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Background and objectives Enumerating circulating tumor cells has been used as a method of monitoring progression of various cancers. Various methods for detecting circulating melanoma cells (CMCs) have been reported, but none has had sufficient sensitivity to determine if the presence of rare CMCs in the blood of Stage I-III melanoma patients predicts if those patients eventually develop metastatic disease. Study design We quantified CMCs in serial blood samples from 38 early stage melanoma patients to determine if CMC numbers predict development of metastatic melanoma. CMCs were enumerated using a photoacoustic flow cytometric detection system that uses a laser to induce high frequency acoustic signals in pigmented CMCs. Results We observed that detection of greater than 2 CMCs/ml of blood from patients with Stage I-III melanoma predicts metastatic disease. Of the 11 patients we studied who had two or fewer CMCs detected at all time points tested, none progressed to metastatic disease over a mean follow-up of 1288 days. In contrast, 18 of the 27 patients (67%) having more than 2 CMCs/ml at one or more time points progressed to metastatic disease over a mean follow-up of 850 days. Conclusions Photoacoustic flow cytometry can detect rare CMCs in the blood of Stage I-III melanoma patients and detectionof these cells is predictive of subsequent development of metastatic disease. Lasers Surg. Med.

Subsets of non-acute promyelocytic leukemia (APL) acute myelogenous leukemia (AML) exhibit aberrant retinoid signaling and demonstrate sensitivity to retinoids in vitro. We present the results of a phase 1 dose-escalation study that evaluated the safety, pharmacodynamics, and efficacy of IRX195183, a novel retinoic acid receptor α agonist, in patients with relapsed or refractory myelodysplastic syndrome (MDS) or AML. In this single center, single arm study, eleven patients with relapsed or refractory MDS/AML were enrolled and treated. Oral IRX195183 was administered at two dose levels: 50 mg daily or 75 mg daily for a total of two 28-day cycles. Patients with stable disease or better were allowed to continue on the drug for four additional 28-day cycles. Common adverse events included hypertriglyceridemia, fatigue, dyspnea, and edema. Three patients at the first dose level developed asymptomatic Grade 3 hypertriglyceridemia. The maximally tolerated dose was not reached. Four of the eleven patients had (36%) stable disease or better. One had a morphological complete remission with incomplete hematologic recovery while on the study drug. Two patients had evidence of in vivo leukemic blast maturation, as reflected by increased CD38 expression. In a pharmacodynamics study, plasma samples from four patients treated at the lowest dose level demonstrated the capacity to differentiate leukemic cells from the NB4 cell line in vitro. These results suggest that IRX195183 is safe, achieves biologically meaningful plasma concentrations and may be efficacious in a subset of patients with MDS/AML. Clinical Trial Registration: clinicaltrials.gov, identifier NCT02749708.

Melanoma is the deadliest skin cancer and is responsible for over 7000 deaths in the US annually. The spread of cancer, or metastasis, is responsible for these deaths, as secondary tumors interrupt normal organ function. Circulating tumor cells, or those cells that spread throughout the body from the primary tumor, are thought to be responsible for metastasis. We developed an optical method, photoacoustic flow cytometry, in order to detect and enumerate circulating melanoma cells (CMCs) from blood samples of patients. We tested the blood of Stage IV melanoma patients to show the ability of the photoacoustic flow cytometer to detect these rare cells in blood. We then tested the system on archived blood samples from Stage III melanoma patients with known outcomes to determine if detection of CMCs can predict future metastasis. We detected between 0 and 66 CMCs in Stage IV patients. For the Stage III study, we found that of those samples with CMCs, two remained disease free and five developed metastasis. Of those without CMCs, six remained disease free and one developed metastasis. We believe that photoacoustic detection of CMCs provides valuable information for the prediction of metastasis and we postulate a system for more accurate prognosis.

CD2, also known as LFA-2, T11, and the E rosette receptor, is a T lymphocyte surface protein functionally important in adhesion to target cells and T cell triggering. LFA-3 is a widely distributed cell surface protein that functions in adhesion on target cells. We find that LFA-3 is expressed on human E, and that CD2 is a receptor for LFA-3 that mediates T cell adhesion to human E. Pretreatment of T lymphocytes with CD2 mAb or of E with LFA-3 mAb inhibits rosetting. Purified CD2 molecules bind to human E and inhibit rosetting. 125I-CD2 binding to E is inhibited by LFA-3 mAb; reciprocally, binding of LFA-3 mAb to human E is inhibited by pretreatment with purified CD2. Higher concentrations of CD2 aggregate human E; aggregation is inhibited by mAb to LFA-3.

Parkinson's disease (PD) is one of the most common movement disorders, and currently there is no effective treatment that can slow disease progression. Preserving and enhancing DA neuron survival is increasingly regarded as the most promising therapeutic strategy for treating PD. IRX4204 is a second generation retinoid X receptor (RXR) agonist that has no cross reactivity with retinoic acid receptors, farnesoid X receptor, liver X receptors or peroxisome proliferator-activated receptor PPARγ. We found that IRX4204 promotes the survival and maintenance of nigral dopaminergic (DA) neurons in a dose-dependent manner in primary mesencephalic cultures. Brain bioavailability studies demonstrate that IRX4204 can cross the blood brain barrier and reach the brain at nM concentration. Oral administration of IRX4204 can activate nuclear receptor Nurr1 downstream signaling in the substantia nigra (SN) andattenuate neurochemical and motor deficits in a rat model of PD. Our study suggests that IRX4204 represents a novel, potent and selective pharmacological means to activate cellular RXR-Nurr1 signaling and promote SN DA neuron survival in PD prevention and/or treatment.

Objective A phase II randomized controlled trial of recombinant human relaxin suggested that a dosage of 25 μg/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 μg/kg/day and 25 μg/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 μg/kg/day or 25 μg/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.

e14095 Background: IRX4204 is a Retinoid X Receptor (RXR) specific agonist (rexinoid). IRX4204 has high potency as a RXR agonist with low binding affinity to Retinoic Acid Receptors (RARs), PPAR, FXR or LXR. Our prior work with the less specific rexinoid bexarotene and erlotinib showed clinical activity in patients with lung cancer cases that harbored KRAS mutations. IRX4204 inhibits lung cancer cell proliferation and can chemoprevent carcinogen-induced lung cancers in mice. We sought to explore the underlying mechanisms engaged by the combination of IRX4204 and erlotinib. Materials and Methods: Human (H1703 and HOP62) and murine (ED1 and LKR13) lung cancer cell lines were treated with IRX4204, erlotinib, IRX4204 plus erlotinib, or vehicle. Reverse phase protein array (RPPA) and mRNA microarray analyses were performed to analyze comprehensively for differentially expressed growth regulatory proteins. Results: Combination of IRX4204 and erlotinib suppressed proliferation of both KRAS mutant (HOP62 and LKR13) and wild-type (H1703 and ED1) lung cancer cell lines. Additive effect was observed as compared to IRX4204 or erlotinib treatment alone. Combining IRX4204 with erlotinib markedly increased inhibition of specific therapeutic targets including Src, phosphorylated Akt and ribosomal S6 proteins. At the mRNA level, Ingenuity Pathway Analysis of species significantly increased or decreased by the combination treatment revealed multiple pathways related to oncogenic signaling. Specifically, we found in H1703 cell line regulation of Granzyme A and AMPK signaling and in HOP62 cell line inhibition of angiogenesis was implicated by altering TSP1 expression. Notably, in ED1 cell line PPARα/RXRα activation, PTEN signaling, PI3K/AKT signaling, TGF-β signaling, and AMPK signaling were each associated with effects of IRX4204 combined with erlotinib. Conclusions: Taken together, these data highlight specific mechanisms and candidate pharmacodynamic biomarkers of response to the combination of this rexinoid and EGFR-TKI in lung cancer. Based on these findings, a clinical trial (NCT02991651) with IRX4204 in combination with erlotinib is underway to treat patients with chemotherapy-refractory non-small cell lung cancer.

e21053 Background: Circulating tumor cells have been correlated with disease state and distant metastatic spread in cancer patients. We postulated that enumerating circulating melanoma cells (CMCs) may predict the onset of distant metastasis in Stage III patients. We detected CMCs using our photoacoustic flow cytometer, in which we irradiated enriched blood samples with nanosecond pulsed laser light. While there is no effect on non-optically active leukocytes, absorption of laser light by pigmented melanoma cells resulted in robust ultrasonic waves that indicated CMCs in the sample. Methods: We tested 32 archived samples from 9 Stage III melanoma patients using our photoacoustic flow cytometer. Each patient had between 2 and 6 serial blood samples. We used a pulsed Nd:YAG laser to irradiate mononuclear cells in suspension and under flow. The number of CMCs detected after testing was recorded, indicating the time sequence of circulating tumor cell activity. Results: The numbers of CTCs for each sample is shown in the table below. The ultimate disease state, whether the patient became metastatic or not, was blinded to the investigators who performed the photoacoustic tests. One sample for patient 3 indicated 63 CMCs, though this test was known to be contaminated and had an unknown number of false detections. Conclusions: We found that patients who had a series of more than 4 CMCs were more likely to become metastatic than those patients who tested for 4 CMCs for fewer, indicating that a sequence of CMC detections in serial blood draws provides a potentially strong predictor of metastasis in Stage III melanoma patients warranting further investigation at this and lower stages of melanoma. We are developing a more rigorous model based on time series analysis of CMCs for prediction of metastasis. [Table: see text]

The function of the immune system is to recognize foreign elements and identify them as normal or potentially harmful. The first step in this discrimination is surveillance by antigen-presenting cells (APCs), which are present throughout the body. Factors that affect the immunogenicity of a molecule include the presence of an adjuvant, the depot effect, the frequency of the antigen administration, and the quantity of the material administered.For the various types of botulinum toxin, the issue of the effect on efficacy is particularly relevant to antibody formation. An antibody to one type of botulinum toxin may potentially bind to an antibody of a different type. Safety issues with therapeutic proteins involve allergic responses, as well as formation of immune complexes and the subsequent immune complex disease. The concerns with botulinum toxins primarily relate to the potential for inhibiting efficacy.Protein therapeutics are important only if the antibodies have been generated against the administered therapeutic. This differs for the types of botulinum toxin. In the development of botulinum toxin type B, the factors that might increase the likelihood or magnitude of an immune response were considered and eliminated as much as possible. Only antibodies to the neurotoxin protein impact efficacy or safety.

9555Background: Circulating tumor cells have been postulated as indicators of the metastatic spread of cancer via the lymphatic and circulatory systems. We have developed a photoacoustic flow cytom...

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.

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.

In a recent report in this journal, Bach et al. (1990) described a method of preparing large numbers of human eosinophils from leukapheresis products obtained from normal donors. We have found the availability of leukapheresis products has enhanced several of the immunology research programs at our institution. Leukapheresis of normal donors for the sole purpose of laboratory research ethically should be constrained by a high degree of safety for the donor. However, the medical literature contains limited information on the safety or risks of leukapheresis of normal donors (Buchholz et al., 1975; Grindon et al., 1982). We have performed over 200 leukapheresis procedures on normal donors without the occurrence of serious adverse reactions. In the process of monitoring the safety of our protocol, we also prospectively evaluated the effects of leukapheresis on hematology, coagulation, and blood chemistry parameters for 40 procedures. We wish to report our safety experience and outline the donor protection features of our apheresis protocol for the benefit of other investigators or human investigation review boards considering the use of this procedure for obtaining leukocytes from healthy volunteers for research purposes. Our protocol is approved and monitored by the institutional review board of the hospital in which

This study was undertaken in an effort to understand the role of cytokines in T lymphocyte trafficking into inflamed synovium and in the potential enhancement of antigen presentation by human synovial fibroblasts. We found that interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN gamma) each increased the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) on human synovial fibroblasts in a dose- and time-dependent manner. Maximal ICAM-1 expression occurred within 8 hours of induction, with the following order of efficacy: IFN gamma greater than TNF alpha greater than IL-1 beta. The number of cells bearing the ICAM-1 antigen also increased, from a basal level of approximately 30% to more than 83% after cytokine induction (for all 3 cytokines). ICAM-1 expression rapidly decreased following cytokine removal. The expression of lymphocyte function-associated antigen 3 was also examined, but it was not changed by any of the 3 cytokines. Class I major histocompatibility complex antigen expression was increased modestly by all 3 cytokines, and expression was maximal by 24 hours after treatment. Only IFN gamma induced HLA class II antigen expression, and this expression persisted for up to 6 days following removal of the lymphokine. IL-6 and granulocyte-macrophage colony-stimulating factor had no effect on any of the parameters examined. Our data support an interactive role for inflammatory cytokines and the expression of adhesion ligands and HLA antigens by human synovial fibroblasts in the pathogenesis of synovial inflammation in rheumatoid arthritis.

In this study we attempted to compare cord blood derived, in vitro differentiated eosinophils to peripheral blood eosinophils with respect to their capacity to respond to various activators and, therefore, their potential ability to contribute to an inflammatory response. The cells were compared with respect to their density, content of eosinophil peroxidase, and eosinophil-derived neurotoxin, and with respect to their responses to various activators. The in vitro cultured, cord blood derived eosinophils were distinctly lighter than the freshly isolated peripheral blood cells. This difference in cell density was reflected in a slightly reduced content of both eosinophil peroxidase (1.17 ± 0.29 compared to 2.03 ± 0.22 arbitrary units/2,000 cells) and eosinophil-derived neurotoxin (18.7 ± 4.0 vs. 26.4 ± 4.5 ng/l04 cells). We compared the cells with respect to two different activation end points; the production of activated oxygen metabolites (superoxide anion) and the secretion of cationic proteins from their granules (eosinophil peroxidase and eosinophil-derived neurotoxin). In general, these responses were either the same in the two cell populations, or they were only slightly lower in the cord blood derived cells. There were, however, a few notable exceptions. Thus the secretory responses of the cultured cells to C5a and C3a anaphylatoxins and O2–– production with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, and with aggregated IgG were consistently greater than those of the normodense eosinophils. The possible implications of these differences on the state of maturation of the in vitro differentiated eosinophils are briefly discussed.

169 Background: IRX4204 is a potent, selective, oral small compound agonist of retinoid X receptor (RXR) nuclear receptor pathways. In preclinical studies IRX4204 synergizes with insulin-like growth factor binding protein-3, to induce apoptosis in human prostate cancer cells and was effective in reducing human prostate tumor burden in a murine xenograft model. The only prostate cancer patient in a phase I trial of IRX4204 trial manifested a sustained greater than 90% prostate-specific antigen (PSA) reduction and a PR lasting over seven months. These findings led to the current open label phase II trial of IRX4204 to evaluate safety and activity or futility, for treatment of advanced castration-resistant metastatic prostate cancer (CRPC). Methods: The trial enrolled 23 men with metastatic CRPC, who had failed a taxane or declined chemotherapy, with ECOG 0-2 with evidence of biochemical or radiographic progression on bone scan, CT and/or MRI. Patients continued with androgen blockade and had to have adequate organ function. Patients were deemed to have demonstrated benefit of IRX4204 treatment if they had PFS greater than 56 days, and/or a 50% PSA decrease, and/or PR or CR by RECIST. Futility defined as four or fewer patients demonstrating benefit in the planned enrollment of 37 patients. Patients received IRX4204 (20 mg/orally daily). PSA was checked Q4 weeks and radiographic assessments Q8 weeks. Radiographic, not PSA progression, was used to determine progression. Results: IRX4204 was well tolerated for up to 11 months. No drug related serious adverse events (SAE) have occurred. Manageable decreases in the thyroid-stimulating hormone (TSH) and increases in triglycerides, both known adverse effects of RXR agonists, have been observed. Protocol defined patient benefit was observed in 13 out of 23 (57%) patients. Progression-free survival (PFS) more than 56 days observed in 13 out of 23 (57%). PFS more than 112 days observed in 9 out of 23 (39%), range 113 to 330 days. PSA 50% response occurred in 3 out of 23 (13%), including one additional PSA 90% response. No objective responses have occurred. Conclusions: This study demonstrates that IRX4204 treatment of taxane-resistant CRPC is well tolerated, and provides an activity signal warranting further evaluation of IRX4204 as a treatment for chemo-refractory CRPC. Funding: Supported by Io Therapeutics, Inc. 1805 East Garry Ave., Suite 110, Santa Ana, 92705. Clinical trial information: NCT01540071.

Objective. To investigate the effects of chimeric CD4 monoclonal antibody cM-T412 treatment in patients with rheumatoid arthritis (RA). Methods. Thirty-two RA patients received daily doses of 10, 50, or 100 mg of cM-T412 intravenously for 7 days. Results. There was a sustained decrease in the number of CD4+ T lymphocytes in all patients. Those who received 50 mg and 100 mg of the antibody experienced significant reductions in disease activity. Conclusion. Treatment with cM-T412 appears to have a dose-dependent beneficial effect in RA patients. The clinical effects of cM-T412 are independent of the depressed numbers of circulating CD4+ T cells.

Objectives. To evaluate the safety, immunogenicity, and biologic effects of chimeric monoclonal anti-CD4 (cM-T412) in patients with refractory rheumatoid arthritis (RA), and to obtain preliminary data on the clinical response to this treatment. Methods. Twenty-five patients with active refractory RA were treated with incremental doses (10 to 700 mg) of cM-T412 in an open-label, escalating-dose phase I trial. Results. Infusion with cM-T412 was followed by an immediate, rapid decline in CD4+ T cells. The level of circulating CD4+ T cells remained depressed in most patients even at 6 months posttreatment. Following antibody infusion, proliferative responses of peripheral blood lymphocytes to mitogens and antigens were determined; mitogen and antigen responses were decreased compared with pretreatment responses. Mitogen responses tended to return to baseline values more rapidly than did responses to antigen. Adverse events included fever (19 patients), which was associated with myalgias, malaise, and asymptomatic hypotension; these symptoms were self-limited and appeared to correlate with transient elevations in interleukin-6. No significant human antibody response to the cM-T412 variable region was detected; only 2 patients developed transiently low levels of antibodies reactive with cM-T412. Significant clinical improvement, defined as ≥50% decrease in tender joint counts compared with baseline, was noted in 43% of patients at 5 weeks and 33% at 6 months following cM-T412 infusion. Conclusions. Treatment of refractory RA with cM-T412 appears to be safe and is associated with sustained decreases in circulating CD4+ T cell counts and depressed in vitro T cell responses. No significant human antichimeric antibody response was detected. Nonblinded assessment of clinical end points suggests that treatment with cM-T412 may have beneficial effects in these patients with refractory RA. A double-blind clinical trial is warranted to determine its clinical efficacy in treating RA.

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN gamma treatment resulted in the largest increase in adhesion, followed by TNF alpha and IL-1 beta. Combinations of IFN gamma + TNF alpha and IFN gamma + IL-1 beta had a synergistic effect on intercellular adhesion molecule 1 (ICAM-1) expression and adhesion. The increase in cellular adhesion induced by cytokines correlated with the up-regulation of the number of cells expressing ICAM-1 and the density of antigen/cell. There was no synergistic effect on leukocyte function-associated antigen 3 (LFA-3) or on HLA class I or class II antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18. These findings suggest the existence of ICAM-1--independent and CD11/CD18-independent adhesion mechanisms. Anti-LFA-3 was completely ineffective as an inhibitor of adhesion. There was no additive or synergistic advantage of using combinations of antibodies to increase the level of inhibition, i.e., anti--ICAM-1 + anti-LFA-3, anti-ICAM-1 + anti-CD18, or anti-ICAM-1 + anti-LFA-1 (CD11a). Our data indicate that proinflammatory cytokines may play a prominent role in the formation and exacerbation of synovial hyperplasia, by regulating the recruitment and retention of T lymphocytes via the up-regulation of adhesion molecules on synovial fibroblasts.

Doses of beta-carotene for cancer-prevention trials have been chosen based on epidemiologic data. Mechanisms of the putative antineoplastic effects by beta-carotene are unknown but may involve modulation of the immune system. We measured plasma carotenoid concentrations and selected immunologic indices at baseline and at 2 and 4 wk in 50 healthy humans (5 groups of 10 each) ingesting 0, 15, 45, 180, or 300 mg beta-carotene/d for 1 mo in this randomized placebo-controlled, open-label, parallel study. Plasma beta-carotene concentrations were markedly increased by 2 wk and were correlated with dose. Beta-carotene concentrations plateaued between 2 and 4 wk except for the 300-mg group. Thus, we developed a dose-concentration curve to optimize beta-carotene-dose selection to achieve target plasma concentrations. We were unable to identify any effects of beta-carotene ingestion on the immunologic indices studied, but modest increases in high-density-lipoprotein cholesterol were observed in all beta-carotene-treated groups.

We are interested in the physiologic mechanisms of eosinophil activation because of the presumed participation of activated eosinophils in the inflammatory sequelae of asthma. Suspecting that other formed elements of the blood may contribute to such an activation, we examined the capacity of platelet-derived growth factor (PDGF), a product of activated platelets, to activate eosinophils. We found that highly purified monkey and human eosinophils, but not guinea pig eosinophils, were activated by PDGF (superoxide anion production) in a dose-dependent fashion. Moreover, this activation was further dependent on a prior ‘priming’ of the cells by a brief exposure to subthreshold concentrations of phorbol ester. The response was specific for the BB homodimer of PDGF suggesting it is receptor-dependent.

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.

BACKGROUND: Relaxin is a pregnancy-related hormone that has tissue remodeling and antifibrotic effects. Systemic sclerosis (scleroderma) is characterized by fibrosis of the skin, vasculature, and internal organs. OBJECTIVE: To assess the efficacy, safety, and dose-response effect of recombinant human relaxin in patients with scleroderma. DESIGN: Multicenter, parallel-group, randomized, double-blind, placebo-controlled trial. SETTING: Academic referral centers. PATIENTS: 68 patients who had had stable, diffuse scleroderma (moderate to severe) for less than 5 years. INTERVENTION: Recombinant human relaxin, 25 or 100 microg/kg of body weight per day, or placebo administered by continuous subcutaneous infusion over 24 weeks. MEASUREMENTS: Modified Rodnan skin score was the primary efficacy measure. Secondary measurements were pulmonary function, the Health Assessment Questionnaire, and other measures of scleroderma that reflected fibrosis. RESULTS: Patients who received 25 microg/kg of recombinant human relaxin per day had significantly lower skin scores than those who received placebo (mean change, -3.6 at 4 weeks [P = 0.021], -7.5 at 12 weeks [P < 0.001], and -8.7 at 24 weeks [P = 0.040]). Similar trends were noted in other outcome measures, including forced vital capacity, measures of oral aperture and hand extension, functional status, and global assessment. Patients who received 100 microg/kg of relaxin per day did not differ from those who received placebo. Drug-related adverse events included menometrorrhagia, reversible anemia, and complications of the subcutaneous drug administration system (site irritation and local infection). CONCLUSIONS: Twenty-four weeks of recombinant human relaxin, 25 microg/kg per day, is associated with reduced skin thickening, improved mobility, and improved function in patients with moderate to severe diffuse scleroderma.

We studied the molecular composition of the complement C5b-9 complex required for optimal killing of Escherichia coli strain J5. J5 cells were incubated in 3.3%, 6.6%, or 10.0% C8-deficient serum previously absorbed to remove specific antibody and lysozyme. This resulted in the stable deposition after washing of 310, 560, and 890 C5b67 molecules per colony-forming unit, respectively, as determined by binding of 125I-labeled C7. Organisms were then incubated with excess C8 and various amounts of 131I-labeled C9. Plots of the logarithm (base 10) of E. coli J5 cells killed (log kill) vs. C9 input were sigmoidal, confirming the multihit nature of the lethal process. When C9 was supplied in excess, 3300, 5700, and 9600 molecules of C9 were bound per organism for cells bearing 310, 560, and 890 C5b-8 complexes, respectively, leading to C9-to-C7 ratios of 11.0:1, 10.8:1, and 11.4:1 and to log kill values of 1.3, 2.1, and 3.9. However, at low inputs of C9 that lead to C9-to-C7 ratios of less than 3.3:1, no killing occurred, and this was independent of the number of C5b-9 complexes bound. Formation of multimeric C9 at C9-to-C7 ratios permissive for killing was confirmed by electron microscopy and by binding of 125I-labeled antibody with specificity for multimeric but not monomeric C9. These experiments are the first to demonstrate a biological function for C9 polymerization and suggest that multimeric C9 is necessary for optimal killing of E. coli J5 cells by C5b-9.

Purpose Central nervous system disease and vasculitis are extraglandular manifestations of Sjogren's syndrome. In our experience, central ner vous system disease develops in approximately 70 percent of patients with Sjogren's syndrome and biopsy documented peripheral vasculitis. In order to further investigate the pathogenesis of central nervous system disease and its relationship to peripheral vasculitis in Sjogren's syndrome, we examined sera of patients with Sjogren's syndrome with and without focal central nervous system involvement for evidence of terminal complement pathway activation. Patients and methods Patients were classified as having active focal central nervous system involvement only when they had focal neurologic deficits on physical examination, plus at least one abnormal neurodiagnostic test result. Two thirds of these patients also had cognitive or psychiatric dysfunction. Patients were classified as having peripheral vasculitis if they had clinical and histopathologic documentation of vascular inflammation. Serum SC5b-9 was measured by a sensitive enzyme-linked immunoabsorbent assay. Total hemolytic complement assay, measurement of serum C3 and C4 by radial immunodiffusion, and determination of immune complexes were performed. Results Fluid-phase terminal complement complexes (SC5b-9) were detected in the sera of 25 of 30 (83 percent) patients with focal central nervous system involvement, but in only seven of 21 (33 percent) patients with Sjogren's syndrome without focal central nervous system disease (p = 0.00084 by Yates' chi-square analysis). Four of these seven patients without focal central nervous system disease, but who had serum SC5b-9, had psychiatric or cognitive dysfunction. SC5b-9 was also detected in sera from 14 of 15 (93 percent) patients with active biopsy-documented peripheral vasculitis in contrast to 18 of 36 (50 percent) patients without clinical evidence of peripheral vasculitis (p = 0.0094). Serum SC5b-9 was a more sensitive indicator of complement activation than circulating immune complex or complement assays. Conclusion These findings suggest that terminal complement activation may participate in the pathophysiology of both central nervous system and peripheral vasculitis in Sjogren's syndrome. Serum SC5b-9 appears to be a useful diagnostic indicator of vascular inflammation in Sjogren's syn drome and appears to identify those patients at risk for central nervous system complications.

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.

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3)) is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,0004 (Mr 50–58 K) which appears early in thymocyte ontogeny and is present on all mature T cells5. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell2–4,6. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression2–4,7–9. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes1, cytotoxic effector function10 and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits resetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes12 suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated anti-gen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.

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 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 neoantigen c 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.

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.

Dermatitis herpetiformis (DH) is associated with a markedly increased frequency of the HLA class II antigens DR3 and DQw2. To investigate a possible role of HLA-DP (or closely associated genes) in the pathogenesis of DH as well as to confirm the previously described alterations of HLA-DR3 and HLA-DQw2 antigen frequency, we have typed 43 patients with DH for HLA-DP, HLA-DQ, and HLA-DR antigens. All patients with DH had typical clinical and histologic features, as well as granular deposits of IgA at the dermal-epidermal junction by direct immunofluorescence. HLA-DR3 was expressed in 41 of 43 (95%) DH patients, whereas HLA-DQw2 was expressed in all 43 (100%). The overall distribution of HLA-DP antigens in patients with DH was significantly different from that seen in all controls and in HLA-DR3 and HLA-DQw2 controls (p less than 0.02). Examination of the frequency of individual DP antigens revealed that HLA-DPw1 was increased (42% of patients with DH vs 11% of all controls and 26% of DR3 positive controls), but this increase was not statistically greater than that expected due to the disequilibrium linkage of DPw1 with DR3/DQw2. Patients with DH, however, did have a statistically significant decreased frequency of DPw2 (14% of patients vs 31% of all controls and 41% of DR3 positive controls) (pc less than 0.05). Studies of three informative families demonstrated that the DPw2 genes of the DH patients were not present on the haplotype thought to carry a DH susceptibility gene (HLA-A1, HLA-B8, HLA-DR3, HLA-DQw2). A role of HLA-DP region genes in the pathogenesis of DH is further suggested by the observation that HLA-DPw1 was expressed in 82% (9 of 11) of DH patients with IgA antibodies against dietary antigens as compared with only 33% (4 of 12) of patients without IgA antibodies. HLA-DP genes or genes closely linked to them may be important in DH either as markers of the disease haplotype or by direct involvement in its pathogenesis.

Cell–cell adhesion is essential for many immunological functions1–4, including interaction of cytotoxic T lymphocytes (CTLs) with their targets5–8. We have explored CTL-target interactions using well-characterized cloned human CTLs9,10. Conjugate formation between these CTLs and many antigen-negative targets is almost as efficient as with specific target cells, but does not lead to target-cell lysis. Thus, on specific target cells, adhesion by antigen-independent pathways may occur concurrently with or precede antigen recognition. The molecules LFA-1, CD2 (Til, LFA-2) and LFA-3 have been shown11–15 to be involved in human CTL conjugation with and lysis of specific target cells. Here we describe monoclonal antibody inhibition studies using individual monoclonal antibodies and mixes which demonstrate (1) that LFA-1, CD2 and LFA-3 are involved in antigen-independent conjugate formation; and (2) suggest that CD2 and LFA-3 are involved in one pathway and LFA-1 in another. We confirmed the existence of distinct pathways by the demonstration that LFA-1-dependent adhesion requires divalent cations and is temperature-sensitive whereas CD2- and LFA-3-dependent adhesion does not require divalent cations and is temperature-insensitive. Together with previous data, our studies suggest that CD2 on the effector interacts with LFA-3 as its ligand on targets.

Previous investigations have defined phenotypic differences between unprimed (naive) and antigen-primed (memory) T cells from human peripheral blood. We now report that memory T cells proliferate much more than naive cells when stimulated with anti-CD3 monoclonal antibody or pairs of anti-CD2 monoclonal antibodies. Enhanced responsiveness to receptor-mediated triggering is a novel mechanism for T cells which could facilitate memory cell response to specific antigen. Furthermore, when triggered via either CD2 or CD3, memory T cells produce substantial amounts of interferon gamma while naive cells produce virtually none; this suggests that differentiation from naive to memory state is accompanied by a stable change in regulation of the gene for interferon-gamma. We conclude that naive and memory T cells are dramatically different in function as well as phenotype.

Recent functional studies indicate that intercellular adhesion molecule-1 (ICAM-1), recognized by monoclonal antibody (MAb) RR1/1, is a ligand for lymphocyte-associated antigen (LFA)-1 in several models of antigen-independent B- and T-cell adhesion and in cytotoxic T-cell recognition. Independent reports have identified other MAbs (LB-2 and 84H10) that inhibit LFA-1-dependent adhesion by B cells or myeloid cells. We analyzed the relationships of these three MAbs. The results show that: 1) all three MAbs inhibit LFA-1-dependent conjugate formation with particular targets and that mixes of these MAbs do not show additive inhibition; 2) LB-2 and 84H10 each inhibit the binding of the other, whereas binding of RR1/1 neither inhibits nor is inhibited by binding of LB-2 or 84H10; 3) all three MAbs bind purified 84H10 protein; and 4) binding of these three MAbs is concordant on 12 different cell types and is similarly enhanced on monocytes with activation. The simplest interpretation of these data is that a single protein, ICAM-1, is recognized by the MAbs RR1/1, LB-2, and 84H10. This finding unifies and extends previous observations; ICAM-1 functions as an adhesion molecule in a variety of leukocyte interactions, apparently as a ligand for LFA-1. Furthermore, MAb binding to either of two distinct ICAM-1 epitopes inhibits ICAM-1-dependent adhesion. Table 1 MAb Inhibition of LFA-1-Dependent Conjugate Mab HeLa U937 U266 TS2/9 MHM23 LB-2 RR1/1 84H10 84H10 + LB-2 84H10 + RR1/1 RR1/1 + LB-2 38 0 ND 2 3 ND 2 ND 24 2 2 2 1 1 4 2 27 2 23 21 23 24 21 24 Conjugate formation was measured between the cytotoxic T-cell clone 8.2 and three antigen-negative targets HeLa, U937, and U266. Enumeration of conjugates was by two-color microfluorometry as described (10,19). The results are expressed as a % of targets that end up in conjugates. All assays were done in the continuous presence of saturating concentrations of the LFA-3 MAb TS2/9(17) to isolate the LFA-1 pathway. MAbs were continuously present during the assay at 100 μg/ml purified IgG except for MHM23, the LFA-1 Mab (20) and TS2/9 which were present at 300 µg/ml of Fab fragment. ND = not determined.

CD2 is a T lymphocyte glycoprotein that functions in adhesion of T lymphocytes and also as a putative receptor for activation signals. Functional data suggest that LFA-3, a widely distributed cell surface glycoprotein, may be the biological ligand of CD2. We have purified LFA-3 from human erythrocytes and characterized the purified protein functionally. LFA-3 bound specifically to CD2+ cells, and this binding was inhibited by CD2 mAb. Conversely, purified LFA-3 inhibited binding of CD2 mAb to cells, and the concentration required for this effect suggests that LFA-3 half-saturated CD2 at 1-5 nM LFA-3. Purified LFA-3 inhibited rosetting of human and sheep erythrocytes with CD2+ T lymphoma cells and T lymphocytes, and mediated aggregation of a CD2+ T lymphoma cell line. Purified LFA-3 reconstituted into planar membranes mediated efficient CD2-dependent adhesion of T lymphoblasts. These data demonstrate that LFA-3 is a ligand for CD2 and that LFA-3 can mediate T lymphocyte adhesion.

Cell-cell adhesion is essential for many immunological functions. The LFA-1 molecule, a member of a superfamily of adhesion molecules, participates in adhesion which is critical to the function of each of the three major subsets of leukocytes: lymphocytes, monocytes and granulocytes. Putative LFA-1 ligands have been identified functionally in different laboratories using three different monoclonal antibodies that inhibit LFA-1-mediated leukocyte adhesion in particular model systems; however, there may be more than one LFA-1 ligand. We have directly compared the three relevant monoclonal antibodies, and show that each binds to the same molecule, intercellular-adhesion molecule-1 (ICAM-1). Most important, B, T and myeloid cells adhere specifically to purified ICAM-1-coated surfaces; such adhesion has distinctive requirements for Mg2+ and Ca2+. This constitutes biochemical evidence that ICAM-1 functions as a ligand for LFA-1-dependent adhesion by a variety of leukocytes.

Although intercellular adhesion molecule-1 (ICAM-1) has been implicated as a ligand in some LFA-1-dependent adhesion, its importance to T cell function has not been established. The present studies investigate the importance of ICAM-1 for human cytotoxic T lymphocytes (CTL), both in their formation of antigen-independent conjugates (AIC) and in their lysis of targets. Analysis of monoclonal antibody (mAb) inhibition of AIC formation indicate that ICAM-1 mAb 1 blocks (a) AIC formation with some but not all targets; (b) the LFA-1 pathway but not the CD2/LFA-3 pathway of adhesion; (c) by binding to the target cell, not the T cell. In studies of cell-mediated lysis (CML) ICAM-1 mAb inhibited lysis of some targets, such as U-937, that use ICAM-1 predominantly in AIC formation; CML on some other targets is not inhibited by ICAM-1 mAb. These data indicate that ICAM-1 is a ligand for AIC formation, antigen-specific CTL recognition and cytolysis of particular target cells. The data also indicate that ICAM-1 is not used in LFA-1-dependent CTL interactions with all kinds of target cells, suggesting the existence of alternative ligands for LFA-1.