Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition.

Case reports of olanzapine treatment of anorexia nervosa.

Two females with severe anorexia nervosa were treated with olanzapine in open trials. Olanzapine was tried because it has caused weight gain in other patient groups. Both anorexic patients had a chronic illness and had failed multiple other treatments. Olanzapine administration was associated with weight gain and maintenance as well as reduced agitation and resistance to treatment. These case histories support further exploration of this class of drugs in anorexia nervosa.

Leptin, neuropeptide Y, and peptide YY in long-term recovered eating disorder patients.

BACKGROUND: Disturbances of leptin, neuropeptide Y (NPY), and peptide YY (PYY) have been found in women who are ill with anorexia or bulimia nervosa. It is not certain whether peptide disturbances are cause or consequence of eating disorders. METHODS: Plasma leptin and cerebrospinal fluid leptin, NPY, and PYY concentrations were measured in women who were recovered from anorexia or bulimia nervosa to determine whether alterations persisted after recovery. RESULTS: NPY, PYY, and leptin concentrations were similar across all diagnostic groups. CONCLUSIONS: Alterations in NPY, PYY, and serum leptin concentrations are probably secondary to pathological eating behaviors. Alterations of these peptides are unlikely to be trait-related disturbances that contribute to the etiology of eating disorders.

Are serotonin selective reuptake inhibitors effective in underweight anorexia nervosa?

OBJECTIVE: Recent data suggest that serotonin selective reuptake inhibiter (SSRI) medication is useful in preventing relapse in weight-restored anorexics. Our clinical impression has been that SSRIs are not effective in patients who are underweight with anorexia nervosa. METHOD: In order to determine whether there was any benefit for SSRI medication in underweight anorexics, we compared two groups of underweight anorexics upon admission to our inpatient hospital using a retrospective chart review. RESULTS: Sixty percent of anorexic patients were taking an SSRI upon admission to our inpatient hospital. The 24 subjects taking an SSRI were compared to the 16 subjects not taking an SSRI. These two groups had similar ages and body weights as well as scores for measures of anxiety and depression and most core eating disorder symptoms. DISCUSSION: These results suggest that SSRI medication had no effect on clinical symptoms of malnourished underweight anorexics.

Long-term exercise patterns and immune function in healthy older women. A report of preliminary findings.

The purpose of this study was to examine the relationship between active versus inactive lifestyle and immunocompetence in older women. A sample of 46 independently dwelling, ambulatory and mentally alert women 60-98 years was examined, 25 who rated themselves as 'active' and 21 who rated themselves as 'inactive'. Lymphocyte subpopulations were analyzed by flow cytometry using selected monoclonal antibodies. The self-reported active subjects (also validated by their current unsolicited participation in a formal exercise class) demonstrated significantly higher percent change in CD25 mitogen stimulated lymphocytes (P = 0.0335) than those who reported themselves to be sedentary.

The uptake of LDL-IC by human macrophages: predominant involvement of the Fc gamma RI receptor.

The incubation of human macrophages with antigen antibody complexes prepared with rabbit anti-LDL and human LDL (LDL-IC) is followed by ingestion of those immune complexes (IC), massive cholesterol ester accumulation, cytokine release and overexpression of the LDL receptor. The massive accumulation of cholesterol esters and overexpression of the native LDL receptor are specifically induced by immune complexes containing native or modified LDL, but not by any other type of IC. We report the results of a series of experiments aimed at defining the receptor preferentially involved in LDL-IC uptake. Flow cytometry studies using CD16, CD32 and CD64 monoclonal antibodies showed a sharp reduction on the expression of CD64 (Fc gamma RI) both by human monocyte-derived macrophages and THP-1 cells after incubation with LDL-IC, suggesting preferential engagement of this type of Fc receptor. Blocking experiments with aggregate-free IgG1 and CD32 monoclonal antibody confirmed that blocking Fc gamma RI prevented both LDL-IC uptake and the upregulation of LDL receptors on THP-1 cells. In contrast, blocking Fc gamma RII did not affect either the uptake of LDL-IC or the expression of LDL receptors on the same cells. The preferential engagement of Fc gamma R-I by LDL-IC suggests a biological difference of LDL-IC relative to other types of IC and opsonized particles. The precise molecular mechanism(s) responsible for the paradoxical upregulation of LDL receptor after the uptake of LDL-IC remain to be elucidated.

Cataplexy and the switch process of multiple personality disorder.

A case history is presented of an 18-year-old male with dissociative disorder and polysubstance abuse. The patient was observed to switch between three personalities, and the personality changes were often associated with symptoms of cataplexy. Both dissociative episodes and cataplexy are associated with strong affective stimuli. Similar reports in the literature are briefly reviewed.

The influence of stress intrusion on immunodepression in generalized anxiety disorder patients and controls.

Previous work from our group has examined the relationship between stress and immunodepression in medical students taking National Boards, Part I, and has described a relationship between stress intrusion scores (SIS) and immunodepression. We have also shown that a high proportion of individuals with generalized anxiety disorders (GAD) and panic disorders (PD) exhibit enhanced stress intrusion (SI) and are more prone to upper respiratory infections (URI). In the present preliminary study, we sought to establish a model to evaluate further the role of SI level on the extent of immunodepression. This would serve to assess in further studies the mechanism(s) of stress-induced immunodepression, its relationship to morbidity, and the role of therapeutic interventions. In 14 GAD patients and 14 controls, we correlated the expression of interleukin-2 receptors (CD25) on T lymphocytes stimulated with anti-CD3 in short term cultures and the frequency of URI and the SIS to assess the relationships among these parameters. A decreased expression of CD25 correlates linearly with increasing SIS and with a higher number of sick days with URI. These results support our previous observations that GAD patients are more susceptible to URI. Moreover, they suggest that there may be a direct relationship between immunodepression and morbidity and between SIS and immunodepression.

Age-related mortality and adherent splenic cell mediator production to endotoxin in the rat.

Rat neonatal mortality to endotoxin and age-related changes in adherent splenic cell mediator production in vitro were investigated. Neonatal rat pups, 24, 48, 96, and 216 h old or maternal adult rats were administered doses of Salmonella enteritidis endotoxin, (.024 mg to 7.5 mg/kg) and survival was monitored for 72 h. Mortality demonstrated high sensitivity (p < .05) of neonates to endotoxin (particularly 24 h old neonates). Endotoxin administration .6 mg/kg intracardiac) produced a 100% lethality in 24 h neonates (p < .05) versus 23% or less lethality in the 48 to 216 h old age group. Endotoxin administration (.4 mg/kg subcutaneous) also produced 100% lethality in 24 h old neonates compared with reduced mortality versus older age groups. Endotoxin in vitro stimulated (p < .05) adherent splenic cell thromboxane (TX)B2, interleukin-6, and nitrite production in most groups. Splenic cell nitrite production was higher (p > .05) in the 24 h old neonates, but lower in 48 h and 96 h old groups compared with maternal adults. Splenic cell TXB2 production was higher (p < .05) in the 24 h and 216 h old neonates relative to maternal adults. In conclusion, 24 h old rat pups are more susceptible to endotoxic shock than older age groups and adults, and exhibit altered production of the cellular mediators nitric oxide and TXB2.

Clinical applications of cytometry: 6th annual meeting.

The Sixth Annual Clinical Applications of Cytometry Meeting was held September 11-14, 1991, in Charleston, SC. Attendance reached a record 470. The meeting provides a forum for interactions among investigators who utilize cytometry as a tool in their clinical immunology, cell biology, hematology, and cancer investigations. Clinical laboratory directors and their technical staff find the meeting of practical value because of the presentation of new applications that they can take home to their own laboratories. The emphasis of the meeting is on advances in the application of cytometry to clinical problems. Often, advances result from new dyes or reagents or improved instrumentation. Sometimes they result from advances in biology that make the studies possible. Occasionally a new way of looking at the same data provides a useful answer. In every case, the effort is to provide a reliable, straightforward way to quantitate biologic information in order to provide improved diagnosis or treatment of human disease.

Psychoneuroimmunology: where are we, where are we going?

We have reviewed briefly the current status of research on central nervous system-immune system interactions, focusing attention on the neural and humoral pathways by which CNS and IS communicate and interact and on the effects of stress and psychiatric illness on immune function. It is evident that CNS-IS communication occurs by direct innervation of lymphoid organs and by means of hormones, neuropeptides and cytokines. There is also clear evidence that humoral substances each of which were thought to be the product of one specific cell type are elaborated and secreted by a variety of cell types. This observation suggests a new unified concept of CNS-IS interactions with mediators of these interactions being produced ubiquitously and acting on cells of the two systems. In examining the effects of stress on IS it has become apparent that stress of various types can have a depressive effect on immune functions, primarily at the level of T lymphocytes and NK cells. This suggests that the defense mechanisms affected by stress are those which are responsible for cytotoxic effector responses. These findings are interesting in that they support older studies implicating stress in the pathogenesis and/or the clinical course of neoplastic diseases. Further support for a role of stress-induced immunodepression in morbidity comes from a very interesting, recent prospective study showing that stress will affect susceptibility to viruses. Finally, exploration of the mechanisms of stress-induced immunodepression, suggests that a variety of mediators which regulate lymphocyte interactions and activation may be affected, perhaps at the level of gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunosuppressive effects of fish oil in normal human volunteers: correlation with the in vitro effects of eicosapentanoic acid on human lymphocytes.

We have studied the effects of dietary supplementation with fish oil on immunological parameters in a group of six normal volunteers, four of whom received a fish oil extract (total EPA dose of 2.4 g/day, which is on the lower range of clinically effective doses) for 6 weeks and two of which received a placebo (olive oil) for an identical period of time. Each volunteer was followed up for a period of 23 weeks after the dietary intervention was ended. All volunteers were boosted with tetanus toxoid (TT) at the onset of the trial. Several immune parameters were followed longitudinally, including NBT reduction and lysozyme release to test neutrophil function; lymphocyte subpopulations; mitogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and anti-CD3; IL-2 release after PHA and pokeweed mitogen (PWM) stimulation; immunoglobulin and anti-TT antibody (ATT) synthesis by stimulated lymphocytes; and serum levels of immunoglobulins and of ATT. No consistent changes were observed in neutrophil function tests, mitogenic responses to PHA and Con A, and lymphocyte subsets. The mitogenic response to anti-CD3 and the release of IL-2 after stimulation with PHA and PWM appeared reduced as a consequence of fish oil ingestion, and levels of serum immunoglobulins decreased in three of the volunteers receiving fish oil supplementation. The systemic humoral response after the TT booster appeared not to be influenced by the ingestion of fish oil. However, in those subjects who were given fish oil supplementation, the specific in vitro response of their peripheral blood lymphocytes to TT appeared to be compromised at Week 3. This could reflect the need for progressive accumulation of EPA in lymphocyte membranes for the suppressive effect to be detectable, but it could also reflect a differential sensitivity to the effects of fish oil of circulating B lymphocytes vs. bone marrow B lymphocytes. All the parameters apparently affected by fish oil ingestion were also affected by the incubation of normal lymphocytes with EPA in vitro. In conclusion, low doses of fish oil may have a mild immunosuppressive effect affecting both T and B cell functions. These observations stress the need for more extensive trials designed to determine whether immunosuppressive effects can be consistently elicited and for studies aimed at determining the mechanisms by which omega-3 fatty acids affect the immune system.

Clinical applications of cytometry: 5th annual meeting.

The 5th annual Clinical Applications of Cytometry meeting was held September 12-15, 1990 in charleston, SC. The theme which emerged repeatedly throughout the meeting was the need to take full advantage of the quantitative power of cytometry to provide the most useful clinically relevant diagnostic and prognostic information. Greater quantitative power is based on careful and reproducible standards and quality control. The same principles, albeit with somewhat different approaches, apply to cell surface immunofluorescence analysis, DNA measurements, and image cytometry assessments. Monoclonal antibody probes against oncogenes, others against lymphokines within the Golgi, and a novel fluorogenic substrate designed to quantitate the activity of a mitochondrial enzyme were exciting developments described at the meeting.

Immunophenotypic analysis of non-Hodgkin's lymphomas.

Flow cytometric cell analysis with fluorescence-labeled antibodies has become a very useful methodology for the immune phenotyping of lymphocytes. The continued evaluation of lymphocyte cell surface antigens has been of value in this respect by providing a clear picture of lymphocyte differentiation steps. Thus it is now possible to precisely identify lymphocytes of abnormal phenotype which may represent malignant cells. Detection of monotypic populations of lymphocytes represents a monoclonal expansion of a lymphocyte subset which is the hallmark of malignancy. In the case of B cell lymphoma, detection of monotypic populations rests on the finding of a monoclonal expansion of a cell type bearing one type of light chain and of heavy chain and/or one of the specific B lymphocyte differentiation antigens. The diagnosis of T cell malignancy is more difficult to establish and a diagnosis of T cell lymphoma rests on the finding of an abnormal phenotype. Thus flow cytometry in combination with histomorphologic examination is a useful technique for the more precise diagnosis of lymphomas and for the establishment of treatment protocols.

Significance of common acute lymphoblastic leukemia antigen (cALLA) positive cells in bone marrow of children off therapy in complete remission.

Bone marrows from 21 children with acute lymphoblastic leukemia in complete remission (CR) and off therapy for 14 months to 10 years were examined by flow cytometry with a panel of monoclonal antibodies. Significant percentages of cALLA+ cells of low fluorescence intensity were present up to 9 years after CR. These results emphasize the need to interpret flow cytometry results in light of the findings that low intensity cALLA+ cells represent a normal population of immature, non-malignant lymphoid cells.

The potentiation of B lymphocyte responses through CD2/LFA-3 interactions involving erythrocytes is IL2 independent.

The response of human B cells to pokeweed mitogen (PWM) stimulation is potentiated when autologous erythrocytes (E) are added to peripheral blood mononuclear cell (PBMC) cultures. This potentiation has been previously shown to be dependent on interactions between the CD2 molecule on T cells and the lymphocyte function-associated antigen 3 (LFA-3) expressed by autologous erythrocytes. Since in other experimental systems the activation of T cells by CD2/LFA-3 interactions has resulted in increased secretion of interleukin 2 (IL2), we were interested in studying the role of IL2 in PBMC cultures stimulated with PWM and autologous E. The addition of autologous E significantly depressed IL2 levels in PWM-stimulated PBMC cultures. This effect was not secondary to increased expression of IL2 receptors by activated cells, since the addition of anti-TAC antibodies did not result in a significant increase in measurable levels of IL2. The addition of anti-IL2 to PBMC failed to abrogate the potentiating effect of E and it actually further enhanced the production of IgM and IgG from cultures stimulated with PWM + E. These results suggest that the potentiation of B cell function induced by autologous E is not mediated by IL2, either directly or indirectly. It is possible that the effect of autologous E either is mediated by other interleukins or is dependent on cell-to-cell contact with directed release of IL2 and/or other lymphokines without detectable secretion to the extracellular compartment.

The interaction of CD2 with its LFA-3 ligand expressed by autologous erythrocytes results in enhancement of B cell responses.

The addition of autologous erythrocytes to unfractionated human mononuclear cell cultures results in enhancement of B cell responses to antigens and mitogens. This costimulating effect of red cells is abrogated by their preincubation with anti-LFA-3 monoclonal antibody. Preincubation of mononuclear cells with anti-CD2 monoclonal antibodies (anti-Leu 5b, OKT11, used singly) has a down-regulating effect on B cell activation and no enhancement of B cell responses is seen when red cells are added to anti-CD2-treated cultures. These results demonstrate a functional effect on B cells of the interaction between the CD2 molecule on T lymphocytes and its natural ligand, LFA-3. The precise mechanism by which this costimulating effect on B lymphocytes takes place is unclear. The study of T cell populations and T cell activation markers shows that the addition of erythrocytes causes a small but reproducible increase in the number of cells expressing the IL-2 receptor and the addition of IL-2 enhances the response of mononuclear cells to antigenic stimulation in the presence of erythrocytes. However, the supernatants of mononuclear cell cultures stimulated with pokeweed mitogen in the presence of autologous erythrocytes show decreased levels of IL-2, compared to supernatants of cells stimulated with pokeweed mitogen alone. The same supernatants show increased levels of interferon-gamma, but the addition of this lymphokine to cultures stimulated with pokeweed mitogen has no potentiating effect. It is possible that the effect of erythrocytes is mediated by other growth and/or differentiation factors, and additional studies will be required to clarify this point.

Subclass specificity of the Fc receptor for human IgG on K562.

The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).