Progress toward analysis of D8/17 binding to B cells in children with obsessive compulsive disorder and/or chronic tic disorder.

BACKGROUND: Previous research has suggested that a subgroup of children with obsessive compulsive disorder (OCD) have neuropsychiatric sequelae of streptococcal pharyngitis, similar to that seen in the neurological manifestation of rheumatic fever (RF). Monoclonal antibody D8/17 demonstrates increased binding to B cells in patients with RF and in patients with neuropsychiatric disorders using immunofluorescent microscopy. OBJECTIVE: The aim of this study was to determine if an earlier immunofluorescent microscopy study of monoclonal antibody D8/17 in childhood-onset OCD and/or chronic tic disorder (CTD) could be replicated using the more objective method of flow cytometric analysis. METHOD: D8/17 binding to B cells was determined in patients with OCD and or CTD (N=32), and healthy controls (N=12) by flow cytometric analysis. RESULTS: Subjects with OCD/CTD showed increased mean cell binding (26.0%) of monoclonal antibody compared with healthy controls (9.1%) (p<0.001). When using the threshold of greater than 19% binding (95% upper confidence interval) as a measure of positivity, 65.6% of patients compared with 8.3% of controls showed increased antibody binding to B cells (p=0.01). CONCLUSIONS: Although this study reports positive results, many methodological issues will need to be addressed before generalized use of assay for diagnostic purposes.

Characterization of corticosteroid receptors in natural killer cells: comparison with circulating lymphoid and myeloid cells.

Lymphocytes mediating natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities are relatively refractory to the changes in circulatory traffic and intrinsic function induced in other cell types by in vivo and in vitro corticosteroids (CS). To investigate if such drug resistance could be attributed to differences in the CS receptor number of affinity (Kd) of these cells, these characteristics were determined in purified populations of large granular lymphocytes (LGL), monocytes, neutrophils (PMN), and T cells. All cell types displayed a single class of CS receptor of uniform affinity; however, LGL resembled monocytes and PMN in receptor number and Kd while T cells had significantly fewer sites per cell with lower Kd. These studies suggest that the unresponsiveness of NK activity to CS is not secondary to differences in CS receptor capacity or affinity.

The effects of in vivo hydrocortisone on lymphocyte-mediated cytotoxicity.

To examine the effects of in vivo hydrocortisone sodium succinate (HC) on natural killer (NK) cell and antibody-dependent cellular cytotoxicity (ADCC), 11 normal adults received a single intravenous bolus of 400 mg hydrocortisone. Lymphocytes were tested for NK activity and ADCC using 51chromium (51Cr)-release and single cell cytotoxicity assays against Molt-4 and sensitized RL O leads to target cells, respectively. Four hours after injection, both NK and ADCC activity were transiently increased in the 51Cr-release system (P less than 0.05). At 4 hours, there was a twofold increase in the relative frequency of potentially cytotoxic target binding cells (P less than 0.001) but the absolute number of these cells did not change (P less than 0.1). However, the percentage lysis of bound targets at 4 hours was not altered (P greater than 0.1). These data suggest that: 1) lymphocytes participating in NK and ADCC reactions are refractory to the kinetic and functional effects of HC; 2) the increased lytic activity observed at 4 hours is due to a selective depletion of noncytotoxic cells from the circulation; and 3) NK and ADCC activity did not differ in their responses to HC.

Mechanisms of human cell-mediated cytotoxicity. III. Dependence of natural killing on microtubule and microfilament integrity.

To date, the mechanisms of human natural killer (NK) cell activity have been poorly defined. Since microtubules and microfilaments are important in many non-NK leukocytic processes, the potential roles of these cellular elements in natural killing were assessed in simultaneously performed 51-chromium-release microcytotoxicity and single cell cytotoxicity assays. The microtubule inhibitors colchicine, vincristine, and vinblastine suppressed chromium-release in a concentration-dependent fashion. This suppression could be prevented by the promoters of microtubule assembly, deuterium oxide and cyclic GMP. Microtubule inhibitors did not affect the binding of natural killer cells to target cells but did suppress the lysis of bound targets. The "recycling" of natural killer cells to repeat the lytic sequence was not altered by microtubule-disrupting agents. Cytochalasin B, a microfilament inhibitor, induced dose-dependent suppression of lytic activity in the chromium-release assay. Concentrations of cytochalasin B greater than 2 micrograms/ml prevented the binding of effector cells to target cells. Concentrations of cytochalasin B less than 2 micrograms/ml depressed killing in the chromium-release assay but did not alter target cell binding or the lysis of bound targets indicative of an inhibition of effector cell recycling. Concentrations of cytochalasin B greater than 2 micrograms/ml did not affect the lysis of bound targets once binding had occurred. The roles of microtubules and microfilaments in the lytic sequence can therefore be subdivided based on these data: 1) the binding of natural killer cells to target cells in dependent on microfilaments; 2) the lysis of bound targets requires microtubule assembly; and 3) the subsequent post-lytic recycling of natural killer cells necessitates a intact microfilament system.

Abnormal natural killer cell activity in systemic lupus erythematosus: an intrinsic defect in the lytic event.

The natural killer (NK) cell activity in fifteen systemic lupus erythematosus (SLE) patients was investigated by employing 51-chromium- (51Cr) release microcytotoxicity and single cell cytotoxicity assays against K562 target cells. Although the SLE patients as a group had depressed NK function in the 51Cr-release assay compared to normal subjects (p less than 0.005), those with clinically active disease displayed the greatest impairment in this activity (p less than 0.001). Active SLE patients were deficient in overall NK activity (Vmax) (p less than 0.005) but had normal percentages of potentially cytotoxic target binding cells (TBC). These TBC, however, were unable to normally kill bound target cells (p less than 0.01), which is indicative of a deficiency of "active" NK cells (p less than 0.005). Those NK cells with intact cytotoxic capabilities could "recycle" and repeat the lytic sequence normally. Exposure of normal lymphocytes to SLE sera did not impair any phase of NK function. These studies indicate that defective NK activity in SLE is secondary to an abnormality in the lytic event itself and is not due to a deficiency of NK cells, an abnormality in target binding, or an inability of NK cells to lyse multiple targets. Additionally, serum factors do not appear to play a major etiologic role in the cytotoxic abnormalities of these patients.

Deficiency of active natural killer cells in the Chediak-Higashi syndrome. Localization of the defect using a single cell cytotoxicity assay.

This study investigated the defective natural killer (NK) cell activity in two patients with the Chediak-Higashi syndrome (CHS) using both a standard 51-chromium release microcytoxicity and a single cell-in-agarose assay against K562 and Molt-4 target cells. CHS patients were deficient in overall maximum NK capacity, but had normal percentages of potentially cytotoxic target bindng cells. the relative number of TBC that could kill bound targets (i.e., "active" NK cells) was significantly depressed in CHS patients when compared with normal controls. The diminished CHS active NK cells that were present, however, were capable of recycling and lysing multiple target cells during the assay period. In vitro interferon (INF) treatment of normal and CHS effector cells did not alter target cell binding, but did increase the maximum NK capacity, percentage of active NK cells and the maximum recycling capacity, as well as the rate of lysis. These studies indicate that the depression of NK activity in patients with CHS is secondary to a deficiency of active NK cells. The CHS active NK cells that are present, however, are capable of normal target lysis and recycling. Potentially cytotoxic pre-NK cells, which can bind but not kill target cells, can be activated by in vitro IFN to develop lytic activity. Thus, IFN treatment may be of potential benefit to the immune surveillance network of CHS patients by activating a population of pre-NK cells to express their cytotoxic potential.