Climate change, uncertainty and allostatic load.

CONTEXT: Humans constantly respond to environmental stressors challenging their somatic stability. Allostasis, an evolved neuroendocrine/physiological stressor response system, is our main pathway for doing so. Effective allostasis returns somatic systems to their current optima; over a lifetime of stressor responses, related systems fail, effectiveness declines, and physiological dysregulation (i.e. allostatic load) increases. Global Climate Change (GCC) multiplies environmental stressors on human populations and is likely to increase allostatic load. OBJECTIVES: As a population-level stressor, GCC increases risks for multiple stressors, including sociocultural instability and food and water insecurity, while also motivating migration. We predict GCC increases risk for elevated allostatic load. Here, we review pathways by which GCC increases climatic and social stressors contributing to greater stress and allostatic load. METHODS: Based upon published sources and primary ethnographic case studies, this review examines how GCC, by multiplying climate-related stressors, likely increases social instability, food and water insecurity, and migration. Thereby, it is proposed that GCC contributes to allostatic load. RESULTS: GCC multiplies stressors on local populations. Those experiencing social insecurity related to GCC during growth and development are expected to show the largest influences on their lifetime allostatic load. Similarly, as GCC increases food and water insecurity, it likely will increase allostatic load in those affected and is likely to propel migrants to seek improved living circumstances. These stressors may be continued among their descendants via historical trauma or epigenetic responses. CONCLUSION: GCC accentuates effects of environmental and sociocultural stressors on human populations. Those exposed to GCC are likely to show lifelong elevated allostatic load.

Comparison of C4d detection on erythrocytes and PTC-C4d to histological signs of antibody-mediated rejection in kidney transplantation.

C4d on erythrocytes (EC4d), C4d peritubular capillary deposition (PTC-C4d) staining and histology were compared in a cross-sectional cohort of 146 renal allograft biopsies (132 patients). EC4d levels paralleled PTC-C4d staining, but were more predictive of peritubular capillaritis (PTC). Donor-specific antibodies (DSA), PTC-C4d, EC4d and PTC were analyzed in an independent longitudinal follow-up cohort (96 biopsies, 76 patients). Seventy-six samples were PTC and EC4d concordant, 11 positive and 65 negative, 7 PTC-EC4d+ and 13 PTC+EC4d-. EC4d levels were related to DSA occurrence. With ABMR defined by PTC and DSA, all apparently discordant patients, EC4d negative, were correctly reassigned comparing EC4d level curves with rejection kinetics, with positive EC4d samples predating biopsy or late biopsies compared with ABMR flare-ups. All EC4d-positive patients without PTC or DSA had permanent high EC4d levels unrelated to rejection. EC4d was more abundant in PTC-positive (mean = 108.5%± 3.4; n = 50) than PTC-negative samples (mean = 88.1%± 1.3; n= 96; p < 0.0001). Sensitivity, specificity, positive predictive value and negative predictive value of PTC-C4d and EC4d for PTC were, respectively, 75%, 79%; 64%, 76% (p < 0.05); 28%, 46% (p < 0.05) and 93%, 94%. Values were similar for DSA. A noninvasive blood test, EC4d, and particularly longitudinally monitoring EC4d levels, may increase surrogate ABMR testing options.

Combination of KIR and HLA gene variants augments the risk of developing birdshot chorioretinopathy in HLA-A*29-positive individuals.

Birdshot chorioretinopathy (BCR), a chronic ocular inflammatory disease with characteristic choroidal lymphocytic infiltrates, has been strongly associated with human leukocyte antigen (HLA)-A29. Although HLA-A29 occurs frequently in all populations, BCR affects only a small percentage of HLA-A29-positive Caucasians, indicating additional susceptibility factors for BCR. Discovery of HLA class I-specific killer cell immunoglobulin-like receptors (KIR) led to a series of epidemiological studies implicating KIR-HLA gene combinations in disease. Here, we characterized KIR-HLA pairs in BCR patients and controls carrying HLA-A*29 as well as controls lacking HLA-A*29. KIR-HLA pairs implicated for weak inhibition (KIR2DL2/3+HLA-C1 and KIR3DL1+HLA-Bw4(T80)) in combination with activating KIR genes associated with autoimmunity (KIR2DS2, 2DS3 and 2DS4) augment the risk of developing BCR in HLA-A*29-positive individuals. The reciprocal association of strong inhibitory pairs (KIR3DL1+HLA-Bw4(I80) and KIR2DL1+HLA-C2) in combination with those implicated in protection from infection (KIR3DS1+HLA-Bw4(I80) and KIR2DS1+HLA-C2) was observed in HLA-A*29-negative controls. These results suggest that a profound effect of KIR2DS2/S3/S4 in the absence of strong inhibition may enhance the activation of natural killer cells and T-cell subsets against intraocular self-antigens, thereby contributing to pathogenesis of BCR.

Fruit and vegetable intakes and prostate cancer risk.

BACKGROUND: There is extensive and consistent evidence that high fruit and vegetable intakes are associated with decreased risks of many cancers, but results for prostate cancer risk have been inconsistent. We studied the associations of fruit and vegetable intakes with prostate cancer risk in a population-based, case-control study of men under 65 years of age. METHODS: Case participants were 628 men from King County (Seattle area), WA, who were newly diagnosed with prostate cancer. Control participants were 602 men recruited from the same underlying population and frequency matched to case participants by age. Self-administered food-frequency questionnaires were used to assess diet over the 3- to 5-year period before diagnosis or recruitment. Daily nutrient intakes were calculated by use of a nutrient database with recently updated analytic values for carotenoids. Odds ratios for prostate cancer risk associated with foods and nutrients were calculated by use of unconditional logistic regression. RESULTS: No associations were found between fruit intake and prostate cancer risk. The adjusted odds ratio (ORs) for the comparison of 28 or more servings of vegetables per week with fewer than 14 servings per week was 0.65 (95% confidence interval [CI] = 0.45-0.94), with a two-sided P for trend =.01. For cruciferous vegetable consumption, adjusted for covariates and total vegetable intake, the OR for comparison of three or more servings per week with less than one serving per week was 0.59 (95% CI = 0.39-0.90), with a two-sided P for trend =.02. The OR for daily intake of 2000 microg or more lutein plus zeaxanthin compared with an intake of less than 800 microg was 0.68 (95% CI = 0.45-1.00). CONCLUSION: These results suggest that high consumption of vegetables, particularly cruciferous vegetables, is associated with a reduced risk of prostate cancer.

B-cell maturation stages of Burkitt's lymphoma cell lines according to Epstein-Barr virus status and type of chromosome translocation.

This study addressed the possible relationship between B-cell maturation stage of Burkitt's lymphoma (BL) cell lines and Epstein-Barr virus (EBV) status, ethnic group, or type of chromosome translocation. Fifty-seven cell lines obtained at the International Agency for Research on Cancer from 51 patients were studied. Cytogenetic analyses of 54 cells lines were available. Cell size, surface immunoglobulins (sIgs), cytoplasmic immunoglobulins (cIgs), mouse red blood cell receptors, and reactivity with various monoclonal antibodies were assessed. Immunoglobulin (Ig) class secretions were measured in the supernatant of 2- and 5-day cultures from 33 cell lines, with the use of a sensitive enzyme-linked immunosorbent assay technique. From this study, BL appears to cover a broad range of the B-cell differentiation sequence, since the following Ig phenotypes were observed: null cells (sIg-, cIg-), large pre-B-cells (intracytoplasmic mu-chains), small B-cells (sIg+, cIg-), and various types of secreting B-cells (sIg+, cIg+). Among the latter, various patterns of cIg could be defined (perinuclear, paranuclear, and vesicular). B-cell maturation stages were correlated with the amount of secreted Ig. In sIg+ cell lines, different classes of Ig were found: 35 IgM, 10 IgM plus IgD, 4 IgG, and 1 IgA. None of the different monoclonal antibodies used was specific to a precise stage of maturation. The stages of maturation were correlated with neither the type of chromosome translocations of BL nor the presence of EBV genome, but the most immature cell lines were all EBV positive and most of them originated from African patients. In contrast with acute lymphoblastic leukemia, the common acute lymphocytic leukemia antigen (CD10) was expressed on nearly all BL cell lines of intermediate maturation stages but only on half of the pre-B ones. In addition, none of the cell lines tested was found to react with CD5 antibodies, which recognize most of the chronic lymphocytic leukemia of the same stage of maturation as that in the B-lymphocyte lineage.

[Nano-biocaptures for research and diagnostics in inflammation diseases and cancer]. / Nano-biocapteurs pour la recherche et les diagnostics des maladies inflammatoires et du cancer.

As part of the ongoing search for ways to decrease the mortality of different pathological conditions related to cancer and inflammatory diseases, nanotechnologies currently under evaluation offer potentially attractive tools for innovative methodologies for early diagnosis, new bioimaging techniques and therapeutic strategies. Nano-tools can be employed for various functions, such as the detection of lesions at very early stages of disease development, extremely precise anatomical localization, or evaluation of the efficacy of medications specifically targeted against cells and pathological tissues. We have synthesized homogeneous CdSe/ZnS (core/shell) highly fluorescent nanocrystals (NC) detectable as individual nanoparticules with a routine fluorescent microscope. These NC are at least 10-fold brighter than the best organic fluorophores and at least 1000-fold more stable against photobleaching than AlexaFluor, for example. When conjugated with proteins, DNA or with drugs, NCs may be excited with the light of any wavelength from UV through visible spectral region providing a range of fluorescence colors depending on their diameter. These properties provide excellent perspectives for high through-put multiplexing and long-term tracking of labeled precursors for days or even weeks. We present here NC applications for ultrasensitive detection of p-glycoprotein, cytokeratins, LCA, Ki67, etc. both on the cellular level and in pathological human surgical specimens.

The C3b/C4b receptor (CR1, CD35) on erythrocytes: methods for study of the polymorphisms.

This report is devoted to methodologies used in analyzing the C3b/C4b receptor (CR1, CD35) on erythrocytes (E), its soluble form, the CRI structural or allotype polymorphism, and CR1 density polymorphism. In primates E CR1 serves as the main system for processing and clearance of complement opsonized immune complexes (IC). CR1 copy numbers decrease with aging of E in normal individuals. Erythrocyte CR1 is also decreased in pathological conditions such as systemic lupus erythematosus (SLE), HIV infection, certain hemolytic anemias, and many other conditions featuring immune complexes. Consequently, CRI on E has an important physiological role in immune complex handling and has interesting alterations in disease.

Genetic analysis of CR1 (the C3b complement receptor, CD35) expression on erythrocytes of HIV-infected individuals.

The number of C3b receptor (CR1) molecules on erythrocytes is genetically determined by two codominant autosomal alleles. The genetic polymorphism of CR1 expression correlates with a Hind III restriction fragment length polymorphism (RFLP) of the CR1 gene. The relative frequency of individuals homozygous for the allele coding for low CR1 numbers is approximately 5% of the normal population. CR1 numbers/erythrocytes are significantly decreased in symptomatic HIV-infected individuals. Decreased CR1 expression correlates with the severity of disease. The present study investigated the CR1 genomic Hind III RFLP-related polymorphism in 79 HIV-infected individuals and 84 healthy subjects. Allele frequencies were found to be similar in both populations. Thus, there is no susceptibility nor resistance to HIV-infected linked to the CR1 gene. Defective expression of CR1 in HIV-infected patients is acquired through central and/or peripheral mechanisms.

Observations on the mechanism of the protective action of sunscreens.

A method is described for measuring the entrance into excised skin of ultraviolet radiation absorbing chemicals (UVRACs) following their application to the cutaneous surface in volatile, partially volatile or nonvolatile vehicles. Also a method is presented for observing changes in optical density (OD) of a sheet of stratum corneum subsequent to the application of an UVRAC and then washing it from the surface. Using these methods, p-aminobenzoic acid (PABA) and 2-ethylhexyl p-dimethyl aminobenzoate (O-PABA) have been studied. In the presence or absence of the nonvolatile vehicle, isopropyl myristate (IM), significant amounts of PABA enter the skin but almost all of the O-PABA remains on the surface. Nevertheless subsequently PABA is more easily removed by water than is O-PABA. When either UVRAC is applied to excised stratum corneum, the OD of the tissue increases immediately; only with PABA is there a further increase as it enters the skin. In vivo, delayed erythemal responses to 280-400 nm radiation of persons to whom the UVRACs are applied correlate well with the observations made on excised skin.

Fasting-associated changes in serum thyrotropin in the rat are influenced by gender.

Studies in the male rat have demonstrated that fasting is associated with a decrease in serum TSH concentrations. The present studies were performed to determine if gender influenced the serum TSH changes associated with fasting. In 8 of 10 experiments in male rats, serum TSH concentrations were significantly reduced in fasted compared to fed groups. In contrast, in none of the 9 experiments in female rats were serum TSH concentrations significantly reduced in the fasted groups. When all experiments were pooled, the decrease in the serum TSH concentration in the fasted rats compared to that in the fed rats was 55 +/- 4% (mean +/- SE) in males and 10 +/- 7% in females (P less than 0.001). In female rats ovariectomy did not result in a pattern in which fasting was associated with a decrease in serum TSH concentrations. Testosterone (T) was administered to male rats during fasting, but this treatment did not prevent the fasting-induced decrease in serum TSH concentrations. In gonadectomized male rats serum TSH concentrations were unchanged by fasting. However, if T was administered to gonadectomized male rats before and during fasting, serum TSH concentrations were significantly decreased in the fasted compared to the fed rats. These studies indicate that there is a sex difference in the serum TSH response to fasting in rats. The decline in serum TSH with fasting in the male rat is not mediated by a decline in serum T concentrations. Rather, T appears to maintain a process which increases the serum TSH concentration, and it is this process that is susceptible to inhibition by fasting.

Vitamin and mineral supplement use is associated with reduced risk of prostate cancer.

This population-based, case-control study in King County, Washington examined supplement use in 697 incident prostate cancer cases (ages 40-64) identified from the Puget Sound Surveillance, Epidemiology and End Results program registry and 666 controls recruited from the same overall population using random-digit dialing sampling. Participants reported their frequency of use of three types of multivitamins and single supplements of vitamins A, C, and E, calcium, iron, and zinc over the 2 years before diagnosis. Logistic regression analyses controlled for age, race, education, family history of prostate cancer, body mass index, number of prostate-specific antigen tests in the previous 5 years, and dietary fat intake. Adjusted odds ratios (95% confidence limits) for the contrast of > or =7/week versus no use were as follows: multivitamins, 0.96 (0.73, 1.26); vitamin A, 0.59 (0.32, 1.06); vitamin C, 0.77 (0.57, 1.04); vitamin E, 0.76 (0.54, 1.08); calcium, 1.04 (0.61, 1.78); iron, 0.50 (0.13, 1.76); and zinc, 0.55 (0.30, 1.00). Odds ratios differed little when cases were stratified by stage of disease at diagnosis or by histopathological grade. There were significant dose-response effects for zinc and ordered dose-response trends for vitamins C and E. Overall, these results suggest that multivitamin use is not associated with prostate cancer risk, but use of individual supplements of zinc, vitamin C, and vitamin E may be protective. Further study is needed to investigate the direct role of these dietary supplements, as well as the role of lifestyle variables associated with supplement use, on prostate cancer risk.

Chemical process synthesis of beta-amino acids and esters.

The importance of beta-amino acids and esters for the synthesis of potential therapeutic agents and biologically active compounds is well known and the subject of this special issue. This review outlines some of the recent approaches reported for the synthesis of both racemic and enantiomeric beta-amino acids and esters with emphasis on those used for large scale production. This compilation is written from a chemical process perspective focusing on the practical application of these processes for large scale synthesis. A survey of procedures described in patent publications as well as current advances from chemical process research groups and results from our laboratory are included with emphasis on asymmetric Michael-type additions, addition of metal enolates to imines/sulfinimines, classical and enzymatic resolutions, and reduction of enantiomeric enamines.

Homogeneous phase pyrophosphate (PPi) measurement (H3PIM). A non-radioactive, quantitative detection system for nucleic acid specific hybridization methodologies including gene amplification.

A one-step, non-radioactive, homogeneous phase revelation system designed to detect and quantify nucleic acid hybridization is described. The principle of the procedure, termed homogeneous phase pyrophosphate (PPi) measurement (H3PIM), is to detect and quantify the release of PPi from nucleotides, which occurs stoichiometrically when nucleic acids are synthesized. The method does not require any special reagents before the H3PIM revelation step. H3PIM is particularly adapted to quantitative measurement of gene amplification or cDNA gene expression using PCR, but can also be used following random priming or simultaneous multi-step nucleic acid amplification. This rapid, sensitive, liquid phase procedure permits the design of low-cost, fully automated devices for gene detection and quantification.

Instantaneous roll-blot from cellulose acetate after electrophoresis. A versatile tool for monoclonal antibody characterization.

An instantaneous blotting method from cellulose acetate to nitrocellulose was developed using a high pressure roll apparatus. This method was applied to the early characterization of monoclonal antibody specificity and to monoclonal immunoglobulin typing in mouse hybridoma supernatants, human sera or unconcentrated urines. Immunoglobulins were then revealed using, successively, anti-isotype specific monoclonal or polyclonal antibodies, avidin-biotinylated peroxidase complexes and cobalt-enhanced diaminobenzidine substrate.

Genomic determination of the CR1 (CD35) density polymorphism on erythrocytes using polymerase chain reaction amplification and HindIII restriction enzyme digestion.

The density of CR1 (the C3b receptor, CD35) on erythrocytes from normal individuals is determined by a codominant bi-allelic system associated with a single base mutation within an intron of the CR1 structural gene, leading to an additional polymorphic HindIII endonuclease site. The CR1 genotype is determined by HindIII digestion of genomic DNA and Southern blotting. We have developed a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of interest followed by HindIII endonuclease digestion and agarose gel electrophoresis which permits a more rapid and reliable determination of the CR1 genotype. The method is suitable for large scale clinical studies in diseases with altered expression of CR1 on erythrocytes.

A simple method for accurate quantification of complement receptor 1 on erythrocytes preserved by fixing or freezing.

The mean number of complement receptor 1 (CR1) molecules on erythrocytes differs between normal individuals within the range of 100-1000 molecules per cell. In some disease states such as systemic lupus erythematosus (SLE), acquired immune deficiency syndrome (AIDS), insulin-dependent diabetes mellitus and malaria, erythrocyte CR1 levels are reduced and CR1 function may be impaired. Current methods for determining erythrocyte CR1 levels by flow cytometry require the use of freshly drawn blood samples because CR1 is lost from erythrocytes during storage. In order to facilitate field studies of associations between erythrocyte CR1 levels and disease, we have developed and validated an assay to quantify CR1 on both healthy and diseased erythrocytes that have been fixed in 5% formaldehyde or frozen in glycerol. These methods enable blood samples to be collected in areas lacking the facilities for flow cytometry and stored for later accurate quantification of CR1. Such procedures will be of particular benefit for future investigations of erythrocyte CR1 expression level and malaria susceptibility.

Enumeration of CR1 complement receptors on erythrocytes using a new method for detecting low density cell surface antigens by flow cytometry.

A sensitive enhancing system was developed for detecting low density cell surface antigen by flow cytometry. The system termed the 'super avidin-biotin system' (SABS) uses biotinylated antibody, phycoerythrin-streptavidin (StreptA-PE), biotinylated goat anti-streptavidin antibody, and StreptA-PE. CR1 complement receptor antigenic sites were quantified on erythrocytes from healthy individuals and patients with antibodies against human immunodeficiency virus (HIV) using SABS and a conventional radioimmunoassay (RIA) with monoclonal anti-CR1 antibody. As little as 50 sites/cell were detected using either SABS or RIA. Accurate quantification of CR1 antigenic sites was achieved within the range of 100-1300 sites/cell. Similar results were obtained using either of the two methods. Intra-assay and day-to-day reproducibilities using SABS were 2% and 12% respectively, comparable to those of conventional RIA measurements. In addition to enumeration of CR1 on erythrocytes for clinical purposes, the use of SABS may probably be extended to a wide number of situations where a sensitive detection of low density cell surface antigen is needed.

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

False-negative results in anti-HLA antibody detection by Sangstat ELISA: complement dependent cytotoxicity is not yet obsolete.

Although ELISA on purified HLA molecules for detecting anti-HLA antibody (P-S ELISA) does detect some antibodies previously missed by the conventional complement dependent cytotoxicity method (C Cytotox), HLA ELISA should not fail to detect antibodies already detected by the conventional reference method to be able to make C Cytotox obsolete and to replace it in routine testing. Among 40 selected sera, 8 false-negative reactions were observed in P-S ELISA. These sera were reanalyzed blind in two laboratories and found to contain non-IgM, warm anti- HLA antibodies. These antibodies were directed in 4 cases against an HLA molecule expressed on a kidney transplant previously rejected by the subject. These antibodies, if missed, would have been potentially harmful in kidney transplantation. Thus P-S ELISA can't yet replace C Cytotox in routine anti-HLA class I detection. The cost/benefit ratio of P-S ELISA as a second-line test remains to be investigated.

Endocytosis of class II histocompatibility antigens and formation of intracytoplasmic granules at the final differentiation stage of human B lymphocytes.

The expression of histocompatibility antigens was investigated using several human lymphoid cell lines representative of different maturation stages of the B-cell lineage. Class II HLA antigens were found at the surface of all cell lines. However, in the myeloma cell line U266, an intracellular macrovesicular pool of these antigens was found in some cells. It originated from microvesicular endocytosis of the surface antigen, subsequently leading to cells bearing HLA class I but not class II antigens. Since the latter play a major role in cellular interactions regulating B-cell differentiation, this phenomenon may be linked to the final stage of maturation of B lymphocytes into plasma cells.