Divergent Profile Research Articles

Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic.

Members of the Opioid Receptor (OR) family of G protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system where they play key roles in nociception and analgesia. Unlike the classical ORs, δ–OR, κ–OR,1 and μ-OR,2 which were delineated by pharmacological criteria in the 1970’s and 1980’s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, aka ORL-1) was discovered relatively recently via molecular cloning and characterization of an orphan GPCR3. Despite its high sequence similarity (~60%) with ORs, NOP has a strikingly distinct pharmacology4,5. Despite high sequence similarity with classical opioid G protein-coupled receptor subtypes, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) has a distinct biological and pharmacological role, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. This study reports the crystal structure of human NOP solved in complex with the peptide mimetic antagonist Banyu Compound-24 (C-24), revealing atomic details of ligand-receptor recognition and selectivity. C-24 mimics the first four N-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to binding of these peptides. The X-ray structure also reveals substantial conformational differences in the pocket regions between NOP and the “classical” opioid receptors κ (Ref. 1) and μ (Ref. 2), which are likely due to a small number of residues that vary between the two receptors. The NOP/C-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

Socioeconomic parameters and mortality in Turner syndrome

Turner syndrome (TS) is characterized by hypogonadism, short adult height, increased morbidity and mortality, contrasted by self-reported normal quality of life and perception of health. Small studies have indicated a similar level of education compared with the background population. To study the socioeconomic profile in TS and the impact of these factors on mortality. Register study using Danish nationwide registries. Nine hundred and seventy-nine TS females and 94,850 controls were included. Information concerning cohabitation, motherhoods, level of education (bachelor degree), income, retirement, and death were obtained. One hundred and three TS and 5989 controls died during the study period. For the socioeconomic parameters, median age at first relevant episode was calculated. Income was analyzed using conditional logistic regression and the other parameters using Cox regression. In comparison with controls, TS had significantly fewer partnerships (hazard ratio (HR): 0.45), fewer motherhoods (HR: 0.18), and retired earlier (HR: 1.8). After the diagnosis of TS, the risk of retiring was increased. Educational attainment (HR: 1.0) as well as risk of unemployment was similar. Before the age of 30, low income was significantly more frequent; hereafter, it was similar to controls. Mortality was significantly increased (HR: 2.9) and slightly lower after adjustment for cohabitation and education (HR: 2.7). A divergent socioeconomic profile is apparent, with a reduced proportion of TS persons finding a partner and becoming mothers. The educational level was similar to controls. The increased mortality in TS was not materially affected after adjustment for cohabitation and education.

Mouse background strain profoundly influences Paneth cell function and intestinal microbial composition.

BackgroundIncreasing evidence supports the central role of Paneth cells in maintaining intestinal host-microbial homeostasis. However, the direct impact of host genotype on Paneth cell function remains unclear. Here, we characterize key differences in Paneth cell function and intestinal microbial composition in two widely utilized, genetically distinct mouse strains (C57BL/6 and 129/SvEv). In doing so, we demonstrate critical influences of host genotype on Paneth cell activity and the enteric microbiota.Methodology and Principal FindingsPaneth cell numbers were determined by flow cytometry. Antimicrobial peptide (AMP) expression was evaluated using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), acid urea-polyacrylamide gel electrophoresis, and mass spectrometry. Effects of mouse background on microbial composition were assessed by reciprocal colonization of germ-free mice from both background strains, followed by compositional analysis of resultant gut bacterial communities using terminal restriction fragment length polymorphism analysis and 16 S qPCR. Our results revealed that 129/SvEv mice possessed fewer Paneth cells and a divergent AMP profile relative to C57BL/6 counterparts. Novel 129/SvEv á-defensin peptides were identified, including Defa2/18v, Defa11, Defa16, and Defa18. Host genotype profoundly affected the global profile of the intestinal microbiota, while both source and host factors were found to influence specific bacterial groups. Interestingly, ileal α-defensins from 129/SvEv mice displayed attenuated antimicrobial activity against pro-inflammatory E. coli strains, a bacterial species found to be expanded in these animals.Conclusions and SignificanceThis work establishes the important impact of host genotype on Paneth cell function and the composition of the intestinal microbiota. It further identifies specific AMP and microbial alterations in two commonly used inbred mouse strains that have varying susceptibilities to a variety of disorders, ranging from obesity to intestinal inflammation. This will be critical for future studies utilizing these murine backgrounds to study the effects of Paneth cells and the intestinal microbiota on host health and disease.

Divergent Pro-Inflammatory Profile of Human Dendritic Cells in Response to Commensal and Pathogenic Bacteria Associated with the Airway Microbiota

Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3–5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota.

The Main Anticancer Bullets of the Chinese Medicinal Herb, Thunder God Vine

The thunder god vine or Tripterygium wilfordii Hook. F. is a representative Chinese medicinal herb which has been used widely and successfully for centuries in treating inflammatory diseases. More than 100 components have been isolated from this plant, and most of them have potent therapeutic efficacy for a variety of autoimmune and inflammatory diseases. In the past four decades, the anticancer activities of the extracts from this medicinal herb have attracted intensive attention by researchers worldwide. The diterpenoid epoxide triptolide and the quinone triterpene celastrol are two important bioactive ingredients that show a divergent therapeutic profile and can perturb multiple signal pathways. Both compounds promise to turn traditional medicines into modern drugs. In this review, we will mainly address the anticancer activities and mechanisms of action of these two agents and briefly describe some other antitumor components of the thunder god vine.

Contradiction between strong lensing statistics and a feedback solution to the cusp/core problem

Standard cosmology has many successes on large scales, but faces some fundamental difficulties on small, galactic scales. One such difficulty is the cusp/core problem. High resolution observations of the rotation curves for dark matter dominated low surface brightness (LSB) galaxies imply that galactic dark matter halos have a density profile with a flat central core, whereas N-body structure formation simulations predict a divergent (cuspy) density profile at the center. It has been proposed that this problem can be resolved by stellar feedback driving turbulent gas motion that erases the initial cusp. However, strong gravitational lensing prefers a cuspy density profile for galactic halos. In this paper, we use the most recent high resolution observations of the rotation curves of LSB galaxies to fit the core size as a function of halo mass, and compare the resultant lensing probability to the observational results for the well defined combined sample of the Cosmic Lens All-Sky Survey (CLASS) and Jodrell Bank/Very Large Array Astrometric Survey (JVAS). The lensing probabilities based on such density profiles are too low to match the observed lensing in CLASS/JVAS. High baryon densities in the galaxies that dominate the lensing statistics can reconcile this discrepancy, but only if they steepen the mass profile rather than making it more shallow. This places contradictory demands upon the effects of baryons on the central mass profiles of galaxies.

Tissue-specific robustness of fatty acid signatures in cultured gilthead sea bream (Sparus aurata L.) fed practical diets with a combined high replacement of fish meal and fish oil1

The present study aimed to determine the tissue-specific robustness of fatty acid (FA) signatures of gilthead sea bream (Sparus aurata L.) by analyzing the changes in lipid class and FA composition of skeletal muscle, brain, liver, and mesenteric adipose tissue. Triplicate groups of fish were fed to visual satiety over a 14-mo production cycle with 2 practical plant protein-based diets formulated with either fish oil or a blend of vegetable oils (66% of fish oil replacement) to contain 53% CP and 21% crude fat on a DM basis. Growth rates (P = 0.22) and tissue lipid class composition were not altered by the dietary treatment (P = 0.34 and 0.52 for neutral lipids and phospholipids, respectively). The FA signatures of neutral lipids reflected the composition of the diet, although the output of principal components analysis revealed a divergent FA profile for liver compared with skeletal muscle, brain, and mesenteric adipose tissue. Because the theoretical EFA needs were met by the 2 diets, the FA composition of phospholipids remained almost unaltered in all tissues. Interestingly, however, the brain showed the greatest robustness and regulatory capacity to preserve the phenotype of fish fed fish oil-based diets. The FA signatures of total lipids are a combinatory result of neutral and polar lipids, and the most relevant fat storage tissues (mesenteric adipose tissue and skeletal muscle) were more easily influenced by dietary FA composition. The present study provides new insights into fish tissue FA composition and reinforces the use of FA signatures as useful criteria in determining whether EFA requirements for a wide range of physiological processes, including those of neural tissues, can be met with practical fish feeds.

Differential Gene Expression Profile in the T Cell Compartment Between Classic PNH and Aplastic Anemia/PNH Syndrome.

Abstract 3011Poster Board II-987Somatic mutation of the X- linked gene PIG-A results in partial or absolute deficiency of cell surface expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs). In humans, this genetic event in a hematopoietic stem cell leads to the blood disease paroxysmal nocturnal hemoglobinuria (PNH). PNH has two major clinical presentations: predominantly hemolytic without overt marrow failure, referred to as classic PNH, and PNH clonal expansion in the setting of marrow failure, or the aplastic anemia/PNH syndrome (AA/PNH). The diagnosis of PNH is based on the presence of GPI-AP-deficient clones mainly within erythrocytes and granulocytes. Since PNH is a stem cell disorder, all hematopoietic lineages are involved and GPI-AP-deficient clones are found among platelets, monocytes and lymphocytes. Except for their different clinical presentations, classic PNH and AA-PNH have never been shown to have specific discriminating biological features. Indeed, we previously reported no difference in the expression profile of CD34 cells in hemolytic and aplastic forms of PNH (Chen G et al, Leukemia 2005; 19:862). As marrow failure in PNH, as in aplastic anemia, is immune-mediated, and T cells are affected by the PIG-A mutation, we hypothesized that the mutant subset of lymphocytes might differ in PNH. We compared by microarray the T cell compartment in both forms of the disease. We sorted peripheral blood GPI-AP-normal and -deficient pooled CD4 and CD8 T cells using CD59 and CD55 as GPI-AP markers, and subjected each subset to array hybridization and analysis. Samples were obtained from 1 patient with classic PNH, 3 patients with AA-PNH syndrome and 3 age-matched normal donors as controls. In preliminary control experiments, we determined using microarray of total RNA derived from healthy donors' T cells, with and without GPI-AP-specific antibodies, the absence of altered gene expression pattern or activation of specific genes due to selective antibody binding to normal cells. By principal component analysis (PCA), the phenotype of the PNH clone in classic PNH was divergent from the AA-PNH clone and from that of normal donors. By consistency test, only 15 probesets/genes resulted common in the comparison between GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome. Using Ingenuity Software, we found that the represented probesets/genes were involved in some major canonical pathways such as cell death, immune and lymphatic system development, immune response, and cell-to-cell signaling interaction. In order to investigate the different gene expression profile derived from specific subset of T cells, we sorted GPI-AP-deficient and GPI-AP-normal CD8 T cells using CD48 and CD52 as markers. When we compared GPI-AP-deficient and GPI-AP-normal cells between classic PNH and AA-PNH syndrome, we found that only 23 probesets/genes were up-regulated and 132 down-regulated confirming the divergent gene expression profile between the two diseases (Figure 1). We conclude that the restricted similarity in the gene expression profile in the GPI-AP-deficient T cells between classic PNH and AA-PNH syndrome leads to a specific “signature” of genes that might support the different clinical presentation and evolution of the two diseases. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Increased amphetamine-induced locomotor activity, sensitization, and accumbal dopamine release in M5 muscarinic receptor knockout mice

Muscarinic M(5) receptors are the only muscarinic receptor subtype expressed by dopamine-containing neurons of the ventral tegmental area. These cells play an important role for the reinforcing properties of psychostimulants and M(5) receptors modulate their activity. Previous studies showed that M(5) receptor knockout (M (5) (-/-) ) mice are less sensitive to the reinforcing properties of addictive drugs. Here, we investigate the role of M(5) receptors in the effects of amphetamine and cocaine on locomotor activity, locomotor sensitization, and dopamine release using M (5) (-/-) mice backcrossed to the C57BL/6NTac strain. Sensitization of the locomotor response is considered a model for chronic adaptations to repeated substance exposure, which might be related to drug craving and relapse. The effects of amphetamine on locomotor activity and locomotor sensitization were enhanced in M (5) (-/-) mice, while the effects of cocaine were similar in M (5) (-/-) and wild-type mice. Consistent with the behavioral results, amphetamine-, but not cocaine, -elicited dopamine release in nucleus accumbens was enhanced in M (5) (-/-) mice. The different effects of amphetamine and cocaine in M (5) (-/-) mice may be due to the divergent pharmacological profile of the two drugs, where amphetamine, but not cocaine, is able to release intracellular stores of dopamine. In conclusion, we show here for the first time that amphetamine-induced hyperactivity and dopamine release as well as amphetamine sensitization are enhanced in mice lacking the M(5) receptor. These results support the concept that the M(5) receptor modulates effects of addictive drugs.

Developmental requirements of T follicular helper cells (90.10)

Abstract After activation, CD4+ helper T (Th) cells differentiate into distinct effector lineages. Th cell subset called T follicular helper (TFH) cells are recently found to be present in germinal centers and are characterized by their expression of chemokine (C-X-C motif) receptor 5 (CXCR5). Despite their potential importance in humoral immunity, the developmental regulation of TFH cells and their relationship with other Th subsets is unclear. We found that mouse TFH cells have a divergent gene expression profile from Th1, Th2 cells, and in vivo developed independently of these cell lineages. TFH cell generation was regulated by ICOS liagand expressed on B cells and was dependent on interleukin (IL)-21, IL-6 and signal transducer and activator of transcription 3 (STAT3). Although TFH cells shared IL-21 expression with Th17 cells, they did not express IL-17, IL-17F, IL-22. Moreover, differentiation of TFH cells did not require TGFβ or Th17-specific orphan nuclear receptors RORα?and RORγ in vivo. Most importantly, T cells activated in vitro in the presence of IL-21 but without TGFβ signaling preferentially acquired TFH gene expression and functioned to promote humoral immunity in vivo. Our data indicate that TFH cells are distinct in their gene expression, developmental regulation and immune function from Th1, Th2 or Th17 lineages. The work is supported by a Scientist Development Grant from the American Heart Association.

“Theory of Mind” and Executive Functioning in Forensic Patients with Schizophrenia

Previous studies in forensic patients with schizophrenia have shown that delinquent patients may outperform nondelinquent patients with regards to "theory of mind" (ToM). Findings were, however, confounded by a lack of control for executive functioning. We examined 33 forensic patients with schizophrenia regarding ToM, intelligence, executive functioning, and psychopathology. Results were compared with a nonforensic schizophrenia sample and a group of healthy controls. Both patient groups performed more poorly on most measures compared with controls. Forensic and nonforensic patients did not differ in task performance. In the forensic group ToM correlated inversely with "excitement" and cognitive symptoms. When "excitement" was covaried out, forensic patients outperformed nonforensic patients with regards to ToM. This study supports the hypothesis that schizophrenic patients with a criminal record are equally impaired in their ability to infer mental states compared with nonforensic patients, but for different reasons associated with a divergent psychopathological profile.

In situ microbial treatment of landfill leachate using aerated lagoons

The aim of this study was to assess the efficiency of leachate treatment by microbial oxidation in four connected on-site aerated lagoons at a landfill site. The landfill site was found to be in an ageing methanogenic state, producing leachate with relatively low COD (mean value 1740 mg l −1) and relatively high ammonium concentrations (mean value 1241 mg l −1). Removal of COD averaged 75%, with retention times varying from 11 to 254 days. Overall 80% of the N load was removed within the plant, some by volatilisation of ammonium. Microbial community profiling of the water from each lagoon showed a divergent community profile, presumably a reflection of the nutrient status in each lagoon. In municipal solid waste landfills under similar conditions, leachate treatment through a facultative aerobic system in which sequential aerobic and anaerobic microbial oxidations occurred can readily be achieved using a simple two-lagoon system, suggesting this technology can be economic to install and simple to run.

S61-04 “Mentalising” and executive functioning in schizophrenia: A comparison between forensic and non-forensic patients

Previous studies in forensic patients with schizophrenia have shown that delinquent patients may outperform non-delinquent patients with regards to their ability to appreciate mental states. Findings were, however, confounded by a lack of control for neurocognitive functioning. We examined 33 forensic patients with schizophrenia regarding their mentalising abilities, intelligence, executive functioning and psychopathology. Results were compared with a non-forensic schizophrenia sample and a group of healthy controls. Both patient groups performed more poorly on most measures compared to controls. Forensic and non-forensic patients did not differ in task performance. In the forensic group mentalising correlated inversely with “excitement” and cognitive symptom scores of the PANSS. When “excitement” was co-varied out, forensic patients outperformed non-forensic patients with regards to their mentalising abilities. This study supports the hypothesis that schizophrenic patients with a criminal record are equally impaired in their ability to infer mental states compared to non-forensic patients, but for different reasons associated with a divergent psychopathological profile.

Involvement of thyroid hormones in chicken embryonic brain development

Thyroid hormones (THs) play an important role in vertebrate brain development by stimulating and coordinating cell proliferation, migration and differentiation. Several TH-responsive genes involved in these processes have been identified, but the information is mainly derived from studies of late brain development, while relatively little is known about the more early stages, prior to the onset of embryonic TH secretion. We have chosen the chick embryo to investigate the role of THs in both late and early brain development. T 4 and T 3 are found in chicken brain from the earliest stages tested (day 4). Indirect clues for the involvement of T 3 in brain development are found in the ontogenetic expression profiles of proteins regulating its bioavailability and action, including TH transporters, deiodinases and TH-receptors. All of them are expressed in whole embryos tested on day 2 of incubation and in developing brain tested from day 4 onwards. Their distribution patterns vary over time and according to the brain area and cell type studied. Hypothyroidism induced during the second half of incubation disturbs cell migration in the cerebellum, providing more direct evidence for the requirement for THs during the later stages of brain development. Direct morphological proof for the requirement for THs during the first half of incubation is still missing, but microarray analysis of telencephalon shows a clearly divergent gene expression profile in hypothyroid embryos. In vivo knockdown of TH transporters and deiodinases in chick embryos cultured ex ovo provides an excellent tool to study the role of THs in early brain development in more detail.

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma

IntroductionDuctal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing invasive ductal carcinoma, and invasive ductal carcinoma.MethodsForty-one samples were evaluated: four non-neoplastic, five pure DCIS, 22 in situ component of lesions with co-existing invasive ductal carcinoma, and 10 invasive ductal carcinoma. Pure cell populations were isolated using laser microdissection. Total RNA was purified, DNase treated, and amplified using the T7-based method. Microarray analysis was conducted using a customized cDNA platform. The concept of molecular divergence was applied to classify the sample groups using analysis of variance followed by Tukey's test.ResultsAmong the tumor sample groups, cells from pure DCIS exhibited the most divergent molecular profile, consequently identifying cells from in situ component of lesions with co-existing invasive ductal carcinoma as very similar to cells from invasive lesions. Additionally, we identified 147 genes that were differentially expressed between pure DCIS and in situ component of lesions with co-existing invasive ductal carcinoma, which can discriminate samples representative of in situ component of lesions with co-existing invasive ductal carcinoma from 60% of pure DCIS samples. A gene subset was evaluated using quantitative RT-PCR, which confirmed differential expression for 62.5% and 60.0% of them using initial and partial independent sample groups, respectively. Among these genes, LOX and SULF-1 exhibited features that identify them as potential participants in the malignant process of DCIS.ConclusionsWe identified new genes that are potentially involved in the malignant transformation of DCIS, and our findings strongly suggest that cells from the in situ component of lesions with co-existing invasive ductal carcinoma exhibit molecular alterations that enable them to invade the surrounding tissue before morphological changes in the lesion become apparent.

Interrelationships between negative energy balance (NEB) and IGF regulation in liver of lactating dairy cows

In dairy cows, negative energy balance (NEB) during the early post-partum period is associated with major alterations in the growth hormone-insulin-like growth factor (GH-IGF) axis. Since the liver mediates nutrient partitioning during lactation, we aimed to determine how NEB alters the endocrine regulation of the insulin-like growth factor (IGF) system by investigating the expression of IGF family members and related steroid receptors. On the second day of lactation, cows were allocated to one of two treatments designed to produce mild (MNEB) or severe NEB (SNEB). MNEB cows ( n = 5) were fed ad lib grass silage supplemented with concentrate and milked x1 daily and SNEB cows ( n = 6) were restricted in dietary intake and milked x3 daily. Energy balance (EB) status was monitored until the second week of lactation when plasma and liver samples revealed a markedly divergent metabolic profile. At this time, plasma protein and hepatic mRNA for IGF-I was reduced in SNEB cows compared with MNEB cows. Both levels of expression correlated highly when data from all animals was pooled ( r = 0.963; P < 0.01). SNEB cows also exhibited reduced hepatic expression for transcripts encoding IGF-1R, IGF-2R, IGF binding proteins (IGFBPs) -3, -4, -5, -6, acid labile subunit, and receptors for oestrogen (ERα) and growth hormone (total GHR and 1A variant), while the expression of IGFBP-2 was elevated. Expression of mRNA for IGF-II, IGFBP-1 and receptors for insulin (A/B) and glucocorticoid (α) was unaffected by EB. Results demonstrate that SNEB affects hepatic synthesis of IGF-I, and other components known to modulate the bioavailability and stability of circulating IGF-I.

Reflections on the Nature of Skills in the Experience Economy: Challenging Traditional Skills Models in Hospitality

This paper addresses the nature of skills in international hospitality. Frequently characterised as 'low skills', it is increasingly recognised that skills bundling in services such as hospitality cannot be solely seen in terms of the technical attributes of work. Emotional and aesthetic dimensions have been added to the services skills bundle. The added dimension of Pine and Gilmore's experience economy suggest a further component within this bundle, namely that of experience skills. This paper explores the role of experiential factors in helping to equip those entering work in the international hospitality industry. Because of the strongly Americanised operating culture of hospitality, the learning demands of the sector for those brought up in a western, developed world environment are relatively small. In addition, those working in hospitality in most developed countries have experience of the sector as both consumers and employees. By contrast, employees in international hospitality in less developed countries do not have similar benefits of experience, either though general acculturation or as consumers of hospitality services. This divergent experience profile has significant implications for the skills demands of hospitality work and leads to the proposition that experience is an important factor in determining the skills demands of hospitality work. This, in turn, leads to this paper's proposal that Experiential Intelligence (ExQ) is an indicator of this difference in terms of workplace skills.

Transitions of serum albumin in patients with glomerulosclerosis ‘in vivo’ characterization by electrophoretic titration curves

HSA functions as a physiological transporter of solutes and small molecules that induce structural transitions 'in vitro'. Analysis of these transitions requires prior purification of HSA that could introduce bias due to conformational changes. We utilized electrophoretic titration curves to describe a neutral to acid (N-A) transition of HSA directly in sera of seven patients with active focal segmental glomerulosclerosis (FSGS). The divergent electrophoretic profile of HSA was characterized by a shift in the range of pHs between 4.5 and 7.5 with an average variation of free electrophoretic mobility corresponding to loss of 1 positive charge in the pKa protonation range of histidyl residues and should involve domain I of HSA. 'In-gel' determination by maleimide-PEO2-biotin of free SH 34 of domain I showed inaccessibility of the dye at this site in pathological HSA and alkylation with the same complex induced N-A transition in normal HSA. Potential binders of free imidazoles such as Ca++ and/or of SH 34 such as NO were excluded on the basis of direct titration and studies on binding stimulation. This is the first report describing a transition of HSA directly 'in vivo', and the utilization of electrophoretic titration curves was critical to this purpose. This transition appears to be specific to FSGS and is unrelated to the nephrotic syndrome, Ca++ and NO binding. Spectroscopic analysis will elucidate the structural implication.

Pimecrolimus and tacrolimus differ in their inhibition of lymphocyte activation during the sensitization phase of contact hypersensitivity

As reported previously, oral administration of the calcineurin inhibitors (CNI) pimecrolimus and tacrolimus resulted in equipotent inhibition of the elicitation phase of contact hypersensitivity (CHS) in mice. The sensitization phase was inhibited by tacrolimus but was unaffected by pimecrolimus, even at higher doses. The kinetics of lymph node hyperplasia and up-regulation of T and B cell activation antigens were analyzed to obtain a better understanding of the divergent CNI profile in CHS. Lymph node (LN) cells of CNI-untreated and treated mice were examined with flow cytometry at various time points after sensitization with oxazolone. LN hyperplasia and drug levels were also determined. Sensitization induced a higher portion of LN cells expressing the activation antigens CD25, CD69 and CD134 and an increase in activated B cells (B220(+)/CD40(+)) compared to naïve mice. Up-regulation of these markers was completely or profoundly blocked with tacrolimus, whereas pimecrolimus at the three-fold higher dose caused significantly less inhibition. Tacrolimus also completely blocked the sensitization-associated increase of CD11c(+) antigen presenting cells (APC) in LN, whereas pimecrolimus showed significantly less inhibition. In contrast to tacrolimus, LN weight and cellularity were not affected by pimecrolimus at any time point after sensitization. Concentration of tacrolimus in blood and in the draining LN substantially exceeded that of pimecrolimus by factors 6.7-14 and 5.6-5.8, respectively, at the same dose levels. In contrast to tacrolimus, systemic treatment of mice with pimecrolimus only weakly interferes with lymphocyte activation and does not affect hyperplasia of the draining lymph nodes during sensitization.

Discordence of HER-2/neu overexpression and estrogen receptor between the primary breast cancer and axillary lymph node with metastasis

20106 Background: Adjuvant hormonal therapy and trastuzumab are known to be associated with a significant reduction in cancer-related mortality in a subset of women with breast cancer. The treatment decision is usually based on immunohistochemistry (IHC) staining of the primary tumor. We reported here a case of discordance of HER-2/neu overexpression and estrogen receptor status between the primary breast cancer and axillary lymph node with metastasis. Methods: IHC and fluorescence in situ hybridization (FISH) analysis was perfromed for the assessment of HER-2/neu. E-cadherin staining was done on the primary tumor and lymph node with metastasis. Results: A 52 year old woman presented with right breast lump. The imaging studies showed a 5 × 5 cm speculated mass. No other abnormalities were noted. An excision biopsy revealed 3 cm moderately differentiated invasive lobular carcinoma. Subsequently a wider excision and axillary lymph node dissection was done. 2/20 lymph nodes were involved by the cancer. IHC was done in the primary tumor and the lymph node with metastasis. IHC of the primary tumor revealed less than 1% nuclei positive for estrogen receptor (ER), 0% nuclei positive for progesterone receptor (PR), and no HER-2/neu overexpression. IHC of the lymph nodes with metastasis revealed 80% nuclei positive for ER, less than 1% nuclei positive for PR, and equivocal HER-2/neu overexpression. FISH analysis for HER-2/neu gene was done on the primary tumor which revealed no amplification in the tumor nuclei (Her2/CEP17 ratio was 3.6/2.7 = 1.3) whereas FISH analysis on the lymph node section revealed amplification in tumor nuclei (Her2/CEP 17 ratio of 7.3/1.9 = 3.9). E-cadherin staining of the primary breast tumor as well as the lymph node with metastasis revealed no membrane staining of the malignant cells in either section. Conclusions: The unusual occurrence of a divergent IHC profile and FISH analysis results between the primary carcinoma and metastatic carcinoma may be due to a subselection of a malignant clone which were not represented in the section of primary tumor chosen for IHC. Future studies are required to determine the utility of IHC staining of metastatic cells involving the lymph nodes if primary tumor is negative for HER-2/neu &amp; ER. No significant financial relationships to disclose.