NSAIDs and Renal Impairment: Deprescribing Chronic NSAID use in General Practice.

Aim NSAIDs are high-risk medicines that can commonly cause adverse renal effects. Recent evidence suggests a rise in the number of patients with acute and chronic renal disease. The aim of this audit is to determine our de-prescribing rate of chronic NSAID use in an Irish general practice. Methods We reviewed NSAID-containing drug prescriptions that were issued over a three month period in 2018. A description analysis was performed to ascertain for the frequency and type of NSAIDs prescribed. An educational session was delivered to clinicians to encourage de-prescribing of NSAIDs if deemed clinically appropriate. Results Fifty-one NSAID-containing prescriptions were identified. Thirty-six (71%) patients, who were prescribed a regular NSAID, were aged between 71-85 years. Meloxicam was used the most (31%), whilst the preferred NSAIDs (naproxen and ibuprofen) were used least (18%). A 37% improvement in de-prescribing of chronic NSAIDs was achieved upon re-auditing. Conclusion NSAIDs are commonly implicated in inappropriate prescribing. Clinicians are encouraged to practice de-prescribing at every opportunity. Recent evidence suggests that pharmacy-led educational interventions can further assist de-prescribing of inappropriate medicines. Thus, a close collaboration between physicians and pharmacists is encouraged to further maximise quality of prescribing and patient care.

Sexual selection drives the evolution of male wing interference patterns.

The seemingly transparent wings of many insects have recently been found to display unexpected structural coloration. These structural colours (wing interference patterns: WIPs) may be involved in species recognition and mate choice, yet little is known about the evolutionary processes that shape them. Furthermore, to date investigations of WIPs have not fully considered how they are actually perceived by the viewers' colour vision. Here, we use multispectral digital imaging and a model of Drosophila vision to compare WIPs of male and female Drosophila simulans from replicate populations forced to evolve with or without sexual selection for 68 generations. We show that WIPs modelled in Drosophila vision evolve in response to sexual selection and provide evidence that WIPs correlate with male sexual attractiveness. These findings add a new element to the otherwise well-described Drosophila courtship display and confirm that wing colours evolve through sexual selection.

Hypercalcaemia due to Hypervitaminosis D in a Self-Supplementing Multiple Sclerosis Patient: A Case Report

There is an increasing literature with regard to vitamin D supplementation in Multiple Sclerosis (MS). We report the case of a 45 year-old male with MS who presented with symptomatic hypercalcaemia secondary to self-supplementation of vitamin D3 purchased online. Treatment was with IV hydration, glucocorticoids, calcitonin and bisphosphonates. This case highlights a lack of consensus guidance regarding safe vitamin D supplementation dosages and the importance of a thorough history with regard to non-prescribed supplements, particularly those easily available online.

Lack of phototoxicity potential with delafloxacin in healthy male and female subjects: comparison to lomefloxacin.

AIMS: Delafloxacin is a fluoroquinolone antibiotic recently approved by the FDA for treatment of acute bacterial skin and skin structure infections (ABSSSI). Delafloxacin was assessed for phototoxicity potential compared with a known phototoxic fluoroquinolone. METHODS: A Phase 1, investigator-blind, placebo/active-controlled, randomized, parallel-group study was conducted in 52 healthy male and female volunteers who received 200 or 400 mg of oral delafloxacin, 400 mg oral lomefloxacin or placebo once daily for 6 days. This study evaluated the photosensitizing potential and possible wavelength dependency of delafloxacin by comparing the response of the skin to ultraviolet A (UVA), ultraviolet B (UVB) and visible radiation prior to and during administration of delafloxacin, lomefloxacin as a positive control, or placebo. Adverse events were monitored throughout the study. RESULTS: Forty-seven subjects completed six days of dosing, and no evidence of phototoxicity was seen with delafloxacin. Delafloxacin at 200 and 400 mg day-1 and placebo did not demonstrate differences in percent change from baseline in minimal erythema dose at all tested wavelengths (295-430 nm) by monochromator and solar simulator. Lomefloxacin, the positive control, had statistically significant differences (p < 0.05) at UVA wavelengths of 335 and 365 ± 30 nm 24 hours after radiation exposure (maximum response). The phototoxic index results were significantly higher for lomefloxacin at 335 nm and 365 nm compared to placebo and delafloxacin. CONCLUSIONS: 200 and 400 mg of delafloxacin administered for 6 days were well tolerated in healthy adult volunteers. Delafloxacin and placebo failed to demonstrate a phototoxic effect but lomefloxacin, the positive control, demonstrated moderate phototoxicity.

Antibacterials dispensed in the community comprise 85%-95% of total human antibacterial consumption.

WHAT IS KNOWN AND OBJECTIVE: Interventions intended to slow the emergence and spread of antibacterial resistance through enhanced antimicrobial stewardship will be more effective if informed by an accurate knowledge of current patterns of antibacterial consumption. For example, knowledge of the relative magnitude of community antibacterial consumption in relation to hospital antibacterial consumption within each nation or region should help guide decisions about the relative importance of community and hospital antimicrobial stewardship programmes. It is commonly stated that community antibacterial consumption comprises approximately 80% of total national antibacterial consumption. We aimed to determine this proportion across a large range of nations. METHODS: We measured community and hospital antibacterial consumption in New Zealand during 2015, from both reimbursement and purchase data, and compared the New Zealand data with those reported from a large range of other nations during similar time periods. RESULTS AND DISCUSSION: Community antibacterial consumption comprised approximately 85%-95% of total antibacterial consumption in all nations for which data were available, and in New Zealand comprised a higher proportion than in any other nation. The proportion of total antibacterial consumption comprised by community consumption was significantly higher in countries with relatively high levels of total antibacterial consumption than in countries with relatively low levels of total antibacterial consumption. WHAT IS NEW AND CONCLUSION: The high proportion of total antibacterial consumption comprised by community antibacterial consumption suggests devoting particular attention to improved community antimicrobial stewardship. These results suggest that improving antimicrobial stewardship in the community may provide greater overall benefits in combating antibacterial resistance than improving antimicrobial stewardship in hospitals.

The CLRX.1/NOD24 (NLRP2P) pseudogene codes a functional negative regulator of NF-κB, pyrin-only protein 4.

Pseudogenes are duplicated yet defunct copies of functional parent genes. However, some pseudogenes have gained or retained function. In this study, we consider a functional role for the NLRP2-related, higher primate-specific, processed pseudogene NLRP2P, which is closely related to Pyrin-only protein 2 (POP2/PYDC2), a regulator of nuclear factor-κB (NF-κB) and the inflammasome. The NLRP2P open-reading frame on chromosome X has features consistent with a processed pseudogene (retrotransposon), yet encodes a 45-amino-acid, Pyrin-domain-related protein. The open-reading frame of NLRP2P shares 80% identity with POP2 and is under purifying selection across Old World primates. Although widely expressed, NLRP2P messenger RNA is upregulated by lipopolysaccharide in human monocytic cells. Functionally, NLRP2P impairs NF-κB p65 transactivation by reducing activating phosphorylation of RelA/p65. Reminiscent of POP2, NLRP2P reduces production of the NF-κB-dependent cytokines tumor necrosis factor alpha and interleukin (IL)-6 following toll-like receptor stimulation. In contrast to POP2, NLRP2P fails to inhibit the ASC-dependent NLRP3 inflammasome. In addition, beyond regulating cytokine production, NLRP2P has a potential role in cell cycle regulation and cell death. Collectively, our findings suggest that NLRP2P is a resurrected processed pseudogene that regulates NF-κB RelA/p65 activity and thus represents the newest member of the POP family, POP4.

Immersion of debrided diabetic foot ulcer tissue in electrochemically generated pH neutral hypochlorous acid significantly reduces the microbial bioburden: whole-genome sequencing of Staphylococcus aureus, the most prevalent species recovered.

BACKGROUND: Diabetic foot ulcer infections (DFUIs) are the leading cause of lower-limb amputations, mediated predominantly by Staphylococcus aureus. pH-neutral electrochemically generated hypochlorous acid (anolyte) is a non-toxic, microbiocidal agent with significant potential for wound disinfection. AIMS: To investigate both the effectiveness of anolyte for microbial bioburden reduction in debrided ulcer tissues and the population of resident S. aureus. METHODS: Fifty-one debrided tissues from 30 people with type II diabetes were aliquoted by wet weight and immersed in 1- or 10-mL volumes of anolyte (200 parts per million) or saline for 3 min. Microbial loads recovered were determined in colony forming units/g (cfu/g) of tissue following aerobic, anaerobic and staphylococcal-selective culture. Bacterial species were identified and 50 S. aureus isolates from 30 tissues underwent whole-genome sequencing (WGS). FINDINGS: The ulcers were predominantly superficial, lacking signs of infection (39/51, 76.5%). Of the 42/51 saline-treated tissues yielding ≥105 cfu/g, a microbial threshold reported to impede wound-healing, only 4/42 (9.5%) were clinically diagnosed DFUIs. Microbial loads from anolyte-treated tissues were significantly lower than saline-treated tissues using 1 mL (1065-fold, 2.0 log) and 10 mL (8216-fold, 2.1 log) immersion volumes (P<0.0005). S. aureus was the predominant species recovered (44/51, 86.3%) and 50 isolates underwent WGS. All were meticillin susceptible and comprised 12 sequence types (STs), predominantly ST1, ST5 and ST15. Whole-genome multi-locus sequence typing identified three clusters of closely related isolates from 10 patients indicating inter-patient transmission. CONCLUSIONS: Short immersions of debrided ulcer tissue in anolyte significantly reduced microbial bioburden: a potential novel DFUI treatment.

Intake estimates of naturally occurring phytosterols using deterministic and probabilistic methods in a representative Irish population.

Intakes of naturally occurring phytosterols were estimated in the Irish population using phytosterol concentration data obtained from the published literature. Both deterministic and probabilistic approaches were used. Concentration data were applied to dietary intake data as assessed in the North/South Ireland Food Consumption Survey (n = 1,379). With the probabilistic approach, a uniform distribution was used in place of fixed values to reflect variability and uncertainty in phytosterol concentrations. Foods consumed were also categorized into 19 food groups, and the phytosterol intake per food group was calculated. Using the deterministic approach, mean phytosterol intake for the total population was 254 mg/day (28.1 mg/1,000 kJ). Using the probabilistic approach, slightly higher mean phytosterol intakes of 287 mg/day were found. In terms of food groups, 'bread and bread-rolls', 'vegetables and vegetable dishes' and 'spreading fat and oils' were the top three contributing food groups to phytosterol intakes. This study has shown how both deterministic and probabilistic approaches may be used to determine phytosterol intakes in a population.

A randomised interventional trial of omega-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus.

OBJECTIVE: To determine the clinical effect of dietary supplementation with low-dose omega-3-polyunsaturated fatty acids on disease activity and endothelial function in patients with systemic lupus erythematosus. METHODS: A 24-week randomised double-blind placebo-controlled parallel trial of the effect of 3 g of omega-3-polyunsaturated fatty acids on 60 patients with systemic lupus erythematosus was performed. Serial measurements of disease activity using the revised Systemic Lupus Activity Measure (SLAM-R) and British Isles Lupus Assessment Group index of disease activity for systemic lupus erythematosus (BILAG), endothelial function using flow-mediated dilation (FMD) of the brachial artery, oxidative stress using platelet 8-isoprostanes and analysis of platelet membrane fatty acids were taken at baseline, 12 and 24 weeks. RESULTS: In the fish oil group there was a significant improvement at 24 weeks in SLAM-R (from 9.4 (SD 3.0) to 6.3 (2.5), p<0.001); in BILAG (from 13.6 (6.0) to 6.7 (3.8), p<0.001); in FMD (from 3.0% (-0.5 to 8.2) to 8.9% (1.3 to 16.9), p<0.001) and in platelet 8-isoprostanes (from 177 pg/mg protein (23-387) to 90 pg/mg protein (32-182), p = 0.007). CONCLUSIONS: Low-dose dietary supplementation with omega-3 fish oils in systemic lupus erythematosus not only has a therapeutic effect on disease activity but also improves endothelial function and reduces oxidative stress and may therefore confer cardiovascular benefits.

Habitual fish consumption does not prevent a decrease in LCPUFA status in pregnant women (the Seychelles Child Development Nutrition Study).

Information on the status of long-chain polyunsaturated fatty acids (LCPUFAs) in pregnancy and breast milk in very high fish-eating populations is limited. The aim of this study was to examine dietary intake and changes in fatty acid status in a population of pregnant women in the Republic of Seychelles. Serum docosahexaenoic acid (DHA) decreased significantly between 28-week gestation and delivery (n=196). DHA status did not correlate significantly with length of gestation and was not associated with self-reported fish intake, which was high at 527 g/week. In breast milk, the ratio of DHA to arachidonic acid (AA) was consistent with those observed in other high fish-eating populations. Overall the data suggest that high exposure to LCPUFAs from habitual fish consumption does not prevent the documented decrease in LCPUFA status in pregnancy that occurs as a result of foetal accretion in the third trimester of pregnancy.

Clinical biochemistry guidelines in Wales: their impact on laboratory services.

BACKGROUND: Evidence of significant variations in clinical biochemistry practice in Wales led to the formation of the All Wales Clinical Biochemistry Audit Group (AWCBAG) in 1993, with the aim of auditing laboratory services to ensure that they are optimally used. As part of this process, clinical guidelines are produced and circulated to all clinical biochemistry departments in Wales. The current aim of the AWCBAG is to assess the extent and impact of adoption of these guidelines across Wales. METHODS: Three surveys were dispatched at intervals over a decade to all clinical biochemistry departments in Wales to investigate practice in: (1) urine albumin testing to screen for diabetic nephropathy; (2) biochemical investigation of menopausal status and the monitoring of hormone replacement therapy; (3) screening for Cushing's syndrome. RESULTS: The results show that laboratories across Wales are generally following guideline criteria and are adapting their practice in-line with changing recommendations. CONCLUSION: The introduction of AWCBAG guidelines has been widely accepted by clinical biochemistry departments in Wales. These guidelines have led to a more efficient and effective use of laboratory services.

Children's experiences following a CBT intervention to reduce dental anxiety: one year on.

Objective: To investigate children's ongoing experiences of dental care and use of strategies to manage their dental anxiety following cognitive behavioural therapy (CBT). Design: A child self-completed postal questionnaire. Settings: Hospital, community and general dental practice. Subjects: Questionnaires were sent to 44 children, aged 10­17 years who had been referred to specialist services due to their dental anxiety. Intervention: Children had all previously received a guided CBT self-help intervention to reduce their dental anxiety and, on completion of treatment, had been discharged to their referring dentist. Questionnaires were sent out 12­18 months later to ascertain dental attendance patterns and application of any strategies learnt from the previous CBT intervention. Results: 22 responses (50%) were received from 16 girls and six boys. Eighty-two percent had subsequently accessed follow-up care with a general dental practitioner and over half of these had undergone a dental procedure, other than a check-up. Ninety-one percent reported feeling less worried about dental visits, than previously, and described a change in cognition, behaviours, and feelings that allowed them to manage their anxiety better. Conclusions: CBT has positive immediate and longitudinal effects in reducing children's dental anxiety. The challenge of adopting this evidence-based approach within primary care settings remains.

Culturally capable and culturally safe: Caseload care for Indigenous women by Indigenous midwifery students.

BACKGROUND: Evidence is emerging of the benefits to students of providing continuity of midwifery care as a learning strategy in midwifery education, however little is known about the value of this strategy for midwifery students. AIM: To explore Indigenous students' perceptions of providing continuity of midwifery care to Indigenous women whilst undertaking a Bachelor of Midwifery. METHODS: Indigenous Bachelor of Midwifery students' experiences of providing continuity of midwifery care to Indigenous childbearing women were explored within an Indigenous research approach using a narrative inquiry framework. Participants were three Indigenous midwifery students who provided continuity of care to Indigenous women. FINDINGS: Three interconnected themes; facilitating connection, being connected, and journeying with the woman. These themes contribute to the overarching finding that the experience of providing continuity of care for Indigenous women creates a sense of personal affirmation, purpose and a validation of cultural identity in Indigenous students. DISCUSSION AND CONCLUSIONS: Midwifery philosophy aligns strongly with the Indigenous health philosophy and this provides a learning platform for Indigenous student midwives. Privileging Indigenous culture within midwifery education programs assists students develop a sense of purpose and affirms them in their emerging professional role and within their community. The findings from this study illustrate the demand for, and pertinence of, continuity of care midwifery experiences with Indigenous women as fundamental to increasing the Indigenous midwifery workforce in Australia. Australian universities should provide this experience for Indigenous student midwives.

Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia.

PURPOSE: Gemtuzumab ozogamicin (Mylotarg; Wyeth Laboratories, Philadelphia, PA) consists of a semisynthetic derivative of calicheamicin, a cytotoxic antibiotic linked to a recombinant monoclonal antibody directed against the CD33 antigen present on leukemic myeloblasts in most patients with acute myeloid leukemia (AML). In this study, we review the preclinical and clinical profiles of this immunoconjugate and the regulatory review that led to marketing approval by the United States Food and Drug Administration. EXPERIMENTAL DESIGN: From the literature and manufacturer's data, we review the activity, tolerability, and pharmacokinetics of gemtuzumab ozogamicin in preclinical and Phase I studies and its activity, efficacy, and side effects in three Phase 2 trials of 142 patients with relapsed AML. RESULTS: In Phase I studies, the major toxicity was myelosuppression, especially neutropenia and thrombocytopenia, resulting from the expression of CD33 on myeloid progenitor cells. The Phase 2 dose was 9 mg/m(2) infused i.v. over 4 h, repeated on day 14. A minority of patients experienced acute infusion-related symptoms, usually transient and occasionally requiring hospitalization. The complete response (CR) rate with full recovery of hematopoiesis was 16%. A subset of patients [CRs with incomplete platelet recovery (CRps)] was identified with blast clearance and neutrophil recovery but incomplete platelet recovery. The duration of responses of CRps appeared to be similar to those of the CRs, although the numbers were small. The question of the equivalence of these response groups was a central issue in the review of this new drug application (NDA). After considerable discussion, the Oncology Drugs Advisory Committee recommended allowing inclusion of CRps resulting in an overall response rate in the Phase 2 studies of 30%. In the subgroup of patients over 60 years of age, the overall response rate was 26%. Response duration was difficult to establish because of the high prevalence of postremission therapies. Tolerability and ease of administration may be improved compared with conventional chemotherapy, except for hepatotoxicity, with 31% of patients exhibiting abnormal liver enzymes. One patient died of liver failure in the Phase 2 trials. CONCLUSIONS: Marketing approval of gemtuzumab ozogamicin was granted on May 17, 2000 by the United States Food and Drug Administration under the Accelerated Approval regulations. Gemtuzumab ozogamicin is indicated for the treatment of patients with CD33 positive AML in first relapse who are 60 years of age or older and who are not considered candidates for cytotoxic chemotherapy. The approved dose was 9 mg/m(2) i.v. over 4 h and repeated in 14 days. Completion of the ongoing studies of gemtuzumab ozogamicin in relapsed AML and initiation of randomized clinical trials comparing the effects of gemtuzumab ozogamicin in combination with conventional induction chemotherapy to conventional chemotherapy alone on survival are mandated to confirm clinical benefit under the accelerated approval Subpart H regulations. Postmarketing reports of fatal anaphylaxis, adult respiratory distress syndrome (ARDS), and hepatotoxicity, especially venoocclusive disease (VOD) in patients treated with gemtuzumab ozogamicin, with and without associated hematopoietic stem cell transplantation (HSCT), have required labeling revisions and the initiation of a registration surveillance program. Tumor lysis and ARDS have been reported in patients with leukocytes above 30,000/ml treated with gemtuzumab ozogamicin; therefore, the reduction of leukocyte counts to below 30,000/ml is recommended prior to treatment. Patients should be carefully monitored for acute hypersensitivity, hypoxia, and delayed hepatotoxicity following treatment with gemtuzumab ozogamicin.

Concentrations of Escherichia coli and genetic diversity and antibiotic resistance profiling of Salmonella isolated from irrigation water, packing shed equipment, and fresh produce in Texas.

Fresh produce has been repeatedly implicated as a vehicle in the transmission of foodborne gastroenteritis. In an effort to assess the risk factors involved in the contamination of fresh produce with pathogenic bacteria, a total of 1,257 samples were collected from cantaloupe, oranges, and parsley (both in the field and after processing) and from the environment (i.e., irrigation water, soil, equipment, etc.). Samples were collected twice per season from two production farms per commodity and analyzed for the presence of Salmonella and Escherichia coli. E. coli was detected on all types of commodities (cantaloupe, oranges, and parsley), in irrigation water, and on equipment surfaces. A total of 25 Salmonella isolates were found: 16 from irrigation water, 6 from packing shed equipment, and 3 from washed cantaloupes. Salmonella was not detected on oranges or parsley. Serotyping, pulsed-field gel electrophoresis (PFGE), and repetitive element sequence-based PCR (rep-PCR) assays were applied to all Salmonella isolates to evaluate the genetic diversity of the isolates and to determine relationships between sources of contamination. Using PFGE, Salmonella isolates obtained from irrigation water and equipment were determined to be different from cantaloupe isolates; however, DNA fingerprinting did not conclusively define relationships between contamination sources. All Salmonella isolates were subjected to antimicrobial susceptibility testing using the disk diffusion method, and 20% (5 of 25) of the isolates had intermediate sensitivity to streptomycin. One Salmonella isolate from cantaloupe was resistant to streptomycin.

Survival of salmonella transformed to express green fluorescent protein on Italian parsley as affected by processing and storage.

To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

Structural Brain Changes following Long-Term 6° Head-Down Tilt Bed Rest as an Analog for Spaceflight.

BACKGROUND AND PURPOSE: Following long-term spaceflight, a subset of the National Aeronautics and Space Administration astronauts present with visual impairment and increased intracranial pressure, known as visual impairment and intracranial pressure syndrome. We investigated structural brain changes following long-term head-down tilt bed rest as a spaceflight analog. MATERIALS AND METHODS: Volumetric analysis was performed on structural pre- and post-bed rest brain MR images. RESULTS: Comparing post-bed rest to pre-bed rest images, we found the following: 1) no significant group differences in GM, WM, CSF, or ventricular volumes; 2) shift of the center of mass of the brain upward and posterior rotation of the brain relative to the skull; 3) a significant correlation between posterior brain rotation and changes in ventricular volume; and 4) significant increases in brain tissue density in regions at the vertex, including the frontoparietal lobes, with contraction of adjacent extra-axial CSF spaces, and significant decreases in tissue density in areas along the base of the brain, including the orbitofrontal cortex. CONCLUSIONS: We observed widespread morphologic changes with brain tissue redistribution in response to gravity changes; possible associated functional changes are unknown. The observation that ventricular change is correlated to posterior brain rotation suggests an alteration in CSF homeostasis. Ultimately, to elucidate any structural changes that may play a role in visual impairment and intracranial pressure syndrome, volumetric analysis of pre- and postflight structural scans of astronauts is needed.

Mechanism-based isocoumarin inhibitors for blood coagulation serine proteases. Effect of the 7-substituent in 7-amino-4-chloro-3-(isothioureidoalkoxy)isocoumarins on inhibitory and anticoagulant potency.

A series of 7-amino-4-chloro-3-(3-isothioureidopropoxy)isocoumarin (NH2-CiTPrOIC) derivatives with various substituents at the 7- and 3-positions have been synthesized as inhibitors of several blood coagulation enzymes. Isocoumarins substituted with basic groups such as guanidino or isothioureidoalkoxy groups were previously shown to be potent irreversible inhibitors of blood coagulation enzymes [Kam et al. Biochemistry 1988, 27, 2547-2557]. Substituted isocoumarins with an isothioureidoethoxy group at the 3-position and a large hydrophobic group at the 7-position are better inhibitors for thrombin, factor VIIa, factor Xa, factor XIa, factor IIa, and factor IXa than NH2-CiTPrOIC (4). PhNHCONH-CiTEtOIC (14), (S)-Ph(CH3)CHNHCONH-CiTEtOIC (25), and (R)-Ph(CH3)CHNHCONH-CiTEtOIC (26) inhibit thrombin quite potently and have kobs/[I] values of (1-4) x 10(4) M-1 s-1. Modeled structures of several isocoumarins noncovalently complexed with human alpha-thrombin suggest that H-bonding between the 7-substituent and the Lys-60F NH3+ relates to the inhibitory potency. Thrombin inhibited by 14, 25, or 26 is quite stable, and only 4-16% of enzymatic activity is regained after incubation for 20 days in 0.1 M Hepes, pH 7.5 buffer. However, 100, 67, and 65% of enzyme activity, respectively, is regained with the addition of 0.38 M hydroxylamine. With normal citrated pig or human plasma, these isocoumarin derivatives prolong the prothrombin time ca. 1.3-3.1-fold and also prolong the activated partial thromboplastin time more than 3-7-fold at 32 microM. Thus, these compounds are effective anticoagulants in vitro and may be useful in vivo.

A 19-year follow-up of tuberculin reactors. Assessment of skin test reactivity and in vitro lymphocyte responses.

Tuberculin skin test reactivity decreases with time such that repeat PPD skin testing may result in reactions of less than 10 mm. This reactivity may be boosted in some individuals with a second tuberculin skin test. The immunologic basis of these observations remains unclear. We studied the relationship between skin-test reactivity and in vitro blastogenic response to PPD in a cohort of 22 individuals (aged 28 to 81 years) known to be tuberculin reactors (induration greater than or equal to 10 mm) in 1970. In 1989, 18 subjects remained reactive to PPD on the first skin test and responded to PPD in vitro (mean incorporation of 3H-thymidine by peripheral blood mononuclear cells, 22,650 cpm). Three subjects reverted (induration in response to PPD less than 10 mm) and lost in vitro reactivity to PPD (mean incorporation of 3H-thymidine, 2,205 cpm). One subject boosted (increase of induration of at least 6 mm to greater than or equal to 15 mm) on the second skin test and showed a concomitant in vitro boost in the blastogenic response to PPD (from 1,008 cpm to 47,837 cpm). In this cohort, interpretation of the two-step tuberculin skin test correlated closely with in vitro proliferative responses. Over a 19-year period, the majority of individuals maintained skin test reactivity and strong in vitro responses to PPD despite a lack of ongoing exogenous exposure to Mycobacterium tuberculosis. The immunologic basis for reversion appears to depend in part on a loss of lymphocyte blastogenic capacity. In the one individual who exhibited the booster phenomenon, repeat antigen stimulation resulted in a dramatic increase in the in vitro blastogenic responses.

Conal enlargement for diffuse subaortic stenosis.

Twelve patients underwent conal enlargement for diffuse subaortic stenosis over a 3 1/2-year period. The subaortic stenosis was due to tunnel outflow in 11 and malattached mitral valve in one. Mean age was 4.4 +/- 4 years and mean subaortic gradient was 50 +/- 21 mm Hg. Three infants had a malalignment ventricular septal defect. In eight patients significant obstruction occurred 2 to 7 years (mean 4 +/- 2) after simple resection of subaortic stenosis (n = 2), ventricular septal defect closure (n = 2), ventricular septal defect closure and subaortic stenosis resection (n = 2), and canal repair (n = 2). In three infants the tunnel outflow distal to a malalignment ventricular septal defect was enlarged and closed with the defect. In three patients with subaortic stenosis proximal to a previously repaired ventricular septal defect, transatrial conal enlargement through the ventricular septal defect was performed. Another patient without a ventricular septal defect had transatrial conal enlargement. The remaining five patients had the modified Konno procedure. Two patients had postoperative complete heart block and one infant had insertion of an apicoaortic conduit for aortic anulus hypoplasia 9 months later. One patient died of pneumonia during the follow-up period. Postoperative echographic outflow gradients up to 3 1/2 years (mean 1.2 +/- 1) ranged up to 25 mm Hg (mean 7 +/- 11) and were mainly at the aortic level. The 11 surviving patients are doing well up to 3 1/2 years of follow-up (mean 1.5 +/- 1). We conclude that conal enlargement procedures with aortic valve preservation are preferable, effective, and can be safely performed for diffuse subaortic stenosis in infants and children.