Expression and functional importance of collagen-binding integrins,alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1)beta(1) parallels that of viral-specific effector CD8(+) T cells (defined by tetramer and IFN-gamma staining). In an adoptive transfer model, mAb-mediated blockade of these integrins on activated effector and memory T cells inhibited Ag-specific delayed-type hypersensitivity responses; similar decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect of alpha(1)beta(1) blockade on the delayed-type hypersensitivity response could be bypassed by direct injection of Ag-specific T cells to inflammatory sites, demonstrating for the first time in vivo that collagen-binding integrins are involved in leukocyte migration into tissues.     

In situ hybridization to the Rdl locus on polytene chromosome 3L of Anopheles stephensi

We are interested in generating a Y-autosome translocation of the Resistance to dieldrin (Rdl) locus in the malaria vector mosquito Anopheles stephensi Liston (Diptera: Culicidae), for use in sterile insect release. To ensure stability of the system, a recombination suppressing inversion can also be induced which encompasses the Rdl locus. As a first step, here we report the cloning of fragments of the Rdl gene from both An. stephensi and An. gambiae Giles using degenerate primers in the polymerase chain reaction. These fragments encode the second membrane-spanning region of the gamma-aminobutyric acid receptor and show high levels of both nucleotide and predicted amino acid identity to other Rdl-like receptors. They confirm that, as in all other arthropod species examined, dieldrin resistance in An. stephensi is associated with replacement of alanine302, in this case with a serine. In situ hybridization of the Rdl probe to polytene chromosomes of An. stephensi localizes the gene to the left arm of chromosome 3 (3L) in region 45C. Rdl localization will enable us to identify chromosomal rearrangements encompassing the Rdl locus and help anchor the genome sequence of An. gambiae to the polytene map.     

Identification of a critical amino acid in the aryl hydrocarbon receptor

Two aryl hydrocarbon receptors (rtAHR2alpha and rtAHR2beta) have been identified in the rainbow trout (Oncorhynchus mykiss). These receptors share 98% amino acid identity, yet their functional properties differ. Both rtAHR2alpha and rtAHR2beta bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dimerize with rainbow trout ARNTb (rtARNTb), and recognize dioxin response elements in vitro. However, in a transient transfection assay the two proteins show differential ability to recognize enhancers, produce transactivation, and respond to TCDD. To identify the sequence differences that confer the functional differences between rtAHR2alpha and rtAHR2beta, we constructed chimeric rtAHRs, in which segments of one receptor form was replaced with the corresponding part from the other isoform. This approach progressively narrowed the region being examined to a single residue, corresponding to position 111 in rtAHR2beta. Altering this residue in rtAHR2beta from the lysine to glutamate found in rtAHR2alpha produced an rtAHR2beta with the properties of rtAHR2alpha. All other known AHRs resemble rtAHR2alpha and carry glutamate at this position, located at the N terminus of the PAS-A domain. We tested the effect of altering this glutamate in the human and zebrafish AHRs to lysine. This lysine substitution produced AHRs with transactivation properties that were similar to rtAHR2beta. These results identify a critical residue in AHR proteins that has an important impact on transactivation, enhancer site recognition, and regulation by ligand.     

Beta-amyloid (Abeta) protein in cerebrospinal fluid as a biomarker for Alzheimer's disease

With the arrival of symptomatic treatment (acetylcholine esterase inhibitors) and the promise of drugs that may delay disease progression, development of diagnostic biomarkers for Alzheimer's disease (AD) are important. Beta-Amyloid (Abeta) protein is the main component of senile plaques. A marked reduction in cerebrospinal fluid (CSF)-Abeta42 in AD has been found in numerous studies. Importantly, reduced CSF-Abeta42 is also found very early in the disease process, before the onset of clinical symptoms. Recent studies suggest that CSF-Abeta42 have a satisfactory performance when used as a diagnostic marker for AD in clinical routine. This paper reviews CSF-Abeta42 as a biomarker for AD.     

Messenger RNA electroporation of human monocytes,followed by rapid in vitro differentiation,leads to highly stimulatory antigen-loaded mature dendritic cells

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.     

Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme.

We localized the epitopes for several murine mAbs to human urokinase-type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so-called 37-loop and 60-loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting uPA-catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI-1, decreasing the second-order rate constant 15- to 6500-fold. There was no correlation between the relative effects of the 37-loop and 60-loop substitutions on the second-order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA-PAI-1 reaction, but rather by steric hindrance of the access of PAI-1 to the active site. The affinity of the SPD antibodies for the uPA-PAI-1 complex was only slightly lower than that for free uPA, indicating that the 37-loop and 60-loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA-polyanion affinity after substitution of the three Arg residues.     

Effects of phospholipase A2 on the binding and ion permeability control properties of the acetylcholine receptor.

An acidic phospholipase A₂ (EC 3.1.1.4) isolated from Naja naja siamensis venom blocks acetylcholine receptor function in excitable post synaptic membrane vesicles from Torpedo californica electroplax. Specifically, the phospholipase acts catalytically to prevent the large increase in sodium efflux induced by carbamylcholine. The efflux inhibition can be correlated with specific hydrolysis of phospholipids in the membrane. During the time course of inhibition, the binding affinity of the receptor for carbamylcholine increases 10-fold, a phenomenon associated with receptor desensitization. Prolonged treatment of the membranes with phospholipase A₂ causes nonspecific lysis of the vesicles. Incorporation of unsaturated fatty acids or lysophosphatidylcholine into Torpedo membranes also blocks carbamylcholine-induced sodium efflux. The fatty acids have no effect on the binding affinity of the receptor, and lysophosphatidylcholine causes a small decrease in receptor affinity for carbamylcholine. Lysophosphatidylethanolamine and most saturated fatty acids have no direct effect on sodium efflux, but the lysophosphatides cause vesicle lysis. All of the inhibitory effects of the phospholipase and the fatty acids can be reversed and/or prevented by treatment of the vesicles with bovine serum albumin.     

Variants of CYP46A1 may interact with age and APOE to influence CSF Aβ42 levels in Alzheimer’s disease

Recent studies have suggested that variants of CYP46A1, encoding cholesterol 24-hydroxylase (CYP46), confer risk for Alzheimers disease (AD), a prospect substantiated by evidence of genetic association from several quantitative traits related to AD pathology, including cerebrospinal fluid (CSF) levels of the 42 amino-acid cleavage product of -amyloid (A42) and the tau protein. In the present study, these claims have been explored by the genotyping of previously associated markers in CYP46A1 in three independent northern European case-control series encompassing 1323 individuals and including approximately 400 patients with measurements of CSF A42 and phospho-tau protein levels. Tests of association in case-control models revealed limited evidence that CYP46A1 variants contributed to AD risk across these samples. However, models testing for potential effects upon CSF measures suggested a possible interaction of an intronic marker (rs754203) with age and APOE genotype. In stratified analyses, significant effects were evident that were restricted to elderly APOE 4 carriers for both CSF A42 (P=0.0009) and phospho-tau (P=0.046). Computational analyses indicate that the rs754203 marker probably does not impact the binding of regulatory factors, suggesting that other polymorphic sites underlie the observed associations. Our results provide an important independent replication of previous findings, supporting the existence of CYP46A1 sequence variants that contribute to variability in -amyloid metabolism.     

Common variants of ACE contribute to variable age-at-onset of Alzheimer’s disease

Studies on the role that genetic variation may play in a complex human disease can be empowered by an assessment of both disease risk in case-control or family models and of quantitative traits that reflect elements of disease etiology. An excellent example of this can be found for the 4 allele of APOE in relation to Alzheimers disease (AD) for which association with both risk and age-at-onset (AAO) is evident. Following a recent demonstration that variants of the gene encoding angiotensin I converting enzyme (ACE) contribute to AD risk, we have explored the potential influence of ACE upon AAO in AD. A total of 2861 individuals from three European populations, including six independent AD samples, have been examined in this study. Three single nucleotide polymorphisms (SNPs) previously demonstrated to have maximum effects upon ACE plasma levels and that span the ACE locus were genotyped in these materials. A strong effect upon AAO was observed for marker rs4343 in exon 17 (P<0.0001), but evidence was also obtained indicating a possible independent effect of marker rs4291 (P=0.0095) located in the ACE promoter. Effects were consistent with data from previous studies suggesting association with AD in case-control models, whereby alleles demonstrated to confer risk to disease also appear to reduce AAO. Equivalent effects were evident regardless of APOE 4 carrier status and in both males and females. These results provide an important complement to existing AD risk data, confirming that ACE harbors sequence variants that contribute to aspects of AD pathology.     

A high-density SNP genomewide linkage scan for chronic lymphocytic leukemia-susceptibility loci

Chronic lymphocytic leukemia (CLL) and other B-cell lymphoproliferative disorders (LPDs) show clear evidence of familial aggregation, but the inherited basis is largely unknown. To identify a susceptibility gene for CLL, we conducted a genomewide linkage analysis of 115 pedigrees, using a high-density single-nucleotide polymorphism (SNP) array containing 11,560 markers. Multipoint linkage analyses were undertaken using both nonparametric (model-free) and parametric (model-based) methods. Our results confirm that the presence of high linkage disequilibrium (LD) between SNP markers can lead to inflated nonparametric linkage (NPL) and LOD scores. After the removal of high-LD SNPs, we obtained a maximum NPL of 3.14 (P=.0008) on chromosome 11p11. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under both dominant (HLOD 1.95) and recessive (HLOD 2.78) models. In addition, four other chromosomal positions (5q22-23, 6p22, 10q25, and 14q32) displayed HLOD scores >1.15 (which corresponds to a nominal P value <.01). None of the regions coincided with areas of common chromosomal abnormalities frequently observed for CLL. These findings strengthen the argument for an inherited predisposition to CLL and related B-cell LPDs.