Diseases in neonatal foals. Part 2: potential risk factors for a higher incidence of infectious diseases during the first 30 days post partum

REASON FOR PERFORMING STUDY: The development of clinical illness in foals is usually predetermined by perinatal history, management or stressful environmental conditions. OBJECTIVES: To determine potential risk factors for an increased incidence of infectious diseases during the first 30 days post partum. METHODS: The population consisted of Thoroughbred foals born on stud farms in the Newmarket (UK) area in 2005 (n = 1031). They were followed for their first 30 days. Factors suspected to influence the incidence of infectious neonatal diseases were examined in a logistic regression approach for each of the 3 outcomes (total infectious diseases, systemic disease with diarrhoea and total infectious diseases excluding diarrhoea). All 28 factors were either foal or mare or stud farm related. RESULTS: Several significant risk factors for a higher disease incidence, such as birth complications, colostrum intake by stomach tube and leucocytosis 12-48 h post partum were identified. The factor 'boarding stud' seemed to be protective against disease. CONCLUSION: Some factors, such as the mare's time at stud before foaling, the mare's rotavirus vaccination schedule and fibrinogen-values that empirically had been linked to the outcome previously were not confirmed as relevant. This included the reported useful prophylactic treatment with antimicrobial drugs. POTENTIAL RELEVANCE: Factors to be considered when evaluating newborn foals include: stud management, the birth process, route of colostrum intake, white and red blood cells, and the date of birth. These may help to detect foals at risk to develop an infection so that targeted prophylactic measures can be initiated.

Functional evaluation of hTM, CD46 and HLA-E transgenes in vitro and in pig leg xenoperfusion experiments

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, and C6 deposition as well as E-selectin and VCAM-1 expression were significantly (P < 0.0001) higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose-dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three- to four-fold increased (P < 0.001) clotting time as compared to wild-type PAEC. The formation of TAT complexes was significantly lower when hTM-transgenic cells (P < 0.0001) were used as compared to wild-type cells. Conclusions Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK-cell tissue infiltration were all down-regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig-to-human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig-to-human xenotransplantation.

Clinical parameters, intestinal function, and IGF1 concentrations in colostrum-deprived and colostrum-fed newborn pony foals.

Colostrum (COL) contains cytokines and growth factors that may enhance intestinal development in neonates. The hypothesis of this study was that besides providing immunoglobulins, COL is important for intestinal function and meconium release in foals. Newborn foals were either fed COL (n = 5) or an equal amount of milk replacer (MR, n = 7) during the first 24 hours of life. To ensure passive immunity, all foals received 1 L plasma. Postnatal development, meconium release, intestinal motility, white blood cell count, insulin-like growth factor 1, and intestinal absorptive function (xylose absorption test) were evaluated. Clinical findings and meconium release were not affected by feeding of COL or MR. Ultrasonography revealed a slightly larger jejunum and stomach in group COL versus MR (P < 0.05). The percentage of polymorphonuclear leucocytes was higher in foals of group MR versus group COL (P < 0.05) and the percentage of lymphocytes was lower in MR compared with COL foals (P < 0.05). Plasma insulin-like growth factor 1 concentration increased during the first 14 days after birth in both groups. A xylose absorption test on Day 5 revealed similar increases in plasma xylose concentrations after oral intake. In conclusion, feeding of COL versus MR was without effect on meconium release and intestinal absorptive function. Differences between foals fed COL and MR with regard to intestinal function are apparently without clinical relevance. In foals that have not received maternal COL, there is no major risk of intestinal problems if they are fed MR and provided with immunoglobulins by transfusion of plasma.

Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene.

Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8 x 10(-5)), establishing a novel phenotype for this genetic variant.

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Phenotype of capillaries in skeletal muscle of nNOS-knockout mice

Because neuronal nitric oxide synthase (nNOS) has a well-known impact on arteriolar blood flow in skeletal muscle, we compared the ultrastructure and the hemodynamics of/in the ensuing capillaries in the extensor digitorum longus (EDL) muscle of male nNOS-knockout (KO) mice and wild-type (WT) littermates. The capillary-to-fiber (C/F) ratio (-9.1%) was lower (P ≤ 0.05) in the nNOS-KO mice than in the WT mice, whereas the mean cross-sectional fiber area (-7.8%) and the capillary density (-3.1%) varied only nonsignificantly (P > 0.05). Morphometrical estimation of the area occupied by the capillaries as well as the volume and surface densities of the subcellular compartments differed nonsignificantly (P > 0.05) between the two strains. Intravital microscopy revealed neither the capillary diameter (+3% in nNOS-KO mice vs. WT mice) nor the mean velocity of red blood cells in EDL muscle (+25% in nNOS-KO mice vs. WT mice) to significantly vary (P > 0.05) between the two strains. The calculated shear stress in the capillaries was likewise nonsignificantly different (3.8 ± 2.2 dyn/cm² in nNOS-KO mice and 2.1 ± 2.2 dyn/cm² in WT mice; P > 0.05). The mRNA levels of vascular endothelial growth factor (VEGF)-A were lower in the EDL muscle of nNOS-KO mice than in the WT littermates (-37%; P ≤ 0.05), whereas mRNA levels of VEGF receptor-2 (VEGFR-2) (-11%), hypoxia inducible factor-1α (+9%), fibroblast growth factor-2 (-14%), and thrombospondin-1 (-10%) differed nonsignificantly (P > 0.05). Our findings support the contention that VEGF-A mRNA expression and C/F-ratio but not the ultrastructure or the hemodynamics of/in capillaries in skeletal muscle at basal conditions depend on the expression of nNOS.

A case of EDTA-dependent pseudothrombocytopenia: simple recognition of an underdiagnosed and misleading phenomenon

1. BMC Clin Pathol. 2014 May 1;14:19. doi: 10.1186/1472-6890-14-19. eCollection 2014. A case of EDTA-dependent pseudothrombocytopenia: simple recognition of an underdiagnosed and misleading phenomenon. Nagler M, Keller P, Siegrist D, Alberio L. Author information: Department of Hematology and Central Hematology Laboratory, Inselspital University Hospital and University of Berne, CH-3010 Berne, Switzerland. BACKGROUND: EDTA-dependent pseudothrombocytopenia (EDTA-PTCP) is a common laboratory phenomenon with a prevalence ranging from 0.1-2% in hospitalized patients to 15-17% in outpatients evaluated for isolated thrombocytopenia. Despite its harmlessness, EDTA-PTCP frequently leads to time-consuming, costly and even invasive diagnostic investigations. EDTA-PTCP is often overlooked because blood smears are not evaluated visually in routine practice and histograms as well as warning flags of hematology analyzers are not interpreted correctly. Nonetheless, EDTA-PTCP may be diagnosed easily even by general practitioners without any experiences in blood film examinations. This is the first report illustrating the typical patterns of a platelet (PLT) and white blood cell (WBC) histograms of hematology analyzers. CASE PRESENTATION: A 37-year-old female patient of Caucasian origin was referred with suspected acute leukemia and the crew of the emergency unit arranged extensive investigations for work-up. However, examination of EDTA blood sample revealed atypical lymphocytes and an isolated thrombocytopenia together with typical patterns of WBC and PLT histograms: a serrated curve of the platelet histogram and a peculiar peak on the left side of the WBC histogram. EDTA-PTCP was confirmed by a normal platelet count when examining citrated blood. CONCLUSION: Awareness of typical PLT and WBC patterns may alert to the presence of EDTA-PTCP in routine laboratory practice helping to avoid unnecessary investigations and over-treatment. PMCID: PMC4012027 PMID: 24808761 [PubMed]

Resistance prediction in AML: analysis of 4601 patients from MRC/NCRI, HOVON/SAKK, SWOG and MD Anderson Cancer Center

Therapeutic resistance remains the principal problem in acute myeloid leukemia (AML). We used area under receiver-operating characteristic curves (AUCs) to quantify our ability to predict therapeutic resistance in individual patients, where AUC=1.0 denotes perfect prediction and AUC=0.5 denotes a coin flip, using data from 4601 patients with newly diagnosed AML given induction therapy with 3+7 or more intense standard regimens in UK Medical Research Council/National Cancer Research Institute, Dutch–Belgian Cooperative Trial Group for Hematology/Oncology/Swiss Group for Clinical Cancer Research, US cooperative group SWOG and MD Anderson Cancer Center studies. Age, performance status, white blood cell count, secondary disease, cytogenetic risk and FLT3-ITD/NPM1 mutation status were each independently associated with failure to achieve complete remission despite no early death (‘primary refractoriness’). However, the AUC of a bootstrap-corrected multivariable model predicting this outcome was only 0.78, indicating only fair predictive ability. Removal of FLT3-ITD and NPM1 information only slightly decreased the AUC (0.76). Prediction of resistance, defined as primary refractoriness or short relapse-free survival, was even more difficult. Our limited ability to forecast resistance based on routinely available pretreatment covariates provides a rationale for continued randomization between standard and new therapies and supports further examination of genetic and posttreatment data to optimize resistance prediction in AML.

Infection rate of emergency bolt-kit vs. non-emergency conventional implanted silver bearing external ventricular drainage catheters.

BACKGROUND Bolt-kit systems are increasingly used as an alternative to conventional external cerebrospinal fluid (CSF) drainage systems. Since 2009 we regularly utilize bolt-kit external ventricular drainage (EVD) systems with silver-bearing catheters inserted manually with a hand drill and skull screws for emergency ventriculostomy. For non-emergency situations, we use conventional ventriculostomy with subcutaneous tunneled silver-bearing catheters, performed in the operating room with a pneumatic drill. This retrospective analysis compared the two techniques in terms of infection rates. METHODS 152 patients (aged 17-85 years, mean=55.4 years) were included in the final analysis; 95 received bolt-kit silver-bearing catheters and 57 received conventionally implanted silver-bearing catheters. The primary endpoint combined infection parameters: occurrence of positive CSF culture, colonization of catheter tips, or elevated CSF white blood cell counts (>4/μl). Secondary outcome parameters were presence of microorganisms in CSF or on catheter tips. Incidence of increased CSF cell counts and number of patients with catheter malposition were also compared. RESULTS The primary outcome, defined as analysis of combined infection parameters (occurrence of either positive CSF culture, colonization of the catheter tips or raised CSF white blood cell counts >4/μl)was not significantly different between the groups (58.9% bolt-kit group vs. 63.2% conventionally implanted group, p=0.61, chi-square-test). The bolt-kit group was non-inferior and not superior to the conventional group (relative risk reduction of 6.7%; 90% confidence interval: -19.9% to 25.6%). Secondary outcomes showed no statistically significant difference in the incidence of microorganisms in CSF (2.1% bolt-kit vs. 5.3% conventionally implanted; p=0.30; chi-square-test). CONCLUSIONS This analysis indicates that silver-bearing EVD catheters implanted with a bolt-kit system outside the operating room do not significantly elevate the risk of CSF infection as compared to conventional implant methods.

Clinical and haematological predictors of antibiotic prescribing for acute cough in adults in Swiss practices - an observational study.

BackgroundAcute cough is a common problem in general practice and is often caused by a self-limiting, viral infection. Nonetheless, antibiotics are often prescribed in this situation, which may lead to unnecessary side effects and, even worse, the development of antibiotic resistant microorganisms worldwide. This study assessed the role of point-of-care C-reactive protein (CRP) testing and other predictors of antibiotic prescription in patients who present with acute cough in general practice.MethodsPatient characteristics, symptoms, signs, and laboratory and X-ray findings from 348 patients presenting to 39 general practitioners with acute cough, as well as the GPs themselves, were recorded by fourth-year medical students during their three-week clerkships in general practice. Patient and clinician characteristics of those prescribed and not-prescribed antibiotics were compared using a mixed-effects model.ResultsOf 315 patients included in the study, 22% were prescribed antibiotics. The two groups of patients, those prescribed antibiotics and those treated symptomatically, differed significantly in age, demand for antibiotics, days of cough, rhinitis, lung auscultation, haemoglobin level, white blood cell count, CRP level and the GP¿s license to self-dispense antibiotics. After regression analysis, only the CRP level, the white blood cell count and the duration of the symptoms were statistically significant predictors of antibiotic prescription.ConclusionsThe antibiotic prescription rate of 22% in adult patients with acute cough in the Swiss primary care setting is low compared to other countries. GPs appear to use point-of-care CRP testing in addition to the duration of clinical symptoms to help them decide whether or not to prescribe antibiotics.

Le purpura thrombotique thrombocytopénique - un diagtnostic méconnu

Le purpura thrombotique thrombocytopénique (PTT) est un diagnostic caractérisé par une hémolyse micro-angiopathique, se traduisant par la présence d’une thrombocytopénie et d’une schizocytose au frottis sanguin. Une déficience de l’enzyme ADAMTS13, enzyme protéolytique du facteur de von Willebrand (vWF), a été caractérisée comme cause pathogénique. L’importance de l’examen visuel du frottis sanguin dans le cadre d’une suspicion clinique ou hématologique d’un PTT est soulignée car il semble que le PTT soit sous-diagnostiqué, surtout parmi les enfants et jeunes adultes. Des superpositions avec le syndrome hémolytique et urémique associé aux diarrhées (SHU D+) et le syndrome hémolytique et urémique atypique (SHUa) sont discutées. Une revue actuelle des démarches diagnostiques, des options thérapeutiques et des facteurs pronostiques du PTT et des SHU est finalement proposée.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

Induction of autophagy is a key component of all-trans-retinoic acid-induced differentiation in leukemia cells and a potential target for pharmacologic modulation

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has achieved success only in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In the study described here, we investigated the functional importance of autophagy in APL cell differentiation. We found that autophagy is increased during all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of the APL cell line NB4 and that this is associated with increased expression of LC3II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we found that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.

An iron stable isotope comparison between human erythrocytes and plasma

We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The 56Fe/54Fe ratio in erythrocytes, expressed as permil difference from the “IRMM-014” iron reference standard (δ56/54Fe), ranges from −3.1‰ to −2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ56/54Fe values measured in two different laboratories range from −3.0‰ to −2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ56/54Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.