Blood cultures and blood microbiota analysis as surrogates for bronchoalveolar lavage fluid analysis in dogs with bacterial pneumonia.

BACKGROUND: Diagnosis of canine bacterial pneumonia relies on airway lavage to confirm septic, suppurative inflammation, and a positive bacterial culture. Considering risks of bronchoalveolar lavage fluid (BALF) collection, minimally invasive methods like culture or next generation sequencing of blood would be appealing. In dogs with bacterial pneumonia, our study aims included (1): determining proportion of agreement between cultivable bacteria in BALF and blood (2); characterizing BALF, blood, and oropharyngeal (OP) microbiota and determining if bacteria cultured from BALF were present in these communities; and (3) comparing relatedness of microbial community composition at all three sites. Bacterial cultures were performed on BALF and blood. After DNA extraction of BALF, blood and OP, 16S rRNA amplicon libraries were generated, sequenced, and compared to a bacterial gene sequence database. RESULTS: Disregarding one false positive, blood cultures were positive in 2/9 dogs (5 total isolates), all 5 isolates were present in BALF cultures (16 total isolates). Based on sequencing data, all sites had rich and diverse microbial communities. Comparing cultured BALF bacterial genera with sequenced taxa, all dogs had ≥1 cultured isolate present in their microbiota: cultured BALF isolates were found in microbiota of BALF (12/16), blood (7/16), and OP (6/11; only 7 dogs had OP swabs). Of 394 distinct taxa detected in BALF, these were present in 75% OP and 45% blood samples. BALF community composition was significantly different than OP (p = 0.0059) and blood (p = 0.0009). CONCLUSIONS: Blood cultures are insensitive but specific for cultured BALF bacteria in canine bacterial pneumonia. Cultivable BALF bacteria were present in BALF, blood and OP microbiota to differing degrees.

The effect of intramammary pirlimycin hydrochloride on the fecal microbiome of early-lactation heifers.

The purpose of this study was to determine the effect of intramammary pirlimycin on the fecal microbiome of dairy cattle. Primiparous heifers were enrolled and assigned to a treatment or control group at a ratio of 2:1. In part 1 of the study, treated heifers (T1) were given intramammary pirlimycin into one infected quarter once daily for 2 d at 24-h intervals, according to the label instructions. Control heifers received no treatment. In part 2 of the study, treated heifers (T2) were given intramammary pirlimycin into one infected quarter once daily for 8 d at 24-h intervals, according to the label instructions. All enrolled heifers (T1, T2, and control) had quarter-level milk samples aseptically collected for bacterial culture and fecal samples collected for 16S rRNA gene sequencing on d 0, 2, 7, 14, 21, and 28. Milk samples were plated on Columbia blood agar and incubated at 37°C for 24 h. Bacteria were identified using MALDI-TOF mass spectrometry. The DNA was extracted from feces using PowerFecal kits (Qiagen, Venlo, the Netherlands). The 16S rRNA gene amplicon library construction and sequencing was performed at the University of Missouri DNA Core facility. Testing for differences in fecal community composition was performed via one-way permutational multivariate ANOVA of Bray-Curtis and Jaccard similarities using Past 3.13 (https://folk.uio.no/ohammer/past/). Mean total count of operational taxonomic units and Chao1, Shannon, and Simpson α-diversity indices were determined and compared via t-test or Wilcoxon rank sum test. A treatment-dependent effect was present in the observed and predicted richness of feces from cows in the T1 group at d 2 posttreatment. Additionally, intramammary pirlimycin induced a significant change in the composition of the fecal microbiota by d 2 in the treated groups. Based on calculated intra-subject similarities, intramammary pirlimycin was associated with a significant acute change in the fecal microbiota of dairy heifers and that chance reversed when the antimicrobial exposure was brief, but sustained following longer exposure. Overall, intramammary pirlimycin administration affected the fecal microbiome of lactating dairy heifers. Further work is necessary to determine the effect of these changes on the heifer and the dairy environment as well as if treatment is influencing antimicrobial resistance among enteric and environmental bacteria.

Hot topic: 16S rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus.

We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.

Species diversity regarding the presence of proximal tubular progenitor cells of the kidney.

The cellular source for tubular regeneration following kidney injury is a matter of dispute, with reports suggesting a stem or progenitor cells as the regeneration source while linage tracing studies in mice seemingly favor the classical theory, where regeneration is performed by randomly surviving cells. We, and others have previously described a scattered cell population localized to the tubules of human kidney, which increases in number following injury. Here we have characterized the species distribution of these proximal tubular progenitor cells (PTPCs) in kidney tissue from chimpanzee, pig, rat and mouse using a set of human PTPC markers. We detected PTPCs in chimpanzee and pig kidneys, but not in mouse tissue. Also, subjecting mice to the unilateral urethral obstruction model, caused clear signs of tubular injury, but failed to induce the PTPC phenotype in renal tubules.

Evaluation of the cost-utility of insulin degludec vs insulin glargine in Sweden.

OBJECTIVE: To evaluate the annual cost-utility of insulin degludec compared with glargine in patients with: type 1 diabetes (T1D), type 2 diabetes receiving basal-only therapy (T2D-BOT), and type 2 diabetes receiving basal-bolus therapy (T2B-BB) in Sweden. METHODS: A cost-utility model was programmed in Microsoft Excel to evaluate clinical and economic outcomes. The clinical trials were designed as treat-to-target, with insulin doses adjusted in order to achieve similar glycemic control between treatments, thus long-term modeling is not meaningful. Basal and bolus insulin doses, incidence of hypoglycemic events, frequency of self-monitoring of blood glucose, and possibility for flexibility in timing of dose administration were specified for each insulin in three diabetes populations, based on data collected in Swedish patients with diabetes and a meta-analysis of clinical trials with degludec. Using these characteristics, the model estimated costs from a societal perspective and quality-adjusted life years (QALYs) in the two scenarios. RESULTS: Use of degludec was associated with a QALY gain compared with glargine in T1D (0.31 vs 0.26 QALYs), T2D-BOT (0.76 vs 0.69 QALYs), and T2D-BB (0.56 vs 0.47 QALYs), driven by reduced incidence of hypoglycemia and possibility for flexibility around timing of dose administration. Therapy regimens containing degludec were associated with increased costs compared to glargine-based regimens, driven by the increased pharmacy cost of basal insulin, but partially offset by other cost savings. Based on estimates of cost and clinical outcomes, degludec was associated with incremental cost-effectiveness ratios of SEK 19,766 per QALY gained, SEK 10,082 per QALY gained, and SEK 36,074 per QALY gained in T1D, T2-BOT, and T2-BB, respectively. LIMITATIONS: The hypoglycemic event rates in the base case analysis were derived from a questionnaire-based study that relied on patient interpretation and recall of hypoglycemic symptoms. The relative rates of hypoglycemia with degludec compared to glargine were derived from a meta-analysis of phase III trials, which may not reflect the relative rates observed in real-world clinical practice. Both of these key limitations were explored in one-way sensitivity analyses. CONCLUSIONS: Based on reduced incidence of hypoglycemia and possibility for flexibility around timing of dose administration, use of degludec is likely to be cost-effective compared to glargine from a societal perspective in T1D, T2-BOT, and T2-BB in Sweden over a 1-year time horizon.

Transferability and characterization of simple sequence repeat markers from Anacardium occidentale to A. humile (Anacardiaceae).

Use of molecular markers can be limited by the high cost and extensive time required for their development. Transfer of simple sequence repeat (SSR) markers reduces the cost and time limitations and has allowed the use of these markers in a larger number of species. We tested 11 SSR markers previously developed for Anacardium occidentale on A. humile. The 11 loci were successfully amplified in A. humile. All loci were polymorphic and generated a mean of 5.4 alleles per locus. The observed heterozygosity was lower than the expected heterozygosity under Hardy-Weinberg equilibrium for most loci, with mean values of 0.463 and 0.696, respectively. The endogamy coefficients were positive and significant for seven loci. However, the combined probability of paternity exclusion was high, and the combined probability of genetic identity was low. None of the pairs of loci were in linkage disequilibrium. The informative power of these loci demonstrates that they are suitable for studies of diversity and genetic structure of natural populations of A. humile. In addition, the loci are suitable for estimating gene flow between populations, assessing species crossing preferences, and performing interspecific comparisons.

Willingness to pay for diabetes drug therapy in type 2 diabetes patients: based on LEAD clinical programme results.

OBJECTIVE: The purpose of this study was to investigate the preferences of people with diabetes for liraglutide vs other glucose lowering drugs, based on outcomes of clinical trials. METHODS: Willingness to pay (WTP) for diabetes drug treatment was assessed by combining results from a recent WTP study with analysis of results from the Liraglutide Effect and Action in Diabetes (LEAD) programme. The LEAD programme included six randomised clinical trials with 3967 participants analysing efficacy and safety of liraglutide 1.2 mg (LEAD 1-6 trials), rosiglitazone (LEAD 1 trial), glimepiride (LEAD 2-3 trials), insulin glargine (LEAD 5 trial), and exenatide (LEAD 6 trial). The WTP survey used discrete choice experimental (DCE) methodology to evaluate the convenience and clinical effects of glucose lowering treatments. RESULTS: People with type 2 diabetes were prepared to pay an extra €2.64/day for liraglutide compared with rosiglitazone, an extra €1.94/day compared with glimepiride, an extra €3.36/day compared with insulin glargine, and an extra €0.81/day compared with exenatide. Weight loss was the largest component of WTP for liraglutide compared with rosiglitazone, glimepiride, and insulin glargine. Differences in the administration of the two drugs was the largest component of WTP for liraglutide (once daily anytime) compared with exenatide (twice daily with meals). A limitation of the study was that it was based on six clinical trials where liraglutide was the test drug, but each trial had a different comparator, therefore the clinical effects of liraglutide were much better documented than the comparators. CONCLUSIONS: WTP analyses of the clinical results from the LEAD programme suggested that participants with type 2 diabetes were willing to pay appreciably more for liraglutide than other glucose lowering treatments. This was driven by the relative advantage of weight loss compared with rosiglitazone, glimepiride, and insulin glargine, and administration frequency compared with exenatide.

Long-term cost-effectiveness of insulin detemir versus NPH insulin in type 2 diabetes in Sweden.

AIM: To evaluate the cost-effectiveness of insulin detemir vs. NPH insulin once daily, in patients with type 2 diabetes in the Swedish setting based on clinical data from a published randomized controlled trial. METHODS: Projections of long-term outcomes were made using the IMS CORE Diabetes Model (CDM), based on clinical data from a 26-week randomized controlled trial that compared once daily insulin detemir and NPH insulin, when used to intensify insulin treatment in 271 patients with type 2 diabetes and body mass index (BMI) 25-40 kg/m(2). Trial results showed that insulin detemir was associated with a significantly lower incidence of hypoglycemic events and significantly less weight gain in comparison with NPH insulin. The analysis was conducted from a third party payer perspective and the base case analysis was performed over a time horizon of 40 years and future costs and clinical outcomes were discounted at a rate of 3% per year. RESULTS: Insulin detemir was associated with higher mean (SD) quality-adjusted life expectancy (5.42 [0.10] vs. 5.31 [0.10] quality-adjusted life years [QALYs]) and lower overall costs (SEK 378,539 [10,372] vs. SEK 384,216 [11,230]; EUR 33,794 and EUR 34,300, respectively, where 1 EUR=11.2015 SEK) compared with NPH insulin. Sensitivity analysis showed that the principal driver of the benefits associated with insulin detemir was the lower rate of hypoglycemic events (major and minor events) vs. NPH insulin, suggesting that detemir might also be cost-saving over a shorter time horizon. Limitations of the analysis include the use of data from a trial outside Sweden in the Swedish setting. CONCLUSIONS: Based on clinical input data derived from a previously published randomized controlled trial, it is likely that in the Swedish setting insulin detemir would be cost-saving in comparison with NPH insulin for the treatment of patients with type 2 diabetes.

Willingness-to-pay for benefits associated with basal insulin treatment of type 2 diabetes.

Data from a 20-week trial comparing insulin detemir and neutral protamine Hagedorn (NPH) insulin in insulin-naïve people with type 2 diabetes were analyzed using willingness-to-pay (WTP) data, a proxy for patient preference. The advantages of insulin detemir relative to NPH insulin with respect to a lower hypoglycemia rate and less weight gain were associated with a value of €27.87 per month.

Willingness to pay for health improvements associated with anti-diabetes treatments for people with type 2 diabetes.

OBJECTIVES: This study aimed to investigate the most important consequences of diabetes medication, as measured by the patients' willingness to pay (WTP). RESEARCH DESIGN AND METHODS: People in Sweden were recruited using existing nationwide e-mail panels if they were adults (>or=18 years) with type 2 diabetes and were receiving pharmacological anti-diabetes treatment(s). Data were collected electronically and results were analysed using a standard statistical model designed for choice games (conditional logit). Six characteristics relating to treatment of diabetes were examined: weight (gain or loss), mean glycated haemoglobin level (HbA(1c)), hypoglycaemic events, nausea, need for injections (with or independently of meals), and blood glucose testing. RESULTS: A total of 461 people with type 2 diabetes (291 males; 170 females) completed an internet questionnaire and were eligible for inclusion. Participants placed high value on weight loss and nausea avoidance; they would pay 176 Swedish Krona (SEK)/euro15.61 per month to lose 1 kg, and would pay SEK 560 (euro49.67) per month to avoid nausea completely. Patients wanting to reduce the number of hypoglycaemic events from three per month to none were willing to pay SEK 419 (euro37.17) per month. Patients valued a 1 percentage point reduction in HbA(1c) at SEK 414 (euro36.72) per month. Participants preferred taking tablets to injections and required a compensation of SEK 376 (euro33.35) to accept one injection/day. Injections independent of meals were preferred to injections with meals (WTP: SEK 140/euro12.42 per month). Potential limitations of this study are that the preferences expressed may not match preferences in real-life situations, and bias through the use of electronic questionnaire, which restricted participation to those with access to, and experience with, the internet. CONCLUSION: People with type 2 diabetes were willing to pay a considerable amount of money each month to lose weight, reduce or avoid hypoglycaemic events and reduce HbA(1C).

MEK-inhibitor U0126 in hyperglycaemic focal ischaemic brain injury in the rat.

BACKGROUND: Hyperglycaemia aggravates ischaemic brain injury, possibly due to activation of signalling pathways involving mitogen-activated protein kinases (MAPK). In this study, the activation of MAPK/ERK was inhibited using the upstream inhibitor of MAPK-ERK-kinase (MEK) U0126, and the effects on focal brain ischaemia were evaluated during normo- and hyperglycaemia. MATERIALS AND METHODS: Temporary (90 min) middle cerebral artery occlusion (MCAO) was induced in five groups of rats. U0126 (400 microg kg(-1)) or vehicle was given as 60-min intravenous infusions starting either 30 min prior to MCAO or 30 min prior to reperfusion. The infarct size was determined by perfusion with tetrazolium red after 24 h of survival, and the neurology was tested with the 4-level scale of Bederson and performance on an inclined plane. The inhibitory effect on the targeted MEK enzyme was investigated by analysing the phosphorylation of the downstream target ERK with western immunoblotting. Two subgroups were investigated with magnetic resonance imaging (MRI), including diffusion-weighted (DWI) and perfusion-weighted imaging (PWI). RESULTS: U0126 effectively reduced the infarct size and improved neurology in hyperglycaemic rats both when given before and after ischemic onset. This effect was not accompanied by any detectable changes in cerebral blood flow on MRI. Normoglycaemic rats had generally milder injuries compared with the hyperglycaemic and there was a nonsignificant trend for U0126 to reduce damage also in the nonhyperglycaemic groups. CONCLUSIONS: In conclusion, U0126 appears to be neuroprotective in this model of hyperglycaemic ischaemic brain injury. The findings support the pathogenic importance of the MEK-ERK pathway in hyperglycaemic-ischaemic brain injury.

Fatigue and blood pressure in primary Sjogren's syndrome.

OBJECTIVE: Primary Sjogren's syndrome (SS) is an autoimmune disease characterized by fatigue. Little is known about the genesis of fatigue. Fatigue is thought to represent a multidimensional concept and it is important to be able to measure it confidently. The aims were to evaluate the reliability and validity of the 20-item Multidimensional Fatigue Inventory (MFI-20) in SS and to search for factors associated with this disabling symptom. METHODS: Forty-eight women with primary SS completed the MFI-20 questionnaire. The results were compared with age-matched women with fibromyalgia (FM) and healthy controls. Convergent construct validity was assessed by correlations to a Visual Analogue Scale (VAS) for global fatigue by Spearman's correlation (r(s)). Test-retest reliability was analysed by the intraclass correlation coefficient (ICC) in 28 women. Associations between clinical variables and subscales of the MFI-20 were analysed. RESULTS: The SS women scored significantly higher in all subscales of the MFI-20 compared to controls but similar to FM. The ICCs were satisfactory, ranging from 0.66 for general fatigue to 0.85 for the total score of MFI-20. All subscales correlated significantly to VAS for global fatigue, general fatigue showing the highest correlation (r(s) = 0.70). The estimated number of hours of sleep/day was significantly associated with many of the fatigue dimensions. All five subscales of the MFI-20 were inversely associated with diastolic blood pressure (BP) and two with systolic BP. CONCLUSIONS: The MFI-20 was found to be a reliable and valid tool for the measurement of fatigue in primary SS. High levels of fatigue were correlated with low BP, suggesting an associated involvement of the autonomic nervous system.

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization.

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX(2)CX(4)HX(4)C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.

Gestational and hormonal regulation of human placental lipoprotein lipase.

The fetal demand for FFA increases as gestation proceeds, and LPL represents one potential mechanism for increasing placental lipid transport. We examined LPL activity and protein expression in first trimester and term human placenta. The LPL activity was 3-fold higher in term (n = 7; P < 0.05) compared with first trimester (n = 6) placentas. The LPL expression appeared lower in microvillous membrane from first trimester (n = 2) compared with term (n = 2) placentas. We incubated isolated placental villous fragments with a variety of effectors [GW 1929, estradiol, insulin, cortisol, epinephrine, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha] for 1, 3, and 24 h to investigate potential regulatory mechanisms. Decreased LPL activity was observed after 24 h of incubation with estradiol (1 micro g/ml), insulin, cortisol, and IGF-1 (n = 12; P < 0.05). We observed an increase in LPL activity after 3 h of incubation with estradiol (20 ng/ml) or hyperglycemic medium plus insulin (n = 7; P < 0.05). To conclude, we suggest that the gestational increase in placental LPL activity represents an important mechanism to enhance placental FFA transport in late pregnancy. Hormonal regulation of placental LPL activity by insulin, cortisol, IGF-1, and estradiol may be involved in gestational changes and in alterations in LPL activity in pregnancies complicated by altered fetal growth.

Placental transport and metabolism in fetal overgrowth -- a workshop report.

Fetal overgrowth in pregnancies complicated by diabetes is the result of an increased substrate availability which stimulates fetal insulin secretion and fetal growth. However, despite strict glycemic control in modern clinical management of the pregnant woman with diabetes, fetal overgrowth remains an important clinical problem. Recent studies in vivo provide evidence for increased delivery of amino acids to the fetus in gestational diabetes (GDM) even when metabolic control is strict. This could be due to that truly normal maternal substrate levels cannot be achieved in diabetic pregnancies and/or caused by altered placental nutrient transport and metabolism. Studies in vitro demonstrate an up-regulation of placental transport systems for certain amino acids in GDM associated with fetal overgrowth. GDM is also characterized by changes in placental gene expression, including up-regulation of inflammatory mediators and Leptin. In type-I diabetes with fetal overgrowth the in vitro activity of placental transporters for both glucose and certain amino acids as well as placental lipoprotein lipase is increased. Furthermore, both clinical observations in type-I diabetic pregnancies and preliminary animal experimental studies suggest that even brief periods of metabolic perturbation early in pregnancy may affect placental growth and transport function for the remainder of pregnancy, thereby contributing to fetal overgrowth. Ultrasound measurements of fetal fat deposits and abdominal circumference as well as 3D ultrasound assessment of placental volume represent non-invasive techniques for in utero diagnosis of fetal and placental overgrowth. It is proposed that these methods represent valuable additions to the clinical management of the diabetic pregnancy. In conclusion, altered placental function may be a mechanism contributing to fetal overgrowth in diabetic pregnancies with apparent optimal metabolic control. It is proposed that detailed information on placental metabolism and transport functions obtained in vitro and in vivo represent a placental phenotype that provides important information and may facilitate diagnosis and improve clinical management of fetal overgrowth.

TcZFP8, a novel member of the Trypanosoma cruzi CCHC zinc finger protein family with nuclear localization

In a 17-kb genomic fragment of Trypanosoma cruzi chromosome XX, we identified three tandemly linked genes coding for CX2CX4HX4C zinc finger proteins. We also showed that similar genes are present in T. brucei and Leishmania major, sharing three monophyletic groups among these trypanosomatids. In T. cruzi, TcZFP8 corresponds to a novel gene coding for a protein containing eight zinc finger motifs. Molecular cloning of this gene and heterologous expression as a fusion with a His-tag were performed in Escherichia coli. The purified recombinant protein was used to produce antibody in rabbits. Using Western blot analysis, we observed the presence of this protein in all three forms of the parasite: amastigote, trypomastigote and epimastigote. An analysis of cytoplasmic and nuclear cell extracts showed that this protein is present in nuclear extracts, and indirect immunofluorescence microscopy confirmed the nuclear localization of TcZFP8. Homologues of TcZFP8 in T. brucei are apparently absent, while one candidate in L. major was identified.

Glucose transporter isoform 4 is expressed in the syncytiotrophoblast of first trimester human placenta.

BACKGROUND: Placental glucose transport mechanisms in early pregnancy are poorly understood. The aims of this study were to investigate the expression of glucose transporter (GLUT) isoforms 1, 3 and 4 in first trimester villous tissue, to assess the effects of insulin on glucose uptake and compare them with term. METHODS: The expression of GLUT isoforms was investigated using immunohistochemistry, Western blot and reverse transcription (RT)-PCR in trophoblast tissue from terminations at 6-13 weeks gestation and term. The effects of insulin (300 ng/ml, 1 h) on glucose uptake were studied in villous fragments. RESULTS: In the first trimester, GLUT1 and GLUT3 were present in the microvillous membrane and the cytotrophoblast, and GLUT4 in perinuclear membranes in the cytosol of the syncytiotrophoblast (ST). GLUT4 protein (48 kDa) and mRNA were identified in trophoblast homogenates. Whereas GLUT1 was expressed abundantly in term placenta, the expression of GLUT3 and 4 was markedly lower at term compared with first trimester. Insulin increased glucose uptake by 182% (n=6, P<0.05) in first trimester fragments, but not in term fragments. CONCLUSIONS: The insulin-regulatable GLUT4 is expressed in the cytosol of first trimester ST compatible with a role for GLUT4 in placental glucose transport in early pregnancy. The placental expression pattern of GLUT isoforms in early pregnancy is distinct from that later in pregnancy.

Src family kinase-inhibitor PP2 reduces focal ischemic brain injury.

OBJECTIVES: To investigate the neuroprotective potential of the Src family kinase (SFK) inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine (PP2) in transient focal cerebral ischemia in the rat. MATERIAL AND METHODS: Sprague-Dawley rats were exposed to transient (90 min) middle cerebral artery occlusion (MCAO) and evaluated after 1 day of survival. PP2 (1.5 mg/kg i.p.) or vehicle was given 30 min after MCAO. The lesions were examined with magnetic resonance imaging (MRI), tri-phenyl tetrazolium chloride (TTC) staining and the functional outcome was determined using neurological scoring according to Bederson et al. RESULTS: PP2-treated rats showed approximately 50% reduction of infarct size on T2-weighted MRI and in TTC staining compared with controls (P < 0.05). Moreover, the neurological score was better in the PP2 group than controls (P < 0.05). CONCLUSION: PP2 is a potential neuroprotective agent in cerebral ischemia-reperfusion. The interference of PP2 with SFKs and/or other pathways remains to be elucidated.

Increased brain injury and vascular leakage after pretreatment with p38-inhibitor SB203580 in transient ischemia.

OBJECTIVES: Focal cerebral ischemia activates intracellular signaling pathways including the mitogen-activated protein kinase p38, which may be involved in the process of ischemic brain injury. In this study, the effect of pretreatment with the p38-inhibitor SB203580 on infarct size and blood-brain barrier (BBB) breakdown was investigated with magnetic resonance imaging (MRI). MATERIALS AND METHODS: Rats were given SB203580 (n = 6) or vehicle (n = 6) in the right lateral ventricle prior to transient (90 min) middle cerebral artery occlusion (MCAO) on the left side. The rats were examined with serial MRI during MCAO, at reperfusion and after 1 and 4 days. RESULTS: The mean infarct size on T2-weighted images after 1 day was significantly higher in the SB203580-treated group than in controls (300 +/- 95 mm3 vs 126 +/- 75 mm3; P < 0.01). Vascular gadolinium leakage, indicating BBB breakdown, was significantly larger in the SB203580-treated group than in controls after 1 day (median leakage score 18.5; range 15-21 vs 6.5; 4-17; P < 0.05) and 4 days (11; 6-15 vs 3.5; 1-9; P < 0.05), although no significant difference was seen initially. CONCLUSION: Pretreatment with SB203580 may aggravate ischemic brain injury and cerebral vascular leakage in the present model of transient ischemia.

Hospitalization in Sweden of children born to immigrants.

AIM: To study whether excess hospitalization occurs among certain groups of children born in Sweden to immigrant parents. METHODS: The study was based on linkage of the Swedish Medical Birth Register 1987-1997 and the Swedish Hospital Discharge Register 1987-1998. RESULTS: Among children whose parents were of Swedish nationality excess hospitalization was found for children of young mothers, parity 3 or more, and if the mother smoked in early pregnancy. These factors were controlled for in the further analysis. Ten years after birth, a large percentage of children born to parents with foreign nationality could not be followed owing to death or emigration (19.9% vs 1.77% of children with Swedish parents). An excess hospitalization of immigrant children up to the age of about 5 y was seen but after that, if anything, these children were hospitalized less. For the whole group of children born to parents of non-Swedish nationality there was no difference in hospitalization rate after stratification for risk factors and age. When analysis was performed for specific nationalities significantly reduced rates were found for several western European countries while significantly increased rates were seen for the Middle East, north Africa and especially sub-Saharan Africa (odds ratio 1.57, 95% confidence interval 1.49-1.64). CONCLUSION: A moderately increased rate of child hospitalization was only observed for some selected immigrant groups.