Untargeted NMR-based metabolomics analysis of kidney allograft perfusates identifies a signature of delayed graft function.

INTRODUCTION: Kidney transplantation (KTx) necessarily conveys an ischemia/reperfusion (I/R) process, which impacts on allograft outcomes. Delayed graft function (DGF) is defined as a non-decrease of serum creatinine by at least 10% daily on 3 consecutive days during the first 7 days post-KTx. DGF significantly conditions both short- and long-term graft outcomes. Still there is a lack of DGF predictive biomarkers. OBJECTIVES: This study aimed to explore the potential of kidney graft perfusate metabolomics to predict DGF occurrence. METHODS: 49 human perfusates from grafts categorized upon donor type [donation after brain death (DBD)/donation after circulatory death (DCD)] and DGF occurrence and 19 perfusates from a murine model classified upon death type (DBD/DCD) were collected and analyzed by NMR-based metabolomics. RESULTS: The multivariate analysis of the murine data highlighted significant differences between perfusate metabolomes of DBD versus DCD. These differences were similarly observed in the human perfusates. After correcting for the type of donor, multivariate analysis of human data demonstrated a metabolomics signature that could be correlated with DGF occurrence. CONCLUSIONS: The metabolome of kidney grafts is influenced by the donor's type in both human and pre-clinical studies and could be correlated with DGF in the human DBD cohort. Thus, metabolomic analysis of perfusate applied prior to KTx may represent a new predictive tool for clinicians in a more personalized management of DGF. Moreover, our data paves the way to better understand the impact of donor's types on the biochemical events occurring between death and the hypothermic storage.

[Diagnosis and management of renal allograft rejection]. / Diagnostic et prise en charge thérapeutique du rejet en transplantation rénale.

Renal allograft rejection involves many mechanisms of innate and adaptive immunity, responsible for parenchymal inflammatory lesions that negatively impact the long-term outcomes of the renal allograft. The heterogeneous presentations of rejections in terms of clinical, biological and histological aspects make them difficult to manage in daily clinical practice. Indeed, current therapeutic strategies are disappointing in term of long-term outcomes, including graft survival. In this article, we will discuss the main effector mechanisms of rejection and their histological classification, as well as the existing treatments and those currently under evaluation.

: Le rejet du greffon rénal fait intervenir de nombreux mécanismes de l'immunité innée et adaptative, responsables de lésions inflammatoires parenchymateuses impactant négativement le devenir au long cours du greffon rénal. La grande hétérogénéité dans la présentation clinique, biologique et histologique des rejets de greffe en fait des entités difficiles à prendre en charge en pratique clinique quotidienne. En effet, les stratégies thérapeutiques actuelles montrent des résultats assez décevants pour le traitement des rejets, ce qui a comme conséquence une diminution significative de la survie des greffons. Nous aborderons dans cet article les principaux mécanismes effecteurs des rejets, leur classification histologique ainsi que les traitements existants et en cours de validation.

Early data on long-term efficacy and safety of inotersen in patients with hereditary transthyretin amyloidosis: a 2-year update from the open-label extension of the NEURO-TTR trial.

BACKGROUND AND PURPOSE: Hereditary transthyretin (hATTR) amyloidosis causes progressive polyneuropathy resulting from transthyretin (TTR) amyloid deposition throughout the body, including the peripheral nerves. The efficacy and safety of inotersen, an antisense oligonucleotide inhibitor of TTR protein production, were demonstrated in the pivotal NEURO-TTR study in patients with hATTR polyneuropathy. Here, the long-term efficacy and safety of inotersen are assessed in an ongoing open-label extension (OLE) study. METHODS: Patients who completed NEURO-TTR were eligible to enroll in the OLE (NCT02175004). Efficacy assessments included the modified Neuropathy Impairment Score plus seven neurophysiological tests composite score (mNIS + 7), the Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) questionnaire total score and the Short-Form 36 Health Survey (SF-36) Physical Component Summary (PCS) score. Safety and tolerability were also assessed. RESULTS: Overall, 97% (135/139) of patients who completed NEURO-TTR enrolled in the OLE. Patients who received inotersen for 39 cumulative months in NEURO-TTR and the OLE continued to show benefit; patients who switched from placebo to inotersen in the OLE demonstrated improvement or stabilization of neurological disease progression by mNIS + 7, Norfolk QOL-DN and SF-36 PCS. No new safety concerns were identified. There was no evidence of increased risk for grade 4 thrombocytopenia or severe renal events with increased duration of inotersen exposure. CONCLUSION: Inotersen slowed disease progression and reduced deterioration of quality of life in patients with hATTR polyneuropathy. Early treatment with inotersen resulted in greater long-term disease stabilization than delayed initiation. Routine platelet and renal safety monitoring were effective; no new safety signals were observed.

Metal-based antimicrobial strategies against intramacrophage Mycobacterium tuberculosis.

The combinatorial chemistry has been an important tool for the development of new strategies against the Mycobacterium tuberculosis. Therefore, we evaluated the antimycobacterial activity of two coordinated metal complexes (Cu(II) and Co(II)) and a free ligand, including in the intramacrophage environment. The complexes were more active than the free ligand, indicating that the complexation favoured the antimicrobial activity. None of the compounds showed cytotoxic effect at the concentration of 200 µg ml-1 and both complexes showed intracellular antimicrobial activity, with results as effective as rifampicin. In this study, it was possible to identify complexes containing benzohydroxamate associated with transition metal ions (Cu2+ and Co2+ ), which were able to inhibit the growth of M. tuberculosis, including in persistence stage. In addition, the docking analysis allows inferring a possible interaction of the metal complexes with the enzyme urease, which has been reported as crucial for the bacillus survival in the intraphagosomal environment. Thus, these set of results demonstrate the potential of these metals in the development of new drugs against M. tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, it was possible to identify complexes containing benzohydroxamate associated with transition metals (Cu2+ and Co2+ ), which were able to inhibit the growth of Mycobacterium tuberculosis, including in the persistence stage. In this context, cobalt and copper can be scaffolds for new drugs against M. tuberculosis.

Presenting phenotype of paediatric inflammatory bowel disease in Wessex, Southern England 2010-2013.

AIM: There has been at least a twofold increase in the incidence of paediatric inflammatory bowel disease (PIBD) over the last 20 years; we report the presenting features from 2010 to 2013 and compare with previous data. METHODS: All patients diagnosed with PIBD at University Hospitals Southampton from 2010 to 2013 were identified from an in-house database. Data were obtained from paper and electronic notes. Height, weight and BMI SDS are presented as median values (95% CI). RESULTS: One hundred and seventy-two patients were included (median age at diagnosis 13.5, 115 male); Crohn's disease (CD) - 107, UC - 50, inflammatory bowel disease unclassified (IBDU) - 15. The most common presenting features of CD were abdominal pain (86%), diarrhoea (78.5%) and weight loss (56.1%); 42.1% of patients had all three. In UC blood in stool (92%), diarrhoea (92%) and abdominal pain (88%) were the most common; all three in 76% of patients. CD presented with ileocolonic disease in 52.5%. UC presented with pancolitis in 64%. There was growth delay in CD: height -0.37 (-0.60 to -0.14); weight -1.09 (-1.35 to -0.83). Growth was maintained in UC: height 0.53 (0.19 to 0.87); weight 0.14 (-0.20 to 0.48). CONCLUSION: Paediatric inflammatory bowel disease phenotype remains as extensive despite increasing incidence. Although the classical phenotype is common, a reasonable proportion present with atypical features, normal growth and normal blood markers.

The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors.

Gene transfer after intra-amniotic injection has, in general, been of low efficiency and limited to epithelial cells in the skin, pulmonary and gastrointestinal system. We have recently shown that early gestational administration results in a more efficient gene transfer to developmentally accessible stem cell populations in the skin and eye. In this study we present a comprehensive analysis of patterns of tissue expression seen after early intra-amniotic gene transfer (IAGT) using lentiviral vectors. To assess the influence of developmental stage on tissue expression, injections were administered from the late head fold/early somite stage (E8) to E18. In early gestation (E8-10), green fluorescent protein (GFP) expression was observed in multiple organs, derived from all three germ layers. Remarkably, GFP expression was observed in tissues derived from mesoderm and neural ectoderm at E8, whereas expression was limited to only epithelial cells of ectoderm- and endoderm-derived organs after E11. The amount and duration of gene expression was much higher after IAGT at early gestational time points. The observed temporal patterns of gene expression correspond to the predicted developmental accessibility of organ-specific cell populations. This model may be useful for the analyses of mechanisms of genetic and/or developmental disease and for the development of prenatal gene therapy for specific disorders.

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients.

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-alpha2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin-alpha2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750-1713_7899-2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin-alpha2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.

Effects of TNF-alpha blockade in monocrotaline-induced pulmonary hypertension.

INTRODUCTION: TNF-alpha blockade in ischemic heart failure is still the subject of debate since clinical trials show conflicting results. However, its benefit in heart failure secondary to pulmonary hypertension has yet to be determined. It has been reported that transgenic rats overexpressing TNF-alpha develop pulmonary hypertension. The aim of this study was to assess the morphologic and hemodynamic effects of administration of an anti-TNF-alpha monoclonal antibody (etanercept) in rats with monocrotaline (MCT)-induced pulmonary hypertension. METHODS: Adult Wistar rats were injected with MCT (60 mg/Kg sc), or vehicle only (day 0). Beginning one day later, the animals were randomly treated with etanercept (ETC, 0.03 mg/Kg sc, three times a week) or with a similar volume of vehicle. The study thus had four groups: Ctrl (n = 6), Ctrl + ETC (n = 6), MCT (n = 6) and MCT + ETC (n = 6). On days 22-23, the rats were instrumented to record right ventricular systolic and end-diastolic pressures, dP/dtmax and tau. At the end of each experiment the heart and lungs were weighed. RESULTS AND CONCLUSIONS: Chronic administration of etanercept induced only a slight increase in relaxation velocity, with no effect on other hemodynamic parameters, including pulmonary hypertension, and no reduction in right ventricular hypertrophy. These results suggest that etanercept does not lead to a significant improvement in heart failure secondary to pulmonary hypertension.

Classification of Paediatric Inflammatory Bowel Disease using Machine Learning.

Paediatric inflammatory bowel disease (PIBD), comprising Crohn's disease (CD), ulcerative colitis (UC) and inflammatory bowel disease unclassified (IBDU) is a complex and multifactorial condition with increasing incidence. An accurate diagnosis of PIBD is necessary for a prompt and effective treatment. This study utilises machine learning (ML) to classify disease using endoscopic and histological data for 287 children diagnosed with PIBD. Data were used to develop, train, test and validate a ML model to classify disease subtype. Unsupervised models revealed overlap of CD/UC with broad clustering but no clear subtype delineation, whereas hierarchical clustering identified four novel subgroups characterised by differing colonic involvement. Three supervised ML models were developed utilising endoscopic data only, histological only and combined endoscopic/histological data yielding classification accuracy of 71.0%, 76.9% and 82.7% respectively. The optimal combined model was tested on a statistically independent cohort of 48 PIBD patients from the same clinic, accurately classifying 83.3% of patients. This study employs mathematical modelling of endoscopic and histological data to aid diagnostic accuracy. While unsupervised modelling categorises patients into four subgroups, supervised approaches confirm the need of both endoscopic and histological evidence for an accurate diagnosis. Overall, this paper provides a blueprint for ML use with clinical data.

Decellularization of placentas: establishing a protocol.

Biological biomaterials for tissue engineering purposes can be produced through tissue and/or organ decellularization. The remaining extracellular matrix (ECM) must be acellular and preserve its proteins and physical features. Placentas are organs of great interest because they are discarded after birth and present large amounts of ECM. Protocols for decellularization are tissue-specific and have not been established for canine placentas yet. This study aimed at analyzing a favorable method for decellularization of maternal and fetal portions of canine placentas. Canine placentas were subjected to ten preliminary tests to analyze the efficacy of parameters such as the type of detergents, freezing temperatures and perfusion. Two protocols were chosen for further analyses using histology, scanning electron microscopy, immunofluorescence and DNA quantification. Sodium dodecyl sulfate (SDS) was the most effective detergent for cell removal. Freezing placentas before decellularization required longer periods of incubation in different detergents. Both perfusion and immersion methods were capable of removing cells. Placentas decellularized using Protocol I (1% SDS, 5 mM EDTA, 50 mM TRIS, and 0.5% antibiotic) preserved the ECM structure better, but Protocol I was less efficient to remove cells and DNA content from the ECM than Protocol II (1% SDS, 5 mM EDTA, 0.05% trypsin, and 0.5% antibiotic).

Differential right and left ventricular diastolic tolerance to acute afterload and NCX gene expression in Wistar rats.

This study evaluated right ventricular (RV) and left ventricular (LV) diastolic tolerance to afterload and SERCA2a, phospholamban and sodium-calcium exchanger (NCX) gene expression in Wistar rats. Time constant tau and end diastolic pressure-dimension relation (EDPDR) were analyzed in response to progressive RV or LV afterload elevations, induced by beat-to-beat pulmonary trunk or aortic root constrictions, respectively. Afterload elevations decreased LV- tau, but increased RV-tau. Whereas LV- tau analyzed the major course of pressure fall, RV- tau only assessed the last fourth. Furthermore, RV afterload elevations progressively upward shifted RV EDPDR, whilst LV afterload elevations did not change LV-EDPDR. SERCA2a and phospholamban mRNA were similar in both ventricles. NCX-mRNA was almost 50 % lower in RV than in LV. Left ventricular afterload elevations, therefore, accelerated the pressure fall and did not induce diastolic dysfunction, indicating high LV diastolic tolerance to afterload. On the contrary, RV afterload elevations decelerated the late RV pressure fall and induced diastolic dysfunction, indicating small RV diastolic tolerance to afterload. These results support previous findings relating NCX with late Ca(2+) reuptake, late relaxation and diastolic dysfunction.

Simultaneous pancreas-kidney transplantation: five-year results from a single center.

We report the 5-year results of our simultaneous pancreas-kidney transplantation (SPKT) program, started on May 2, 2000. Forty-two SPKT were performed on 42 type I diabetic patients with chronic renal failure. The procedure was performed with enteric diversion and vascular anastomosis to the iliac vessels. Immunosuppressive protocol included antithymocyte globulin, tacrolimus, mycophenolate mofetil, and steroids. The 24 women and 18 men had a mean age of 33.5 +/- 6.3 years and mean 22.8 +/- 14.2 years time of diabetes evolution. Forty patients had been on dialysis for 34.3 +/- 24.1 months, and two were preemptive transplantations. Acute rejection episodes were treated in eight patients (19.1%): in three cases they affected both organs; in two only the kidney was affected; and the other three were pancreas graft rejections. The incidence of postoperative complications requiring re-operation was 42.9%, mostly pancreas graft related. Two patients died, one due to cardiovascular disease; the other was transplant related. Three kidney grafts were lost, and the causes were immunologic, thrombosis, and patient death. Pancreas graft loss occurred in seven patients: thrombosis (n = 3); infection (n = 3); immunologic (n = 1). The patients with surviving grafts were doing well, with normal kidney and pancreas function: serum creatinine = 0.89 +/- 0.15 mg/dL; fasting blood glucose = 79 +/- 16 mg/dL; HbA1c = 4.7 +/- 1.1%. The 1-year patient, kidney, and pancreas survival rates were 97.3%, 94.6%, and 83.8% and 5-year values, 91.7%, 89.2%, and 78.7%, respectively. In conclusion, these results are similar to the most recent UNOS/IPTR reports, leading us to consider our experience with SPKT very positive.

Morphologic, molecular and hemodynamic cardiac alterations in a model of nephropathy induced by puromycin aminonucleoside.

INTRODUCTION: Proteinuria and decreased glomerular filtration rate are assuming increased importance in defining cardiovascular risk in chronic renal insufficiency. The aim of this work was to study morphologic, molecular and hemodynamic cardiac alterations in an animal model of proteinuria and renal insufficiency induced by puromycin aminonucleoside (PAN). METHODS: Normotensive rats (n = 14) were injected with PAN (150 mg/kg, i.p.) or with vehicle. Blood pressure was measured daily and the animals were placed in metabolic cages for evaluation of urinary excretion of sodium, protein and creatinine. Fourteen days after PAN administration left ventricular hemodynamics were evaluated through a pressure tip micromanometer and heart morphology was examined. Transmural samples of left ventricle were then taken for mRNA quantification of SERCA2a, phospholamban (PLB), insulin-like growth factor 1 (IGF-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). RESULTS: The animals treated with PAN presented a decrease in creatinine clearance (14th day: 2.24 +/- 0.32 vs. 4.51 +/- 1.08 ml/min) and an increase in proteinuria (14th day: 51.0 +/- 9.0 vs. 3.8 +/- 0.7 mg/mg creatinine), without changes in systolic (14th day: 151 +/- 7 vs. 141 +/- 6 mmHg) or diastolic blood pressure (14th day: 85 +/- 7 vs. 86 +/- 3 mmHg), These alterations were accompanied by cardiac atrophy with decreased left ventricular contractility. A reduction in the SERCA2a/PLB mRNA ratio was observed without significant alteration in the expression of IGF-1 in the left ventricle. CONCLUSIONS: PAN-induced nephropathy is accompanied by cardiac atrophy, left ventricular dysfunction and alterations in the expression of genes involved in myocardial calcium kinetics. These findings were not accompanied by increases in blood pressure and may contribute to our understanding of the increased cardiovascular risk in chronic renal insufficiency.

A Comparative, Prospective and Randomized Evaluation of Roux-en-Y Gastric Bypass With and Without the Silastic Ring: A 2-Year Follow Up Preliminary Report on Weight Loss and Quality of Life.

BACKGROUND: Currently, Roux-en-Y gastric bypass (RYGB) is one of the most widely used bariatric surgeries. Banding the pouch forms a banded gastric bypass operation, an accepted and frequently used variant. Placing a silastic ring around the pouch to band the gastric bypass operation increases the restriction mechanism. However, the ubiquitous use of the banded gastric bypass remains controversial. One of the controversies is the effect of the silastic ring on patients' perception of their well being after surgery because of the frequency of vomiting. A prospective, blindly randomized, comparative trial was undertaken to resolve this controversy. METHOD: Four hundred subjects scheduled for gastric bypass surgery were randomized into two arms of the trial, 200 with a silastic ring (WR) and 200 without (NR). After 2-year follow-up, the variables associated with the scores of Bariatric Analysis and Reporting Outcome System (BAROS) were analyzed. RESULTS: The initial median weight (125 kg), BMI (47), and age (36 years) were the same in both the NR and WR groups. The median excess weight loss, weight regain, and incidence of vomiting were 71, 10.5, and 7.75%, respectively, in the NR group vs. 75.4 and 1.1, and 24.4% in the WR group. The mean QOL score was 79% in the NR group vs. 80% in the WR group. CONCLUSION: After 2-year follow-up, silastic ring placement in the RYGB resulted in greater weight loss and weight stability and a threefold greater incidence of vomiting. There was no difference in the scores in the quality of life analysis.

Minimally invasive repair of Morgagni hernia - A multicenter case series.

Children may benefit from minimally invasive surgery (MIS) in the correction of Morgagni hernia (MH). The present study aims to evaluate the outcome of MIS through a multicenter study. National institutions that use MIS in the treatment of MH were included. Demographic, clinical and operative data were analyzed. Thirteen patients with MH (6 males) were operated using similar MIS technique (percutaneous stitches) at a mean age of 22.2±18.3 months. Six patients had chromosomopathies (46%), five with Down syndrome (39%). Respiratory complaints were the most common presentation (54%). Surgery lasted 95±23min. In none of the patients was the hernia sac removed; prosthesis was never used. In the immediate post-operative period, 4 patients (36%) were admitted to intensive care unit (all with Down syndrome); all patients started enteral feeds within the first 24h. With a mean follow-up of 56±16.6 months, there were two recurrences (18%) at the same institution, one of which was repaired with an absorbable suture; both with Down syndrome. The application of MIS in the MH repair is effective even in the presence of comorbidities such as Down syndrome; the latter influences the immediate postoperative recovery and possibly the recurrence rate. Removal of hernia sac does not seem necessary. Non-absorbable sutures may be more appropriate.

Thoracoscopy in the management of pediatric empyemas.

INTRODUCTION: Thoracoscopy is increasingly being used in the treatment of empyema. This study assesses feasibility, efficacy and safety in children. MATERIAL AND METHODS: Clinical files of patients who underwent primary thoracoscopy for empyema between 2006 and 2014 were reviewed. Demographic, clinical and surgical data were analyzed and a comparison between the period before (period1) and after (period2) the learning curve was performed. RESULTS: Ninety-one patients (53 males, 58%) were submitted to thoracoscopy at a median age of 4 years. There were 19 conversions to thoracotomy with a steady decrease of conversion rate until 2009 (period1) and no conversions thereafter (period2). There was no difference in any of the analyzed parameters between patients submitted to thoracoscopy alone and those requiring conversion in period1. Six cases (6.6%) needed redo-operation (five in period2) and thoracotomy was the elected approach in four. Necrotizing pneumonia was present in 60% of the reoperated cases; in other words, in period2 3 out of 9 cases with necrotizing pneumonia required reintervention (p=0.07). Thoracotomy was avoided in sixty-eight (75%) patients (62% in period1 versus 92% in period2, p=0.001). DISCUSSION AND CONCLUSIONS: Thoracoscopic approach for empyema is feasible and safe avoiding a significant number of thoracotomies after a short learning curve. An increase of reintervention rate should be expected, but throracoscopy alone is effective in the great majority of the cases. Necrotizing pneumonia may be associated with a higher risk of reintervention, as it is a contra-indication to thoracoscopy and probably surgery.

IgA antibody responses to Mycobacterium tuberculosis recombinant MPT-64 and MT-10.3 (Rv3019c) antigens in pleural fluid of patients with tuberculous pleurisy.

SETTING: Microbiological tests lack sensitivity for pleural tuberculosis (TB) and histopathology is expensive, time consuming and needs specialised personnel. Immunoassay (ELISA) may be a promising approach in this respect. OBJECTIVE: To evaluate the reactivity of IgA antibody to MPT-64 and MT-10.3 recombinant mycobacterial protein antigens in pleural fluid as a marker of pleural TB, based on the fact that IgA is the main antibody in the mucosa/serosa of the gastrointestinal and respiratory tract. METHOD: Anti-MPT-64 and MT-10.3 IgA response was determined by ELISA in 72 patients with pleural TB and 27 with other pleural conditions. RESULTS: High sensitivities for IgA were measured against MPT-64 (52/72, 72%) and MT-10.3 (52/72,72%) antigens. Combining the sensitivities of both antigens, further increase in sensitivity (55/72, 76%) was obtained with no loss of specificity (96%). Similar IgA reactivity was obtained from culture-negative and culture-positive specimens. In eight pleural TB patients with human immunodeficiency virus (HIV) co-infection, the sensitivity was 88% (7/8). CONCLUSION: To our knowledge, this is the first description of the presence of IgA antibody pleural TB effusion reactive to MPT-64 and MT-10.3, with sensitivity similar to histopathological examination, which is presently considered the gold standard for pleural TB.

Thyroxine binding in a TTR Met 119 kindred.

Recently, a transthyretin variant, TTR Met 119, in which methionine substitutes for threonine 119, a component of the protein's iodothyronine binding site, was identified in individuals with transient euthyroid hyperthyroxinemia. Healthy carriers of Met 119 have normal serum thyroid hormone concentrations, but two studies of Met 119 carriers have differed as to whether T4 binding to TTR is increased. An additional kindred has been identified by hybrid isoelectric focusing in an ongoing screening program for TTR variants in the Portuguese population with TTR Met 30 associated familial amyloidotic polyneuropathy. Cyanogen bromide peptide mapping and DNA restriction length polymorphism analyses showed that the propositus was a compound heterozygote for two TTR variants: Asn 90 and Met 119. Family analysis revealed that he inherited the TTR Met 119 variant from the mother and the TTR Asn 90 variant from the father. Neither the compound heterozygote nor his parents had symptoms of familial amyloidotic polyneuropathy. Serum dialysis with stepwise saturation of iodothyronine binding sites confirmed that TTR binding of T4 is increased in TTR Met 119. The increased binding is due to a higher TTR concentration rather than an increased association constant for T4. Because of the small proportion of serum T4 bound by TTR, increased T4 binding by TTR did not affect the ratio of free to bound T4 or T4 concentrations. In contrast, plasma retinol binding protein, almost all of which is bound by TTR, was elevated. The Asn 90 mutation does not affect either the concentration or the hormone binding characteristics of the protein. Possible long-term effects of these mutations and the combined heterozygotic state remain to be determined.

PACAP(6-38) inhibits the growth of prostate cancer cells.

The effects of pituitary adenylyl cyclase activating polypeptide (PACAP) analogs on prostate cancer cell lines was investigated. 125I-PACAP-27 bound with high affinity to PC-3 cells (Kd = 10 nM) to a single class of sites (Bmax = 30000/cell). By RT-PCR, a major 305 bp band was observed using cDNA derived from PC-3, LNCaP or DU-145 cells. Specific 125I-PACAP binding was inhibited with high affinity by PACAP-27, PACAP-38 and PACAP(6-38) (IC50 values of 15, 10 and 300 nM, respectively) but not by PACAP(28-38). PACAP elevated cAMP and the increase caused by PACAP-27 was reversed by PACAP(6-38). PACAP transiently increased c-fos gene expression and the increase in c-fos mRNA was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in PC-3 cells, whereas PACAP(6-38) reduced colony number and size. In nude mice bearing PC-3 xenografts, PACAP(6-38) significantly slowed tumor growth. These data suggest that biologically active type 1 PACAP receptors are present on human prostate cancer cells and that prostate cancer cell growth is inhibited by PACAP(6-38).