Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation.

Tissue transglutaminase (tTG) is a Ca2+-dependent cross-linking enzyme that participates in the apoptotic machinery by irreversibly assembling a protein scaffold that prevents the leakage of intracellular components. In the present study a single-chain antibody fragment (scFv) detecting tTG is described. We demonstrate that TG/F8 scFv, selected from a phase display library of human V-gene segments by binding to guinea-pig liver tTG, can react with human tTG both in Western blot and in immunohistochemistry. The specific detection of tTG by TG/F8 in human thymocytes is verified by mass spectrometric analysis of the purified protein. Furthermore, we demonstrate that in lymphoid cells tTG is cleaved by caspase 3 during the late phase of apoptotic death, concomitant to DNA fragmentation, and that such cleavage causes loss of cross-linking function. We propose tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.

A simplified procedure for the preparation of antibodies to serum fibronectin.

In the present paper we describe in detail a simple procedure for the preparation of monospecific antisera to human and mouse serum fibronectin. A similar procedure could also be used to prepare antibodies to fibronectin from other species. The procedure, based on the recently reported affinity of fibronectin for gelatin, essentially consists of two steps (1) Immunization of rabbits with fibronectin purified from serum by affinity chromatography using gelatin coupled to CNBr-activated Sepharose 4B. (2) Absorption of the antiserum obtained by an immunoabsorbent prepared using fibronectin-free serum proteins that remained after absorbing serum with gelatin-Sepharose. The antisera obtained were monospecific, as determined by immunoelectrophoresis and did not show any difference with respect to antisera prepared by different procedures.

Different expansions of T lymphocyte subpopulations in the lung and corticosteroid-induced changes in patients with active pulmonary sarcoidosis.

Pulmonary sarcoidosis is a disease characterized by increased numbers of T lymphocytes within the alveolar structures and the consequent spontaneous release of a variety of mediators relevant to the pathogenesis of this disorder. This phenomenon is associated with a different expansion of the T cell subpopulations present in the lung. Using monoclonal antibodies specific for T cell subsets with helper functions, we have evaluated the different T lymphocyte subpopulations present in patients with active and inactive pulmonary sarcoidosis. The small fraction of helper T cells recognized by the 5/9 monoclonal antibody appear to be preferentially expanded in the lung of patients with active disease. The functions of the 5/9+ lung T cells in pulmonary sarcoidosis were evaluated by considering the spontaneous release of monocyte chemotactic factor, the help in immunoglobulin production, and the spontaneous production of interleukin-2 by the 5/9+ lung T cells from patients with active disease. These T cell functions appeared to be restricted to the 5/9+ T cell subset. The sensitivity of the 5/9+ lung T cells to corticosteroid treatment in pulmonary sarcoidosis was studied by performing bronchoalveolar lavage in patients with active disease before and after oral prednisone therapy. Evaluation of lymphocyte subsets after 3 months of therapy showed a marked reduction of 5/9+ T cell percentages even though the overall proportion of lavage cells that were T lymphocytes was still elevated. Thus the 5/9 monoclonal antibody may be considered a good marker in gauging the activity of the alveolitis in pulmonary sarcoidosis because it recognizes the T cell subsets responsible for many activities relevant to the pathogenesis of this disorder. In addition, analysis of the proportions of 5/9+ lung T cells may result in a useful means to evaluate the early response to therapy.

Immunoglobulin secretion by mouse myeloma cells; involvement of an energy-requiring step.

The process of Immunoglobulin secretion has been extensively studied but the cellular mechanisms underlying assembly and transport of these molecules are still poorly understood. Evicence is presented in this paper for the presence, in the secretory pathway of mouse myeloma cells, of an energy requiring step, as indicated by the strong inhibition of Immunoglobulin secretion by a variety of respiratory chain inhibitors and of oxidative phosphorylation uncouplers.

Presence of serum IgD and IgD-containing plasma cells in the mouse.

A new technique which makes use of anti-delta-specific allo-or heteroantisera coupled to Sepharose 4 B has been employed to investigate the presence of IgD molecules in the sera of three mouse strains. The sera were reacted with the insoluble immunosorbents, and the eluted material was labeled with 125I prior to antigenic or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This material reacted with anti-delta or anti-L chain reagents, but not with other anti-class-specific antisera and, after reduction, was resolved in three discrete bands in SDS-PAGE. The fastest of these bands comigrated with L chain, whereas the other two moved identically to mouse lymphocyte surface delta chains. Immunofluorescence studies on fixed smears of spleen and Peyer's patches' cells revealed the presence of a small but consistent number IgD-containing plasma cells.

CD16 and CR3 receptors distinguish between the two mechanisms of tumour cytotoxicity in neutrophils.

Previous studies have suggested that phorbol ester-activated neutrophils kill both antibody non-coated and antibody-coated K562 target cells. In this report the contribution of the receptors Fc gamma III (CD16) and CR3 (CD11b/CD18) in the lytic process was investigated. In neutrophils CD16 and CR3 are up-regulated by the phorbol ester up to 4 and 10 times, respectively. As expected, lysis of non-immunized K562 targets is not affected by the treatment of neutrophils with anti CD16, AB8.28, whereas lysis of immunized targets is decreased by 50%. In addition, the interaction of CD16 and AB8.28 induces calcium mobilization and increases granule secretion. Surprisingly, the simultaneous binding of AB8.28 and anti-CR3 OKM1 to neutrophils completely abolishes the lysis of antibody-coated targets. Unlike CD16, CR3 does not possess a functional role and binding of OKM1 to CR3 does not affect cytotoxicity of immunized K562 targets, but it blocks lysis of non-coated target almost completely, indicating a function as adhesion protein for CR3. These studies demonstrate a distinct role of CD16 and CR3 in mediating antibody-dependent and antibody-independent cellular cytotoxicity, respectively.

Helper T-lymphocytes in pulmonary sarcoidosis. Functional analysis of a lung T-cell subpopulation in patients with active disease.

Pulmonary sarcoidosis is a disease characterized by increased numbers of T-lymphocytes in the alveolar structures, which through the production of lymphokines modulate granuloma formation and polyclonally activate B cells to secrete immunoglobulins. The T-lymphocyte alveolitis is associated with a different expansion of various T-cell subpopulations identified by different monoclonal antibodies. Patients with active disease have increased numbers of helper T cells in the lungs, recognized by the OKT4 monoclonal antibody and decreased numbers of suppressor OKT8-positive lung T cells, whereas patients with inactive disease have increased numbers of OKT8-positive T cells and decreased numbers of OKT4-positive T cells in the lungs. Using the IgG fraction of a monoclonal antibody called 5/9, which reacts in normal subjects with approximately 30% of the OKT4-positive T-lymphocytes, it has been shown that the 5/9-positive T cells appear preferentially expanded in pulmonary sarcoidosis at sites of disease activity. To evaluate the functions of the T-cell subpopulation identified by the 5/9 monoclonal antibody in pulmonary sarcoidosis, we studied the unfractionated T-lymphocytes and the 5/9-positive and the 5/9-negative T-cell fractions in bronchoalveolar lavage of 12 patients with active lung disease. On T-cell suspensions, the spontaneous release of monocyte chemotactic factor and the polyclonal activation of autologous peripheral blood lymphocytes were determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of IgD. II. Molecular forms of IgD in human B cells.

Endogenous and surface labeling techniques were used on human lymphoid cells to characterize intracytoplasmic, membrane and secreted IgD, IgD synthesized by lymphocytes and inserted into the cell membrane displayed a single molecular form with the same mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as the previously described slow migrating serum IgDl. Plasma cells produced and secreted IgDl and another IgD corresponding to the faster-moving serum IgD2. Conversion of one molecular form into the other was never observed, thus indicating that neither molecule is a precursor or a degradation product of the other.

Characterization of IgD. I. Isolation of two molecular forms from human serum.

Human IgD present in the serum of normal individuals or of patients with Hodgkin's disease (having high IgD concentrations) was characterized and compared with five IgD myeloma proteins. IgD was isolated using a highly specific anti-delta insoluble immunoabsorbent from which the bound material was eluted with sodium dodecyl sulphate (SDS) or urea. The latter reagent could be removed by extensive dialysis, thus making possible the renaturation of the eluted molecules. The purity of the IgD thus isolated was confirmed by antigenic analysis. Both kappa and lambda light chain determinants were present on serum IgD, although lambda light chain was predominant with a ratio over the kappa chain of 2:1. SDS-polyacrylamide slab gel electrophoresis analysis revealed two different molecular forms of serum IgD, one (IgD) migrating identically to monoclonal IgD, the other (IgD2) having a faster mobility. The difference between the two molecules was entirely, due to the different sizes of their constituent delta chains. Peptide mapping of the two chains (delta1 and delta2 respectively) and of the delta chain of an IgD myeloma protein was carried out using 125I-labelled material. The three molecules displayed a high degree of homology, the delta2 chain differing by the presence of three characteristic extra peptides. The significance of these extra peptides is discussed in the light of the peptide mapping technique employed.

Abnormalities of circulating T cell subsets in atopy: influence of specific immunotherapy.

Blood samples of patients with severe respiratory allergic diseases contain increased numbers of T cells bearing surface HLA-DR antigens, indicating the presence of activated T cells. In the same group of patients, MLR3 and MLR4, two monoclonal antibodies (Mab) directed to subsets of activated peripheral T cells, recognize T cell percentages within the normal range. Thus, it seems possible that specialized subsets of activated T cells (HLA-DR+/MLR3-MLR-) are represented in the peripheral blood of atopic patients. Such cells are lacking in patients after specific immunotherapy. Similar results--an increased percentage of 5/9+ T cells in untreated patients and normal counts of 5/9+ T cells in treated ones--were obtained in the two groups of patients by using another Mab, 5/9, which serves as a reliable marker of helper T cells in resting peripheral T lymphocytes. These data further support the concept of a T cell imbalance in allergic patients and suggest a possible role of specific immunotherapy in correcting the modification of peripheral T cell abnormalities.

Late apoptotic effects of taxanes on K562 erythroleukemia cells: apoptosis is delayed upstream of caspase-3 activation.

The efficacy of taxanes on human leukemia cells is the object of intensive in vitro investigation concerning the influence of cell-type-specific characteristics on cytotoxic response to drugs. The present study dissects the response to taxanes of HL60 acute myelomonocytic leukemia and of K562 chronic myelogenous leukemia, in parallel over a 72-hr time-span. The kinetics of cytotoxicity following pulsed and continuous exposure to either taxol or taxotere showed a delayed response of K562 cells independently of dose and type of exposure. In K562 cells, apoptosis became evident at 48 hr and prominent at 72 hr of treatment. These events were mirrored by delayed kinetics of caspase-3 activation. Comparable microtubule targeting was demonstrated in HL60 and in K562 cell lines, as bcl-2 and raf-1 were phosphorylated following treatment with taxanes. These observations indicate that early activation processes were responsible for apoptosis, but that the delay was determined by other factors. In addition, cell-free-system experiments excluded the presence of excess nuclear and/or cytoplasmic inhibitory factors and demonstrated that K562 cells possess a fully competent caspase system which can be readily activated. Processing of caspase-3 pro-enzyme was in fact increased by addition of cytochrome c. These results extend to taxol and taxotere the notion that drug-induced apoptosis is delayed upstream of caspase-3 activation in K562 cells, that such kinetics is independent of drug concentration and exposure time, and that it is linked to intrinsic cellular characteristics mapping between bcl-2 phosphorylation and cytochrome c release.

The interstitial lung disease associated with rheumatoid arthritis: evidence for imbalance of helper T-lymphocyte subpopulations at sites of disease activity.

Rheumatoid arthritis (RA) is a generalized disorder characterized by chronic inflammation of peripheral joints and by involvement of many organs, including the lung parenchyma. The inflammatory infiltrates of the rheumatoid synovial membranes are associated with increased numbers of T-lymphocytes, with increased proportions of helper (OKT4 positive) T-cells and decreased percentages of suppressor/cytotoxic (OKT8 positive) T-cells, and since patients with interstitial lung disease associated with RA often have increased numbers of lymphocytes in the alveolar structures, it seemed possible that rheumatoid lung disease could also be associated with an imbalance of T-lymphocyte subpopulations. To test this hypothesis, patients with chronic interstitial lung disease and RA were evaluated by lung biopsy, gallium-67 scanning and bronchoalveolar lavage to assess the activity of the lung disorder and the T-lymphocyte subpopulations were identified with the OKT4, OKT8 and Tec T-5.9 monoclonal antibodies. The Tec T-5.9 is a recently described monoclonal antibody which recognizes a small T-cell fraction of the OKT4 positive T-lymphocytes, responsible for many helper T-cell functions, including the response to allogenic antigens and help in immunoglobulin production by B-cells. Histologic evaluation of the biopsies demonstrated active lung inflammation in all patients and gallium-67 scans showed an increased lung uptake in five of the six patients studied.(ABSTRACT TRUNCATED AT 250 WORDS)

T-derived colony-inhibiting activity: partial characterization and mechanism of action.

A soluble inhibitor of granulocyte macrophage colony growth, to which we shall refer to as T-derived colony-inhibiting activity (Td/CIA), was obtained from the supernatant of T cells from 5 healthy donors and 5 patients with severe aplastic anemia (SAA) in remission, following immunosuppressive therapy. The supernatants were purified by an ACA 44 column and the suppressor activity found in fractions of 70,000-80,000 daltons. Experiments were then performed to test for endogenous productions of Td/CIA in normal marrow cells (NBM), reversibility of suppression, and competitive inhibition with human placenta-conditioned medium (HPCM). The results of this study can be summarized as follows: the endogenous production of Td/CIA can be elicited by addition of mitogens to NBM, and is prevented if the marrow is T-depleted or treated with cyclosporin A; suppression is completely reversible if Td/CIA is removed from NBM by washing at 1, 48, 72 and 96 h; CFU-c which have been exposed to Td/CIA once, and freed from Td/CIA by washing, are still sensitive to a second exposure of Td/CIA; there is no clear competitive inhibition between Td/CIA and HPCM. These experiments represent an in vitro model of a lymphokine-mediated regulation of CFU-c growth not associated with death of progenitor cells.

Symptomatic treatment of recurrent malignant pleural effusions with intrapleurally administered Corynebacterium parvum. Clinical response is not associated with evidence of enhancement of local cellular-mediated immunity.

Intrapleural injection of Corynebacterium parvum (CBP) has been recently proposed as a useful symptomatic treatment of recurrent malignant effusions. Although the result is often a fibrotic thickening of the pleura, CBP is thought to stimulate the effector cells present in the effusion and, possibly, to activate the antitumor cytotoxic activity of the pleural fluid mononuclear cells. To test this hypothesis, we studied 7 patients with recurrent malignant pleural effusions caused by lung cancer and evaluated the cellular composition, the proportions of lymphocyte subpopulations, and the cytotoxic activity of mononuclear cells in the pleural fluid before and 7 days after injection of CBP in the pleural space. The CBP treatment induced a marked decrease in the rate of accumulation of pleural fluid (p less than 0.01) and in the concentration of immune effector cells in the pleural exudate (p less than 0.001). These changes were associated with a decrease in the percentages of pleural fluid monocytes and lymphocytes present (p less than 0.01, each comparison) and to a marked increase in the percentages of pleural fluid neutrophils (p less than 0.001). No significant changes in the proportions of T- and B-lymphocytes or in the proportions of helper/inducer and suppressor/cytotoxic T-cells or of natural killer cells were observed in the pleural exudate after CBP treatment (p greater than 0.2, each comparison). In addition, the cytotoxic activity of pleural fluid mononuclear cells was similar before and after CBP treatment (p greater than 0.2), and the levels of interferon, as a marker of immunoactivation of mononuclear cells, were not changed after treatment (p greater than 0.2).(ABSTRACT TRUNCATED AT 250 WORDS)

CD38 expression distinguishes two groups of B-cell chronic lymphocytic leukemias with different responses to anti-IgM antibodies and propensity to apoptosis.

The expression of CD38 by B cells chronic lymphocytic leukemia (B-CLL) was studied in 20 untreated patients. The cells expressed abundant CD38 (relative fluorescence intensity range, 6 to 15) in 6 cases (group I patients), whereas CD38 expression was low to absent (relative fluorescence intensity range, 0 to 3) in the remaining cases (group II patients). Exposure of the cells from group I patients to goat antihuman mu chain antibodies (Ga mu-ab) resulted in the elevation of intracellular free Ca2+ concentration([Ca2+]i) followed by apoptosis. In contrast, exposure of group II cells to Ga mu-ab was not followed by increased levels of [Ca2+]i, programmed cell death or cell proliferation. No differences in the expression of surface IgM were noted in the two groups of B-CLL cells. Normal peripheral blood B cells, which expressed low to absent CD38, were capable of mobilizing [Ca2+]i and of proliferating after exposure to Ga mu-ab. The collected data suggest that, although group I B-CLL cells were able to transduce the signals delivered by IgM crosslinking, this pathway was severely impaired in group II B-CLL cells. However, unlike that observed in normal circulating B cells, stimulation of group I cells with Ga mu-ab resulted in apoptosis rather than proliferation. CD38 did not appear to be directly involved in [Ca2+]i mobilization induced by Ga mu-ab in group I B-CLL cells because their exposure to anti-CD38 monoclonal antibodies failed to cause [Ca2+]i mobilization or to block the [Ca2+]i response induced by Ga mu-ab. These data indicate that CD38 expression identified a particular subset of B-CLL cells with defined functional properties, including the propensity to undergo apoptosis.

A possible cytotoxicity inducer role of 5/9 positive lymphocytes infiltrating the liver in CAH patients.

The presence, in chronic active hepatitis (CAH) patients, of an inflammatory infiltrate basically composed of T lymphocytes suggested the hypothesis that these lymphocytes could play a role in the pathogenesis of the disease. The aim of this study has been to characterize, in a group of carefully selected CAH patients, the liver-infiltrating T lymphocyte, utilizing commercial monoclonal antibody (anti-Leu 1, anti-Leu 2a, anti-Leu 3a) and 5/9 monoclonal antibody that recognizes a further lymphocyte subset within T4 cells. Our data show that both T4 positive subpopulation and T8 positive subpopulation are represented in the infiltrate in the same ratio; furthermore the distribution of 5/9 positive lymphocytes is prevalent where the infiltrate is mainly composed of T8 positive lymphocytes. Moreover, there is a positive correlation between 5/9 positive cells in the liver and GPT and the patients with high percentages of infiltrating 5/9 positive lymphocytes show a low T4/T8 ratio with respect to patients with low percentages of 5/9 positive cells. These data support the hypothesis that 5/9 positive lymphocytes may present an inducer role on cytotoxic cells.

Pulmonary sarcoidosis: excess of helper T lymphocytes and T cell subset imbalance at sites of disease activity.

Different lymphocyte subpopulations have been evaluated in bronchoalveolar fluid and blood obtained from six patients with active and six with inactive pulmonary sarcoidosis and from six normal subjects by means of two recently described monoclonal antibodies, 5/9 and MLR4. The percentages of OKT4 positive (helper) and OKT8 positive (suppressor) T cells were also determined. Patients with active sarcoidosis had significantly higher proportions of 5/9 positive T cells in the bronchoalveolar fluid than patients with inactive disease (p less than 0.01) or normal subjects (p less than 0.001). In contrast, the proportions of 5/9 positive blood T cells were similar in the three groups studied. Patients with active sarcoidosis had also a greater proportion proportion of MLR4 positive T lymphocytes in bronchoalveolar fluid than patients with inactive disease or normal subjects (p less than 0.01 for each comparison), but similar proportions of MLR4 positive blood T cells were found in each group. The ratio of 5/9 positive to MLR4 positive T cells was higher in the bronchoalveolar fluid (but not in the blood) in patients with either active or inactive sarcoidosis than in normal subjects. These observations suggest that the MLR4 negative fraction rather than the MLR4 positive fraction of the 5/9 positive T cells is preferentially expanded in the lungs of patients with pulmonary sarcoidosis and may indicate a secondary role for the MLR4 positive T cells in producing lung injury in this disorder. Comparisons of the OKT4 positive and 5/9 positive T cells showed that in patients with active disease most of the lung T lymphocytes expressed both the OKT4 and the 5/9 surface antigens, so the 5/9 monoclonal antibody may be considered a good marker of activity in this disorder. Pulmonary sarcoidosis may be characterised by the preferential expansion of helper T cell subsets at sites of disease activity.

Systemic administration of autologous, alloactivated helper-enriched lymphocytes to patients with metastatic melanoma of the lung. A phase I study.

A phase I study was carried out to test the feasibility and toxicity of infusing large numbers of autologous, alloactivated helper lymphocytes into patients with metastatic melanoma. Patient peripheral blood lymphocytes (Pt-PBL) obtained by lymphopheresis and expressing the helper phenotype BT5/9 were separated and stimulated for 48 or 72 h with a pool of PBL from four to six healthy donors. Patients were then infused with such activated lymphocytes over a 2-3 h period. A total of 4 phereses and infusions (2/week for 2 weeks) were carried out for each cycle in each patient. Of the five patients treated, two received a second round of infusions. Infusion of autologous PBL stimulated in vitro for 48 h caused chills, fever, headache, and increased blood pressure. All symptoms disappeared in 2-3 h and were easily controlled by appropriate therapy. When lymphocytes were given after 72 h of allostimulation, no or very mild toxicity was observed. Serum chemistry, coagulation, autoimmunity, and urine analysis showed no gross abnormalities during therapy or follow-up of the patients. Immunological parameters (OKT4/OKT8 ratio, NK activity and cytotoxic T cell activity to autologous melanoma) were evaluated before starting the therapy, during its course and during the 3 to 6 months follow-up. The OKT4/OKT8 ratio increased significantly but transiently soon after the first course of infusions in one of the two patients tested. NK activity increased after 75-100 days in the three patients tested and in one of them it was high even after 180 days. No correlation between NK activity and prognosis was apparent. Cytotoxicity to autologous tumor was assessed in two patients, only of one of whom exhibited an increased activity from 75 to 180 days, which was associated with a prognosis better than that of the negative patient. Five patients were treated: two had progressive disease, two had stable disease for 5 and 6 months, respectively. In the first of these patients, a new cycle of lymphocyte infusions was carried out which caused a measurable reduction of lung tumor nodules whose growth, however, resumed 4 months later. This patient died 14 months after the onset of therapy. The fifth patient had a partial regression of pulmonary and intracranial metastases after therapy, but eventually died 3 months later. These results indicate that infusion of a high numbers of autologous, allostimulated helper PBL is a feasible and safe procedure, which could therefore be used in future studies of adoptive immunotherapy of cancer.