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Human biobanks are collections of biological samples and health information that allow the organization of biomedical research for upgrading the knowledge of human disorders from different diseases (cancer, allergies, rare diseases, etc.), and reach real answers for diagnosis and treatment. A wide range of samples can be stored in these biorepositories such as hair, nails, urine, tissue, whole blood, red blood cells, buffy coat, plasma, serum, DNA, and RNA. Among these, buffy coat and whole blood are widely used by researchers because they can obtain DNA and RNA from these matrices. Some preliminary studies have been performed on animals to evaluate the quality and functionality of the nucleic acids obtained from some of these matrices, although more in-depth studies are needed in this area. In this study, blood samples extracted by venipuncture from 30 healthy volunteers were used to obtain DNA from buffy coat and whole blood. The purity and integrity of the nucleic acids obtained were assessed by spectrophotometry, fluorimetry, and agarose electrophoresis, and functionality was assessed by PCR and real-time PCR. Another aspect tested in this study was based on the comparison between short-term and long-term storage at -80°C and fresh samples from both matrices to evaluate the storage conditions at the biobank. Results showed differences in the yield obtained from both matrices as a function of the storage time, although the functionality of all the obtained DNA remained intact.

Background Due to their poor immune system, cancer patients undergoing chemotherapy and hemodialysis patients are more at risk for toxoplasmosis and its complications than the healthy people. The current study aimed to determine prevalence of toxoplasmosis in these patients and comparing it with healthy subjects in southwest Iran. Methods This cross-sectional study was performed on sera and buffy coat of 100 cancer patients, 47 hemodialysis patients, and 170 healthy subjects. IgG and IgM anti-Toxoplasma gondii antibodies in serum were measured by ELISA method. Molecular diagnosis was conducted by PCR method on buffy coat of the seropositive samples. Results The seroprevalence of T. gondii in cancer, hemodialysis patients and healthy subjects were 13%, 27.7% and 15.9% respectively. Moreover, seropositivity for IgM antibody was 2.1% in hemodialysis, 2% in cancer patients and 0.6% in healthy individuals. Our results were showed there was no significant difference between prevalence toxoplasmosis in case and control group. In molecular survey, only one case (cancer patient) was positive for Toxoplasma DNA. Contact with cats and consumption of undercooked meat were two studied risk factors which had significant associations with T. gondii seropositivity in the hemodialysis patients (odds ratio [OR] = 14.667; 95% confidence interval [CI] = 1.453–148.045) and control (odds ratio [OR] = 3.07; 95% confidence interval [CI] = 1.093–8.639) respectively. Conclusion Seroprevalence of toxoplasmosis in hemodialysis patients was higher than Healthy Individuals; however, the seroprevalence of toxoplasmosis in cancer patients was similar to the Healthy Individuals.

Monócitos são leucócitos mononucleares fagolíticos circulantes com a capacidade de se diferenciarem em macrófagos, desempenhando múltiplas funções no sistema imune. Os monócitos têm sido descritos como células apresentadoras de antigénios (APC) competentes, uma vez que são capazes de adquirir, preservar e processar antigénios em tecidos periféricos e transportá-los para a zona das células T nos nódulos linfáticos, onde encontram células T cognatas. As células invariantes NKT (iNKT) são células T restritas a CD1d caracterizadas pela expressão de um TCR semi-invariante, uma vez que contém uma cadeia α invariável. Este TCR especial permite o reconhecimento de um vasto espectro de antigénios lipídicos. O antigénio protótipo usado para ativação de iNKT é a α-Galactosilceramida (αGalCer). Após a estimulação por parte do antigénio, as células iNKT rapidamente produzem grandes quantidades de citocinas. O Buffy Coat é um produto do processamento do sangue total e consiste numa unidade residual, contudo enriquecida em leucócitos. O Buffy Coat é uma fonte de células, nomeadamente monócitos, que está disponível diariamente para utilização em investigação. De acordo com o intervalo de tempo entre a colheita e o processamento da dádiva de sangue, os Buffy Coats são processados por dois protocolos distintos dando origem a: Buffy Coats processados em sangue fresco (Fresh Blood), em que a colheita e o processamento ocorrem no mesmo dia; ou Buffy Coats processados em sangue de pernoite (Overnight) onde o processamento do sangue total só ocorre no dia seguinte à colheita. Esta tese tem como objetivo avaliar se as diferenças no tempo de processamento de Buffy Coats poderão ter impacto na viabilidade, no fenótipo e na função dos monócitos. Em particular, a função dos monócitos como célula apresentadora de antigénios foi investigada. Como CD1d é uma molécula apresentadora de antigénio não polimórfica, ao contrário das moléculas HLA altamente polimórficas, a apresentação de antigénios lipídicos (αGalCer e α-GalGalCer) a células iNKT restritas a CD1d foi avaliada. Foram analisados Buffy Coats com diferentes tempos de processamento e diferentes tempos de espera nomeadamente: Buffy Coat processado em Fresh Blood e que aguardou 24h para ser analisado; Buffy Coat processado em Overnight que esperou 0h para ser analisado e Buffy Coat processado em Fresh Blood que esperou 48h pelos ensaios. Os ensaios de ativação realizados levaram à conclusão de que os monócitos provenientes de Buffy Coats processados em Fresh Blood e que esperam 24h para ser analisados têm uma melhor capacidade de ativar células iNKT do que aqueles que advêm de Buffy Coats processados em Overnight e que esperam 0h para serem analisados. Esta diferença não pode ser justificada por diferenças na viabilidade dos monócitos, nem pela expressão à superfície de CD1d ou pelo estado de ativação apurado pela expressão à superfície de CD40 e CD80 quando comparados os Buffy Coats processados em tempos distintos. Tendo em conta estas diferenças, foi investigado se o aumento do tempo de espera para o ensaio de ativação se poderia traduzir numa melhor capacidade de apresentar antigénios lipídicos por parte dos monócitos. Foi verificado que a capacidade de apresentação de antigénios por parte dos monócitos de Buffy Coats processados em Fresh Blood que aguardam 48h não supera a capacidade de ativar iNKT pelos monócitos de Buffy Coats processados em Fresh Blood que esperam 24h pelas experiências. Com o objetivo de tentar explicar as diferenças na capacidade de apresentação de antigénios lipídicos por parte dos monócitos isolados dos Buffy Coats com diferentes tempos de processamento, analisou-se a expressão de genes associados à ativação de monócitos ou genes importantes para a apresentação de antigénios. Não foram observadas diferenças na expressão dos genes IL-6, IL-8, IL12p-40, IL-18 e XBP1s entre os monócitos de Buffy Coats processados em Fresh Blood que esperam 24h para serem analisados e os monócitos de Buffy Coats processados em Overnight que aguardam 0h para serem analisados. Monócitos provenientes de Buffy Coats processados em Fresh Blood que aguardam 48h até serem analisados tiveram maior expressão dos genes IL-6 e IL-18 quando comparados aos monócitos de Buffy Coats processados em Overnight que esperam 0h para serem analisados. Ainda foi realizada a ativação de monócitos com Lipopolissacarídeo (LPS) para avaliar se as diferenças observadas na capacidade de ativação das iNKT por monócitos dos diferentes Buffy Coats também seriam observadas ao usar um estímulo mais geral como o LPS. Os resultados mostram que a capacidade de ativar células iNKT e a ativação com LPS podem estar relacionadas, mas mais experiências são necessárias para obter conclusões mais fiáveis. Além disso, colocou-se a hipótese de que as diferenças na capacidade de ativação de células iNKT demonstradas pelos monócitos provenientes de dois tipos de Buffy Coats poderiam dever-se a diferentes capacidades de captação do antigénio lipídico. Para testar esta hipótese, monócitos foram incubados com αGalCer fluorescente (αGalCer-BODIPY) e a captação de αGalCer pelos monócitos foi quantificada por citometria de fluxo. Quatro experiências foram realizadas, mas os resultados não mostraram uma diferença clara na captação de αGalCer entre monócitos dos dois tipos de Buffy Coats que justificassem as diferenças observadas na capacidade de ativação de iNKT. Esta tese contribui para uma melhor caracterização dos monócitos isolados de Buffy Coats. Este trabalho indica que os diversos protocolos de processamento de Buffy Coats influenciam as funções dos monócitos como APC. Os resultados mostram que monócitos de Buffy Coats processados em Fresh Blood que esperam 24h têm melhor capacidade de ativar células iNKT. Portanto, o tipo de protocolo para o processamento de Buffy Coats deve ser cuidadosamente considerado na investigação diária, uma vez que pode levar a resultados diferentes. Monocytes are circulating mononuclear phagocytes with the capacity to differentiate into macrophages, carrying out multiple functions in the immune system. Monocytes have been described as competent antigen-presenting cells (APC), as they are able to acquire, preserve and process antigens in peripheral tissues and transport them into the T cell zone of the draining lymph nodes where they encounter cognate T cells. Invariant NKT cells (iNKT) are CD1d-restricted T cells characterized by the expression of a semi-invariant TCR, as it contains an invariable α chain. This special TCR enables the recognition of a vast spectrum of lipid antigens. The prototype antigen used for activation of iNKT is α-Galactosylceramide (αGalCer). Upon antigen stimulation, iNKT cells rapidly produce large amounts of cytokines. The Buffy Coat is a product of the whole blood processing and consists of a residual but leukocyte enriched unit. The Buffy Coat is a source of cells, namely monocytes, that is daily available for research use. According to the time interval between collection and processing of the blood donation, Buffy Coats are processed by two different protocols giving rise to: Fresh Blood Buffy Coats, in which the blood collection and processing occurs in the same day; or Overnight Buffy Coats where the processing of the whole blood only happens in the following day of the blood harvest. This thesis aimed to assess if the differences in the processing time of Buffy Coats could have an impact on monocyte viability, phenotype and function. In particular, monocyte function as an antigen-presenting cell was investigated. As CD1d is a non-polymorphic antigen-presenting molecule in opposition to the high polymorphic HLA molecules, lipid antigen (αGalCer and α-GalGalCer) presentation to CD1d-restricted iNKT cells was investigated. Buffy Coats with different processing and waiting times were analysed: Buffy Coat Fresh Blood processed which waited 24h to be analysed; Buffy Coat Overnight processed which waited 0h to be analysed and Buffy Coat Fresh Blood processed that waited 48h for the assays. We found that monocytes from Buffy Coats Fresh Blood processed 24h analysed have a better capacity to activate iNKT than those from Buffy Coats Overnight processed 0h analysed. This difference was not justified by different monocyte viability, CD1d cell surface expression, or activation state measured by CD40 and CD80 cell surface expression comparing the different time processed Buffy Coats. Considering these differences, we investigated whether increasing the waiting time for the activation assay could translate into a better capacity to present lipid antigens by the monocytes. We found that the antigen presenting capacity of monocytes from Buffy Coats Fresh Blood processed that waits 48h to be analysed does not exceed the capacity of monocytes from the Buffy Coats Fresh Blood processed 24h analysed in iNKT activation. In order to try to explain the different capacity of monocytes isolated from Buffy Coats with different processing times in lipid antigen presentation, the expression of genes associated with monocyte activation or genes important for antigen presentation was investigated in monocytes isolated from the three different Buffy Coats. No differences in expression of IL-6, IL-8, IL12p-40, IL-18 and XBP1s genes were observed between monocytes from Buffy Coats Fresh Blood processed 24h analysed and monocytes from Buffy Coats Overnight processed 0h analysed. Monocytes from Buffy Coats Fresh Blood processed 48h analysed had higher expression of the genes IL-6 and IL-18 when compared to monocytes from Buffy Coats Overnight processed 0h analysed. Monocyte activation assays with Lipopolysaccharide (LPS) were also performed to assess whether the differences observed in the capacity to activate iNKT by monocytes from different Buffy Coats were also observed when using a more general stimulus as LPS. Our results show that capacity to activate iNKT and activation with LPS might be related but more experiments are needed to retrieve more reliable conclusions. In addition, we postulated that the differences in the capacity to activate iNKT demonstrated by the monocytes from the two Buffy Coats types could be due to different capacity to uptake the lipid antigen. To test this hypothesis monocytes were incubated with fluorescent αGalCer (αGalCer-BODIPY) and αGalCer uptake by monocytes was quantified by flow cytometry. Four experiments were carried out, the results did not show a clear difference in αGalCer uptake between monocytes from the two types of Buffy Coats that justified the differences observed in the capacity to activate iNKT. This thesis contributes to a better characterization of the monocytes isolated from Buffy Coats. This work indicates that the various protocols of processing of the Buffy Coats influence monocytes’ function as APC. Our results show that monocytes from Buffy Coats Fresh Blood processed that waits 24h to the activation assay have a better capacity to activate iNKT cells. Hence, the type of protocol for Buffy Coats processing should be carefully considered in daily research, since it may lead to different outcomes. Mestrado em Biomedicina Molecular

This study was designed to examine the potentials of telomere length, mitochondria, and acute phase protein genes as novel biomarkers of gastrointestinal (GI) tract pathologies and meat quality traits. Chickens were fed a diet containing corticosterone (CORT) for 4 weeks and records on body weight, telomere length, GI tract and muscle histopathological test, meat quality traits, mitochondria, and acute phase protein genes were obtained at weeks 4 and 6 of age. The body weight of CORT-fed chickens was significantly suppressed (p &lt, 0.05). CORT significantly altered the GI tract and meat quality traits. The interaction effect of CORT and age on body weight, duodenum and ileum crypt depth, pH, and meat color was significant (p &lt, 0.05). CORT significantly (p &lt, 0.05) shortened buffy coat telomere length. UCP3 and COX6A1 were diversely and significantly expressed in the muscle, liver, and heart of the CORT-fed chicken. Significant expression of SAAL1 and CRP in the liver and hypothalamus of the CORT-fed chickens was observed at week 4 and 6. Therefore, telomere lengths, mitochondria, and acute phase protein genes could be used as novel biomarkers for GI tract pathologies and meat quality traits.

Background Racial/ethnic disparities in health reflect a combination of genetic and environmental causes, and DNA methylation may be an important mediator. We compared in an exploratory manner the blood DNA methylome of Japanese Americans (JPA) versus European Americans (EUA). Methods Genome-wide buffy coat DNA methylation was profiled among healthy Multiethnic Cohort participant women who were Japanese (JPA; n = 30) or European (EUA; n = 28) Americans aged 60–65. Differentially methylated CpGs by race/ethnicity (DM-CpGs) were identified by linear regression (Bonferroni-corrected P Results We identified 174 DM-CpGs with the majority of hypermethylated in JPA compared to EUA (n = 133), often in promoter regions (n = 48). Half (51%) of the genes corresponding to the DM-CpGs were involved in liver function and liver disease, and the methylation in nine genes was significantly correlated with gene expression for DM-CpGs. A total of 156 DM-CpGs were associated with rs7489665 (SH2B1). Methylation of DM-CpGs was correlated with blood levels of the cytokine MIP1B (n = 146). We confirmed some of the DM-CpGs in the TCGA adjacent non-tumor liver tissue of Asians versus EUA. Conclusion We found a number of differentially methylated CpGs in blood DNA between JPA and EUA women with a potential link to liver disease, specific SNPs, and systemic inflammation. These findings may support further research on the role of DNA methylation in mediating some of the higher risk of liver disease among JPA.

Chronic inflammation is a pathological process where cells of the mesenchymal lineage become a major source of inflammatory mediators. Platelet-rich fibrin (PRF) has been shown to possess potent anti-inflammatory activity in macrophages, but its impact on mesenchymal cells has not been investigated. The aim of this study was, therefore, to expose mesenchymal cells to inflammatory cytokines together with lysates generated from liquid platelet-poor plasma (PPP), the cell-rich buffy coat layer (BC, concentrated-PRF or C-PRF), and the remaining red clot layer (RC), following centrifugation of blood. Heating PPP generates an albumin gel (Alb-gel) that when mixed back with C-PRF produces Alb-PRF. Membranes prepared from solid PRF were also subjected to lysis. We report here that lysates of PPP, BC, and PRF decreased the cytokine-induced expression of interleukin 6 (IL6) and nitric oxide synthase (iNOS) in the bone marrow-derived ST2 cells. Consistently, PPP, BC, and PRF greatly decreased the phosphorylation and nuclear translocation of p65 in ST2 cells. The inflammatory response caused by Pam3CSK4 was reduced accordingly. Moreover, PPP, BC, and PRF reduced the enhanced expression of inflammatory mediators IL6 and iNOS in 3T3-L1 pre-adipocyte mesenchymal cells, and iNOS and CCL5 in murine calvarial cells. Surprisingly, PRF lysates were not effective in reducing the inflammatory response of human gingival fibroblasts and HSC2 epithelial cells. The data from the present study suggest that both liquid PRF and solid PRF exert potent anti-inflammatory activity in murine mesenchymal cells.

Introduction Red blood cell (RBC) transfusion (RBCT) is one of the most frequently performed procedures in inpatients within modern hospital systems. However transfusion practices may vary by an institution or even by a prescribing physician. In order to assess the current RBCT practice in our institution, we performed a detailed analysis of RBCTs. Material and methods We performed a retrospective analysis of all RBCTs in our institution between January 2018 and December 2019. The data collected included: age, sex, type of RBC, number of single and multiple unit RBCTs in bleeding and nonbleeding patients, number of multiple unit RBCTs in non-bleeding patients with and without hemoglobin (Hb) concentration determination after each RBC, primary indication for RBCT, pre-post RBCT Hb concentration in non-bleeding patients, lactate concentration pre-post RBCT in non-bleeding patients. Results The indications for RBCT were anemia (2244, 56.2 % RBC) and bleeding (1463, 36.6 % RBC). The most frequently used types of RBCs were RBCs without buffy coat (75.1 %) and leucodepleted RBCs (20.9 %). In non-bleeding patients 45.7 % were multiple unit RBCTs, only 3% were performed with Hb determination following the first unit of RBC, 508 (20.2 %) RBCT were performed with pre-post RBCT lactate concentration determination. Conclusions Analysis of the local RBCT practice showed significant room for improvement. Areas of concern were type of RBC ordered, multiple unit transfusions in non-bleeding patients, lack of laboratory control of oxygenation pre-post transfusion or not taking it into account in RBCT decision making. Deficiencies are planned to be addressed by a comprehensive blood utilization review programme.

Resumen Introducción: los concentrados plaquetarios (CPQ) son hemocomponentes lábiles afectados por varios factores, desde el método de obtención hasta las condiciones de almacenamiento, que provocan una paulatina pérdida de funcionalidad, por lo que es necesario evaluar parámetros de calidad que garanticen la viabilidad de las plaquetas durante los días de almacenamiento, con el propósito de monitorear el mantenimiento de las características funcionales de las plaquetas. Materiales y métodos: estudio descriptivo transversal, con un tamaño muestral de 64 CPQ, evaluados a los 3, 5 y 7 días de almacenamiento. Los parámetros monitoreados fueron físicos, de almacenamiento y porcentaje de la activación plaquetaria mediante la medición de P-selectina (CD62) por citometría de flujo. Se aplicó el estadístico de chi cuadrado, Anova de un factor, Kruskall-Wallis y correlación de Pearson. Resultados: existen diferencias significativas al séptimo día con relación al tercer y quinto día de almacenamiento, especialmente en el parámetro de formación de remolino plaquetario o swirling (p < 0.005) y agregados plaquetarios (p = 0.001). La activación plaquetaria aumentó significativamente (p = 0.001) desde el quinto día. Conclusiones: la viabilidad de los CPQ difiere con los días de almacenamiento, por lo que es necesario evaluar pH, formación de remolinos y agregados a todos los CPQ antes de ser transfundidos como indicativos de activación plaquetaria y disminución de su funcionalidad. Abstract Introduction: Platelet concentrates (CPQ) are labile blood components affected by several factors from the method of production to storage conditions that cause a gradual loss of functionality. For this reason, it is necessary to evaluate the platelet quality parameters that guarantee the viability during the storage days, with the purpose of monitoring the maintenance of the functional characteristics of the platelets. Materials and methods: This cross-sectional descriptive study had a sample size of 64 platelet concentrates, evaluated at 3, 5, and 7 days of storage. The monitored parameters were the physical storage parameters and percentage of platelet activation by measuring P-selectin (CD62) via flow cytometry. The chi-square statistic, one-way Anova, Kruskal-Wallis test, and Pearson correlation were applied. Results: Significant differences were observed on the 7th day in relation to the 3rd and 5th day of storage, especially in the swirling parameter (p < 0.005) and platelet aggregates (p = 0.001). The platelet activation increased significantly (p = 0.001) on the 5th day. Conclusions: Based on the findings of this study, the viability of the platelet concentrates differs with the days of storage. For this reason, it is necessary to evaluate the swirling, pH, and aggregates to all platelet concentrates before being transfused as an indication of platelet activation and decreased functionality. Resumo Introdução: os concentrados de plaquetas (CPQ) são hemocomponentes lábeis afetados por diversos fatores, desde o método de obtenção até as condições de armazenamento que ocasionam uma perda gradativa de funcionalidade, sendo necessário avaliar os parâmetros de qualidade que garantem a viabilidade das plaquetas ao longo dos dias de armazenamento, a fim de monitorar a manutenção das características funcionais das plaquetas. Materiais e métodos: estudo descritivo transversal, com tamanho de amostra de 64 CPQ, avaliados aos três, cinco e sete dias de armazenamento. Os parâmetros monitorados foram físicos, de armazenamento e porcentagem de ativação plaquetária pela dosagem de P-selectin (CD62) por citometria de fluxo. Os testes estatísticos aplicados incluíram o teste qui-quadrado, ANOVA de um fator, teste de Kruskall-Wallis e correlação de Pearson. Resultados: há diferenças significativas no sétimo dia em relação ao terceiro e quinto dia de armazenamento, principalmente no parâmetro formação de redemoinhos ou swirling de plaquetas (p < 0.005) e agregados plaquetários (p = 0.001). A ativação plaquetária aumentou significativamente (p = 0.001) a partir do quinto dia. Conclusões: a viabilidade dos concentrados de plaquetas difere com os dias de armazenamento, por isso é necessário avaliar o pH, a formação de redemoinhos e agregados a todos os concentrados de plaquetas antes de serem transfundidos como indicativo de ativação plaquetária e diminuição de sua funcionalidade.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

Background: Circulating tumor DNA (ctDNA) and exosome have been widely researched in the field of medical technology and diagnosis platforms. The purpose of our study was to improve the capturing properties of ctDNA and exosome, which involved combining two beads using approaches that may provide a new method for cancer diagnoses. Methods: We present a dual isolation system including a polydopamine (PDA)–silica-coated alginate bead for circulating tumor DNA (ctDNA) capture and an anti-CD63 immobilized bead for exosome capture. We examined the ctDNA mutation in pre-operative plasma samples obtained from 91 colorectal cancer (CRC) patients using a droplet digital PCR (ddPCR). Results: The area under the curve (AUROC) of ctKRAS G12D mutation in the buffy coat was 0.718 (95% CI: 0.598−0.838, p = 0.001). Patients with CRC that had unmethylation of MLH1 and MSH2 showed significantly higher buffy coat ctKRAS G12D mutations, ascites ctKRAS G12D mutations, miR-31-5, and mixed scores than the patients with a methylation of MLH1 and MSH2. Conclusion: Our proposed alginate bead using the specific gravity-free method suggests that the screening of mutated ctKRAS DNA and miR-31-5 by liquid biopsy aids in identifying the patients, predicting a primary tumor, and monitoring in the early detection of a tumor.

Trypanosomosis is highly reliant on the distribution of vectors responsible for transmission. A cross-sectional study was conducted to determine the prevalence and associated risk factors of bovine trypanosomosis as well as the distribution of vectors in the Jimma zone, Ethiopia. Blood samples from a total of 2088 cattle were collected and tested using a buffy coat and Giemsa techniques. An overall 13.36% prevalence of trypanosomosis was recorded in study areas. The highest proportion of the infections was caused by T. vivax (44.80%) followed by T. congolense (36.92%) and mixed infection (18.28%) of both species. The study also revealed that trypanosomosis was associated with anemia as the mean PCV was significantly lower among trypanosome-infected animals (20.34 ± 4.39) than non-infected ones (27.98 ± 3.68). Moreover, anemia was more pronounced with T. congolense infection (19.54 ± 3.22) than T. vivax (21.07 ± 3.96) and mixed infection of both species (20.16 ± 2.71). This study identified age, body condition, and agro-ecology as risk factors for the occurrence of trypanosomosis in cattle. Vector survey was conducted by deploying 377 mono-pyramidal traps in selected districts. Accordingly, Glossina species and other biting flies (Stomoxys and Tabanus) were identified with an apparent density of 5.27 and 1.74 fly/trap/day, respectively. Moreover, a higher 4.49 fly/trap/day of G. tachinoides than G. morsitans submorsitans (0.79 fly/trap/day) was noted in study areas. The present study indicated that trypanosomosis is the major cattle production constraint in the areas. Hence, applicable management techniques of the disease and its vector should be implemented and further investigation involving molecular technique should be conducted in different seasons.

Evaluation of the in vitro human liver microsome and hepatocyte metabolism of ketotifen demonstrated that norketotifen (NK) is the major demethylated hepatic metabolite of ketotifen. It is here reported that NK is completely devoid of the severe and dose-limiting sedative effects of ketotifen. Thus, while ketotifen is clinically dose-limited to 1 mg, bid, there are no dose-limiting sedative effects elicited by NK, even after the highest single-dose (16 mg) or after repeat-doses (8 mg × 7 days) in humans or after the highest doses given to dogs in repeat-dose toxicological studies (40 mg/kg × 14 days). In addition, NK-but not ketotifen-was found to express potent and dose-dependent inhibition of the release of the pro-inflammatory cytokine TNFα from activated human buffy coat preparations. Thus, when used as an anti-inflammatory drug, ketotifen is the sedating prodrug which is converted to NK a nonsedating metabolite with anti-inflammatory activity.

Background Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. Methodology/Principal findings Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. Conclusions/Significance Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance., Author summary Visceral leishmaniasis (VL) is a widespread parasitic disease caused by sand fly-borne parasites of the Leishmania donovani complex (L. donovani and L. infantum). Without early diagnosis and successful chemotherapy symptomatic human VL is fatal. Dogs are reservoir hosts of L. infantum, and canine visceral leishmaniasis (CVL) often precedes outbreaks in local human populations. With dogs sampled within a region of emergent CVL in northern Argentina, we compared a range of diagnostic techniques, including microscopy, serology by enzyme linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs), and detection and identification of Leishmania DNA. Novel serological assays based on the rK28 antigen were more sensitive than those based on rK39, and without evidence of cross reaction with six other canine pathogens. A commercial DNA detection kit (LAMP; Eiken Loopamp), used for the first time with CVL, was more sensitive than PCR on lymph node samples. Unexpectedly, we also found canine infection with the Viannia sub-genus of Leishmania. Our results reinforce the need for improved diagnosis, vigilance, surveillance and control of CVL.

Objective: The use of Pap tests as a molecular diagnostic for high-grade serous ovarian carcinoma (HGSC) is a promising concept, but studies to detect tumor DNA report limited sensitivity. “Cancer-like” somatic mutations from clonal expansions in non-neoplastic tissue can be detected in Pap tests and blood, and could potentially be utilized for cancer risk-assessment. We aimed to determine whether TP53 somatic mutations in blood and Pap test DNA are associated with HGSC or with inherited ovarian cancer susceptibility. Methods: Peripheral blood and Pap samples were collected from 26 women with HGSC and 35 women without cancer, all of whom underwent gynecologic surgery at a single academic institution. Blood samples alone were available for an additional four women as part of an ongoing study. DNA was extracted from the blood buffy coat layer and Pap samples. The coding region of TP53 was deeply sequenced (mean depth ~3800x) using duplex sequencing, an ultra-accurate error-correction sequencing approach. Mutations frequently found in the UMD TP53 Cancer Database were termed “cancer-like”. Mutation frequencies (total coding mutations divided by nucleotides sequenced, MF) were compared between groups using t-tests. Age-related trends were explored with fitted linear regression models. Results: 572 coding-region TP53 mutations were identified in DNA from Pap tests from 26 women with HGSC (300 mutations) and 35 women without cancer (272 mutations). 287 mutations were identified in the DNA from blood from 29 women with HGSC (161 mutations) and 36 without cancer (226 mutations). DNA from Pap tests demonstrated a higher MF in patients with HGSC than those without cancer (2.5x10-6 vs. 1.6x10-6, p=0.03). Cancer-like MF was also significantly higher in patients with HGSC (8.8x10-7 vs. 5.0x10-7, p=0.01). There were no significant differences in MF amongst cases without cancer with or without germline BRCA1 or BRCA2 mutations. Cancer-like mutations increased with age in Pap test samples from women without cancer (p Citation Format: Talayeh Ghezelayagh, Jeanne Fredrickson, Jeffrey D. Krimmel-Morrison, Brendan Kohrn, Marc R. Radke, Enna Manhardt, Barbara S. Norquist, Elizabeth M. Swisher, Rosa Ana Risques. Evaluation of TP53 mutations in Pap test and blood DNA as novel biomarkers of ovarian cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB046.

Mycobacterium avium subsp paratuberculosis (MAP) is the causative agent of Johne's disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

Human basophils are terminally differentiated granulocytes that are least abundant in the peripheral blood but play important roles in allergic diseases. Studies on human basophils are limited by the high cost on the isolation of human basophils by magnetic-activated cell sorting (MACS) for negative depletion of non-basophils, followed by CD123-based positive selection of basophils. Moreover, such CD123-based purification of basophils may be limited by blocking of the binding of IL-3/anti-CD123 to the surface CD123. Here we identified SSClow CD4- CD127- HLA-DR- CRTH2high as unique markers for the identification of human basophils through stringent flow cytometric analysis of leukocytes from buffy coat. We established an efficient and cost-effective method for isolating human basophils from buffy coat based on positive magnetic selection of CRTH2+ cells followed by flow cytometric sorting of SSClow CD4- CD127- HLA-DR- CRTH2high cells. Approximately 1 to 1.5 million basophils were isolated from one buffy coat with a purity of >97%. Basophils purified by this method were viable and efficiently responded to key regulators of basophils including IL-3 and anti-IgE. This method can be used for purifying human basophils for subsequent functional studies.

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

Introduction: The objective of the present study was to describe the experience of the Blood and Tissues Bank of Aragon with the Reveos® Automated Blood Processing System and Mirasol® Pathogen Reduction Technology (PRT) System, comparing retrospectively routine quality data obtained in two different observation periods. Methods: Comparing quality data encompassing 6,525 blood components from the period 2007–2012, when the semi-automated buffy coat method was used in routine, with 6,553 quality data from the period 2014–2019, when the Reveos system and subsequently the Mirasol system were implemented in routine. Results: Moving from buffy coat to Reveos led to decreased discard rates of whole blood units (1.2 to 0.1%), increased hemoglobin content (48.1 ± 7.6 to 55.4 ± 6.6 g/unit), and hematocrit (58.9 ± 6.5% to 60.0 ± 4.9%) in red blood cell concentrates. Platelet concentrates (PCs) in both periods had similar yields (3.5 ×1011). Whereas in the earlier period, PCs resulted from pooling 5 buffy coats, in the second period 25% of PCs were prepared from 4 interim platelet units. The mean level of factor VIII in plasma was significantly higher with Reveos (92.8 vs. 97.3 IU). Mirasol PRT treatment of PCs reduced expiry rates to 1.2% in 2019. One septic transmission was reported with a non-PRT treated PCs, but none with PRT-treated PCs. Conclusion: Automation contributed to standardization, efficiency, and improvement of blood processing. Released resources enabled the effortless implementation of PRT. The combination of both technologies guaranteed the self-sufficiency and improvement of blood safety.

A cross-sectional study was conducted in December 2018 in four purposively selected villages of Bedele district, Oromia Regional State, Southwest Ethiopia. The study aimed to determine the prevalence of bovine trypanosomosis and associated risk factors of trypanosome infections in cattle. A total of 384 blood samples were collected from systematically selected cattle and examined using buffy coat and thin blood smear examination methods. The overall prevalence of bovine trypanosomosis was 8.3%. Trypanosoma congolense (68.8%) and T. vivax (31.2%) were the prevailing trypanosome species identified in the area. A statistically significant difference (P 0.05). Similarly, no statistically significant difference (P > 0.05) was observed between age categories of cattle. The mean PCV value of trypanosome infected cattle (21.4 ± 3.6) was significantly (P