Modification of T cell antinuclease idiotype expression by in vivo administration of anti-idiotype.

Immunization of BALB/c mice with nuclease leads to the production of anti-nuclease antibodies bearing a set of cross-reactive idiotypes (Id) distinct from those produced by B10.D2 mice after similar immunization. In both strains, such immunization with nuclease also leads to the production of splenic T helper cells (TH), which provide nuclease-specific help in an in vitro plaque-forming cell response to nuclease-TNP. Pig anti-(BALB/c antinuclease) anti-idiotypic antibodies (pig anti-BALB/c Id) react only with TH of nuclease-primed BALB/c and not with B10.D2 animals. After administration of pig anti-BALB/c Id in complete Freund's adjuvant to BALB/c and B10.D2 mice, Id-bearing nonantigen-binding molecules were induced in both strains. Such treatment also resulted in the induction of nuclease-specific splenic TH cells in both strains. BALB/c TH cells induced by anti-Id, like the majority of nuclease-primed BALB/c TH cells, bore BALB/c Id, as shown by their functional elimination with anti-Id plus complement. B10.D2 TH cells induced by anti-Id, unlike TH cells from nuclease-primed B10.D2 mice, also bore BALB/c idiotypic determinants by the same criterion. Thus, it appears that one can manipulate the expression of Id on serum immunoglobulins and on antigen-specific TH cells by administration of exogenous anti-Id reagents. These results have implications both for network interactions in the immune response and for the genetic basis of Igh-C linked Id expression.

Induction of idiotype-bearing, nuclease-specific helper T cells by in vivo treatment with anti-idiotype.

Treatment of BALB/c mice with purified pig anti-(BALB/c anti-nuclease) anti-idiotypic antibodies has been found to induce the appearance of idiotype-bearing immunoglobulins (Id') in the serum of these mice in the absence of detectable antigen binding activity. This phenomenon appeared to require T cells in the hosts because no Id' was detected in the serum of nude mice similarly treated. Furthermore, the spleens of BALB/c mice treated with anti-idiotype were found to contain helper T cells capable of providing help in an in vitro plaque-forming cell response to trinitrophenyl-nuclease equivalent to that provided by helper T cells from the spleens of nuclease-primed animals. Helper T cells from both anti-idiotype-treated and nuclease-treated animals were found to be antigen-specific and to be similarly susceptible to elimination by treatment with anti-idiotype plus complement. Therefore, treatment with both antigen and anti-idiotype appeared to prime similar populations of antigen-specific helper T cells, while having different effects on the induction of antibody. These findings are consistent with the network theory of receptor interactions in the immune response, and may provide a means for studying individual cell populations involved in such interactions.

Clinical trials with human tumor necrosis factor: in vivo and in vitro effects on human mononuclear phagocyte function.

The purpose of this investigation was to understand the biological effects of recombinant human tumor necrosis factor used as therapy for cancer. We studied changes in mononuclear phagocyte function following exposure to this cytokine in vitro or in vivo. Tumor necrosis factor increased phorbol myristate acetate-induced hydrogen peroxide production 8- to 20-fold in peripheral blood monocytes and peritoneal macrophages in vitro in a dose-dependent manner. Similarly, tumor necrosis factor increased phorbol myristate acetate-induced peroxide production 2.3-fold in monocytes isolated from nine patients following an i.v. infusion of this cytokine (40 to 200 micrograms/m2). In addition, tumor necrosis factor induced a 2.3-fold increase in tissue factor-like activity in mononuclear phagocytes in vitro. In vivo, tumor necrosis factor induced a trend toward higher procoagulant activity in monocytes, although this change was not statistically significant. We also noted a trend toward increased activated partial thromboplastin times and the presence of fibrin D-dimer in patients treated with tumor necrosis factor, demonstrating activation of the coagulation and fibrinolytic systems. Thus, in vivo treatment of humans with i.v. recombinant human tumor necrosis factor induced functional changes in mononuclear phagocytes similar to those noted with in vitro treatment.

Biologic response (antiviral) to recombinant human interferon alpha 2a as a function of dose and route of administration in healthy volunteers.

The kinetics of the antiviral effect of intramuscular and intravenous injections of recombinant human interferon alpha 2a were investigated in healthy volunteers. Cohorts of eight to 11 subjects received single intramuscular injections of either 0.3 X 10(6), 3 X 10(6), or 18 X 10(6) U or an intravenous infusion of 18 X 10(6) U over 30 minutes. Serial samples of peripheral blood mononuclear cells were analyzed for antiviral effects including both (2'-5') oligoadenylate synthetase activity and resistance to vesicular stomatitis virus infection in vitro. A dose-response relationship was established between recombinant human interferon alpha 2a dose and both vesicular stomatitis virus resistance and (2'-5') oligoadenylate synthetase activity. At the 0.3 X 10(6) U dose an antiviral effect occurred without clinical side effects. The presence of clinical side effects is not necessary for an antiviral effect.

Interferon kinetics and adverse reactions after intravenous, intramuscular, and subcutaneous injection.

Three groups of six subjects each received a single 36 X 10(6) U dose of recombinant leukocyte A interferon (rIFN-alpha A) as a 40-min infusion, an intramuscular injection, or a subcutaneous injection. Blood samples were collected at specific times after dosing for analysis of rIFN-alpha A serum concentrations by an enzyme immunoassay method, ELISA. The rIFN-alpha A was rapidly distributed and moderately eliminated (t 1/2 = 5.1 hr) after intravenous infusion. The maximum concentrations at the end of intravenous infusion were tenfold the maximum concentrations after intramuscular and subcutaneous injections. Renal tubular secretion or extrarenal elimination was suggested by clearance values of 1.8 times the glomerular filtration rate. After intramuscular and subcutaneous injection, rIFN-alpha A was absorbed slowly (time to reach maximum concentration ranged from 4 to 8 hr), which resulted in prolonged serum concentrations. Estimated bioavailability was more than 80% for both intramuscular and subcutaneous injection shares qualitatively the same adverse reactions, the reactions differ in severity and duration. The adverse effects appear to be related to route of administration of herpes labialis were also noted. There were no significant clinical laboratory abnormalities of medical concern. Although rIFN-alpha A injected by intravenous infusion or intramuscular or subcutaneous injection shares qualitatively the same adverse reactions, the reactions differ in severity and duration. The adverse effects appear to be related to route of administration.

Effects of prednisone, aspirin, and acetaminophen on an in vivo biologic response to interferon in humans.

In healthy volunteers receiving a single intramuscular dose of 18 X 10(6) U interferon alone or after 24 hours of an 8-day course of prednisone (40 mg/day), aspirin (650 mg every 4 hours), or acetaminophen (650 mg every 4 hours), the magnitude of the biologic response to interferon was quantified by measuring the time course of the induction of 2'-5'-oligoadenylate synthetase and resistance to vesicular stomatitis virus infection in human peripheral blood mononuclear cells. Prednisone decreased the AUC of 2'-5'-oligoadenylate synthetase activity (p less than 0.05), whereas administration of aspirin or acetaminophen did not affect this biologic response. No measurable effect was seen during administration of prednisone, aspirin, or acetaminophen on the duration or intensity of vesicular stomatitis virus yield reduction. The side effects seen with interferon administration at the dose tested were not altered in a clinically meaningful manner by prednisone, aspirin, or acetaminophen.

Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients.

This study examined the safety and pharmacokinetic profile of a potentially therapeutic and fully human anti-CMV monoclonal antibody (SDZ MSL-109) in a phase I dose escalation trial in patients receiving allogeneic bone marrow transplants. Fifteen adult marrow transplant patients, twelve with chronic myelogenous leukemia and three with acute nonlymphocytic leukemia, in cohorts of five patients each, were administered monoclonal antibody intravenously at doses of 50, 250, and 500 micrograms/kg at approximately three-week intervals for six months. Administration of the monoclonal antibody was associated with minimal side effects and no dose-related toxicity. Antibody elimination curves in all dose groups were consistent with a two-compartment model with an alpha half-life at the low, middle, and high dose groups of 1.03, 0.82, and 0.79 days, and a beta half life of 13.9, 14.0, and 16.5 days, respectively. The volume of distribution decreased with repetitive dosing to approximate the plasma volume in each patient and the pharmacokinetic profile was comparable to that of human IgG. There was no host antiidiotypic or antiallotypic antibody formation, indicating that MSL-109 was not immunogenic. Further studies are warranted to assess the potential efficacy of human monoclonal anti-CMV disease in marrow transplant recipients and other patients with immunodeficiency disorders.

Human monoclonal anti-cytomegalovirus (CMV) antibody (MSL 109): enhancement of in vitro foscarnet- and ganciclovir-induced inhibition of CMV replication.

Human CMV causes a number of diseases that cause considerable morbidity and that can be life-threatening in immunocompromised patients, particularly those with AIDS. Ganciclovir (GCV) and Foscarnet (PFA) are currently the drugs of choice for management of CMV disease. Both are not without side effects and have a relatively narrow margin of safety. In this report the effects of a human IgG1 neutralizing monoclonal antibody MSL-109 (MSL, Sandoz Pharmaceuticals) on CMV replication was examined both alone or in combination with either GCV or PFA. Human embryonic lung fibroblasts were infected with CMV strain AD169 with a multiplicity of infection of 3 plaque forming units/cell for 1 h. Prior to infection the virus was incubated for 30 min at 37 degrees C with serial concentrations of the MSL Ab (0.1-3.0 micrograms/ml). Concentrations of GCV (0.3 to 30 microM) or PFA (50-400 microM) were added to CMV-infected cells that had been either previously incubated with MSL or not. Four days after infection CMV replication was measured by DNA/DNA probe hybridization using the Hybriwix system. MSL in combination with GCV had an additive effect that was observed at concentrations of GCV of 3-10 microM and MSL of 1-10 micrograms/ml. On the other hand, MSL (3-10 micrograms/ml) together with PFA (100-400 microM) produced a synergistic effect on CMV replication. The data suggest that MSL at doses achievable in humans, enhanced GCV- and PFA-induced antiviral effect in a dose-dependent manner and that the combination might be clinically useful in the treatment of CMV disease.

Safety and tolerability of two percent cyclosporine (Sandimmune) ophthalmic ointment in normal volunteers.

Thirty-six healthy male volunteers were enrolled in two sequential double-masked, placebo-controlled trials with the objective of assessing the safety and local tolerability of 2% cyclosporine ophthalmic ointment. Subjects were randomly assigned to active or placebo groups and dosed once, twice, or thrice daily for 14 days. Safety and tolerability were assessed through patient interviews, ophthalmologic examinations, routine laboratory testing, and blood cyclosporine assays. Relative to placebo, cyclosporine ointment was associated with higher frequencies of ocular burning, tearing, redness, itching, and headache. These intolerances were dose-related and reported predominantly in the TID group; QD and BID cyclosporine ophthalmic ointment were better tolerated than the placebo control. Symptoms were usually mild, were reported only once beyond Day 2 in the QD-BID groups, and never required interruption of the study. Transitory, asymptomatic, and unexplained elevations of serum transaminases were seen in five subjects in the first study, but were not confirmed in the second and are not felt to be drug-related. Cyclosporine blood levels were uniformly below the limits of detection. We conclude that the tolerability profile of 2% cyclosporine ointment, dosed once or twice daily in normal volunteers, is acceptable and supportive of trials in patient populations.

T cell recognition in the mixed lymphocyte response. I. Non-T, radiation-resistant splenic adherent cells are the predominant stimulators in the murine mixed lymphocyte reaction.

The ability of subpopulations of murine spleen cells to stimulate a mixed lymphocyte response (MLR) was studied. It was found that T cells (nylon-nonadherent spleen cells) and B cells [G-10 passed and treated with rabbit anti-mouse brain serum (RAMB) and complement (C)] were poor stimulators of an MLR. In contrast, whole spleen cells or B cells plus adherent cells (RAMB +C-treated spleen cells) produced good stimulation. However, a non-T, radiation-resistant splenic adherent cell (SAC) population was up to 20 to 50 times more efficient as a stimulator of an MLR on a per cell basis than an unseparated spleen population. These SAC were shown to express Ia determinants encoded by genes in I-A and I-E/C. These results suggest that Ia+ SAC may be the predominant stimulating cells in spleen cell populations, and the preferential target for T cell recognition in cell interaction events.

The murine Kupffer cell. II. Accessory cell function in in vitro primary antibody responses, mitogen-induced proliferation, and stimulation of mixed lymphocyte responses.

The ability of murine Kupffer cells to function in several in vitro immunologic systems was investigated. These cells have been shown previously to function as accessory cells in antigen-stimulated T cell proliferation in response to protein antigens. In the present study it has been demonstrated that murine Kupffer cells also are competent as accessory cells in in vitro primary antibody responses to TNP-KLH and for T cell proliferative responses to concanavalin A. In addition, murine Kupffer cells were found to be potent stimulators of mixed lymphocyte responses. These studies extend previous observations by demonstrating that Kupffer cells are competent accessory cells in several distinct in vitro correlates of in vivo immune responses. The role of Kupffer cells in in vivo immune responses, particularly those to enterically derived antigens, may require re-evaluation in the light of these findings.

High-resolution zone electrophoresis, combined with immunofixation, in the detection of an occult myeloma paraprotein.

A high-resolution agarose gel electrophoretic technique, coupled with immunofixation, was used to follow paraprotein concentrations retrospectively in a patient with multiple myeloma of nine years' duration. Although the patient's IgA lambda gammopathy "disappeared" shortly after the initiation of therapy, as judged by routine cellulose acetate electrophoresis and immunoelectrophoresis, high-resolution zone electrophoresis demonstrated a monoclonal band that we identified by immunofixation as the IgA lambda paraprotein. The combination of the two simple, inexpensive, and reliable techniques of high-resolution agarose electrophoresis and immunofixation thereby permitted detection and identification of a myeloma protein in a patient otherwise thought to be in complete remission. We believe this approach is useful in assessing persistent or recurrent disease in patients with a known history of myeloma; this combination of techniques may also prove beneficial in the early diagnosis of multiple myeloma.

The expression and functional involvement of nuclease- specific idiotype on nuclease-primed helper T cells.

The expression and functional significance of idiotypic determinants on antigen-specific helper T (Th) cell populations for responses to Staphylococcal nuclease (Nase) were evaluated in an in vitro antibody response system. Trinitrophenyl (TNP)- specific plaque-forming cell responses to TNP-conjugates of Nase (TNP-Nase) were shown to require the cooperation of Nase-primed Th cells as well as unprimed B and accessory cells. The expression on these antigen-primed Th cells of idiotypic determinants cross-reactive with those on anti-Nase antibodies was demonstrated by the specific elimination of Th cells for TNP-Nase by treatment with affinity-purified anti-idiotypic antibodies plus complement. The susceptibility of Nase-primed Th cells to elimination by such treatment was specific in that anti-idiotypic antibodies affected Th cells only from strains normally expressing the same (or a cross-reactive) idiotype on anti-Nase antibodies. A functional role of the idiotypes expressed on Nase-primed Th cells was suggested by the fact that anti-idiotypic antibody present throughout the period of culture, in the absence of complement, suppressed responses to TNP-Nase in an antigen- and strain-specific manner. It was further shown, by cell mixing experiments, that this inhibition appeared to occur at the level of the Th cells and was not dependent on the strain of origin of the B cells. Thus, antigen-specific Nase-primed Th cells express strain-specific idiotypic determinants cross-reactive with, or identical to, those of anti-Nase antibodies. These cell surface idiotypic determinants appear to be functionally involved in the activity of Th cells for the induction of antibody responses to TNP-Nase in vitro.

T cell recognition in the mixed lymphocyte response. II. Ia-positive splenic adherent cells are required for non-I region-induced stimulation.

Ia-positive splenic adherent cells (SAC) have been shown to be the predominant stimulators of a mixed lymphocyte response (MLR) to whole H-2 differences, in which most of the proliferative response is directed against I region-encoded determinants. The present studies were undertaken to examine the ability of several purified lymphoid subpopulations to activate T cells in response to the non-H-2-linked MIs products or to products of the K or D regions of H-2. The results demonstrated that adherent cell-depleted populations of T and B cells were nonstimulatory, whereas SAC were potent stimulators for responses involving each of these genetic differences. Treatment of these SAC with anti-Ia and C abrogated their MLR-stimulating ability. In contrast, whereas treatment of SAC with anti-Ia and C abrogated their ability to stimulate an MLR directed against K or D region-encoded determinants, this treatment had no effect on their ability to generate a cytotoxic T lymphocyte response against these same determinants. These findings suggest that in addition to presenting allogeneic I region-encoded determinants, Ia-positive SAC also play a unique role in the presentation of non-I region-encoded alloantigens to proliferating T cells.

In vivo administration of tumor necrosis factor-alpha is associated with antiviral activity in human peripheral mononuclear cells.

Tumor necrosis factor-alpha (TNF-alpha) has a spectrum of biologic effects and has been shown to exert antiviral effects in fibroblasts in vitro. The in vivo administration of TNF-alpha (40-160 micrograms/m2 intravenously over 2 hr) and its effects on vesicular stomatitis virus (VSV) replication in peripheral blood mononuclear cells (PBMC) from patients with malignancy was investigated. Blood was obtained before, during, and after infusion. The PBMC were separated and infected with VSV at a multiplicity of infection of 0.005 plaque-forming units/cell and virus yields were determined 72 h later. The TNF-alpha inhibited VSV yields by as much as 99% in a dose-dependent manner with the inhibition initially observed during the first hour of infusion. Despite a rapid reduction in TNF-alpha serum levels, the higher doses still produced antiviral effects 4 hr after the infusion. Sera obtained at identical times had no interferon activity. Human gamma-interferon (25 micrograms/ml) added in vitro augmented the TNF-alpha-induced inhibitory activity in both magnitude and duration. Percentages of lymphocytes and monocytes in peripheral blood were reduced at 4 hr after TNF-alpha administration and the monocyte to lymphocyte ratio was diminished and temporally coincided with the loss of TNF-induced antiviral state. These data suggest that the in vivo administration of TNF has a direct inhibitory activity on VSV replication in human peripheral blood mononuclear cells that was enhanceable by gamma-interferon and possibly monocyte mediated.

Genetic control of immune response to staphylococcal nuclease. XII: Analysis of nuclease antigenic determinants using anti-nuclease monoclonal antibodies.

SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG1, kappa isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1-126 and 49-149 but failed to bind to fragment 99-149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.

A phase I pharmacokinetic study of recombinant human tumor necrosis factor administered by a 5-day continuous infusion.

Nineteen patients with advanced cancer were entered into a phase I clinical trial of Tumor Necrosis Factor (TNF) which was designed to determine the pharmacokinetic profile, safety, and maximal tolerated dose (MTD) of the recombinant human cytokine in vivo. TNF was administered by continuous infusion for 24 hours followed by pharmacokinetics and a 120-hour infusion repeated every 3 weeks. The initial dose was 40 micrograms/m2 and was ultimately escalated to 200 micrograms/m2. A total of forty 5-day cycles were administered to 18 of these patients; and all were evaluable for toxicity. Toxicities in this trial included fever, chills, rigors, hypotension, headaches, seizures, lethargy, weight loss, and malaise. At all dose levels, but more significantly at the highest doses, hematological toxicities were observed and grade 3 neurotoxicity (headache and confusion), and hypotension were noted. Two patients expired during the study, and this was felt to be related to septic episodes. Because of these severe toxicities, 160 micrograms/m2 was defined as the MTD. At 160 micrograms/m2 peak serum levels occurred within 5-20 minutes of initiation and were not detectable 1 hour later. No anti-tumor responses were observed. No measurable plasma levels of TNF were observed with the administration of doses of 80 micrograms/m2. This dose level could be further studied in phase II studies alone and in combination with other agents, utilizing a continuous infusion schedule.

Therapy of genital herpes with topically applied interferon.

Ninety-four patients with recurrences of genital herpes were randomized in a double-blind trial to receive topical therapy for 5 days with either alpha-2a interferon at 30 X 10(6) IU/ml or 10 X 10(6) IU/ml or placebo six times daily. No differences were noted between either interferon dose and placebo with respect to the duration of viral shedding, the time to crusting, or the time to healing of herpetic lesions. Aqueous solutions of alpha-2a interferon applied topically to unroofed vesicles do not appear to be clinically useful in the treatment of recurrences of genital herpes.

Time course of interferon levels, antiviral state, 2',5'-oligoadenylate synthetase and side effects in healthy men.

The kinetics of the biologic response following a single intramuscular injection of 18 X 10(6) units of recombinant human interferon-alpha 2a (rHuIFN-alpha 2a) was investigated during 11 courses in 10 healthy individuals. Serial peripheral blood mononuclear cell (PBM) samples were assayed for their biologic responsiveness to rHuIFN-alpha 2a by measuring both their 2',5'-oligoadenylate (2-5A) synthetase activity and their resistance to in vitro vesicular stomatitis virus (VSV) infection. A significant increase in 2-5A synthetase levels occurred at 6 h, and enzyme levels returned to baseline values between 96 and 104 h postinjection. Protection of PBMs from VSV infectivity began within 1 h and lasted up to 144 h postinjection. The clinical side effects induced by IFN administration and serum IFN levels were not parallel over time with the antiviral effects observed. This study defines the time course of the biologic response induced by rHuIFN-alpha 2a in healthy volunteers. A parallel time course between the induction of 2-5A synthetase activity and the development of the antiviral state in PBMs was demonstrated.