Comparative NMR studies of diffusional water permeability of erythrocytes from some animals introduced to Australia: Rat rabbit and sheep

The diffusional water permeability (P _d) of the red blood cell (RBC) membrane of rat, rabbit and sheep, representing some animal species introduced to Australia, has been monitored, by a Mn²⁺-doping ¹H nuclear magnetic resonance (NR) technique using a high-field spectometer operating at 400 MHz. In order to make comparisons with previous measurements on the same species (performed at 25 MHz) an analysis of the influence of instrumental parameters and of the state of blood oxygenation was performed on samples of rat and rabbit blood. It was found that by using a short interpulse delay (around 100 µs) in the Carr-Purcell-Meiboom-Gill sequence, and by performing the measurements of the transverse relaxation time of the water in the cell interior (T _2i) on packed cells (haematocrit >95%), the values of the water exchange time (T _e) obtained with a high-field spectrometer are comparable with those obtained by using a low-field NMR spectrometer. The values of the diffusional water permeability (P _d), calculated from the T _e values, were, at 24.6°C around 10 × 10^–3 cm/s in case of rat and rabbit RBC and around 5 × 10^–3 cm/s for sheep RBC; at 37°C the P _d values were around 16 × 10^–3 cm/s for rat RBC, 14 × 10^–3 cm/s for rabbit RBC and 7 × 10^–3 cm/s for sheep RBC. These values are very close to the P _d values previously reported for the European counterparts of these species. There were no significant differences in the P _d values between laboratory rabbits and wild rabbits, or between castrated males, pregnant or nonpregnant females. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of animal species introduced to Australia or by sex hormone status. If the NMR instrument-parameters are carefully chosen then comparative studies of RBC water permeability in different laboratories, at separate locations and using different instruments are meaningful.     

Mental health and the city: intra-urban mobility among individuals with schizophrenia

Intra-urban residential mobility of a cohort with schizophrenia was compared to a matched cohort with no mental illness using population-based administrative data. The percentage of individuals with one or more changes in postal code in the three-year mobility study period was examined, along with measures of the movement between different intra-urban areas. The schizophrenia cohort was more likely to move than the matched cohort; however, this depends on their age, income level, and area of residence at baseline. Age, gender, marital status, income quintile, and use of physicians and hospitalizations were associated with mobility. Individuals in the schizophrenia cohort were significantly more likely to move from the suburb to the inner city, and significantly less likely to move from the inner city to the suburb than those with no mental illness. Implications of the findings and directions for future research are discussed, with particular attention paid to the utility of administrative data for further mental health research.     

Sequential changes in in vivo muscle and liver protein synthesis and plasma and tissue glutamine levels in sepsis in the rat

We have investigated sequential changes in skeletal muscle and hepatic protein synthesis following sepsis, and their relationship to changes in circulating and tissue glutamine concentrations. Male Wistar rats underwent caecal ligation and puncture (CLP) or sham operation, with starvation, and were killed 24, 72 or 96 h later. A group of non-operated animals were killed at the time of surgery. Protein synthesis was determined using a flooding dose of L-[4-(3)H] phenylalanine, and glutamine concentrations were measured by an enzymic fluorimetric assay. Protein synthesis in gastrocnemius muscle fell in all groups. Gastrocnemius total protein content was reduced after CLP and at 72 and 96 h after sham operation. After CLP, protein synthesis was lower at 24 h, and total protein content was lower at 72 and 96 h, than in sham-operated animals. CLP was associated with increased liver protein synthesis at all time points, whereas there was no change after sham operation. Liver protein content did not change after CLP, but was lower at 72 and 96 h after sham operation than in non-operated animals. Plasma glutamine concentrations were reduced at 24 h after sham operation, and at 72 and 96 h after CLP. Muscle glutamine concentrations were reduced in all groups, with the decrease being greater following CLP than after sham operation. In the liver, glutamine concentrations were unchanged after CLP, but increased after sham operation. In rats with sepsis, decreases in muscle protein synthesis and content are associated with markedly reduced muscle glutamine concentrations. Plasma glutamine concentrations are initially maintained, but fall later. In liver, protein synthesis is increased, while glutamine concentrations are preserved. These results support a peripheral-to-splanchnic glutamine flux in sepsis.     

PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis.

A novel controlled release formulation has been developed with PEGylated human insulin encapsulated in PLGA microspheres that produces multi-day release in vivo. The insulin is specifically PEGylated at the amino terminus of the B chain with a relatively low molecular weight PEG (5000 Da). Insulin with this modification retains full biological activity, but has a limited serum half-life, making encapsulation necessary for sustained release beyond a few hours. PEGylated insulin can be co-dissolved with PLGA in methylene chloride and microspheres made by a single o/w emulsion process. Insulin conformation and biological activity are preserved after PEGylation and PLGA encapsulation. The monolithic microspheres have inherently low burst release, an important safety feature for an extended release injectable insulin product. In PBS at 37 degrees C, formulations with a drug content of approximately 14% show very low (< 1%) initial release of insulin over one day and near zero order drug release after a lag of 3-4 days. In animal studies, PEG-insulin microspheres administered subcutaneously as a single injection produced < 1% release of insulin in the first day but then lowered the serum glucose levels of diabetic rats to values < 200 mg/dL for approximately 9 days. When doses were given at 7-day intervals, steady state drug levels were achieved after only 2 doses. PEG-insulin PLGA microparticles show promise as a once-weekly dosed, sustained release basal insulin formulation.     

Clinical Application of Whole-Genome Sequencing To Inform Treatment for Multidrug-Resistant Tuberculosis Cases

The treatment of drug-resistant tuberculosis cases is challenging, as drug options are limited, and the existing diagnostics are inadequate. Whole-genome sequencing (WGS) has been used in a clinical setting to investigate six cases of suspected extensively drug-resistant Mycobacterium tuberculosis (XDR-TB) encountered at a London teaching hospital between 2008 and 2014. Sixteen isolates from six suspected XDR-TB cases were sequenced; five cases were analyzed in a clinically relevant time frame, with one case sequenced retrospectively. WGS identified mutations in the M. tuberculosis genes associated with antibiotic resistance that are likely to be responsible for the phenotypic resistance. Thus, an evidence base was developed to inform the clinical decisions made around antibiotic treatment over prolonged periods. All strains in this study belonged to the East Asian (Beijing) lineage, and the strain relatedness was consistent with the expectations from the case histories, confirming one contact transmission event. We demonstrate that WGS data can be produced in a clinically relevant time scale some weeks before drug sensitivity testing (DST) data are available, and they actively help clinical decision-making through the assessment of whether an isolate (i) has a particular resistance mutation where there are absent or contradictory DST results, (ii) has no further resistance markers and therefore is unlikely to be XDR, or (iii) is identical to an isolate of known resistance (i.e., a likely transmission event). A small number of discrepancies between the genotypic predictions and phenotypic DST results are discussed in the wider context of the interpretation and reporting of WGS results.     

Thyroid-stimulating immunoglobulin bioassay using cultured human thyroid cells

Modifications are described in the cultured thyroid cell cAMP assay for TSH which make it suitable for the measurement of thyroid-stimulating immunoglobulins. Comparison was made between this assay and two others measuring cAMP responsiveness in human thyroid tissue, namely the thyroid slice and thyroid plasma membrane adenylate cyclase assays, all performed with the same tissue sample. Of immunoglobulin G (IgG) samples from 7 unselected patients with untreated hyperthyroidism associated with Graves' disease, 5 produced significant stimulation of cAMP content in cultured thyroid cells when compared to pooled normal IgG. None of these 7 produced a statistically significant increase in thyroid slice cAMP content when assayed in triplicate, the same replicate number used in the cultured thyroid cell assay. Similarly, none of the same Graves' IgG samples produced significant stimulation (vs. control IgG) in the membrane adenylate cyclase assay, in which sensitivity to TSH stimulation was very poor. With a scaled-down modification of the assay using microtiter wells and acetylation to enhance detection of cAMP in the RIA, significant TSI activity was observed in 15 of 18 (83%) IgG samples from patients with untreated Graves' disease. The data indicate the excellent sensitivity and precision of the thyroid cell cAMP assay, as well as its convenience.     

Microarray analysis of Mycobacterium microti reveals deletion of genes encoding PE-PPE proteins and ESAT-6 family antigens

Mycobacterium microti is the agent of tuberculosis in wild voles and has been used as a live vaccine against tuberculosis in man and cattle. To explore the M. microti genome in greater detail, we used a M. tuberculosis H37Rv genomic DNA microarray to detect gene deletions among M. microti isolates. A number of deletions were identified that correlated with those described previously (Infect. Immun. 70 (2002) 5568) but a novel M. microti deletion was also found (MiD4) which removes 5 genes that code for ESAT-6 family antigens and PE-PPE proteins. Southern blot experiments showed that this region was also deleted from M. pinnipedii, a mycobacterium isolated from seals that is closely related to M. microti. Genes encoding ESAT-6 antigens and PE-PPE proteins appear to be frequently deleted from M. microti, and the implications of this are discussed.     

A pilot study of continuous infusion heparin for the prevention of hepatic veno-occlusive disease after bone marrow transplantation

Twenty-eight patients undergoing marrow transplantation participated in a pilot study to determine the safety of continuous infusion heparin for the prevention of veno-occlusive disease (VOD) of the liver. Four doses of continuous infusion heparin were administered, ranging from a dose prolonging the partial thromboplastin time (PTT) to 1.5-2.0 times the patients' baseline value, to a dose prolonging the PTT to less than 1.2 times the patients' baseline value. Seven patients (25%) received a full course of heparin, beginning from the day the preparative therapy started through day 14 post-transplant (range 20-26 days on heparin). In 21 patients infusions were ended before day 14 post-transplant, a median of 16 days on heparin (range 1-26 days). Of these, 14 patients were withdrawn from heparin because of bleeding and seven were withdrawn because of anticipated bleeding. Bleeding was observed in 27 patients and was minor in 25. Two patients developed major bleeding in the gastrointestinal tract which was not fatal. Minor bleeding was observed in 27 of 28 case control patients who did not receive heparin. The sites of bleeding were similar in control and heparin treated patients. VOD developed in 20 patients (71%) and was sever or fatal in four (14%). The prevalence of VOD was not influenced by the dosage of heparin or the duration of its administration. We conclude that low dose heparin resulting in marginal prolongation of the PTT may be infused into patients undergoing marrow transplantation with a low risk of serious bleeding. Further studies are needed to evaluate its efficacy.     

Identification of false positive exercise tests with use of electrocardiographic criteria: a possible role for atrial repolarization waves

Atrial repolarization waves are opposite in direction to P waves, may have a magnitude of 100 to 200 mu V and may extend into the ST segment and T wave. It was postulated that exaggerated atrial repolarization waves during exercise could produce ST segment depression mimicking myocardial ischemia. The P waves, PR segments and ST segments were studied in leads II, III, aVF and V4 to V6 in 69 patients whose exercise electrocardiogram (ECG) suggested ischemia (100 mu V horizontal or 150 mu V upsloping ST depression 80 ms after the J point). All had a normal ECG at rest. The exercise test in 25 patients (52% male, mean age 53 years) was deemed false positive because of normal coronary arteriograms and left ventricular function (5 patients) or normal stress single photon emission computed tomographic thallium or gated blood pool scans (16 patients), or both (4 patients). Forty-four patients with a similar age and gender distribution, anginal chest pain and at least one coronary stenosis greater than or equal to 80% served as a true positive control group. The false positive group was characterized by 1) markedly downsloping PR segments at peak exercise, 2) longer exercise time and more rapid peak exercise heart rate than those of the true positive group, and 3) absence of exercise-induced chest pain. The false positive group also displayed significantly greater absolute P wave amplitudes at peak exercise and greater augmentation of P wave amplitude by exercise in all six ECG leads than were observed in the true positive group.(ABSTRACT TRUNCATED AT 250 WORDS)