18FDG PET/CT in the early assessment of non-small cell lung cancer response to immunotherapy: frequency and clinical significance of atypical evolutive patterns.

PURPOSE: This prospective study aimed (1) to assess the non-small cell lung cancer (NSCLC) evolutive patterns to immunotherapy using FDG-PET and (2) to describe their association with clinical outcome. DESIGN: Fifty patients with metastatic NSCLC were included before pembrolizumab or nivolumab initiation. FDG-PET scan was performed at baseline and after 7 weeks of treatment (PETinterim1) and different criteria/parameters of tumor response were assessed, including PET response criteria in solid tumors (PERCIST). If a first PERCIST progressive disease (PD) without clinical worsening was observed, treatment was continued and a subsequent FDG-PET (PETinterim2) was performed at 3 months of treatment. Pseudo-progression (PsPD) was defined as a PERCIST response/stability on PETinterim2 after an initial PD. If a second PERCIST PD was assessed on PETinterim2, a homogeneous progression of lesions (termed immune homogeneous progressive-disease: iPDhomogeneous) was distinguished from a heterogeneous evolution (termed immune dissociated-response: iDR). A durable clinical benefit (DCB) of immunotherapy was defined as treatment continuation over a 6-month period. The association between PET evolutive profiles and DCB was assessed. RESULTS: Using PERCIST on PETinterim1, 42% (21/50) of patients showed a response or stable disease, most of them (18/21) reached a DCB. In contrast, 58% (29/50) showed a PD, but more than one-third (11/29) were misclassified as they finally reached a DCB. No standard PETinterim1 criteria could accurately distinguished responding from non-responding patients. Treatment was continued in 19/29 of patients with a first PERCIST PD; the subsequent PETinterim2 demonstrated iPDhomogeneous, iDR and PsPD in 42% (8/19), 26% (5/19), and 32% (6/19), respectively. Whereas no patients with iPDhomogeneous experienced a DCB, all patients with iDR and PsPD reached a clinical benefit to immunotherapy. CONCLUSION: In patients with a first PD on PERCIST and treatment continuation, a subsequent PET identifies more than half of them with iDR and PsPD, both patterns being strongly associated with a clinical benefit of immunotherapy.

An experimental test of density-dependent selection on temperament traits of activity, boldness and sociability.

Temperament traits are seen in many animal species, and recent evolutionary models predict that they could be maintained by heterogeneous selection. We tested this prediction by examining density-dependent selection in juvenile common lizards Zootoca vivipara scored for activity, boldness and sociability at birth and at the age of 1 year. We measured three key life-history traits (juvenile survival, body growth rate and reproduction) and quantified selection in experimental populations at five density levels ranging from low to high values. We observed consistent individual differences for all behaviours on the short term, but only for activity and one boldness measure across the first year of life. At low density, growth selection favoured more sociable lizards, whereas viability selection favoured less active individuals. A significant negative correlational selection on activity and boldness existed for body growth rate irrespective of density. Thus, behavioural traits were characterized by limited ontogenic consistency, and natural selection was heterogeneous between density treatments and fitness traits. This confirms that density-dependent selection plays an important role in the maintenance of individual differences in exploration-activity and sociability.

Disruptive viability selection on a black plumage trait associated with dominance.

Traits used in communication, such as colour signals, are expected to have positive consequences for reproductive success, but their associations with survival are little understood. Previous studies have mainly investigated linear relationships between signals and survival, but both hump-shaped and U-shaped relationships can also be predicted, depending on the main costs involved in trait expression. Furthermore, few studies have taken the plasticity of signals into account in viability selection analyses. The relationship between signal expression and survival is of particular interest in melanin-based traits, because their main costs are still debated. Here, we first determined the main factors explaining variability in a melanin-based trait linked to dominance: the bib size of a colonial bird, the sociable weaver Philetairus socius. We then used these analyses to obtain a measure representative of the individual mean expression of bib size. Finally, we used capture-recapture models to study how survival varied in relation to bib size. Variation in bib size was strongly affected by year and moderately affected by age, body condition and colony size. In addition, individuals bearing small and large bibs had higher survival than those with intermediate bibs, and this U-shaped relationship between survival and bib size appeared to be more pronounced in some years than others. These results constitute a rare example of disruptive viability selection, and point towards the potential importance of social costs incurred by the dominance signalling function of badges of status.

Evidence- and consensus-based (S3) Guidelines for the Treatment of Actinic Keratosis - International League of Dermatological Societies in cooperation with the European Dermatology Forum - Short version.

BACKGROUND: Actinic keratosis (AK) is a frequent health condition attributable to chronic exposure to ultraviolet radiation. Several treatment options are available and evidence based guidelines are missing. OBJECTIVES: The goal of these evidence- and consensus-based guidelines was the development of treatment recommendations appropriate for different subgroups of patients presenting with AK. A secondary aim of these guidelines was the implementation of knowledge relating to the clinical background of AK, including consensus-based recommendations for the histopathological definition, diagnosis and the assessment of patients. METHODS: The guidelines development followed a pre-defined and structured process. For the underlying systematic literature review of interventions for AK, the methodology suggested by the Cochrane Handbook for Systematic Reviews of Interventions, the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was adapted. All recommendations were consented during a consensus conference using a formal consensus methodology. Strength of recommendations was expressed based on the GRADE approach. If expert opinion without external evidence was incorporated into the reasoning for making a certain recommendation, the rationale was provided. The Guidelines underwent open public review and approval by the commissioning societies. RESULTS: Various interventions for the treatment of AK have been assessed for their efficacy. The consenting procedure led to a treatment algorithm as shown in the guidelines document. Based on expert consensus, the present guidelines present recommendations on the classification of patients, diagnosis and histopathological definition of AK. Details on the methods and results of the systematic literature review and guideline development process have been published separately. CONCLUSIONS: International guidelines are intended to be adapted to national or regional circumstances (regulatory approval, availability and reimbursement of treatments).

Contribution of increased ISG15, ISGylation and deregulated type I IFN signaling in Usp18 mutant mice during the course of bacterial infections.

Host genetics has a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss-of-function mutation within the gene ubiquitin-specific peptidase 18 (Usp18(Ity9)), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I interferon (IFN) signaling and as a protease to remove ISG15 from substrate proteins. Usp18(Ity9) mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18(Ity9) mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18(Ity9) mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers and elevated interleukin (IL)-1ß and IL-17. Furthermore, Usp18(Ity9) mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in the lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18(Ity9) mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.

Systematic review of oral treatments for seborrheic dermatitis.

Seborrheic dermatitis (SD) is normally treated with topical corticosteroids and antifungals. Oral therapies can be prescribed in severe or unresponsive cases. This review aims to assess the quantity and quality of published reports on oral therapies for SD. MEDLINE and Embase databases and the reference listings of publications were searched for any publication using oral treatment for SD. The quality of the included publications was assessed using a modified 27 item checklist by Downs and Black. Twenty-one publications (randomized controlled trials, open trials and case reports) covering eight oral therapies (itraconazole, terbinafine, fluconazole, ketoconazole, pramiconazole, prednisone, isotretinoin and homeopathic mineral therapy) were identified. Most of the publications investigated oral antifungals and the quality of the evidence was generally low. The clinical efficacy outcome reported varied considerably between the studies, preventing statistical analysis and direct comparison between treatments. However, ketoconazole therapy was associated with more relapses compared with other treatments. Itraconazole dosing regimen for SD was generally 200 mg/day for the first week of the month followed by 200 mg/day for the first 2 days for 2-11 months. Terbinafine was prescribed at 250 mg/day either as a continuous (4-6 weeks) or as an intermittent regimen (12 days per month) for 3 months. Fluconazole has administered daily (50 mg/day for 2 weeks) or weekly (200-300 mg) for 2-4 weeks. Ketoconazole dosing regimen was 200 mg daily for 4 weeks. Finally, a single 200 mg dose of pramiconazole was administered to patients. This review also highlights key areas for consideration when designing future studies.

Network meta-analysis of the outcome 'participant complete clearance' in nonimmunosuppressed participants of eight interventions for actinic keratosis: a follow-up on a Cochrane review.

The conclusions of pairwise meta-analyses of interventions for actinic keratosis (AK) are limited due to the lack of direct comparison between some interventions. Consequently, we performed a network meta-analysis for eight treatments [5-aminolaevulinic acid (ALA)-photodynamic therapy (PDT), cryotherapy, diclofenac 3% in 2·5% hyaluronic acid (DCF/HA), 5-fluorouracil (5-FU) 0·5% or 5·0%, imiquimod (IMI) 5%, ingenol mebutate (IMB) 0·015-0·05%, methyl aminolaevulinate (MAL)-PDT and placebo/vehicle (including placebo-PDT)] to determine their relative efficacies. As part of a prior Cochrane systematic review, different databases and grey literature were searched for randomized controlled trials up to April 2012. The inclusion criteria were parallel-group studies with nonimmunosuppressed participants: (i) reporting 'participant complete clearance' and (ii) comparing at least two of the interventions. Thirty-two publications met the criteria and they included the following number of individual or pooled studies (n) and total number of participants (N) for the different interventions: 5-FU 0·5% (n = 4, N = 169), 5-FU 5·0% (n = 2, N = 44), ALA-PDT (n = 6, N = 739), cryotherapy (n = 2, N = 174), DCF/HA (n = 5, N = 299), IMI (n = 14, N = 1411), IMB (n = 3, N = 560), MAL-PDT (n = 7, N = 557) and placebo (n = 32, N = 2520). Network analyses using a random-effects Bayesian model were carried out with the software ADDIS v1.16.1. The interventions were ranked as follows based on calculated probabilities and odd ratios: 5-FU > ALA-PDT ≈ IMI ≈ IMB ≈ MAL-PDT > cryotherapy > DCF/HA > placebo. This efficacy ranking was obtained based on the current available data on 'participant complete clearance' from randomized controlled trials and the analysis model used. However, several other factors should also be considered when prescribing a treatment for AK.

Terbinafine in the treatment of dermatophyte toenail onychomycosis: a meta-analysis of efficacy for continuous and intermittent regimens.

OBJECTIVE: To compare mycological and complete cures of terbinafine continuous and intermittent regimens in the treatment of toenail onychomycosis. METHODS: The PubMed database was searched using the terms "terbinafine", "onychomycosis", "continuous" and "pulse(d)" or "intermittent". The inclusion criteria were head-to-head comparison of terbinafine pulse and continuous regimens for dermatophyte toenail infections. Risk ratios were calculated for intention-to-treat and evaluable patient analyses, when possible. Pooled estimates for total and subgroup analyses were calculated using a random effect model, Mantel-Haenszel method and their probabilities were calculated with z-statistics. RESULTS: Nine studies from eight publications were included. Two continuous regimens and four intermittent regimens were investigated. A pooled risk ratio of 0.87 was obtained for intention-to-treat (95% CI: 0.79-0.96, P = 0.004, n = 6) and evaluable patient (95% CI: 0.80-0.96, P = 0.003, n = 8) analyses of mycological cure, favouring continuous terbinafine. For complete cure, pooled risk ratios of 0.97 (95% CI: 0.77-1.23, P = 0.82, n = 7) for intention-to-treat and 0.93 (95% CI: 0.76-1.13, P = 0.44, n = 9) for evaluable patient analyses showed equality of the two regimens. The pulse regimen that demonstrated consistently comparable results to the continuous terbinafine regimen was two pulses of terbinafine 250 mg/day for 4 weeks on/4 weeks off. CONCLUSIONS: Meta-analysis of published studies of toenail onychomycosis showed that a continuous terbinafine regimen is generally significantly superior to a pulsed terbinafine regimen for mycological cure. In contrast, some pulse terbinafine regimens were as effective as continuous terbinafine regimens for complete cure.

Impact of Usp18 and IFN signaling in Salmonella-induced typhlitis.

In humans, Salmonella infection causes two major clinical diseases, typhoid fever and a self-limiting gastro-enteritidis. Salmonella transmission occurs by the fecal-oral route and the interactions between the bacteria and the digestive tract epithelium are central to the outcome of the infection. Using a mouse model of typhoid fever, we previously identified a mutation in USP18 affecting type I interferon (IFN) signaling resulting in increased susceptibility to systemic Salmonella infection. In this study, we demonstrate the effects of this mutation during the early response to Salmonella using a model of typhlitis. Mutant Usp18 mice showed a minimal inflammatory response early after Salmonella Typhimurium infection that was associated with low pathologic scores and low IFN-γ production. This resulted in an increased interaction of Salmonella with the cecal epithelium and earlier systemic dissemination of the bacteria. The global transcriptional signature in the cecum of mouse during Salmonella infection showed normal expression of tissue specific genes and upregulation of type I IFN pathway in mutant mice. In control mice, there was a significant over-representation of genes involved in cellular recruitment and antibacterial activity paralleling the histopathological features. These results show the impact of USP18 in the development of Salmonella-induced typhlitis.

Identification and characterization of Cri1, a locus controlling mortality during Citrobacter rodentium infection in mice.

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ∼16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.

Renal tumour suppressor function of the Birt-Hogg-Dubé syndrome gene product folliculin.

BACKGROUND: Renal cell carcinoma (RCC) comprises five major molecular and histological subtypes. The Birt-Hogg-Dubé (BHD) syndrome is a hereditary human cancer syndrome that predisposes affected individuals to develop renal carcinoma of nearly all subtypes, in addition to benign fibrofolliculomas, and pulmonary and renal cysts. BHD is caused by loss-of-function mutations in the folliculin (FLCN) protein. The molecular function of FLCN is still largely unknown; opposite and conflicting evidence of the role of FLCN in mammalian target of rapamycin signalling/phosphorylated ribosomal protein S6 (p-S6) activation had recently been reported. RESULTS AND METHODS: Here, the expression pattern of murine Flcn was described, and it was observed that homozygous disruption of Flcn results in embryonic lethality early during development. Importantly, heterozygous animals manifest early preneoplastic kidney lesions, devoid of Flcn expression, that progress towards malignancy, including cystopapillary adenomas. A bona fide tumour suppressor activity of FLCN was confirmed by nude mouse xenograft assays of two human RCC cell lines with either diminished or re-expressed FLCN. It was observed that loss of FLCN expression leads to context-dependent effects on S6 activation. Indeed, solid tumours and normal kidneys show decreased p-S6 upon diminished FLCN expression. Conversely, p-S6 is found to be elevated or absent in FLCN-negative renal cysts. CONCLUSION: In accordance with clinical data showing distinct renal malignancies arising in BHD patients, in this study FLCN is shown as a general tumour suppressor in the kidney.

[Management of pregnancies after adjustable gastric banding]. / Prise en charge des grossesses après anneau gastrique ajustable.

OBJECTIVES: To report our experience of the association adjustable gastric banding and pregnancy. To define a management for a such association. MATERIALS AND METHODS: Retrospective and descriptive study on two centers over a 3-year follow-up of pregnancies begun with a Lap-Band gastric banding placed by laparoscopic way. RESULTS: Twenty-one pregnancies, 22 newborns resulting from 18 women were identified. Eleven patients were hospitalized. The motive of the hospitalization was severe epigastralgia for four patients requiring three deflations for mechanical complication. No case of preeclampsia was identified. Seven bands were deflated. In the group of the deflated bands, the mean maternal weight gain was 19 vs 10 kg (P=0.008), the mean birth weight was 3700 vs 3204 g (P=0.09) with a rate of fetal macrosomia increased, 50 vs 29% (P=0.038). The difference between the rates of cesarean delivery was not significant (NS) between the two groups. The childbirth term was appreciably the same, 39.4 vs 38.6 weeks of gestation (NS). The only case of gestational diabetes was found in the deflated band group. Three intrauterine growth restrictions whose one fetal death occurred in the not deflated band group. CONCLUSION: Results obtained were comparable to those of the literature. This series confirms that adjustable gastric banding limits the usual complications of the morbid obesity during pregnancy. It is generally well tolerated and must not be thus deflated by principle, but only on symptoms. That will be a total dysphagia, severe epigastric pains, vomiting after the first trimester of pregnancy or an intrauterine growth restriction.

Tumor cell survival dependence on the DHX9 DExH-box helicase.

The NTP-dependent DExH/D-box helicase DHX9 is a key participant in a number of gene regulatory steps, including transcriptional, translational, and microRNA-mediated control, DNA replication and maintenance of genomic stability. DHX9 has also been implicated in tumor cell maintenance and drug response. Here we report that inhibition of DHX9 expression is lethal to human cancer cell lines and murine Eµ-Myc lymphomas. Using a novel conditional shDHX9 mouse model, we demonstrate that sustained and prolonged (6 months) suppression of DHX9 does not result in any deleterious effects at the organismal level. Body weight, blood biochemistry and histology of various tissues were comparable to control mice. Global gene expression profiling revealed that, although reduction of DHX9 expression resulted in multiple transcriptome changes, these were relatively benign and did not lead to any discernible phenotype. Our results demonstrate a robust tolerance for systemic DHX9 suppression in vivo and support the targeting of DHX9 as an effective and specific chemotherapeutic approach.

Differential subcellular localization of mGluR1a and mGluR5 in the rat and monkey Substantia nigra.

Neurons in the rat substantia nigra (SN) are enriched in group I metabotropic glutamate receptor (mGluR) subtypes and respond to group I mGluR activation. To better understand the mechanisms by which mGluR1 and mGluR5 mediate these effects, the goal of this study was to elucidate the subsynaptic localization of these two receptor subtypes in the rat and monkey substantia nigra. At the light microscope level, neurons of the SN pars reticulata (SNr) displayed moderate to strong immunoreactivity for both mGluR1a and mGluR5 in rats and monkeys. However, mGluR1a labeling was much stronger in monkey than in rat SN pars compacta (SNc) neurons, whereas a moderate level of mGluR5 immunoreactivity was found in both species. At the electron microscope level, the immunoreactivity for both group I mGluR subtypes was primarily expressed postsynaptically, although light mGluR1a labeling was occasionally seen in axon terminals in the rat SNr. Immunogold studies revealed a striking difference in the subcellular distribution of mGluR1a and mGluR5 immunoreactivity in SNr and SNc neurons. Although the bulk of mGluR1a was attached to the plasma membrane, >80% of mGluR5 immunoreactivity was intracellular. Plasma membrane-bound immunoreactivity for group I mGluRs in both SNc and SNr neurons was mostly extrasynaptic or in the main body of symmetric, putative GABAergic synapses. On the other hand, asymmetric synapses either were nonimmunoreactive or displayed perisynaptic labeling. These data raise important questions about the trafficking, internalization, and potential functions of group I mGluRs at extrasynaptic sites or symmetric synapses in the substantia nigra.

The interaction of bovine milk galactosyltransferase with lipid and alpha-lactalbumin.

The activation of galactosyltransferase (UDPgalactose: N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase, EC 2.4.1.38) by alpha-lactalbumin has been studied at low concentrations of alpha-lactalbumin where the relationship is sigmoidal. The sigmoidal shape of the activation curve was eliminated by neutral lipids such as phosphatidylcholine and phosphatidylethanolamine, detergents such as Triton X-100 or by an aggregated form of alpha-lactalbumin generated by crosslinking alpha-lactalbumin with dithiobissuccinimidylpropionate. It is proposed that these different reagents present a hydrophobic surface to the enzyme which is necessary for lactose synthase activity. In competition experiments, large amounts of alpha-lactalbumin were able to displace lipid from the enzyme as suggested by the loss of the lipid-activating effect in the presence of an excess of alpha-lactalbumin. Optimal lactose synthase activity was obtained when the ratio of lipid/alpha-lactalbumin/enzyme was 60:6:1. The mechanism by which the lipid effect was obtained probably involved a phase transition in the enzyme which was detected as a sharp break in the Arrhenius curve. The presence of phosphatidylcholine abolished the break demonstrating that full activity of the enzyme required both alpha-lactalbumin and lipid.

Glucose transport and glucose transporters in muscle and their metabolic regulation.

Skeletal muscle is the primary tissue responsible for insulin-dependent glucose uptake in vivo; therefore, glucose uptake by this tissue plays an important role in determining glycemia. Glucose uptake in muscle occurs by a system of facilitated diffusion involving at least two distinct glucose transporters, GLUT-1 and GLUT-4. Both bind the fungal metabolite and inhibitor of glucose transport cytochalasin B. In human skeletal muscle, both types of transporters are detected immunologically, and corresponding mRNA transcripts of both transporter forms are detected. In human skeletal muscle cells in culture, in which contamination by other tissues is ruled out, a 50,000-Mr polypeptide is photolabeled with cytochalasin B. In rat skeletal muscle, acute treatment with insulin in vivo increases glucose-transport activity and the number of specific cytochalasin B-binding sites at the plasma membrane. In mildly diabetic (streptozocin-induced) rats, the number of cytochalasin B-binding sites is decreased in total membranes, and preferentially in the plasma membrane. In response to acute insulin treatment, however, there is still recruitment of glucose transporters to the plasma membrane from an intracellular membrane store. Hence, migration of transporters does occur in this form of diabetes. In L6 muscle cells in culture, acute treatment (1 h) with insulin causes recruitment of glucose transporters to the plasma membrane, and prolonged exposure to insulin or to glucose-deprived medium causes increased expression of GLUT-1 mRNA and GLUT-1 protein. Prolonged exposure (24 h) to high glucose in the medium causes a decrease in the number of glucose transporters in the plasma membrane. Hence, in those cells the expression of the GLUT-1 glucose transporter is modulated by insulin.

Loss of Cdh1 and Trp53 in the uterus induces chronic inflammation with modification of tumor microenvironment.

Type II endometrial carcinomas (ECs) are estrogen independent, poorly differentiated tumors that behave in an aggressive manner. As TP53 mutation and CDH1 inactivation occur in 80% of human endometrial type II carcinomas, we hypothesized that mouse uteri lacking both Trp53 and Cdh1 would exhibit a phenotype indicative of neoplastic transformation. Mice with conditional ablation of Cdh1 and Trp53 (Cdh1(d/d)Trp53(d/d)) clearly demonstrate architectural features characteristic of type II ECs, including focal areas of papillary differentiation, protruding cytoplasm into the lumen (hobnailing) and severe nuclear atypia at 6 months of age. Further, Cdh1(d/d)Trp53(d/d) tumors in 12-month-old mice were highly aggressive, and metastasized to nearby and distant organs within the peritoneal cavity, such as abdominal lymph nodes, mesentery and peri-intestinal adipose tissues, demonstrating that tumorigenesis in this model proceeds through the universally recognized morphological intermediates associated with type II endometrial neoplasia. We also observed abundant cell proliferation and complex angiogenesis in the uteri of Cdh1(d/d)Trp53(d/d) mice. Our microarray analysis found that most of the genes differentially regulated in the uteri of Cdh1(d/d)Trp53(d/d) mice were involved in inflammatory responses. CD163 and Arg1, markers for tumor-associated macrophages, were also detected and increased in the uteri of Cdh1(d/d)Trp53(d/d) mice, suggesting that an inflammatory tumor microenvironment with immune cell recruitment is augmenting tumor development in Cdh1(d/d)Trp53(d/d) uteri. Further, inflammatory mediators secreted from CDH1-negative, TP53 mutant endometrial cancer cells induced normal macrophages to express inflammatory-related genes through activation of nuclear factor-κB signaling. These results indicate that absence of CDH1 and TP53 in endometrial cells initiates chronic inflammation, promotes tumor microenvironment development following the recruitment of macrophages and promotes aggressive ECs.

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins.

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.

Primary structure and expression of a naturally truncated human P2X ATP receptor subunit from brain and immune system.

A novel member of the ionotropic ATP receptor gene family has been identified in human brain. This 422 amino acid long P2X receptor subunit has 62% sequence identity with rat P2X5. Several characteristic motifs of ATP-gated channels are present in its primary structure, but this P2X5-related subunit displays a single transmembrane domain. Heterologous expression of chimeric subunits containing the C-terminal domain of rat P2X5 leads to the formation of desensitizing functional ATP-gated channels in Xenopus oocytes. The developmentally regulated mRNA, found in two splicing variant forms, is expressed at high levels in brain and immune system.

Presynaptic NMDA receptor subunit immunoreactivity in GABAergic terminals in rat brain.

N-methyl-D-aspartate (NMDA) receptors are commonly found post-synaptically; they mediate fast excitatory neurotransmission in the central nervous system. In this study, we provide immunocytochemical data supporting the existence of presynaptic NMDA receptors in GABAergic terminals using polyclonal antisera raised against the C-terminus of the NMDAR1 subunit. At the light microscope level, rich plexuses of NMDAR1-positive varicose fibers were found in various nuclei in the basal forebrain (bed nucleus of stria terminalis, septum, parastrial nucleus, vascular organ of the lamina terminalis), thalamus (paraventricular nucleus, midline nuclei), and hypothalamus (parvocellular paraventricular nucleus, arcuate nucleus, preoptic nucleus, suprachiasmatic nucleus). In the brainstem, labeled fibers were much less abundant and were confined to the ventral tegmental area, periaqueductal gray, parabrachial nucleus, and locus coeruleus. At the electron microscope level, NMDAR1-immunoreactive terminals examined in the bed nucleus of stria terminalis, parvocellular paraventricular hypothalamic nucleus, and arcuate nucleus formed symmetric synapses, contained darkly stained large dense-core vesicles, and displayed gamma-aminobutyric acid (GABA) immunoreactivity. Terminals with similar ultrastructural features were found in the paraventricular thalamic nucleus. These findings demonstrate the existence of NMDAR1 subunit immunoreactivity in subsets of GABAergic terminals, which raises questions about the potential roles and mechanisms of activation of presynaptic NMDA heteroreceptors in the rat central nervous system. The pattern of distribution and ultrastructural features of these boutons suggest that they may arise from local GABAergic projections interconnecting a group of brain structures mediating stress responses and/or other endocrine, autonomic, and limbic functions.