Allium sativum (garlic) treatment for murine transitional cell carcinoma

BACKGROUND: Currently, immunotherapy with Bacillus Calmette-Guerin (BCG) is the most effective treatment for superficial bladder carcinoma, but treatment-related toxicity may limit its use in some patients. Alternative treatments are needed for patients who fail to respond to BCG immunotherapy. Allium sativum (AS), or garlic, is known to have a broad range of biologic activities, including immune stimulation and reported antitumor activity. For these reasons, the authors conducted a series of experiments designed to explore the possible therapeutic effects of AS in the MBT2 murine bladder carcinoma model. METHODS: C3H/HeN mice were randomized prior to initiation of each experimental protocol. Mice received 1 x 10(3) MBT2 cells in 0.1 mL RPMI-1640, administered subcutaneously into the right thigh, on Day 0 of the experiment. AS was injected at the site of tumor transplantation on Day 1 and at 2- to 7-day intervals up to Day 28. To evaluate the effects of oral AS in this model, treatment was initiated 30 days prior to tumor inoculation and continued for 30 days after tumor inoculation. Animals in all experiments were followed for tumor incidence, tumor growth, and survival. RESULTS: In the initial experiments, subcutaneous AS significantly reduced tumor volume compared with the saline control (P < 0.05). Unfortunately, treatment-related death was also observed, requiring reduction in the total dose of AS. Animals that received 5 weekly immunizations of AS (5 mg, 5 mg, 1 mg, 1 mg, and 1 mg; cumulative dose = 13 mg) had significantly reduced tumor incidence, tumor growth, and increased survival when compared with animals that received the saline control. No treatment-related deaths were observed with this treatment schedule. To determine whether systemic AS administration might be effective, orally administered AS was tested at doses of 5 mg, 50 mg, and 500 mg per 100 mL of drinking water. Mice that received 50 mg oral AS had significant reductions in tumor volume (P < 0.05) when compared with animals that received the saline control, and mice that received 500 mg oral AS had significant reductions in both tumor volume and mortality (P < 0.05). CONCLUSIONS: The significant antitumor efficacy of subcutaneous and oral AS warrants further investigation and suggests that AS may provide a new and effective form of therapy for transitional cell carcinoma of the bladder.     

Severe defects in immunity and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency

Spontaneous mouse mutations that cause severe immunodeficiency or autoimmunity are invaluable tools with which to investigate the mammalian immune system. Mutations at the 'motheaten' locus result in severe immunological dysfunction due to disruption of the structural gene encoding Src-homology 2-domain phosphatase-1 (SHP-1). This natural model for a specific protein-tyrosine-phosphatase deficiency is being widely utilized to determine the role of SHP-1 in the negative regulation of multiple signaling pathways in a number of hematopoietic lineages.     

Induction of alloantigen-specific T cell tolerance through the treatment of human T lymphocytes with wortmannin

Signaling through the CD28 molecule on T cells by its natural ligand, B7, on APCs has recently been shown to require the presence of an active phosphatidylinositol 3-kinase pathway to mediate some of its costimulatory activities (1-7). Using the phosphatidylinositol 3-kinase inhibitor, wortmannin (WN) (8), on human and murine T cells, we have inhibited B7-1-mediated T cell activation and induced Ag-specific tolerance. The addition of WN and/or the B7-1 antagonist, CTLA4Ig, to primary human T cell cultures stimulated with B7-1-transfected allogeneic melanoma cell lines inhibited the generation of alloantigen-specific proliferative and cytolytic responses in vitro. Subsequent examination of these WN- and CTLA4Ig-treated primary T cell cultures revealed that these lymphocyte populations were tolerized to rechallenge with the priming alloantigens in secondary cultures in the absence of additional inhibitor(s). However, reactivity to a third party allogeneic stimulator remained intact. This WN-induced tolerance was reversed by the addition of high dose IL-2, but not IL-4 or IL-7, to the primary cultures, indicating that T cell anergy, not deletion, was responsible for this phenomenon. In vivo studies using a murine graft-vs-host disease (GVHD) model demonstrated that WN treatment of allogeneic donor lymphocytes in vitro failed to generate a significant GVHD in irradiated mouse recipients compared with control allogeneic donor lymphocytes. These findings suggest potentially novel therapeutic strategies for the prevention of GVHD.     

Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.     

Detecting vascular problems in patients with diabetes treated with an intra-aortic balloon pump

Individuals with diabetes are at increased risk for both peripheral vascular disease and coronary artery disease. In patients with severe coronary artery disease, a cardiac assist device called an intra-aortic balloon pump (IABP) often is used to aid the failing heart and prevent further cardiac ischemia. Because this device is inserted via the femoral artery, patients are at risk of limb ischemia distal to the insertion site. Patients with diabetes are particularly prone to this complication. Detecting the early signs and symptoms of ischemia is crucial to preventing serious sequelae. Standard vascular examination techniques, in addition to being subjective and not easily reproducible, may be misleading in patients with diabetes. This article provides a review of the signs and symptoms of lower limb ischemia and noninvasive vascular tests that clinicians can use to evaluate lower extremity circulation. Also included are protocols for patient care during and after hospitalization, and two case studies of cardiac patients with diabetes who were treated with an IABP.     

The rate of fibrinolysis is increased by free retraction of human gingival fibroblast populated fibrin lattices

This study describes the development and comparison of two HPLC methods for the analysis of the antihypertensive drug captopril. The first method is based on a precolumn derivatization of captopril with the fluorescent label monobromobimane (MBB). The second method is based on a postcolumn reaction with the fluorescent reagent o-phthaldialdehyde (OPA). Since the disulfide metabolites of captopril can be reconverted to the active drug in vivo, the bioanalysis of captopril should involve both the determination of its free thiol form (free captopril) and the total amount of free thiol and reducible disulfides (total captopril). For total captopril analysis, disulfides were reduced with tributylphosphine (TBP) prior to protein precipitation. Since the reducing agent interfered with the MBB derivatization reaction, this method was not suitable for total captopril analysis. Both methods were validated for the bioanalysis of free captopril in human plasma. After removal of plasma proteins, samples were analyzed without an additional extraction procedure. The limit of quantitation in plasma was 12.5 ng/ml for the MBB method (limit of detection 30 pg) and 25 ng/ml for the OPA method (limit of detection 50 pg). The OPA method was also validated for total captopril analysis in human plasma and urine. The limit of quantitation was 25 ng/ml in plasma and 250 ng/ml in urine (limit of detection 50 pg). We conclude that for the analysis of free captopril the precolumn MBB method is superior to the OPA method since only the derivatization reaction has to be carried out immediately. The postcolumn OPA method is especially suitable for the analysis of total captopril since reducing reagents and high concentrations of endogenous thiols do not interfere with the derivatization reaction.     

Effects of a low-dose amino acid supplement on adaptations to cycling training in untrained individuals

The purpose of this study was to determine if amino acid supplementation influences blood and muscle lactate response to exercise and the time course of the metabolic adaptations to training. Two groups of untrained males (n = 7 each) were given (double-blind) a daily supplement (2.9 g.day-1) containing a mixture of leucine, isoleucine, valine, glutamine, and carnitine (EXP) or 3 g.day-1 of lactose (CON). Following 7 days of supplementation there was no significant change in VO2peak, time to exhaustion (TTX) at 120% VO2peak, or muscle and blood lactate in either EXP or CON. Subjects then initiated 6 weeks of combined aerobic and anaerobic training on a Monark cycle ergometer. It was found that amino acid supplementation had no effect on either blood or muscle lactate accumulation during exercise, while supplementation resulted in a faster adaptation in buffer capacity. Performance during intense exercise was not improved with amino acid supplementation.