Repetitive intrathecal injection of human NMO-IgG with complement exacerbates disease severity with NMO pathology in experimental allergic encephalomyelitis mice.

Neuromyelitis optica (NMO) is recognized as a different CNS autoimmune disease from multiple sclerosis (MS). Whether NMO-IgG contributes directly to the pathogenesis of NMO or is just a serologic marker of autoimmune responses of the disease needs to be clarified. We created MOG-induced experimental autoimmune encephalomyelitis (EAE) mice by passively transferring NMO-IgG to model the pathogenic findings in NMO patients. The mice were divided into three groups and administered intrathecal PBS, human complement with IgG from normal subjects, or IgG from AQP4(+) patients on days 8 and 11 after immunization. The EAE scores of EAE mice with intrathecal NMO-IgG injection were significantly elevated 14 days post-immunization. All of the mice were sacrificed for brain and spinal cord pathology analysis on day 21 post-immunization. Compared to mice given normal human IgG, EAE mice injected with NMO-IgG had markedly decreased AQP4 and glial fibrillary acidic protein (GFAP) expression and fluorescent intensity in the brain and spinal cord but more scattered deposition of complement (C9neo). Thus, our studies not only support the pathogenic role of NMO-IgG with complement in NMO disease but also provide a platform for the development of future therapeutics.

Suppression of tumor growth by a heterologous antibody directed against multiple myeloma dominant CD38 antigen in SCID mice injected with multiple myeloma cells.

Employing passive immunization - using a heterologous anti-CD38 IgG antibody containing serum - in SCID mice injected subcutaneously with human multiple myeloma cells, we have shown that treatments with the antiserum - especially in the presence of complement - significantly decreased cancer growth. However, administered antibody and complement was not sufficient in amount to prevent cancer cell multiplication and cancer growth expansion to a satisfactory degree. Larger volumes of the same components more than likely would have further reduced cancer growth and prolonged the life of mice. In control mice, cancer growth progressed faster proving that lytic immune response against multiple myeloma cells is necessary for cancer cell kill.

Complement deficiency states and associated infections.

A major function of the immune system is to protect the host from microbial infections. The complement system plays important roles in both the innate and the adaptive immune defense and also acts as a bridge between these arms of immunity. This is obvious from complement deficiencies which in varying degree, depending on which factor is missing, are associated with increased infection susceptibility and also increased risk for other, mainly autoimmune diseases. Genetically determined deficiencies are described for almost all complement proteins but the consequences show a wide variation. Here the genetic defects and molecular abnormalities in complement deficient persons, related clinically relevant infections and the options for prevention and therapy are reviewed. The roles of complement in host defense against common infections are also discussed.

T cell deficiency does not reduce lesions in mice produced by intracerebral injection of NMO-IgG and complement.

We reported recently that intracerebral administration of NMO-IgG with human complement produces neuromyelitis optica (NMO) lesions in mice. We examined the role of T cells in the formation of NMO lesions by comparing brain histopathology in wildtype and nude mice. Brains were co-injected with IgG from NMO patients and human complement. At 24h and 5days, wildtype vs. nude mouse brains had comparable inflammation (CD45 immunoreactivity), loss of myelin (Luxol Fast Blue staining) and loss of AQP4 immunoreactivity. We conclude that T cells are not required for the formation of NMO lesions in this mouse model.

Intra-cerebral injection of neuromyelitis optica immunoglobulin G and human complement produces neuromyelitis optica lesions in mice.

Neuromyelitis optica is an inflammatory demyelinating disease of the central nervous system associated with autoantibodies against the glial water channel protein aquaporin-4. It has recently been reported that immunoglobulin from neuromyelitis optica patients injected peripherally does not cause lesions in naive rats, but only when pre-existing central nervous system inflammation is present. Here, we investigated whether immunoglobulin G from aquaporin-4-autoantibody-positive neuromyelitis optica patients has the potential to damage the central nervous system either alone or in the presence of human complement. Immunoglobulin G from neuromyelitis optica patients did not activate mouse complement and was not pathogenic when injected into mouse brain. However, co-injection of immunoglobulin G from neuromyelitis optica patients with human complement produced neuromyelitis optica-like lesions in mice. Within 12 h of co-injecting immunoglobulin G from neuromyelitis optica patients and human complement, there was a striking loss of aquaporin-4 expression, glial cell oedema, myelin breakdown and axonal injury, but little intra-parenchymal inflammation. At 7 days, there was extensive inflammatory cell infiltration, perivascular deposition of activated complement components, extensive demyelination, loss of aquaporin-4 expression, loss of reactive astrocytes and neuronal cell death. In behavioural studies, mice injected with immunoglobulin G from neuromyelitis optica patients and human complement into the right hemisphere preferentially turned to the right at 7 days. No brain inflammation, demyelination or right-turning behaviour was seen in wild-type mice that received immunoglobulin G from non-neuromyelitis optica patients with human complement, or in aquaporin-4-null mice that received immunoglobulin G from neuromyelitis optica patients with human complement. We conclude that co-injection of immunoglobulin G from neuromyelitis optica patients with human complement reproduces the key histological features of neuromyelitis optica and that aquaporin-4 is necessary and sufficient for immunoglobulin G from neuromyelitis optica patients to exert its effect. In our mouse model, immunoglobulin G from neuromyelitis optica patients does not require pre-existing central nervous system inflammation to produce lesions.

Formation of complement membrane attack complex in mammalian cerebral cortex evokes seizures and neurodegeneration.

The complement system consists of >30 proteins that interact in a carefully regulated manner to destroy invading bacteria and prevent the deposition of immune complexes in normal tissue. This complex system can be activated by diverse mechanisms proceeding through distinct pathways, yet all converge on a final common pathway in which five proteins assemble into a multimolecular complex, the membrane attack complex (MAC). The MAC inserts into cell membranes to form a functional pore, resulting in ion flux and ultimately osmotic lysis. Immunohistochemical evidence of the MAC decorating neurons in cortical gray matter has been identified in multiple CNS diseases, yet the deleterious consequences, if any, of MAC deposition in the cortex of mammalian brain in vivo are unknown. Here we demonstrate that the sequential infusion of individual proteins of the membrane attack pathway (C5b6, C7, C8, and C9) into the hippocampus of awake, freely moving rats induced both behavioral and electrographic seizures as well as cytotoxicity. The onset of seizures occurred during or shortly after the infusion of C8/C9. Neither seizures nor cytotoxicity resulted from the simultaneous infusion of all five proteins premixed in vitro. The requirement for the sequential infusion of all five proteins together with the temporal relationship of seizure onset to infusions of C8/C9 implies that the MAC was formed in vivo and triggered both seizures and cytotoxicity. Deposition of the complement MAC in cortical gray matter may contribute to epileptic seizures and cell death in diverse diseases of the human brain.

Synergism between fludarabine and rituximab revealed in a follicular lymphoma cell line resistant to the cytotoxic activity of either drug alone.

We have shown previously that the anti-CD20 chimaeric monoclonal antibody rituximab exerts its effects on neoplastic B-lymphoma cell lines in part via complement-dependent cytotoxicity. In addition, membrane expression levels of complement inhibitory proteins CD55 and CD59 play a role in determining susceptibility to lysis. We have identified one t(14;18)-positive human B-cell non Hodgkin's lymphoma cell line (Karpas 422) that is resistant to rituximab and complement and used it for subsequent studies on the possible interaction between this novel therapeutic agent and established antineoplastic drugs. We have exposed Karpas to several chemotherapeutic agents (doxorubicin, idarubicin, cisplatin, taxol) for different time periods and subsequently exposed the cells to rituximab and human complement. The combination of these drugs with rituximab induced an additive cytotoxic effect. In contrast, exposure to fludarabine (1 microg/ml for 48-72 h) showed a synergistic effect, with cell lysis increasing from 10% to 20% using fludarabine or rituximab and complement alone to about 70% with both cytotoxic agents. Analysis of the mechanism for this synergistic effect showed that fludarabine downmodulates the membrane expression of CD55 (from 96% to 55% positive cells) without significantly altering CD20 levels. Northern analysis demonstrated that fludarabine induced a general downmodulation of steady state mRNA levels with no change in transcription rate detected in run-off assays. The study of the effect of fludarabine and rituximab in six freshly isolated B-cell chronic lymphocytic leukaemia (B-CLL) samples showed that, in most cases, fludarabine has an additive cytotoxic activity with rituximab and complement. This report gives a rational support for clinical studies with combinations of drugs, including monoclonal antibodies and fludarabine.

Immunomodulation in progeny from thymectomized primiparous mice exposed to benzo(a)pyrene during mid-pregnancy.

Previous studies have shown that Benzo(a)pyrene (B(a)P3) given to non-thymectomized (NTX) female mice alters expression of T cell subsets and suppresses cell mediated immunity (CMI) and humoral immunity (HI) in the progeny. Thus, maternal exposure to B(a)P may influence changes in progeny immune status. To understand how maternal cellular and humoral factors influence embryonic development of progeny immunity, adult female mice were thymectomized (TX) at 6 weeks, mated and injected with 150 microg B(a)P)/g body weight at 12 days of pregnancy. After B(a)P exposure, the following studies were performed: (A) Maternal reproductive capacity and survival rate of progeny; (B) Detection of T cells in progeny thymus; (C) Functional characteristics of progeny thymus or spleen. Maternal thymectomy and B(a)P exposure reduced average litter size by 40%. Serological sensitivity of thymus cells with anti-Thyl + complement occurred at a higher dilution of mAb in progeny from TX mothers exposed to B(a)P, suggesting that B(a)P-thymectomy led to increased sensitivity of developing thymocytes to mAb plus complement. Progeny from TX mothers exposed to B(a)P showed enhanced thymic CMI, but suppressed splenic CMI and HI. Thus, thymectomy prevents CMI immunosuppression by B(a)P, while HI is still suppressed. These results indicate that the maternal thymus is necessary for incurring the effect of B(a)P on progeny CMI.

In vitro and in vivo responses of murine granulocytes to human complement-derived, haemolytically inactive C5b67 (iC5b67).

Haemolytically inactive C5b67 (iC5b67), which was made from purified human components and decayed to a haemolytically inactive form, was evaluated as an agonist for murine leucocytes both in vitro and in vivo. In an in vitro assay, iC5b67 stimulated chemotaxis for both neutrophils purified from mouse bone marrow and splenic eosinophils of IL-5 transgenic mice. The stimulation was dose-dependent, with high dose inhibition. As with human neutrophils, iC5b67 also failed to up-regulate CR3 (CD11b/CD18) expression and to stimulate superoxide generation in murine bone marrow neutrophils, in vitro. In vivo, iC5b67 elicited an inflammatory response in a mouse model of pleuritis. A marked infiltration of neutrophils, which peaked at 4 h, was followed by an infiltration of eosinophils and mononuclear leucocytes. This inflammatory response was dose- and time-dependent. However, the protein concentration in the pleural wash fluid did not increase, indicating that iC5b67 did not induce a capillary leak. Although the infiltration of neutrophils could not be reproduced by pure C7 or human serum albumin (HSA), C5b6 did induce an influx of neutrophils. We were able to document the existence of C7, both antigenically and functionally, in pleural washes of normal mice, making it likely that the activity of C5b6 resulted from the in situ formation of C5b67 and iC5b67. The mouse model of pleuritis promises to be a useful in vivo system in which to evaluate the pro- and anti-inflammatory effects of iC5b67 that have been noted in vitro.

Pre-clinical evaluation of anti-lacto-N-fucopentaose III (CD15) monoclonal antibodies for ex vivo bone marrow purging in acute myeloid leukemia.

In order to eliminate residual leukemic cells from the marrow of patients with acute myeloid leukemia (AML) prior to autologous bone marrow transplantation, the optimal conditions of utilization of three CD15 murine monoclonal antibodies (MoAb) were investigated. The VIM-D5 MoAb was used with rabbit complement (C'), whereas the 8.27 and SMY15A MoAbs were used in the presence of human C'. These antibodies were also tested after fixation on magnetic beads. In a culture assay in semi-solid medium with a mixture of normal marrow and 1% HL60 cells, a lysis of clonogenic cells greater than 99% was achieved with the three antibodies and two rounds of complement, or with antibody-coated magnetic beads. Cultures of leukemic clonogenic cells (CFU-L) were performed in 47 cases. An inhibition equal to or greater than 90% was achieved in seven cases with VIM-D5, 16 cases with 8.27 and 11 cases with SMY15A and C'. The correlation with cytotoxicity of fresh cells was low. Twenty cases were purged with antibody-coated beads. An inhibition equal to or greater than 90% was observed in 10 cases with VIM-D5, 11 cases with 8.27 and 12 cases with SMY15A. The mean recovery of normal CFU-GM was higher than 70% and that of BFU-E higher than 95% with any method of treatment. It is concluded that efficient marrow purging of clonogenic AML cells can be achieved in some cases without toxicity for normal progenitors. The addition of other MoAbs seems necessary to obtain a significant purge in a majority of cases.

In vitro comparison of two methods of T cell depletion associated with different rates of graft failure after allogeneic marrow transplantation.

Clinical trials in another center have shown a substantially lower risk of graft failure associated with T cell depletion by treatment of donor marrow with the use of an antibody against CD6 compared to depletion with a mixture of eight antibodies previously used for clinical trials in our center. In order to evaluate mechanisms possibly responsible for this difference, we compared lymphoid cell surface phenotypes and in vitro functions in marrow cells treated by complement-mediated lysis with anti-T12 (CD6) or with the eight antibody mixture. Treatment with the eight antibody mixture produced more than three log depletion of precursors for IL-2-producing cells (pIL-2) and approximately one log depletion of precursors for NK cells. On the other hand, treatment with anti-T12 produced approximately one log depletion of pIL-2 and had no effect on NK precursors. Additional studies were carried out with treated marrow cells cultured in medium containing recombinant IL-2. Compared to cells treated with the eight antibody mixture, the marrow cells that remained after anti-T12 treatment had more cytotoxic activity against K562, Daudi and an EBV-transformed human B cell line during the first 6 days of culture, but marrows treated by the two methods showed similar cytotoxic activity after 10 days of culture. Cultures from marrow treated with anti-T12 contained more CD3+ and CD6+ cells than cultures from marrow treated with the eight antibody mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Early induction of immune resistance against leukemia in lethally total body irradiated mice reconstituted with syngeneic bone marrow cells obtained from previously immunized donor mice.

BALB/c x DBA/2 F1 (CD2F1) mice were lethally irradiated and reconstituted with syngeneic bone marrow cells (SBMC) obtained from normal or previously immunized (against L1210 lymphatic leukemia) donors. These recipient mice are called TBI + SBMT or TBI + Imm-SBMT mice, respectively. TBI + Imm-SBMT, but not TBI + SBMT mice, were able to develop strong immune resistance against L1210 leukemia, but not against MOPC 104E plasmacytoma, if the immunization procedure (four i.p. injections at weekly intervals of immunogenic L1210 cells) was started as early as 7 days posttransplantation. Incubation of Imm-SBMC with mafosfamide (ASTA Z7654) before grafting abrogated the ability of the recipient mice to develop early resistance against the leukemia. Treatment of Imm-SBMC with monoclonal or polyclonal antibodies plus complement showed that two or three subpopulations of Imm-SBMC were necessary for the transfer of immune information against leukemia: T lymphocytes with phenotype Thy 1.2+, Lyt 1+2-, I-Ad-, macrophages with phenotype Mac-1+, I-Ad-, and probably asialo-GM 1+ cells. Recipient mice immunized against L1210 leukemia before TBI + SBMT do not develop early resistance to the leukemia.

Prostaglandin production in chronic progressive multiple sclerosis.

Peripheral blood monocytes have been implicated in the immune reactions that accompany demyelination in patients with multiple sclerosis (MS). We measured prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) release from peripheral monocytes exposed in vitro to complement. Our studies suggest that there is a significantly higher production of PGE2 in monocytes from patients with chronic progressive MS than in those with exacerbation or remitting MS and healthy controls. No significant differences in TxB2 release were noted between the three groups.

Continuation of chemotherapy versus supportive care alone in patients with inoperable non-small cell lung cancer and stable disease after two or three cycles of MACC. Results of a randomized prospective study.

Recommendations concerning the continuation of chemotherapy in nonresponding patients with non-small cell lung cancer (NSCLC) and stable disease after the first chemotherapy test are empirical and often conflicting. Between 1984 and 1987, 116 inoperable NSCLC patients were treated with methotrexate, doxorubicin, cyclophosphamide, and lomustine (MACC regimen). After two or three cycles of therapy, 74 patients were judged to have stable disease and assigned at random either to chemotherapy maintenance with the same regimen (38 subjects) or to chemotherapy discontinuation (36 subjects). The two study groups were comparable for all the major prognostic factors. Median time to progression was 26 weeks for the chemotherapy group compared to 24 weeks for the nonchemotherapy group (P = NS). Median survival was prolonged in the treatment arm (47 weeks) compared to nontreatment arm (30 weeks), which was statistically nonsignificant. A patient self-assessment of the quality of life revealed a significantly worse tolerance to therapy and a better physical condition in the chemotherapy group. Objective MACC toxicity was significant with two treatment-related deaths. This study failed to demonstrate sufficient therapeutic benefits to justify the increased cost and toxicity of continuing treatment in nonresponding NSCLC patients.

Autologous bone marrow transplantation in non-Hodgkin's lymphoma: monoclonal antibodies plus complement for ex vivo marrow treatment.

PURPOSE: Patients with non-Hodgkin's lymphoma who fail to achieve a complete remission or who relapse are rarely cured with conventional therapies. For this group of patients, intensive therapy and bone marrow rescue may be curative. Our goal was to assess the effects of autologous transplantation in patients with B-cell lymphomas and a poor prognosis. To avoid occult or overt contamination with lymphoma, monoclonal antibodies BA-1, BA-2, and BA-3 were used for ex vivo marrow treatment. PATIENTS AND METHODS: Seventeen patients underwent intensive therapy and autologous bone marrow transplant using the aforementioned marrow. Ten of the 17 patients (Group I) had disease that was in complete or partial remission. The other seven patients either had disease that was not responsive to treatment or had bone marrow transplant as their initial therapy at relapse. RESULTS: The ex vivo treatment did not adversely affect engraftment. For those patients who could be evaluated, the median time to white cell engraftment was 24 days; the median durations of red cell and platelet support were 24 and 29 days, respectively. Eleven of the 17 patients had complete remissions at the evaluation 28 days after transplant. Three patients subsequently experienced a relapse, three died while their disease was in complete remission, and five are alive and disease-free 405 to 1,674 days after transplant. Group I patients had an estimated 40 percent disease-free survival rate at three years compared with 0 percent for Group II patients (p less than 0.01). CONCLUSION: Our data support autologous bone marrow transplantation as an important treatment modality for the non-Hodgkin's lymphomas. With the current preparative regimens available, however, its use should be limited to patients with disease that is still responding to conventional therapies.

Current chemotherapy of small cell lung cancer.

Since the advent of effective cytotoxic combinations in the early 1970s, results from chemotherapy for small cell lung cancer have improved very little. Maintenance chemotherapy appears of no benefit. Although attractive theoretically, "non-cross-resistant" combinations may not yet exist, and most data do not support alternating 1 regimen with another. Anticoagulant therapy with warfarin probably does not have a meaningful impact on survival, at least in extensive stage disease. To date the addition of VP-16, an active new agent, has not produced improvement in survival over earlier programs. The most promising leads to date involve dose escalation, especially with cyclophosphamide. Moderate "outpatient" escalation in limited disease induction therapy produced survival benefit in a randomized trial, and several studies indicate that the incidence of complete response can be increased by more intensive, inpatient "consolidation" with cyclophosphamide with or without other drugs after the induction period. Some form of local therapy, however, will be necessary to control disease in the chest, even with maximal dose intensification.

A controlled evaluation of combined 5-fluorouracil, doxorubicin, and mitomycin C (FAM) for the treatment of advanced non-small-cell lung cancer.

One hundred eighty-six patients with advanced non-small-cell lung cancer were randomly assigned to treatment with combined 5-fluorouracil, doxorubicin, and mitomycin C (FAM) or combined methotrexate, doxorubicin, cyclophosphamide, and lomustine (MACC). Respective objective regression rates were comparable at 20% and 16%. Distribution of intervals to progression (overall median, 2.8 months) and survival times (overall median, 5.0 months) were essentially identical between the two regimens. The comparability of therapeutic effect was also evident within the subset of 81 patients who had adenocarcinoma cell type, although MACC showed a small advantage in survival after covariate analysis. In large-cell carcinoma, MACC showed a higher regression rate than that of FAM as well as a small advantage in survival. In squamous-cell carcinoma, however, FAM was superior to MACC in regression rates (32% v 4%) and also provided somewhat longer survival. With regard to toxicity, MACC produced a higher incidence of nausea and vomiting, whereas FAM produced more frequent and severe thrombocytopenia. From an overall standpoint, the therapeutic accomplishments of both regimens were disappointing. Our study does, however, provide additional evidence that mitomycin C-containing regimens may be selectively effective for squamous-cell carcinoma of the lung.

Role of inflammatory neutrophils in antitumor effects induced by intraperitoneal administration of Corynebacterium parvum in mice.

We studied the role of inflammatory neutrophils in the antitumor effects that follow i.p. injection of Corynebacterium parvum (1400 micrograms) into C3HeB/FeJ mice challenged with the murine ovarian teratocarcinoma. Peritoneal neutrophils, obtained from mice 6 hr after injection of C. parvum, exerted significant antitumor effects when injected admixed with murine ovarian terato-carcinoma cells into the peritoneal cavities of normal mice. Treatment of recipient mice with whole-body irradiation or repeated injections of silica prevented the antitumor effects, indicating that neutrophils were activating a second effector mechanism in recipient mice. Peritoneal cells obtained at 24 or 72 hr or at 7 or 11 days following C. parvum injection were considerably less effective in activation of this effector mechanism. Heat-killed C. parvum (6 hr)-induced neutrophils activated antitumor responses, but thioglycolate-induced cells were without effect. Antitumor responses in mice receiving peritoneal neutrophils were not due to simple transfer of C. parvum organisms in the inocula. These results indicate that inflammatory neutrophils, elicited into the peritoneal cavity by injection of C. parvum, play an important role in the activation of subsequent antitumor effects.

In vivo reduction of NK activity with anti-NK 1 serum: direct evaluation of NK cells in tumor clearance.

We have developed a new model to test the in vivo functions of NK cells. Injection of B6 mice with as little as 25 microliter of an NK-specific alloantiserum, anti-NK 1.1, significantly reduced the recipient's NK activity. The reduction, monitored in vitro as a decrease in the ability of spleen cells (SC) to lyse 51Cr-labelled YAC-1 target cells, occurred rapidly, within 2 h of administration of the anti-NK 1.1 serum. NK activity gradually returned to control levels but still was significantly depressed at 48 h. Comparable decreases were observed whether the serum was injected by the intravenous (i.v.) or the intraperitoneal (i.p.) route. Injection of the mice with exogenous complement (newborn rabbit serum) did not significantly increase the antiserum's effect. To test the requirement for NK cells in tumor clearance in vivo, mice were pre-treated with anti-NK 1.1 serum and subsequently injected i.v. with 125IdUrd (5-iodo 2'deoxyuridine)-labelled YAC-1 or RBL-5 lymphoma cells. Tumor cell clearance in lungs, liver and spleen was reduced two to four-fold in the anti-serum-treated mice compared to injection controls. These results provide direct evidence that NK cells are involved in the elimination of tumor cells in vivo.