Recombinant human interleukin 6 is a potent inducer of the acute phase response and elevates the blood platelets in nonhuman primates.

Human interleukin 6 (IL-6) produced by molecular cloning was administered to nonhuman primates to assess its biological activities in vivo. Rhesus monkeys were treated s.c. with recombinant human (rh) IL-6 at 3 and 30 micrograms/kg body weight/day for 11 days, followed by the administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) at 5.5 micrograms/kg/day for 5 days. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) increased, whereas negatively regulated APP (prealbumin) decreased in response to rhIL-6 treatment in a dose-dependent manner. Platelet counts rose after a latent period of 4-5 days following the start of rhIL-6 treatment, resulting in a maximum twofold increase above normal levels 2-3 days after the termination of the rhIL-6 treatment. Recombinant human IL-6 treatment induced a two to threefold rise in myeloid progenitor blood cell levels. The subsequent administration of rhGM-CSF to rhIL-6-pretreated animals did not increase the progenitor cell levels in blood above those found with rhGM-CSF treatment alone, indicating that rhIL-6 compared to recombinant human interleukin 3 (rhIL-3) has a minor proliferative effect on hematopoietic precursors in vivo. In conclusion, rhIL-6 was shown to be a potent stimulator of APP and was able to increase the number of platelets in circulation in nonhuman primates.

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates.

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on platelet survival and activation using a nonhuman primate model.

In humans and nonhuman primates, the in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) consistently results in marked increase of megakaryocyte ploidy and size similar to that observed with interleukin-6 (IL-6). However, whereas the administration of IL-6 also results in an increase in circulating platelets, there is no predictable corresponding increase in peripheral blood platelets following treatment with rhGM-CSF. To determine whether the failure of rhGM-CSF to produce thrombocytosis is secondary to cytokine-related increased platelet activation and consumption in vivo, we quantified autologous platelet survival time and in vivo platelet activation before and during 5 days of administration of rhGM-CSF to two rhesus monkeys. Platelet survival was measured using autologous platelets labeled with 111Indium-oxine. Platelet activation was assessed by flow cytometric determination of the expression of the major platelet membrane glycoprotein (GP) IIb/IIIa complex, and an activation-dependent epitope on GPIIb/IIIa (recognized by monoclonal antibodies [MABs] LJ-P4 and PAC1, respectively). Platelet activation was also assessed by dose-response aggregometry using adenosine diphosphate (ADP). While megakaryocyte ploidy increased during rhGM-CSF administration, peripheral platelet counts were 418 x 10(9)/L and 525 x 10(9)/L before and 402 x 10(9)/L and 508 x 10(9)/L during cytokine treatment in animals 1 and 2, respectively. No changes were observed in the mean platelet volume. 111Indium-labeled platelet recovery in circulation was similar before (94.7%, 91.8%) and during (92.9%, 92.8%) rhGM-CSF administration, which indicates that cytokine-related in vivo sequestration of platelets does not occur. Autologous platelet survival was 5.6 and 6.2 days before and 5.0 and 5.4 days during the rhGM-CSF treatment (p = 0.07), without significant change in the corresponding platelet turnover rate (derived from the platelet count and survival time). The flow cytometric analysis showed no increase in the binding of either LJ-P4 or PAC1 MABs to the platelet membrane during rhGM-CSF administration. The aggregometry studies demonstrated similar concentrations of ADP inducing half-maximal aggregation (ED50). Overall, the above data indicate that treatment with rhGM-CSF is not associated with in vivo activation, sequestration, or increased consumption of platelets. The data suggest that the failure of rhGM-CSF-stimulated megakaryocytes to increase peripheral platelet count is a manifestation of ineffective megakaryocytopoiesis resulting from inability to increase platelet delivery to the circulation.

Studies of an improved rhesus hematopoietic progenitor cell assay.

We have improved Rhesus monkey marrow cell growth in semisolid media by means of substituting supplemented calf serum for fetal bovine serum. The cloning efficiency of light-density marrow cells separated on 60% Percoll was 126 (+/- 54)/10(5) (n = 12, +/- SD), and for light-density peripheral blood cells 60 (+/- 46)/10(6) (n = 11). Thirty-five percent of the colonies were multilineage, whereas the remainder were unilineage colonies composed of erythrocytes, megakaryocytes, and neutrophilic or monocytic granulocytes. Unilineage megakaryocyte colonies comprised 12% of the total marrow progenitor cells. The [3H]TdR suicide index of marrow progenitor cells was 47% +/- 9% (n = 12). This progenitor cell assay should prove useful in preclinical studies of the effect of recombinant hematopoietic growth factors on the number and cycling status of Rhesus hematopoietic progenitor cells.

Differential induction of cytokine-specific mRNA in human PBL after in vitro culture with either IL2 or IL4.

The present study was designed to investigate whether human equivalents of murine T helper cell subsets can be demonstrated by propagation of peripheral blood lymphocytes (PBL) with either recombinant human (rh) interleukin (IL) 2 or rhIL4 in the presence of neutralising antibodies. Cells of both cultures, termed T-IL2 or T-IL4, respectively, were challenged on day 8 using a combination of phorbol-12-myristate-13-acetate (PMA) and the Ca2(+)-ionophore A23187 (Io). Total cellular RNA was isolated at different time points after PMA/Io-stimulation and the expression of 7 distinct cytokine genes was assessed by Northern analysis. Whereas maximal accumulation of mRNA species for IL2, GM-CSF, TNF alpha and TNF beta did not reveal major differences between cells of T-IL2 and T-IL4 cultures, substantial differences emerged for the induction of IFN gamma and IL3 messages. Accumulation of IFN gamma-mRNA consistently was 2- to 13-fold higher in T-IL2 than in T-IL4 cells, depending on the time point of RNA harvest. In contrast, IL3-specific mRNA levels induced in T-IL4 cells were 2-5 times greater than those in T-IL2. If PBL cultured with IL2 for 7-8 days were subsequently shifted to IL4 and further propagated until day 14, the mRNA induction pattern seen for IFN gamma and IL3 was similar to that obtained if cells had continuously been propagated with IL2. Collectively, these results indicate a selective outgrowth of distinct responder phenotypes by IL2 or IL4 rather than a direct modulation of cytokine expression by these factors.

Efficacy of recombinant human granulocyte-macrophage colony-stimulating factor in rhesus monkeys.

The glycosylated and the non-glycosylated recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and E. coli, respectively, were administered in rhesus monkeys either by the subcutaneous (three times daily) or intravenous route (6-hr infusions) for seven consecutive days. Within 24 hr peripheral white blood cells (WBC) increased 2-3 fold over normal values. Thereafter, the WBC increased steadily in a dose-dependent manner to reach maximum levels on the last day of or one day after the treatment period. The differential counts showed that neutrophils contributed to 50-80%, eosinophils to 10-20%, monocytes to 2-7%, and lymphocytes to 15-30% of the WBC rise. No effect was found on platelets and erythrocytes. After termination of treatment, WBC counts returned to normal levels within one week. Subcutaneously administered CSF was more effective in inducing leukocytosis than that injected intravenously In addition to the rise in WBC, the administered rh GM-CSF also enhanced the oxidative metabolism and bactericidal activity of the circulating mature granulocytes isolated from the blood of monkeys treated with rh GM-CSF. These results show that glycosylated or non-glycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the functional activity of mature granulocytes in vivo.

Lipid A analogs aimed at preventing the detrimental effects of endotoxin.

Evidence has been presented for two potential methods of administering lipid A derivatives for the reduction of endotoxicity: 1. Use of low doses of agonists to induce early-phase tolerance for a sufficiently long period to protect patients at risk of endotoxin shock. 2. Administration of high doses of antagonists to the LPS-induced release of proinflammatory cytokines. The strengths and weaknesses of both approaches can be summarized as follows: Approach 1 appears promising for patients at risk for septicemias, based on iatrogenic induction of neutropenias or genetically caused neutropenic states, e.g., in cancer patients receiving aggressive chemotherapy or irradiation and in patients receiving immunosuppressive therapy (transplantations, myelodysplastic syndromes, and so forth.) Strengths. A long lasting effect can be expected. Broad protection against many types of infectious organisms. Strong potentiation of antibiotic chemotherapy anticipated irrespective of resistance patterns to antibiotics. Weaknesses. Only prophylactic treatment appears possible. Potential for endotoxic side-effects remains. Approach 2, the administration of LPS antagonists, appears most promising in clinical situations when interference with acute endotoxic shock symptoms subsequent to polytrauma is necessary. Strengths. Immediate onset of activity would be expected. Lower risk of side-effects. Weaknesses. Therapy may already be too late. Activity is restricted to endotoxicity, there being no anti-infectious potential. High drug levels might be required for a prolonged period. Synergism with antibiotics is not yet established. Together, these new lipid A derivatives open up new potential therapeutic avenues for the prophylaxis and therapy of septic shock, septicemias, and infections. Clinical studies will soon show whether the exciting pharmacologic effects observed in animals can be translated into humans.

Long-term studies of microencapsulated and adsorbed influenza vaccine nanoparticles.

Incorporation of antigens into nanometer-sized polymer particles was recently shown to lead to a good adjuvant effect. An optimal antibody response with killed influenza virus antigens was achieved with 0.5% poly(methyl methacrylate). Long-term experiments showed prolonged antibody response of polymer adjuvants with incorporated or adsorbed influenza virus. Adsorption also yielded an optimal adjuvant effect with 0.5% poly(methyl methacrylate). The antibody response was accompanied by protection of the mice against infection with mice-adapted influenza virus. In addition, the polymer vaccines were more stable against temperature inactivation than were vaccines with aluminum hydroxide or without adjuvants.

Distribution and elimination of poly(methyl methacrylate) nanoparticles after subcutaneous administration to rats.

Poly(methyl [1-14C]methacrylate) nanoparticles were injected subcutaneously into rats. Almost all of the radioactivity stayed at the injection site. After an initial urinary and fecal excretion of approximately 1% of the administered dose per day, the rate of elimination dropped to a low level (approximately 0.005%/day via the feces and approximately 0.0005%/day via the urine) within 70 days. After 200 days, the fecal elimination increased exponentially until a greater than 100-fold increase was observed after 287 days in one rat. After this time, a tendency for an increase in fecal elimination was also observed in the other animals, and the radioactivity in all organs and tissue increased by approximately 100 times in all animals in comparison with the organ radioactivity determinations at earlier times.

Distribution and elimination of polymethyl methacrylate nanoparticles after peroral administration to rats.

Polymethyl [1-14C]methacrylate nanoparticles were administered orally to bile cannulated rats. Ten to fifteen percent of the administered radioactivity was absorbed and found in the bile and urine. Within 48 h, 94-97% of the absorbed radioactivity had been eliminated from the body. After 8 d, the highest residual radioactivity was found in the bone marrow, fatty renal tissue, stomach, liver, and lymph nodes.

In vitro IgE but not IgG production of canine peripheral blood B cells is inhibited by CD40 ligation.

The aim of this study was to investigate in vitro IgE induction in peripheral canine B cells. CD21(+) B cells were purified from the peripheral blood of beagle dogs by positive selection via magnetic separation to a purity of >/=95%. Subsequently, proliferation, and IgG and IgE production of canine B cells were investigated after stimulation with human recombinant Interleukin-4 (hrIL-4) and human recombinant Interleukin-2 (hrIL-2) in the presence or absence of CD40L-CD8 fusion protein (CD40L) of mouse origin. We could demonstrate that canine B cells react on hrIL-2 alone by proliferation and IgG production but not by IgE secretion, whereas activation with hrIL-4 induced proliferation and mainly IgE production. Together, both cytokines synergistically increased B cell proliferation as well as IgG and IgE production. We could also show that mouse CD40L induces proliferation of dog B cells, which is further enhanced by addition of hrIL-4. Unexpectedly, CD40L led to a dramatic decrease in the IL-4 mediated IgE secretion (82% inhibition on an average). In contrast, IgG production was not affected significantly by CD40L. The same effects of CD40L were observed when B cells were stimulated by a combination of IL-2 and IL-4 and this inhibition could not be abrogated by increasing the amounts of IL-4. In summary, activation of canine B cells from peripheral blood by hrIL-4 in the presence or absence of hrIL-2 led to marked IgE production that is strongly and in a dose-dependent manner inhibited by CD40L. Stimulation of IgG production is not influenced by CD40L.

Allergic pulmonary and ocular tissue responses in the absence of serum IgE antibodies (IgE) in an allergic dog model.

Allergen-specific serum IgE may be insensitive as a marker for IgE-mediated reactions at the mucosal level. Five of six atopic beagle dogs developed high ovalbumin (OVA)-specific serum IgE levels after sensitization. This study aimed to show that these dogs still express allergen-specific IgE at the pulmonary and ocular mucosal levels and in the skin even when corresponding serum IgE was below the detection limit. When serum IgE levels were negative, all dogs exhibited allergic reactions at the tissue level. Specifically, they displayed positive ocular reactions after an ocular OVA challenge. After airway challenge with aerosolized OVA, five out of six animals reacted with decreased compliance and increased resistance of the lungs. Furthermore, an eosinophilia in the bronchoalveolar lavage fluid (BALF) was observed. Four weeks after the last exposure to OVA, IgE-positive BALF cells were seen in all animals. Six weeks on, all dogs still displayed positive skin reactions to OVA. This indicates that not only skin testing but also detection of ocular and pulmonary allergic tissue reactions including cell-bound IgE in BALF can serve as more sensitive and lasting surrogate markers of hypersensitivity in the allergic dog model than detection of allergen-specific serum IgE levels.

Protection induced by inactivated influenza virus vaccines with polymethylmethacrylate adjuvants.

Nanocapsules from a copolymer of polymethylmethacrylate and polyacrylamide were tested for adjuvant activity in mouse protection experiments with inactivated influenza virus as antigen. Viruses were either adsorbed on the capsules after polymerization or added to the monomers and incorporated by copolymerization after X-ray initiation. Both preparations showed enhanced immunity as compared to fluid vaccine, if the adjuvant content was 1%. The adjuvant effect was comparable to that caused by the mineral adjuvant Al(OH)3. After dilution of the polymer the adjuvant effect was lost. Such synthetic polymers at suitable concentrations could serve as alternatives to mineral adjuvants.

Viral antigens and impurities in influenza vaccines. Evaluation by immunoelectrophoretic methods.

Laurell electrophoretic methods were used to evaluate internal influenza virus antigens and immunogenic impurities (from the fertile egg) in influenza whole virus and subunit preparations. It could be found that subunit preparations obtained after selective solubilization of the surface antigens with cetyltrimethylammoniumbromide are essentially free from internal structure proteins, ribonucleoprotein, and matrix protein. In addition impurities (non viral antigen substances) are considerably reduced in comparison with whole virus preparations, when the core particle is separated by centrifugation. By testing hyperimmune sera against whole virus and subunit preparations, concerning antibody specificities to host components, it could be demonstrated that whole viruses stimulate more antibodies to the host components, than subunit preparations. This is especially true if antigens are tested which are associated with the chorioallantoic membrane.

Potency of influenza vaccines: mouse protection experiments in correlation to field studies in man.

The seroconversion rates have been studied following vaccination of human volunteers with two commercial influenza vaccines. Vaccine A did not give a significant increase of hemagglutination-inhibition titers. Vaccine B, on the other hand, raised the titers 2- to 8- fold, depending on the pretiters of the individuals. The potency of the same vaccines has been tested using mouse protection experiments: vaccine B gave significantly better protection rates, as measured by survival as well as by reduction of lung lesions. These results give additional evidence that the use of mouse protection experiments for the evaluation of different influenza vaccines is meaningful.

Killer T cell responses to influenza A during a drift period: studies in mice.

After intravenous immunization of mice with any influenza A H3N2 drift strain attempts to restimulation of cytotoxic T cell (CTL) activities with the same virus or other drift period variants were unsuccessful for up to 6 weeks. Cross-stimulation 4-5 months after primary sensitization yielded, in most situations, positive but lower--as compared to primary--secondary cytotoxic T cell responses. Homotypic challenge was also effective after priming with some influenza A subtypes (A/E/72, A/PC/73, A/T/77) at this time.

Protection of mice from mortality caused by living and heat-killed bacteria by SDZ MRL 953.

Protective effects of SDZ MRL 953, a monosaccharidic lipid A analog with a reduced toxicity, were investigated in models of experimental septic shock caused by injections of LPS, and inoculations of heat-killed or live bacteria. Female B6D2F1 mice were challenged with a combination of galactosamine (800 mg/kg) plus various doses of heat-killed isolates of Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus or LPS from Salmonella abortus equi. In some experiments, isolates of living bacteria at sublethal inocula were also combined with galactosamine. More than 90% of the animals died within 24 hr when the challenge was performed either simultaneously with or up to 4 hr after an intraperitoneal administration of galactosamine. No death was observed when galactosamine was omitted or administered after the microbial or LPS challenge. Pretreatment of the animals with SDZ MRL 953 (1-10 mg/kg) rendered the animals resistant to the lethal effects of both bacterial and LPS challenge in a time- and dose-dependent manner. The levels of TNF-alpha in control mice rose to greater than 600 pg/ml 2 hr postbacterial or LPS challenge, but were below detection in animals pretreated with SDZ MRL 953. Protection against both the infection and the toxicity of heat-killed bacteria or LPS was also achieved when murine anti-TNF-alpha monoclonal antibody was administered prophylactically. Together, these data suggest that SDZ MRL 953 enhances the resistance of mice against the toxicity of heat-killed gram-negative bacteria and S. aureus, and attenuates host responses to living bacteria which may lead to irreversible shock and death.

Effect of SDZ MRL 953 on the survival of mice with advanced sepsis that cannot be cured by antibiotics alone.

Stimulation of nonspecific immunity as an additional modality for therapy of sepsis that cannot be cured by antibiotics alone was investigated. SDZ MRL 953, a novel monosaccharidic lipid A analog as a prototype immunostimulant, and cefotaxime or gentamicin were administered to normal or myelosuppressed mice in a state of advanced sepsis caused by Escherichia coli or Staphylococcus aureus. In this novel model, antibiotic therapy was initiated when the infected mice appeared moribund. At this stage, neither pretreatment with the immunostimulant nor therapy with high doses of cefotaxime or gentamicin was effective in protecting the animals from fatal sepsis. However, pretreatment with a single dose of SDZ MRL 953 1 day prior to microbial inoculation dramatically improved the curative effects of the antibiotics. Hence, long-term survival was significantly enhanced with increasing doses of the immunostimulant in the combined therapy. Peritoneal macrophages from SDZ MRL 953-pretreated animals were primed for enhanced production of microbicidal reactive oxygen metabolites in vitro. In conclusion, the results of the present study indicate that SDZ MRL 953 is a potential candidate for use in a clinical setting as an adjunct to antimicrobial therapy for infections that cannot be treated successfully with appropriate antibiotics alone.

Prediction of the role of granulocyte-macrophage colony-stimulating factor in animals and man from in vitro results.

The possibility of predicting the clinical effects of cytokines from in vitro data is discussed, using GM-CSF as an example. GM-CSF incubated with bone marrow cells has been shown to induce proliferation and colony formation, predominantly of the colony-forming unit granulocyte and granulocyte-macrophage types. Daily treatment of normal monkeys with GM-CSF resulted in transient neutropenia followed by neutrophilia. After withdrawal of GM-CSF the neutrophil levels returned to baseline. Predictably, GM-CSF administration results in accelerated neutrophil recovery in patients with chemotherapy-induced neutropenia. GM-CSF has also been shown to induce microbial killing by neutrophils and monocytes in vitro. This activity translated into a dose-related protection of GM-CSF-pretreated mice infected with lethal doses of micro-organisms. Interleukin-3 (IL-3) increases the cellularity of the bone marrow and GM-CSF can induce mobilization of bone marrow cells into the peripheral blood. Therefore, it was predicted and subsequently proved that a combination of these cytokines is synergistic, increasing the yields of peripheral blood progenitor cells which could be collected and then retransplanted into patients undergoing myeloablative chemotherapy. Monkeys injected with recombinant human IL-3 and GM-CSF had increased antibody titres to human IL-3 compared with monkeys given IL-3 alone, suggesting a potential use of GM-CSF which was not predicted from its in vitro results, that of vaccine adjuvancy.(ABSTRACT TRUNCATED AT 250 WORDS)