A phase 2 study of MK-0457 in patients with BCR-ABL T315I mutant chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia.

Aurora kinase overexpression has been observed in patients with hematologic malignancies. MK-0457, a pan-aurora kinase inhibitor that also inhibits the ABL T315I mutant, was evaluated to treat patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome (Ph+) acute lymphoblastic leukemia (ALL) with the T315I mutation. Adults with Ph+ chronic phase (CP)-, accelerated phase (AP)- or blast phase (BP)-CML, or ALL and documented BCR-ABL T315I mutation were treated with a 5-day continuous infusion of MK-0457 administered every 14 days at 40 mg/m(2)/h, 32 mg/m(2)/h or 24 mg/m(2)/h. Fifty-two patients (CP, n=15; AP, n=14; BP, n=11; Ph+ ALL, n=12) were treated. Overall, 8% of patients achieved major cytogenetic response; 6% achieved unconfirmed complete or partial response; 39% had no response. Two patients (CP CML) achieved complete hematologic response. No patients with advanced CML or Ph+ ALL achieved major hematologic response. The most common adverse event (AE) was neutropenia (50%). The most common grade 3/4 AEs were neutropenia (46%) and febrile neutropenia (35%). MK-0457 demonstrated minimal efficacy and only at higher, intolerable doses; lower doses were tolerated and no unexpected toxicities were observed. These data will assist in the development of future aurora kinase inhibitors and in the selection of appropriate target patient populations.

Differential expression of nitric oxide synthases in EGF-responsive mouse neural precursor cells.

Nitric oxide (NO) is a gaseous, radical molecule that plays a role in various physiological processes in the nervous system such as learning and hippocampal plasticity. It is generated from l-arginine by nitric oxide synthases (NOS), which come in three isoforms depending on the tissue of origin, namely inducible-NOS (iNOS in macrophages), endothelial-NOS (eNOS in endothelial cells) and neural-NOS (nNOS in neural cells). We used epidermal growth factor (EGF)-responsive nestin-positive neural precursor cells originating from the mouse E16 embryonic striatum, and studied the relative expression of NOS isoforms probed with isoform-specific antibody using the avidin-biotin immunohistochemical method. Our data revealed both nNOS and eNOS to be expressed in both neurospheres and desegregated neural precursor cells. However, iNOS signals were virtually undetectable in both cell categories. When the neural precursor cells were carried in the presence of poly-l-ornithine (PLO), there was a strong induction of the expression of iNOS proteins, indicating the possibility that this isoform is amenable to modulation by extracellular cues. These preliminary results suggest both nNOS and eNOS to be important in the physiology of neural precursor cells, and that iNOS might also play a role at certain stages in the life of these cells.

A 21-mer synthetic peptide of toxic shock syndrome toxin 1, TSST-1[58-78], activates T cells by binding to MHC class II and by an MHC unrestricted xenostimulatory pathway.

Toxic shock syndrome toxin-1 (TSST-1) is a "superantigen" which binds to MHC class II molecules and induces a polyclonal stimulation of T cells. In this communication by using a FACS technique and a 21-mer synthetic peptide from the primary sequence of TSST-1 (KGEKVDLNTKRTKKSQHTSEG), designated TSST-1(58-78), we demonstrated binding of the peptide only to cells bearing MHC class II. The proliferative effect of TSST-1(58-78) on human T cells was shown to be inhibited much more by anti-HLA-DR than by anti-HLA class I antibody. Furthermore, human monocytes were able to present TSST-1(58-78) to a mouse VSV specific T cell clone by a xenostimulatory mechanism. These data indicate this peptide to contain an active site of the TSST-1 holotoxin.

Cellular expression of a GPI-linked T cell activation protein.

A T cell activation protein was identified by generating a monoclonal antibody (anti-D2) against a gamma delta T cell receptor bearing gibbon ape T cell line (MLA144). Immunoprecipitation studies revealed three polypeptides of 180, 150, and 120 kDa. The antigen was also found to be expressed on endothelial cells in vivo and in vitro and on tumor cell lines from a variety of tissues. Studies performed using a variety of antibodies reveal this protein to be identical to an endothelial cell protein previously identified by several antibodies to T cell activation proteins (CDw109). We demonstrate that this protein is anchored in the membrane via a glycosylphosphatidylinositol (GPI) tail in T cells, tumor cells, and endothelial cells. An analysis of tissue sections reveals this protein to be normally highly expressed on vascular endothelial cells.

Significantly reduced salivary nitric oxide levels in smokers.

BACKGROUND: NO is a free radical gas with manifold physiologic functions primarily as a biologic messenger. Recently we reported the presence of NO in freshly secreted human saliva. We report here a significant reduction of salivary NO production in current smokers compared to non-smokers. PATIENTS AND METHODS: Saliva was collected from smokers (27 donors) and non-smokers (21) after rinsing the mouth with an antiseptic. Freshly secreted saliva, about 200-300 microliters, was collected within the first 30-60 seconds after mouth rinsing. NO was assayed using the Griess reagent which measures its byproduct NO2. The values thus obtained were statistically analysed using the Wilcoxon test. RESULTS: Our studies indicate significantly decreased NO levels as measured by its product NO2 in freshly secreted saliva in smokers compared to non-smokers (p = 0.0042). The median NO2 level in smokers was 64.85 microM, in non-smokers 194.45 microM, respectively. CONCLUSIONS: The role of salivary NO in normal physiology is as yet unknown, however, the finding of reduced salivary NO levels in smokers suggests a potential role in the pathogenesis of smoking related morbidity of the upper aerodigestive tracts.

Autocrine regulation of the production of the gaseous messenger nitric oxide in a glioblastoma cell line.

We used a glioblastoma multiform (GBM) cell line to study the mechanism of cellular regulation of nitric oxide (NO) production. Our experiments indicate a confluent monolayer of GBM cells to release NO as measured through its oxidized NO2 form which gradually accumulates and reaches a peak by 7 to 10 days of culture. the addition of the L-arginine analogs L-NG-monomethyl-L-arginine and L-N omega-nitro-L-arginine and dexamethasone to the GBM cultures caused a substantial inhibition of NO production. The addition of monoclonal antibodies against IL-1 and TNF alpha to the cultures resulted in an inhibition of NO production, whereas the addition of anti-TGF beta monoclonal antibodies resulted in an increase in NO production. These findings suggest the presence of an autocrine regulatory mechanism for NO production in some tumor cell lines.

Evidence for the release and possible neural regulation of nitric oxide in human saliva.

We report the presence of nitric oxide (NO) as measured through its product NO2 in human saliva. The presence of NO is not due to oral bacterial activity but is due to a genuine release of NO from the salivary glands and can be detected in freshly secreted saliva collected from a mouth freshly rinsed with an antiseptic preparation to reduce the bacterial content significantly. In order to study the regulation of salivary NO we used lemon juice to increase salivary flow and detected an immediate transient reduction of salivary NO2 which rose to levels equal to and sometimes higher than basal levels in five minutes in most donors indicating salivary NO production might be under neural control. Salivary NO production may play a physiological role in both the natural antibacterial properties of salivary secretion and possibly in detoxification of oral carcinogens.

Murine T cell hybridoma-derived products regulate the growth of skeletal muscle myoblasts.

We have analyzed the effects of T cell products on the growth of murine myoblasts. Supernatants from some T cell hybridomas were shown to have either growth factor activity or growth inhibitory activity on two myoblast lines raised in our laboratory from two different strains of mice and also on the C2C12 myoblast line. The hybridoma-derived growth factor activity was shown not to require cofactors contained in serum as its effects could be demonstrated in assays done in serum-free media. Further studies on the effects of purified lymphokines on myoblasts using recombinant IL-2, IL-3, IL-4, IL-6, and GM-CSF showed these lymphokines not to have any growth stimulatory activity indicating that factor(s) other than the ones tested may be operative. Characterization by dialysis and column chromatography of hybridoma supernatants revealed the presence of a low-molecular-weight inhibitor and a higher-molecular-weight growth factor that was found in several hybridoma supernatants.

Guanidine group specific ADP-ribosyltransferase in murine cells.

We have identified a guanidine group specific ADP-ribosyltransferase activity, capable of transferring an ADP-ribose group from NAD to a low molecular weight guanidine compound [p-(nitrobenzylidine)amino]guanidine and proteins such as histone and poly-L-arginine, in a variety of murine cell lines. The enzyme activity appears to be associated with an integral membrane protein of apparent molecular weight 30-33 kDa. Incubation of the viable cells in isotonic phosphate buffered saline with [32P]NAD results in the incorporation of label into cellular proteins. Dimethyl sulfoxide treatment of the cells downregulates the transferase activity as well as the ADP-ribosylation of cell proteins with extracellular NAD.

A mycobacterial heat-shock protein-responsive gamma delta T cell clone also responds to the homologous human heat-shock protein: a possible link between infection and autoimmunity.

Prokaryotic and eukaryotic cells respond to a variety of stress conditions by increasing the synthesis of a family of proteins collectively known as heat-shock proteins (HSP). One of these, a 65-kDa HSP that is highly conserved in many bacteria, is a major target of the immune response to mycobacteria. A gamma delta T cell clone from a healthy donor that recognizes not only the 65-kDa mycobacterial HSP but also the recombinant human homologue of this HSP protein was raised. Like alpha beta T cell clones, which recognize mycobacterial HSP, the clone requires antigen-presenting cells for antigen-induced proliferation and can also be directly activated via receptor cross-linking through CD3 or the delta chain of the gamma delta T cell receptor. These data suggest that the induction of a gamma delta T cell response by bacterial antigens could lead to the expansion of cells that respond to autologous proteins and, therefore, may result in the development of autoimmunity.

Human gamma delta+ T cells respond to mycobacterial heat-shock protein.

Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.

Clinical evaluation of efficacy, pharmacokinetics, and safety of teicoplanin for serious gram-positive infections.

Nineteen patients hospitalized for serious gram-positive infections were treated with teicoplanin, a new glycopeptide antibiotic. A variety of infections were treated, including endocarditis, septic thrombophlebitis, osteomyelitis, pyogenic arthritis, and soft tissue infection. Of 13 infections that could be evaluated in 12 patients, there were 8 clinical cures, 2 improvements, 1 recurrence, and 2 failures. Of the eight patients with Staphylococcus aureus bacteremia, seven were clinically cured or improved with teicoplanin therapy. Of the nine patients in whom the bacteriological response to treatment could be fully evaluated, six were cured; there was recurrence of infection in one, and treatment failed in two patients. In vitro testing showed the 13 bacterial isolates (9 S. aureus, 3 S. epidermidis, and 1 group B streptococcus) to be uniformly susceptible to teicoplanin, with MICs ranging from 0.12 to 0.5 microgram/ml. Every isolate was more susceptible in vitro to teicoplanin than to vancomycin. Three of the staphylococcal isolates were resistant to methicillin. Pharmacokinetic studies demonstrated that after an initial drug-accumulation period, a single daily dose adequately maintained the teicoplanin concentrations in serum within therapeutic ranges. Teicoplanin also penetrated well into synovial fluid. The drug was well tolerated by either intravenous or intramuscular administration. The most significant adverse reaction was an urticarial rash which required discontinuation of therapy in one patient; a second patient experienced a modest decrease in high-frequency auditory threshold. Asymptomatic eosinophilia and mild elevation of serum transaminases were noted as well. The results of this study suggest that teicoplanin is a safe and effective new agent for treatment of serious infections caused by gram-positive organisms.

In vivo responses to Mycobacterium leprae: antigen presentation, interleukin-2 production, and immune cell phenotypes in naturally occurring leprosy lesions.

To investigate the immune defect in lepromatous leprosy we studied immune cell phenotypes, lymphocyte activation states, and interleukin-2 (IL-2) production in naturally occurring leprosy skin lesions. Mouse hybridoma monoclonal antibodies reacting with the IL-2 receptor (anti-Tac), unbound IL-2 (DMS-1), antigen-presenting Langerhans' cells (OKT6) and the OKT4-Leu3 and OKT8 T-lymphocyte subpopulations were used with indirect horseradish peroxidase and alkaline phosphatase techniques on frozen biopsy sections. The percentage of Tac+ lymphocytes and the number of OKT6+ cells in the epidermis and dermal granuloma were significantly correlated in naturally occurring lesions (correlation coefficient 0.79) and were higher in tuberculoid than in lepromatous lesions. Leu3 antigen was expressed by 70-90% of Tac+ cells in tuberculoid lesions. Although the percentage of cells producing IL-2 was low in lesions of both lepromatous and tuberculoid patients, it was about 15 times greater in tuberculoid than in lepromatous lesions (0.032 +/- 0.037 tuberculoid vs 0.0019 +/- 0.023 lepromatous). There was an association between the number of OKT6+ cells and the percentage of IL-2-producing cells, but the association was weaker than that of OKT6+ cells and the percentage of IL-2 receptor-bearing cells (r = 0.2), implying that IL-2 production is not an intervening variable in the latter association. The absolute number of OKT4-Leu3+ lymphocytes was significantly different in different clinical leprosy groups and was positively correlated with host resistance (mean OKT4-Leu3+ cells/mm2 in 6 micron sections; 1412 +/- 288 tuberculoid, 400 +/- 93 borderline lepromatous, 200 +/- 100 polar lepromatous; r = 0.95). Absolute numbers of OKT8+ cells/mm2 in lesions were not significantly different. We conclude that there is a relative paucity of OKT4-Leu3+ cells as well as IL-2-producing cells at the local level in lepromatous leprosy lesions. Possible functional relationships between these findings and the failure of macrophage activation and destruction of Mycobacterium leprae in lepromatous leprosy are discussed.

The role of interleukin-2 (IL-2) in the specific unresponsiveness of lepromatous leprosy to Mycobacterium leprae: studies in vitro and in vivo.

The role of IL-2 in the immunological deficiency of lepromatous leprosy patients towards Mycobacterium leprae have been studied further. After initial stimulation with M. leprae + IL-2, lepromatous lymphocytes could be restimulated with M. leprae alone. The specificity of the responses obtained varied. Some patients gave a stronger response to BCG as compared to M. leprae, while in others a stronger response to M. leprae as compared to BCG was obtained. Studies of the composition of lymphocytes in dermal infiltrates subsequent to injection of killed M. leprae revealed that in both tuberculoid and lepromatous patients, early accumulation of cell staining for both IL-2 receptor and IL-2 were seen. However, with time IL-2 receptor and IL-2 staining lymphocytes diminished in lepromatous infiltrates, while these were maintained in tuberculoid lesions.

Reversal by interleukin-2 of the T cell unresponsiveness of lepromatous leprosy to Mycobacterium leprae.

In some subjects Mycobacterium leprae causes disseminated (lepromatous) disease. Such subjects show both in vivo and in vitro deficient T cell responses to M. leprae, but not to other antigens. We have recently shown that lepromatous peripheral blood mononuclear cells (PBMC) failed to produce interleukin 2 (IL-2) in response to M. leprae and that T cell-conditioned media (TCM) can reverse the T cell unresponsiveness in a majority of lepromatous leprosy patients (Haregewoin et al. 1983). Here we show that highly purified and recombinant IL-2 had effects similar to TCM. On the other hand, lepromatous PBMC produced IL-1, and IL-1 had no restorative effect. These findings provide further evidence that the unresponsiveness in lepromatous leprosy often results from a deficiency in IL-2 production. After initial stimulation with TCM + M. leprae, lepromatous PBMC could be restimulated with M. leprae alone, providing clear evidence that M. leprae-reactive lymphocytes were generated in the presence of TCM. The present findings are discussed in relation to the possible mechanisms involved in the failure of IL-2 production. If our findings can be reproduced in vivo, IL-2 may offer a novel approach to therapy in lepromatous leprosy.

Characterization and functional studies of the murine T-lymphocyte response to Mycobacterium leprae antigen.

Mice were immunized with Mycobacterium leprae in incomplete Freund's adjuvant, and sensitized lymphocytes were obtained from draining lymph nodes. The lymphocytes thus obtained proliferated specifically in vitro in the presence of M. leprae antigen, and this response was shown to be both T-cell and macrophage dependent. T-cell blasts generated in vitro in response to M. leprae antigen were grown in the presence of interleukin-2 (IL-2). The proliferative response of these blasts to M. leprae antigen was strictly dependent on the presence of syngeneic spleen cells as antigen-presenting cells. M. leprae-immune F1 blasts responding to the antigen in the context of either parental H-2 haplotype-bearing accessory cell could be obtained by positive selection from an F1 hybrid-responding cell population. By means of flow microfluorometry the T-cell phenotype of the M. leprae-specific T-cell blasts was found to be Thy-1+ and to be composed of Lyt-1+ and Lyt-2+ subpopulations. Functionally, the blasts were shown to transfer delayed-type hypersensitivity locally to non-immunized recipients and to have cytolytic activity. Limiting dilution analysis showed the frequency of M. leprae-responding cells from blasts grown in IL-2 to be approximately 1/333.