Hot under the collar: A 14-year association between temperature and violent behavior across 436 U.S. counties.

OBJECTIVES: Violent behavior is influenced by individual and societal characteristics, but the role of environmental factors is less understood. Our aims were to use national-level data to identify the association between criminal behavior and short-term temperature conditions, including the departure of daily temperatures from normal conditions. METHODS: We conducted a multi-stage hierarchical time-series model across 436 U.S. counties and 14-years representing 100.4 million people to investigate the association between daily mean temperature and daily mean temperatures departing from normal conditions with violent and non-violent crime counts. First-stage comparisons were made within counties to control for population and geographic heterogeneities, while a second stage combined estimates. We evaluated differences in risk based on county sociodemographic characteristics and estimated non-linear exposure-response relationships. RESULTS: We observed a total of 9.0 million violent crimes and 20.9 million non-violent property crimes between 2000 through 2013. We estimated that each 10 °C increase in daily temperature or daily departure from long-term normal temperatures were associated with 11.92% (95% PI: 11.57, 12.27) and 10.37% (95% PI: 10.05, 10.69) increase in the risk of violent crime, respectively. Similar, but lower in magnitude trends, were observed for property crime risks. We found that crime risk plateaus and decreases at high daily temperatures, but for temperatures departing from normal, the association with crime increased linearly. Seasonal variations showed that anomalously warm temperatures days during cool months had the greatest risk. CONCLUSIONS: Our study revealed an association between higher temperatures and high departure from normal temperatures with both violent and non-violent crime risk, regardless of community-type. However, our findings on seasonal and daily trends suggest that daily mean temperature may impact crime by affecting routine activities and behavior, as opposed to a temperature-aggression relationship. These results may advance public response and planning to prevent violent behavior.

Metabolism of formycin B by Leishmania amastigotes in vitro. Comparative metabolism in infected and uninfected human macrophages.

Formycin B is metabolized by cutaneous Leishmania amastigotes within cultured human macrophages to give formycin B 5'-monophosphate and formycin A 5'-mono-, di-, and triphosphates. Formycin A is also incorporated into RNA. The activity of formycin B against amastigotes was correlated with the levels of formycin A metabolites formed in the parasites. Uninfected macrophages also convert formycin B into the same products, but the levels are markedly lower than those seen in infected macrophages. The results suggest that a sufficient therapeutic index exists to warrant consideration of formycin B as an anti-leishmanial drug in humans.

Developing a geostatistical simulation method to inform the quantity and placement of new monitors for a follow-up air sampling campaign.

Sampling campaign design is a crucial aspect of air pollution exposure studies. Selection of both monitor numbers and locations is important for maximizing measured information, while minimizing bias and costs. We developed a two-stage geostatistical-based method using pilot NO2 samples from Lanzhou, China with the goal of improving sample design decision-making, including monitor numbers and spatial pattern. In the first step, we evaluate how additional monitors change prediction precision through minimized kriging variance. This was assessed in a Monte Carlo fashion by adding up to 50 new monitors to our existing sites with assigned concentrations based on conditionally simulated NO2 surfaces. After identifying a number of additional sample sites, a second step evaluates their potential placement using a similar Monte Carlo scheme. Evaluations are based on prediction precision and accuracy. Costs are also considered in the analysis. It was determined that adding 28-locations to the existing Lanzhou NO2 sampling campaign captured 73.5% of the total kriged variance improvement and resulted in predictions that were on average within 10.9 µg/m3 of measured values, while using 56% of the potential budget. Additional monitor sites improved kriging variance in a nonlinear fashion. This method development allows for informed sampling design by quantifying prediction improvement (accuracy and precision) against the costs of monitor deployment.

School environmental conditions and links to academic performance and absenteeism in urban, mid-Atlantic public schools.

School facility conditions, environment, and perceptions of safety and learning have been investigated for their impact on child development. However, it is important to consider how the environment separately influences academic performance and attendance after controlling for school and community factors. Using results from the Maryland School Assessment, we considered outcomes of school-level proficiency in reading and math plus attendance and chronic absences, defined as missing 20 or more days, for grades 3-5 and 6-8 at 158 urban schools. Characteristics of the environment included school facility conditions, density of nearby roads, and an index industrial air pollution. Perceptions of school safety, learning, and institutional environment were acquired from a School Climate Survey. Also considered were neighborhood factors at the community statistical area, including demographics, crime, and poverty based on school location. Poisson regression adjusted for over-dispersion was used to model academic achievement and multiple linear models were used for attendance. Each 10-unit change in facility condition index, denoting worse quality buildings, was associated with a decrease in reading (1.0% (95% CI: 0.1-1.9%) and math scores (0.21% (95% CI: 0.20-0.40), while chronic absences increased by 0.75% (95% CI: 0.30-1.39). Each log increase the EPA's Risk Screening Environmental Indicator (RSEI) value for industrial hazards, resulted in a marginally significant trend of increasing absenteeism (p < 0.06), but no association was observed with academic achievement. All results were robust to school-level measures of racial composition, free and reduced meals eligibility, and community poverty and crime. These findings provide empirical evidence for the importance of the community and school environment, including building conditions and neighborhood toxic substance risk, on academic achievement and attendance.

Development of miltefosine for the leishmaniases.

The leishmaniases consist of visceral and cutaneous syndromes present in > 30 endemic regions of the world. Miltefosine (hexadecylephosphocholine) is the first oral agent that is effective and tolerated for both visceral and cutaneous disease in several endemic regions, and represents a major advance in the treatment of these diseases.

Cytostatic effect of TNFalpha on cancer cells is independent of p21WAF1.

Tumor necrosis factor alpha (TNFalpha) is a cytotoxic/cytostatic compound for a variety of human cancer cells. The p21WAF1 protein is a cyclin-dependent kinase inhibitor (CDKI) that binds to cyclin/cyclin-dependent kinase (CDK) complexes and inhibits their kinase activities, thereby leading to cell cycle arrest. We found that the cytostatic effect of TNFalpha on the cervical cancer cell line, ME180, was concomitant with an arrest of these cells in the G0/G1 phase of the cell-cycle. This corresponded with an increase in both p21WAF1 mRNA and protein levels which likely occurred via a p53-independent pathway since ME180 is infected with the human papilloma virus. To elucidate the role of p21WAF1 in the TNFalpha-mediated growth and cell cycle arrest, we stably transformed ME180 cells with an antisense p21WAF1 expression vector. Two clones with reduced levels of p21WAF1 both in their basal state as well as after their exposure to TNFalpha were selected. The growth of these cells was still inhibited by TNFalpha and they arrested in G0/G1 similar to wildtype or empty vector transfected cells. These results indicate that although p21WAF1 expression increases dramatically with TNFalpha treatment, it may not play a critical role in the cytostatic effect of TNFalpha on ME180 cervical cancer cells.

Inhibition of growth of human leukemia cell lines by retrovirally expressed wild-type p16INK4A.

Loss of the p16INK4A gene by homozygous deletions or point mutations is attributed to the development of many types of cancers including leukemia. T cell acute lymphoblastic leukemias (T-ALLs) and B-cell ALLs show a remarkable rate of 75 and 20% homozygous deletion of this gene, respectively. Restoration of p16 expression in p16-deficient solid tumor cell lines results in a dramatic reduction of growth and maligant phenotype. To test the hypothesis that p16INK4A suppresses the growth of p16-deficient leukemias, we utilized a retroviral system to restore wild-type (wt) or mutant p16 protein expression. We tested the efficacy of our system by expressing the wt or mutant p16 genes in the osteosarcoma cell line, U20S, which lacks p16 and retains functional retinoblastoma protein (pRb). The wt p16 protein formed complexes with both cyclin-dependent kinases (CDK) 4 and 6 and inhibited U20S growth by 30-fold. The p16 mutants E120K and R144C formed complexes with CDK4 and CDK6 in cells and inhibited cell growth as effectively as wt p16 (20-fold) while the mutant proteins that did not complex with detectable levels of CDK4 or CDK6 only inhibited growth 0.25- and five-fold (G101W and D141, respectively) or not at all (H83Y and DA4). The COOH-terminal 'tail' of the wt p16 protein (amino acid residues 141-156), missing in mutant D141, enhanced the growth suppressive capability of p16. The amino acid substitutions in mutants G101W and H83Y not only disrupted CDK4 and CDK6 binding, but decreased the protein half-lives by two- and three-fold, respectively, compared to wt p16. The wt, but not mutant p16 genes, effectively inhibited the growth of T cell acute lymphoblastic (CEM) and myeloid leukemia (NB-4 and K562) cell lines that lacked the p16 gene, but retained functional pRb. Growth of the T-ALL cell line, HSB-2, which lacked both p16 and pRb, was not inhibited, indicating the growth suppression involved the pRb pathway. These results define regions critical for the function of p16 and demonstrate that restoration of wt p16 expression in p16-deficient leukemias significantly reverted their transformed phenotype and inhibited their growth.

Noninvasive management of Indian visceral leishmaniasis: clinical application of diagnosis by K39 antigen strip testing at a kala-azar referral unit.

Firm diagnosis of visceral leishmaniasis (kala-azar) requires organ aspiration and microscopic examination of tissue specimens. To determine the usefulness of noninvasive diagnosis by strip test detection of anti-K39 immunoglobulin (Ig) G antibody in blood specimens obtained by fingerstick, 143 Indian patients with suspected kala-azar (fever, splenomegaly, anemia) were studied. Of 120 strip test-positive subjects (subjects with presumed kala-azar [group A]), amphotericin B treatment induced clinical cure in 119. Of 23 strip test-negative subjects (subjects presumed to have other diseases [group B]), 16 had other disorders diagnosed at entry, 4 responded to empiric antimalarial therapy, 2 were proven to have kala-azar, and 1 died elsewhere after undergoing splenic aspiration. Six months after treatment ended, all 120 patients in group A and the 18 assessable patients in group B were healthy. In a region in India where visceral infection is prevalent, strip test detection of anti-K39 IgG is a clinically promising diagnostic guide in persons with suspected kala-azar.

Miltefosine for new world cutaneous leishmaniasis.

The oral agent miltefosine has demonstrated a >95% cure rate in Indian visceral leishmaniasis. We performed a large, placebo-controlled study of miltefosine therapy (2.5 mg/kg per day orally for 28 days) against cutaneous leishmaniasis in Colombia and Guatemala. In regions in Colombia where Leishmania vianna panamensis is common, the per-protocol cure rates for miltefosine and placebo were 91% (40 of 44 patients) and 38% (9 of 24). These values are similar to historic values for the antimony standard of care and placebo. In regions in Guatemala where L. v. braziliensis and L. mexicana mexicana are common, the per-protocol cure rates were 53% (20 of 38) for miltefosine and 21% (4 of 19) for placebo. The miltefosine rate was lower than historic antimony cure rates of >90%. Miltefosine was well tolerated. Miltefosine is a useful oral agent against cutaneous leishmaniasis due to L. v. panamensis in Colombia but not against leishmaniasis due to L. v. braziliensis in Guatemala.

Diminished adrenocorticotropin response to insulin-induced hypoglycemia in nondepressed, actively drinking male alcoholics.

Although changes in hypothalamic-pituitary-adrenal axis function have frequently been reported in alcoholics, the majority of studies have used recently detoxified subjects in whom abstinence phenomena and clinical depression may contribute to observed stress axis alterations. To isolate the primary effects of alcohol dependence on the stress axis, the ACTH and cortisol responses to insulin-induced hypoglycemia were measured in seven actively drinking male alcoholics recruited from the general public through a newspaper advertisement along with eight age-matched male controls. The alcoholic subjects met current American Psychiatric Association diagnostic criteria for alcohol dependence, were stably employed, and had no concurrent psychiatric disorders, cognitive impairment, or psychometric evidence of depression. While relatively young (30.0 yr; range, 22-48 yr), they had lengthy histories of alcohol-related problems (11.9 yr; range, 5-30 yr). Insulin administration resulted in similar nadirs in blood sugar in both alcoholic and control groups. However, the plasma ACTH response was markedly blunted in the alcoholics (P = 0.040, by Mann-Whitney U test). There was a nonsignificant trend toward increased cortisol levels in the alcoholic group. The findings suggest that altered hypothalamic-pituitary-adrenal axis function in alcoholics is a primary results of chronic ethanol exposure rather than a confounding effect of clinical depression or recent detoxification.

Efficacy of ketoconazole against Leishmania braziliensis panamensis cutaneous leishmaniasis.

PURPOSE, PATIENTS, AND METHODS: The classic agent for cutaneous leishmaniasis is pentavalent antimony. However, there are no reports of the efficacy of antimony versus placebo or of the efficacy of any alternative therapy versus either antimony or placebo. In the present report, the oral antifungal agent ketoconazole (600 mg/day for 28 days) was compared to a recommended regimen of intramuscular Pentostam (20 mg antimony/kg, with a maximum of 850 mg antimony/day, for 20 days) in a randomized study of the treatment of Panamanian cutaneous leishmaniasis due to Leishmania braziliensis panamensis. A separate group of patients with this disease was administered placebo. RESULTS: Ketoconazole clinically cured 16 of 21 (76%) patients. The lesions on nine patients healed by 1 month after therapy, and the lesions healed by 3 months after therapy on the other seven patients. Side effects were limited to a 27% incidence of mild, reversible hepatocellular enzyme elevation and an asymptomatic, reversible, approximately 70% decrease in serum testosterone in all patients. Pentostam cured 13 of 19 (68%) patients; the lesions on seven patients healed by the end of therapy, and the lesions on four other patients healed by 1 month after the end of therapy. Side effects were a 47% incidence of mild, reversible hepatocellular enzyme elevation and the morbidity due to 20 intramuscular injections in almost all patients. The placebo group of 11 patients had a 0% cure rate. By 1 month after therapy, all placebo-treated patients demonstrated new lesions or one lesion that was 23% to 875% larger than before therapy. CONCLUSION: Both ketoconazole and Pentostam were more effective than placebo against L. braziliensis panamensis cutaneous leishmaniasis. Oral ketoconazole is comparable in efficacy to this parenteral Pentostam regimen and can be recommended as initial treatment for this disease.

Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture.

Ketoconazole, a clinically effective antimycotic agent active in vitro against the amastigote stage of Leishmania mexicana Walter Reed 227 in human monocyte-derived macrophages, was found to inhibit growth and impair sterol biosynthesis of the cultured promastigote stage by approx. 50% at a concentration of approx. 10(-8)M. Sterol biosynthesis was interfered with at the level of the removal of the 14 alpha-methyl group of lanosterol, as judged by changes in the distribution of [2-14C]mevalonate radioactivity among desmethyl sterol and methyl sterol thin-layer chromatography fractions, by the loss of 4-desmethyl sterols (mainly 5-dehydroepisterol), and by the accumulation of 14 alpha-methyl sterols. The growth inhibition and sterol changes were evident in promastigotes cultured in a cholesterol-rich medium and in a cholesterol-poor medium, even though promastigotes incorporated cholesterol. The mechanism of action of ketoconazole against promastigotes may be that postulated for Candida albicans: interference with membrane permeability secondary to loss of desmethyl sterols and accumulation of 14 alpha-methyl sterols.

Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells.

Murine macrophage tumor cells infected with Leishmania mexicana mexicana were exposed to the antimycotic drug ketoconazole and to [2-14C]mevalonate, then the amastigotes were isolated, collected, purified, and their free sterols were analyzed by chromatographic and mass spectrometric methods. Control amastigotes contained as products of de novo biosynthesis C28 4-desmethyl sterols (episterol, 5-dehydroepisterol), C29 4-desmethyl sterols (stigmasta-7,24 (28)-dien-3 beta-ol, stigmasta-5,7,24(28)-trien-3 beta-ol), 4-methyl sterols (4 alpha, 14 alpha-dimethylzymosterol, obtusifoliol) and a 4,4-dimethyl sterol (lanosterol). Present also were macrophage sterols (cholesterol, desmosterol) and a putative product of the C-24 alkylation of desmosterol by amastigotes (24-methylenecholesterol). Amastigotes from macrophages exposed to ketoconazole showed notable changes in the proportions, concentrations and specific activities of their free sterols; increased for 4 alpha, 14 alpha-dimethylzymosterol and decreased for the endogenous C28 and C29 4-desmethyl sterols. Such changes were observed at a ketoconazole concentration as low as 0.01 microgram ml-1. By contrast, uninfected macrophages accumulated only small amounts of lanosterol of high specific activity at a ketoconazole concentration of 10 micrograms ml-1. the ketoconazole-induced alterations in amastigote sterols parallel those previously reported in fungi and L. m. mexicana promastigotes, and suggest a biochemical mechanism for the anti-leishmanial activity of the drug in which changes in sterol composition are linked to disturbances of cell membrane structure and function, and hence to cytotoxicity.

Pharmacokinetic justification of antiprotozoal therapy. A US perspective.

Infections with parasitic protozoa have always been problems for the developing world and are becoming of greater importance to the developed world in this age of easy international travel. The major human protozoal diseases are summarised with an emphasis on their presentation in normal hosts and in immunocompromised individuals and current US drug treatment recommendations are discussed. Present antiprotozoal regimens are based either on a pharmacokinetic rationale or on clinical trial and error. Regimens based on trial and error include amphotericin B against leishmaniasis and arsenic against African trypanosomiasis. Regimens which are to some extent driven by pharmacokinetic or biochemical considerations include paromomycin and metronidazole against amoebiasis, sodium stibogluconate against leishmaniasis, halofantrine and mefloquine against malaria, dihydrofolate reductase (DHFR) inhibitors against Pneumocystis carinii and toxoplasmosis and aerosolised pentamidine against P. carinii pneumonia. The majority of pharmacokinetic studies have been performed only on agents which have some therapeutic activity against other diseases of the developed world. Despite the trend toward rational treatment regimens, no studies have been performed that permit optimisation of antiprotozoal treatment regimens on the basis of clinical conditions such as renal failure.

Sodium stibogluconate (Pentostam) inhibition of glucose catabolism via the glycolytic pathway, and fatty acid beta-oxidation in Leishmania mexicana amastigotes.

The biochemical mechanism of action of antimony (Sb) in pentavalent form complexed to gluconic acid (sodium stibogluconate)--the drug of choice for the leishmaniases--has been only slightly investigated. We recently reported that, in stibogluconate-exposed Leishmania mexicana amastigotes, there is a dose-dependent decrease in the ATP/ADP ratio [Berman et al., Antimicrob. Agents Chemother. 27, 916 (1985)]. To investigate mechanisms by which ADP phosphorylation to ATP might be inhibited, stibogluconate-exposed amastigotes were incubated with [14C]glucose, fatty acid, or acetate, and 14CO2 production was determined. In organisms exposed to 500 micrograms Sb/ml, formation of 14CO2 from [6-14C]glucose and [1-14C]palmitate was inhibited 69 and 67% respectively. In comparison, formation of 14CO2 from [1-14C]glucose and [2-14C]acetate was inhibited less than 15%. These results suggest that glucose catabolism via glycolytic enzymes and fatty acid beta-oxidation, but not glucose metabolism via the hexosemonophosphate shunt or the citric acid cycle, is specifically inhibited in stibogluconate-exposed Leishmania mexicana amastigotes. Inhibition of these pathways suggests a mechanism for the inhibition of ADP phosphorylation previously reported.

Acetylcholine exocytosis in PC12 cells deficient in SNAP-25.

Stimulus-induced acetylcholine (ACh) exocytosis from presynaptic nerve terminals involves two important steps: fusion of ACh loaded vesicles at presynaptic release sites, followed by release into the synaptic cleft. We studied the role of the putative vesicle fusion protein SNAP-25 in this process. The nerve growth factor-differentiated PC12 cell line was used as an experimental model. A bee venom tetradecapeptide (INLKALAALAKKIL-NH2) phospholipase A2 (PLA2) activator, mastoparan, was used to induce ACh release. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K+ induced ACh exocytosis. The results indicate that in PC12 cells, ACh exocytosis due to mastoparan plus K+ can occur in the absence of SNAP-25.

Inhibition of leishmanial protein kinase by antileishmanial drugs.

Protein kinase was isolated from both amastigotes and promastigotes of Leishmania mexicana amazonensis. Unlike the previously described enzyme from L. donovani promastigotes, activity of the L. mexicana kinases was 2-3 times higher at low ionic strength than at high ionic strength, and was 3-10-fold augmented by removal of endogenous low molecular weight inhibitors. The Km of the L. mexicana kinases was 123-223 microM, compared to the value of 70 microM for the beef heart kinase. Purine nucleoside analogs are potent antileishmanial agents that are phosphorylated to their respective triphosphates. The mechanism of the analogs is thought to involve competition of the triphosphates with ATP. Cordycepin triphosphate inhibited the amastigote, promastigote, and beef heart protein kinases approximately equally. However, the Kis of formycin A triphosphate for the leishmanial kinases (Ki 40-120 microM) were far less than that of the beef heart kinase (Ki 1,380 microM). The mechanisms of certain chemotherapeutic purine nucleosides may involve specific inhibition of leishmanial protein kinase by the nucleoside triphosphate.

Activity of imidazoles against Leishmania tropica in human macrophage cultures.

The anti-leishmanial activity of four imidazoles has been determined in Leishmania tropica-infected human monocyte-derived macrophage cultures. One of the imidazoles, hydrolyzed ketoconazole [cis-1-[4-[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl] methoxyphenyl]piperazine], eliminated 80 and 95% of the parasites at drug concentrations (2.0 and 2.5 microgram/ml) that are achievable in vivo by a structurally similar compound, ketoconazole. These results demonstrate that an imidazole has anti-leishmanial activity in a model system, and suggests that hydrolyzed ketoconazole should be considered for in vivo trials in animal models of the disease.

Recommendations for treating leishmaniasis with sodium stibogluconate (Pentostam) and review of pertinent clinical studies.

Pentavalent antimonial compounds have been the mainstay of the treatment of visceral, cutaneous, and mucosal leishmaniasis for approximately half a century. Pentostam (sodium stibogluconate) is the pentavalent antimonial compound available in the United States (through the Centers for Disease Control). As dosage regimens for treating leishmaniasis have evolved, the daily dose of antimony and the duration of therapy have been progressively increased to combat unresponsiveness to therapy. In the 1980s, the use of 20 mg/kg/day (instead of 10 mg/kg/day) of antimony was recommended, but only to a maximum daily dose of 850 mg. The authors have concluded on the basis of recent efficacy and toxicity data that this 850-mg restriction should be removed; the evidence to date, which is summarized here, suggests that a regimen of 20 mg/kg/day of pentavalent antimony, without an upper limit on the daily dose, is more efficacious and is not substantially more toxic than regimens with lower daily doses. We recommend treating all forms of leishmaniasis with a full 20 mg/kg/day of pentavalent antimony. We treat cutaneous leishmaniasis for 20 days and visceral and mucosal leishmaniasis for 28 days. Our judgment of cure is based on clinical criteria.

Activity of oral drugs against Leishmania tropica in human macrophages in vitro.

Because of the need for orally active antileishmanial agents, orally administrable drugs have sometimes been used to treat human leishmaniases without prior demonstration of efficacy in experimental models. The antileishmanial activity of such agents was tested against Leishmania tropica (a cause of cutaneous leishmaniasis) within human macrophages in vitro. Although trimethoprim + sulfamethoxazole and isoniazid + rifampin have been reported as efficacious orally in certain human studies of cutaneous disease, these drugs were ineffective in vitro (less than or equal to 40% parasite elimination) at peak achievable serum levels. The combination of allopurinol and Pentostam is being tested in humans. In vitro, allopurinol (5 micrograms/ml) augmented the antileishmanial effect of a low concentration of Pentostam (5 micrograms/ml) but not of a higher concentration of Pentostam (20 micrograms/ml). Nifurtimox is a nitrofuran which has questionable activity against human cutaneous disease. Nifurtimox was similarly only 50% effective in vitro at peak achievable serum levels (1.0-3.0 micrograms/ml). However, furazolidone, another orally administered nitrofuran, eliminated 92% of parasites at 1.0 micrograms/ml. Chlorpromazine and quinacrine are concentrated in tissues that are susceptible to infection by Leishmania. Chlorpromazine and quinacrine eliminated only 15% and 35% of organisms in vitro at achievable serum levels (less than or equal to 0.3 microgram/ml), but eliminated virtually all organisms in vitro at possible achievable tissue levels. Both the negative and the positive data of this report may aid in selection of effective orally active agents for in vivo trials.