Autoimmunity and glomerulonephritis in mice with targeted deletion of the serum amyloid P component gene: SAP deficiency or strain combination?

Human serum amyloid P component (SAP) binds avidly to DNA, chromatin and apoptotic cells in vitro and in vivo. 129/Sv x C57BL/6 mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoantibodies and immune complex glomerulonephritis. SAP-deficient animals, created by backcrossing the 129/Sv SAP gene deletion into pure line C57BL/6 mice and studied here for the first time, also spontaneously developed broad spectrum antinuclear autoimmunity and proliferative immune complex glomerulonephritis but without proteinuria, renal failure, or increased morbidity or mortality. Mice hemizygous for the SAP gene deletion had an intermediate autoimmune phenotype. Injected apoptotic cells and isolated chromatin were more immunogenic in SAP(-/-) mice than in wild-type mice. In contrast, SAP-deficient pure line 129/Sv mice did not produce significant autoantibodies either spontaneously or when immunized with extrinsic chromatin or apoptotic cells, indicating that loss of tolerance is markedly strain dependent. However, SAP deficiency in C57BL/6 mice only marginally affected plasma clearance of exogenous chromatin and had no effect on distribution of exogenous nucleosomes between the liver and kidneys, which were the only tissue sites of catabolism. Furthermore, transgenic expression of human SAP in the C57BL/6 SAP knockout mice did not abrogate the autoimmune phenotype. This may reflect the different binding affinities of mouse and human SAP for nuclear autoantigens and/or the heterologous nature of transgenic human SAP in the mouse. Alternatively, the autoimmunity may be independent of SAP deficiency and caused by expression of 129/Sv chromosome 1 genes in the C57BL/6 background.     

Classical and alternative pathway complement activation are not required for reactive systemic AA amyloid deposition in mice

During induction of reactive systemic amyloid A protein (AA) amyloidosis in mice, either by chronic inflammation or by severe acute inflammation following injection of amyloid enhancing factor, the earliest deposits form in a perifollicular distribution in the spleen. Because the splenic follicular localization of immune complexes and of the scrapie agent are both complement dependent in mice, we investigated the possible complement dependence of AA amyloid deposition. In preliminary experiments, substantial depletion of circulating C3 by cobra venom factor had little effect on experimental amyloid deposition. More importantly, mice with targeted deletion of the genes for C1q or for both factor B and C2, and therefore unable to sustain activation, respectively, of either the classical complement pathway or both the classical and alternative pathways, showed amyloid deposition similar to wild type controls. Complement activation by either the classical or alternative pathways is thus not apparently necessary for the experimental induction of systemic AA amyloid in mice.     

Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Are primed CD4+ T lymphocytes different from unprimed cells?

Primed and unprimed lymphocytes are usually classified as separate subsets of cells, based on phenotypic and functional distinctions. In the case of CD4⁺ T lymphocytes, primed cells are thought to proliferate more vigorously, quickly and easily, and to release a different profile of cytokines, than their naive equivalent. However, most of these data were obtained from studies in which populations of lymphocytes were compared before and after antigenic stimulation, and therefore did not distinguish between the effects resulting from the clonal expansion of specific precursor cells within such populations and those due to cell differentiation per se. We have investigated the contribution of precursor cell frequency to some of the functional changes observed in populations of CD4⁺ T cells following antigenic stimulation, using approaches in which antigen-specific precursor frequencies are high in both primary and secondary stimulations: mixed leukocyte reaction responses and cells from αβ T cell receptor transgenic mice. Our data suggest that when equivalent numbers of antigen-specific naive and previously primed CD4⁺ responder T cells are compared, there is no difference in their potency to proliferate but only the previously activated subset can generate cytokines such as interferon-γ.     

Multiplex RT-nested PCR differentiation of gill-associated virus (Australia) from yellow head virus (Thailand) of Penaeus monodon

A multiplex RT-nested PCR has been developed to detect and differentiate the closely related prawn viruses, gill-associated virus (GAV) from Australia and yellow head virus (YHV) from Thailand. RT-PCR using primers to conserved sequences in the ORF1b gene amplified a 794 bp region of either GAV or YHV. Nested PCR using a conserved sense primer and either a GAV- or YHV-specific antisense primer to a divergent sequence differentially amplified a 277 bp region of the primary PCR amplicon. Multiplexing the YHV antisense primer with a GAV antisense primer to another divergent sequence allowed the viruses to be distinguished in a single nested PCR. Nested PCR enhanced detection sensitivity between 100- and 1000-fold and GAV or YHV RNA was detectable in approximately 10 fg lymphoid organ total RNA. The multiplex RT-nested PCR was also able to co-detect GAV and YHV RNA mixed over a wide range of concentrations to simulate potential dual-infection states. The robustness of the test was examined using RNA samples from Penaeus monodon prawns infected either chronically or acutely with GAV or YHV and collected at different locations in Eastern Australia and Thailand between 1994 and 1998. GAV- (406 bp) or YHV-specific (277 bp) amplicons were differentially generated in all cases, including five YHV RNA samples in which no primary RT-PCR amplicon was detected. Sequence analysis of GAV and YHV PCR amplicons identified minor variations in the regions targeted by the virus-specific antisense primers. However, none occurred at positions that critically affected the PCR.     

Neurophysiological responses to face, facial regions and objects in adults with Asperger's syndrome: an ERP investigation.

Face processing differences have been observed between AS and control subjects at the behavioural and neurological levels. The purpose of the present study was to investigate the neurophysiological basis of processing faces and facial features (eyes and mouths) in adults with AS relative to age- and gender-matched typically-developing controls. These results were compared with ERPs generated to objects in both groups to determine if any differences were specific to facial stimuli. Although both groups elicited earlier N170 latencies to faces than to face parts and to eyes relative to mouths, adults with AS exhibited delayed N170 latencies to faces and face parts relative to controls. This difference was not observed to objects. Together these findings suggest that adults with AS may be slower to process facial information.     

Recognition of a novel naturally processed, A2 restricted, HCV-NS4 epitope triggers IFN-gamma release in absence of detectable cytopathicity

Using short term CTL lines derived from HLA A2/K^b transgenic mice and IFN-gamma release assays we demonstrate that the NS4.1769 epitope, is generated from natural processing of the NS4 antigen, and presented in the context of the A2/K^b molecules. Interestingly, T cell recognition of the naturally processed form of the NS4.1769 epitope was associated with significant IFN-gamma release, but no direct cytolytic activity. Epitopes of this phenotype might be of interest, in terms of therapy of chronic HCV infection by associating the benefit of localized lymphokine release with low or absent direct cytopathicity.     

Predominant type 1 CMV-specific memory T-helper response in humans: evidence for gender differences in cytokine secretion

Cell-mediated memory immune responses to viral antigens are important for protection against viruses causing persistent or acute infections. This study compared the cytokine profile of memory T-helper cells specific for cytomegalovirus (CMV) in healthy CMV-seropositive men and women. The cytokine response reflected T(H)1 bias, with dominant secretion of interferon (IFN)-gamma along with moderate levels of tumor necrosis factor-alpha, interleukin (IL)-10, and IL-2. Analyzed by gender, women had higher and significant spontaneous release of IFN-gamma and CMV-specific IL-2 secretion. Similar analysis with herpes simplex virus-1 showed a trend toward higher cytokine responsiveness in women, but the differences were not statistically significant. In contrast, men had statistically significant higher influenza virus-specific tumor necrosis factor-alpha secretion. IL-4 and IL-5, both T(H)2 cytokines, were low for all three viruses. The results show a predominant T(H)1 antiviral cytokine T-help memory response with significant differences linked to gender. Such differences may have an impact in the design of immunization strategies against CMV.     

Ventral medulla pHi measured in vivo by 31P NMR is not regulated during hypercapnia in anesthetized rat

Chemoreceptors in the ventral medulla contribute to the respiratory response to hypercapnia. Do they ‘sense’ intracellular pH (pH_i)? We measured pH_i in the ventral medulla or cortex (control) using ³¹P-NMR obtained via a novel 3×5 mm² surface coil in anesthetized rats breathing air or 7% CO₂. During air breathing over 240 min, pH_i decreased slightly from 7.13±0.02 to 7.05±0.02 (SEM; n=5; 2 cortex, 3 ventral medulla). During 180 min of hypercapnia, cortical pH_i (n=4) decreased from 7.17±0.02 to 6.87±0.01 by 90 min and recovered by 150 min. Ventral medulla pH_i showed no such regulation. It decreased from 7.11±0.02 to 6.88±0.02 at 90 min and recovered only after cessation of hypercapnia (n=5), results consistent with pH_i being the chemoreceptor stimulus. However, non-chemoreceptor neurons that contribute to our medullary NMR signal also do not appear to regulate pH_i in vitro. Regional differences in pH_i regulation between cortex and ventral medulla may be due to both chemosensitive and non-chemosensitive neurons.