Reuven Porat

Pharmacokinetics, safety and immunomodulatory effects of human recombinant interleukin-1 receptor antagonist in healthy humans

A phase I study of human recombinant interleukin-1 receptor antagonist (IL-1ra) was conducted in healthy males between the ages of 18 and 30. Twenty-five volunteers received a single, 3 h continuous intravenous infusion of doses ranging between 1 mg/kg and 10 mg/kg IL-1ra. At 3 h into the infusion, plasma IL-1ra levels were 3.1 micrograms/ml and 29 micrograms/ml for the 1 mg/kg and 10 mg/kg doses, respectively. Post-infusion plasma IL-1ra levels declined rapidly, exhibiting an initial half-life of 21 min and a terminal half-life of 108 min. Clinical, hematological, biochemical, endocrinological and immunomodulatory effects were monitored over 72 h and compared to those of four subjects receiving a 3 h infusion of saline. There were no clinically significant differences between the drug and saline groups in symptoms, physical examinations, complete blood counts, mononuclear cell phenotypes, blood chemistry profiles, serum iron and serum cortisol levels. Peripheral blood mononuclear cells (PBMC) obtained after completion of the IL-1ra infusion synthesized significantly less interleukin 6 ex vivo than PBMC from saline-injected controls. These data suggest that transient blockade of interleukin 1 receptors is safe and does not significantly affect homeostasis.

Plasma Cytokines, Cytokine Antagonists, and Disease Progression in African Women Infected With HIV-1

Wasting and chronic fever are prominent manifestations of human immunodeficiency virus (HIV) infection in Africa [1] and are commonly found in patients with the acquired immunodeficiency syndrome (AIDS) in the United States and Europe. Although many factors contribute to fever and wasting, cytokines such as interleukin-1 and tumor necrosis factor- (TNF-) may be particularly relevant because they are associated with cachexia [2], fever [3], and inflammation [4] both in experimental animals and humans. Tumor necrosis factor- and interleukin-1 enhance HIV-1 replication in vitro, suggesting that cytokines may contribute to the pathogenesis and progression of HIV infection [5, 6]. Infections resulting in cytokine responses, increased HIV replication, and immune destruction may therefore heighten susceptibility to new infections and drive clinical progression toward AIDS. Cytokine antagonists such as the interleukin-1 receptor antagonist (interleukin-1Ra) and the soluble form of the p55 (type I) TNF- receptor (TNFsRp55) block the inflammatory action of their agonists, both in vitro [7] and in vivo [8]. High levels of the p75 (type II) TNF receptor were associated with undetectable levels of TNF- in a study of 7 asymptomatic HIV-infected patients or patients with AIDS [9]. Elevated plasma levels of cytokine inhibitors have been reported to correlate with rapid disease progression in HIV-1-infected patients [10]. We compared plasma levels of interleukin-1 , TNF-, interleukin-6, interleukin-8, interferon-, interleukin-1Ra, and TNFsRp55 among HIV-seropositive asymptomatic women from Zaire who had been infected with HIV for at least 5 years [11], women with advanced AIDS, and healthy HIV-seronegative controls. Our data suggest the clinical importance of cytokine antagonists in modulating symptoms during the early stages of HIV-1 infection. Methods Study Site and Patients We did this study in Kinshasa, Zaire, together with Projet SIDA (a collaboration among the Zairian Ministry of Health, the Centers for Disease Control and Prevention [CDC], the National Institute of Allergy and Infectious Diseases, the Armed Forces Institute of Pathology, and the Institute of Tropical Medicine, Antwerp, Belgium). We did a cross-sectional comparison of plasma cytokine levels using a convenience sample of 51 randomly selected HIV-seropositive clinically asymptomatic women from a well-described cohort enrolled in a longitudinal study of perinatal HIV-1 transmission [11]. Eleven female HIV-seronegative controls from the same cohort were also enrolled. Each control and asymptomatic patient denied having symptoms suggestive of infectious or inflammatory disease, and none had clinical evidence of current or recent acute illness. We randomly selected a third group of 48 women with advanced AIDS from a new cohort developed for a study of diarrhea and wasting. None of the 99 HIV-1-seropositive patients had ever received antiretroviral therapy. All participants gave written informed consent for the study, which was approved by the Human Investigation Research Committees at New England Medical Center and Mama Yemo Hospital. We gave each participant a comprehensive medical questionnaire, which elicited a history of fever, and a physical examination, which included anthropometry (midarm circumference and skin-fold thickness at three sites) and was done by study physicians trained by staff from the U.S. Department of Agriculture Nutrition Research Center at Tufts University, Boston, Massachusetts. Bioelectric impedance analysis (BIA) was done while the patient was in the recumbent position after rehydration using a BIA 101-A Impedance Analyzer (RJL Systems, Mt. Clemens, Michigan). Impedance data were transformed to body composition estimates using the BodyComp program (version 1.5). At the study clinic, we drew blood into ethylenediamine tetraacetic acid tubes between 9 a.m. and 12 a.m. for complete blood count and lymphocyte phenotyping. Serum samples were obtained and rapidly frozen at 60 C until assayed in batches to reconfirm HIV serology and to determine -carotene and albumin levels. Samples for cytokine analysis were collected in heparin, centrifuged, rapidly divided into aliquots, and frozen at 60 C. Laboratory Testing Routine Laboratory Tests We assayed carotene and albumin levels using standard colorimetric laboratory methods. We did complete blood counts using routine automated methods (Coulter Electronics, Luton, United Kingdom) and determined leukocyte differential counts manually. We examined Giemsa-stained thin smears of peripheral blood for malaria parasites. Serologic testing of HIV-1 and enumeration of CD4+ and CD8+ lymphocyte counts were done as previously described [12] according to CDC-established criteria [13]. Cytokine Assays Frozen samples were packed in dry ice, sent by courier to Boston, and stored at 70 C until analyzed. Samples were coded and tested in a single assay-blinded fashion. We measured levels of TNF- [14], TNFsRp55 [15], interleukin-1 [16], interleukin-1Ra [17], interleukin-6 [18], and interleukin-8 [19] by radioimmunoassay after chloroform extraction as described previously. The lower limit of detection for the cytokines was 40 to 156 pg/mL. We measured immunoreactive interferon- levels in a 1:10 dilution of plasma by commercial enzyme-linked immunosorbent assay kit (Endogen, Boston, Massachusetts) with a detection limit of 0.14 pg/mL. We reported the lower limit of detection for each sample in which cytokine levels were undetectable. Statistical Analysis We determined descriptive statistics for each clinical and laboratory variable using EPI-INFO software (EPI-INFO, Stone Mountain, Georgia). Pairwise analyses using the Wilcoxon rank-sum test were done to assess the significance of differences in the means and medians between groups. We considered P values less than 0.05 to be statistically significant. Results Clinical Findings Patients with AIDS were a mean of 4.1 years older than asymptomatic HIV-positive patients and controls. These patients reported having had fever for 12.6 days in the previous 2 months compared with 3.1 days in the asymptomatic HIV-positive group and 2.2 days in the control group (P < 0.001). Forty-five of 48 patients with AIDS reported losing more than 10% of their normal body weight, and 15 of these 45 reported having had fever for more than 2 weeks in the previous month. Thickness of the biceps, triceps, subscapular skin folds, and midarm circumference were markedly reduced in the patients with AIDS compared with the patients in the other two groups (Table 1). Patients with AIDS also had a profound decrease in fat (46.2%) and lean body mass (17.9%) as calculated from bioelectric impedance analysis data (P < 0.001). Asymptomatic HIV-positive patients had detectable but mild reductions in body weight, skinfold thickness, midarm circumference, and lean body mass compared with controls. Table 1. Nutritional and Hematologic Characteristics* Clinical Laboratory Variables Patients with AIDS had significantly lower mean albumin and carotene levels than asymptomatic HIV-positive patients or controls (Table 1). Levels in the asymptomatic HIV-positive group were close to the lower limit of the normal range but were still significantly lower than levels in the control group (P = 0.04). Patients with AIDS had a greater reduction in absolute CD4 cell count and CD4:CD8 ratio than did the asymptomatic HIV-positive patients. Cytokine Levels Interleukin-1 and Tumor Necrosis Factor- Median circulating levels of interleukin-1 and TNF- were greater in asymptomatic HIV-infected patients than in patients with AIDS or in controls (Table 2). These cytokine levels were below the limit of detection in all 11 HIV-negative controls. We detected interleukin-1 in only 2 of the patients with AIDS; only 4 patients with AIDS had detectable TNF-. Table 2. Cytokine and Cytokine Antagonist Levels* Interleukin-1Ra and Soluble p55 Tumor Necrosis Factor- High levels of the two naturally occurring cytokine antagonists, interleukin-1Ra and TNFsRp55, were seen in the HIV-positive asymptomatic women (Table 2). When calculated on a weight-for-weight basis, this group had a nearly 17-fold greater mean interleukin-1Ra level and an 8-fold greater mean TNFsRp55 level than patients with AIDS. The relative excess of antagonist over agonist in each patient was also markedly increased in the HIV-positive asymptomatic group compared with the patients with AIDS, with a greater than 73-fold median excess (range, 3.4-fold to 243.6-fold) of TNFsRp55 over TNF- and a 14-fold excess (range, 0.1-fold to 60.9-fold) of interleukin-1Ra over interleukin-1 . We could not further compare patients with AIDS with HIV-positive asymptomatic patients, because most of the patients with AIDS had plasma cytokine values below the level of detection for the assay. If the TNF- and interleukin-1 levels are assumed to have been at the threshold level of detection, then the excess antagonist to agonist was 17.8-fold (range, 5.8-fold to 28.2-fold) for TNFsRp55 over TNF- and 1.7-fold (range, 0.6-fold to 26.5-fold) for interleukin-1Ra over interleukin-1 in the patients with AIDS. Interleukin-6, Interleukin-8, and Interferon- We detected interleukin-6 in only 3 of 48 patients with AIDS, 2 of 51 HIV-positive asymptomatic patients, and 4 of 11 HIV-negative controls. Median circulating interleukin-6 levels were similar in all three groups (Table 2). Interleukin-8 levels, however, were significantly elevated in the HIV-positive asymptomatic women; levels were higher than 40 pg/mL in 43 of these patients and in 12 of the women with AIDS, but none of the controls had elevated levels (P < 0.001). Interferon- levels could be measured in approximately half of the combined asymptomatic HIV-infected and AIDS groups. Eleven of the 13 highest values (> 11 pg/mL), however, were found in patients with AIDS (P = 0.005). Patients with AIDS who had detectable interferon- also reported a me

Interleukin-1 (IL-1) receptor blockade reduces endotoxin and Borrelia burgdorferi-stimulated IL-8 synthesis in human mononuclear cells.

Interleukin‐1 (IL‐1) is a potent stimulator of IL‐8 production by fibroblasts and monocytes. In the present study, we asked how much of endotoxin (LPS)‐induced IL‐8 production by human peripheral blood mononuclear cells was due to IL‐1 induced by LPS. Cells were stimulated with either IL‐1β, LPS, or Borrelia burgdorferi, and total IL‐8 was determined by a specific radioimmunoassay. The addition of saturating concentrations of IL‐1 receptor antagonist protein (IRAP) reduced the IL‐1β‐, LPS‐, and B. burgdorferi‐induced IL‐8 synthesis by 85, 50, and 40%, respectively. Increasing the concentration of LPS did not affect the reduction in IL‐8 synthesis observed in the presence of IRAP. Significant inhibition of the IL‐1β‐induced IL‐8 synthesis was observed when IRAP was added 60 or 90 min after IL‐1β; similarly, IL‐8 synthesis after LPS was also reduced by delayed addition of IRAP. These data suggest that the ameliorative effects of IL‐1 receptor blockade in models of inflammation and infection may be due, in part, to suppression of IL‐1‐induced IL‐8.—Porat, R.; Poutsiaka, D. D.; Miller, L. C.; Granowitz, E. V.; Dinarello, C. A. Interleukin‐1 (IL‐1) receptor blockade reduces endotoxin and Borrelia burgdorferi‐stimulated IL‐8 synthesis in human mononuclear cells. FASEB J. 6: 2482‐2486; 1992.

Glycosylated recombinant human tumor necrosis factor binding protein-1 reduces mortality, shock, and production of tumor necrosis factor in rabbit Escherichia coli sepsis.

OBJECTIVE To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. DESIGN Prospective, randomized, controlled trial. SETTING University hospital research laboratory. SUBJECTS Eighteen female, New Zealand white rabbits. INTERVENTIONS Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. MEASUREMENTS AND MAIN RESULTS Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p < .05). Treatment with r-hTNF binding protein-1 was associated with 100% survival, as compared with 55.6% of the saline-treated rabbits (p < .05). Approximately 85% of r-hTNF binding protein-1 was cleared from the circulation 1 hr after the bolus injection (from 171 +/- 27 micrograms/mL at time = 0, to 27 +/- 4 micrograms/mL at 60 mins, decreasing to 6 +/- 2 micrograms/mL for the next 3 hrs). The r-hTNF binding protein-1-treated rabbits had lower serum TNF bioactivity during the first 2 hrs (p < .01). The decreased bioactivity of TNF was confirmed by a specific radioimmunoassay for rabbit TNF. However, at 4 hrs, the vehicle-treated rabbits had lower serum bioactive TNF concentrations (p < .05). The decrease in TNF concentrations in the r-hTNF binding protein-1-treated rabbits resulted from decreased production and, in part, from carry-over of r-hTNF binding protein-1 into the bioassay. CONCLUSIONS Treatment with r-hTNF binding protein-1 improved hemodynamic variables and survival of E. coli-challenged rabbits. Administration of r-hTNF binding protein-1 suppressed bioactivity of TNF in the circulation of these rabbits, and the production of TNF as well.

Circulating leptin during experimental endotoxemia in humans.

Figure 1. Changes in plasma leptin levels after intravenous injection of endotoxin. Healthy human volunteers were injected with 3 ng/kg endotoxin (A, ) or saline (B, ). At time of injection and at n 5 3 n 5 3 different times afterwards, plasma was isolated. Leptin levels were measured by ELISA. There were no significant changes in leptin levels over time in either group, nor were there significant differences between groups. Circulating Leptin during Experimental Endotoxemia in Humans

Cytokine production by human peripheral blood mononuclear cells stimulated by a Pseudomonas aeruginosa culture filtrate: Role of plasma and polymyxin B

The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out. In this study, we examined the production of interleukin-1 alpha (IL-1α), interieukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate. Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized. The results of our study indicate that the Ps. aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components. The enhancing effect of plasma was most evident at low concentrations of the filtrate. The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma. There was a direct correlation between the production of IL-1α and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced. There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes

Effects of bactericidal/permeability-increasing protein on endotoxin-induced fever and Escherichia coli-induced shock in rabbits

Binding of bactericidal/permeability-increasing protein (BPI) to endotoxin inhibits endotoxin-triggered responses. We investigated the effects of BPI on endotoxin fever and E. coli-induced septic shock in rabbits. Pre-incubation of endotoxin with BPI blocked fever compared to control rabbits (n = 6). A marked reduction in fever was also observed when BPI was injected before endotoxin. E. coli-challenge resulted in 66% mortality (n = 6); pre-treatment with BPI resulted in survival of all animals (n = 3). Mean arterial blood pressure was higher in BPI-treated compared to control rabbits. Comparable leukopenia and thrombocytopenia was observed with either BPI or vehicle treatment. Tumor necrosis factor (TNF) and interleukin-1 receptor antagonist were similarly elevated in both BPI- and saline-treated rabbits. However, in BPI treated rabbits, peak TNF levels were 34 % lower compared to saline controls (P < 0.05). Further studies are warranted to assess whether BPI may have therapeutic potential for the treatment of septic shock.