Dissociation of caspase-mediated events and programmed cell death induced via HLA-DR in follicular lymphoma.

Human leukocyte antigens (HLA) class II antigen-mediated apoptosis has been documented in antigen-presenting cells and B lymphoproliferations. Characteristics of the apoptosis include rapidity and selectivity for mature cells. Follicular lymphomas are particularly refractory to apoptosis. The B-cell lymphoma Ramos shares characteristics of this subgroup and is insensitive to apoptosis via simple HLA-DR engagement. However, oligomerization of HLA-DR antigens induced caspase activation followed by phosphatidylserine externalization, activation of PKC-delta and cleavage of nuclear lamin B. Mitochondrial injury was also detected. However, inhibition of caspase activation simply delayed the apoptotic phenotype but neither protected against cell death nor prevented mitochondrial injury. The data in this report demonstrate that the requirements for the initiating signal (oligomerization versus engagement) as well as the molecular pathways varies between different B lymphoproliferations despite their common expression of HLA-DR. Finally, blockade of caspase activation in parallel with HLA-DR mAb stimulation could provide a potent autovaccination stimulus by leading to necrotic death of B-cell lymphomas.

A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.

MHC class II molecules have a crucial role in thymic selection and in generating Ag-specific T cell responses. There is extensive evidence for second messenger generation via MHC class II molecules, which can lead to apoptosis of B lymphocytes. We have examined HLA class II-mediated apoptosis in both normal and tumoral human B lymphocytes. Phosphatidylserine exposure and DNA fragmentation were observed in B cells within 24 h of stimulation via HLA class II. In marked comparison with Fas, the cell-permeable and irreversible caspase inhibitors zVAD-fmk and DEVD-fmk failed to inhibit HLA-DR-mediated apoptosis. No direct activation of caspase 3 was detected, and cleavage of pro-caspase 3 was not observed. Cleavage of poly(ADP-ribose) polymerase was detected via Fas but not via HLA class II. Although phosphatidylinositol-3-kinase has been implicated in HLA class I-mediated apoptosis, neither wortmannin nor LY294002 affected HLA class II-mediated apoptosis. CD95-sensitive cells were used to reveal that death occurred independently of CD95-CD95 ligand interactions. Overall, these data reveal a pathway of HLA-DR-mediated apoptosis that neither requires nor involves caspases. Moreover, it is phosphatidylinositol-3-kinase independent and Fas/CD95 independent. This pathway of HLA class II-mediated apoptosis could have an important role in the regulation of APC populations or in the control of malignant B lymphocyte proliferations.

Maine Carpal Tunnel Study: outcomes of operative and nonoperative therapy for carpal tunnel syndrome in a community-based cohort.

A prospective, community-based, observational study of the outcome of surgical and nonoperative management was conducted. The study included 429 patients with carpal tunnel syndrome recruited in physicians' offices throughout Maine. Patients were assessed at baseline and at 6, 18, and 30 months following presentation using validated scales that measured symptom severity, functional status, and satisfaction. Seventy-seven percent of eligible survivors from the original cohort were monitored for 30 months. Surgically treated patients demonstrated improvements of 1.2 to 1.6 points on the 5-point Symptom Severity and Functional Status scales (23% to 45% improvement in scores), which persisted over the 30-month follow-up period. The nonoperatively managed patients showed little change in clinical status at 6, 18, and 30 months. While workers' compensation recipients had worse outcomes than nonrecipients, 36 of 68 (53%) workers' compensation recipients were completely or very satisfied with the results of the procedure 30 months after surgery. There were no significant differences in outcome between patients treated with endoscopic versus open carpal tunnel release. Among worker's compensation recipients, 12 of 68 (18%) surgical patients and 4 of 32 (13%) nonoperatively treated patients remained out of work because of carpal tunnel syndrome at 30 months. Thus, carpal tunnel surgery offered excellent symptom relief and functional improvement in this prospective community-based sample, irrespective of the surgical approach, even in workers' compensation recipients. Work absence remained high in both surgically and nonoperatively managed workers' compensation recipients.

HLA class II-induced translocation of PKC alpha and PKC beta II isoforms is abrogated following truncation of DR beta cytoplasmic domains.

Several studies have attempted to identify regions of the MHC class II molecule that participate in signal transduction. The importance of intact murine I-A cytoplasmic domains, either to tether the class II molecule to the cytoskeleton or to recruit signal-transducing proteins, is now well established. Recent data have also suggested that residues of the I-A beta transmembrane are involved in a second distinct signaling pathway. In the light of data suggesting that the second messengers activated on ligation of human and mouse class II molecules may differ, we set out to investigate whether the structural requirements for signaling for the human DR molecule are similar to those already described for the murine I-A molecule. We show that mutant DR class II molecules lacking 12 amino acids of the beta-chain cytoplasmic tail fail to translocate the protein kinase C alpha (PKC alpha) and PKC beta II isoenzymes following stimulation with an anti-class II mAb. In contrast, truncation of either or both cytoplasmic domains of the DR molecule has no effect on class II-induced tyrosine kinase activity. PKC translocation following class II stimulation has been observed in both human and murine B lymphocytes, whereas tyrosine kinase activation is present in human B lymphocytes but absent in resting murine B lymphocytes. Therefore, we conclude that the DR beta cytoplasmic tail is requisite for the principal signaling pathway initiated via MHC class II. These data suggest that the signaling pathways seen in resting vs primed murine B cells may also operate in human APCs.

The Maine Lumbar Spine Study, Part I. Background and concepts.

STUDY DESIGN: This paper describes the background and factors that led to the development and implementation of the Maine Lumbar Spine Study, a prospective cohort study of patients undergoing surgical and nonsurgical treatment of herniated lumbar disc with sciatica and symptomatic spinal stenosis. OBJECTIVES: To define the factors leading to the study and the methods of designing and implementing a community-based effectiveness study to evaluate the outcomes of herniated lumbar intervertebral disc and spinal stenosis. SUMMARY OF BACKGROUND DATA: Variations in the utilization of surgery for these conditions and physicians' uncertainty regarding the best way to manage them resulted in support of a community-based study of the effectiveness of treatment alternatives. METHODS: A prospective cohort design was used. Methods of patient enrollment, data collection, management, and analysis are described. An innovative method of ascertaining the representativeness of the enrolled versus nonenrolled patient population is presented. RESULTS: The importance of developing community-based networks of physicians is discussed. CONCLUSIONS: These networks play an important role in analyzing practice pattern variations and in stimulating and participating in effectiveness research. Because effectiveness studies must be conducted at the community level, mechanisms must be developed with which to support and implement these efforts.

Conformation of human leukocyte antigen class II molecules. Evidence for superdimers and empty molecules on human antigen presenting cells.

Subpopulations of human leukocyte antigen (HLA) class II molecules were studied in antigen presenting cells. We present evidence for double dimers or "superdimers" of HLA class II molecules that were stable in an SDS solution at room temperature but dissociated when heated to 50 degrees C into 60-kDa alphabeta heterodimers. Development of an immunofluorescence assay allowed us to quantify the expression of HLA antigens as reflected by the number of bound isotype-specific monoclonal antibodies per cell. The total expression of class II (DR, DQ, and DP) augmented 6-fold after a 36-h interferon-gamma (IFNgamma) treatment of freshly isolated monocytes. Next, we used a recombinant and fluorescein-conjugated form of the class II-associated invariant chain as a quantitative probe for empty peptide-binding sites. The fraction of empty class II molecules was 0.73-2.9% in resting monocytes but was reduced to 0. 12-0.5% of the total after IFNgamma treatment. The fraction of empty sites in B lymphocytes was 0.09-0.36%. The mean number of empty sites per cell were: 6.3 x 10(3) (monocytes), 7.2 x 10(3) (IFNgamma-activated monocytes), 5.2 x 10(2) (B lymphocytes), and 3.6 x 10(3) (Raji B cells). A minor population (4.3-7.4% of total cells), which expressed a much higher number of empty sites, was consistently present in all cell types studied.

Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1.

The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli. The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography [correction of chromatorgraphy]). Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions. The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay. The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).

Bacterial superantigen signaling via HLA class II on human B lymphocytes.

Staphylococcus enterotoxins and toxic shock syndrome toxin-1 (TSST-1) are members of the family of staphylococcal exoproteins (SE) which binds specifically to HLA class II molecules and certain V beta T cell receptor phenotypes. These bacterial products have been termed "superantigens" due to their capacity to stimulate a greater proportion of T lymphocytes than peptide antigens without a requirement for antigen processing. The SE stimulate monocytes to secrete IL-1 and TNF-alpha and affect B lymphocyte proliferation in response to anti-human IgM and Ig production by PBMC. The current study concerns the transmission of signals in human B lymphocytes following fixation of TSST-1. Activation of both PLC and PKC are observed while intracellular calcium levels remain unchanged. Levels of HLA class II mRNA were increased suggesting that a pathway leading to activation was triggered. This study therefore identifies some of the second messengers involved after SE fixation on HLA class II molecules and suggests that the signals transmitted via class II antigens as well as those via the TCR may have a role in the physiological responses to bacterial superantigens.

Lymphocyte programmed cell death is mediated via HLA class II DR.

HLA class II molecules are constitutively expressed on human B lymphocytes and are induced on human T lymphocytes after activation, through which signal transduction via these molecules has been extensively described. We have observed cell death of as many as 60% after stimulation of lymphocytes via HLA class II DR molecules, but not via DP or DQ. Propidium iodide fluorescence, DNA fragmentation and morphology of the dead cells were examined. The reported cell death was very rapid, and independent of Fc receptors and of complement. A morphologically distinct sub-population of activated B cells was sensitive to HLA-DR mediated death, while resting B lymphocytes from the same donor did not die as a result of HLA class II mediated stimulation. Death via HLA-DR was distinguishable from necrosis. Cytoskeletal integrity, serine/threonine phosphatase activity and endonucleases were required for the pathway leading to HLA-DR mediated death. The 'ladder' pattern of DNA fragmentation which typically characterizes apoptosis was not observed, despite the observation of cell and nuclear shrinkage normally associated with apoptosis. These data suggest that HLA class II mediated death is a means of rapidly removing either T or B lymphocytes which have already served their role in the immune response, thereby avoiding the inflammatory responses associated with necrosis and concentrating the ligands for new TCR and/or CD4 interactions.

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes.

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.

Lymphocyte activation via MHC class II antigens.

Class II antigens of the major histocompatibility complex (MHC) have a clear role in restricting cellular interactions and presenting processed antigen to T cells. In addition, a fundamental role for class II antigens in cellular activation has been suggested, following studies which demonstrated that class II antigen binding altered signal transduction in various cell types. We have investigated the role of HLA class II antigens in signal transduction of human B lymphocytes. Resting B lymphocytes proliferate in response to immobilized anti-class II antibodies and intracellular free calcium is increased following cross-linking of anti-class II antibodies. The two dimensional PAGE pattern of resting B lymphocytes was examined and differences were noted after stimulation with either anti-class II antibodies, anti-IgM or PMA. The 2D PAGE pattern following stimulation with anti-class II antibodies was not identical to that observed following stimulation with either anti-IgM or PMA. These data suggest that the activation of B-Go via the class II antigens shares part of the pathway of anti-IgM induced activation but does not follow an identical pathway.

Effect of extremely-low-frequency pulsed magnetic fields on the mitogenic response of peripheral blood mononuclear cells.

The effect of extremely-low-frequency pulsed magnetic fields (PMF) on the response of human peripheral blood mononuclear cells to mitogenic stimulation is reported. We investigated 25 healthy control subjects. Mitogen-stimulated mononuclear cells were exposed to PMF for 72 h and an inhibition of 3H-thymidine uptake was observed in all but one subject. The degree of inhibition of 3H-thymidine uptake was as much as 60%. There was no significant difference between the blastogenic responses of mononuclear cells exposed to PMF for 12 h and control cultures. This study establishes an inhibitory effect of PMF on an in vitro measure of immune function.

Characteristics of immune complexes detectable by two independent assays in gynaecological malignancies.

Immune complexes from patients with ovarian and endometrial cancer have been examined using the C1q solid phase (C1qSP) and polyethylene glycol (PEG) precipitation assays. The amount of IgG in the PEG precipitates was inversely correlated with the amount of IgM whilst the IgM values correlated positively with the C1qSP assay values. Separation studies revealed that most of the C1qSP activity was not precipitated from cancer sera by 2% PEG. The immune complexes were fractionated on Sephacryl S-300. Both the PEG precipitation and C1qSP assays detected high molecular weight immune complex like activity (greater than 19S). The C1qSP assay also detected a second peak of activity at approximately 7-8S. Most of the PEG detectable immune complexes from sera dissociated during column chromatography, whereas the C1qSP detectable complexes were relatively stable. Furthermore the IgG from fractionated PEG precipitates emerged as a monomer (7S) component. The PEG assay appeared to be detecting a high molecular weight complex containing mostly IgG rather than IgM with a low affinity for C1q, which was easily dissociated. The C1qSP assay detected a more stable high molecular weight complex containing a relatively high proportion of IgM and a low molecular weight complex, both with a high affinity for C1q.

A comparative study of complement components in polyethylene glycol precipitated immune complexes from patients with ovarian cancer and patients with rheumatoid arthritis.

Circulating immune complexes and complement components C1q, C3 and C4 were measured following polyethylene glycol precipitation of serum from patients with ovarian cancer (OC), patients with rheumatoid arthritis (RA) and control patients (CP). The C3 and C4 content of the precipitated material was significantly greater in those patients with OC in comparison to those with RA or CP, while the C1q content did not differ significantly between the two groups of patients. A statistically significant correlation between IgG, C3 and C4 levels was found in the immune complexes of both groups of patients. C1q levels correlated significantly with IgG immune complex levels in patients with RA and CP, but did not in patients with OC. These data illustrate that immune complexes from patients with OC differ from complexes precipitated from RA sera and normal sera in their complement content. It is suggested that the largely unsuccessful attempts to detect immune complexes in sera from patients with ovarian cancer, using assays dependent on the binding of C1q may be related to differences in solubility of complexes from these patients in comparison to complexes present in non-neoplastic conditions.

An assessment of sequential measurements of immune complex levels in ovarian cancer patients with respect to clinical progress.

An evaluation of immune-complex levels as an additional parameter in the clinical follow-up of ovarian cancer patients is described. These patients were treated aggressively with monthly chemotherapy following surgery. Sequential measurements of immune-complex levels have been carried out concurrently and clinical findings were recorded in a uniform manner suitable for comparison with the levels of immune complexes. The relationships between changes in tumor mass and changes in levels of immune complexes were investigated. A significant relationship between decreasing immune-complex levels and simultaneously decreasing tumor mass was found. Chemotherapy was associated with a decrease in immune-complex levels which increased again within 1 month. These data did not support an effective role for immune-complex levels in the evaluation of these patients.

Sequential studies of circulating immune complexes in gynecological malignancies.

Immune complexes have been assayed sequentially in seventeen patients with gynecological malignancies, using a polyethylene glycol precipitation assay and a Clq solid phase assay. The PEG assay demonstrated a good correlation between PEG assay levels and the course of disease in nine out of eleven patients with ovarian adenocarcinoma and all four patients with endometrial carcinoma. Three of the patients with ovarian cancer were not treated with aggressive surgery and exhibited equivocal signs of progressive disease during subsequent follow-up. There was no clear relationship between immune complex levels and the clinical condition in two of these patients. The ClqSP assay result did not correlate with disease progression and, in several instances, progressive disease was associated with a fall in ClqSP values. In a further series, twelve out of twenty patients with ovarian cancer who were initially in remission from disease but subsequently relapsed demonstrated elevated levels of PEG immune complexes between one and three months before clinical detection of recurrence whereas patients who stayed in remission maintained normal levels of PEG immune complexes. These data suggest that the PEG assay may be of clinical value in the monitoring of ovarian cancer patients with minimal residual disease following surgery.

Nucleoside and glucose transport in erythrocytes from new-born lambs.

1. Glucose and inosine transport by erythrocytes from new-born lambs and adult sheep were compared. Uptake of both permeants was considerably faster in the new-born. Inosine uptake by erythrocytes from nucleoside-permeable and impermeable lambs were not significantly different at birth. The difference between the two phenotypes was first apparent 30 days after birth.2. The post-natal changes in glucose and inosine transport activity closely paralleled the progressive decrease in the percentage of fetal erythrocytes (i.e. cells containing fetal haemoglobin) in the circulation. Cell fractionation studies confirmed that the permeability changes were directly related to changes in the relative proportions of fetal and adult haemoglobin containing erythrocytes.3. The results demonstrate that fetal cells are highly permeable to both glucose and inosine. These cells are replaced by erythrocytes which contain adult haemoglobin and which have a much lower, but still significant, glucose permeability and either low or negligible inosine transport activity depending on the genotype of the animal.4. Inosine transport by fetal erythrocytes from both nucleoside-permeable and impermeable animals was mediated by a nucleoside transport system which had similar properties to that responsible for nucleoside transport in adult nucleoside-permeable cells. Glucose transport in both fetal and adult cells was highly stereo-specific, indicating the presence of a selective transport system.5. It is suggested that the regulatory mechanism responsible for initiating the switch from fetal to adult haemoglobin synthesis may also be responsible for the changes in glucose and nucleoside transport activity.