The Frequencies of IFNγ+IL2+TNFα+ PPD-Specific CD4+CD45RO+ T-Cells Correlate with the Magnitude of the QuantiFERON? Gold In-Tube Response in a Prospective Study of Healthy Indian Adolescents

Background

QuantiFERON-TB Gold In-Tube (QFT) is an IFNγ-release assay used in the diagnosis of Mycobacterium tuberculosis (MTB) infection. The risk of TB progression increases with the magnitude of the MTB-specific IFNγ-response. QFT reversion, also associated with low Tuberculin Skin Test responses, may therefore represent a transient immune response with control of M. tuberculosis infection. However, studies at the single cell level have suggested that the quality (polyfunctionality) of the T-cell response is more important than the quantity of cytokines produced.

Objective

To explore the quality and/or magnitude of mycobacteria-specific T-cell responses associated with QFT reversion and persistent QFT-positivity.

Methods

Multi-color flowcytometry on prospectively collected peripheral blood mononuclear cells was applied to assess mycobacteria-specific T-cell responses in 42 QFT positive Indian adolescents of whom 21 became QFT negative (reverters) within one year. Ten QFT consistent negatives were also included as controls.

Results

There was no difference in the qualitative PPD-specific CD4+ T-cell response between QFT consistent positives and reverters. However, compared with QFT consistent positives, reverters displayed lower absolute frequencies of polyfunctional (IFNγ+IL2+TNFα+) CD4+ T-cells at baseline, which were further reduced to the point where they were not different to QFT negative controls one year later. Moreover, absolute frequencies of these cells correlated well with the magnitude of the QFT-response.

Conclusion

Whereas specific polyfunctional CD4+ T-cells have been suggested to protect against TB progression, our data do not support that higher relative or absolute frequencies of PPD-specific polyfunctional CD4+ T-cells in peripheral blood can explain the reduced risk of TB progression observed in QFT reverters. On the contrary, absolute frequencies of these cells correlated with the QFT-response, suggesting that this readout reflects antigenic load.     

Expression level of c-FLIP versus Fas determines susceptibility to Fas ligand-induced cell death in murine thymoma EL-4 cells

The caspase-8 inhibitor c-FLIP blocks death receptor-mediated cell death and plays an essential role in the regulation of lymphocyte homeostasis and the immune escape of tumors. The murine thymoma cell line EL-4 was resistant to Fas ligand (FasL)-induced apoptosis by constitutive expression of FLIP (L). Cycloheximide downregulated the expression of FLIP (L) and markedly sensitized EL-4 cells to FasL-induced apoptosis. In contrast, DNA-damaging agents sensitized EL-4 cells to FasL-induced cell death via an increase of cell-surface Fas without any influence on FLIP (L) expression. Enforced expression of transfected Fas rendered EL-4 cells highly susceptible to FasL-induced cell death. These findings demonstrate that susceptibility to FasL-induced cell death mainly depends on the expression level of c-FLIP versus cell-surface Fas.     

Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes

Cytolytic granules from a T-cell line with specific cytolytic activity were isolated. Granules were solubilized and fractionated on a TSK 4000 gel filtration column. Lytic activity was eluted as a single retarded peak. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the lytic fractions contained a single protein (perforin) with an apparent molecular weight of approximately 66 kDa. It separated well from the other proteins present in the granules. Isolated perforin polymerized and inserted into lipid bilayers in the presence of Ca2+, forming tubular structures with inner diameters varying from 6 to 16 nm. Lipid insertion of perforin was demonstrated using a membrane-restricted, photoactivatable probe. The lytic properties of perforin suggest an important role of this particular protein during cytolytic T-cell-mediated target cell lysis.     

Isolation and complete structure of the lymphocyte serine protease granzyme G, a novel member of the granzyme multigene family in murine cytolytic T lymphocytes. Evolutionary origin of lymphocyte proteases

A cDNA clone that is closely related to the granule-associated serine proteases of cytolytic T lymphocytes (CTL), called granzymes A-F, was isolated from a CTL expression library. The encoded serine protease, granzyme G, shows 70%-89% nucleotide identities to the granzymes C-F and, like those, consists of 228 amino acids preceded by the short propeptide Glu-Glu and a 18 residue long signal peptide. Granzyme G was identified by amino-terminal sequence analysis as a correctly processed and sorted protein stored in lysosome-like granules. The phylogenetic history of the granzyme multigene family was reconstructed by two tree-making methods and by Southern blot analyses of human, rat, and mouse DNA. Our results indicate differences in the evolutionary pathway between these species. The murine granzymes C-G descended from a progenitor present at the time of mammalian radiation. Granzyme C branched off first after the primate-rodent split and was involved in a recombination event with granzyme B before the rat-mouse divergence. Granzymes D and E have diverged after the mouse-rat speciation. However, no experimental evidence for the existence of a granzyme C-D-E-F-G equivalent was found in humans, and loss of the ancestral gene in the primate lineage is discussed. In view of the species differences in the number of granzyme gene copies during recent evolution, we propose that the murine granzymes B-G play several distinct roles in CTL-mediated effector functions as a response to quite recent changes of the biochemical environment.     

Is excessive running predictive of degenerative hip disease? Controlled study of former elite athletes.

OBJECTIVE--To determine the effects of regular long distance running on the state of the hips in later life. DESIGN--Retrospective study of a cohort of elite athletes and a group of normal, healthy, untrained controls examined 15 years after initial testing. SETTING--Research project at school for physical education and sports. SUBJECTS--27 Former long distance runners (mean age 42), nine former bobsleigh riders (mean age 42), and 23 normal, healthy, untrained men (mean age 35) who had been examined in 1973 and who agreed to re-examination in 1988. MAIN OUTCOME MEASURE--Radiological evidence of degenerative hip disease in 1988. RESULTS--Physiological and exercise characteristics of all subjects had been recorded in 1973, and in 1988 these measurements were repeated together with radiological examination of the hips. An additive radiological index of hip disease based on grades of subchondral sclerosis, osteophyte formation, and joint space narrowing was significantly increased among runners as compared with bobsleigh riders and untrained controls. After adjustment for age the significant effect of type of sports activity remained (p = 0.032). In multivariate analyses age and milage run in 1973 (97 km/week) emerged as independent, significant, and positive predictors of radiological signs of degenerative hip disease in 1988 (p = 0.017 and p = 0.024 respectively). Among runners alone running pace in 1973 rather than milage run was the stronger predictor of subsequent degenerative hip disease. The milage run in 1988 was not particularly predictive of the radiological index, but endurance in 1988 was inversely related to degenerative hip disease seen radiologically. CONCLUSION--Long term, high intensity, high milage running should not be dismissed as a potential risk factor for premature osteoarthritis of the hip.     

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.     

Membranolysis by the ninth component of human complement

The lytic property of isolated C9 was shown by using heat (48°C) to induce polymerization of C9, which was then exposed to egg lecithin vesicles containing carboxyfluorescein. In this environment, C9 penetrated the vesicles and released the marker. C5b-9 and C5b-8 also released carboxyfluorescein at 48°C, as did C5b-7 to a lesser extent. However, neither isolated C5b-6, C7, C8 nor albumin caused such release. At 30°C, the C9 remained in the monomeric form and could not lyse the vesicles.C9 polymers formed at 48°C were ring-shaped with an internal diameter of approximately 100 Å and an outer diameter of approximately 180 Å. These rings of C9 polymers strikingly resembled the ultrastructures formed by the membrane attack complex of complement, viewed from the top.Our results indicate that polymeric C9 causes the membranolysis of phospholipid vesicles and, hence, that C9 alone is a cytotoxic molecule.     

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.     

Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma

Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.     

Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement

Poly(C9), the tubular 27 S complex forming the transmembrane channel of the membrane attack complex of complement, was purified to homogeneity by gel filtration and sucrose density gradient ultracentrifugation. The molecular weight of poly(C9) was determined by two independent methods in addition to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. First, sedimentation equilibrium analysis using 0.2% SDS or 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 X 10(6) and a weight average molecular weight of all poly(C9) complexes of 1,050,000 +/- 40,000 (S.D.). SDS and deoxycholate binding to poly(C9) was measured in an air-driven ultracentrifuge and was determined to be 0.53 +/- 0.065 (S.D.) g of SDS and 0.26 +/- 0.015 (S.D.) g of deoxycholate/g of poly(C9), respectively. Second, the mass of 27 S poly(C9) devoid of bound detergent was determined by electron scattering of unstained specimens in the scanning transmission electron microscope. The molecular weight obtained by this method was 1,078,000 +/- 194,000. The inner diameter of poly(C9) tubules imaged in top view projections by negative staining electron microscopy varied between 9 and 12 mm. The accumulated data suggest a true heterogeneity of the molecular weight of poly(C9) due to polymers with varying protomer numbers. Using a mean value of 73,500 for the molecular weight of monomeric C9, the protomer number of poly(C9) tubules appear to vary between 12 and 18 C9 subunits. Approximately 50-75% of the tubules have 14 to 16 subunits as deduced from the mass distribution determined by electron scattering and from ring size measurements. It is suggested that poly(C9) tubules with various protomer numbers may arise due to limited flexibility in the C9-C9 interaction.