A model agreement for genetic research in socially identifiable populations

Genetic research increasingly focuses on population-specific human genetic diversity. However, the naming of a human population in public databases and scientific publications entails collective risks for its members. Those collective risks can be evaluated and protections can be put in place by the establishment of a dialogue with the subject population, before a research study is initiated. Here we describe an agreement to undertake genetic research with a Native American tribe. We identified the culturally appropriate public and private social units within which community members are accustomed to make decisions about health. We then engaged those units in a process of communal discourse. In their discourses about our proposed study, community members expressed most concern about culturally specific implications. We also found that, in this population, private social units were more influential in communal decision making than were public authorities. An agreement was reached that defined the scope of research, provided options for naming the population in publications (including anonymity), and addressed the distribution of royalties from intellectual property, the future use of archival samples, and specific cultural concerns. We found that informed consent by individuals could not fully address these collective issues. This approach may serve as a general model for the undertaking of population-specific genetic studies.     

Tumor cell cytotoxicity of a novel metal chelator

We have synthesized a novel six-coordinate metal chelator from the triamine cis-1,3,5-triaminocyclohexane by the addition of a 2-pyridylmethyl pendant arm on each nitrogen, which we term tachpyr. The experiments described here were designed to explore whether this compound exhibits potential antitumor activity. When added to MBT2 or T24 cultured bladder cancer cells, tachpyr was profoundly cytotoxic, with an IC50 of approximately 4.6 micromol/L compared with 70 micromol/L for desferioxamine. To explore the mode of action of tachpyr, several metal complexes were prepared, including Fe(II), Ca(II), Mn(II), Mg(II), Cu(II), and Zn(II) tachpyr complexes. Of these, the Zn(II), Cu(II), and Fe(II) complexes were without toxic effect, whereas the Ca(II), Mn(II), and Mg(II) complexes remained cytotoxic. To further probe the role of Zn(II) and Cu(II) chelation in the cytotoxicity of tachpyr, sterically hindered tachpyr derivatives were prepared through N-alkylation of tachpyr. These derivatives were unable to strongly bind Fe(III) or Fe(II) but were able to bind Zn(II) and Cu(II). When added to cells, these sterically hindered tachpyr derivatives were nontoxic, consistent with a role of iron depletion in the cytotoxic mechanism of tachpyr. Further, the addition of tachpyr to proliferating cultures resulted in an early and selective inhibition of ferritin synthesis, an iron storage protein whose translation is critically dependent on intracellular iron pools. Taken together, these experiments suggest that tachpyr is a cytotoxic metal chelator that targets intracellular iron, and that the use of tachpyr in cancer therapy deserves further exploration.     

Pulmonary function test outcomes in healthy Navajo Native American adolescents

Prediction equations for estimating lung volumes have been determined for Caucasians, African-Americans, and Mexican-Americans. These separate equations were determined because of differences in thoracic morphology between people of various racial groups, making it impossible to use one prediction formula to accurately estimate lung volumes for all individuals. One hundred ninety-one adolescent Navajo children (males, n = 110; females, n = 81) between 11 and 18 yr of age volunteered for the study and underwent a series of pulmonary function tests (PFT). New pulmonary function prediction equations for Navajo youth were generated for estimating pulmonary volumes and capacities that more accurately predict expected PFT outcomes than formulas in common use for Caucasian, Mexican-American, or African-American youth.     

Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria

Tannic acid, propyl gallate and methyl gallate, but not gallic acid, were found to be inhibitory to the growth of intestinal bacteria Bacteroides fragilis ATCC 25285, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputrificum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Salmonella typhimurium TA98 and S. typhimurium YG1041 at 100-1000 microg/ml in culture broth. Neither Bifidobacterium infantis ATCC 15697 nor Lactobacillus acidophilus ATCC 4356 was inhibited by any of the above compounds up to 500 microg/ml. Tannic acid has a much greater relative binding efficiency to iron than propyl gallate, methyl gallate or gallic acid. The inhibitory effect of tannic acid to the growth of intestinal bacteria may be due to the strong iron binding capacity of tannic acid; whereas the effect of propyl gallate and methyl gallate probably occurs by a different mechanism. The growth of E. coli was restored by the addition of iron to the medium after the precipitate caused by tannic acid was removed. Neither B. infantis nor L. acidophilus require iron for growth. This probably contributes to their resistance to tannic acid. Because tannins are abundant in the human diet, tannins may affect the growth of some intestinal bacteria and thus may have an impact on human health.     

Unfolding mechanism of rubredoxin from Pyrococcus furiosus

As part of our studies on the structural and dynamic properties of hyperthermostable proteins, we have investigated the unfolding pathways of the small iron-sulfur protein rubredoxin from Pyrococcus furiosus (RdPf) at pH 2. Unfolding has been initiated by temperature jump, triggered by manual mixing of a concentrated protein solution into a thermally preequilibrated buffer. The process has been followed in real time by absorption, tryptophan fluorescence emission, and far-UV circular dichroism. Unlike the case of the mesophilic rubredoxin from Clostridium pasteurianum (RdCp), RdPf displays a complex unfolding kinetics, pointing to the formation of at least three intermediates. All of the steps, including the one involving metal ion release, are extremely slow. However, hydrophobic core relaxation--not Fe3+ loss--is rate-determining for RdPf unfolding. This clearly rules out the fact that Fe3+ is solely responsible for the kinetic stability of RdPf. Results have been discussed in terms of sequential vs parallel pathways, and the possible role of irreversible phenomena has been taken into consideration. Aggregation does not appear to play a significant role in the observed kinetic complexities. According to a proposed sequential mechanism, partial release of secondary structure elements precedes iron loss, which is then followed by further loss of beta-sheet content and, finally, by hydrophobic relaxation. Although the main features of the RdPf unfolding mechanism remain substantially unchanged over the experimentally accessible temperature range, final hydrophobic relaxation gets faster, relative to the other events, as the temperature is decreased. A qualitative assessment of the unfolding activation parameters suggests that this arises from the very low activation energies (Ea) that characterize this step.     

Ethical and practical issues involved in behavioral pharmacology research that administers drugs of abuse to human volunteers

Researchers carrying out non-therapeutic research that involves the administration of drugs of abuse to human volunteers can be faced with many ethical and practical questions. The history of this type of research is relatively brief, with little in the way of published information relevant to carrying out behavioral pharmacological research with human participants. The aim of this article is to raise issues that occur in most studies of this type and to provide solutions that we have found acceptable and which have been approved by a variety of institutions and regulatory agencies. Clearly, there are other approaches that would work equally as well, and we are not attempting to provide 'the' answer to many of the issues raised. We believe that raising these issues and providing our perspectives is important for stimulating others to discuss them and for all of us to strive, where possible, to reach a consensus concerning ethical practices and to become aware of gaps and pitfalls. The topics discussed range from the nuts and bolts of acquiring and keeping track of drugs, to selecting research participants and designing ethical studies that protect our volunteers while still collecting scientifically useful data.     

A randomized controlled trial of filgrastim during remission induction and consolidation chemotherapy for adults with acute lymphoblastic leukemia: CALGB study 9111

Recombinant human granulocyte colony-stimulating factor (G-CSF; filgrastim) shortens the time to neutrophil recovery after intensive chemotherapy, but its role in the treatment of adults with acute lymphoblastic leukemia (ALL) is uncertain. We randomly assigned 198 adults with untreated ALL (median age, 35 years; range, 16 to 83) to receive either placebo or G-CSF (5 microgram/kg/d) subcutaneously, beginning 4 days after starting intensive remission induction chemotherapy and continuing until the neutrophil count was >/=1, 000/microL for 2 days. The study assignment was unblinded as individual patients achieved a complete remission (CR). Patients initially assigned to G-CSF then continued to receive G-CSF through 2 monthly courses of consolidation therapy. Patients assigned to placebo received no further study drug. The median time to recover neutrophils >/=1,000/microL during the remission induction course was 16 days (interquartile range [IQR], 15 to 18 days) for the patients assigned to receive G-CSF and 22 days (IQR, 19 to 29 days) for the patients assigned to placebo (P < .001). Patients in the G-CSF group had significantly shorter durations of neutropenia (<1, 000/microL) and thrombocytopenia (<50,000/microL) and fewer days in the hospital (median, 22 days v 28 days; P = .02) compared with patients receiving placebo. The patients assigned to receive G-CSF had a higher CR rate and fewer deaths during remission induction than did those receiving placebo (P = .04 by the chi-square test for trend). During Courses IIA and IIB of consolidation treatment, patients in the G-CSF group had significantly more rapid recovery of neutrophils >/=1,000/microL than did the control group by approximately 6 to 9 days. However, the patients in the G-CSF group did not complete the planned first 3 months of chemotherapy any more rapidly than did the patients in the placebo group. Overall toxicity was not lessened by the use of G-CSF. After a median follow-up of 4. 7 years, there were no significant differences in either the disease-free survival (P = .53) or the overall survival (P = .25) for the patients assigned to G-CSF (medians, 2.3 years and 2.4 years, respectively) compared with those assigned to placebo (medians, 1.7 and 1.8 years, respectively). Adults who received intensive chemotherapy for ALL benefited from G-CSF treatment, but its use did not markedly affect the ultimate outcome.     

Diagnosis and rehabilitation strategies for patients with hysterical hemiparesis: a report of four cases

BACKGROUND: Sulphidoleukotrienes (slt) are important mediators in allergic diseases that are synthesized after allergen-specific stimulation. OBJECTIVES: The aim of this study is to determine in vitro slt production after allergen-specific (Dermatophagoides pteronyssinus) stimulus of peripheral blood leucocytes and to observe whether histamine release in whole blood with the same allergen correlates with slt production. We also wanted to evaluate whether a correlation exists between the release of slt and histamine and other diagnostic procedures as well as various clinical situations. METHODS: We studied 62 patients sensitive to Dermatophagoides pteronyssinus (Der p), 30 atopic controls and 12 healthy donors. We determined slt production using the CAST-ELISA technique and histamine release using two concentrations of Der p extract (20 and 2 ng/mL). We also carried out quantification of specific and total IgE levels, skin tests and pulmonary function test on each patient. RESULTS: We observed a significantly increased slt release after in vitro stimulation with Der p. There was a significant difference in the slt release between controls and sensitive patients (P < 0.001) and between atopic controls and sensitive patients (P < 0.001). The data are similar to those obtained with histamine release. We noted a positive correlation (P < 0.001) between slt and histamine release (r = 0.71, at 2 ng/mL and r = 0.83 at 20 ng/mL). We also found a positive (P<0.001), although weak (r=0.4 with at 2ng/mL, and r = 0.34 with P = 0.003 at 20 ng/mL) correlation between slt release and specific IgE levels as well as between slt release and skin-test reactivity (r = 0.49 at 2 ng/mL and r = 0.45 at 20 ng/mL; P < 0.001). No significant correlation between slt release and asthma severity was observed, although a trend toward higher slt production in severe and moderate asthma was detected. We found a significant (P<0.001) but weak (r=-0.3) negative correlation between age and slt release. With respect to sex-related differences, we found significant differences (P < 0.05) in slt release between the sexes with a higher slt release in men than in women. CONCLUSION: We conclude that CAST-ELISA for quantification of slt production is a useful in vitro method for diagnosing sensitization to Der p. There also exists a close correlation between slt release and other parameters of allergic sensitization in vitro as well as in vivo.     

Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans

The FUNGITEST method (Sanofi Diagnostics Pasteur, Paris, France) is a microplate-based procedure for the breakpoint testing of six antifungal agents (amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and miconazole). We compared the FUNGITEST method with a broth microdilution test, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 180 isolates of Candida spp. (50 C. albicans, 50 C. glabrata, 10 C. kefyr, 20 C. krusei, 10 C. lusitaniae, 20 C. parapsilosis, and 20 C. tropicalis isolates) and 20 isolates of Cryptococcus neoformans. Overall, there was 100% agreement between the methods for amphotericin B, 95% agreement for flucytosine, 84% agreement for miconazole, 83% agreement for itraconazole, 77% agreement for ketoconazole, and 76% agreement for fluconazole. The overall agreement between the methods exceeded 80% for all species tested with the exception of C. glabrata (71% agreement). The poorest agreement between the results for individual agents was seen with C. glabrata (38% for fluconazole, 44% for ketoconazole, and 56% for itraconazole) and C. tropicalis (50% for miconazole). The FUNGITEST method misclassified as susceptible 2 of 12 (16.6%) fluconazole-resistant isolates, 2 of 10 (20%) itraconazole-resistant isolates, and 4 of 8 (50%) ketoconazole-resistant isolates of several Candida spp. Further development of the FUNGITEST procedure will be required before it can be recommended as an alternative method for the susceptibility testing of Candida spp. or C. neoformans.