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PubMed1992

Didanosine and zidovudine resistance patterns in clinical isolates of human immunodeficiency virus type 1 as determined by a replication endpoint concentration assay.

Reports of in vitro resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (AZT) have raised concerns about the development of resistance to other dideoxynucleosides in clinical use. To address this, we have developed a screening assay which supports the growth of clinical isolates and have applied this to a series of paired isolates from patients entered into a phase I trial of didanosine (DDI). Thirteen patients (10 with AIDS, 3 with AIDS-related complex) who had been exposed to AZT for a mean of 6.5 months (range, 1 to 13 months) were treated with DDI at 750 mg/day. Paired isolates were obtained pretherapy and after a mean of 58 weeks (range, 21 to 90) of DDI therapy by coculture of peripheral blood mononuclear leukocytes (PBLs) with phytohemagglutinin-stimulated donor PBLs. Isolates were passaged only one additional time in PBLs and then tested in parallel in a microtiter assay with phytohemagglutinin-stimulated donor PBLs as targets. PBLs were infected with 10(5) 50% tissue culture infectious doses per 10(7) cells and exposed to DDI (1 to 50 microM) or AZT (0.01 to 100 microM), and supernatants were assayed for the HIV p24 antigen at 7 days postinfection. Control AZT-susceptible and resistant isolates were included. The median pre- and posttherapy DDI susceptibilities of the 13 pairs of isolates were 10.0 microM (range, 1 to 25 microM) and 17.5 microM (range, 2.5 to 50 microM), respectively (P = 0.036; Wilcoxon signed-rank test). These studies thus indicated that (i) the susceptibility to DDI tends to mildly decrease with drug exposure; (ii) the susceptibility to AZT improves with time off AZT; (iii) baseline susceptibilities to DDI have a wide range, and the CD4 response may correlate with the initial susceptibility; and (iv) a PBL-based microtiter assay is useful for screening clinical isolated for dideoxynucleoside susceptibility profiles.

Didanosine↗
PubMed2002

Performance characteristics of the PolyTiter Immunofluorescent Titration system for determination of antinuclear antibody endpoint dilution.

Conventional screening for circulating antinuclear antibodies (ANA) is generally performed by immunofluorescent (IF) microscopy with a 1:40 dilution of serum. Intensity of IF staining is then semiquantitated by using twofold serial dilutions, where the highest dilution in which staining intensity equals the endpoint control is expressed as an endpoint titer. The PolyTiter Immunofluorescent Titration system (Polymedco, Inc.) facilitates ANA-IF assay (IFA) testing by relating the intensity of IF staining to reference calibrators (defined in PolyTiter units), providing an endpoint titer directly from a 1:40 dilution. This study was conducted to assess the performance characteristics of the PolyTiter system. Two technologists each evaluated 10 replicates of three specimens and two controls on five sequential days. Endpoint dilution agreement (defined as +/-2 dilutions) with the reference was 100% for all controls and for all specimens by one technologist. The second reader reported agreement of 98, 88, and 100% for the low, medium, and high specimens, respectively. Analysis of PolyTiter unit values yielded between-reader, between-run, and within-run precision coefficients of variation of less than 10%. The variance component in the lot-to-lot analysis was zero, indicating all of the variation was due to run-to-run differences. Overall endpoint dilution agreement between PolyTiter and serial dilution in the evaluation of 125 specimens at three sites was 90, 93, and 86%. Pattern identification with the PolyTiter was similar to that with serial dilution. The PolyTiter system demonstrates acceptable performance for routine ANA-IFA testing in the clinical laboratory.

Antibodies, Antinuclear↗
PubMed1975

[Biosynthesis of glutathione. VI. Ocurrence and level of gamma-glutamylcysteine in human red cells (author's transl)].

The concentration of the glutathione precursor gamma-glutamylcysteine in hemolysates of human red cells was determined using three different methods. a) The [14C]N-ethylmaleimide derivatives of the intracellular sulfhydryl compounds were separated on a cation exchange column. The amount of [14C]N-ethylmaleimide glutamycysteine was radiochemically determined. b) With purified glutathione synthetase, an enzymatic endpoint metabolite determination was performed with [14C]glycine as one substrate. The amount of labelled glutathione formed from the gamma-glutamylcysteine present was measured radiochemically.

Cysteine↗
PubMed2002

Determination of a humane endpoint in the L1210 model of murine leukemia.

The purpose of our study was to derive an alternate end-point to death or moribund appearance for the frequently used L1210 model of murine leukemia. In reviewing the published literature, we were unable to identify a suitable intermediate marker of substantive disease that predicted outcome in the BDF1 recipient of the L1210 leukemia. In an attempt to refine the use of animals in our laboratory, we developed a scoring sheet for behavioral and physical changes that followed intravenous injection of L1210 lymphocytic leukemia cells into BDF1 recipients. At 12-h intervals for the first 2 days after tumor-cell injection and at 6-h intervals thereafter, animals were observed and scored for each parameter. When death was imminent, animals were euthanized by inhalation of methoxyflurane followed by decapitation. Changes in physical and behavioral characteristics then were correlated with the end-point of death. Changes occurred in the mice approximately 7 days after tumor cell inoculation and 24 h before death. The earliest of these signs was hunched posture, followed by one or more other characteristics including decreased activity, increased facial swelling, ears in backward position, abdominal swelling, squinting eyes, and labored breathing. From these data, we were able to develop criteria for early euthanasia. Use of these intermediate end-points likely will substantially reduce the stress on the animals without compromising scientific outcomes in experiments using this or related preclinical models of cancer.

Animal Testing Alternatives↗
PubMed2004

Estimating suicidality as an outcome measure in clinical trials of suicide in schizophrenia.

A Suicide Monitoring Board (SMB) evaluated putative events of suicidal behavior to determine whether they should be counted as outcome data (endpoints) in the International Suicide Prevention Trial (InterSePT) study. The InterSePT study compared clozapine and olanzapine prospectively, on reduction of suicidality in schizophrenia and schizoaffective disorder. SMB members, blinded to patient identification, evaluated packages of clinical information and the forms used to summarize the putative events and their context. The SMB members met regularly by international teleconference to discuss ratings they found problematic and to achieve consensus. Exactly 243 of the 980 patients enrolled (24.8%) were considered to have experienced 577 suicidal events. Of these events, 483 (83.7%) were confirmed by the SMB as real or valid endpoints. Before the consensus discussions, acceptable levels of agreement (K = 0.52) were reached among the SMB members regarding ratings considered as valid suicidal events; agreement increased (K = 0.64) when consensus ratings and those of on-site psychiatrists also blinded to treatment were also included. This article discusses our experience of evaluating schizophrenia patients for suicidality in general and in particular from reviewing chart information. The process of making clinical judgments regarding the seriousness of suicidal behavior in schizophrenia patients and their suicidal risk warrants further study.

Adult↗
PubMed2006

Short-term outcome after anti-tumor necrosis factor-alpha therapy in rheumatoid arthritis: do we need to revise our assessment criteria?

OBJECTIVE: To evaluate the Disease Activity Score (DAS) using various aggregated dimensions to quantify treatment outcome in patients with rheumatoid arthritis (RA), in order to determine the best instrument to be used as an endpoint that indicates good response in terms of EULAR response criteria and DAS28 remission criteria, and which satisfies the demands of clinical rheumatology. METHODS: Using raw data for each patient subjected to anti-tumor necrosis factor-a therapy (81 patients), before and 6 months after treatment, DAS28 was calculated 4 times using the standard equation, as follows: (1) DAS 1 (the standard DAS28): tender joint count (TJC), swollen joint count (SJC), patient global assessment (PGA), erythrocyte sedimentation rate (ESR); (2) DAS 2: TJC, SJC, PGA, C-reactive protein (CRP); (3) DAS 3: TJC, SJC, physician global assessment (PhGA), ESR; and (4) DAS 4: TJC, SJC, PhGA, CRP. Disease activity was identified if DAS score exceeded 5.1. A clinically significant response was recorded if there had been improvement of > 1.2 of the DAS score. RESULTS: DAS 2, DAS3, and DAS4 were superior to the current DAS score used for assessment of RA activity (effect size differences were -0.35, -0.13, and -0.48, respectively). Assessment of disease activity using TJC, SJC, PhGA, and CRP was the best tool to assess response to therapy. ESR was marginally superior to CRP in its sensitivity to monitor disease activity changes (effect sizes 1.08 and 1.03, respectively). CONCLUSION: These results suggest that self-report indices on their own, such as PGA and pain score, are inadequate indicators of disease activity. The DAS might profitably be amended by one or 2 continuous measures for better quantification of the degree of improvement of patients on a given therapeutic modality. Using PhGA and CRP instead of PGA and ESR, respectively, in the DAS equation discriminated better between different patients' responses than the traditional DAS score.

Adult↗
PubMed2005

Factors determining the optimal body site and method for obtaining punch biopsies of human skin as a tissue in which to assess pharmacodynamic and pharmacokinetic endpoints in drug development studies.

There are potential advantages to detecting pharmacodynamic (PD) and pharmacokinetic (PK) endpoints in a tissue-based compartment such as the skin during the development of molecularly targeted drugs. We explored regional differences between inner arm, inner thigh, lower back and buttocks in 12 healthy male Caucasian volunteers in the tolerability of skin biopsy procedures; the Ki67 proliferation index; the frequency of detecting hair follicles and sweat glands; and the percentage of melanocytes. We also explored the amounts of tissue and protein obtained, and two separate methods of splitting biopsies for processing in mutually exclusive media. Biopsies from all body sites were well tolerated. The subjective ranking order was inner arm > buttocks = back > thigh. There were no statistically significant differences in the Ki67 labelling index (P > 0.05). The frequency of detecting sweat glands was the same in all body sites, but the frequency of detecting hair follicles was higher in back and buttock, compared to arm and thigh. The percentage of melanocytes was significantly lower in the buttocks compared to the back and thigh (P < 0.05), but not compared to the arm (P = 0.07). A 4-mm punch biopsy yielded a mean of 16.8 mg of tissue (range: 9-28 mg) and 160 microg of protein (range: 80-270 microg). In vivo sample splitting, by following a 2-mm punch with a 4-mm overpunch, had a shorter time from devascularisation to immersion into processing medium than ex vivo dissection of a 4-mm sample, which may be of importance to the assessment of labile endpoints. We conclude that multiple punch biopsies of the skin are feasible, with the buttocks representing the studied body site with the optimal balance between tolerability, hair follicle density and melanocyte density for obtaining tissue in which to assess PD and PK endpoints during drug development studies.

Adolescent↗
PubMed1993

Chemoprevention strategies: the relevance of premalignant and malignant lesions of the upper aerodigestive tract.

Patients with premalignant and malignant lesions of the upper aerodigestive tract have historically been the focus of chemoprevention trials within the United States. Experience with this population has formed the basis for trials involving other environmentally induced cancers such as lung and bladder. Given that head and neck cancer patients are at risk for second primary malignancies, prevention strategies can be directed towards decreasing mortality from these metachronous neoplasias. Validity of these strategies, including risk determination, intermediate endpoints, and preventive efficacy of single and combination agents, can be determined. Current limitations in chemoprevention trials involving these patients relate to the sporadic nature of the disease. In fact, the prevalence of oral premalignancy within the United States has not been clearly defined. Individual physician experience with this disease process is limited. Organizational efforts should therefore be directed towards facilitating clinical trials involving dentists, oral surgeons, head and neck surgeons, and other primary health care providers in the community. Risk factors which identify clinically defined normal or premalignant tissue at risk for malignant progression need to be better defined.

Anticarcinogenic Agents↗
PubMed2005

Correlation between 96-h mortality and 24-h acetylcholinesterase inhibition in three grass shrimp larval life stages.

Three life stages of larval grass shrimp were tested to determine whether acetylcholinesterase (AChE) activity expressed as 24-h sublethal effect endpoints (EC20 and EC50) could be used to predict 96-h mortality (lowest observable effect concentration (LOEC) and LC50) for shrimp exposed to three organophosphate insecticides. With regard to mortality, newly hatched larvae and 18-day-old larvae were the most sensitive in the malathion and azinphosmethyl exposures. In the chlorpyrifos exposures, newly hatched larvae and postlarvae were the most sensitive life stages. Results of the 24-h AChE inhibition tests showed that newly hatched larvae were generally more sensitive in the three organophosphate exposures. A regression analysis of the EC50's and LC50's yielded the strongest correlation with R2=0.987 (correlation coefficient=0.994 and 95% confidence intervals 0.969-0.999). The LOEC/EC20 relationship yielded R2=0.962. For these grass shrimp life stages and pesticides, sublethal effect endpoints could be used as a predictor of 96-h mortality.

Acetylcholinesterase↗
PubMed2003

Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae).

A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC(50) values, using microalgal growth inhibition as the endpoint. The obtained results were as follows: Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC(50) values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C. closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media.

Diatoms↗
PubMed2001

In vitro susceptibilities of Bartonella and Rickettsia spp. to fluoroquinolone antibiotics as determined by immunofluorescent antibody analysis of infected Vero cell monolayers.

The in vitro susceptibilities of Bartonella and Rickettsia spp. to different concentrations of ciprofloxacin, levofloxacin, ofloxacin and sparfloxacin in Vero cell cultures, were determined by enumeration of immunofluorescent-stained bacilli. After incubation in a CO(2)-enriched atmosphere, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate for each species and the monolayers were re-incubated. Growth status was determined by evaluation of immunofluorescent staining bacilli. Effective inhibitory antibiotic dilution endpoints were determined by counting Bartonella- and Rickettsia-specific fluorescent foci across a range of antibiotic dilutions with an epi-fluorescent microscope, and were compared with an antibiotic-negative control. Based upon the use of C(max):MIC and AUC:MIC data, levofloxacin exhibited activity against Bartonella elizabethae and B. quintana.

Animals↗
PubMed2005

In vitro activities of voriconazole, fluconazole, itraconazole and amphotericin B against non Candida albicans yeast isolates.

Antifungal susceptibility testing was performed on 197 yeast isolates from the BCCM/IHEM biomedical fungi and yeasts collection (Belgian Co-ordinated Collections of Micro-organisms / IPH-Mycology) to study the in vitro activity of voriconazole against fluconazole, itraconazole and amphotericin B. MICs of the four antifungal agents were determined by an adapted NCCLS M27-A microdilution reference method. MIC readings were visually and spectrophotometrically determined. Optical density data were used for calculation of the MIC endpoints. For amphotericin B, the MIC endpoint was defined as the minimal antifungal concentration that exerts 90% inhibition, compared to the control growth. The azoles endpoints were determined at 50% inhibition of growth. The MIC distribution of voriconazole susceptibilities showed that 193 isolates had a MIC < or = 2 microg/ml and 185 a MIC < or = 1 microg/ml. Cross-tabulation of voriconazole, fluconazole, and itraconazole MICs indicated that voriconazole MICs raised with fluconazole and itraconazole MICs. The in vitro data obtained in this study suggest that voriconazole may also be effective treating yeast infection in patients infected with fluconazole or itraconazole resistant isolates.

Amphotericin B↗
PubMed1988

Rapid and overnight microdilution antibiotic susceptibility testing with the Sensititre Breakpoint Autoreader system.

The Sensititre Breakpoint Autoreader system (SBAS) is a broth microdilution method with one to three concentrations of each antibiotic and innovative fluorescence technology to define inhibitory endpoints. We tested 248 gram-negative bacilli and 80 gram-positive cocci using both the rapid (5 h) and overnight (18 h) SBAS procedures. Inhibitory endpoints were also determined by visual inspection of the microdilution trays after 18 h of incubation. SBAS results were compared with those obtained by a standardized disk diffusion (SDD) procedure. Agreement between the rapid SBAS and SDD results for all antibiotic-organism combinations was found in 3,730 of 4,571 (81.6%) tests, with 3.9% very major, 6.5% major, and 7.9% minor discrepancies noted. Data analysis by organism group revealed 86.8, 57.3, 71.4, and 62.3% agreement for members of the family Enterobacteriaceae, Pseudomonas spp., staphylococci, and enterococci, respectively. The results of the overnight SBAS and SDD agreed in 4,154 of 4,654 (89.2%) tests, with 2.3% very major, 1.3% major, and 7.1% minor discrepancies recorded. Concordance was noted in 90.4, 78.1, 90.6, and 83.3% of the comparisons for the members of the Enterobacteriaceae, Pseudomonas spp., staphylococci, and enterococci, respectively. The inhibitory endpoints determined with the Autoreader were as reliable as those determined by visual inspection after 18 h of incubation.

Anti-Bacterial Agents↗
PubMed2001

The measurement of blood pressure and the detection of hypertension in children and adolescents.

Despite the publication of several expert committee guidelines for the measurement of blood pressure (BP) and the diagnosis of hypertension in children and adolescents, it was our perception and clinical experience that there still appeared to be a general lack of standardisation of BP measurement techniques and little consensus on the criteria for diagnosing hypertension. To investigate this further, we have conducted a postal survey of consultant-grade paediatricians who were members of the British Paediatric Association (BPA). A total of 1500 questionnaires were sent out and 708 analysable replies were received (47.1%). This showed that 68.6% of paediatricians routinely measured BP, at least on one occasion, in children or adolescents attending their outpatient clinics, 17.7% started at or soon after birth, 12.3% started at the age of 1 year, 20.0% at 3 years, 12.0% from 7 years of age and 3.5% from the age of 13. Only 60.5% reported that they had a choice of four or more different cuff sizes in their clinic. Forty-one percent of respondents reported that the BP was always or sometimes measured by nurses. Fifty-one percent of respondents measured diastolic BP at the phase of muffling of sound (Korotkoff phase IV), 31.9% used the disappearance of sound (phase V) whilst 15.9% claimed that they measured both end-points. The criteria for diagnosing a child as being hypertensive varied greatly; 17.9% reported that they responded to the systolic BP alone, 13.5% to the diastolic BP alone, 65.9% relied on both pressures, and 2.7% responded to either the systolic or diastolic pressure if it was raised. Furthermore, 12.9% diagnosed hypertension if the BP exceeded the 90th percentile in relation to age and 41.8% used the 95th percentile. However 45.3% of respondents employed a higher dividing line. In hospitalised children, leg blood pressures were measured routinely by 30.3%, although a further 44.0% would do so if aortic coarctation or other vascular diseases were suspected. Despite considerable variation in clinical practice, techniques and criteria, only 11.4% of clinicians would manage the patients themselves, with the remainder referring the child on to the appropriate specialist. The survey suggests a general lack of standardisation of BP measurement techniques and little consensus on the criteria for diagnosing hypertension amongst paediatricians. Simplified, shortened and updated guidelines on hypertension in paediatric practice and research are needed.

Adolescent↗
PubMed2004

Developmental effects of embryonic exposure to toxaphene in the American alligator (Alligator mississippiensis).

A variety of organochlorine pesticides have been shown to adversely affect embryonic development. A number of abnormalities have been documented in alligators (Alligator mississippiensis) from highly-contaminated Lake Apopka, FL, USA that are similar to the results of experimental studies exposing embryos to pesticides. In the current study, we exposed developing alligator embryos to varying concentrations of toxaphene, a broad-spectrum pesticide found in relatively high concentration in Lake Apopka alligator egg yolk. The toxaphene, dissolved in 50 microl of ethanol, was applied topically to the eggshell just prior to the sex-determining period of development. Shortly after hatching, we examined a number of morphological and physiological endpoints to determine the consequences of sub-lethal embryonic exposure to toxaphene. Our results indicate that toxaphene had little or no effect on the morphological endpoints examined including body mass (BM) and size, liver, thyroid, and gonad development. In addition, toxaphene failed to affect sexual differentiation, or in vitro thyroxin, testosterone (T), and estradiol production. However, male plasma T concentration was higher in animals treated with 10 and 0.01 microg toxaphene/kg (based on mean egg mass) than control males. Because in vitro T production was not different among control groups, we suggest the difference in plasma T could be due to differences in hypothalamic-pituitary stimulation of the gonad or hepatic steroid degradation. This study indicates that technical grade toxaphene, at the applied doses, does not induce the same developmental abnormalities associated with alligators living in Lake Apopka. Future studies should consider the effects of embryonic exposure to a mixture of chemicals, including toxaphene metabolites, on development in alligators to better evaluate the consequences of environmental contamination.

Alligators and Crocodiles↗
PubMed2002

Structure-activity relationships and dose responses of polychlorinated dibenzo-p-dioxins for short-term effects in 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant and -sensitive rat strains.

Dose responses of the characteristic short-term effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD), and 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) were compared in the resistant Han/Wistar (Kuopio) (H/W) rats and the sensitive Long-Evans (Turku/AB) (L-E) rats. The resistance of H/W rats is linked to the altered H/W-type aryl hydrocarbon receptor (AHR). Exceptionally, in terms of acute lethality, the most potent congener for H/W rats is HxCDD, followed by HpCDD, PeCDD, and TCDD. The study objectives were to find out if this exceptional sensitivity of H/W rats also holds for nonlethal toxic endpoints and to compare potency and efficacy (magnitude of effect) of PCDDs between L-E and H/W rats. Dose responses for several endpoints were determined, modeled, and used for ED50 and relative potency (REP) calculations. For all endpoints measured, TCDD was the most potent congener, followed by PeCDD, HxCDD, and HpCDD in both strains, and the REP estimates were consistent with the current toxic equivalency factors (TEFs). For most endpoints, H/W rats showed smaller responses to all congeners than L-E rats, and this difference was due to lower efficacy rather than lower potency. H/W rats showed lower efficacy to body weight loss, serum aspartate aminotransferase activity, and serum concentrations of total bilirubin, free fatty acids, and thyroxine. In contrast, effects on cytochrome P4501A1 induction, thymus atrophy, and dental defects were similar in both strains. In conclusion, the results are in agreement with the current WHO-TEFs and imply that relative potency values derived from mortality are not necessarily valid for other endpoints. The results support our previous observations about two different types of AHR-mediated mechanisms. Type I effects are similar in both strains, and type II effects show decreased efficacy of toxic response in relation with the altered H/W-type AHR.

Animals↗