Informing the use of a supplementary immunisation programme for the management of a community cluster of invasive meningococcal disease, Yorkshire, 2022.

OBJECTIVES: To outline the management of a community cluster of serogroup B invasive meningococcal disease (IMD) cases, including key factors for decision making and the choice and implementation of control measures. STUDY DESIGN: Descriptive report of cluster management. METHODS: Subtyping of IMD cases identified a number of potentially linked cases in a defined geographical area. An Incident Management Team (IMT) was convened to coordinate the public health response. A case definition was developed in order to identify further cases within the cluster. RESULTS: Four cases of IMD met the case definition and were initially considered as part of this cluster. Three resided in the same small town, which was the focus for public health management. The IMT agreed that it would be proportionate to instigate additional control measures. The population at higher risk of infection were identified, and a supplementary vaccination programme was rolled out in the community. Over five clinics, 45.6% (639/1401) of the target cohort received at least one dose of the vaccine, with 34.7% (486/1401) receiving both doses. Inequalities in uptake were observed by sex, age and deprivation. CONCLUSIONS: Decision making for public health responses to IMD clusters is complex. Informed by epidemiological evidence, numerous partners engaged in collaborative decision making, which was critical for the effective implementation of the community response. Links between the local authority public health team and the community enabled the use of existing structures and relationships to maximise the number of vaccinations delivered. No further cases of IMD linked to this cluster were identified.

Molecular dissection of TNFR-TNFα bidirectional signaling reveals both cooperative and antagonistic interactions with p75 neurotrophic factor receptor in axon patterning.

During neural development, complex organisms rely on progressive and regressive events whereby axons, synapses, and neurons are overproduced followed by selective elimination of a portion of these components. Tumor necrosis factor α (TNFα) together with its cognate receptor (Tumor necrosis factor receptor 1; TNFR1) have been shown to play both regressive (i.e. forward signaling from the receptor) and progressive (i.e. reverse signaling from the ligand) roles in sympathetic neuron development. In contrast, a paralog of TNFR1, p75 neurotrophic factor receptor (p75NTR) promotes mainly regressive developmental events in sympathetic neurons. Here we examine the interplay between these paralogous receptors in the regulation of axon branch elimination and arborization. We confirm previous reports that these TNFR1 family members are individually capable of promoting ligand-dependent suppression of axon growth and branching. Remarkably, p75NTR and TNFR1 physically interact and p75NTR requires TNFR1 for ligand-dependent axon suppression of axon branching but not vice versa. We also find that p75NTR forward signaling and TNFα reverse signaling are functionally antagonistic. Finally, we find that TNFα reverse signaling is necessary for nerve growth factor (NGF) dependent axon growth. Taken together these findings demonstrate several levels of synergistic and antagonistic interactions using very few signaling pathways and that the balance of these synergizing and opposing signals act to ensure proper axon growth and patterning.

Four-month outbreak of invasive meningococcal disease caused by a rare serogroup B strain, identified through the use of molecular PorA subtyping, England, 2013.

Molecular PorA subtyping provides information that increasingly requires the adaptation of standard public health approaches to outbreak management. We report an outbreak of a rare subtype of meningococcal infection not previously identified in the United Kingdom (UK). The outbreak occurred in the Warwickshire area in England between February and June 2013. Molecular subtyping allowed the identification of additional cases, prompting an enhanced public health response that included efforts to identify potential social networks that might benefit from chemoprophylaxis. It also prompted swabbing to define nasopharyngeal carriage in the focal nursery and helped explain the unusual epidemiological pattern. Without subtyping to identify a link, the additional cases would have been managed as sporadic cases in accordance with current UK guidance.

Exploring genetic variation in potential indicators of resilience in sheep using fibre diameter measured along the wool staple.

Production animals are increasingly exposed to a wide variety of disturbances that can compromise their productivity, health and well-being. As a result, there is a growing need to be able to select animals that are more resilient to environmental disturbances. Fibre diameter variation measured along a wool staple is expected to contain information about how resilient sheep are to the disturbances of their internal and external environment. This study aimed to develop potential resilience indicators from fibre diameter variation, estimate their genetic parameters and assess whether these traits are genetically correlated across three age stages. The study used 6 140 Merino sheep from the Sheep Cooperative Research Centre Information Nucleus Flocks recorded at yearling, 2 years old, and adult ages. Eight potential traits were defined based on theory, literature and exploratory analysis, which were suggested to capture the animal's ability to resist, respond and recover from potential disturbances. Genetic evaluation of the traits was conducted using pedigree-based animal models. The traits were shown to be low to moderately heritable (0.01-0.33) when examined at each of the three age stages. The potential indicators were generally well correlated with one another within age stages. Further, the genetic correlation between the same trait measured at different age stages was moderate to high between yearling and 2 years old (0.35-0.94) and between 2 years old and adults (0.18-0.70), while slightly lower between yearling and adult estimates (0.09-0.62). These results suggest that selection for resilience indicators from fibre diameter is possible; however, further studies are warranted to refine the trait definitions and validate these indicators against other measures of health, fitness and productive performance.

Short communication: Accuracy of whole-genome sequence imputation in Angus cattle using within-breed and multi breed reference populations.

Genotype imputation is a standard approach used in the field of genetics. It can be used to fill in missing genotypes or to increase genotype density. Accurate imputed genotypes are required for downstream analyses. In this study, the accuracy of whole-genome sequence imputation for Angus beef cattle was examined using two different ways to form the reference panel, a within-breed reference population and a multi breed reference population. A stepwise imputation was conducted by imputing medium-density (50k) genotypes to high-density, and then to the whole genome sequence (WGS). The reference population consisted of animals with WGS information from the 1 000 Bull Genomes project. The within-breed reference panel comprised 396 Angus cattle, while an additional 2 380 Taurine cattle were added to the reference population for the multi breed reference scenario. Imputation accuracies were variant-wise average accuracies from a 10-fold cross-validation and expressed as concordance rates (CR) and Pearson's correlations (PR). The two imputation scenarios achieved moderate to high imputation accuracies ranging from 0.896 to 0.966 for CR and from 0.779 to 0.834 for PR. The accuracies from two different scenarios were similar, except for PR from WGS imputation, where the within-breed scenario outperformed the multi breed scenario. The result indicated that including a large number of animals from other breeds in the reference panel to impute purebred Angus did not improve the accuracy and may negatively impact the results. In conclusion, the imputed WGS in Angus cattle can be obtained with high accuracy using a within-breed reference panel.

Genomic evaluations using similarity between haplotypes.

Long-range phasing and haplotype library imputation methodologies are accurate and efficient methods to provide haplotype information that could be used in prediction of breeding value or phenotype. Modelling long haplotypes as independent effects in genomic prediction would be inefficient due to the many effects that need to be estimated and phasing errors, even if relatively low in frequency, exacerbate this problem. One approach to overcome this is to use similarity between haplotypes to model covariance of genomic effects by region or of animal breeding values. We developed a simple method to do this and tested impact on genomic prediction by simulation. Results show that the diagonal and off-diagonal elements of a genomic relationship matrix constructed using the haplotype similarity method had higher correlations with the true relationship between pairs of individuals than genomic relationship matrices built using unphased genotypes or assumed unrelated haplotypes. However, the prediction accuracy of such haplotype-based prediction methods was not higher than those based on unphased genotype information.

Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

Comparison of two live-animal ultrasound systems for genetic evaluation of carcass traits in Angus cattle.

The improvement of carcass traits is an important breeding objective in beef cattle breeding programs. The most common way of selecting for improvement in carcass traits is via indirect selection using ultrasound scanning of selection candidates which are submitted to genetic evaluation programs. Two systems used to analyze ultrasound images to predict carcass traits are the Pie Medical Esaote Aquila (PIE) and Central Ultrasound Processing (CUP). This study compared the ability of the two systems to predict carcass traits for genetic evaluation in Australian Angus cattle. Genetic and phenotypic parameters were estimated using data from 1,648 Angus steers which were ultrasound scanned twice with both systems, first at feedlot entry and then following 100 d in the feedlot. The traits interpreted from ultrasound scanning included eye muscle area (EMA), rib fat (RIB) rump fat (RUMP), and intramuscular fat (IMF). Abattoir carcass data were collected on all steers following the full feedlot feeding period of 285 d. For all ultrasound scan traits, CUP resulted in higher phenotypic and genetic variances compared to the PIE. For IMF, CUP had higher heritability at feedlot intake (0.51 for CUP compared to 0.37 for PIE) and after 100 d feeding (0.54 for CUP compared to 0.45 PIE). CUP predicted IMF also tended to have stronger correlations with the breeding objective traits of carcass IMF and marbling traits, both genetically (ranging from 0.59 to 0.75 for CUP compared to 0.45-0.63 for PIE) and phenotypically (ranging from 0.27 to 0.43 for CUP compared to 0.19-0.28 for PIE). Ultrasound scan EMA was the only group of traits in which the heritabilities were higher for PIE (0.52 for PIE compared to 0.40 for CUP at feedlot intake and 0.46 for PIE compared to 0.43 for CUP at 100 d of feeding), however with similar relationships to the breeding objective carcass EMA observed. For subcutaneous fat traits of ultrasound RIB and RUMP, the heritabilites and genetic correlations to the related carcass traits were similar, with the exception being the higher heritability observed for CUP predicted RUMP at feedlot intake at 0.52 compared to 0.38 for PIE. The results from this study indicates that the CUP system, compared to PIE, provides an advantage for genetic evaluation of carcass traits in Angus cattle, particularly for the IMF and associated marbling traits.

Estimated strain coverage of serogroup B meningococcal vaccines: A retrospective study for disease and carrier strains in Greece (2010-2017).

Invasive meningococcal disease (IMD) is associated with high case fatality rates and long-term sequelae among survivors. Meningococci belonging to six serogroups (A, B, C, W, X, and Y) cause nearly all IMD worldwide, with serogroup B meningococci (MenB) the predominant cause in many European countries, including Greece (~80% of all IMD). In the absence of protein-conjugate polysaccharide MenB vaccines, two protein-based vaccines are available to prevent MenB IMD in Greece: 4CMenB (Bexsero™, GlaxoSmithKline), available since 2014; and MenB-FHbp, (Trumenba™, Pfizer), since 2018. This study investigated the potential coverage of MenB vaccines in Greece using 107 MenB specimens, collected from 2010 to 2017 (66 IMD isolates and 41 clinical samples identified solely by non-culture PCR), alongside 6 MenB isolates from a carriage study conducted during 2017-2018. All isolates were characterized by multilocus sequence typing (MLST), PorA, and FetA antigen typing. Whole Genome Sequencing (WGS) was performed on 66 isolates to define the sequences of vaccine components factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA), and Neisseria adhesin A (NadA). The expression of fHbp was investigated with flow cytometric meningococcal antigen surface expression (MEASURE) assay. The fHbp gene was present in-frame in all isolates tested by WGS and in 41 MenB clinical samples. All three variant families of fHbp peptides were present, with subfamily B peptides (variant 1) occurring in 69.2% and subfamily A in 30.8% of the samples respectively. Sixty three of 66 (95.5%) MenB isolates expressed sufficient fHbp to be susceptible to bactericidal killing by MenB-fHbp induced antibodies, highlighting its potential to protect against most IMD in Greece.

Brain target sites for 1,25-dihydroxyvitamin D3.

Autoradiographic studies with 3H-labeled 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] demonstrate, in certain neurons of rat forebrain, hindbrain, and spinal cord, a nuclear retention and concentration of radioactivity, which can be prevented by treatment with 1,25(OH)2D3, but not with 25-hydroxyvitamin D3. These results indicate the presence of brain receptors in addition to pituitary receptors for 1,25(OH)2D3 and suggest a central modulation of calcium homeostasis and other central effects for this hormone. The existence of a brain-pituitary axis for certain 1,25(OH)2D3-mediated endocrine-autonomic effects is postulated.

Tuberculosis antigen-specific immune responses can be detected using enzyme-linked immunospot technology in human immunodeficiency virus (HIV)-1 patients with advanced disease.

There are limited data on the efficacy of T cell-based assays to detect tuberculosis (TB) antigen-specific responses in immune-deficient human immunodeficiency virus (HIV) patients. The aim of this study is to determine whether TB antigen-specific immune responses can be detected in patients with HIV-1 infection, especially in those with advanced disease (CD4 T cell count < 300 cells/microl). An enzyme-linked immunospot (ELISPOT) assay, which detects interferon (IFN)-gamma secreted by T cells exposed to TB antigens, was used to assess specific immune responses in a prospective study of 201 HIV-1-infected patients with risk factors for TB infection, attending a single HIV unit. The performance of the ELISPOT assay to detect TB antigen-specific immune responses is independent of CD4 T cell counts in HIV-1 patients. The sensitivity and specificity of this assay for the diagnosis of active tuberculosis does not differ significantly from values obtained in immunocompetent subjects. The negative predictive value of the TB ELISPOT test is 98.2%. A positive predictive value of 86% for the diagnosis of active tuberculosis was found when the combined number of early secretory antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) IFN-gamma spots to CD4 T cell count ratio was > 1.5. TB antigen-specific immune responses can be detected in HIV patients with low CD4 T cell counts using ELISPOT technology in a routine diagnostic laboratory and is a useful test to exclude TB infection in immune-deficient HIV-1 patients. A combination of TB antigen-specific IFN-gamma responses and CD4 T cell counts has the potential to distinguish active tuberculosis from latent infection.

Muscle Na+-K+-ATPase activity and isoform adaptations to intense interval exercise and training in well-trained athletes.

The Na+ -K+ -ATPase enzyme is vital in skeletal muscle function. We investigated the effects of acute high-intensity interval exercise, before and following high-intensity training (HIT), on muscle Na+ -K+ -ATPase maximal activity, content, and isoform mRNA expression and protein abundance. Twelve endurance-trained athletes were tested at baseline, pretrain, and after 3 wk of HIT (posttrain), which comprised seven sessions of 8 x 5-min interval cycling at 80% peak power output. Vastus lateralis muscle was biopsied at rest (baseline) and both at rest and immediately postexercise during the first (pretrain) and seventh (posttrain) training sessions. Muscle was analyzed for Na+ -K+ -ATPase maximal activity (3-O-MFPase), content ([3H]ouabain binding), isoform mRNA expression (RT-PCR), and protein abundance (Western blotting). All baseline-to-pretrain measures were stable. Pretrain, acute exercise decreased 3-O-MFPase activity [12.7% (SD 5.1), P < 0.05], increased alpha1, alpha2, and alpha3 mRNA expression (1.4-, 2.8-, and 3.4-fold, respectively, P < 0.05) with unchanged beta-isoform mRNA or protein abundance of any isoform. In resting muscle, HIT increased (P < 0.05) 3-O-MFPase activity by 5.5% (SD 2.9), and alpha3 and beta3 mRNA expression by 3.0- and 0.5-fold, respectively, with unchanged Na+ -K+ -ATPase content or isoform protein abundance. Posttrain, the acute exercise induced decline in 3-O-MFPase activity and increase in alpha1 and alpha3 mRNA each persisted (P < 0.05); the postexercise 3-O-MFPase activity was also higher after HIT (P < 0.05). Thus HIT augmented Na+ -K+ -ATPase maximal activity despite unchanged total content and isoform protein abundance. Elevated Na+ -K+ -ATPase activity postexercise may contribute to reduced fatigue after training. The Na+ -K+ -ATPase mRNA response to interval exercise of increased alpha- but not beta-mRNA was largely preserved posttrain, suggesting a functional role of alpha mRNA upregulation.

A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts.

Effect of 1,25 dihydroxyvitamin D3 on insulin secretion.

Recent autoradiographic studies demonstrated that B-cells concentrate 1,25 (OH)2 D3 in their nuclei, suggesting a genomic action on B-cell function. This study was undertaken to investigate the effects of 1,25 (OH)2 D3 on insulin secretion in vitamin D-deficient rats. Mature vitamin D-deficient rats were injected with 1,25 (OH)2 D3 or the ethanol-isotonic saline vehicle. Administration of 1,25 (OH)2 D3 to 10 rats resulted in a 17 microunits/ml (113%) increase in insulin levels and 0.9 mg/dl (16%) increase in plasma calcium. No changes were found in insulin or calcium levels in 5 control rats given vehicle alone. A group of vitamin D-deficient rats with plasma calcium levels of 5.4 +/- 0.1 mg/dl had insulin levels that were the same as those observed in a group of vitamin D-deficient rats with plasma calcium levels of 6.3 +/- 0.1 mg/dl. The difference in calcium levels between these two groups is similar to the increase in plasma calcium found after 1,25 (OH)2 D3 administration. The results of these studies indicate that 1,25 (OH)2 D3 action on pancreatic B-cells affects insulin secretion. Since insulin increases synthesis of 1,25 (OH)2 D3, the existence of a feedback loop between B-cells and kidney proximal tubule cells is suggested.

Regulation of insulin secretion from novel engineered insulinoma cell lines.

In the accompanying article, we describe the creation of novel cell lines derived from RIN 1046-38 rat insulinoma cells by stable transfection with combinations of genes encoding human insulin, GLUT2, and glucokinase. Herein we describe the regulation of insulin secretion and glucose metabolism in these new cell lines. A cell line (betaG I/17) expressing only the human proinsulin transgene exhibits a clear increase in basal insulin production (measured in the absence of secretagogues) relative to parental RIN 1046-38 cells. betaG I/17 cells engineered for high levels of GLUT2 expression and a twofold increase in glucokinase activity (betaG 49/206) or engineered for a 10-fold increase in glucokinase activity alone (betaG 40/110) exhibit a 66% and 80% suppression in basal insulin secretion relative to betaG I/17 cells, respectively. As a result, betaG 49/206 and betaG 40/110 cells exhibit potent insulin-secretory responses to glucose alone (6.1- and 7.6-fold, respectively) or to glucose plus isobutylmethylxanthine (10.8- and 15.1-fold, respectively) that are clearly larger than the corresponding responses of betaG I/17 or parental RIN 1046-38 cells. betaG 49/206 and betaG 40/110 cells also exhibit a rapid and sustained response to glucose plus isobutyl-methylxanthine in perifusion studies that is clearly larger in magnitude than that of the two control lines. Glucose dose-response studies show that both engineered and non-engineered lines respond maximally to submillimolar concentrations of glucose and that betaG 49/206 cells are the most sensitive to low concentrations of the hexose, consistent with their clearly elevated rate of [5-3H]glucose usage. Finally, 5-thioglucose, a potent inhibitor of low-K(m) hexokinases, most effectively normalizes glucose concentration dependence for insulin secretion in the cell line with highest glucokinase expression (betaG 40/110). We conclude that GLUT2 and/or glucokinase expression imposes tight regulation of basal insulin secretion in cell lines that overexpress human proinsulin, allowing a marked improvement in the range of secretagogue responsiveness in such cells.

Novel insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase, and human insulin expression.

Cellular engineering studies in our group are directed at creating insulin-secreting cell lines that simulate the performance of the normal islet beta-cell. The strategy described in this article involves the stepwise stable introduction of genes relevant to beta-cell performance into the RIN 1046-38 insulinoma cell line, a process that we term "iterative engineering." RIN cells stably engineered to contain multiple copies of the human insulin gene exhibit a large increase in insulin content, such that they approach the content of human islets assayed in parallel. Analysis by high-performance liquid chromatography demonstrates that these engineered cell lines process human proinsulin to mature insulin with high efficiency. Cell lines that are further engineered to express the GLUT2 and glucokinase genes demonstrate stable expression of the three transgenes for the full lifetime of the lines produced to date (6 months to 1 year in continuous culture). Transplantation of the engineered cell lines into nude rats reveals that stably integrated genes are expressed at constant levels in the in vivo environment over the full duration of experiments performed (48 days). Several endogenous genes expressed in normal beta-cells, including rat insulin, amylin, sulfonylurea receptor, and glucokinase, are stably expressed in the insulinoma lines during these in vivo studies. Endogenous GLUT2 expression, in contrast, is rapidly extinguished during in vivo passage. The loss of GLUT2 is overcome in engineered cell ines in which transporter expression is provided by a stably transfected transgene. These results suggest that a potential advantage of the iterative engineering approach may be to preserve stability of function and phenotype, particularly in the in vivo setting.

Chronic intermittent hypoxia and incremental cycling exercise independently depress muscle in vitro maximal Na+-K+-ATPase activity in well-trained athletes.

Athletes commonly attempt to enhance performance by training in normoxia but sleeping in hypoxia [live high and train low (LHTL)]. However, chronic hypoxia reduces muscle Na(+)-K(+)-ATPase content, whereas fatiguing contractions reduce Na(+)-K(+)-ATPase activity, which each may impair performance. We examined whether LHTL and intense exercise would decrease muscle Na(+)-K(+)-ATPase activity and whether these effects would be additive and sufficient to impair performance or plasma K(+) regulation. Thirteen subjects were randomly assigned to two fitness-matched groups, LHTL (n = 6) or control (Con, n = 7). LHTL slept at simulated moderate altitude (3,000 m, inspired O(2) fraction = 15.48%) for 23 nights and lived and trained by day under normoxic conditions in Canberra (altitude approximately 600 m). Con lived, trained, and slept in normoxia. A standardized incremental exercise test was conducted before and after LHTL. A vastus lateralis muscle biopsy was taken at rest and after exercise, before and after LHTL or Con, and analyzed for maximal Na(+)-K(+)-ATPase activity [K(+)-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase)] and Na(+)-K(+)-ATPase content ([(3)H]ouabain binding sites). 3-O-MFPase activity was decreased by -2.9 +/- 2.6% in LHTL (P < 0.05) and was depressed immediately after exercise (P < 0.05) similarly in Con and LHTL (-13.0 +/- 3.2 and -11.8 +/- 1.5%, respectively). Plasma K(+) concentration during exercise was unchanged by LHTL; [(3)H]ouabain binding was unchanged with LHTL or exercise. Peak oxygen consumption was reduced in LHTL (P < 0.05) but not in Con, whereas exercise work was unchanged in either group. Thus LHTL had a minor effect on, and incremental exercise reduced, Na(+)-K(+)-ATPase activity. However, the small LHTL-induced depression of 3-O-MFPase activity was insufficient to adversely affect either K(+) regulation or total work performed.

Sleep in athletes undertaking protocols of exposure to nocturnal simulated altitude at 2650 m.

A popular method to attempt to enhance performance is for athletes to sleep at natural or simulated moderate altitude (SMA) when training daily near sea level. Based on our previous observation of periodic breathing in athletes sleeping at SMA, we hypothesised that athletes' sleep quality would also suffer with hypoxia. Using two typical protocols of nocturnal SMA (2650 m), we examined the effect on the sleep physiology of 14 male endurance-trained athletes. The selected protocols were Consecutive (15 successive exposure nights) and Intermittent (3x 5 successive exposure nights, interspersed with 2 normoxic nights) and athletes were randomly assigned to follow either one. We monitored sleep for two successive nights under baseline conditions (B; normoxia, 600 m) and then at weekly intervals (nights 1, 8 and 15 (N1, N8 and N15, respectively)) of the protocols. Since there was no significant difference in response between the protocols being followed (based on n=7, for each group) we are unable to support a preference for either one, although the likelihood of a Type II error must be acknowledged. For all athletes (n=14), respiratory disturbance and arousal responses between B and N1, although large in magnitude, were highly individual and not statistically significant. However, SpO2 decreased at N1 versus B (p<0.001) and remained lower on N8 (p<0.001) and N15 (p<0.001), not returning to baseline level. Compared to B, arousals were more frequent on N8 (p=0.02) and N15 (p=0.01). The percent of rapid eye movement sleep (REM) increased from N1 to N8 (p=0.03) and N15 (p=0.01). Overall, sleeping at 2650 m causes sleep disturbance in susceptible athletes, yet there was some improvement in REM sleep over the study duration.

Ultrastructural localization of tartrate-resistant, purple acid phosphatase in rat osteoclasts by histochemistry and immunocytochemistry.

The intracellular localization of the tartrate-resistant purple acid phosphatase in osteoclasts of developing rat bone has been determined immunocytochemically using an antiserum to the purified bone-derived purple acid phosphatase. The localization of the immunoreactivity was compared with the results of enzyme histochemistry using p-nitrophenylphosphate as substrate and 10 mM tartrate. Both methods revealed the presence of the enzyme in numerous vesicles of various sizes up to 2-3 microns in diameter and in granules. There was no immunoreactivity in the Golgi apparatus, and tartrate completely inhibited the histochemical activity of this organelle. No consistent extracellular activity could be detected, nor was any reaction product observed at the ruffled border. The localization of the tartrate-resistant purple acid phosphatase in osteoclasts is consistent with an intracellular function for this enzyme.

Islet cell culture in defined serum-free medium.

A serum-free, hormone- and factor-supplemented, defined medium was developed which maintains functional activity of primary cultures of adult islet cells and continuous islet cell lines. Medium supplements examined included proteose peptone (PP), transferrin (TrFe), insulin-like growth factor-I (IGF-I), an insulinotropic fragment of human GH [hGH-(6-13)], ethanolamine (EA), phosphoethanolamine (PEA), and human serum albumin (HSA). Glucose-stimulated insulin secretion from islet monolayers was determined after culture in serum-free supplemented medium by either 2- to 3-h static incubations or in a superfusion system with either low (2.8-8.3 mM) or stimulatory (16.7-19.4 mM) glucose concentrations. Glucose-induced secretion was not sustained after 3-4 days of culture in medium supplemented with PP (0.5 mg/ml) and TrFe (10 micrograms/ml) alone. Addition of T3 did not restore glucose-induced secretion, although a combination of T3 and IGF-I or of T3, IGF-I, and PRL (10(-10)-10(-9) M) maintained glucose-induced insulin secretion for 1 month. No beneficial effects were noted with hGH-(6-13). The beta-cell lines HIT-T15 and RINr 1046-38 were used to screen for a potential replacement for PP, the undefined component of the serum-free medium. A combination of HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) provided a replacement for PP. In fact, insulin secretion from HIT-T15 cells was significantly better after culture in medium supplemented with HSA, EA, PEA, TrFe, T3, IGF-I, and PRL than in medium with PP, TrFe, T3, IGF-I, and PRL. HSA (1 mg/ml), EA (50 microM), and PEA (50 microM) in combination with TrFe (10 micrograms/ml), T3 (0.1 nM), IGF-I (0.65 nM), and PRL (1 nM) were used in studies with primary islet monolayers. After 3 weeks of culture islet monolayers were superfused, and the biphasic glucose-induced insulin secretion of cells maintained in defined medium was indistinguishable from the insulin secretion of cells maintained in medium with 5% fetal bovine serum. These studies indicate that adult rat beta-cells retain biphasic glucose-induced insulin secretion after extended culture in defined serum-free medium. The defined medium was also useful for cultures of RINr 1046-38 and HIT-T15 cells and should provide a basis for formulating media for islet cells from higher mammals, including man.