Death of peripheral CD8+ T cells in the absence of MHC class I is Fas-dependent and not blocked by Bcl-xL

Productive immune responses require an appropriate environment to support peripheral CD8(+) T cell survival. Although host MHC class I molecules appear to be required for this process, the cellular and molecular requirements have not been comprehensively studied. Using adoptive transfer of 2C/recombinase-activating gene-2 (RAG-2)(-/-) TCR-transgenic T cells, we found that the survival of both naive and effector CD8(+) T cells was dependent upon host expression of the same MHC class I alleles that supported thymic selection. Expression of appropriate MHC class Iby either bone marrow- or non-bone-marrow-derived cells was sufficient, suggesting that professional antigen-presenting cells were not mandatory. In contrast to MHC class I, neither T cell expression of CD28 nor host expression of ICAM-1 was required for peripheral T cell survival. Finally, T cell death in the absence of appropriate host MHC class I was overcome by elimination of Fas signaling but not by overexpression of Bcl-x(L) by CD8(+) T cells. These results suggest that, in the absence of a survival signal provided by engagement of host MHC/self peptide complexes, CD8(+) T cells die via a Fas-dependent, mitochondria-independent pathway.     

Spontaneous aberrant crypt foci in Apc1638N mice with a mutant Apc allele

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.     

Comparison of Digene hybrid capture 2 and conventional culture for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in cervical specimens

Digene's Hybrid Capture 2 (HC2) CT/GC, CT-ID, and GC-ID DNA tests were evaluated by comparison to traditional culture methods for detecting Chlamydia trachomatis and Neisseria gonorrhoeae infections in 669 cervical specimens from high-risk female populations attending two sexually transmitted disease clinics. For detection of either or both infections, the HC2 CT/GC test algorithm had 93.8% sensitivity and 95.9% specificity compared to those of culture. After resolution of discrepant results by direct fluorescent-antibody (DFA) staining or PCR assay, the relative sensitivity and specificity of the HC2 CT/GC test algorithm increased to 94.8 and 99.8%, while the values for culture were 83.6% (McNemar's P value, 0.0062) and 100%, respectively. For detection of the individual pathogens, the relative sensitivities for the HC2 CT-ID and GC-ID tests were 97.2 and 92.2% and the specificities were greater than 99% compared to culture adjucated by DFA staining and PCR. Test performance varied at the two clinics: the HC2 CT/GC algorithm, CT-ID, and GC-ID tests had significantly higher sensitivities (McNemar's P value, <0.05) than that of culture for the population at one clinic as well as for the combined populations. At the other clinic, the HC2 tests performed as well as culture.     

Autoantibodies to C-reactive protein is a common finding in SLE,but not in primary Sjögren's syndrome,rheumatoid arthritis or inflammatory bowel disease

The occurrence of antibodies to human C-reactive protein (CRP) was analysed by enzyme-linked immunosorbent assay (ELISA) in 56 patient sera known to contain antibodies to double-stranded DNA (dsDNA) and in 16 sera from patients with primary Sj?gren's syndrome (SS), 15 rheumatoid arthritis, 31 Crohn's disease, and 37 ulcerative colitis. Eighty-seven per cent of the patients with anti-dsDNA antibodies had systemic lupus erythematosus (SLE) and the remaining had autoimmune hepatitis. The cut-off for positive anti-CRP test was set at the 95th percentile of 100 healthy blood donors. Twenty of 56 anti-dsDNA sera (36%) and two of 16 SS sera (13%) had antibodies reactive with human CRP, whereas all other samples were negative. Thirteen of 27 SLE patients (48%) were positive on at least one occasion. The sera containing anti-CRP antibodies only reacted with surface-bound antigen, but not with native CRP in solution. In conclusion, we found that autoantibodies to CRP are common in sera from patients with anti-dsDNA antibodies. It is not likely that this explains the relative failure of CRP response in patients with active SLE. However, it cannot be excluded that anti-CRP autoantibodies have other biological potentials of pathophysiological interest in SLE, for instance by binding to CRP deposited on cell and tissue surfaces.     

Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by the Cryptococcus neoformans Capsule

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.Cryptococcus neoformans is the etiological agent of cryptococcal meningitis, a life-threatening infection of particular importance in patients with deficiencies in cellular immunity, most notably patients with the AIDS. The yeast is surrounded by a polysaccharide capsule that is composed primarily of glucuronoxylomannan (GXM), which has a linear (1→3)-α-d-mannopyranan backbone bearing β-d-xylopyranosyl, β-d-glucopyranosyluronic acid, and O-acetyl substituents (3, 9, 54). The cryptococcal capsule occurs as four major serotypes (A, B, C, and D) and is an essential virulence factor for the yeast.One of the most striking features of the cryptococcal capsule is its ability to activate the alternative complement pathway. Incubation of encapsulated cryptococci in normal human serum (NHS) leads to the deposition of 10⁷ to 10⁸ C3 fragments on the yeast (28, 56). The C3 is deposited at the surface and throughout the capsule (30). Available evidence indicates that the amount of anti-GXM antibodies found in NHS is not sufficient to initiate the classical pathway (24); consequently, activation and binding of C3 to the cryptococcal capsule are mediated entirely by the alternative complement pathway (29, 30, 55). One of the hallmark features of alternative pathway deposition of C3 onto encapsulated cryptococci is a delay of 5 to 8 min before readily detectable amounts of C3 are found on yeast cells incubated in NHS (29, 55). Once past the initial lag, C3 fragments rapidly accumulate on the yeast cells as incubation proceeds for an additional 10 min.Recently, there has been interest in antibody-mediated resistance to cryptococcosis. Monoclonal antibodies (MAbs) have been proposed for treatment of cryptococcosis (7), and immunization with GXM-protein conjugates has been suggested for prevention of cryptococcosis (6, 12, 13). However, it is becoming increasingly clear that anti-GXM MAbs may have distinct specificities and biological activities. Anti-GXM MAbs which differ in (i) reactivities with GXM of the four major serotypes (2), (ii) apparent binding sites in the cryptococcal capsule (32, 37), and (iii) abilities to provide protection in a murine model of cryptococcosis (32, 37) have been described. Some differences in biological activity are related to differences in the epitope specificities of the various MAbs (32, 37).One means by which antibodies could enhance resistance to cryptococcosis is through accelerated deposition of opsonic C3 fragments via the action of the classical pathway. Such an acceleration would reduce or eliminate the 5- to 8-min lag that occurs during alternative pathway-mediated deposition of C3 fragments. The objectives of our study were to evaluate the effects of anti-GXM MAbs on the kinetics and sites for deposition of C3 fragments into the cryptococcal capsule. We examined several well-characterized antibodies that differed in the epitope specificity of the MAbs. The results showed that MAbs with different isotypes and epitope specificities had distinctly different effects on activation and binding of C3 via the classical and alternative pathways; many antibodies markedly suppressed C3 binding, some antibodies accelerated C3 binding, and other antibodies had little or no effect.     

Multicontrast MRI of remyelination in the central nervous system

Although magnetic resonance imaging (MRI) represents the most sensitive tool for the detection of white matter abnormalities in patients with multiple sclerosis (MS), the heterogeneity of MS placques severely hampers the elucidation of specific pathophysiological processes. In order to identify putative MRI markers for de- and remyelination, we employed the cuprizone mouse model which leads to a selective and reversible demyelination of the corpus callosum with little or no axonal damage. Apart from histopathology, animals were studied with high-resolution three-dimensional MRI in vivo using multiple contrasts. While individual MRI findings significantly correlated with electron microscopy, the differentiation of regions with normal, demyelinated or remyelinated white matter by one contrast alone was less specific than by histology or electron microscopy. However, an accurate MRI prediction of the in vivo myelin status was achieved by a discriminant function analysis using a combination of T1, T2 and magnetization transfer contrast. With a correct assignment of 95% of all animals examined, the procedure will allow for the survey of new therapeutic approaches aiming at improved remyelination.