Chemoimmunotherapy reduces the progression of multiple myeloma in a mouse model.

Multiple myeloma (MM) is a B-cell malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow. Recently, we showed a correlation between increased ratios of functional regulatory T cells (Treg) and disease progression in a unique mouse model that mimics the human disease. Cyclophosphamide (CYC) is a cytotoxic alkylating agent widely used in chemotherapeutic regimens. Low-dose CYC was previously reported to selectively reduce Treg levels and to contribute to immunostimulation. Our objectives were (a) to determine whether treatment using a low-dose CYC could reduce MM progression and (b) to further characterize the modes of action underlying these effects. We found that both low- and high-dose CYC given to sick mice with hind limb paralysis resulted in the disappearance of the paralysis, the replacement of plasma tumor cells in the bone marrow by normal cell populations, and a significant prolongation of survival. However, only low-dose CYC treatment decreased the incidence of MM. Low-dose CYC rendered Tregs susceptible to apoptosis because of the downregulation of Bcl-xL and CTLA-4 in these cells, and a decreased production of interleukin 2 by effector CD4 cells. Moreover, using this treatment, we noted the recovery of IFN-γ-producing natural killer T cells and maturation of dendritic cells. Treatment of tumor-bearing mice with repeated administrations of low-dose CYC at longer time intervals (coinciding with the blocked renewal of Tregs) resulted in reduced tumor load, and the prevention or delay of disease recurrence, thereby breaking immune tolerance against MM tumor cells.

Amelioration of brain pathology and behavioral dysfunction in mice with lupus following treatment with a tolerogenic peptide.

OBJECTIVE: Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations. METHODS: Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test. RESULTS: Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests. CONCLUSION: Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.

A daidzein-daunomycin conjugate improves the therapeutic response in an animal model of ovarian carcinoma.

The use of daunomycin against neoplasms is limited due to its severe cardiotoxicity. The cytotoxicity of daunomycin can be minimized by linking it to an affinity tag. Since ovarian cancer cells are sensitive to isoflavone action, we synthesized a daidzein daunomycin conjugate. In MLS human ovarian cancer cells, the conjugate was shown to have a larger cytotoxic effect than daunomycin per se at a low concentration. The conjugate was then tested in vivo in mice carrying MLS xenografts. Tumour growth in the groups of conjugate and daunomycin was inhibited by >50% as compared to vehicle treated mice. In contrast to daunomycin treated mice, no weight reduction or death was seen in mice treated with the conjugate. In vivo imaging of the fluorescence signal generated by daunomycin indicated uptake of both conjugate and daunomycin by the tumour. Tumour fluorescence was, however, higher in the conjugate treated mice than in the daunomycin treated mice, thus suggesting specific delivery of the drug to the tumour. Histological examination of myocardial tissue indicated that only the daunomycin, but not conjugate treated mice showed cardiac damage. These results indicate that targeting of daunomycin via carboxymethyldaidzein retains daunomycin's cytotoxic effects while averting its toxicity in an ovarian xenograft.

Peptides based on the complementarity-determining regions of a pathogenic autoantibody mitigate lupus manifestations of (NZB x NZW)F1 mice via active suppression.

Two peptides based on the complementarity-determining regions (CDR) 1 and 3 (pCDR1 and pCDR3) of a murine monoclonal anti-DNA autoantibody that expresses the common idiotype 16/6Id were shown to down-regulate systemic lupus erythematosus (SLE)-associated T cell responses and to prevent the development of clinical symptoms in the SLE-prone mice, (NZB x NZW)F(1). In the present study the ability of the CDR-based peptides to treat an already established disease was tested. Mice were given 10 weekly injections of peptides either i.v. or s.c. The treatment led to a moderate reduction in the anti-DNA autoantibody titer, and a significant decrease in proteinuria and kidney pathology. The CDR-based peptides affected the pathogenic isotypes (IgG2a and IgG3) of the anti-DNA antibodies in the serum and in immune complexes in the kidneys. Both peptides mitigated disease manifestations and prolonged the survival of mice that were treated starting at the age of 7 months when full-blown disease was already developed. Furthermore, some beneficial effects of treatment with the CDR-based peptides could be adoptively transferred to diseased recipients. A reduction in the secretion of IL-2, IFN-gamma, IL-4 and IL-10 was detected in supernatants of splenocytes of the treated mice. In contrast, treatment up-regulated the immunosuppresive cytokine-transforming growth factor-beta. Thus the ameliorating effect of the CDR-based peptides on SLE manifestations is at least partially via the immunomodulation of the cytokine profile.

Exogenous Dp71 is a dominant negative competitor of dystrophin in skeletal muscle.

Dystrophin, the protein which is absent or non-functional in Duchenne muscular dystrophy, consists of four main domains: an N-terminal actin binding domain, a rod shaped domain of spectrin-like repeats, a cysteine-rich domain and a unique C-terminal domain. In muscle, dystrophin forms a linkage between the cytoskeletal actin and a group of membrane proteins (dystrophin associated proteins). The N-terminal domain binds to the cytoskeletal actin and the association with the dystrophin associated proteins is mediated mainly by the cysteine-rich and C-terminal domains of dystrophin. The dystrophin gene also encodes two isoforms of non-muscle dystrophins and a number of smaller products consisting of the two C-terminal domains with different extensions into the spectrin-like repeat domain. Dp71, which consist of the C-terminal and the cysteine-rich domains of dystrophin, is the major product of the gene in all non-muscle tissues tested so far, but it is absent in differentiated skeletal muscle. In an attempt to understand the functions of Dp71, we produced transgenic mice over-expressing this protein in several tissues. The highest levels of exogeneous Dp71 were detected in skeletal muscle, in association with the sarcolemma. This resulted in muscle damage similar to that found in mice which lack dystrophin. The data indicates that Dp71 competes with dystrophin for the binding to the dystrophin associated proteins. Since Dp71 lacks the actin binding domain, it cannot form the essential linkage between the dystrophin associated proteins complex and the cytoskeleton.

4-Nitrobenzylidene malononitrile reduces apoptosis-mediated liver injury in mice.

BACKGROUND: Apoptosis plays a role in experimental and clinically related liver damage. Inhibitors of tyrosine kinases were shown to modulate apoptosis induced by different agents in various cell types. AIMS: Investigation of the effect of 4-nitrobenzylidene malononitrile (belonging to the tyrphostins family which are selective inhibitors of protein tyrosine kinases) on apoptosis-mediated acute liver injury. METHODS: Two murine experimental models exhibiting apoptosis-mediated liver injury were used: (1) mice treated with tumor necrosis factor-alpha and D-galactosamine; and (2) mice treated with anti-Fas antibody. Liver injury was assessed by serum levels of transaminases and by microscopic analysis. Apoptosis was assessed by labeling of apoptotic cells in the liver by the TUNEL assay and by determination of caspase-3 activity. RESULTS: Pretreatment of mice with 4-nitrobenzylidene malononitrile reduced tumor necrosis factor-alpha/D-galactosamine-induced hepatotoxicity. TUNEL positive cells in sections from livers treated with vehicle (control), 4-nitrobenzylidene malononitrile, tumor necrosis factor-/d-galactosamine and tumor necrosis factor-alpha/D-galactosamine and 4-nitrobenzylidene malononitrile, were >0.2, >0.2, 49+/-2.3 and 4+/-0.2 per mm(2), respectively. 4-Nitrobenzylidene malononitrile also reduced hepatotoxicity induced by anti-Fas antibody. Caspase-3 activation induced by either tumor necrosis factor-alpha/D-glactosamine or by anti-Fas treatment, was reduced by pretreatment with N-nitrobenzylidene malononitrile. CONCLUSIONS: The findings may provide a base for development of a new therapeutic modality to reduce apoptosis-mediated liver damage.

Oral treatment of mice with copolymer 1 (glatiramer acetate) results in the accumulation of specific Th2 cells in the central nervous system.

Mucosal administration of copolymer 1 (Cop 1, Copaxone(R), glatiramer acetate) suppresses experimental autoimmune encephalomyelitis (EAE), and is currently tested for its efficacy in the treatment of multiple sclerosis (MS). Here we demonstrate that oral treatment with Cop 1 induces, in mice, specific Th2 cells in the central nervous system (CNS), as manifested by their isolation from brains of actively sensitized Cop 1-fed mice, as well as, by the localization of orally induced Cop 1 specific suppressor cells in the brain, after their passive transfer to the periphery. Feeding with Cop 1 results in the accumulation in the CNS of cells that secrete Th2 cytokines in response to either Cop 1 or the autoantigen myelin basic protein (MBP), even in Th1 shifting environment, which consequently would lead to therapeutic effect in the MS diseased organ.