Angiotensin converting enzyme intron 16 insertion/deletion genotype is associated with plasma C-reactive protein concentration in uteroplacental dysfunction

Introduction: Disturbance of the uteroplacental circulation (UPC) and the renin-angiotensin system are involved in the pathogenesis of preeclampsia. In women with history of preeclampsia persistently elevated C-reactive protein (CRP) levels have been described. The angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) genotype is associated with ACE activity and assumed to be a risk factor for preeclampsia. As ACE generates proinflammatory angiotensin II, we analysed, whether ACE intron 16 I/D genotype is associated with CRP and whether this association exhibited a relation to uteroplacental dysfunction. Materials and methods: A total of 639 women have been followed during pregnancy with repeated measurements of CRP levels (observations: n=2333). ACE intron 16 I/D genotype was determined, and its association with CRP was assessed with adjustment for non-independent observations. Results: CRP levels of ACE D allele carriers were significantly higher than those of the ACE II (wild-type) genotype (p=0.0003, p adj=0.04). This relation was allele-dose dependent (p<10−4, p adj<0.02). Association between ACE I/D and CRP was significantly restricted to patients presenting with impaired UPC in univariate (p<0.04) and multivariate analyses (p=0.01). Conclusions: The ACE I/D genotype is significantly associated with CRP elevations during pregnancies complicated by disturbed UPC. Whether this effect on CRP is involved in pathogenesis of preeclampsia has to be elucidated

an In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP- BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration

Adding a piece to the puzzle of Latin American blood donors and the potential risk of Trypanosoma cruzi transmission in Germany

Introduction: Chagas disease (CD) is caused by the Trypanosoma cruzi (T. cruzi) infection and has become a global health concern due to population mobility, as well as non-vectorial transmission routes. Several countries outside Latin America (LA) have reported transfusion-associated transmission, but equivalent studies in Germany are lacking. This study aims to collect first data on the risk of transfusion associated transmission as well as LA blood donors originating from CD endemic countries in Germany Materials and methods: A total of 305 blood donors who were assumed to be at risk for T. cruzi infection were retrospectively (267) as well as prospectively (38) selected at German blood donation sites in Bavaria and Berlin, and all retrospectively as well as 27 prospectively selected were serologically screened. Prospective study subjects additionally filled out a questionnaire. Results: All samples tested seronegative for T. cuzi specific antibodies. Prospectively enrolled study subjects all had high socio-economic status including good education. Knowledge regarding CD was limited but willingness to donate frequently was high. Blood donation rates from donors born in LA countries seem to increase from 2015. Discussion: Although no transfusion associated T. cruzi infection has been documented in Germany, it has likely already happened unnoticed, or will do in the near future. Performing risk-adapted serology-based blood donor screenings in Germany could avoid transfusion-associated transmission events as well as contribute to active case detection. Moreover, larger, and ongoing studies are needed to increase the evidence base as well as end the neglect of CD in Germany

Disease Severity, Fever, Age, and Sex Correlate With SARS-CoV-2 Neutralizing Antibody Responses

Clinical trials on the use of COVID-19 convalescent plasma remain inconclusive. While data on safety is increasingly available, evidence for efficacy is still sparse. Subgroup analyses hint to a dose-response relationship between convalescent plasma neutralizing antibody levels and mortality. In particular, patients with primary and secondary antibody deficiency might benefit from this approach. However, testing of neutralizing antibodies is limited to specialized biosafety level 3 laboratories and is a time- and labor-intense procedure. In this single center study of 206 COVID-19 convalescent patients, clinical data, results of commercially available ELISA testing of SARS-CoV-2 spike-IgG and -IgA, and levels of neutralizing antibodies, determined by plaque reduction neutralization testing (PRNT), were analyzed. At a medium time point of 58 days after symptom onset, only 12.6% of potential plasma donors showed high levels of neutralizing antibodies (PRNT50 >= 1:320). Multivariable proportional odds logistic regression analysis revealed need for hospitalization due to COVID-19 (odds ratio 6.87; p-value 0.0004) and fever (odds ratio 3.00; p-value 0.0001) as leading factors affecting levels of SARS-CoV-2 neutralizing antibody titers in convalescent plasma donors. Using penalized estimation, a predictive proportional odds logistic regression model including the most important variables hospitalization, fever, age, sex, and anosmia or dysgeusia was developed. The predictive discrimination for PRNT50 >= 1:320 was reasonably good with AUC: 0.86 (with 95% CI: 0.79-0.92). Combining clinical and ELISA-based pre-screening, assessment of neutralizing antibodies could be spared in 75% of potential donors with a maximal loss of 10% of true positives (PRNT50 >= 1:320)

SARS-CoV-2 T Cell Response in Severe and Fatal COVID-19 in Primary Antibody Deficiency Patients Without Specific Humoral Immunity

Morbidity and mortality of COVID-19 is increased in patients with inborn errors of immunity (IEI). Age and comorbidities and also impaired type I interferon immunity were identified as relevant risk factors. In patients with primary antibody deficiency (PAD) and lack of specific humoral immune response to SARS-CoV-2, clinical disease outcome is very heterogeneous. Despite extensive clinical reports, underlying immunological mechanisms are poorly characterized and levels of T cellular and innate immunity in severe cases remain to be determined. In the present study, we report clinical and immunological findings of 5 PAD patients with severe and fatal COVID-19 and undetectable specific humoral immune response to SARS-CoV-2. Reactive T cells to SARS-CoV-2 spike (S) and nucleocapsid (NCAP) peptide pools were analyzed comparatively by flow cytometry in PAD patients, convalescents and naive healthy individuals. All examined PAD patients developed a robust T cell response. The presence of polyfunctional cytokine producing activated CD4(+) T cells indicates a memory-like phenotype. An analysis of innate immune response revealed elevated CD169 (SIGLEC1) expression on monocytes, a surrogate marker for type I interferon response, and presence of type I interferon autoantibodies was excluded. SARS-CoV-2 RNA was detectable in peripheral blood in three severe COVID-19 patients with PAD. Viral clearance in blood was observed after treatment with COVID-19 convalescent plasma/monoclonal antibody administration. However, prolonged mucosal viral shedding was observed in all patients (median 67 days) with maximum duration of 127 days. PAD patients without specific humoral SARS-CoV-2 immunity may suffer from severe or fatal COVID-19 despite robust T cell and normal innate immune response. Intensified monitoring for long persistence of SARS-CoV-2 viral shedding and (prophylactic) convalescent plasma/specific IgG as beneficial treatment option in severe cases with RNAemia should be considered in seronegative PAD patients

Effects of phlebotomy-induced reduction of body iron stores on metabolic syndrome: results from a randomized clinical trial

<p>Abstract</p> <p>Background</p> <p>Metabolic syndrome (METS) is an increasingly prevalent but poorly understood clinical condition characterized by insulin resistance, glucose intolerance, dyslipidemia, hypertension, and obesity. Increased oxidative stress catalyzed by accumulation of iron in excess of physiologic requirements has been implicated in the pathogenesis of METS, but the relationships between cause and effect remain uncertain. We tested the hypothesis that phlebotomy-induced reduction of body iron stores would alter the clinical presentation of METS, using a randomized trial.</p> <p>Methods</p> <p>In a randomized, controlled, single-blind clinical trial, 64 patients with METS were randomly assigned to iron reduction by phlebotomy (n = 33) or to a control group (n = 31), which was offered phlebotomy at the end of the study (waiting-list design). The iron-reduction patients had 300 ml of blood removed at entry and between 250 and 500 ml removed after 4 weeks, depending on ferritin levels at study entry. Primary outcomes were change in systolic blood pressure (SBP) and insulin sensitivity as measured by Homeostatic Model Assessment (HOMA) index after 6 weeks. Secondary outcomes included HbA1c, plasma glucose, blood lipids, and heart rate (HR).</p> <p>Results</p> <p>SBP decreased from 148.5 ± 12.3 mmHg to 130.5 ± 11.8 mmHg in the phlebotomy group, and from 144.7 ± 14.4 mmHg to 143.8 ± 11.9 mmHg in the control group (difference -16.6 mmHg; 95% CI -20.7 to -12.5; <it>P </it>< 0.001). No significant effect on HOMA index was seen. With regard to secondary outcomes, blood glucose, HbA1c, low-density lipoprotein/high-density lipoprotein ratio, and HR were significantly decreased by phlebotomy. Changes in BP and HOMA index correlated with ferritin reduction.</p> <p>Conclusions</p> <p>In patients with METS, phlebotomy, with consecutive reduction of body iron stores, lowered BP and resulted in improvements in markers of cardiovascular risk and glycemic control. Blood donation may have beneficial effects for blood donors with METS.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01328210">NCT01328210</a></p> <p>Please see related article: <url>http://www.biomedcentral.com/1741-7015/10/53</url></p

Evaluation of vardenafil for the treatment of subjective tinnitus: a controlled pilot study

<p>Abstract</p> <p>Background</p> <p>Vardenafil (Levitra^®) represents a potent and highly selective phosphodiesterase type 5 (PDE5) inhibitor, which is established for treatment of various diseases. There are several unpublished reports from patients stating that vardenafil has a considerable therapeutic effect on their concomitant tinnitus. This pilot study was conducted to specifically assess the effect of vardenafil in patients with chronic tinnitus.</p> <p>Methods</p> <p>This trial was based on a prospective, randomized, double-blind, placebo-controlled, parallel group design. Fourty-two consecutive subjects with mon- or binaural chronic tinnitus received 10 mg vardenafil (N = 21) or matching placebo tablets (N = 21) administered orally twice a day over a period of 12 weeks. Clinical examination and data acquisition took place at each visit: at baseline, after 4 weeks, after 12 weeks (end of treatment with study medication), and at non-medicated follow-up after 16 weeks. Assessment of clinical effectiveness was based on a standardized tinnitus questionnaire (TQ), the Short Form 36 health survey (SF-36), audiometric measurements (mode, pitch and loudness of tinnitus; auditory thresholds) and biomarkers of oxidative stress in patients' blood (malondialdehyde, protein carbonyl, homocysteine and total antioxidative status). Therapeutic efficacy was evaluated by comparison of subjective and objective parameters with baseline data between both treatment groups (ANCOVA).</p> <p>Results</p> <p>Vardenafil had no superior efficacy over placebo in the treatment of chronic tinnitus during this study. The primary efficacy criterion 'TQ total score' failed to demonstrate significant improvement compared to placebo. Subjective reports of TQ subscales and general quality of life areas (SF-36), objective audiometric examinations as well as investigated biomarkers for oxidative stress did not reveal any significant treatment effects. The safety profile was favorable and consistent with that in other vardenafil studies.</p> <p>Conclusion</p> <p>Although hypoxia and ischemia play a special role in the pathogenesis of tinnitus, the PDE5-inhibitor-induced increase of nitric oxide-mediated vasodilatation exerted no specific influence on tinnitus symptomatology. Considering the unclear risk of rarely associated hearing impairment, systemic application of vardenafil or other PDE5 inhibitors prove to be not appropriate for therapy of chronic tinnitus.</p

Validation of serological and NAT-testing for HIV, HBV, HCV and Treponema pallidum in postmortem blood

Das Hauptrisiko im Rahmen von Gewebetransplantationen besteht in der potenziellen Übertragung klinisch relevanter Infektionserreger. Zur weiteren Minimierung dieses Restrisikos hat sich im Tissue Banking ein aus der Transfusionsmedizin abgeleitetes Sicherheitsstufenkonzept etabliert und umfasst gesetzlich vorgeschriebene Qualitätskriterien für Spenderauswahl, Labortestung, Gewebegewinnung und - verarbeitung, mikrobiologische Inaktivierungsverfahren und Qualitätssicherung. Dieses Konzept kann nicht unverändert von Lebend- auf Leichenspender übertragen werden, da die relevanten Untersuchungen bei Lebendspendern ausschließlich aus frischen Blutproben durchgeführt werden. Dementsprechend sind die im Blutspendewesen verwendeten infektionsserologischen bzw. Nukleinsäure-Amplifikations-Technik (NAT)-Teste nur für prämortale aber nicht für postmortale Proben validiert und lizenziert. Bei Leichenspendern muss jedoch häufig postmortales Blut getestet werden, insbesondere dann, wenn es sich nicht um Multiorganspender handelt (z.B. Spender aus Rechts-medizinischen Instituten) oder wenn keine frischen (<7 Tage) prämortalen Blutproben verfügbar sind. Die Ergebnisse vereinzelter Studien in diesem Zusammenhang sind widersprüchlich und erlauben keine Schlussfolgerungen. Insofern sind die Themen der Vergleichbarkeit prä- und postmortaler Probentestung, der Entwicklung von Algorithmen zur Validierung aus postmortalem Blut und Untersuchungen zur Stabilität von Antigenen und Antikörpern für HIV, HBV und HCV in Leichenblut von ho-hem wissenschaftlichem und klinischem Interesse. Die Schwerpunkte dieser Arbeit umfassen folgende Fragestellungen: Gibt es Unterschiede zwischen der prä- und postmortalen Infektionstestung aus Blutproben desselben Gewebespenders? Wie kann eine Validierung von infektionsserologischen Testsystemen für postmortale Blutproben durchgeführt werden? und Ist der sichere Nachweis von Anti-HIV 1/2, HBsAg, Anti-HBc und/oder Anti-HCV aus Blutproben von potenziell infektiösen Spendern bis zu 48 Stunden postmortal möglich? Im ersten Teilprojekt der hier vorgelegten Arbeit wurde bei 487 Hornhautspendern eine Ü-bereinstimmung der infektionsserologischen Testergebnisse auf Anti-HIV 1/2, HBsAg, Anti-HBc und Anti-HCV zwischen prä- und postmortalen Proben in 95,7% ermittelt. Es kam ins- gesamt bei 21 Fällen zu Diskrepanzen (4,3%). Bei 17 (3,5%) postmortalen Proben waren die Ergebnisse falsch positiv und bei 4 (0,8%) falsch negativ. Im zweiten Teilprojekt wurde eine prospektive Untersuchung des Verlaufs serologischer Pa-rameter für HIV-, HBV- und HCV-Infektionen bei 30 kühl gelagerten Verstorbenen mit diesen Infektionskrankheiten bis zu 48 Stunden postmortem (0, 12, 24, 36, 48 Std.) vorgenommen. Mit dieser erstmals durchgeführten Studie konnte bei allen untersuchten Parametern eine Antigen- bzw. Antikörperstabilität bis zu 48 Stunden postmortem nachgewiesen werden. Im dritten Teilprojekt wurden 20 postmortale Blutproben von Augenhornhautspendern in zwei unterschiedlichen Konzentrationen (Iow/high) mit definierten PEI/WHO- Standards für Anti-HIV 1/2, HBsAg, Anti-HBc und Anti-HCV gespikt und unter Mitführung einer Negativkontrolle am BEP-Ill-Laborautomaten (Siemens) untersucht. Im Ergebnis wurden alle ungespikten Proben richtig negativ und alle gespikten Proben richtig positiv bestimmt. Bei der unter vergleichbarer Logistik durchgeführten Untersuchung auf Treponema pallidum wurden in zwei Fällen falsch positive Ergebnisse ermittelt. Beide Proben waren makroskopisch auffällig und stark hämolytisch. Schließlich wurde auch der Genomnachweis für HIV, HBV, HCV und HAV unter Verwendung von PEI-Standards validiert. Es konnten für sämtliche untersuchten Viren in allen Proben richtige Ergebnisse ermittelt werden. Ein Einfluss der p.m. Entnahmezeit oder einer Hämolyse auf die Messergebnisse zeigte sich nicht. Mit den vorgestellten Studien konnten wir aufzeigen, dass Infektionen mit HIV, HBV, HCV und Treponema pallidum aus p.m. Blut bis zu zwei Tagen postmortem valide detektiert wer-den. Dabei können falsch positive und falsch negative Ergebnisse durch optimierte präanalytische Vorgehensweisen weitgehend verhindert werden.The transmission of viral and non-viral infectious pathogens continues to be the most serious of the potential adverse effects of allogenic tissue transplantations. The EU directive 2006/17/EC stipulates that cadaveric blood specimens for serology testing in the context of post-mortal tissue donation must be taken not later than 24 hours post-mortem. An expanded time slot would significantly improve availability of tissue donations, but there are no significant data on the stability of infectious serology assays for anti-HIV, anti-HCV, HBsAg and anti-HBc in samples collected more than 24 hours post- mortem. Post-mortem modifications might give false negative or false positive results in infectious disease testing due to hemoly-sis, autolysis and bacterial growth. Most CE-marked test equipments for infectious serology testing are not validated for testing post-mortal blood. Up to now no study compares the re-sults of infection testing in pre-mortal and post-mortal samples of the same donors. Our thematic priority was the comparison of infectious testing of pre- and postmortem blood samples and investigations in the methodology that may be used as guidance for validation of serological infection diagnosis of post-mortem blood samples. In a first study pre- and postmortem findings from the same cornea donors of the University Tissue Bank of the Charité in the years 2004-2009 (n = 487) were retrospectively analyzed and compared. The test results from pre-mortem blood samples were defined as the refer-ence for the post-mortem blood test. Of 487 cornea donors, there were a total of 21 cases (4.3%) with discrepancies between serological test results from pre- and post-mortem blood samples. Of these, false negative results within post-mortem serology occurred in 4 of 487 cases (0.8%), false positive results within the post-mortem blood samples occurred at a much more frequent rate, with 17 of 487 cases (3.5%). In a second prospective study, serum samples of 30 deceased persons were taken upon admission to the Institute of Forensic Medicine, and after a further 12, 24, 36 and 48 hours post-mortem. All samples were measured twice. First, using the Abbott Axsym® system, and on average, after 9-month storage at -70°C using the BEP- III®-System with Siemens and Ortho reagents. All samples (0, 12, 24, 36, 48 h) were reactive. Indeterminate or false nega-tive results did not occur. In a third study negative postmortem sera were spiked with standard sera (PEI/WHO) for Anti-HIV 1/2, HBsAg, Anti-HBc und Anti-HCV in concentrations which give low and high posi-tive results for the respective marker. None of the postmortem samples was false positive. None of the spiked post-mortem samples was false negative. The testing for Anti-Treponema pallidum was performed with postmortem sera, which were spiked with anti-Treponema pallidum-positive standard sera in concentrations which give low and high positive results at the respective dilution. Two of the unspiked postmortem sera were false positive most likely due to intense hemolysis. None of the spiked samples were false negative after 0, 24 and 60 hours, respectively. Similarly to serological testing commercial available NAT systems are usually not validated for screening of post mortem blood samples. We spiked post mortem serum and plasma samples from cornea donors and control samples from blood donors, serologically and NAT negative for all investigated parameters, with defined concentrations of WHO reference material and tested for HIV-1, HCV, HBV and HAV by NAT using DRK Baden-Würtemberg-Hessen CE PCR kits. The analytical sensitivity was 100% for control and post mortem specimens when spiked with virus standards at concentrations of 3 x level of detection (LOD). Invalid results did not occured. The analytical specificity rate for all assays was 100%. Based on the presented validations post mortem donor samples can be tested valid for for infectious deseases up to a minimum of 48 hours postmortem. The methodologies described in the studies may be used as guidance for validation of serological testing in postmortem blood samples

Algorithms for the Testing of Tissue Donors for Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus

Background: Although transmission of pathogenic viruses through human tissue grafts is rare, it is still one of the most serious dreaded risks of transplantation. Therefore, in addition to the detailed medical and social history, a comprehensive serologic and molecular screening of the tissue donors for relevant viral markers for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) is necessary. In the case of reactive results in particular, clear decisions regarding follow-up testing and the criteria for tissue release must be made. Methods: Based on the clinical relevance of the specific virus markers, the sensitivity of the serological and molecular biological methods used and the application of inactivation methods, algorithms for tissue release are suggested. Results: Compliance with the preanalytical requirements and assessment of a possible hemodilution are mandatory requirements before testing the blood samples. While HIV testing follows defined algorithms, the procedures for HBV and HCV diagnostics are under discussion. Screening and decisions for HBV are often not as simple, e.g., due to cases of occult HBV infection, false-positive anti-HBc results, or early window period positive HBV NAT results. In the case of HCV diagnostics, modern therapies with direct-acting antivirals, which are often associated with successful treatment of the infection, should be included in the decision. Conclusion: In HBV and HCV testing, a high-sensitivity virus genome test should play a central role in diagnostics, especially in the case of equivocal serology, and it should be the basis for the decision to release the tissue. The proposed test algorithms and decisions are also based on current European recommendations and standards for safety and quality assurance in tissue and cell banking