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Background: Inflammation and immune dysregulation have been implicated in the pathogenesis of pediatric-onset obsessive-compulsive disorder (OCD) and tic disorders such as Tourette syndrome (TS). Though few replicated studies have identified markers of immune dysfunction in this population, preliminary studies suggest that serum immunoglobulin A (IgA) concentrations may be abnormal in these children with these disorders. Methods: This observational retrospective cohort study, conducted using electronic health records (EHRs), identified 206 children with pediatric-onset OCD and 1024 adults diagnosed with OCD who also had testing for serum levels of IgA. IgA deficiency and serum IgA levels in pediatric OCD were compared with IgA levels from children diagnosed with autism spectrum disorders (ASD; n = 524), tic disorders (n = 157), attention-deficit/hyperactivity disorder (ADHD; n = 534), anxiety disorders (n = 1206), and celiac disease, a condition associated with IgA deficiency (n = 624). Results: Compared with ASD and anxiety disorder cohorts, the pediatric OCD cohort displayed a significantly higher likelihood of IgA deficiency (OR = 1.93; 95% CI = 1.18–3.16, and OR = 1.98; 95% CI = 1.28–3.06, respectively), though no difference was observed between pediatric OCD and TS cohorts. Furthermore, the pediatric OCD cohort displayed similar rates of IgA deficiency and serum IgA levels when compared with the celiac disease cohort. The pediatric OCD cohort also displayed the highest percentage of IgA deficiency (15%,) when compared with TS (14%), celiac disease (14%), ADHD (13%), ASD (8%), and anxiety disorder (8%) cohorts. When segregated by sex, boys with OCD displayed a significantly higher likelihood of IgA deficiency when compared with all comparison cohorts except for celiac disease and tic disorders; no significant difference in IgA deficiency was observed between female cohorts. Pediatric OCD subjects also displayed significantly lower adjusted serum IgA levels than the ASD and anxiety disorder cohorts. Adults with OCD were also significantly less likely than children with OCD to display IgA deficiency (OR = 2.71; 95% CI = 1.71–4.28). When compared with children with celiac disease, no significant difference in IgA levels or rates of IgA deficiency were observed in the pediatric OCD cohort. Conclusions: We provide further evidence of IgA abnormalities in pediatric-onset OCD. These results require further investigation to determine if these abnormalities impact the clinical course of OCD in children.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.

CD8+ lymphocytes are believed to be important in host defence against the human immunodeficiency virus (HIV)-1, inhibiting HIV-1 replication through both cytolytic and non-cytolytic pathways. The cytolytic pathway involves calcium-dependent exocytosis of perforin and granzyme proteases, as well as Fas-mediated programmed cell death, whereas the noncytolytic pathway involves the release of chemokines that prevent viral entry. Using granzyme A as a marker of cytolytic granule proteins, and macrophage inflammatory protein (MIP)-1alpha and RANTES as markers of HIV-1 inhibitory chemokines, we show that these two very different mediators of viral inhibition are both localized in the cytolytic granules of HIV-1-specific CD8+ cytotoxic T lymphocytes (CTL). Following antigen-specific activation, these mediators are secreted together, facilitating both lysis of virion-producing cells and the inhibition of free virus. In addition, RANTES, MIP-1alpha and MIP-1beta are secreted by CTL as a macromolecular complex containing sulphated proteoglycans. This association appears to have a functional significance, because heparan sulphate facilitates RANTES inhibition of HIV-1 infection of monocytes.

Members of the CED-3/interleukin-1beta-converting enzyme (ICE) protease (caspase) family are synthesized as proforms, which are proteolytically cleaved and activated during apoptosis. We report here that caspase-2 (ICH-1/NEDD-2), a member of the ICE family, is activated during apoptosis by another ICE member, a caspase-3 (CPP32)-like protease(s). When cells are induced to undergo apoptosis, endogenous caspase-2 is first cleaved into three fragments of 32-33 kDa and 14 kDa, which are then further processed into 18- and 12-kDa active subunits. Up to 50 microM N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), a caspase-3-preferred peptide inhibitor, inhibits caspase-2 activation and DNA fragmentation in vivo, but does not prevent loss of mitochondrial function, while higher concentrations of DEVD-CHO (50 microM) inhibit both. In comparison, although the activity of caspase-3 is very sensitive to the inhibition of DEVD-CHO (50 nM), inhibition of caspase-3 activation as marked by processing of the proform requires more than 100 microM DEVD-CHO. Our results suggest that the first cleavage of caspase-2 is accomplished by a caspase-3-like activity, and other ICE-like proteases less sensitive to DEVD-CHO may be responsible for activation of caspase-3 and loss of mitochondrial function.

Background Most infants with congenital Toxoplasma gondii infection have no symptoms at birth, but many will have retinal disease or neurologic abnormalities later in life. Early detection and treatment of congenital toxoplasmosis may reduce these sequelae. Methods In Massachusetts since January 1986, and in New Hampshire since July 1988, newborns have been screened for intrauterine infection with T. gondii by means of an IgM capture immunoassay of blood specimens routinely collected for screening for metabolic disorders. Congenital infection is confirmed by assays for specific IgG and IgM antibodies in serum from infants and their mothers. For this study, infants with serologic evidence of infection underwent extensive clinical evaluation and received one year of treatment. Results Through June 1992, 100 of 635,000 infants tested had positive screening tests. Congenital infection was confirmed in 52 infants, 50 of whom were identified only through neonatal screening and not through initial clinical exami...

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.

Human haptoglobin (Hp) is synthesized at hepatic and extrahepatic sites as an acute-phase reactant protein (APP). We investigated the effects of Hp on granulocyte function. The chemotaxis of freshly isolated human granulocytes and differentiated HL-60 cells in response to the bacterial tripeptide, f-met-leu-phe, was inhibited in the presence of a physiological concentration of Hp, but chemotaxis in the presence of the proinflammatory cytokine interleukin-8 (IL-8) was not inhibited. Phagocytosis of viable Escherichia coli, as well as fluoresceinated nonviable E. coli, was inhibited. Hp also reduced granulocyte intracellular bactericidal activity against E. coli. The observed inhibitory effects of Hp on granulocyte function are similar to those reported for C-reactive protein and suggest that APPs dampen the acute inflammatory response.

Despite considerable information regarding the structure and expression of granzymes, the serine proteases that reside in the cytotoxic granules of cytotoxic T lymphocytes (CTL), little is known about their physiologic substrates or their role in the economy of these effector cells. From a conceptual framework, such proteases may act on the effector cells themselves (“cis” action), on the target cells (“trans”), or in a more nonspecific fashion; current information remains sketchy and controversial. In this chapter, the functional properties and possible physiologic roles of CTL granzymes will be reviewed, with particular reference to granzyme A.

Clones of cytotoxic T lymphocytes (CTL) differ in their specific reactivity with diverse target cell antigens. To learn about the uniqueness of individual CTL clones we injected rats and mice with cloned CTL in an effort to prepare clone-specific antisera and to analyze the CTL surface molecules that were immunoprecipitated by these antisera. Three clones were studied. They were all derived from BALB.B mice and were specific for antigens encoded by the major histocompatibility complex of the H-2d haplotype. Antisera raised in rats against individual clones contained antibodies to lymphocyte function-associated antigen type 1 (LFA-1) and inhibited the cytotoxic activity of all of the clones. In contrast, BALB/c and BALB.K mice injected with individual clones consistently yielded alloantisera that were clone specific in their ability to inhibit CTL-mediated lysis of target cells (P815). In addition, these alloantisera immunoprecipitated from extracts of 125I-radiolabeled CTL a disulfide-bonded dimer consisting of approximately equal to 45-kilodalton subunits. This dimer resembles the putative T-cell antigen-recognition receptor recently identified in several laboratories. The alloantisera also immunoprecipitated CTL surface molecules that were associated with beta 2-microglobulin and that differed in apparent molecular mass (37-38 kilodaltons) in different clones.

The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells1 and natural killer cells2 cytoplasmic granules that contain perform, a cytolytic protein that resembles the ninth component of complement (C9)3. Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper–killer or Th/c cells4–6. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines7. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that (1) it is a membrane-associated, disulphide-linked dimer, (2) it has trypsin-like properties but is not a general protease, (3) in density gradient centrifugation it sediments with perforin, (4) it is secreted by Tc cells during their cytolytic attack on target cells, and (5) antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.

A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of gamma-interferon and in blocking the effector functions of the cells-namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by alpha-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, alpha- and beta-chains, and (on some clones) Lyt-2. Other components with Mr congruent to 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.

Interferon (IFN) was found to be secreted by cloned lines of murine cytotoxic T lymphocytes in response to mitogenic or antigen specific stimulation. The IFN produced was shown to be of the immune type based on its sensitivity to pH 2.0 and on the ability of an antiserum to immune IFN to neutralize the antiviral activity.

Immune responses begin with the binding of antigen to surface receptors on lymphocytes. In this reaction, called ‘antigen recognition’, lymphocyte receptors discriminate by variations in the strength of the binding reaction among vast numbers of antigens, including many that differ from each other only very slightly in structure. Antigen recognition by B lymphocytes is now well understood: their recognition molecules are cell surface antibodies (Warner, 1974) and antigen binding by antibodies has been characterized, for many antigens, at a highly sophisticated chemical level. However, antigen recognition by T cells (i.e. thymus-derived lymphocytes) is poorly understood and remains one of the most pressing challenges of modern immunology.