Safety and Efficacy of Prolonged Use of Dalbavancin in Bone and Joint Infections.

Dalbavancin is a lipoglycopeptide with potent activity against Gram-positive microorganisms, a long half-life, a favorable safety profile, and a high concentration in bone, which makes it an interesting alternative for treatment of osteoarticular infections. We performed a multicentric retrospective study of all patients with an osteoarticular infection (septic arthritis, spondylodiscitis, osteomyelitis, or orthopedic implant-related infection) treated with at least one dose of dalbavancin between 2016 and 2017 in 30 institutions in Spain. In order to evaluate the response, patients with or without an orthopedic implant were separated. A total of 64 patients were included. Staphylococcus epidermidis and Staphylococcus aureus were the most frequent microorganisms. The reasons for switching to dalbavancin were simplification (53.1%), adverse events (25%), or failure (21.9%). There were 7 adverse events, and no patient had to discontinue dalbavancin. In 45 cases, infection was related to an orthopedic implant. The implant material was retained in 23 cases, including that in 15 (65.2%) patients that were classified as cured and 8 (34.8%) that presented improvement. In 21 cases, the implants were removed, including those in 16 (76.2%) cases that were considered successes, 4 (19%) cases were considered improved, and 1 (4.8%) case that was considered a failure. Among the 19 cases without implants, 14 (73.7%) were considered cured, 3 (15.8%) were considered improved, and 2 (10.5%) were considered failures. The results show that dalbavancin is a well-tolerated antibiotic, even when >2 doses are administered, and is associated with a high cure rate. These are preliminary data with a short follow-up; therefore, it is necessary to gain more experience and, in the future, to establish the most appropriate dose and frequency.

Qualitative behavioral assessment of transport-naive and transport-habituated sheep.

Objective and issue-neutral qualitative assessments of livestock behavior could provide a powerful assessment of welfare, augmenting quantitative measures such as autonomic and endocrine changes, which are often difficult to assess under many commercial livestock conditions. We set out to validate the use of qualitative behavioral assessment (QBA) in sheep using controlled experimental conditions (transport as a challenge) and comparing assessments against physiological variables. The behavioral expression of 14 Merino wethers, which had never experienced land transport, were assessed during their first road event (naïve to transport), and then again on their seventh event, 8 d later (habituated to transport). Blood samples were collected immediately before loading and after unloading, and heart rate and core body temperature were measured continuously throughout each event. Continuous video footage recorded during each event was used to provide clips of individual animals that were shown to observers for QBA. There was significant consensus (P < 0.001) amongst 63 observers in terms of their assessment of the behavioral expression of the sheep. Transport-naïve sheep were assessed as being more 'alert', 'anxious', and 'aware', whereas transport-habituated sheep were more 'comfortable', 'tired', and 'confident' (P = 0.015). Heart rate and heart rate variability, core body temperature and a stress leukogram were greater (P < 0.05) in sheep during the first (naïve) event compared with the habituated event, and were significantly correlated with the QBA scores (P < 0.05). In conclusion, QBA is a valid, practical and informative measure of behavioral responses to transport.

Physiological responses of Bos taurus and Bos indicus cattle to prolonged, continuous heat and humidity.

Two experiments were conducted to investigate the physiological responses of Bos taurus (Angus cross, n = 6) and Bos indicus (Brahman, n = 6) cattle to prolonged heat and humidity, as can occur during live export by sea. Each experiment was carried out in climate-controlled rooms, where heifers were exposed to 15 d of sustained heat and humidity. The treatment was designed to be representative of a long-haul, live-export voyage leaving a southern Australian winter and traveling to a Middle Eastern summer. Wet bulb temperature (WBT) was used to give a combined measure of dry bulb temperature and relative humidity and was increased over several days, culminating in 5 d at 32 degrees C WBT between d 7 and 11. By d 11, the respiratory rate and core body temperature increased (P < 0.001) compared with values at lower ambient temperature on d 1 and 2 when climate-controlled rooms were not operating. Feed intake of Bos taurus was reduced (P < 0.001) by d 11, whereas that of Bos indicus did not change (P = 0.14). Despite no diurnal variation in climatic conditions, core body temperature of both Bos taurus and Bos indicus continued to show a circadian amplitude of approximately 1 degrees C throughout the hottest period. This amplitude increased during the recovery period after heat was removed (up to 1.8 degrees C for Bos indicus and 1.6 degrees C for Bos taurus). Water intake for both Bos taurus and Bos indicus increased when WBT increased (P < 0.01 on d 11). Significant acid-base and blood electrolyte imbalances occurred in both Bos taurus and Bos indicus, with changes in Bos taurus being more substantial and prolonged. The increase in respiratory rate coincided with a decrease in the partial pressures of carbon dioxide and bicarbonate in venous blood. However, during the hottest period, average daily venous blood pH remained unchanged. When the heat load was reduced after d 11, the blood pH decreased, indicating metabolic acidosis. Blood pH declined from 7.44 to 7.36 for Bos taurus (P < 0.001) and from 7.44 to 7.38 for Bos indicus (P < 0.001). Other parameters measured include heart rate; packed cell volume; plasma and urine Na, K, and Cl; urine pH; and specific gravity. Our results suggest that Bos taurus cattle experience significant physiological changes during exposure to prolonged and continuous high heat and humidity, with alterations persisting for some days after the heat-stress conditions subside. Bos indicus experience similar but less pronounced physiological changes.

Enzyme-linked immunospot assay responses to early secretory antigenic target 6, culture filtrate protein 10, and purified protein derivative among children with tuberculosis: implications for diagnosis and monitoring of therapy.

BACKGROUND: The ability to detect tuberculosis-specific lymphocytes by enzyme-linked immunospot (ELISPOT) assay may have important implications for the diagnosis and monitoring of tuberculosis in children, for which routine methods lack sensitivity. We conducted a study to determine the presence and time course of ELISPOT responses in children with tuberculosis. METHODS: Blood samples were obtained from children with a clinical diagnosis of tuberculosis, and interferon-gamma ELISPOT assays were performed using purified protein derivative (PPD), early secretory antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP10) as stimulants. A subset of children were retested after 1, 3, and 6 months of therapy. RESULTS: Detectable responses to ESAT-6 or CFP10 were found in 49 of 70 children with clinical tuberculosis but were more frequently found in those with culture-proven disease (P = .05). The number of subjects with responses to PPD increased after 1 month of therapy (P = .0004) and decreased at 3 and 6 months. CONCLUSION: Tuberculosis-specific ELISPOT testing is a promising tool that should be evaluated as a potential diagnostic test for childhood tuberculosis. We caution against the use of an early decrease in response as a marker of successful antituberculous chemotherapy.

HIV infection of undetermined origin during infancy.

The case histories of two children with horizontally acquired HIV infection are described. These children were diagnosed at a paediatric hospital in sub-Saharan Africa. Although the source(s) of infection was not identified, both children had had several contacts with the health service, experienced invasive procedures and ingested expressed milk from their own mothers during hospital admission. Health-care institutions, particularly those located in high HIV prevalence areas, must implement effective infection control measures to ensure that the risk of horizontal infection is minimized. Attention should be given to practices that are unique to each clinical discipline.

Decline in structural examination compliance in the hospital medical record with advancing level of training.

A retrospective review of 115 randomly pulled hospital charts of patients admitted to the care of osteopathic physicians at an American Osteopathic Association-accredited primary care hospital revealed a significant decline in the frequency of structural examinations in history and physical examinations during a merger and relocation, as well as with increasing level of training of the examiner (chi 2 test, P < .001). Attending physicians completed fewer structural examinations (45% of their history and physical examinations) compared to house staff (70%) and students (92%). A follow-up anonymous survey of 100 osteopathic physicians and students (response rate, 58%) revealed that the low overall frequency of structural examination completion (60%) was due primarily to attending physicians who considered this examination less relevant or impractical for their hospitalized patients. These results indicate that education on relevance of structural examination and manipulative treatment for acutely ill patients needs to be directed not only to house staff and students but also to attending physicians to preserve and improve the use of osteopathic principles at osteopathic hospitals.

Constitutive expression of functional GABA(B) receptors in mIL-tsA58 cells requires both GABA(B(1)) and GABA(B(2)) genes.

Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.

X-linked agammaglobulinaemia and the underlying genetics in two kindreds.

OBJECTIVE: Molecular analysis of the Bruton's tyrosine kinase (Btk) gene in two unrelated families, with a combined total of seven boys, affected by X-linked agammaglobulinaemia (XLA). METHODS: Protein electrophoresis and western blotting were used for the examination of Btk protein synthesis in blood leucocytes. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction (RT-PCR) technique. Sequence alterations were screened for by the single-stranded conformation polymorphism (SSCP) method and characterized by standard sequencing protocols. RESULTS: Western blotting revealed Btk protein to be absent in leucocytes of affected males from both families. A novel 3 b.p. deletion in exon 3 of the Btk gene was found to be responsible for the XLA phenotype in the affected proband in one family (kindred I). A diagnostic PCR assay was established to detect this mutation in other affected male siblings and carrier females. For the second family (kindred II), the coding sequence of the Btk gene and the promoter region were found to be normal. CONCLUSIONS: The present study has demonstrated genetic heterogeneity in the Btk gene in South African XLA patients and has identified a novel mutation in this gene in the largest of the affected kindreds. The gene mutation in the second kindred was undetermined and may be indicative of a defect in some other gene associated with Btk function or stability. Western blotting was found to be informative in establishing a deficiency of Btk protein in both probands and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

Family perceptions of support interventions in the intensive care unit.

Family needs of critically ill patients have been well documented in nursing literature. However, few researchers have examined support interventions intended to meet these needs. This study examines which interventions provide the greatest benefit to critically ill patients' families and recommends nursing actions to meet families' support needs.

Dopamine D2-receptor isoforms expressed in AtT20 cells inhibit Q-type high-voltage-activated Ca2+ channels via a membrane-delimited pathway.

Dopamine D2 receptors both acutely and chronically inhibit high-voltage-activated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D2L (long) and D2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D2L and D2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D2-receptor regulation of Ca2+ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca2+ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D2-agonist quinpirole (250 microM) to the K+/CXR solution significantly increased the lag times for D2-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D2L- and D2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.

The role of ultrasonography in the detection and management of adnexal masses during the second and third trimesters of pregnancy.

OBJECTIVE: Our purpose was to determine the effect of routine second-trimester and third-trimester ultrasonographic examinations on the prevalence of detectable and operable adnexal disease. STUDY DESIGN: The study group consisted of 7996 pregnant women between 13.0 and 42.8 weeks' gestation. The size and architectural pattern of any detectable adnexal masses were noted. RESULTS: A total of 328 of the 7996 (4.1%) women in the study group had 335 ultrasonographically detectable adnexal masses; 309 of the masses were unilocular or had a single thin septation and 26 were architecturally complex. Of the ovarian cysts 252 of 309 (81.6%) had a mean diameter < 3.0 cm; 60% of the 252 patients in this subgroup had serial ultrasonographic examinations; 43 of the unilocular cysts resolved, and 17 have persisted for up to 2 years. There is a statistically significant trend toward decreasing frequency of ovarian cysts with increasing gestational age (chi2 for linear trend; P < .00001). Eighteen of the 7996 had an exploratory laparotomy (1 operation per 444 deliveries) during pregnancy or in the postpartum period. In addition, 1 patient had a paratubal cyst excised at the time of postpartum bilateral tubal ligation. Pathologically confirmed lesions included 8 benign cystic teratomas, 3 mucinous cyst adenomas, 2 paratubal cysts, 2 corpus lutea, 1 serous cystadenoma, 1 follicular cyst, 1 endometrioma, and 1 ovarian fibroma. CONCLUSION: Ovarian cysts are found in 4.1% of second-trimester and third-trimester obstetric ultrasonographic examinations. Most ultrasonographically detectable cysts are < 3.0 cm in diameter and usually resolve. The frequency of exploratory laparotomy for adnexal disease is not significantly different from that in reports before the widespread use of obstetric ultrasonography.

GABA(B)R1a/R1b-type receptor antisense deoxynucleotide treatment of melanotropes blocks chronic GABA(B) receptor inhibition of high voltage-activated Ca2+ channels.

GABA(B) and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA(B), but not GABA(A), agonist treatment also resulted in HVA-CC inhibition. Two GABA(B) receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA(B) antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA(B) receptor, which, like the D2 receptor, is coupled to chronic HVA-CC inhibition in melanotropes.

Disseminated histoplasmosis in an 'immunocompetent' child.

A rare case of severe disseminated histoplasmosis in a 7-year-old boy with apparently normal immune function is described. Current recommendations for diagnostic investigations, monitoring and the treatment of this disease with amphotericin B and itraconazole are reviewed.

Amyloid beta-peptide(25-35) changes [Ca2+] in hippocampal neurons.

Insoluble aggregates of the amyloid beta-peptide (A beta) is a major constituent of senile plaques found in brains of Alzheimer disease (AD) patients. The detrimental effects of aggregated A beta is associated with an increased intracellular Ca2+ concentration ([Ca2+]i). We examined the effects of A beta(25-35) on [Ca2+]i and intracellular H+ concentration ([H+]i) in single hippocampal neurons by real time fluorescence imaging using the Ca(2+)- and H(+)-specific ratio dyes, indo-1 and SNARF-1. Incubation of these cultures with A beta(25-35) for 3-12 days in vitro increased [Ca2+]i and [H+]i in large, NMDA-responsive neurons.

Heterogeneity in POMC expression among explanted melanotropes decreases with time in culture and bromocriptine treatment.

The biosynthetic activity of rat intermediate lobe melanotropes in vivo is inhibited by stimulation of dopamine D2 receptors. Individual melanotropes are innervated differentially by dopaminergic axons and vary in their levels of pro-opiomelanocortin (POMC) mRNA. We tested the hypothesis that placement of the lobe in primary culture, which removes the inhibitory innervation, would increase POMC mRNA levels and abolish the heterogeneity in POMC expression. POMC mRNA levels increased successively in untreated melanotropes when tested on culture Days 10, 16, and 20; however, some heterogeneity in POMC expression persisted. If treated with a D2 receptor agonist (1 microM bromocriptine) from culture Day 1, POMC mRNA levels were decreased significantly throughout the testing period when compared to untreated cells with the same time in culture. Although some melanotropes still expressed high POMC levels, preparations appeared more homogeneous by Day 20. Melanotrope responses were reversible, since POMC mRNA levels were down-regulated by application and up-regulated by withdrawal of a D2 receptor agonist. A short agonist treatment resulted in subpopulations that responded differently to the agonist, possibly representing a mechanism for fine-tuning peptide hormone release.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

Primary immunodeficiency diseases at Red Cross War Memorial Children's Hospital.

OBJECTIVE: To describe the spectrum of primary immunodeficiency diseases (PIDs) diagnosed at Red Cross War Memorial Children's Hospital. DESIGN: Retrospective, descriptive study. SETTING: Tertiary, referral hospital. PATIENTS: All patients investigated by the immunology service because of suspected PIDs, between January 1983 and December 1996. METHODS: Review of immunology service database and hospital case records. RESULTS: During the 14-year review period, 515 patients were investigated, a mean of 36.8 new patients per annum. Ninety-three patients with PIDs were diagnosed, a mean of 6.6 new patients per annum. The spectrum of PIDs was similar to that reported in developed countries. As in other series, antibody deficiencies predominated, accounting for 56% (52/93) of diagnoses. The male/female ratio was 1.5:1; 73% (62/85) came from the Western Cape, the remaining 27% (23/85) resided in five other provinces. Eighty per cent (70/87) presented with recurrent or atypical infection, with or without failure to thrive. Sinopulmonary infections (80%), diarrhoeal disease (19%) and candidiasis (18%) were the most common preceding infections. By the age of 5 years, only 60% had been diagnosed, compared with about 80% in developed countries. During the study period, 20% (19/93) were known to have died. CONCLUSIONS: The results show a pattern of PIDs incidence similar to that in developed countries. Diagnosis was delayed in many patients, which probably contributed to morbidity. To facilitate earlier diagnosis and to improve outcome, children should be considered for an immunological assessment if they exhibit increased susceptibility to infection.

Optimal statistical design for toxicokinetic studies.

Optimal statistical design strategies are applied to toxicokinetic experiments, for determining proper allocations of subjects and/or spacings of sampling times under a variety of nonlinear concentration-time models. The strategies include: (i) optimal allocations of subjects assuming the placement of time points is fixed, (ii) optimal spacing of design time points while assuming an equal allocation of subjects per time points and (iii) allocations/time-point spacings optimized jointly. Emphasis is placed on the first case, where a variance-minimization method is illustrated for optimizing the allocations when estimating specific toxicokinetic parameters. Appeals to forms of D-optimality are also considered, for cases when no specific toxicokinetic parameter is of specialized interest.

Types and activities of voltage-operated calcium channels change during development of rat pituitary neurointermediate lobe.

Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through omega-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age-groups. This effect was decreased by nifedipine, an L-type channel blocker, at all ages, and by omega-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO4, had no effect. In glial-like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca2+ channels.