Abstract: The alpha1beta1 integrin, very late antigen (VLA)-1, characterizes collagen adherent interferon (IFN) gamma producing memory T cells in inflamed synovium. We now report that the mean percentage of VLA-1+ T cells is significantly lower among peripheral blood mononuclear cells of rheumatoid patients responsive to antitumor necrosis factor (TNF) alpha therapy than of those with active disease not receiving therapy. Neutralization of TNFalpha during in vitro polyclonal activation of VLA-1- T cells reduced differentiation to expression of VLA-1 and inhibited secretion of IFNgamma, but did not affect integrin expression on in vivo differentiated VLA-1+ T cells. Moreover, synovial fluids of patients relapsing during and after therapy were enriched in VLA-1+ T cells and lines derived from VLA-1+ T cells in peripheral blood of treated patients retained collagen binding and secreted IFN gamma. Thus, whereas therapy decreases VLA-1+ T cells in rheumatoid arthritis patients, a subset is resistant and contributes to residual and recurring inflammation.

Abstract: The expression of the collagen receptor alpha(1)beta(1) integrin (VLA-1) on CD4(+) T cells is largely restricted to CCR7(-)CD45RO(+) cells that localize to inflamed tissues. Moreover, neutralizing alpha(1) integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4(+) T cells is restricted to conventional effectors. In contrast, Foxp3(+) T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4(+) T cells even upon polyclonal anti-CD3-induced activation. Because TNFalpha blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFalpha affected the balance between the proinflammatory VLA-1(+) effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFalpha blockade, inhibited the typical activation-dependent generation of CD4(+)VLA-1(+) Th1 cells. In contrast, it augmented the outgrowth of VLA-1(neg/dim)CD25(high) and Foxp3(+)CD4(+) T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFalpha blockade generated CD4(+) T cell lines enriched for VLA-1(-)Foxp3(+) Tregs. Importantly, these CD4(+) T cells displayed potent suppressive functions toward autologous CD4(+) PBL, including the suppression of the activation-dependent induction of VLA-1(+) effectors. Thus, we propose a novel mechanism by which anti-TNFalpha therapy may restore self-tolerance, by shifting the balance between VLA-1(+) effectors and Foxp3(+) Tregs, during immune activation, in favor of the latter suppressor cell population.

Abstract: In contrast to pleural effusion or ascites, there are few data regarding the chemical and cell-count parameters of pericardial effusions (PEs) to aid diagnosis. In the present work, all patients who underwent pericardiocentesis during a 9-year period (1995 to 2004) at a tertiary hospital and who had available fluid laboratory results were retrospectively identified. Causes of PE were diagnosed using predetermined criteria. The results of pericardial fluid biochemical and hematologic tests were compared with blood test results and analyzed to identify cut-off points that could distinguish among the various causes or among various groups of causes. Of 173 patients who underwent pericardiocentesis in the study period, 120 had available fluid laboratory results, and these patients constituted the study population. The most common causes of PE were neoplastic, idiopathic, and effusion related to acute pericarditis (accounting for 42, 22, and 17 of 120 patients, respectively). Most fluids (118 of 120) would have been classified as exudates by adopting Light's pleural effusion criteria. Moreover, in all parameters examined, there was a considerable overlap of test results among the different pericardial disorders. Thus, no biochemical or cell-count parameter was found useful at reasonable accuracy for differentiating among the individual causes or among various groups of pericardial disorders. In conclusion, most PEs are exudates. The analysis of pericardial fluid biochemical and cell-count composition is generally not helpful for the diagnosis of most PEs.

Abstract: Autologous stem cell transplantation is being considered as treatment of severe refractory autoimmune disorders, including MS. Stem cell mobilization is achieved with granulocyte-colony stimulating factor (G-CSF), however, G-CSF administration resulted in cases of worsened clinical MS status. We studied autoreactive T-cell properties, which can promote CNS inflammation in MS. We show that G-CSF enhances MS autoreactive T cell line adhesion to the ECM proteins collagen IV and fibronectin as effectively as the proinflammatory IFNgamma and TNFalpha, known to exacerbate MS symptoms. We propose a link between clinical worsening of MS symptoms induced by G-CSF and the hyperstimulation of T cell adhesion to ECM elicited by G-CSF.

Abstract: Celiac disease (CD) is a chronic, immune-mediated disorder of the gut, driven by T cells reacting locally to a distinct antigen, gliadin. Thus, CD offers the opportunity to study the T cell memory response to gliadin and whether gut tropism and T helper cell type 1 (Th1) polarization, which characterize the effector phase, are preserved in the memory progeny. It is notable that previous studies yielded conflicting results as to the presence of gliadin-specific memory CD4+ T cells in the peripheral blood of CD patients. However, we used a different and highly sensitive approach based on fluorescein-derived label dilution, whereby the memory cells are identified operationally by their greater capacity to proliferate upon re-encounter with antigen. Thus, using flow cytometry, we could resolve multiple successive generations as well as immunophenotype the dividing cells. Here, we show that the peripheral blood lymphocyte of some CD patients on a gliadin-free diet, but not healthy donors, contains a detectable population of CD4+ memory T cells specific for deamidated gliadin. Moreover, these gliadin-specific memory T cells are marked by a distinctive phenotype: They express high levels of the gut-homing beta7 integrins and primarily produce interferon-gamma and tumor necrosis factor alpha. We conclude that memory for gliadin-derived antigens within the circulating CD4+ T cells is linked with gut tropism as well as Th1 polarization.

Abstract: Large symptomatic pericardial effusion (PE)-PE that causes hemodynamic compromise-can be the initial presentation of an unrecognized underlying malignancy. However, the prevalence and features of this association have not been thoroughly characterized.We performed a retrospective study of all patients with hemodynamically significant PE who underwent pericardiocentesis in a 9-year period (1995-2004) in a tertiary hospital. Etiologies of pericardial disease were diagnosed using predetermined criteria. Demographic and clinical data of patients with hemodynamically significant PE as the presentation of their malignant disease were compared to those with established neoplastic disease, and to those with other etiologies.We identified 173 patients who underwent pericardiocentesis during the study period. Neoplastic PE was found in 58 patients (33%), 45 of whom had a known malignant disease at the time of pericardiocentesis. Pericardial disease was found to be the presentation of an unrecognized underlying neoplastic disease, mostly a lung tumor, in 13 patients (7.5% of all etiologies). After exclusion of pericardial effusions with easily attributable causes by clinical circumstances, physical examination, and simple laboratory tests (traumatic, uremic, post-pericardiotomy, rheumatic, and effusions related to known neoplasia), newly found cancer accounted for 18% of the remaining 74 cases. No epidemiologic or clinical parameter was found useful to differentiate between cancerous and noncancerous effusions.In conclusion, a large symptomatic PE may be the presentation of an unrecognized underlying malignancy in approximately one-fifth of the patients with a nonrevealing basic workup. This grave diagnosis cannot be ruled out on the basis of any clinical parameter. Thus, a more extensive workup should probably be considered in this patient group.

Abstract: The role of platelets in T-lymphocytes adhesion is not clear yet. Herpesvirus saimiri (HVS)-infected CD4(+) T-lymphocytes were placed into polystyrene plates pre-coated with fibronectin. The adherent T-cells were enumerated by image analysis. Under static condition, 38+/-10cells/mm(2) adhered and addition of gel-filtered platelets (GFP) and PMA enhanced cell adhesion 4.3- and 4.1-fold. Using PMA plus GFP 11.9-fold enhancement in cell adhesion was achieved. In contrast, under flow (200s(-1)), neither basal adhesion nor following separate addition of PMA or GFP was observed, whereas combined addition of PMA and GFP induced noticeable adhesion (34cells/mm(2)). The adhesion was inhibited by blockade of alpha(5)-integrin (CD49e, 87%), beta(2)-integrin (CD18, 78%), CD40L (60%), PSGL-1 (CD162, 60%), and CD40L plus PSGL-1 (83%). Thus, activated platelets promote activated T-cell adhesion to fibronectin under flow via integrins (alpha(5)beta(1), and alpha(L)beta(2)), CD40-CD40L and P-selectin-PSGL-1 mediated interactions.

Abstract: The purpose of this study was to examine the role of platelets in CD4+ T lymphocyte adhesion to subendothelial extracellular matrix (ECM). Herpesvirus saimiri (HVS)-infected CD4+ T cells were incubated on ECM. An image analysis was used to evaluate T cell adhesion. Under static condition, T cell activation with 4-alpha-Phorbol 12-myristate 13-acetate (PMA) resulted in a 2.6-fold increase in cell adhesion. However, adhesion was not affected by platelets. In contrast, under flow (200s(-1)), platelets markedly enhanced both resting and PMA-activated T cell adhesion (33- and 48-fold), forming lymphocyte-platelet co-aggregates that contain approximately 90% of the adherent T cells. Abrogation of platelet aggregation with tirofiban inhibited formation of platelet-T cell co-aggregates under flow and reduced T cell adhesion by 74%. Separate and combined blockade of CD40L and P-selectin glycoprotein-1 (PSGL-1) on PMA-activated lymphocytes reduced adhesion under flow in the presence of platelets by 28%, 33%, and 55%, respectively. Blockade of beta1-integrins decreased adhesion under both static and flow conditions (by 35% and 44%, respectively), while blockade of beta2-integrin reduced adhesion only under static condition (by 23%). A similar adhesion pattern was observed using CD4+ T cells isolated from normal donor peripheral blood. In conclusion, platelets support CD4+ lymphocyte adhesion to ECM under flow by formation of heterotypic platelet-lymphocyte coaggregates involving alphaIIbbeta3 integrin and beta1-related integrins, as well as CD40L and PSGL-1.

Abstract: OBJECT: Gastrointestinal involvement in adult dermatomyositis (DM) and polymyositis (PM) is usually mild, resulting from myoenteric dismotility. Severe inflammation of the alimentary tract in cases of adult DM and PM is rare. The purpose of this study was to examine the prevalence and clinical characteristics of inflammatory gastrointestinal involvement in patients with DM. METHODS: The charts of all cases with polymyositis or dermatomyositis, registered in our rheumatology clinic between 1984 and 2004, were reviewed retrospectively for documentation of severe gastrointestinal involvement. The clinical course and the histopathologic findings in all the patients were noted, and the prevalence of this disorder was computed. RESULTS: Among 48 patients with DM or PM, 3 patients with DM and severe gastrointestinal tract manifestations were identified (6% of the study population). Edematous hyperemic bowel wall, with multiple erosions and ulcerous lesions were the prominent endoscopic findings, whereas diffuse mucosal inflammation and multiple vascular ectasias without vasculitis dominated the histologic picture. The resulting clinical course was notable for recurrent abdominal pain and bloody diarrhea, ending catastrophically in two patients with fatal gastrointestinal perforations, despite aggressive immunosuppressive therapy. CONCLUSIONS: Severe inflammatory gastrointestinal tract disease should be recognized as a grave, albeit rare, manifestation of adult DM that portends a poor prognosis and carries a high rate of fatal complications. The role of vasculopathy in the pathogenesis of this syndrome remains to be determined.

Abstract: BACKGROUND: Benign prostatic hypertrophy is the most common benign tumor in males, resulting in prostatectomy in 20-30% of men who live to the age of 80. There are no data on the association of prostatectomy with autoimmune phenomena in the English-language medical literature. OBJECTIVES: To report our experience with three patients who developed autoimmune disease following prostatectomy. PATIENTS: Three patients presented awith autoimmune phenomenon soon after a prostectomy for BPH or prostatic carcinoma: one had clinically diagnosed temporal arteritis, one had leukocytoclastic vasculitis, and the third patient developed sensory Guillian-Barré syndrome following prostatectomy. CONCLUSIONS: In view of the temporal association between the removal of the prostate gland andthe autoimmune process, combined with previously known immunohistologic features of BPH, a cause-effect relationship probably exists.

Abstract: The alpha1beta1 integrin, also known as "very late antigen" (VLA)-1, is normally expressed on mesenchymal cells, some epithelial cells, activated T cells, and macrophages, and interacts, via the I-domain of the extracellular domain of the alpha1 subunit, with collagen molecules in the extracellular matrix (ECM). By "outside-in" transmembranal signaling to the interior of the cell, it mediates adhesion, migration, proliferation, remodeling of the ECM, and cytokine secretion by endothelial cells, mesangial cells, fibroblasts, and immunocytes. Importantly, its expressions and functions are enhanced by inflammatory cytokines including interferon (IFN)gamma and tumor necrosis factor (TNF)alpha, thus augmenting angiogenesis and fibrosis linked, in particular, to inflammation. Moreover, within the immune system, VLA-1 marks effector memory CD4+ and CD8+ T cells that are retained in extralymphatic tissues by interactions of the integrin with collagen and produce high levels of IFNgamma. Thus, immune-mediated inflammation in vivo is inhibited by blockade of the VLA-1-collagen interaction in experimental animal models of arthritis, colitis, nephritis, and graft versus host disease (GVHD), suggesting that inhibiting the interaction of the alpha1 I-domain with its ligands or modulating "outside-in" signaling by VLA-1 would be a useful approach in the human diseases simulated by these experimental models.

Abstract: Gammadelta T-cells participate in the immune response to infections and in autoimmunity by recognizing bacteria-derived and autologous antigens. The goal of this study was to evaluate the involvement of gammadelta T-cells in Behçet's disease (BD). Gammadelta T-cells in the peripheral-blood mononuclear cells (PBMCs) of Israeli patients with definite BD (n = 23), normal controls (n = 16), and patients with familial Mediterrranean fever (FMF; n = 20) were evaluated by means of flow cytometry. The responses of patient and control gammadelta T-cells to medium conditioned by microorganisms cultured from an oral ulcer of a patient with active BD were compared. The proportions of CD3(+) and CD8(+) cells in the PBMCs were not significantly different between groups. In contrast, gammadelta-T-cells accounted for 7.01% +/- 4.42% of the PBMCs in BD compared with 3.56% +/- 3.45% in FMF (P < .005) and 3.7% +/- 3.15% in normal individuals (P < .009). Their numbers were significantly higher during active disease than in remission (9.45% +/- 5.08% versus 2.27% +/- 3.3%; P < .009). The number of T-cell-receptor gammadelta(+) and Vdelta2(+) cells of BD patients, but not of controls, increased after 96 hours of culture in medium containing supernatant of microorganisms cultured from an oral ulcer in a patient with BD relative to their proportions in control medium: 58.2% vs 13.9% (P < .05) and 28% vs 9% (P < .04), respectively, of the cultured T-cells (n = 4).gammadelta T-cells are expanded in BD PBMCs during active disease. An exaggerated proliferative response to products released by microorganisms present in oral ulcers may play a role in this phenomenon.

Abstract: We previously found that the peripheral blood (PB) mononuclear cells (MCs) (PBMCs) of a patient with chronic neutropenia contained an expanded population of cytotoxic CD8+ T cells using a variable (V) region delta1 gene product in the T-cell receptor-alpha (TCR-alpha) polypeptide [Vdelta1-constant(C)alpha+ T cells]. Sequencing of polymerase chain reaction (PCR) amplification products have now revealed a productive Vdelta1/joining (J)alphaIGRJa03/Calpha rearrangement of the TCR-alpha gene, predominantly associated with a Vbeta16/Dbeta2.1/Jbeta2.1/Cbeta2 TCR-beta gene, in these cells. Furthermore, we detected a markedly deficient proliferative response of the patient PBMCs to triggering with monoclonal antibodies (MoAbs) to the CD3 molecule, contrasting with a substantial response to the Vbeta3, 12, 14, 15, 17 and 20-specific staphylococcal enterotoxin B (SEB) superantigen, suggesting defective TCR-mediated activation of the Vdelta1+/Vbeta16+ clone. Moreover, whereas triggering of Vdelta1- T cells cultured with interleukin-2 (IL-2) by MoAb to the CD3 molecule enhanced proliferation, Vdelta1-Calpha+ T cells were inhibited by MoAbs to either CD3 or Vdelta1. Vdelta1-Calpha+ T-cell clones spontaneously secrete interferon-gamma (IFN-gamma) and were further induced to release tumour necrosis factor (TNF-alpha) when triggered by anti-CD3 plus phorbol ester. Aberrant signalling by the clonotypic TCR together with the functional properties of the CD8+ Vdelta1+/Vbeta16+ clone may thus contribute to the immunohaematological abnormalities observed in this patient.

Abstract: We previously reported that human interleukin (IL)-2 dependent T cell lines derived from very late antigen (VLA)-1(+) CD45RO(+) peripheral blood (PB) T-cells adhere constitutively to collagen type IV, whereas lines from VLA-1(-) PB lymphocytes (L) adhere weakly. Here we report that the latter are induced to adhere by phorbol 12-myristate 13-acetate (PMA). Both PMA dependent and constitutive adhesion, including that of a Herpes Virus Saimiri (HVS) infected CD4(+)VLA-1(+) clone (HVST) were inhibited by anti-VLA-1 monoclonal antibodies (mAb), by inhibitors of phospholipase C (PLC)gamma and by lovastatin but not by a MEK1 inhibitor, whereas only PMA induced adhesion was blocked by inhibition of protein-kinase (PK) C. Furthermore, lovastatin enhanced PLCgamma and anti VLA-1 mAb blockade, and its effect was not reversed by mevalonic acid (MVA). Lovastatin also inhibited interferon (IFN)gamma secretion by T cells triggered with anti-CD3 and in cells detaching from collagen IV. These results suggest new ways for functional modulation of activated T-cells interacting with collagen.

Abstract: The alpha1beta1 integrin, very late antigen-1 (VLA-1), is a collagen receptor expressed in many CD4+ T cells localizing to inflamed tissues. Here we show that the expression of VLA-1 is a stable marker of a distinct subset of CD4+ memory T cells. Thus, in human peripheral blood lymphocytes (PBLs), approximately 1-4% of the CD4+ T cells express VLA-1, and following T cell receptor activation ex vivo, the percentage of VLA-1+ cells increases within the CD45RO+ population. Importantly, the activated VLA-1+ and VLA-1- cells can be isolated and maintained in culture as phenotypically stable subsets. Functionally, CD4+ memory T cells, operationally defined as the cells that divide rapidly following stimulation with a recall antigen, are highly enriched for VLA-1+ cells. Moreover, depletion of the small fraction of VLA-1+ cells present in CD4+ PBLs prior to stimulation significantly abrogated the proliferative response to recall antigens. Notably, the VLA-1+ cells in fresh CD4+ PBLs are composed of resting CD45RO+/RA-, CCR7-, CD62L+, CD25-, and VLA-4hi cells. Interestingly, this VLA-1+ subset is enriched for Th1-type cells, and Th1-polarizing conditions during T cell activation favor the emergence of VLA-1+ cells. Thus, VLA-1 expression is a stable marker of a unique subset of human memory CD4+ T cells that predominantly differentiates into Th1 cells.

Abstract: BACKGROUND: An abnormal distribution of subsets of gammadelta T cells, which are a component of the inflammatory infiltrate in arthritic synovium, has been demonstrated in the peripheral blood (PB) of patients with arthritis and neutropenia. OBJECTIVE: To evaluate whether the clinical manifestations of patients with arthritis and neutropenia are related to the specific gammadelta T cell subset predominant in the PB. METHODS: Flow cytometry of PB lymphocytes in six consecutive patients with chronic neutropenia and arthritis was performed. Variable (V) gamma and delta gene families were analysed by polymerase chain reaction. cDNA was subjected to direct automated sequencing of T cell receptor (TCR) genes. RESULTS: Three patients had non-deforming and non-erosive rheumatoid factor (RF)(+) polyarticular rheumatoid arthritis, RF(+) oligoarticular arthritis, or RF(-) non-deforming oligoarticular psoriatic arthritis with persistent expansions of Vgamma1(+)/Vdelta2(+), Vgamma2(+)/Vdelta2(+), or Vgamma1(+)/Vdelta (undetermined (2- 1-)) T cells, respectively. The other three patients, without persistent expansion of gammadelta T cells, had either non-deforming and non-erosive oligo- or polyarthritis with a balanced distribution of several Vdelta and Vgamma genes, or severe erosive RF(+) arthritis with deficiency of all but Vgamma1(+)/Vdelta1(+) T cells. CONCLUSIONS: gammadelta T cell lymphoproliferations in chronic neutropenia and arthritis use different Vgamma and Vdelta gene families, often forming T cell receptor (TCR) structures that are infrequent in normal adult PB. Arthritis with Vgamma1(+)/Vdelta2(+), Vgamma2(+)/Vdelta2(+), or Vgamma1(+)/Vdelta2(-)/Vdelta1(-) gammadelta T cells in the PB is non-deforming and non-erosive, suggesting a protective effect of these cells, as opposed to a more pathogenic contribution of Vgamma1(+)/Vdelta1(+) cells.

Abstract: Multiple sclerosis (MS), a chronic demyelinating disease, is thought to be initiated by pathogenic T cells that transmigrate the vascular endothelium and enter the brain through vascular and parenchymal basement membranes (BM). Vaccination with T-cell lines reactive with myelin basic protein (MBP) and myelin oligodendrocytic glycoprotein (MOG) epitopes, expanded with interleukin-2 (IL-2), and attenuated by ionizing radiation is currently being evaluated as a therapeutic modality for this disease. We examined mechanisms potentially involved in pathogenic cell migration into the central nervous system (CNS) and the influence of irradiation on these processes. Seven of 7 autoantigen-responsive T-cell lines from MS patients adhered to collagen IV, the major collagenous constituent of BMs. This adhesion was inhibited almost completely by monoclonal antibody (MAb) to very late antigen (VLA)-1 and partially by anti-VLA-2. T-cell lines from healthy donors adhered more variably to collagen IV. Furthermore, patient derived T cells actively transmigrated through a collagen IV gel toward medium containing TNF-a, in a process that was inhibited by MAbs to VLA-1. Ionizing radiation at the dose used in vaccine preparation, inhibited morphological polarization associated with migratory capability, induced integrin clustering on the cell membrane, and abrogated adhesion to collagen IV. These findings may have important implications for understanding the pathogenesis of MS and how irradiation of potentially pathogenic T cells produces a reagent with possible therapeutic effects in T-cell vaccination (TCV).

Abstract: BACKGROUND: Wandering spleen is a spleen lacking its normal ligamentous attachments, and thus subjected to free movement in the abdominal cavity, and even torsion around its pedicle. Surgical treatment includes either fixation (splenopexy) or resection (splenectomy). Both procedures can now be accomplished using the laparoscopic approach. METHODS AND RESULTS: We describe a case of a torsion of a wandering spleen, leading to recurrent episodes of abdominal pain, and eventually to splenic ischemia, necessitating splenectomy. The diagnosis was complicated by associated angiographic findings of celiac axis occlusion, possibly by median arcuate ligament compression. Laparoscopic splenectomy was successful, and led to complete resolution of symptoms. CONCLUSIONS: Although a rare condition, wandering spleen can be diagnosed accurately by imaging studies, mainly CT scan and angiography. Nowadays, the laparoscopic approach is preferred and enables the surgeon to perform either splenopexy or splenectomy, depending on the vascular status of the spleen.

Abstract: We report that very late antigen-1 (VLA-1(+)) CD3(+)CD45RO(+) T-cells are selectively segregated from VLA-1(-) peripheral blood (PB) mononuclear cells (MC), in which CD3(+) T-cells are evenly CD45RO(+) and CD45RO(-), when PBMC are stained with a monoclonal antibody (mAb) to VLA-1 and passaged on immunomagnetic columns. In contrast, both VLA-1(+) and VLA-1(-) MC isolated from synovial fluid (SF) are mainly CD45RO(+)CD3(+) T-cells. VLA-1(+) MC formed 13 +/- 5.3% of MC eluting from columns loaded with PBMC of patients with seropositive rheumatoid arthritis (n = 6) and 2.3 +/- 1.6% of patients (n = 4) with other arthritides (P < 0.022). Importantly, only the VLA-1(+) MC from PB and SF adhered to collagen IV upon triggering with phorbol 12-myristate 13-acetate. Moreover, adhesion and migration on collagen IV were preferentially maintained in lines cultured from VLA-1(+) T-cells, and both were inhibited by mAb to the VLA-1 alpha1 I domain. These results suggest that VLA-1(+) CD45RO(+) T-cells in patients with arthritis could play a role in both systemic and local inflammation by rapidly adhering to collagen IV.

Abstract: A monoclonal antibody (MAb), CH11, was developed by immunizing mice with CD4+ gammadelta T-cell receptor (TCR)+ cells. It recognized an antigen expressed in the surface membrane of T-cell lines, but not of U937, lymphoblastoid B cells (LBC), K562, Raji or Daudi cells, indicating selectivity for the T-cell lineage. In addition, it labelled 70-80% of normal peripheral blood mononuclear cells (PBMC), with high expression on the erythrocyte rosetting (E+) fraction, and low/absent expression on E- cells. However, CD4+ T cells expressed higher levels of reactivity than CD8+ or gammadelta+ T-cell receptor (TCR)+ lymphocytes in PB. Furthermore, in 7 of 10 individuals tested, 7.34+/-3.88% of unselected PBMC were CH11- CD3+ and were relatively enriched in CD8+ and in gammadelta TCR+-cells. In addition, thymic gammadelta T cells, and gammadelta lymphoproliferations from two patients were nonreactive or weakly reactive with the MAb. Activation of E+ cells with phorbol-12-myristate-13-acetate (PMA) enhanced CH11 expression uniformly, whereas activation with phytohemagglutinin (PHA) selectively down-regulated expression of the antigen on the CD8+ subset. In Western blots performed in nonreducing (NR) conditions, MAb CH11 detected a 100 kDa molecule in PBMC and Jurkat T-cell lysates. Preincubation of T cells with MAb CH11 specifically abrogated their subsequent reactivity with MAb to CD6, suggesting that MAb CH11 is recognizing an epitope of CD6. Given its function as a receptor for ligands on thymic epithelium, activated leukocytes and synoviocytes, this newly defined heterogeneity of expression and regulation of the CD6 molecule on subsets of T cells may help determine their functional repertoire in vivo.

Abstract: We describe herein the clinical and laboratory manifestations of a unique group of patients (pts) presenting with hypereosinophilic syndrome (HES) who were treated in our medical centers for 4-13 years. Skin biopsies, flow cytometry of peripheral blood mononuclear cells (PBMC), assays for cytokines and immunoglobulin (Ig) production in vitro, and Southern blots of T-cell receptor (TCR) genes were performed. All four pts had a persistent hypereosinophilia (> 1.9 x 10(9)/L) and chronic skin rash. Three of four had elevated IgE, thrombotic manifestations and lung involvement (asthma and/or infiltrates), and one had deforming sero-negative arthritis of the hands. 66-95% of their peripheral T-cells expressed CD4 but not CD3 or TCR molecules on the cell surface membrane. Activated CD4+CD3- cells secreted interleukin (IL)-4 and/or 5, and were required for maximal IgE secretion by autologous B-cells. Two pts had evidence of rearrangement of TCR genes of the CD4+CD3- cells, one of whom died of anaplastic lymphoma. In conclusion, HES with CD4+CD3- lymphocytosis may be associated with high serum IgE, dermatological, pulmonary, thrombotic and rheumatic manifestations which may be due to Th2 effects of CD4+CD3- cells migrating to end organs. Fatal systemic lymphoid malignancy may also develop in some pts with monoclonal expansion of the CD4+CD3- T-cells.

Abstract: The alpha1 beta1 integrin, an inserted (1) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human alpha1beta1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human alpha1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg2+ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV or alpha1 I-domain. MAb I B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free alpha1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.

Abstract: Cell-mediated cytotoxicity is thought to play an important role in the immunopathogenesis of inflammatory myopathies. We determined whether lymphocytes circulating in patient peripheral blood contain automyocytotoxic precursors. Peripheral blood mononuclear cells, sheep red blood cell rosetting (E+) and nonrosetting (E-) cells, were isolated from patients with polymyositis or dermatomyositis and tested for their ability to kill autologous-cultured myotubes derived from diseased muscle biopsies. Patient-derived as well as normal allogeneic mononuclear cells lysed both polymyositis-dermatomyositis and nonrelated myotubes. However, whereas patient E+ cells were preferentially cytotoxic to autologous myotubes, their E-cells did not discriminate autologous from allogeneic targets. Furthermore, cultures of patient E+ cells triggered by phytohemagglutinin and interleukin-2 maintained myocytotoxic potential. In these cultures, virtually all autologous but only 50% of allogeneic killing was mediated by CD3+ T cells. Moreover, autologous cell-mediated killing was abrogated by anti-CD3 monoclonal antibodies. In conclusion, both myocytotoxic CD3+ T-cell clones specific for autologous myotubes, as well as non-T cells, which are nonspecifically myocytotoxic, are present in the peripheral blood of patients with inflammatory myopathies.

Abstract: During the past decade knowledge about the etiology of venous thromboembolism has increased tremendously. Inherited and acquired risk factors for venous thromboembolism are common in patients as well as in the general population. Whether the presence of most of these risk factors has consequences for the management of symptomatic and asymptomatic individuals is not fully clear at present. Therefore, while searching for new thrombophilic defects, it is crucial to determine the absolute risk for (recurrent) venous thromboembolism as well as other clinical manifestations in carriers. However, tentative guidelines for managing patients and their families are given in this review.

Abstract: Rheumatoid arthritis is a chronic inflammatory disease affecting about 1% of the adult population. The pathophysiology of rheumatoid arthritis remains incompletely understood. An infectious aetiology of the disease has long been postulated, but not proved. Despite insufficient evidence for the infectious nature of this disorder, several antibacterials, such as sulfa compounds, tetracyclines and rifampicin, have been investigated in the treatment of rheumatoid arthritis. In the last few years, minocycline, a semi-synthetic derivative of tetracycline, has been extensively studied as a therapeutic agent for rheumatoid arthritis. The antirheumatic effect of minocycline can be related to its immunomodulatory and anti-inflammatory, rather than to its antibacterial properties. Its efficacy in rheumatoid arthritis has been reported in 2 open trials and in 3 double-blind controlled studies. The first 2 double-blind studies, 1 in The Netherlands and 1 in the US, were performed in patients with advanced disease. Both studies showed a modest, but statistically significant improvement in the clinical parameters of disease activity and in the erythrocyte sedimentation rate in the minocycline-treated patients. The US study also reported that patients in the minocycline group developed fewer erosions than those in the placebo group. This finding supports the role of minocycline as a disease modifying agent. The common adverse effects of minocycline reported in these 2 studies included gastrointestinal adverse effects, dizziness, rash and headaches. Less common adverse effects were intracranial hypertension, pneumonitis, persistent skin and mucosal hyperpigmentation, lupus-like syndrome and acute hepatic injury. The third double-blind study enrolled only seropositive rheumatoid arthritis patients with early disease (less than 1 year duration), and showed very encouraging results of significant improvement in the disease activity parameters in the minocycline treated group of patients. The same authors later reported that about half of these patients were in or near remission after 3 years of follow up. No adverse effects were reported in this study. Summarising the data of these 3 double-blind studies, we may conclude that minocycline may be beneficial in patients with rheumatoid arthritis, especially when given early in the disease course or in patients with a mild disease.

Abstract:

Abstract: In mice, monoclonal antibody (mAb) to the alpha1 integrin abrogate gastro-intestinal damage during graft-versus-host-disease (GVHD), suggesting anti alpha1 mAb as candidates for treatment in humans as well. Our current data show that one such reagent, mAb 1B3.1, when immobilized to plastic wells via rabbit- anti murine (ram) immunoglobulin (Ig) induces a protein kinase-dependent spreading of activated human T cells. Furthermore, it significantly increases the proliferative response, and expression of interleukin-2 (IL-2) receptors (R) and CD69, of resting T cells, expressing minimal integrin on the cell surface, to sub-optimal stimulation by anti-CD3 mAb. We found, in addition, that mAb 1B3.1 a) immuno-precipitates alpha1beta1 integrins from cell-surface iodinated canine epithelial cells b) is highly reactive with canine T cells after their activation and c) inhibits adhesion of canine T cells to collagen IV. Despite the potential ability of the mAb to co-activate T cells in vitro, two dogs that received 4 injections of 0.5-0.3 mg/Kg of mAb 1B3.1 remained healthy, developing only marginal transient lymphopenia. Injection of 0.75mg/Kg in a third dog induced a more marked lymphopenia, and an additional dose of 1.0 mg/Kg 2 weeks later was followed by gastrointestinal hemorrhage. importantly, the lymphopenia was associated with a greater and more persistent decrease of CD8+ than of CD4+ T cells, leading to an increase in the CD4/CD8 ratio 24 hours after the injection. Thus, despite it's co-activating effects in vitro, administration of this mAb in vivo is feasible when appropriately dosed, and may have immuno-modulatory effects.

Abstract: We have identified and characterized the tissue distribution of the antigen recognized by a novel monoclonal antibody (mAb) 1B10, raised against an activated gammadelta T cell clone. Immunohistochemistry of tissue sections, and analysis of single cell suspensions by flow cytometry revealed that mAb 1B10 weakly reacted with <6% of normal human peripheral blood mononuclear cells (PBMC). After 5-6 days of in vitro culture of PBMC activated with phytohemagglutinin (PHA), 55% of the CD4+ and 25% of the CD8+ T cells became 1B10+. 1B10 expression was maintained on long term cultured interleukin 2 (IL-2)-dependent T cell receptor (TCR) alphabeta+ and gammadelta+ clones, and importantly, in contrast to resting T cells, the majority of in vivo activated synovial T lymphocytes from a patient with rheumatoid arthritis were 1B10+. In addition, myelo-monocytic U927 cells, tissue macrophages and some epithelia and fibroblasts were found to react with mAb 1B10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of molecules immuno-precipitated by mAb 1B10 from radio-iodinated cell surface membrane lysates of T lymphocyte and U937 cells revealed 26 and 29 kiloDalton (kDa) glycoproteins respectively. In conclusion, mAb 1B10 recognizes a novel <<late>> appearing 26 kDa T cell activation antigen that may be useful for further studies of activated T cells in health and disease.

Abstract: Several of the beta1 integrin receptors [very late antigen (VLA) molecules] for extracellular matrix (ECM) proteins are expressed by malignant T cells in cutaneous T-cell lymphoma (CTCL). We evaluated the function of VLA-1, a beta1 integrin specifically expressed in epidermotropic mycosis fungoides (MF), in CD4+ leukemic T cells Jurkat line). We found that Jurkat cells adhere significantly to collagens only after their activation with phorbol 12-myristate 13-acetate (PMA). However, the adhesion to collagen IV (but not to collagen I) of Jurkat cells selected for expressing increased levels of VLA-1 (with unchanged levels of VLA-2, the second collagen integrin receptor) was significantly enhanced relative to that of "VLA-1 low" cells. Monoclonal antibody (mAb) 1B3.1, directed against the collagen binding domain of VLA-1, inhibited adhesion to collagen IV and to collagen I by 36.67%+/-5.25% and 18%+/-4.32%, respectively (p<0.05), whereas the inhibition by anti-VLA-2 mAb PIE6 was comparable on both collagens (25%+/-7.48% and 36.3%+/-0.94%, respectively; p<0.09). Immuno-histochemical studies of skin biopsies from 10 untreated MF patients showed that in all cases at least 10% of the lymphocytes residing in the epidermis are VLA-1+VLA-2-. While not directly applicable to MF, the demonstrated functions of VLA-1 in leukemic Jurkat cells, together with its expression in MF skin, suggest a role for VLA-1 integrins in epidermotropism in a small proportion of leukemic MF cells.

Abstract: An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of soluble alpha1beta1 integrins (salpha1) in human serum samples was developed. Solid phase-bound anti-alpha1 integrin monoclonal antibody (mAb) TS2/7 was used to capture salpha1, and mAb 1B3.1 was used to detect the immobilized integrin. An extract of human placenta (PE) containing 340 ng/mL of VLA-1 molecules served as a positive control, and serum samples from normal donors and patients were assayed. Optimal binding of anti-alpha1 integrin mAb 1B3.1, expressed as specific optical density (OD), was obtained when a 5 microng/mL solution of anti-alpha1 integrin "capture" mAb TS2/7 was immobilized to the wells and the PE was added. Solutions of albumin or collagen, in contrast, did not result in binding, confirming the specificity of the assay for sal. Furthermore, the specific OD of the wells correlated directly with the concentration of PE. A concentration of salpha1 above that of a 1:100 dilution of PE--that is, >3.4 ng/mL of integrin, in which the intra-assay correlation of variance was <5.7%, was found in 5 of 8, 3 of 8, and 6 of 9 serum samples from normal individuals, patients with connective tissue diseases (CTD), and patients with liver diseases (LD), respectively. These results suggest, for the first time, that salpha1 are present in healthy and diseased human serum.

Abstract: A patient with end-stage renal failure, due to IgA nephropathy, was found to have a mediastinal mass. Biopsy specimen of the mass showed a necrotizing vasculitis. Antineutrophil antibodies to myeloperoxidase were strongly positive. To our knowledge, no case of a mediastinal mass due vasculitis has been reported in the literature, and our observation should lead to broadening of the spectrum of clinical manifestations of vasculitis.

Abstract: OBJECTIVES: Dermatomyositis and polymyositis (DM/PM) are associated with neoplasms. The aim of the present study is to compare our experience in Israel with other published data. METHODS: Thirty-five adult patients with DM/PM, admitted to Sheba Medical Center during the 11-year interval between 1984 and 1994, were studied for the prevalence and features of malignant diseases. Patients with DM/PM alone and with DM/PM and malignancy were identified by using the hospital computer system. The manifestations of DM/PM and features of the malignant diseases were abstracted from the patients' charts. The presence or absence of malignancy and the type of cancer were verified in the National Cancer Registry. RESULTS: There were 15 men and 20 women. The mean age at the onset of the disease was 53 +/- 18 years. A total of 15 had PM and 20 DM. Malignancies occurred in four patients with PM (27%) and in nine with DM (45%) a frequency 12.6 times higher than in the general population. In six patients, the malignancy and the DM/PM were diagnosed simultaneously; in four before and in three after the appearance of the DM/PM. Hematologic, gastrointestinal, breast, ovarian, and lung tumors, malignant melanoma, and metastatic carcinoma of unknown primary were found among our patients. Eight DM/PM patients with malignancy died during the study period of infection, pulmonary embolism, and tumor spread. CONCLUSIONS: Our study found that DM/PM is associated with high rates of malignancy and mortality.

Abstract: BACKGROUND: CD4+CD3- T cells have previously been shown to play a pathogenic role in the hypereosinophilic syndrome by secreting IL-5 and IL-4. OBJECTIVES: The goal of this study was to study the role of CD4+CD3- and other T-cell subsets in a patient with eosinophilia, dermatitis, and a high level of IgE (100,000 IU/mL) in the serum. METHODS: We isolated PBMCs and performed flow cytometry, cell cultures, and in vitro assays of Ig, lymphokine production, and cell-mediated cytotoxicity. RESULTS: Flow cytometric and immunohistochemical analysis of the PBMCs revealed a major population (consisting of approximately 85% of the CD4+ T cells) that lacked expression of CD3 and T-cell receptors on the cell surface (CD4+CD3- T cells), but did express CD3 peptides in the cytoplasm. Activation of the PBMCs in vitro resulted in a 100-fold greater than normal release of IL-4, whereas IFN-gamma production was less than normal, suggesting a predominantly type 2 helper functional phenotype of the CD4+CD3- T cells. Importantly, both CD4-CD8(low) Vdelta1+ T-cell receptor gammadelta+ and CD4+CD3- T cells were cultured from the PBMCs. The former secreted IFN-gamma exclusively, whereas the latter secreted both IL-4 and IFN-gamma. Furthermore, only the T-cell receptor gammadelta+ lymphocytes were cytotoxic to autologous B-lymphoblastoid cells and specifically inhibited IgE production in cultures of autologous polyclonally stimulated PBMCs. CONCLUSIONS: The results suggest that CD8(low) Vdelta1+ T-cell receptor gammadelta+ clones functionally counteract IgE-inducing effects of type 2 CD4+CD3- helper cells in this patient with hypereosinophilic syndrome.

Abstract: A patient with a severe bleeding tendency due to acquired von Willebrand disease (VWD) is presented. Although no underlying disorder has emerged during 6 years of follow-up, an immune-mediated mechanism was responsible for acquired VWD in this patient as demonstrated by detection of von Willebrand factor (VWF)/anti-VWF complexes in the patient's plasma and their removal by protein A-sepharose beads and resumption of normal haemostasis with correction of VWF antigen, VWF activity and VWF multimeric pattern after treatment of the patient with high-dose gammaglobulin. Detection of anti-VWF antibodies in the patient's plasma had a significant impact on the choice of therapeutic intervention to control bleeding.

Abstract: Recently we encountered a patient (p1) with a Clark's level IV melanoma associated with recurrent cutaneous metastasis who subsequently experienced a complete remission after a period of therapy with dichloronitrobenzene (DCNB) and interferon-alpha (IFNalpha). Another patient (p2) with a similar Clark's level of disease but with a fatal prognosis and melanoma cells from the Sk-Mel 28 and MeWo cell lines served as control groups. Since cytokines and members of the alpha1 integrin family have been implicated in the growth and metastatic behaviour of melanomas, we evaluated the cytokine effects and integrins expressed by melanoma cells in the patients' tumours. P1, but not p2 nor MeWo melanoma cells, expressed HLA-DR, alpha1beta1 and the tumour necrosis factor receptor (TNF-R). Culturing p1 melanoma cells with TNFalpha, but not MeWo or p2 melanoma cells, increased their expression of alpha1beta1 integrin and was cytotoxic for the cells. Significant in vivo growth of metastatic p1 and p2 melanoma explants as well as MeWo cells grafted subcutaneously onto nude mice was noted over 36 days. Intralesional injection of TNFalpha induced complete regression of p1 explants, but not of p2 or MeWo explants. Intralesional injection of IFNalpha or anti-alpha1beta1 integrin arrested p1 graft growth. These results suggest that the slow growth of the melanoma cells in nude mice, as well as the susceptibility to tumour killing by TNFalpha and the dependence of the tumour on signals mediated by the alpha1beta1 integrin are features that may have contributed to the beneficial therapeutic response in patient 1.

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Abstract: T lymphocytes that express T-cell receptors encoded by the gamma and delta T-cell receptor genes (gamma delta T cells), and preferentially those expressing the V gamma 9 and V delta 2 gene segments, are activated by microbial and parasitic organisms in vitro and have been implicated in the pathogenesis of the fever and rigors during acute malaria. We have found, in a cohort of nine nonimmune patients who contracted malaria during travel to endemic areas (five with Plasmodium falciparum and four with P. vivax infections) that gamma delta T lymphocytes expanded to comprise 17.92% +/- 11% of the peripheral blood mononuclear cells (vs 3.08% +/- 2.4% gamma delta cells in normal control subjects). Although V delta 2+ cells predominated among the gamma delta subset, gamma delta lymphocytes expressing the V delta 1 gene segment also expanded significantly in some patients. Importantly, the gamma delta cells continued to expand for 2 months after the infection, and the mean level of gamma delta cells peaked during the second month after the acute clinical syndrome, when patients were free of symptoms. Thus although gamma delta T cells may contribute to the pathogenesis of the acute clinical syndrome, our findings suggest that gamma delta lymphocytes could also play a role in generating an immune response to plasmodia.

Abstract: Infection is the major cause of morbidity and mortality in systemic lupus erythematosus (SLE). Although various fungi account for a substantial number of these lethal infections, aspergillosis, an important opportunistic infection in immunosuppressed patients, is described rarely. Only 23 cases have been reported in the English-language medical literature. Risk factors for acquiring aspergillosis in these patients were high grade disease activity, granulocytopenia, use of steroids and other immunosuppressive treatment and presence of bacterial infection. The diagnosis in most patients was delayed and they died. Here, we describe three SLE patients with invasive aspergillosis. Features of our patients' diseases were similar to those reported previously. Aspergillosis appeared while they had active SLE treated with high dose corticosteroids. In 2 patients the fungal infection was systemic and diagnosed post mortem. Both were leukopenic and had concurrent bacterial infection and one received amphotericin B prior to death. In the third, the infection was localized to a transplanted kidney and was cured by nephrectomy. Aspergillosis should be suspected in patients with active SLE, who are immunocompromised and sustain concomitant bacterial infections. The currently poor prognosis may be improved with more aggressive diagnostic investigation and treatment.

Abstract: Directed migration of keratinocytes and fibroblasts is a fundamental prerequisite in wound healing. Cation-dependent affinity changes of integrins are responsible for cell adhesion to and deadhesion from extracellular matrix proteins and have been implicated in driving cell migration. The specific requirements for divalent cations in the integrin-dependent migration of human dermal fibroblasts and human epidermal keratinocytes to various extracellular matrix proteins have been studied in vitro using blindwell Boyden chambers. The migration of the tested cells to collagen type I was mediated by the alpha 2 beta 1 integrins, to fibronectin by the combined action of the alpha 3 beta 1 and the alpha 5 beta 1 integrin, and the migration of fibroblasts to laminin dependent both on the alpha 2 beta 1 and the alpha 6 beta 1 integrins. No migration of keratinocytes to laminin was detected. Mg2+ alone induced cell migration with an optimum at 2 mM for fibroblasts and at 10 mM for keratinocytes. Ca2+ alone at 2 mM only marginally enhanced fibroblast and keratinocyte migration. At higher concentrations Ca2+ suppressed the stimulatory Mg2+ effect. 2 mM Ca2+ combined with 2 mM Mg2+ showed an additive stimulatory effect on the migration of fibroblasts to fibronectin. These data suggest that extracellular divalent cations differentially influence the integrin-mediated cell migration. A concentration gradient of Mg2+/Ca2+, as reported in tissue injury, thus may play a regulatory role in cell migration required for tissue remodelling.

Abstract: Expanded populations of T lymphocytes bearing gamma delta T cell receptors have been detected in several patients with Felty syndrome. The goal of this study was to functionally characterize these lymphocytes in a newly described patient with this disease. For this, fluorescence-activated cell sorter analysis of T cell surface antigens, proliferation, and tumor necrosis factor alpha enzyme-linked immunosorbent assays, as well as quantitative assays of immunoglobulins secreted by pokeweed mitogen-driven B cells were performed. The gamma delta cells, that expressed a CD3+CD4-V gamma 9-V delta 2+C delta + phenotype, and constituted 60% of the peripheral blood T cells, did not proliferate after triggering with anti-CD3, but did secrete tumor necrosis factor alpha and the addition of these cells to pokeweed mitogen-stimulated B cells from the patient decreased their secretion of immunoglobulin M while augmenting IgG secretion. These data suggest that the expanded anergic V gamma 9-V delta 2+ gamma delta cells can play an immunoregulatory role in the patient.

Abstract: The reorganization of extracellular matrix (ECM) is an important function in many biological and pathophysiological processes. Culture of fibroblasts in a three-dimensional collagenous environment represents a suitable system to study the underlying mechanisms resulting from cell-ECM interaction, which leads to reprogramming of fibroblast biosynthetic capacity. The aim of this study was to identify receptors that transduce ECM signals into cellular events, resulting in reprogramming of connective tissue metabolism. Our data demonstrate that in human skin fibroblasts alpha 1 beta 1 and alpha 2 beta 1 integrins are the major receptors responsible for regulating ECM remodeling: alpha 1 beta 1 mediates the signals inducing downregulation of collagen gene expression, whereas the alpha 2 beta 1 integrin mediates induction of collagenase (MMP-1). Applying mAb directed against different integrin subunits resulted in triggering the heterodimeric receptors and enhancing the normal biochemical response to receptor ligation. Different signal transduction inhibitors were tested for their influence on gel contraction, expression of alpha 1(I) collagen and MMP-1 in fibroblasts within collagen gels. Ortho-vanadate and herbimycin A displayed no significant effect on any of these three processes. In contrast, genistein reduced lattice contraction, and completely inhibited induction of MMP-1, whereas type I collagen down-regulation was unaltered. Calphostin C inhibited only lattice contraction. Taken together, these data indicate a role of tyrosine-specific protein kinases in mediating gel contraction and induction of MMP-1, as well as an involvement of protein kinase C in the contraction process. The data presented here indicate that different signaling pathways exist leading to the three events discussed here, and that these pathways do not per se depend upon each other.

Abstract: The functions of VLA-1 (CD49a, alpha 1 beta 1 integrin), a potential ECM protein receptor on activated human T lymphocytes in vivo, were investigated. Within a panel of 25 long-term cultured IL-2-dependent T cell lines, tau delta and CD8+ alpha beta cells expressed significantly higher levels of alpha 1 beta 1 than CD4+ alpha beta cells. While VLA-1-high tau delta or CD8+ cells adhered to plastic wells coated with collagen IV, collagen I, or fibronectin, moAb 1B3.1 to VLA-1 only inhibited the adherence to collagen IV. tau delta and CD8+ VLA-1-high T cells layered upon collagen IV in the presence of Mg2+ also spread elongated cytoplasmic extensions, which were abrogated by moAb 1B3.1. In contrast, spreading on fibronectin or spontaneous non-ECM-related spreading were not inhibited. Crosslinking of surface VLA-1 molecules with plastic-bound moAb 1B3.1 selectively induced expression of IL-2R on two of six VLA-1+ clones, both of which expressed tau delta TCR. Thus, CD49a is a specific collagen IV receptor in VLA-1-high tau delta and CD8+ alpha beta cells and can transmit signals to these lymphocytes to spread and express IL-2R.

Abstract: We report here the analysis of potential ligand binding domains within the human integrin alpha 1 subunit, a known collagen/laminin receptor. This integrin is effectively blocked by the mouse monoclonal antibody 1B3.1. A truncated version of the alpha 1 subunit lacking the NH2-terminal half of the extracellular domain is not recognized by monoclonal antibody 1B3.1. Furthermore, we have isolated a cDNA containing the I domain from chicken alpha 1 bearing significant homology to the human and rat alpha 1 sequences. Replacing the human I domain with its chicken counterpart led to the surface expression of a functional heterodimer with endogenous mouse beta 1 on NIH 3T3 cells. However, 1B3.1 does not bind to the chicken/human chimera, demonstrating that the human alpha 1 I domain is required for epitope recognition. Mutation of Asp253 within the I domain to alanine resulted in surface expression of an alpha beta heterodimer recognized by 1B3.1 but with markedly reduced binding to collagen IV or laminin. Since a previously reported mutation of a homologous Asp in the Mac-1 I domain has similar consequences, these results suggest a central role for the I domain in ligand recognition for all integrin alpha subunits containing this domain.

Abstract: The specific requirements for divalent cations in the integrin-dependent adhesion and deadhesion of human dermal fibroblasts and human epidermal keratinocytes to various extracellular matrix proteins have been studied in vitro. The adhesion of both cell types to collagen type I and to laminin was enhanced by Mg2+ in a concentration-dependent manner, while Ca2+ dose-dependently antagonized this effect, thus promoting deadhesion. The cation-dependent conversion between adhesion and deadhesion occurred already at 2 to 10 min after addition of the alternate cation and was almost completed at 20 min. Interestingly, Ca2+ could not reverse the Mg(2+)-enhanced adhesion of both cell types to fibronectin. Inhibition studies with function-blocking antibodies directed against distinct beta 1 integrins showed that the Mg(2+)-enhanced fibroblast adhesion to collagen type I was mediated by the alpha 1 beta 1 and the alpha 2 beta 1 integrins, whereas keratinocyte adhesion to collagen type I was mediated by the alpha 2 beta 1 integrin. Both cell types utilized the alpha 2 beta 1 and the alpha 6 beta 1 integrins for Mg(2+)-dependent adhesion to laminin and the alpha 5 beta 1 integrin for the adhesion to fibronectin. Integrin expression at the cell surface was not altered, indicating that divalent cation-dependent conformational changes of beta 1 integrins most likely regulate their functional activity.

Abstract: In order to study the potential role of gamma delta T lymphocytes in cytotoxicity against breast carcinoma cells, normal peripheral blood gamma delta cells were isolated and triggered with an alloantigenic lymphoblastoid B cell line and recombinant interleukin-2 and cloned. The clones expressed a CD3+V gamma 9+V delta 2+CD4-CD8- (or CD8+) phenotype. Five clones were cytotoxic to the Molt 4 T cell leukemia, but not to the alloantigen, whereas one clone lysed the alloantigen, but not the Molt 4 line. Clones that were cytotoxic to Molt 4 were either spontaneously cytotoxic against MCF 7 breast carcinoma cells or could be induced to kill MCF 7 cells by monoclonal antibodies specific for the gamma delta T cell receptor. In situ staining demonstrated that V delta 2+ as well as other subsets of gamma delta T cells can be detected in the lymphocytic infiltrate within breast carcinomas. These data showing that V delta 2+ T cells can recognize and kill cells of breast carcinoma lineage in vitro, and that cells expressing V delta 2 genes in their T cell receptor structure can be detected in the tumor, suggest that further studies of the nature of the interactions between V delta 2 T cells and breast carcinoma cells are warranted.

Abstract: CD8+ T-lymphocyte populations may be expanded in the peripheral blood of patients with chronic idiopathic neutropenia and may be involved in suppression of granulopoiesis. In this report, we have analyzed the T-cell receptor (TCR) used by the T lymphocytes of a patient with chronic severe neutropenia. Using specific oligonucleotides in the polymerase chain reaction (PCR) to amplify cDNA specific for the different families of the V alpha, V beta, and V delta TCR genes, and monoclonal antibodies (MoAbs) to examine T-lymphocyte subsets and their TCR, a persistent expansion of CD3+CD8+ T lymphocytes and a reduced repertoire of TCR V alpha and V beta genes were found in the patient's peripheral blood mononuclear cell (PBMC) preparations. A predominant portion of the T lymphocytes expressed a unique TCR structure. Thus, we found that, despite the fact that 98% of the T cells expressed alpha beta TCR on the surface membrane and less than 2% expressed tau delta TCR, nonetheless, 40% to 60% of the T cells stained positively with anti V delta 1 MoAb. Using the PCR analysis, the V delta 1 gene segment was found to be rearranged to C alpha, rather than to C delta genes. The expanded C alpha V delta 1+ cells, which are found only rarely in normal PB, expressed CD8 and were cytotoxic, and the C alpha V delta 1 receptor was functional in cytotoxicity. This constitutes the first description of an expansion of cytotoxic CD8+ lymphocytes expressing a functional "hybrid" C alpha V delta 1 gene in vivo, and suggests a pathogenic role for CD8+ C alpha V delta 1+ cells in some patients with idiopathic neutropenia.

Abstract: In a 48-week open trial, 18 patients with active rheumatoid arthritis (RA), resistant to second line agents, received 200 mg minocycline daily. Twelve patients completed 48 weeks of therapy. Statistically significant improvement was noted in almost all variables of disease activity. Three patients discontinued therapy because of lack of improvement, 2 patients because of side effects and one patient was lost to followup. Cytofluorographic analysis revealed a significant decrease in expression of a T cell activation antigen (gp 26). Our data suggest that minocycline could be a useful therapeutic agent in RA.

Abstract: In the human immune system, very late antigen 1 (VLA-1), a putative collagen receptor, is expressed on the surface of T lymphocytes that have undergone mitogenic or antigenic stimulation. A new VLA-1-specific monoclonal antibody, 1B3.1, was used to probe the expression and function of VLA-1 on T lymphocytes in patients with arthritis. Synovial mononuclear cells from the joints of patients with rheumatoid arthritis or other joint diseases contained 32.9 +/- 13.8% 1B3.1-positive cells (42.8 +/- 10.4% in patients with rheumatoid arthritis and 28 +/- 12.6% in non rheumatoid patients). In the peripheral blood, patients with active rheumatoid arthritis expressed VLA-1 on 11.7 +/- 6.0% of their mononuclear cells, compared to 1.9 +/- 1.5% in controls (P less than 0.001). Using dual fluorescence analysis, virtually all the 1B3.1-positive synovial cells were CD3+ T lymphocytes and included both CD4+ and CD8+ T cells. When 1B3.1-expressing synovial mononuclear cells or in vitro activated T lymphocytes were triggered with anti-CD3 antibodies, marked augmentation of their proliferation occurred if they were simultaneously cross-linked with mab 1B3.1. Collagen type IV, a putative ligand of VLA-1, also augmented T-cell proliferation to anti-CD3. The data suggest that the VLA-1 molecule could play an important role in the pathophysiology of arthritis by modulating T-cell activation in these diseases.

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Abstract: We describe a case of pulmonary nocardiosis in a patient with rheumatoid arthritis (RA) receiving treatment with combined immunosuppressive agents and prednisone. This infection is still considered rare, hard to diagnose, and difficult to treat. To the best of our knowledge, such a case has not been described in a patient with RA.

Abstract: A monoclonal antibody, 1B3.1, was raised against a cloned IL-2-dependent T cell line that expresses the T gamma delta T cell receptor. MoAb 1B3.1 reacted with long-term cultured T cell lines of both T gamma delta and T alpha beta lineage, and with in vivo-stimulated T cells, derived from synovial fluid, but not with resting or short-term activated T cells, B cells, or macrophages. Immunoprecipitation of the 1B3.1 target antigens showed that 1B3.1 recognizes a 200/110 kDa molecule that is identical to the VLA-1 heterodimer precipitated by MoAb TS2/7. 1B3.1, however, binds to an epitope of VLA-1 that is distinct from the TS2/7 binding site. This new MoAb could be useful in further studies of the functions of VLA-1, and of the cells that express this molecule.

Abstract: A subpopulation of the CD3+ peripheral T lymphocytes express the TCR-gamma/delta complex. Three distinct TCR-gamma forms that differ in size and in the ability to form a disulfide bridge with the TCR-delta subunit have been described. In this study we analyze the structural difference between the non-disulfide-linked 55-kD and 40-kD TCR-gamma chains. The 40-kD TCR-gamma form contains a smaller polypeptide backbone and carries less carbohydrate compared with the 55-kD TCR-gamma form. A cDNA clone corresponding to the 40-kD TCR-gamma subunit lacks one copy of the second exon of the constant region that is present in the other TCR-gamma subunit. This exon copy encodes part of the connector region that is located between the constant domain and the membrane spanning region. We show that the number of potential N-linked glycan attachment sites are the same for the two TCR-gamma forms. Since these attachment sites are located in the connector region we conclude that the connector region influences the amount of N-linked carbohydrates added to the core TCR-gamma polypeptide, probably by affecting the conformation of the protein. In contrast to the TCR-beta constant region usage, the TCR-gamma constant regions are unequally expressed. Virtually exclusive usage of disulfide-linked complexes were found in some individuals, while both the disulfide-linked and the 40-kD, non-disulfide-linked TCR-gamma forms were detected in other subjects. The ability to distinguish these TCR-gamma/delta forms now makes it possible to study the mechanisms that govern their selection and to determine if they correspond to functionally distinct isotypes.

Abstract: The CD3+, IL-2-dependent normal human thymocyte clone, CII, expresses on its surface a CD3-associated gamma/delta TCR. We have further elucidated the structure of this receptor from the nucleotide sequence of cDNA and genomic clones from CII that encode functional TCR-gamma and -delta chains. We find that the CII line expresses a C gamma 2 constant region that is a polymorphic form lacking a copy of an internal exon; the sequence of this constant region accounts for the size of the gamma chain and noncovalent linkage of gamma and delta chains in the CII TCR. The V gamma region used for the CII TCR is identical to the several previously characterized expressed human V gamma segments. Possible implications of this finding are discussed.

Abstract: Tumor necrosis factor/cachectin (TNF) has been implicated as a mediator of the host response in sepsis and neoplasia. Recent work has shown that TNF can modulate endothelial cell hemostatic properties, suggesting that endothelium is a target tissue for TNF. This led us to examine whether endothelial cells have specific binding sites for TNF and augment the biological response to TNF by elaborating the inflammatory mediator, IL-1. Incubation of 125I-recombinant human TNF with confluent, cultured human umbilical vein endothelial cells resulted in time-dependent, reversible, and saturable binding. Binding was half-maximal at a TNF concentration of 105 +/- 40 pM, and at saturation 1,500 molecules were bound per cell. Heat-treated TNF, which is biologically inactive, did not bind to endothelium. In addition to surface binding, TNF induced the elaboration of IL-1 activity by endothelial cells in a time-dependent manner. Generation of IL-1 activity required protein synthesis and was half-maximal at a TNF concentration of 50 +/- 20 pM. IL-1 activity from TNF-treated endothelium could be adsorbed by an immobilized antibody to IL-1. Heat-treated TNF was ineffective in eliciting endothelial cell IL-1. These data indicate that TNF can bind specifically to endothelium and initiate a cascade of inflammatory and coagulant events on the vessel surface potentially central to the host response to neoplasia and sepsis.

Abstract: The known T-cell receptors (TCRs) involved in the recognition of antigen and major histocompatibility complex (MHC) molecules are glycoproteins comprised of polymorphic disulphide-linked alpha- and beta-chains. The genes encoding these chains are homologous to immunoglobulin genes and consist of V (variable), J (joining) and C (constant) regions that rearrange during development. TCRs are expressed relatively late in thymocyte development and only in association with an invariant molecular complex of proteins termed T3. Immature thymocytes do not express the TCR-T3 complex but do express messenger RNA encoding a third rearranging T-cell receptor-like gene, termed T gamma. Here we report a clone of normal immature T4-T8- human thymocytes, designated CII, which does not express mature mRNA for T alpha or T beta genes, but does express high levels of T gamma mRNA. This clone also expresses high levels of surface T3, and antibodies to T3 induce immunologically relevant functions in CII cells. Immunoprecipitation of CII surface-labelled proteins with anti-T3 co-precipitates a T3 molecular complex together with two additional and novel peptides of relative molecular mass (Mr), 44,000 (44K) and 62,000 (62K).

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Abstract: Interleukin 1 (IL-1) is a potent mediator of inflammatory and immunologic phenomena. In addition, IL-1 may be intimately involved in the regulation of hemostasis, since interaction of IL-1 with endothelial cells has been reported to induce tissue factor activity. We demonstrate that perturbation of the endothelial cell induces augmented IL-1 release. Human umbilical vein endothelial cells perturbed by treatment with lipopolysaccharide produced enhanced amounts of IL-1 activity. IL-1 activity from lipopolysaccharide-treated endothelial cell supernatants could be absorbed by an antibody to IL-1 coupled to Sepharose. Elaboration of IL-1 activity was dependent on the dose of lipopolysaccharide and occurred in a time-dependent manner. Addition of cycloheximide blocked generation of IL-1 activity. A physiological vessel wall perturbant, the coagulation enzyme thrombin, induced comparable amounts of IL-1 activity in endothelial cell cultures. This effect was specific for the enzyme, since active site-blocked thrombin and prothrombin had no effect on IL-1. In addition, IL-1-containing supernatants from thrombin-stimulated endothelial cells induced tissue factor procoagulant activity in fresh endothelial cell cultures. Thus, in contrast to the multiple, known inhibitory mechanisms that block thrombin procoagulant activity, these data suggest a circle of interaction in which thrombin induces endothelial cell elaboration of IL-1, a mediator of endothelial cell procoagulant activity. Endothelial cell production of IL-1 in response to perturbation allows these cells to play an integral role in the regulation of the inflammatory and coagulation systems.

Abstract: We investigated the functional role of the T4 molecule in the activation of T cells by OKT3. T4+ cells were induced to proliferate by OKT3 and erythrocyte rosette-negative accessory cells in the presence or absence of OKT4C, OKT4, and OKT1. OKT4C (IgG1), and not OKT4 (IgG2) or OKT1 (IgG1) inhibited proliferation when OKT4C was added during the first 24 h of cell culture. The inhibition of OKT3 activation by OKT4C did not require Ia+ accessory cells, since T4+ cells could be activated by OKT3 in the presence of Ia- U937 cells, and this activation was markedly inhibited by OKT4C. Furthermore, T4+ cells could be induced to proliferate by OKT3 covalently linked to Sepharose beads, in the absence of any accessory cells. Under these conditions, OKT4C, but not OKT4 or OKT1 significantly inhibited proliferation. These data demonstrate that at least one mechanism by which anti-T4 antibodies inhibit T cell activation is independent of any putative role of T4 molecules in the recognition of Ia on target cells. The data are compatible with the idea that perturbation of the T4 molecules can transmit a negative signal to T4+ cells.

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Abstract: Adenosine deaminase (adenosine aminophydrolase, ADA) activity was found to be low in lymphocytes of chronic lymphatic leukaemia (CLL) patients compared to normal lymphocytes. This was determined on 42 controls and 49 CLL patients. The mean activity in normal lymphocytes was found to be 4.35 +/- 3.34 mumol/h/10(8) cells while in CLL cells it was 2.45 +/- 2.54 mumol/h/10(8) cells.