Immunocytochemical and electron-microscopic features of tooth pulp innervation in hereditary sensory and autonomic neuropathy

Increases in urine drug concentration that result from changes in urinary output may be mistakenly interpreted as new drug use rather than carryover from previous drug exposure. Normalization of drug excretion to urine creatinine concentration reduces the variability of drug measurement attributable to urine dilution. A specimen ratio of 1.5 or greater between two creatinine normalized positive urine cannabinoid tests was previously proposed as an indicator of new marijuana use. This approach has received wide attention for potential use in treatment and employee assistance programs associated with workplace drug testing. Unfortunately, there has been limited evaluation of the usefulness of this ratio under controlled-dosing conditions with marijuana smokers. A controlled clinical study was conducted to examine the excretion profile of creatinine and marijuana metabolites in a group of six marijuana users who smoked two different doses of marijuana over a 4-week period. A relative operating characteristic curve was constructed from sensitivity and specificity data for 26 different specimen ratios ranging from 0.1 to 2.0. The most accurate specimen ratio (85.4%) for differentiating new use from residual excretion was 0.5. Use of this ratio provided a sensitivity of 80.1%, a specificity of 90.2%, and 5.6% false-positive and 7.4% false-negative predictions. To substantiate the validity of the 0.5 specimen ratio, urine cannabinoid and creatinine data from a controlled clinical trial specifically addressing water dilution as a means of specimen adulteration were evaluated. Sensitivity, specificity, accuracy, and percent false-positive and percent false-negative predictions were 71.9%, 91.6%, 83.9%, 5.4%, and 10.7%, respectively. These data compared favorably with the results from the first clinical study, with the exception of slightly lower sensitivity and higher false-negative percentages in the water dilution study. This would be expected because of the ingestion of large amounts of water and consequent dilution of urine drug concentration. These data indicated that selection of a specimen ratio to evaluate sequential creatinine normalized urine drug concentrations can improve the ability to distinguish residual excretion from new marijuana usage. The selection of an appropriate specimen ratio can be made based on the needs of a specific urine drug-testing program taking into account sensitivity, specificity, and accuracy data.     

Resistance to antimicrobial agents used for animal therapy in pathogenic-, zoonotic- and indicator bacteria isolated from different food animals in Denmark: a baseline study for the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP)

This study describes the establishment and first results of a continuous surveillance system of antimicrobial resistance among bacteria isolated from pigs, cattle and broilers in Denmark. The three categories of bacteria tested were: 1) indicator bacteria (Escherichia coli, Enterococcus faecalis, Enterococcus faecium), 2) zoonotic bacteria (Campylobacter coli/jejuni, Salmonella enterica, Yersinia enterocolitica), and 3) animal pathogens (E. coli, Staphylococcus aureus, coagulase-negative staphylococci (CNS), Staphylococcus hyicus, Actinobacillus pleuropneumoniae). A total of 3304 bacterial isolates collected from October 1995 through December 1996 were tested for susceptibility to all major classes of antimicrobial agents used for therapy in Denmark. Bacterial species intrinsically resistant to an antimicrobial were not tested towards that antimicrobial. Acquired resistance to all antimicrobials was found. The occurrence of resistance varied by animal origin and bacterial species. In general, resistance was observed more frequently among isolates from pigs than from cattle and broilers. The association between the occurrence of resistance and the consumption of the antimicrobial is discussed, as is the occurrence of resistance in other countries. The results of this study show the present level of resistance to antimicrobial agents among a number of bacterial species isolated from food animals in Denmark. Thus, the baseline for comparison with future prospective studies has been established, enabling the determination of trends over time.     

The effects of adenine dinucleotides on epileptiform activity in the CA3 region of rat hippocampal slices

Alpha, omega-adenine dinucleotides (Ap(n)A) consist of two adenosine molecules linked at the 5' position by phosphate groups, the number of which is denoted by n and can range from 2 to 6. The aim of this study was to investigate the effect of Ap4A and Ap5A on the rate of epileptiform activity. Hippocampal slices (450 microm), when perfused with a medium containing no added magnesium and 4-aminopyridine (50 microM), generate epileptiform activity of an interictal nature. Ap4A and Ap5A at 1 microM depressed the discharge rate to a significant extent. At this concentration adenosine (1 microM) did not produce any effect. However at 10 microM adenosine, Ap4A and Ap5A all decreased the burst frequency. Adenosine deaminase (0.2 U/ml) totally annulled the inhibition of epileptiform activity produced by 10 microM adenosine or 1 microM Ap4A and Ap5A. Adenosine deaminase did not significantly change the maximum depression of activity produced by 10 microM Ap4A and Ap5A. 8-cyclopentyl-1,3-dimethylxanthine, an A1, receptor antagonist, increased the basal rate of epileptiform activity and prevented the depression of burst discharges by Ap4A. 5'-adenylic acid deaminase converts AMP into IMP which is inactive. 5'-adenylic acid deaminase did not prevent the inhibitory effects of Ap4A. The results suggests that in the CA3 region of the hippocampus, Ap4A and Ap5A act partly by stimulating xanthine-sensitive receptors directly and partly through the formation of the metabolite, adenosine.     

Stringent allele/epitope requirements for MART-1/Melan A immunodominance: implications for peptide-based immunotherapy

The exclusiveness of the relationship between peptide and HLA alleles, combined with their extensive polymorphism, emphasizes the need for immunization strategies based on endogenous processing of full length proteins (containing multiple epitopic determinants) for presentation to T cells. This could allow vaccination regardless of the patient's HLA phenotype, assuming that individual molecules can be efficient T cell Ags in association with various HLA alleles. An endogenous system of Ag presentation was developed using dendritic cells infected with recombinant viral vectors expressing the melanoma-associated Ag MART-1/Melan A. CD8+ T cells from melanoma patients were activated in vitro by coincubation with infected dendritic cells and tested for recognition of HLA-A-matched melanoma targets. This allowed the analysis of T cell induction in association with any HLA-A allele of a given patient's phenotype. In this system, MART-1/Melan A could not efficiently immunize in association with HLA-A alleles other than A*0201, including the one residue variant from A*0201: HLA-A*0226. Clonal analysis of MART-1/Melan A-specific CTL confirmed that MART-1/Melan A immunodominance is strongly restricted to the AAGIGILTV/HLA-A*0201 combination. The stringent epitope/allele requirements for MART-1/Melan A/TCR interactions were not associated with limitations in the TCR repertoire. In conclusion, autologous induction of MART-1/Melan A CTL by whole Ag processing and presentation is restricted to a unique allele/ligand combination and is excluded by minimal changes in HLA structure. Thus, whole protein vaccination for small m.w. Ags may provide no further advantage over a peptide-based approach.     

Results of a randomized phase III trial of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C alone in patients with superficial bladder cancer

PURPOSE: We study toxicity and efficacy of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin (BCG) in patients with intermediate or high risk superficial bladder cancer compared to the use of intravesical mitomycin C alone. MATERIALS AND METHODS: Patients with intermediate and high risk papillary superficial bladder cancer and carcinoma in situ were randomized after transurethral resection between 4 weekly instillations with 40 mg. mitomycin C followed by 6 weekly instillations with BCG (group 1, 90 patients) or 10 weekly instillations with mitomycin C (group 2, 92 patients). RESULTS: The frequency of bacterial and chemical cystitis, and other local side effects was similar in both groups. Allergic reactions, including skin rash, were more frequent in the mitomycin C only group (12 of 92 patients versus 5 of 90, p = 0.08), and other systemic side effects were more frequent in the sequential group (16 of 90 versus 8 of 92, p = 0.07). After a median followup of 32 months the number of recurrences (sequential 35 of 90 patients versus mitomycin C only 42 of 92, p = 0.36) and progression (5 of 90 versus 4 of 92 respectively, p = 0.70) were similar in both groups. CONCLUSIONS: We did not find any major differences in toxicity or treatment efficacy with intravesical mitomycin C and the sequential use of BCG or mitomycin C for intermediate and high risk superficial papillary bladder cancer.     

In vivo toxicity, pharmacokinetics, and anticancer activity of Genistein linked to recombinant human epidermal growth factor

Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have vital anti-apoptotic functions in human breast cancer cells. We have shown previously that targeting the naturally occurring PTK inhibitor genistein to the EGFR-associated PTK complexes using the EGF-Genistein (Gen) conjugate triggers rapid apoptotic cell death in human breast cancer cells and abrogates their in vitro clonogenic growth. In the present study, we examined the in vivo toxicity profile, pharmacokinetics, and anticancer activity of EGF-Gen. No toxicities were observed in mice treated with EGF-Gen at dose levels as high as 40 mg/kg administered i.p. as a single dose or 140 mg/kg administered i.p. over 28 consecutive days. EGF-Gen significantly improved tumor-free survival in a severe combined immune deficiency (SCID) mouse xenograft model of human breast cancer, when it was administered 24 h after inoculation of tumor cells. At 100 microg/kg/day x 10 days (1 mg/kg total dose), which is >100-fold less than the highest tested and nontoxic cumulative dose (ie., 140 mg/kg) in mice, EGF-Gen was more effective than cyclophosphamide (50 mg/kg/day x 2 days), Adriamycin (2.5 mg/kg x 1 day), or methotrexate (0.5 mg/kg x 1 day), the most widely used standard chemotherapeutic drugs for breast cancer, and resulted in 60% long-term tumor-free survival. Furthermore, treating SCID mice with established s.c. human breast cancer xenografts of 0.5-cm diameter with EGF-Gen at this dose level resulted in disappearance of the tumors in two of five mice and >50% shrinkage in three of five mice within 10 days, whereas all of the control tumors in five PBS-treated mice as well as five mice treated with unconjugated Gen (1 mg/kg/day x 10 days) showed >200% increase in diameter during the same observation period. EGF-Gen treatment reduced the growth rate of breast cancer xenografts of 1.0-cm diameter, but unlike with tumors of 0.5-cm diameter, it failed to cause shrinkage or disappearance of these larger tumors. The level of EGF-Gen systemic exposure that was effective in SCID mice was achieved in cynomolgus monkeys without any significant side effects detectable by clinical observation, laboratory studies, or histopathological examination of multiple organs. EGF-Gen might be useful in the treatment of breast cancer as well as other EGFR-positive malignancies.     

Alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl] propenamide: an inhibitor of the epidermal growth factor receptor tyrosine kinase with potent cytotoxic activity against breast cancer cells

Epidermal growth factor receptor (EGF-R) tyrosine kinase is known to be overexpressed in several malignancies and is an important target for anticancer drug design. We constructed a homology model to represent the structure of EGF-R and propose that this model can be used to design potent inhibitors of EGF-R. We used our EGF-R model and a docking procedure to rationally design compounds predicted to bind favorably to EGF-R. This approach led to the successful design of a leflunomide metabolite analogue, which was found to have an IC50 value of 1.7 microM in EGF-R inhibition assays and killed >99% of human breast cancer cells in vitro by triggering apoptosis. The reported studies may provide the basis for the development of a new class of potent and clinically useful anti-breast cancer agents.     

Cross-reactions between mouse Ia and human HLA-D/DR antigens analyzed with monoclonal alloantibodies

Two mouse monoclonal anti-I-E/Ck alloantibodies (H7-8.26 and H10-81.10) directed against 2 distinct determinants of the specificity Ia-7 and 1 anti-I-Ak alloantibody (H8-15.9) directed against a public determinant common to the I-A subregion products of the H-2k, H-2b, H-2d, H-2q, and H-2ja haplotypes identified cross-reactive determinants on lymphoid cells from various mammalian species, including rat, dog, pig, cow, hamster, and guinea pig. In man, these antibodies detected nonpolymorphic determinants of DR antigens on B cell-enriched peripheral blood lymphocytes from 50 unrelated individuals. These cross-reactive DR determinants were also detected on lymphoblastoid B cell lines, on PHA-activated peripheral T lymphocytes, and on allospecific cytolytic T cell clones, but not on various DR-negative human T leukemia cell lines. Two chains of 29,000 and 35,000 daltons m.w., corresponding to DR antigens, were precipitated by H7-8.26 and H8-15.9 antibodies from radiolabeled membrane extracts of Raji cells. Competitive binding experiments indicated that the 3 mouse anti-Iak antibodies identified 3 distinct cross-reactive determinants on human cells. The results indicate that: a) The cross-reactivity described between mouse I-E/C gene products (Ia-7) and human DR antigen(s) involves, in fact, several distinct and topologically distant determinants. b) At least 1 determinant cross-reacting with DR can be identified on I-Ak gene products. c) The intriguing genetic problem of mouse MHC allotypic determinant(s) being nonpolymorphic in man cannot be simply explained by the deletion of an I-E alpha chain in some strains of mice.     

The effect of thalidomide on BCG-induced granulomas in mice

Granuloma proliferation is the result of a series of complex biological events in which a variety of cell types and cytokines are involved. Tumor necrosis factor alpha (TNF-alpha) plays a central role. In the present study, we investigated the effect of thalidomide (alpha-N-pthalimidoglutarimide), a selective inhibitor of TNF-alpha synthesis, on granuloma formation during BCG infection in Oncins France 1 (OF-1) mice. Subcutaneous injections of 30 mg/kg body weight of thalidomide daily for 14, 21 or 28 days into the mice resulted in the reduction of the size and total number of liver granulomas. The most striking effect of thalidomide was observed after 28 days, when the total number of liver granulomas was reduced by as much as 40% (P < 0.05). Serum TNF-alpha levels of thalidomide-treated mice were significantly lower (85%) than control mice on day 14 and remained lower (55%) on days 21 and 28. Positive immunohistochemical staining specific for TNF-alpha were demonstrable only in well-developed granulomas in which central mononuclear cells presented extensive differentiation into epithelioid cells. Daily administration of thalidomide for 21 to 28 days to the BCG-infected mice inhibited local TNF-alpha expression in well-developed granulomas. The mechanisms by which thalidomide modulates the granuloma proliferation are discussed.     

Ebulitins: a new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin I in its leaves. Ebulitins are basic proteins of M(r) 32,000, 29,000 and 29,000 for ebulitins alpha, beta and gamma, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.