A doença hemolítica perinatal (DHPN) é caracterizada pela destruição das hemácias fetais por anticorpos da classe IgG presentes na circulação materna. Esses anticorpos, dirigidos contra antígenos eritrocitários presentes nas hemácias do feto, atravessam a barreira placentária e promovem a hemólise prematura dos eritrócitos, podendo levar à anemia fetal. O presente trabalho teve como objetivo principal determinar a prevalência dos anticorpos irregulares em gestantes atendidas em maternidades públicas da hemorrede de Santa Catarina por meio de um estudo observacional, com coorte retrospectiva, entre janeiro de 2012 a dezembro de 2015, englobando a análise de dados arquivados no laboratório de Imunohematologia do Centro de Hematologia e Hemoterapia de Santa Catarina, situado na cidade de Florianópolis. As variáveis investigadas nas amostras de cordão e de recém nascido (RN) foram: Anticorpo(s) materno(s) identificado(s) no teste de eluato de amostras com teste de antiglobulina direto positivo; fenotipagem ABO/RhD da mãe do RN. No caso das amostras de gestantes com suspeita de aloimunização e mães dos RNs, avaliaram-se as seguintes variáveis: anticorpo(s) antieritrocitário(s) identificado (s); fenotipagem ABO/RhD da gestante. O número total de casos analisados ao longo dos 4 anos foi equivalente a 585, sendo que o número de casos de anticorpos irregulares maternos na população estudada foi equivalente a 45. A distribuição do total de anticorpos irregulares maternos (TAIM) para cada ano demonstrou forte tendência de crescimento linear (coeficiente de correlação linear igual a 0,9311). Os dados coletados apontaram as seguintes especificidades de anticorpos, citados de acordo com a ordem decrescente de distribuição de frequência : anti-D (57,8%); anti-D,-C (11,1%); anti-c (6,7%); anti-c,-E (4,4%); anti-D,-E (4,4%); anti-M (4,4%); anti-C (2,2%); anti-E (2,2%); anti-K (2,2%); anti-Fy(b) (2,2%); anti-Jk(a) (2,2%) com prevalência de 41% do anti-D sob as outras classes de anticorpos. A associação entre o fenótipo RhD da gestante e a realização do teste de Pesquisa de Anticorpos Irregulares (PAI) demonstrou que o fato do fenótipo RhD ser positivo ou negativo não influencia na execução do teste (p=0,1672; Coeficiente de Cramer equivalente a 0,1783), entretanto, foi possível observar uma predominância do número de casos de testes de PAI não realizados sob os realizados, em especial em gestantes RhD positivo. Os resultados encontrados contribuíram para a elaboração do material clínico na forma de cartilha intitulado: DHPN - DOENÇA HEMOLÍTICA PERINATAL E TESTES IMUNOHEMATOLÓGICOS PARA DIAGNÓSTICO LABORATORIAL. A cartilha foi elaborada na forma de perguntas e respostas englobando principalmente esclarecimentos sobre os testes imunohematológicos que auxiliem no diagnóstico da DHPN. / Perinatal hemolytic disease is characterized by the destruction of fetal red blood cells by IgG antibodies present in the maternal circulation. These antibodies directed against erythrocyte antigens present in red blood cells of the fetus, cross the placental barrier and promote the premature haemolysis of red blood cells and can lead to fetal anemia. This study aimed to determine the prevalence of irregular antibodies in pregnant women in public hospitals in hemorrede of Santa Catarina through an observational study with retrospective cohort from January 2012 to December 2015, involving the analysis of archived data in Immunohematology laboratory of Center of Hematology and Hemoterapy of Santa Catarina, located in Florianópolis. The variables to be investigated in the cord samples and newborn (NB) were: maternal antibody identified in eluate test of samples with a positive direct antiglobulin test; phenotyping ABO / RhD of newborn`s mother. For samples of pregnant women with suspected alloimmunization and mothers of newborns, the following variables will be evaluated: Antierythrocyte(ies) antibody(ies) identified; phenotyping ABO / RhD of the pregnant woman. The total number of cases analyzed over the four years was equivalent to 585, and the number of cases of maternal irregular antibodies in the population studied was equivalent to 45. The distribution of total maternal irregular antibodies (TMIA) for each year showed strong linear growth trend (linear correlation coefficient of 0.9311). The data collected showed the following specific antibodies, cited according to the descending order of frequency distribution: Anti-D (57.8%); anti-D, -C (11,1%); anti-c (6.7%); anti-c E (4.4%); anti-D, E (4.4%); Anti-M (4.4%); Anti-C (2.2%); Anti-E (2.2%); anti-K (2.2%); anti-Fy (b) (2.2%); antiJk(a) (2.2%) with a prevalence of 41% of anti-D in relation to other antibody specificities. The association between RhD phenotype of pregnant women and the realization of Irregular antibody screening test (PAI) showed that the fact of the RhD phenotype be positive or negative does not influence the test execution (p = 0.1672; Cramer coefficient equivalent to 0.1783), however, it was possible to observe a predominance of the number of cases of PAI tests not performed under performed, especially in RhD positive pregnant women. The results contributed to the development of the clinical material in the form of spelling book entitled: DHPN - PERINATAL HEMOLYTIC DISEASE AND IMMUNOHEMATOLOGICAL TESTS FOR LABORATORY DIAGNOSTICS. The spelling book was prepared in the form of questions and answers covering mainly clarifications on immunohematological tests to assist in the diagnosis of DHPN.

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes microorganismos em diferentes habitats tais como: água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de C. jejuni de origem avícolas para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino, equino e bovino, sendo cada sangue testado isoladamente. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2 , MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de Campylobacter jejuni em placas de TSA – sangue, foi possível observar que houve hemólises em 48,89% das amostras quando utilizado sangue eqüino, em 40% em sangue de bovino e em 31,11% quando de ovino. Quanto à influência de agentes quelantes e íons em caldo TSB na atividade de hemolisinas em amostras de Campylobacter jejuni semeadas em placas de TSA – sangue ovino, foi observada atividade hemolítica em 26,67% quando utilizado CaCl2, 15,55% (FeCl3), 22,22% (EDTA), 11,11% (MgCl2) e apenas 2,22% (ácido acético). No tocante à atividade hemolítica, o TSA - sangue bovino apresentou 15,55% (CaCl2), 24,44% (FeCl3), 26,26% (EDTA), 20% (MgCl2) e 11,11% (ácido acético). A atividade hemolítica para o sangue equino foi de 24,44% (CaCl2), 22,22% (FeCl3), 28,89% (EDTA), 28,89% (MgCl2) e 8,89% (ácido acético). Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação da Polimerase em Cadeia (PCR), foram utilizadas 119 amostras de C. jejuni de origem avícolas. Foi possível observar que 38% possuíam os três genes, e foram identificados somente os genes cdtA e cdtC em 19% do total de amostras, sendo que o gene cdtB foi encontrado em 14%, o gene cdtC foi observado em 12%, os genes cdtA e cdtB em somente 1%, os genes cdtB e cdtC em 1% e para cdtA em 1%. Observou-se que os resultados são dignos de atenção, pois demonstraram em amostras avícolas a presença de estirpes de C. jejuni com potencial virulento. A atividade hemolítica apresentou significativo aumento quando utilizado sangue de origem equina. A mesma foi diminuída quando utilizados agentes quelantes ou íons, nos três tipos de sangue. / Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and also for other animals. The great diversity of ecological habitats in different organisms such as water, food, and animals may promote new virulence factors. This study aimed at detecting the distending cytolethal toxin (CDT) encoding genes by PCR, studying the activity of hemolysin and also the influence of chelation solutions and ions. A total of 45 samples of C. jejuni from poultry origin, grown in Tryptone Soy Broth (TSB) were used for investigating hemolytic activity. After bacterial growth, samples were plated on Tryptic Soy Agar (TSA) containing 5% sheep, equine or bovine blood, being each blood tested individually. In order to check the influence of chelation agents and ions solution on the hemolytic activity, samples of C. jejuni strains were grown in TSB containing chelation agents individually: EDTA, acetic acid, CaCl2 ion, MgCl2 and FeCl3 solutions, all in microaerophilic atmosphere. Regarding the detection of Campylobacter jejuni hemolysin activity on TSA plates, blood hemolysis were observed in 48.89 % of samples when equine blood was used; in 40% of samples when bovine blood was used and in 31.11 % when the blood used was of sheep origin. The influence of ions and chelation agents in hemolysin activity in TSB when Campylobacter jejuni was plated on TSA with sheep blood can be described as: hemolytic activity was observed at 26.67% of samples when CaCl2 was used, at 15.55 % for FeCl3, 22 22 % for EDTA, 11.11 % for MgCl2 and only 2.22% when acetic acid was used. The hemolytic activity detected when bovine blood - TSA was used indicated 15.55% for CaCl2, 24.44% for FeCl3, 26.26 % for EDTA, 20 % for MgCl2 and 11.11% for acetic acid. In terms of the hemolytic activity when equine blood was used, the results indicated 24.44% for CaCl2, 22.22 % for FeCl3, 28.89 % for EDTA, 28.89 % for MgCl2 and 8.89% for acetic acid. Finally, regarding the detection of cdtA, cdtB and cdtC through PCR, 119 samples of C. jejuni from poultry origin were used. The results indicated that all three genes were present in 38 % of the samples, whereas only two genes were identified in 19 % of samples, while the cdtB gene was singly found in 14%, the cdtC gene was independently observed in 12%, cdtA and cdtB genes together were found in 1% of the samples; the cdtB and cdtC genes associated were detected in 1%, while cdtA alone answered for 1% of detections. The results also showed the presence of C. jejuni strains with virulence potential. The hemolytic activity increased significantly when blood of equine origin was used, and that this activity was reduced when ions or chelating agents were used in combination with the three types of blood cells.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal disorder characterized by thrombotic microangiopathy, thrombocytopenia, and acute kidney injury. Its pathogenesis has been attributed to a ‘triggering' event that leads to dysregulation of the complement cascade at the level of the endothelial cell surface. Consistent with this understanding of the disease, mutations in complement genes have been definitively implicated in aHUS. However, the existence of other genetic contributors is supported by two observations. First, in ~50% of cases, disease-causing variants are not identified in complement genes, and second, disease penetrance is typically incomplete and highly variable. To test this hypothesis, we identified pathways established to have crosstalk with the complement cascade, focusing initially on the coagulation pathway. Using targeted genomic enrichment and massively parallel sequencing we screened 36 European-American patients with sporadic aHUS patients for genetic variants in 85 complement and coagulation genes, identifying deleterious rare variants in several coagulation genes. The most frequently mutated coagulation gene in our study cohort was PLG, which encodes a zymogen of plasmin and plays key role in fibrinolysis. These results implicate the coagulation pathway in the pathogenesis of aHUS. Based on this outcome, we developed a clinical genetic testing panel to screen disease-related genes in a group of ultra-rare complement-mediated diseases that includes, in addition to aHUS, thrombotic thrombocytopenic purpura (TTP), C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) patients. Data from 193 patients validate the usage of this panel in clinical practice and also provide confirmatory insight into the pathogeneses of these diseases. Specifically, we found that in aHUS and TTP patients, variants were frequently identified in complement regulator genes, while in C3GN and DDD patients, variants were additionally found in C3 convertase genes. To understand variability in disease penetrance, we completed targeted genetic screening in two aHUS families grossly discordant for disease penetrance, identifying in one family a co-segregating Factor X-deficiency variant (F10 p.Glu142Lys) that abrogated the effect of the complement mutation. Functional studies of the F10 p.Glu142Lys variant show that it decreases Factor X activity predicting to a hypo-coagulable state and further illustrating the importance of complement-coagulation crosstalk in exacerbating, but also mitigating the aHUS phenotype. In our final studies, we have sought to complete a comprehensive analysis for other potentially related pathways by using bioinformatics to identify candidate pathways coupled with whole exome sequencing. Preliminary data from 43 aHUS patients and 300 controls suggest that pathways for dermatan and heparan sulfate synthesis, which are relevant to the formation of the extra-cellular matrix and cell surface adhesion, may be implicated in the aHUS.

BACKGROUND: Over the past 5 years, 634 cases of Shigatoxin E. coli (STEC) infection were reported to Tennessee Health Department 1. At our local children’s hospital, 4-5 children are hospitalized with STEC infection each year. Some of these children had no history of ingesting food items that could have placed them at risk to develop STEC infection; however, there are other ways that humans could get infected, such as exposure to contaminated water from cattle farms 2. GOALS: To determine if bodies of water in the city are contaminated with STEC. METHODS: Fifty (50) ml of water samples were collected from selected areas of Johnson City, TN. Samples were inoculated to Sorbitol McConkey Agar (SMA) plates under sterile techniques & incubated at 36C for 18 hours under aerobic conditions. RESULTS: Table 1 E. coli Strains Isolated from Water Samples Colony Types Founders Park Sinking Creek Carroll Creek Cherokee Creek Colorless (STEC) 14 (3.5) 24 (6) 32 (8) 35 (8.75) Pink (Non-STEC ) 8 (2) 3 (0.75) 7 (1.75) 4(1) DISCUSSION/ CONCLUSION: All sampled sites were positive for STEC. STEC is a normal flora of the gastrointestinal tract of cattle. Around city neighborhoods are pastures, as cattle farming is a major livelihood in Northeastern, TN. It is highly possible that water runoff from these pastures contaminates the waters around the city. Public health measures should be undertaken to inform the community that these waters are contaminated with STEC to prevent STEC infection. References: Reportable Conditions. TN Epi-news, TN Health Dept Issue 3, Volume 9, 2016 Escherichia coli O157:H7 Infections in Children Associated with Raw Milk & Raw Colostrum From Cows—California, 2006. MMWR Weekly, 57(23); 625-628, June 23, 2008.

Enterohemorrhagic E. coli (EHEC) infections are a leading cause of foodborne illness in the United States. Shiga-like toxins are produced that can cause hemorrhagic colitis and can lead to dangerous complications, such as acute kidney injury and hemolytic uremic syndrome (HUS). There are currently no specific treatments for HUS, and therefore more research into EHEC and HUS needs to be done. Our study focuses on Shiga-like toxin induction of endoplasmic reticulum (ER) stress in in vitro and in vivo systems, using human monocyte-like THP-1 cells and a non-human primate model of HUS. We used qPCR to determine the levels of ER stress marker expression induced by both Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) challenges. We also looked at ER stress marker expression in non-human primates that survived a lethal Stx2 challenge after being given a Stx2 binding tetravalent peptide. We expected to see increased ER stress marker expression in THP-1 cells challenged with both Shiga-like toxins and in animals that received lethal doses of the toxins. Although results were inconclusive for THP-1 cell experiments, our preliminary non-human primate data suggest that the timing of ER stress marker production is important, and Shiga-like toxins may suppress the unfolded protein response (UPR) in some baboon tissues. We also show that the therapeutic peptide TVP may reverse this UPR suppression and relieve ER stress leading to animal survival. Our study, along with the current literature, shows that Shiga-like toxin induced ER stress is a promising area for future study.

BACKGROUND: Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) are emerging food- and water- borne pathogens and a leading cause of acute renal failure in otherwise healthy children. Ribotoxic Shiga toxins are the primary virulence factors and are responsible for the potentially lethal EHEC complication of hemolytic uremic syndrome (HUS). HUS, defined clinically by microangiopathic hemolytic anemia, thrombocytopenia and thrombotic microangiopathy which contribute to acute kidney injury or renal failure, is associated with significant patient morbidity. No pathogen- or toxin- specific therapeutic exists, and antibiotic use is contraindicated. Understanding the molecular mechanisms of Stx toxicity could lead to the development of Stx specific therapies. HYPOTHESIS: Experimental evidence suggests a role for leukocytes in systemic Stx2 trafficking and in Stx2 mediated kidney pathology. Cell stress responses, such as the ER stress response and ribosomal stress response, are hypothesized to induce apoptosis, and ultimately cell death, contributing to kidney injury; however these processes have only been described in vitro. If leukocyte and kidney cell stress responses are playing significant roles in in vivo Stx2 kidney injury, then down-regulation of these processes may provide therapeutic benefit. RESULTS: Mice injected with Stx2 or infected with Stx2-producing bacteria developed lethal kidney injury as judged by biomarkers and histopathology. Experimentally induced leukopenia did not alter kidney injury in either model, but did cause striking increases in the intestinal bacterial colonization which was dependent on the presence of Stx2. No Stx binding capacity was observed for either murine or human leukocytes ex vivo. Transcriptional evidence of kidney ER stress and apoptotic biomarkers were observed in both models of Stx2-mediated kidney injury, but down-regulation of these processes did not yield therapeutic benefit. CONCLUSIONS: Contrary to the current disease paradigm, no major role for leukocytes in systemic Stx2 trafficking or kidney injury was observed in vivo, but a novel role for host immune responses to Stx2 in the control of intestinal colonization by Stx2-producing bacteria was identified. Cell stress and apoptosis is induced by Stx2 in vivo but prevention of these is not sufficient to appreciably alter organ injury or survival in the murine models.

Autoimmune hemolytic anemia (AIHA) is a disease process that involves the destruction of red blood cells mediated by the humoral immune system. It can be characterized as a cold agglutinin syndrome, paroxysmal cold hemoglobinuria, and warm, mixed type, and drug-induced AIHA. Although a well-established relationship exists between the presence of AIHA and lymphoproliferative malignancy, AIHA rarely presents in association with solid malignancies. An analysis of the limited number of published cases of AIHA in association with solid malignancies performed showed that AIHA may present before the diagnosis of a solid malignancy, concurrently with the presence of a solid malignancy, or even on resolution of a solid malignancy. Few cases of solid cancers associated with AIHA have been reported. AIHA rarely presents as a paraneoplastic syndrome indicating existence of a solid cancer. We report a case of inflammatory breast cancer with AIHA.

No description available.

Tumor necrosis factor-alpha (TNF-α) is a cytokine produced primarily by macrophages during acute inflammation. In this study we examined the differential effect of retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) on the induction of TNF-α secretion from U937 monocytic cell populations by using the reverse hemolytic plaque assay (RHPA). The RHPA will allow us to investigate both changes in TNF-α secreting populations as well as monitor the relative amount of TNF-α released from individual cells. Our results indicate that treatment of U937 cells with RA (10-6M) moderately increases the secreting cell populations, and dramatically enhances the amount of TNF-α secreted from cells already committed to secretion. In contrast, treatment with PMA (250ng/ml) drastically increased the secreting population, but only slightly increasing the amount of TNF-α released. These results suggest that induction of TNF-α secretion from U937 cells occurs by different pathways.

Enterohemorrhagic E. coli (EHEC) infection is a leading cause of acute kidney failure in otherwise healthy children, and a leading cause of foodborne illness with an outsized economic impact from outbreaks. EHEC secrete two Shiga-like toxins (Stx1 and Stx2) which are AB5 holotoxins that inhibit protein synthesis in cells expressing the toxin receptor Gb3. Infection with EHEC typically begins with a diarrheal prodrome that can progress in 5-15% of cases to hemolytic uremic syndrome (HUS), a clinical diagnosis characterized by thrombocytopenia, hemolytic anemia, and thrombotic microangiopathy. Historically, strains of EHEC expressing Stx2 have been associated with more severe disease. We hypothesized that tissue injury due to the toxins leads to the release of damage-associated molecular patterns (DAMPs), which act through inflammatory receptors to promote the endothelial dysfunction that drives this disease alongside the inciting Shiga toxins. Here we demonstrate that two well-characterized DAMPs, extracellular histones and HMGB1, are produced in two different mouse models when Stx2 is present; one model represents challenge with the toxin alone, and the second model introduces toxin through secretion with a lysogenized bacterium, C. rodentium, mimicking EHEC colonization. We investigate whether Stx1, Stx2, or histones affect the endothelial expression of well-characterized members of the protein C pathway, namely the endothelial protein C receptor (ECPR), protease-activated receptor 1 (PAR1), and thrombomodulin (TM), on human aortic (HAEC) and human renal glomerular endothelial cells (HRGEC). We show that Stx and/or histones reduce endothelial expression of these anti-coagulant molecules and histones dramatically increase expression of pro-thrombotic PAR-1. These changes lead to physiologically important decreases in activated protein C (APC), a critical anti-coagulant and cytoprotective molecule. Finally, we demonstrate that histones exacerbate thrombin's barrier-disruptive effects on the endothelium, and prevent APC's protective effects. These data provide novel mechanistic insight into the endothelial dysfunction that characterizes HUS and also provide a new perspective on systemic consequences of the bacterial Shiga toxins that might drive organ injury in susceptible patients.