Martin E. Sanders

Human naive and memory T cells: reinterpretation of helper-inducer and suppressor-inducer subsets

Abstract The subsets of human peripheral blood T cells identified by CD45R antibodies (such as 2H4) and by CDw29 antibodies (such as 4B4) are assuming increasing importance in studies of both basic immunology and clinical medicine. Here, Martin Sanders and colleagues propose that these subsets represent naive and memory (previously activated) T cells, respectively, and discuss the current understanding of these subsets in the light of this reinterpretation.

The CD2-LFA-3 and LFA-1-ICAM pathways: relevance to T-cell recognition

No process is more central to T-lymphocyte function than cell-cell adhesion, yet it is only recently that interest in lymphocyte adhesion has burgeoned. Neglect of adhesion is particularly surprising since immunologists are surrounded by a veritable sea of adhesive interactions of lymphocytic cells: transformed lymphocytes grow in aggregates, stimulated lymphocytes aggregate and T cells conjugate with their targets. In retrospect, it is obvious that all lymphocyte adhesion (both antigen-specific and seemingly non-specific adhesive interactions) has to be based on specific receptor-ligand interactions. In this review Malegapuru Makgoba, Martin Sanders and Stephen Shaw focus primarily on the two molecular pathways of lymphocyte adhesion that have been shown to play a critical role in facilitation of antigen-specific recognition, namely CD2 and its ligand, lymphocyte function associated antigen-3 (LFA-3), and LFA-1 and its ligand, intercellular adhesion molecule-1 (ICAM-1). A variety of excellent recent reviews have dealt with this and related aspects of T-cell adhesion. Of particular interest is the review that follows in this issue: it deals with the CD44 molecule which has also been implicated in both adhesion and activation of T cells.

Recombinant human relaxin in the treatment of scleroderma. A randomized, double-blind, placebo-controlled trial.

Relaxin, a heterodimer protein with a molecular weight of 6000, is secreted by the corpus luteum and placenta during pregnancy (1, 2). It is structurally related to insulin and insulin-like growth factor I, and its principal physiologic role seems to be fostering the growth and remodeling of the uterus. Relaxin also loosens the pelvic ligaments and ripens the uterine cervix in preparation for parturition (3). The availability of recombinant human relaxin has permitted focused investigations of its effects on connective tissue. Recombinant human relaxin alone reduces synthesis of dermal fibroblast collagen and enhances the effects of interferon- (4). Relaxin attenuates the actions of profibrotic cytokines, including transforming growth factor- and interleukin-1 (5), and increases secretion of dermal fibroblast collagenase while reducing levels of tissue inhibitor of metalloproteinase (5). Of interest, the effect of relaxin on reduced secretion of collagen and tissue inhibitor of metalloproteinase is dose-dependent, whereas its effect on collagenase is optimal in a narrow range of concentrations (5). Finally, recombinant human relaxin prevents the development of bleomycin-induced pulmonary fibrosis in rodents (6), as well as dermal fibrosis in rodent irritant models (7). In vitro and animal studies suggest that recombinant human relaxin might be therapeutically useful for diseases characterized by fibrosis. Systemic sclerosis (scleroderma) is the prototypical fibrosing disease in humans. Although the pathogenesis of systemic sclerosis is not completely understood, tissue fibrosis dominates the clinical features of the disease and largely determines its morbidity and mortality (8). Scleroderma-related fibrosis includes both the fibrotic intimal hyperplasia of small arteries and arterioles (the Raynaud phenomenon, renal crisis, and pulmonary hypertension), as well as extravascular tissue fibrosis (skin, interstitial lung disease, and tendon involvement) (8). The long-term clinical benefit of preventing or reversing fibrosis in systemic sclerosis has not been tested, and no therapies to date have demonstrated such effects (9). Before porcine relaxin was withdrawn from the market in the early 1960s in response to reformed policies of the U.S. Food and Drug Administration (FDA), open case studies showed that it improved scleroderma-related skin change and healed cutaneous ulcers (10). Phase I studies of recombinant human relaxin in patients with diffuse scleroderma have demonstrated that steady-state serum concentrations of relaxin up to 60 times higher than those seen in normal pregnancy could be safely achieved with continuous subcutaneous infusion (11, 12). The most common drug-related adverse events associated with relaxin treatment have been menometrorrhagia and moderate reversible reductions in hemoglobin. In phase I studies, extent and severity of skin thickening as well as patient global assessment and functional status improved over periods of up to 1 year. However, interpretation of these findings has been hampered by short duration of treatment (11) or inadequacies of open-label design (12). We report the results of a randomized, double-blind, controlled clinical trial comparing placebo with recombinant human relaxin, 25 g/kg of body weight per day and 100 g/kg per day, given for 24 weeks in patients with stable, diffuse, moderate to severe scleroderma. Methods Patients Before screening, all patients gave informed consent according to the principles of the Declaration of Helsinki and in compliance with FDA requirements. Patients were recruited through 13-member institutions of the Scleroderma Clinical Trials Consortium. Men and women 18 to 70 years of age were included if they had a history of systemic sclerosis with diffuse scleroderma (defined as skin involvement proximal to the elbows or knees, excluding the face and neck) and less than 5 years had elapsed since onset of the first non-Raynaud sign or symptom. A baseline modified Rodnan skin score of at least 20, or of at least 16 in the case of truncal involvement, was required for inclusion in the treatment phase of the study. Patients were excluded from this phase if their skin score varied by more than 5 points from screening to the first treatment day. We excluded patients who had systemic sclerosis with limited scleroderma (skin involvement restricted to face and neck and sites distal to elbows and knees); eosinophilic fasciitis; eosinophilia myalgia syndrome; or scleroderma in conjunction with any other definable connective tissue disease, such as rheumatoid arthritis, systemic lupus erythematosus, polymyositis, or dermatomyositis. We also excluded patients with a substantial history of environmental exposure to tainted rapeseed oil, vinyl chloride, trichloroethylene, or silica. In addition, patients with renal crisis in the previous 6 months; chronic renal failure; or severe cardiovascular, gastrointestinal, or pulmonary disease were excluded. Patients were required to discontinue putative disease-modifying treatments for scleroderma (including d-penicillamine, cyclophosphamide, cyclosporine, azathioprine, methotrexate, potassium aminobenzoate, photopheresis, colchicine, or any other experimental treatment) at least 4 weeks before beginning treatment with the study drug. Patients were excluded if they were receiving more than 10 mg of prednisone per day or an equivalent dose of another glucocorticoid. Intervention We administered recombinant human relaxin, 25 g/kg per day or 100 g/kg per day, or placebo for 24 weeks by continuous subcutaneous infusion, using microinfusion pumps (Panomat T-Series 5 mL, Disetronic Medical Systems, Inc., Minneapolis, Minnesota). Recombinant human relaxin was produced by Connetics Corp. (Palo Alto, California) in Escherichia coli (13). The placebo was a sterile acetate buffer solution that was identical in composition to the buffer used for relaxin. Patients were randomly assigned to receive placebo or recombinant human relaxin (25 g/kg per day or 100 g/kg per day). Randomization was performed at a centralized data management organization (Pacific Research Associates, Los Altos, California). Biased coin randomization (14, 15) was used to stratify patients on the basis of disease duration ( 2.5 years or>2.5 to 5 years) and use of d-penicillamine in the previous 6 months (16). The same randomization procedure was used to replace patients who withdrew before completing 4 weeks of treatment. Patient prescriptions for the study medication were forwarded to a centralized pharmacy (Coram Healthcare of Northern California, Hayward, California) for preparation of blinded supplies of the study drug. Each patients dose was based on screening body weight. The dose was adjusted only if body weight changed by 10% or more during the study. Treatment was administered over 24 hours for 24 weeks. The infusion site and needle were changed at least every 72 hours. The dosage of 25 g/kg per day was selected on the basis of pharmacokinetic results from earlier studies. We anticipated that it would be safe and well tolerated and would produce steady-state serum concentrations of relaxin that were approximately three- to fivefold greater than those found in human pregnancy (11). On the basis of preclinical and earlier clinical studies, we hypothesized that this serum concentration would have antifibrotic effects. To measure the potential for a doseresponse effect, we selected the dosage of 100 g/kg per day on the basis of safety and tolerability data from earlier clinical studies (11, 12). Continuous subcutaneous infusion was chosen as the mode of administration to eliminate the need for six daily subcutaneous injections, to conserve drug supply, and to mimic the constancy of relaxin concentrations that are usually seen in pregnancy (11). Study Design The objectives of the study were to assess the efficacy, safety, and doseresponse effect of recombinant human relaxin in patients with diffuse scleroderma. The study was conducted as a randomized, double-blind, placebo-controlled, parallel-treatment clinical trial. Assessments The primary measure of efficacy was the modified Rodnan skin score, a clinical evaluation by palpation of skin thickness in 17 body areas (face, chest, abdomen, right and left fingers, hands, forearms, upper arms, thighs, legs, and feet). Each area receives a score of 0 to 3 for degree of thickness (0=normal, 1=mild thickening, 2=moderate thickening, and 3=severe thickening). The total score ranges from 0 to 51. The modified Rodnan skin score has been the standard measure of outcome in recent clinical trials involving scleroderma (16-18). Many recent studies have confirmed that total skin scoring is both accurate (with an interobserver variability of 4.6 units) and reproducible (with an intraobserver variability of 3.1 units) (19, 20). Skin scoring is in many ways an ideal outcome measure for scleroderma because it is accessible, cost-effective, sensitive to change, and, as a measure of fibrosis, directly relevant to the biological process of disease (21). Before the study began, investigators were trained according to the standards of one experienced observer. All skin scoring for each individual patient was performed by a single investigator. Secondary measures of efficacy were the following: maximal oral aperture; maximal hand extension (18); tenderness and swelling of metacarpophalangeal joints (as a unit), wrists, and knees; enumeration of cutaneous ulcers; functional status according to the Health Assessment Questionnaire (HAQ) (22); global disease assessments by patients and investigators; and pulmonary function tests, including lung diffusion capacity and forced vital capacity. Serum relaxin levels were determined by using enzyme immunoassay (6). The presence of antirelaxin antibody was measured in an enzyme immunoassay that used purified recombinant relaxin and affinity-purified antihuman immunoglobulin as the

Recombinant Human Relaxin in the Treatment of Systemic Sclerosis With Diffuse Cutaneous Involvement : A Randomized, Double-Blind, Placebo-Controlled Trial

OBJECTIVE A phase II randomized controlled trial of recombinant human relaxin suggested that a dosage of 25 microg/kg/day was safe and clinically effective in improving skin disease and reducing functional disability in scleroderma (systemic sclerosis; SSc). We undertook a large randomized, double-blind, placebo-controlled clinical trial to compare placebo with 10 microg/kg/day and 25 microg/kg/day recombinant human relaxin, given for 24 weeks in patients with stable, diffuse, moderate-to-severe SSc. METHODS Men and women ages 18-70 years with diffuse cutaneous SSc (dcSSc) were administered recombinant human relaxin (10 microg/kg/day or 25 microg/kg/day) or placebo for 24 weeks as a continuous subcutaneous infusion. There was a followup safety visit at week 28. RESULTS The primary outcome measure, the modified Rodnan skin thickness score, was similar among the 3 groups at baseline and at weeks 4, 12, and 24. Secondary outcomes such as functional disability were similar in all 3 groups, while the forced vital capacity decreased significantly in the relaxin groups. The discontinuation of both doses of relaxin at week 24 led to statistically significant declines in creatinine clearance and serious renal adverse events (defined as doubling of serum creatinine, renal crisis, or grade 3 or 4 essential hypertension) in 7 patients who had received relaxin therapy but in none who had received placebo. CONCLUSION Recombinant relaxin was not significantly better than placebo in improving the total skin score or pulmonary function or in reducing functional disability in patients with dcSSc. In addition, relaxin was associated with serious renal adverse events, the majority of which occurred after stopping the infusion. If relaxin is used therapeutically for any conditions other than scleroderma, close monitoring of blood pressure and renal function must be performed.

Plasma thromboplastin component deficiency: I. Studies on its inheritance and therapy

Abstract 1.1. A case is presented of plasma thromboplastin component deficiency in a fifteen year old male, clinically resembling true hemophilia (antihemophilic globulin deficiency). Routine coagulation studies differed in no way from that of classical hemophilia. 2.2. Mixtures of the patients plasma with that of true hemophilic plasma, and plasma from a patient with plasma thromboplastin antecendent deficiency upon recalcification showed mutual correction. No such correction was evident with plasma from two other cases of plasma thromboplastin component deficiency. 3.3. Hemophilic plasma, PTA deficient plasma, normal plasma, normal serum and normal plasma stored at 4 °c. for periods as long as nineteen days could correct the coagulation defect of the patient in vitro. Barium sulfate-adsorbed plasma, antihemophilic globulin and plasma from other patients with plasma thromboplastin component deficiency were lacking in corrective effect. 4.4. Therapeutic trials with frozen fresh normal plasma, normal plasma stored at 4 °c. for seven days and fourteen days, citrated normal serum stored at 4 °c. for seven days and even hemophilic plasma stored for seven days at 4 °c. indicated the effectiveness of these preparations. Although the serum prothrombin activity became quite high seventy-two hours after administration of such plasmas, clinical disease did not appear for at least one week after administration. Antihemophilic globulin had no therapeutic or laboratory effect, restricting its use to cases of true hemophilia only. 5.5. A family history of hemorrhagic disturbance on the maternal side was elicited, the disease appearing exclusively in males in its overt form. The carrier state in the female was essentially asymptomatic except in the patients mother where spontaneous ecchymoses and post-operative bleeding appeared. Serum prothrombin consumption in the mother was borderline and her ability to correct the defect in her son was poorer than other relatives although the correction did occur. 6.6. The need for routine identification of the specific deficiency in each patient now suspected of hemophilia is stressed, especially as a prerequisite to a rational approach to therapy. A terminology based upon such identification is proposed and should do much to dispell the confusion surrounding the use of the term hemophilia in many related and non-related coagulation disturbances.

Activation of Human Eosinophils by Platelet-Derived Growth Factor

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.

Quantitation of activation of the human terminal complement pathway by ELISA.

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

Pitfalls in the quantitative estimation of the secretion of granule proteins by eosinophils

The secretion of preformed granule proteins by eosinophils is an important correlate of eosinophil activation. However, a review of the literature reveals large disparities in the amounts of these substances which were reportedly secreted when eosinophils were activated. In the present study we report that our attempts to quantitate the secretion of eosinophil peroxidase and eosinophil-derived neurotoxin from activated eosinophils by measuring these substances in the incubation supernatants were uniformly unsuccessful. We found that, once they were secreted, both eosinophil peroxidase and eosinophil-derived neurotoxin were promptly lost to assay and presumably destroyed. Thus the measurement of the difference in the concentration of these substances in eosinophils prior to and after activation, revealed that as much as 65% of the eosinophil-derived neurotoxin and 62% of the peroxidase in the eosinophils were lost to assay during activation of the cells whereas the largest amount of these substances which could be measured in the incubation supernatants never exceeded 2%. Evidence is presented that the destruction of eosinophil-derived neurotoxin must occur prior to the release of this substance into the medium. Attempts to inhibit the destruction of eosinophil peroxidase and of eosinophil-derived neurotoxin by incorporating various inhibitors into the incubations were unsuccessful. These results emphasize the need to monitor the overall recoveries of secreted products from activated eosinophils and suggest that meaningful estimates of the secretion of these granule proteins from activated eosinophils can only be obtained by measuring the residual content of these substances in eosinophils after they have been activated and comparing these values to the contents of eosinophils prior to activation.

Preparation of large numbers of highly purified normodense human eosinophils from leukapheresis

Methods have been developed for the preparation of large numbers of virtually pure, normodense eosinophils from the peripheral blood of normal human volunteers by means of leukapheresis. The purification depends on the sequential removal of mononuclear cells using a one-step discontinuous density gradient, lysis of erythrocytes, enrichment of eosinophils by centrifugation through discontinuous Percoll gradients and, finally, passive selection of the eosinophils by removal of the remaining polymorphonuclear neutrophils with a monoclonal antibody to CD16. The purity of the isolated eosinophils was consistently in excess of 95%. Recovery into the normodense eosinophil fraction ranged between 10 and 87% (average 31.6 +/- 4.2) and recovery during the monoclonal antibody step averaged 80.3 +/- 8.6%. These methods have made it possible, for the first time, to isolate 2-20 x 10(7) virtually pure normodense eosinophils from the peripheral blood of a single donor for further biochemical or pharmacological experimentation.

Case reportPlasma thromboplastin component deficiency: I. Studies on its inheritance and therapy☆

Abstract 1.1. A case is presented of plasma thromboplastin component deficiency in a fifteen year old male, clinically resembling true hemophilia (antihemophilic globulin deficiency). Routine coagulation studies differed in no way from that of classical hemophilia. 2.2. Mixtures of the patients plasma with that of true hemophilic plasma, and plasma from a patient with plasma thromboplastin antecendent deficiency upon recalcification showed mutual correction. No such correction was evident with plasma from two other cases of plasma thromboplastin component deficiency. 3.3. Hemophilic plasma, PTA deficient plasma, normal plasma, normal serum and normal plasma stored at 4 °c. for periods as long as nineteen days could correct the coagulation defect of the patient in vitro. Barium sulfate-adsorbed plasma, antihemophilic globulin and plasma from other patients with plasma thromboplastin component deficiency were lacking in corrective effect. 4.4. Therapeutic trials with frozen fresh normal plasma, normal plasma stored at 4 °c. for seven days and fourteen days, citrated normal serum stored at 4 °c. for seven days and even hemophilic plasma stored for seven days at 4 °c. indicated the effectiveness of these preparations. Although the serum prothrombin activity became quite high seventy-two hours after administration of such plasmas, clinical disease did not appear for at least one week after administration. Antihemophilic globulin had no therapeutic or laboratory effect, restricting its use to cases of true hemophilia only. 5.5. A family history of hemorrhagic disturbance on the maternal side was elicited, the disease appearing exclusively in males in its overt form. The carrier state in the female was essentially asymptomatic except in the patients mother where spontaneous ecchymoses and post-operative bleeding appeared. Serum prothrombin consumption in the mother was borderline and her ability to correct the defect in her son was poorer than other relatives although the correction did occur. 6.6. The need for routine identification of the specific deficiency in each patient now suspected of hemophilia is stressed, especially as a prerequisite to a rational approach to therapy. A terminology based upon such identification is proposed and should do much to dispell the confusion surrounding the use of the term hemophilia in many related and non-related coagulation disturbances.