109 research outputs found

    Carbonic Anhydrase 5 Regulates Acid-Base Homeostasis in Zebrafish

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    The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5) mutation, collapse of fins (cof), which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders

    Association of CD40 Gene Polymorphisms with Sporadic Breast Cancer in Chinese Han Women of Northeast China

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    BACKGROUND: Breast cancer is a polygenetic disorder with a complex inheritance pattern. Single nucleotide polymorphisms (SNPs), the most common genetic variations, influence not only phenotypic traits, but also interindividual predisposition to disease, treatment outcomes with drugs and disease prognosis. The co-stimulatory molecule CD40 plays a prominent role in immune regulation and homeostasis. Accumulating evidence suggests that CD40 contributes to the pathogenesis of cancer. Here, we set out to test the association between polymorphisms in the CD40 gene and breast carcinogenesis and tumor pathology. METHODOLOGY AND PRINCIPAL FINDINGS: Four SNPs (rs1800686, rs1883832, rs4810485 and rs3765459) were genotyped by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method in a case-control study including 591 breast cancer patients and 600 age-matched healthy controls. Differences in the genotypic distribution between breast cancer patients and healthy controls were analyzed by the Chi-square test for trends. Our preliminary data showed a statistically significant association between the four CD40 gene SNPs and sporadic breast cancer risk (additive P = 0.0223, 0.0012, 0.0013 and 0.0279, respectively). A strong association was also found using the dominant, recessive and homozygote comparison genetic models. In the clinical features analysis, significant associations were observed between CD40 SNPs and lymph node metastasis, human epidermal growth factor receptor 2 (C-erbB2), estrogen receptor (ER), progesterone receptor (PR) and tumor protein 53 (P53) statuses. In addition, our haplotype analysis indicated that the haplotype C(rs1883832)G(rs4810485), which was located within the only linkage disequilibrium (LD) block identified, was a protective haplotype for breast cancer, whereas T(rs1883832)T(rs4810485) increased the risk in the studied population, even after correcting the P value for multiple testing (P = 0.0337 and 0.0430, respectively). CONCLUSIONS AND SIGNIFICANCE: Our findings primarily show that CD40 gene polymorphisms contribute to sporadic breast cancer risk and have a significant association with clinicopathological features among Chinese Han women from the Heilongjiang Province

    Structural and Thermodynamic Approach to Peptide Immunogenicity

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    In the conventional paradigm of humoral immunity, B cells recognize their cognate antigen target in its native form. However, it is well known that relatively unstable peptides bearing only partial structural resemblance to the native protein can trigger antibodies recognizing higher-order structures found in the native protein. On the basis of sound thermodynamic principles, this work reveals that stability of immunogenic proteinlike motifs is a critical parameter rationalizing the diverse humoral immune responses induced by different linear peptide epitopes. In this paradigm, peptides with a minimal amount of stability (ΔGX<0 kcal/mol) around a proteinlike motif (X) are capable of inducing antibodies with similar affinity for both peptide and native protein, more weakly stable peptides (ΔGX>0 kcal/mol) trigger antibodies recognizing full protein but not peptide, and unstable peptides (ΔGX>8 kcal/mol) fail to generate antibodies against either peptide or protein. Immunization experiments involving peptides derived from the autoantigen histidyl-tRNA synthetase verify that selected peptides with varying relative stabilities predicted by molecular dynamics simulations induce antibody responses consistent with this theory. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the development/evolution of humoral immune responses

    Impaired distal nephron acidification in chronically phosphate depleted rats

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    Renal tubular bicarbonate reabsorption and acidification were evaluated in phosphate depleted rats (PD) and controls. After 33 days of phosphate depletion, urine pH of PD rats ( N =5, 6.36±0.15) was significantly higher than control ( N =5, 5.64±0.09, P <0.005) following an NH 4 Cl load. Urinary titratable acid of PD rats (9.6±1.8) was significantly reduced compared to control (117.2±19.7 μEq/3 h, P <0.001), whereas NH 4 + excretion was not different. The plasma HCO 3 − thresholds at which bicarbonaturia occurred (approximately 25 mEq/l) were identical in controls and phosphate depleted rats during isotonic bicarbonate infusion. The higher urine pH of phosphate depleted rats following NH 4 Cl administration was not due to low urinary phosphate as 3-day phosphate depleted rats could normally acidify urine after NH 4 Cl (pH=5.86±0.09, N =6 vs. control 5.87±0.08, N =6, P =N.S.) despite urinary phosphate excretion as low as in 33-day PD rats. These data indicate the presence of impaired distal tubular acidification in chronically phosphate depleted rats.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47445/1/424_2004_Article_BF00584277.pd

    Proliferation of Acid-Secretory Cells in the Kidney during Adaptive Remodelling of the Collecting Duct

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    The renal collecting duct adapts to changes in acid-base metabolism by remodelling and altering the relative number of acid or alkali secreting cells, a phenomenon termed plasticity. Acid secretory A intercalated cells (A-IC) express apical H+-ATPases and basolateral bicarbonate exchanger AE1 whereas bicarbonate secretory B intercalated cells (B-IC) express basolateral (and apical) H+-ATPases and the apical bicarbonate exchanger pendrin. Intercalated cells were thought to be terminally differentiated and unable to proliferate. However, a recent report in mouse kidney suggested that intercalated cells may proliferate and that this process is in part dependent on GDF-15. Here we extend these observations to rat kidney and provide a detailed analysis of regional differences and demonstrate that differentiated A-IC proliferate massively during adaptation to systemic acidosis. We used markers of proliferation (PCNA, Ki67, BrdU incorporation) and cell-specific markers for A-IC (AE1) and B-IC (pendrin). Induction of remodelling in rats with metabolic acidosis (with NH4Cl for 12 hrs, 4 and 7 days) or treatment with acetazolamide for 10 days resulted in a larger fraction of AE1 positive cells in the cortical collecting duct. A large number of AE1 expressing A-IC was labelled with proliferative markers in the cortical and outer medullary collecting duct whereas no labeling was found in B-IC. In addition, chronic acidosis also increased the rate of proliferation of principal collecting duct cells. The fact that both NH4Cl as well as acetazolamide stimulated proliferation suggests that systemic but not urinary pH triggers this response. Thus, during chronic acidosis proliferation of AE1 containing acid-secretory cells occurs and may contribute to the remodelling of the collecting duct or replace A-IC due to a shortened life span under these conditions

    Down selecting adjuvanted vaccine formulations: a comparative method for harmonized evaluation.

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    The need for rapid and accurate comparison of panels of adjuvanted vaccine formulations and subsequent rational down selection, presents several challenges for modern vaccine development. Here we describe a method which may enable vaccine and adjuvant developers to compare antigen/adjuvant combinations in a harmonized fashion. Three reference antigens: Plasmodium falciparum apical membrane antigen 1 (AMA1), hepatitis B virus surface antigen (HBsAg), and Mycobacterium tuberculosis antigen 85A (Ag85A), were selected as model antigens and were each formulated with three adjuvants: aluminium oxyhydroxide, squalene-in-water emulsion, and a liposome formulation mixed with the purified saponin fraction QS21. The nine antigen/adjuvant formulations were assessed for stability and immunogenicity in mice in order to provide benchmarks against which other formulations could be compared, in order to assist subsequent down selection of adjuvanted vaccines. Furthermore, mouse cellular immune responses were analyzed by measuring IFN-γ and IL-5 production in splenocytes by ELISPOT, and humoral responses were determined by antigen-specific ELISA, where levels of total IgG, IgG1, IgG2b and IgG2c in serum samples were determined. The reference antigens and adjuvants described in this study, which span a spectrum of immune responses, are of potential use as tools to act as points of reference in vaccine development studies. The harmonized methodology described herein may be used as a tool for adjuvant/antigen comparison studies

    Antibody isotype analysis of malaria-nematode co-infection: problems and solutions associated with cross-reactivity

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    <p>Abstract</p> <p>Background</p> <p>Antibody isotype responses can be useful as indicators of immune bias during infection. In studies of parasite co-infection however, interpretation of immune bias is complicated by the occurrence of cross-reactive antibodies. To confidently attribute shifts in immune bias to the presence of a co-infecting parasite, we suggest practical approaches to account for antibody cross-reactivity. The potential for cross-reactive antibodies to influence disease outcome is also discussed.</p> <p>Results</p> <p>Utilising two murine models of malaria-helminth co-infection we analysed antibody responses of mice singly- or co-infected with <it>Plasmodium chabaudi chabaudi </it>and <it>Nippostrongylus brasiliensis </it>or <it>Litomosoides sigmodontis</it>. We observed cross-reactive antibody responses that recognised antigens from both pathogens irrespective of whether crude parasite antigen preparations or purified recombinant proteins were used in ELISA. These responses were not apparent in control mice. The relative strength of cross-reactive versus antigen-specific responses was determined by calculating antibody titre. In addition, we analysed antibody binding to periodate-treated antigens, to distinguish responses targeted to protein versus carbohydrate moieties. Periodate treatment affected both antigen-specific and cross-reactive responses. For example, malaria-induced cross-reactive IgG1 responses were found to target the carbohydrate component of the helminth antigen, as they were not detected following periodate treatment. Interestingly, periodate treatment of recombinant malaria antigen Merozoite Surface Protein-1<sub>19 </sub>(MSP-1<sub>19</sub>) resulted in increased detection of antigen-specific IgG2a responses in malaria-infected mice. This suggests that glycosylation may have been masking protein epitopes and that periodate-treated MSP-1<sub>19 </sub>may more closely reflect the natural non-glycosylated antigen seen during infection.</p> <p>Conclusions</p> <p>In order to utilize antibody isotypes as a measure of immune bias during co-infection studies, it is important to dissect antigen-specific from cross-reactive antibody responses. Calculating antibody titre, rather than using a single dilution of serum, as a measure of the relative strength of the response, largely accomplished this. Elimination of the carbohydrate moiety of an antigen that can often be the target of cross-reactive antibodies also proved useful.</p
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