Analysis of the Status of the Cutaneous Endogenous and Exogenous Antioxidative System of Smokers and the Short-Term Effect of Defined Smoking Thereon.

The daily consumption of tobacco products leads to a boost in free radical production in tissues, promoting the risk for malignancies, metabolic alterations and chronic-inflammatory diseases. This study aimed to broaden the knowledge of the status of the antioxidative (AO) system in the skin, compared to the blood, of healthy appearing smokers. Both, the basic status compared to non-smokers and the short-term impact of controlled cigarette consumption in smokers were analyzed. Our study showed that the basic level of the AO system of smokers significantly differed from that of non-smokers. As determined by resonant Raman spectroscopy (RRS), the levels of exogenous AOs were decreased in both, the skin, in vivo (ß-carotene and lycopene), and blood plasma (ß-carotene only). In contrast, the levels of glutathione (GSH), the prototypical endogenous AO, which were analyzed by fluorimetric assays in cutaneous tape strips and blood plasma, were increased in the skin, although unchanged in the blood of smokers. Elevated cutaneous GSH levels were reflected by an elevated overall radical scavenging activity in the skin, as quantified by non-invasive electron paramagnetic resonance (EPR) spectroscopy. Analysis of the expression of selected stress-associated genes in blood immune cells by quantitative RT-PCR in subgroups of non-smokers and smokers additionally demonstrated the downregulation of AKR1C2 in smokers, and its negative correlation with blood plasma levels of the protective immune mediator interleukin-22, assessed by the ELISA technique. Controlled cigarette consumption did not alter exogenous or endogenous AOs in the skin of smokers, but decreased lycopene levels in blood plasma. Moreover, there was a decline in blood IL-22 levels, while no relevant response of blood cell gene expressions was found after the considered short time. Our data therefore demonstrate a strengthened endogenous AO status in the skin of smokers, which may indicate a long-term adaptation to chronic oxidative stress in this specific organ. While this effect was not clearly visible in the blood, this compartment seems to be useful as an immediate indicator of the body's AO consumption. Moreover, decreased levels of AKR1C2, which we show for the first time to be expressed in immune cells, may be a candidate marker for long-term smoking. In addition, this study demonstrates that the rate constant of a spin probe decline determined by EPR spectroscopy mainly represents the endogenous AO status of a tissue.

Spatial distribution and incidence of bovine neonatal pancytopenia in Bavaria, Germany.

BACKGROUND: Bovine neonatal pancytopenia (BNP) is a haemorrhagic disease of neonatal calves. BNP was first described in Germany in 2009, later on also in other European countries, and in New Zealand in 2011. The disease is characterised by spontaneous bleeding, pancytopaenia in the bone marrow, and a high case fatality ratio. The causal role of a specific bovine viral diarrhoea virus (BVDV) vaccine (PregSure®BVD, then Pfizer Animal Health, now Zoetis, Berlin, Germany) has been established over the last years, causing the production of alloantibodies in some vaccinated cattle, which in the case of pregnant cattle, are transferred to the newborn calf via the colostrum. However, striking regional differences in the incidence of the disease were observed within Germany and other countries, but as the disease was not notifiable, no representative data on the spatial distribution are available. In this study, we address the spatial distribution and incidence of BNP using the results of two representative surveys amongst cattle practitioners in Bavaria, Germany. The surveys, asking about the occurrence of BNP, were conducted in 2009 and 2010. Answers were analysed spatially by testing for clusters using space-time models. Practitioners were also asked how many cows they serve in their practice and this number was used to estimate the incidence of BNP. Furthermore, in the survey of 2010, practitioners were also asked about usage of vaccine against BVDV. RESULTS: From the results of the surveys, three clusters were identified in Bavaria. These clusters also coincided with the usage of the specific BVDV vaccine as indicated by the veterinary practices. Furthermore, the representative surveys allow the estimation of the incidence of BNP to be in the order of 4 cases per 10,000 calves at risk. CONCLUSIONS: The study is the only representative survey conducted on BNP. Despite the fact that BNP is a non-infectious disease, regional clusters were identified.

Diagnosing bovine parafilariosis: utility of the cytochrome c oxidase subunit 1 gene and internal transcribed spacer region for PCR detection of Parafilaria bovicola in skin biopsies and serohemorrhagic exudates of cattle.

BACKGROUND: Parafilaria bovicola (Nematoda: Filariidae) causes cutaneous bleedings in bovine species. Flies serve as intermediate hosts. In recent years, reports on bovine parafilariosis have become more frequent, corroborating the necessity of reliable diagnostic interventions especially since no molecular or serological test has been available. We aimed to establish a polymerase chain reaction assay to detect DNA of P. bovicola in flies, skin biopsies and serohemorraghic exudates of bleeding spots. METHODS: PCRs targeting the cytochrome c oxidase subunit 1 (cox1) gene and the internal transcribed spacer region (ITS) of the ribosomal RNA gene cluster were evaluated for their diagnostic sensitivity as well as performance and specificity on biopsy and serohemorrhagic exudate samples from P. bovicola-infected cattle. RESULTS: Using serohemorrhagic exudates (n = 6), biopsies (n = 2) and flies (n = 1), the PCR targeting the cox1 gene resulted in a gel band of almost 700 bp. Cloning, sequencing, and removal of primer sequences yielded a 649-bp fragment of the P. bovicola cox1 gene. The PCR targeting the ITS region showed a band of about 1100 bp. Cloning, sequencing, and removal of primer sequences resulted in a 1083 bp stretch of the P. bovicola ITS region. Testing samples from presumably affected animals, the cox1-PCR resulted in bands with the expected size and they were all confirmed as P. bovicola by sequencing. In contrast, the ITS-PCR proved to be less sensitive and less specific and additionally amplified the ITS region of Musca domestica or buttercup DNA. When analysing for sensitivity, the cox1-PCR yielded visible bands up to 2 ng of genomic DNA, whereas the ITS-PCR produced bands up to 3 ng. In a plasmid dilution series, the minimum number of target DNA copies was 102 for the cox1-PCR and 101 in the ITS-PCR. CONCLUSIONS: The evaluated cox1-PCR enables reliable detection of P. bovicola DNA in skin biopsies and serohemorrhagic exudates. This PCR and, to a limited extent, the ITS-PCR, may help evaluate different therapeutic approaches. Furthermore, the cox1-PCR may be useful for epidemiological studies on the geographical distribution of P. bovicola. Further understanding of the epidemiology of this parasite will help develop and implement effective control strategies.

Risk-adjusted intraventricular hemorrhage rates in very premature infants: towards quality assurance between neonatal units.

BACKGROUND: The incidence of intraventricular hemorrhage (IVH) in very low birth weight infants can be used as an index of the quality of care in neonatal intensive care units as long as it is adjusted to reflect the infants' risk profiles on admission to the unit, which may vary systematically from one institution to another. Adjustment for gestational, birth-related, and neonatological risk factors enables a fair comparison of IVH rates across neonatal intensive care units. METHODS: Data on 1782 neonates born at less than 32 weeks of gestation or weighing less than 1500 g at birth were retrieved from the 26 744 anonymous data sets collected in the Peri- and Neonatal Survey of the German state of Saxony in the years 2001-2005. An analysis of 30 putative risk factors with stepwise logistic regression analysis enabled the construction of a specific risk predictor for severe (grade 3-4) IVH. Risk-adjusted institutional incidence rates were then calculated. RESULTS: Five independent risk factors (low gestational age, low Apgar scores at 1 min, early infection, absence of pathological Doppler findings during pregnancy, and the use of tocolytic agents) were found to be relevant to the prediction of IVH. A risk predictor incorporating them was found to have a correct prediction rate (ROC(AUC) value) of 87.7%. The crude incidence of severe IVH in different institutions ranged from 1.92% to 15.02% (mean, 8.55%); after adjustment, the range was 5.14% to 11.58%. When the institutions studied were ranked in order of their incidence of IVH before and after adjustment for risk factors, individual institutions rose or fell by as many as 4 places in the ranking because of the adjustment. CONCLUSION: These findings reveal the importance of adjusting the incidence of IVH in very low birth weight infants by the patients' risk profiles to enable valid comparisons between institutions for the purpose of quality surveillance.

Immunophenotyping and characterization of BNP colostra revealed pathogenic alloantibodies of IgG1 subclass with specifity to platelets, granulocytes and monocytes of all maturation stages.

Bovine neonatal pancytopenia (BNP) is mainly characterized by multiple haemorrhages, thrombocytopenia and leukocytopenia as a result of bone marrow depletion. BNP can be induced in healthy calves through application of colostrum from BNP donors, proofing that BNP is mediated to maternal alloantibodies. Alloantibody binding to bovine blood cells is present in sera and colostra of BNP donors and is probably initialized by vaccination with a certain BVD vaccine. To understand etiology and pathomechanisms of BNP, we closely characterized disease inducing antibodies regarding immunoglobulin subclass and binding specificities to peripheral blood derived leukocytes and platelets. By exact phenotyping the targeted blood cell subsets, including platelets for the first time, we investigated that BNP alloantibodies are exclusively of IgG1 subclass. Interestingly, IgG1 of BNP colostra bound to 70% leukocytes and 100% platelets irrespective of different bovine breeds and cellular maturity of all specimens tested. Furthermore, staining pattern on platelets as well as leukocyte subsets by BNP-IgG1 alloantibody exposed 100% reactivity to platelets, granulocytes and monocytes. Interestingly, the main part of T-helper cells was not bound by colostral alloantibodies. Our results point to a crucial role of IgG1 antibodies in BNP and to a target antigen that is expressed by all cells of myeloid lineage, but only partially by the lymphoid lineage.

Case control study to investigate risk factors for bovine neonatal pancytopenia (BNP) in young calves in southern Germany.

A case control study on farm level was conducted at the Clinic for Ruminants, LMU Munich, to identify possible risk factors associated with the observed increase in numbers of calves showing clinical signs of Bovine Neonatal Pancytopenia (BNP) since 2006 in southern Germany. Interviews were conducted between August 2008 and June 2010. The characteristics of 56 dairy farms with at least one confirmed case of BNP (thrombocytopenia and leukocytopenia and/or typical findings in post-mortem examination and bone marrow histology) were compared with those of two sets of 50 control dairy farms each, with no history of BNP. The first set of 50 control farms was selected randomly from veterinary practices which had never observed a BNP case on the farms they serviced. The second set of 50 control farms was matched by the veterinary practices which had provided case farms. Two separate analyses were conducted: (1) case farms (n=56) vs. randomly selected control farms (n=50) and (2) case farms (n=56) vs. a matched set of control farms (n=50). All variables with p<0.2 in the univariable analysis were included in stepwise logistic regression models. In the first analysis, only the use of PregSure(®) BVD vaccine was positively associated with BNP with an odds ratio of 1292 (95% CI: 114-14707). In the second analysis, conditional logistic regression models did not converge, therefore non-conditional logistic regression models were conducted. In the non-conditional analysis five variables remained in the model, three of which were negatively associated with BNP: the use of vitamin E and selenium, the frequent use of mastitis tubes, and the use of stem growth regulators in grain production. The use of prophylactic measures (such as control of parasites or vaccination of calves against respiratory disease) was positively associated with BNP with an odds ratio of 14.3 as well as the use of PregSure(®) BVD vaccine with an odds ratio of 426 (95% CI: 20-9095).

[Risk-adjusted assessment: late-onset infection in neonates]. / Risikoadjustierte Qualitätsbeurteilung am Beispiel der neonatalen Spätinfektion.

OBJECTIVE: The weak point of the countrywide perinatal/neonatal quality surveillance is the ignorance of interhospital differences in the case mix of patients. As a result, this approach does not produce reliable benchmarking. The objective of this study was to adjust the result of the late-onset infection incidence of different hospitals according to their risk profile of patients by multivariate analysis. METHOD: The perinatal/neonatal database of 41,055 newborns of the Saxonian quality surveillance from 1998 to 2004 was analysed. Based on 18 possible risk factors, a logistic regression model was used to develop a specific risk predictor for the quality indicator "late-onset infection". RESULTS: The developed risk predictor for the incidence of late-onset infection could be described by 4 of the 18 analysed risk factors, namely gestational age, admission from home, hypoxic ischemic encephalopathy and B-streptococcal infection. The AUC(ROC) value of this quality indicator was 83.3%, which demonstrates its reliability. The hospital ranking based on the adjusted risk assessment was very different from hospital rankings before this adjustment. The average correction of ranking position was 4.96 for 35 clinics. CONCLUSION: The application of the risk adjustment method proposed here allows for a more objective comparison of the incidence of the quality indicator "late onset infection" among different hospitals.

[Risk-adjusted assessment of neonatology wards by the new quality indicator "transfer rate of mature newborns"]. / Risikoadjustierte Qualitätsbeurteilung neonatologischer Einrichtungen an Hand des neuen Qualitätskriteriums "Verlegerate von reifen Neugeborenen".

OBJECTIVE: 1. The transfer rate of mature newborns will be presented as a new quality indicator. 2. Another objective of this study was to adjust the transfer rate of mature newborns of different hospitals according to their "risk" profile of patients by multivariate analysis. METHOD: The perinatal database of 118,416 newborns of the Saxonian quality surveillance from 2001 to 2004 was analysed. Based on 17 clinical and 3 structural factors, a logistic regression model was used to develop a specific "risk" predictor for the quality indicator "transfer rate". RESULTS: For care level III (basic care) a "risk" predictor for the transfer rate was developed, which consists of 15 factors. The AUC(ROC)-value of this quality indicator was 78.6%, which is sufficient. The hospital ranking based on the adjusted risk assessment was different from the hospital ranking prior to this adjustment. The average correction of ranking position was 10.4 for 43 clinics. CONCLUSION: 1. The new quality indicator "transfer rate of mature newborns" can be recommended. 2. The application of the risk adjustment method proposed here allows for a more objective comparison of the quality indicator "transfer rate" among different hospitals.

Ingestion of colostrum from specific cows induces Bovine Neonatal Pancytopenia (BNP) in some calves.

BACKGROUND: Since 2006, cases of haemorrhagic diathesis in young calves have been observed with a much higher incidence than previously known. The syndrome, now uniformly called Bovine Neonatal Pancytopenia (BNP), is characterized by multiple (external and internal) haemorrhages, thrombocytopenia, leukocytopenia, and bone marrow depletion. Although various infectious and toxicological causes of bleeding disorders in calves have been ruled out, the aetiology of BNP remains unknown. However, field observations have led to the hypothesis that the aetiological principle may be transmitted to calves via colostrum.The objective of the present study was to verify whether ingestion of colostrum from dams of known BNP calves can elicit signs of BNP and typical haematological findings in conveniently selected neonatal calves. Six such calves received one feeding of colostrum (or a mixture of colostrum batches) from dams of known BNP calves. As controls, another six conveniently selected calves from herds which had never had a BNP case received one feeding of colostrum from their own dams. Haematological and clinical parameters were monitored. RESULTS: One of the six experimental calves never showed any haematological, clinical or pathological evidence of BNP. In the other five calves, thrombocyte and leukocyte counts dropped within a few hours following ingestion of colostrum. Of those, three calves developed clinical signs of BNP, their post-mortem examination revealed bone marrow depletion. Of the remaining two calves, a pair of mixed twins, marked thrombocytopenia and recurrent leukocytopenia was evident in one, in which only slight changes in the bone marrow were detected, while in the other thrombocyte counts dropped, but rebounded later, and no bone marrow changes were noted. Thrombocyte counts of the experimental calves were statistically significantly lower than those of the control calves at 2 hours post ingestion of colostrum and at every sampling point between 9 hours and 8 days postcolostral. Leucocyte counts of the experimental calves were statistically significantly lower than those of control calves at 2 hours post ingestion of colostrum and 3-7 days postcolostral. CONCLUSIONS: BNP can be induced in some calves by ingestion of colostrum from cows that have given birth to BNP calves.