pulsatile perfusion (PP) might be a cost-effective cold preservation technique to reduce the incidence of delayed graft function (DGF) in kidneys from deceased donors. With the aim to address whether PP can reduce the incidence of DGF in kidneys from controlled donors after cardiac death (cDCD), we compared the clinical outcome of 30 recipients of kidneys from cDCD preserved by static cold storage (cDCD-SCS) with 30 recipients of cDCD kidneys preserved by PP (cDCD-PP). The end-points were the incidence of primary nonfunction (PNF), DGF and acute rejection (AR), the length of hospitalization, 1, 3, 6 and 12-months graft function, graft survival and patient survival. Donor, recipient and preimplantation data were well matched. DGF was significantly lower (53.3% vs. 86.6% P<0.001) and the length of hospitalization shorter (10 vs. 14 days P<0.033) in the cDCD-PP group. Similarly, postoperative and short-term graft function (7 and 30 days and 6 and 12 months, respectively) was statistically better in the cDCD-PP than in the cDCD-SCS. In summary, in this cohort, clinical introduction of PP was associated with a significant reduction of DGF, shorter hospitalization and better graft function than SCS.

Background: There are reasons to expect an association with Alzheimer's disease (AD) within the HLA region. The HLA-B & C genes have, however, been relatively understudied. A geographically specific association with HLA-B7 & HLA-Cw*0702 had been suggested by our previous, small study., Methods: We studied the HLA-B & C alleles in 196 cases of 'definite' or 'probable' AD and 199 elderly controls of the OPTIMA cohort, the largest full study of these alleles in AD to date., Results: We replicated the association of HLA-B7 with AD (overall, adjusted odds ratio = 2.3, 95% confidence interval = 1.4-3.7, p = 0.001), but not the previously suggested interaction with the epsilon4 allele of apolipoprotein E. Results for HLA-Cw*0702, which is in tight linkage disequilibrium with HLA-B7, were consistent with those for the latter. Homozygotes of both alleles appeared to be at particularly high risk of AD., Conclusion: HLA-B7 and HLA-Cw*0702 are associated with AD in the Oxford population. Because of the contradictions between cohorts in our previous study, we suggest that these results may be geographically specific. This might be because of differences between populations in the structure of linkage disequilibrium or in interactions with environmental, genetic or epigenetic factors. A much larger study will be needed to clarify the role of homozygosity of HLA alleles in AD risk.

Recently we showed that the neurotensin polyplex is a nanoparticle carrier system that targets reporter genes in nigral dopamine neurons in vivo. Herein, we report its first practical application in experimental parkinsonism, which consisted of transfecting dopamine neurons with the gene coding for human glial cell line-derived neurotrophic factor (hGDNF). Hemiparkinsonism was induced in rats by a single dose of 6-hydroxydopamine (30 microg) into the ventrolateral part of the striatum. We showed that transfection of the hGDNF gene into the substantia nigra of rats 1 week after the neurotoxin injection produced biochemical, anatomical, and functional recovery from hemiparkinsonism. RT-PCR analysis showed mRNA expression of exogenous hGDNF in the transfected substantia nigra. Western blot analysis verified transgene expression by recognizing the flag epitope added at the C-terminus of the hGDNF polypeptide, which was found mainly in dopamine neurons by double immunofluorescence techniques. These data indicate that the neurotensin polyplex holds great promise for the neuroprotective therapy of Parkinson disease.

The rodent dentate gyrus (DG) is formed in the embryo when progenitor cells migrate from the dentate neuroepithelium to establish a germinal zone in the hilus and a secondary germinal matrix, near the fimbria, called the hippocampal subventricular zone (HSVZ). The developmental plasticity of progenitors within the HSVZ is not well understood. To delineate the migratory routes and fates of progenitors within this zone, we injected a replication-incompetent retrovirus, encoding the enhanced green fluorescent protein (EGFP), into the HSVZ of postnatal day 5 (P5) mice. Between P6 and P45, retrovirally-infected EGFP(+) of progenitors migrated into the DG, established a reservoir of progenitor cells, and differentiated into neurons and glia. By P6-7, EGFP(+) cells were observed migrating into the DG. Subsets of these EGFP(+) cells expressed Sox2 and Musashi-1, characteristic of neural stem cells. By P10, EGFP(+) cells assumed positions within the DG and expressed immature neuronal markers. By P20, many EGFP(+) cells expressed the homeobox prospero-like protein Prox1, an early and specific granule cell marker in the CNS, and extended mossy fiber projections into the CA3. A subset of non-neuronal EGFP(+) cells in the dentate gyrus acquired the morphology of astrocytes. Another subset included EGFP(+)/RIP(+) oligodendrocytes that migrated into the fimbria, corpus callosum, and cerebral cortex. Retroviral injections on P15 labeled very few cells, suggesting depletion of HSVZ progenitors by this age. These findings suggest that the early postnatal HSVZ progenitors are multipotent and migratory, and contribute to both dentate gyrus neurogenesis as well as forebrain gliogenesis.

Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.

Background: There is mounting evidence from experimental and clinical studies that the quality of organs from cadaver donors may be influenced by events occurring around the time of brain death, and that these may affect transplant outcome. The aim of this study is to investigate the influence of donor factors on renal allograft outcome in a homogeneous cohort of 518 patients transplanted in a single centre over a 9 year period., Methods: Endpoints of the study were delayed graft function (DGF), acute rejection (AR), 1 year graft survival and long-term survival of those grafts that reached 1 year. Multivariate analysis was performed to determine factors that may have influenced the graft outcome indicators., Results: DGF was the major predictor of graft failure overall with cold ischaemia time (CIT) as an important independent factor. The level of histocompatibility did not influence graft survival. DGF was the major factor affecting 1 year graft survival (P<0.0005) with effects persisting beyond 1 year. DGF was significantly influenced by CIT, donor age, female kidney into male recipient and donor creatinine (P<0.05). Other donor factors and factors associated with donor management were not risk factors for DGF, rejection episodes or graft survival. The risk factors for a number of AR episodes were HLA-DR mismatch and DGF (P<0.005). When grafts surviving for 1 year were considered, only CIT, recipient age and creatinine at 1 year (P<0.05) were found to affect graft survival significantly., Conclusions: The results of this analysis of well-matched transplant recipients show that CIT and DGF are the most important predictors of poor short and long-term graft survival. Therefore, in order to improve the long-term survival of renal allografts efforts should focus on limiting CIT and the damage that occurs during this period and on improving our understanding of DGF.

Background: MICA and MICB (MHC class I-related chain A and B) are polymorphic genes that encode molecules related to MHC class I and are expressed on epithelial cells in response to stress. Incompatible donor MIC antigens can stimulate antibody production in transplant recipients. This study was designed to determine MICB expression in kidney pretransplant and any subsequent changes in expression following transplantation and to correlate changes with inflammatory markers and clinical events., Methods: Paired renal biopsies obtained from living donor (n=10) and cadaveric allografts (n=50) before and 7 days posttransplant were stained for MICB, leukocytic infiltration, and HLA class II antigens., Results: Variable tubular MICB expression was evident in donor biopsies [high 6/60 (10%), low/negative 13/60 (22%), intermediate 41/60 (68%)]. Following transplantation, MICB was up-regulated on renal tubules of 17/60 (28%) biopsies and was associated with MHC class II antigen induction (P=0.02) and leukocyte infiltration (P=0.01). Acute tubular necrosis leading to delayed graft function (DGF) and acute rejection (AR) cause cellular stress within the transplanted kidney. We found a strong association between up-regulation of MICB and cellular stress, 15/17 biopsies with up-regulated MICB expression had AR and/or DGF (P=0.003)., Conclusions: This is the first study demonstrating variable levels of MICB expression in kidneys before transplantation and induction of MICB expression following renal transplantation. MICB expression is associated with HLA class II antigen induction, leukocytic infiltration of the graft and cellular stress in the transplanted kidney. Expression of MICB could contribute significantly to the alloimmune response in mismatched donors and recipients.

DNA repair plays a critical, but imprecisely defined role in excitotoxic injury and neuronal survival throughout adulthood. We utilized an excitotoxic injury model to compare the location and phenotype of degenerating neurons in mice (strain 129-C57BL) deficient in the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), an enzyme required for nonhomologous end joining (NHEJ). Brains from untreated adult heterozygous and DNA-PKcs null mice displayed comparable cytoarchitecture and undetectable levels of cell death. By day 1, and extending through 4 days following kainic acid-induced seizures, brains from DNA-PKcs null mice showed widespread neurodegeneration that encompassed the entire hippocampal CA1-CA3 pyramidal cell layer, entorhinal cortex, and lateral septum, with relative sparing of the dentate gyrus granule cell layer and hilus, as judged by toluidine blue, Fluoro-Jade B, and terminal dUTP nick end labeling staining. In contrast, seizure-related neurodegeneration in heterozygous littermates was limited to the CA3 region of the hippocampus. NeuN and calbindin staining revealed a selective decrease in the number and density of NeuN-positive neurons in the pyramidal layers of degenerating regions in both heterozygous and DNA-PKcs null mice. To elucidate the mechanisms leading to cell death, we examined an involvement of the p53 pathway, known to be induced by DNA damage. Addition of pifithrin-alpha, a p53 inhibitor, or expression of a dominant-negative p53 rescued neurons from kainate-induced excitotoxic cell death in primary cortical cultures derived from wildtype, DNA-PKcs heterozygous, or DNA-PKcs null neonatal mice. Moreover, pifithrin-alpha prevented kainate-induced loss of mitochondrial membrane potential, dendrite degeneration, and cell death. Results suggest that NHEJ plays a neuroprotective role in excitotoxicity, within the perforant, Schaffer collateral, hippocampal-septal, and temperoammonic pathways, in part by repairing DNA damage that would otherwise result in activation of a p53-dependent apoptotic cascade., ((c) 2005 Wiley-Liss, Inc.)

Background: Deposition of the complement protein C4d in renal allograft biopsies obtained during graft dysfunction and rejection has been proposed to be a sensitive marker of antibody-mediated acute rejection. To determine the diagnostic specificity of C4d deposition, it is important to study biopsies from allografts with no evidence of dysfunction. In this study, we examined C4d deposition in protocol biopsies obtained irrespective of clinical status., Methods: Immunohistochemistry for C4d was performed on routine protocol biopsies preimplantation and on day 7 posttransplantation from 48 unselected renal allografts. Serum samples obtained up to 1 month after transplantation were assayed for donor-reactive antibodies (DRA). Results were correlated with histopathology and clinical outcome measures., Results: Diffuse C4d deposition was detected in the peritubular capillaries of 6 of 48 (13%) biopsies. C4d deposition was present in 5 of 15 (33%) biopsies that showed acute rejection (Banff 97, category 4) but only in 1 of 33 (3%) biopsies with no rejection (P=0.003, 97% specificity). Posttransplant DRAs were detected in 21 of 48 (44%) patients. All five recipients with C4d deposition and rejection had posttransplant DRA; the recipient whose biopsy showed C4d positivity, but not rejection, did not have detectable DRA. C4d deposition was not treated with plasmapheresis or intravenous immunoglobulin and was not associated with poor posttransplant graft outcome at 1-year follow-up., Conclusions: Our results show that in early posttransplant protocol biopsies, C4d is a specific marker for the presence of humoral rejection, as indicated by its association with DRA and acute histologic rejection.

Recently we reported that neurotensin-SPDP-poly-L-lysine (NT-vector) is able to bind plasmid DNA (NT-polyplex) and polyfect cells expressing the high-affinity neurotensin receptor (NTRH) although with low transfecting efficiency: in vitro, 6.5+/-1.5%, and in vivo, 5+/-4%. In this work, we attempted to increase the transfecting efficiency by integrating the hemagglutinin HA2 fusogenic peptide and the Vp1 nuclear localization signal of SV40 to the NT-polyplex (fusogenic-karyophilic-NT-polyplex). Confocal microscopy and flow cytometry analysis showed that the fusogenic-karyophilic-NT-polyplex produced mostly nuclear localization of the plasmid DNA in NTRH-bearing N1E-115 cells. About 50% of N1E-115 cells internalized and expressed the reporter gene when the plasmid DNA was transferred by the fusogenic-karyophilic-NT-polyplex. Although to a less extent, the addition of each viral peptide separately to NT-polyplex (fusogenic-NT-polyplex or karyophilic-NT-polyplex) improved polyfection. Fusogenic-NT-polyplex produced 22.41+/-5.96% of internalization and 20.35+/-0.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75+/-3.88% and 10.94+/-2.04%, respectively. Basal internalization and expression were detected in N1E-115 cells in the presence of 100 nM SR-48692 and in NTRH-lacking cells. The fusogenic-karyophilic-NT-polyplex was microinjected into the substantia nigra to test its ability for gene transfer in vivo. Fusogenic-karyophilic-NT-polyplex internalization was observed within dopamine neurons only. Reporter gene expression was observed in a high proportion of dopamine neurons up to 60 days after NT-polyfection. Both internalization and expression were prevented by SR-48692. Our results show that the fusogenic-karyophilic-NT-polyplex is a highly efficient and specific gene vector and encourage its use to transfer gene of physiological interest to NTRH-bearing neurons., (Copyright 2002 Elsevier Science B.V.)

Background: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase., Materials and Methods: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression., Results: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist., Conclusions: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases.

We describe herein a method for synthesizing a non-viral gene vector that exploits the internalization properties of neurotensin (NT), as well as the procedures for a successful gene transfer to cells via the high-affinity NT receptor. The gene vector is NT cross-linked with poly-L-lysine via N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP). The SPDP-derivatives containing either NT or poly-L-lysine are purified by gel filtration. The non-viral vector resulting from the reaction of NT-SPDP with HS-SPDP-poly-L-lysine is purified on Biogel A-1.5 m. This vector is complexed with plasmid DNA at a specific molar ratio to form the NT-polyplex, which ensures the delivery of the gene of interest to cells under conditions of receptor-mediated internalization. The NT-polyplex has shown ability to mediate transient gene expression in vitro [Brain Res. Mol. Brain Res. 69 (1999) 249] and in vivo [Soc. Neurosci. Abstr. 25 (1999) 67. 7]. This approach holds great promise for research and therapy.

In this paper, the results of mercury concentrations in two abiotic compartments (river water and suspended particles) and two biotic compartments (fish and human hair) from the upper Madeira rivers of the Bolivian Amazon basin are presented. Because of the local hydrological regimes and a high deposition rate in the plain, due to the presence of a subsidence zone at the bottom of the Andean piedmont, in the dry season, the highest mercury concentrations and fluxes were not found in rivers where mining activities took place (2.25-6.99 ng l(-1); and 1.07-8.67 mg Hg d(-1) km(-2)), but at the outlet of the Andean basins exploited for their alluvial gold (7.22-8.22 ng l(-1); and 9.47-9.52 mg Hg d(-1) km(-2)). The total mercury concentrations measured in surface waters of the upper Beni basin varied during the dry season, from 2.24 to 2.57 ng l(-1) in the glacial waters of the Zongo river, to 7.00 ng l(-1) in the Madeira River at Porto Velho and 9.49-10.86 ng l(-1) at its confluence with the Amazon. The results obtained from fish indicate, on one hand, that 86% of the piscivorous fishes collected in the Beni river were contaminated, and, on the other hand, their high mercury concentrations could exceed by almost four times the WHO (1976) safety limit. In the Beni River, the mercury concentrations found in omnivorous and mud-feeding fish ranged from 0.02 to 0.19 microg g(-1) (wet wt.), and in piscivorous fish, from 0.33 to 2.30 microg Hg g(-1) (wet wt.). The mercury accumulated by carnivorous fishes was mainly present in its organic form; methylmercury represented 73-98% of the total mercury analysed. Eighty persons were studied in the entire Bolivian Amazonian basin. Unlike the gold miners, who are more affected by tropical diseases, such as malaria and yellow fever, the indigenous people living on the banks of the Beni river, present elevated levels of mercury (9.81 microg g(-1) on average). We observed an increase in contamination in young children still being breast-fed, confirming that hair mercury concentration in babies was significantly affected by maternal mercury contamination during pregnancy. These results show that the major health impacts caused by mercury affect people who are not working directly in gold mining activities but who have a regular fish diet.

We report herein the synthesis of a novel DNA delivery system and in vitro evidence of its ability to transfect cell lines by binding to the high-affinity neurotensin receptor and subsequent internalization of ligand-receptor complexes. The targeting vehicle consisted of neurotensin crosslinked with poly-L-lysine via N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The SPDP-derivatives with either neurotensin or poly-L-lysine were purified by gel filtration. The conjugate resulting of the reaction of neurotensin-SPDP with HS-SPDP-poly-L-lysine was purified through Biogel A 1.5. The neurotensin-SPDP-poly-L-lysine conjugate was able to bind plasmidic DNAs (pSV2cat and pGreen Lantern-1) at optimal molar ratios of 1:5 and 1:6 (DNA: conjugate), respectively. The conjugate internalized those plasmids in the cell lines (N1E-115 and HT-29) bearing the high-affinity neurotensin receptor. Expression of the plasmid products, chloramphenicol acetyltransferase and green fluorescent protein, was observed in such cell lines. Both internalization and expression of the plasmids transferred by the neurotensin-SPDP-poly-L-lysine conjugate were prevented by neurotensin (1 microM) and SR-48692 (100 nM), a specific antagonist of the high-affinity neurotensin receptor. The neurotensin-SPDP-poly-L-lysine conjugate was unable to transfect cell lines lacking the neurotensin receptor (COS-7 and L-929). In rat brain, the high-affinity neurotensin receptor is expressed by specific neurons such as those of the nigrostriatal and mesolimbic dopaminergic systems. Therefore, the neurotensin-SPDP-poly-L-lysine conjugate could be a useful tool for gene delivery to those neuronal systems., (Copyright 1999 Elsevier Science B.V.)