[The reasons for the formation of tolerance to food antigens].

One of the main issues of the peculiarities of the immune reactions of the gastrointestinal tract is the mechanisms of ensuring tolerance to food antigens. Concentrations of antibodies to food antigens actually reflect the state of the intestinal mucosa barrier function, and the degree of penetration of antigens into the blood determines the level of immune response to them. The aim of the study was to determine the risk criteria for violation of tolerance to food antigens. Material and methods. The study included the results of a survey and examination of 1334 adults living in the north of the European part of the Russian Federation, including 1100 born in the North, of which 970 were women and 364 were men. The average age of the respondents was 45.5±1.0 years. The comparison group consisted of 344 patients with pathology of the gastrointestinal tract who applied to the medical company "Biocor". The content of immunoglobulins (Ig) G to food antigens, total IgA, cytokines (tumor necrosis factor α, interleukin-6, interleukin-4) in blood serum were determined by enzyme immunoassay. Results. Rural residents often (more than 28%) have elevated concentrations of IgG to potato, river fish, wheat and rye antigens. Urban residents have the most pronounced decrease in tolerance to food antigens of chicken, cod, beef and pork. In healthy individuals, elevated (>100 ME/ml) concentrations of antibodies to meat products are recorded in the range of 11.3-13.9%, to dairy antigens - 11.5-14.1%, cereals - 11.9-13.4%. Slightly less frequently, elevated concentrations of antibodies to fish antigens (7.5-10.1%), vegetables (3.8-7.0%) and fruits (4.9-6.5%) are detected. In inflammatory and oncological diseases of the gastrointestinal tract, the content of antibodies to food antigens increases sharply. On average, the frequency of impaired tolerance to food antigens in patients is 2.7-6.1 times higher than in healthy individuals. Conclusion. Violation of tolerance to food antigens is associated with an increase in blood pro-inflammatory cytokines, mainly interleukin-6. In practically healthy individuals, a decrease in tolerance to food antigens is associated with a deficiency of blood IgA. The risk criteria for violation of the diet or consumption of low-quality foods may be an increase in the frequency of detection of elevated concentrations of antibodies to meat products in 14.6±3.0%, fish - 10.7±2.3%, cereals - 13.7±1.6%, dairy products - 14.8±1.5%, vegetables - 7.8±2.4% and fruits - 6.9±5.8%.

Probing an Ixodes ricinus salivary gland yeast surface display with tick-exposed human sera to identify novel candidates for an anti-tick vaccine.

In Europe, Ixodes ricinus is the most important vector of human infectious diseases, most notably Lyme borreliosis and tick-borne encephalitis virus. Multiple non-natural hosts of I. ricinus have shown to develop immunity after repeated tick bites. Tick immunity has also been shown to impair B. burgdorferi transmission. Most interestingly, multiple tick bites reduced the likelihood of contracting Lyme borreliosis in humans. A vaccine that mimics tick immunity could therefore potentially prevent Lyme borreliosis in humans. A yeast surface display library (YSD) of nymphal I. ricinus salivary gland genes expressed at 24, 48 and 72 h into tick feeding was constructed and probed with antibodies from humans repeatedly bitten by ticks, identifying twelve immunoreactive tick salivary gland proteins (TSGPs). From these, three proteins were selected for vaccination studies. An exploratory vaccination study in cattle showed an anti-tick effect when all three antigens were combined. However, immunization of rabbits did not provide equivalent levels of protection. Our results show that YSD is a powerful tool to identify immunodominant antigens in humans exposed to tick bites, yet vaccination with the three selected TSGPs did not provide protection in the present form. Future efforts will focus on exploring the biological functions of these proteins, consider alternative systems for recombinant protein generation and vaccination platforms and assess the potential of the other identified immunogenic TSGPs.

Detection of Borrelia burgdorferi antigens in tissues and plasma during early infection in a mouse model.

Borrelia burgdorferi is the causative agent of Lyme borreliosis, which is the most common tick-borne human disease in Europe and North America. Currently, the diagnosis of Lyme borreliosis is based on serological tests allowing indirect detection of anti-Borrelia antibodies produced by patients. Their main drawback is a lack of sensitivity in the early phase of disease and an incapacity to prove an active infection. Direct diagnostic tests are clearly needed. The objectives of this study were to produce tools allowing sensitive detection of potential circulating Borrelia antigens and to evaluate them in a mouse model. We focused on two potential early bacterial makers, the highly variable OspC protein and the conserved protein FlaB. High-affinity monoclonal antibodies were produced and used to establish various immunoassays and western blot detection. A very good limit of detection for OspC as low as 17 pg/mL of sample was achieved with SPIE-IA. In infected mice, we were able to measure OspC in plasma with a mean value of 10 ng/mL at 7 days post-inoculation. This result suggests that OspC could be a good blood marker for diagnosis of Lyme borreliosis and that the tools developed during this study could be very useful.

Different mechanisms of oxygenator failure and high plasma von Willebrand factor antigen influence success and survival of venovenous extracorporeal membrane oxygenation.

OBJECTIVE: Failure of membrane oxygenator (MO) function of venovenous extracorporeal membrane oxygenators (VV ECMO) remains problematic. The development of device-induced coagulation disorder (COD) or worsened gas transfer (WGT) necessitates a system exchange. The aim was to correlate von Willebrand factor antigen (vWF:Ag) with the predisposition to MO failure and mortality. METHODS: Laboratory parameters (inflammation, coagulation) and ECMO-related data from 31 VV ECMO patients were analyzed before and after the first MO exchange. Study groups were identified according to the exchange reasons (COD, WGT) and the extent of vWF:Ag (low, ≤425%; high, >425%). RESULTS: vWF:Ag remained unchanged after system exchange. High vWF:Ag was associated with systemic endothelial activation of older and obese patients with elevated SOFA score, increased norepinephrine and higher requirement of continuous renal replacement therapy without an effect on MO runtime and mortality. Including the mechanism of MO failure (COD, WGT), various patient group emerged. COD/low vWF:Ag summarized younger and less critically ill patients that benefit mainly from ECMO by a significant improvement of their inflammatory and coagulation status (CRP, D-dimers, fibrinogen) and highest survival rate (91%). Instead, WGT/high vWF:Ag presented older and more obese patients with a two-digit SOFA score, highest norepinephrine, and aggravated gas transfer. They benefited temporarily from system exchange but with worst survival (33%). CONCLUSIONS: vWF:Ag levels alone cannot predict early MO failure and outcome in VV ECMO patients. Probably, the mechanism of clotting disorder in combination with the vWF:Ag level seems to be essential for clot formation within the MO. In addition, vWF:Ag levels allows the identification different patient populations In particular, WGT/high vWF:Ag represented a critically ill population with higher ECMO-associated mortality.

Proper definition of the set of autoantibody-targeted antigens relies on appropriate reference group selection.

Autoimmune diseases are frequently associated with autoantibodies. Recently, large sets of autoantibody-targeted antigens ("autoantigen-omes") of patient and control sera have been revealed, enabling autoantigen-omic approaches. However, statistical standards for defining such autoantigen-omes are lacking. The z-score indicates how many standard deviations an antigen reactivity of a given sample is from the mean reactivity of the corresponding antigen in a reference group. Hence, it is a common measure to define significantly positive reactivity in autoantigen profiling approaches. Here, we address the risk of biased analyses resulting from unbalanced selection of the reference group. Three study groups were selected. Patients-of-interest were chronic inflammatory demyelinating polyneuropathy (CIDP); controls were other neuropathies (ONP); and healthy controls (HC). Each serum was screened for significant autoantigen reactivity using HuProt™ protein arrays. We compared three possible selections of reference groups for statistical z-score calculations: method#1, the control groups (ONP + HC); method #2, all groups together; method #3, the respective other groups (e.g., CIDP + HC for the ONP autoantigen-ome). The method selection seriously affected the size of the autoantigen-omes. Method #1 introduced a bias favoring significantly more antigens per patient in the CIDP group (for z >4: 19 ±â€¯3 antigens) than in the control groups (ONP: 2 ±â€¯1; HC: 0 ±â€¯0). The more balanced methods #2 and #3 did not result in significant differences. This contribution may help to avoid interpretation biases and to develop guidelines for population studies revealing autoantigen-omes via high throughput studies such as protein microarrays, immunoprecipitation with mass spectrometry, or phage display assays.

Simultaneous testing of immunological sensitization to multiple antigens in sarcoidosis reveals an association with inorganic antigens specifically related to a fibrotic phenotype.

Organic and inorganic antigens were studied simultaneously in the same cohort of sarcoidosis patients to investigate whether correlations between clinical characteristics and immunological sensitization could reveal new phenotypes. Sensitization to antigens of mycobacteria, Propionibacterium acnes catalase and vimentin was investigated in 201 sarcoidosis and 51 obstructive sleep apnoea patients, serving as control group. Sensitization to aluminium, beryllium, silica and zirconium was also studied in 105 of the sarcoidosis patients and in 24 of the controls. A significantly higher percentage of sarcoidosis patients (27·6%) than controls (4·2%) had an immunological response to metals or silica (P = 0·014). A higher percentage of these sarcoidosis patients showed fibrosis on chest X-ray 5 years after the diagnosis (69·2 versus 30·3%, P = 0·016). No significant differences in mycobacterial or vimentin enzyme-linked immunospot (ELISPOT) assay results were observed between sarcoidosis and control patients. A significantly lower percentage of sarcoidosis patients (3·5%) than control patients (15·7%) had a positive ELISPOT for P. acnes catalase (P = 0·003). However, sarcoidosis patients sensitized to P. acnes catalase were more likely to have skin involvement, while sarcoidosis patients sensitized to mycobacterial antigens were more likely to have cardiac involvement. Our study suggests a more prominent role for inorganic triggers in sarcoidosis pathogenesis than previously thought. Immunological sensitization to inorganic antigens was associated with development of fibrotic sarcoidosis. No association was found between sensitization to bacterial antigens or vimentin and sarcoidosis in Dutch patients. However, our data suggest that trigger-related phenotypes can exist in the heterogeneous population of sarcoidosis patients.

Distinct expression trend of signature antigens of Staphylococcus aureus osteomyelitis correlated with clinical outcomes.

The major limitations of clinical outcome predictions of osteomyelitis mediated by Staphylococcus aureus (S. aureus) are not specific and definitive. To this end, current studies aim to investigate host immune responses of trend changes of the iron-regulated surface determinant (Isd) of IsdA, IsdB, IsdH, cell wall-modifying proteins of amidase (Amd) and glucosaminidase (Gmd), and secreted virulence factor of chemotaxis inhibitory protein S. aureus (CHIPS) and staphylococcal complement inhibitor (SCIN) longitudinally to discover their correlationship with clinical outcomes. A total of 55 patients with confirmed S. aureus infection of the long bone by clinical and laboratory methods were recruited for the study. Whole blood was collected at 0, 6, 12 months for the serum that was used to test IsdA, IsdB, IsdH, Gmd, Amd, CHIPS, and SCIN using a customized Luminex assay after clinical standard care parameters were collected. The patients were then divided into two groups: (1) infection controlled versus (2) adverse outcome based on clinical criteria for statistical analysis. We found that standard clinical parameters were unable to distinguish therapeutic outcomes. Significant overexpression of all antigens was confirmed in infection patients at 0-, 6-, and 12-month time points. A distinct expression trend and dynamic changes of IsdB, Amd, Gmd, and CHIPS were observed between infection controlled and adverse outcome patients, while the IsdA, IsdH, SCIN remained demonstrated no statistical significance. We conclude that dynamic changes of specific antigens could predict clinical outcomes of S. aureus osteomyelitis. Clinical Relevance: The trend changes of host immune responses to S. aureus specific antigens of IsdB, Gmd, Amd, and CHIPS could predict clinical outcomes of S. aureus osteomyelitis.

Prozone phenomenon observed in indirect immunofluorescence assay by antibodies against neuronal antigens.

A marked prozone effect was observed in indirect immunofluorescence with human sera and human cerebrospinal fluid in two clinical cases involving breast carcinoma with paraneoplastic neuronal antibodies, and anti- N-methyl-D-aspartic acid (NMDA) receptor antibodies. Anti-Yo antibodies and anti-NMDA antibodies were not detectable under high concentrations (1:10 serum dilution and neat CSF respectively) but showed a true effect when sufficiently diluted at 1:80 and 1:5 respectively. This paper demonstrates that prozone effects have their occurrences in indirect immunofluorescence, and clinicians and laboratory technicians should be wary of its implications during screening of autoantibody markers in neurological diseases.

Congenital dysfibrinogenaemia presented with preterm premature rupture of the membranes and vaginal bleeding.

A 26-year-old woman was found to have congenital dysfibrinogenaemia after presenting to our hospital with premature rupture of the membranes and vaginal bleeding. Given the absence of clear guidelines for the management of pregnancy complicated by dysfibrinogenaemia, we followed expert consensus that exists among published works, with some modifications. This case was managed by a multidisciplinary team of obstetrics-gynaecology, haematology and paediatric haematology. Here we review how the patient presented, the investigations that led to the diagnosis and the treatment options.

Characterization of circulating immune cells in acute Kawasaki disease suggests exposure to different antigens.

Kawasaki disease (KD) is an acute pediatric vasculitis of unknown etiology that can cause coronary artery aneurysms, and is the leading cause of acquired heart disease in children. We studied aspects of the innate and adaptive immune response in 17 acute KD children prior to treatment with intravenous immunoglobulin. Distinct patterns within the innate immune response correlated with specific clinical features. Proinflammatory myeloid dendritic cells (mDC) were abundant in four of 17 (23·5%) subjects who were older and manifested severe inflammation with clinical myocarditis and elevated hepatobiliary enzyme levels. Of the nine subjects with low levels of anti-inflammatory, tolerogenic mDC, six had enlarged cervical lymph nodes at diagnosis. In contrast, the adaptive immune repertoire varied greatly with no discernible patterns or associations with clinical features. Two subjects with aneurysms had numerous circulating CD8+ T cells. Ten subjects showed low CD4+ T cell numbers and seven subjects had CD4+ T cells in the normal range. CD4+ T cells expressed interleukin-7 receptor (IL-7R), suggesting repeated antigenic stimulation. Thymic-derived regulatory T cells (nTreg ) and peripherally induced regulatory T cells (iTreg ) were also enumerated, with the majority having the nTreg phenotype. Natural killer (NK) and NK T cell numbers were similar across all subjects. Taken together, the results of the immune monitoring suggest that KD may have multiple triggers that stimulate different arms of the innate and adaptive compartment in KD patients. Thus, it is possible that diverse antigens may participate in the pathogenesis of KD.

Sensitivity and specificity of malaria rapid diagnostic test (mRDT CareStatTM) compared with microscopy amongst under five children attending a primary care clinic in southern Nigeria.

BACKGROUND: Malaria diagnosis using microscopy is currently the gold standard. However, malaria rapid diagnostic tests (mRDTs) were developed to simplify the diagnosis in regions without access to functional microscopy. AIM: The objective of this study was to compare the diagnostic accuracy of mRDT CareStatTM with microscopy. SETTING: This study was conducted in the paediatric primary care clinic of the Federal Medical Centre, Asaba, Nigeria. METHODS: A cross-sectional study for diagnostic accuracy was conducted from May 2016 to October 2016. Ninety-eight participants were involved to obtain a precision of 5%, sensitivity of mRDT CareStatTM of 95% from published work and 95% level of confidence after adjusting for 20% non-response rate or missing data. Consecutive participants were tested using both microscopy and mRDT. The results were analysed using EPI Info Version 7. RESULTS: A total of 98 children aged 3-59 months were enrolled. Malaria prevalence was found to be 53% (95% confidence interval [CI] = 46% - 60%), whilst sensitivity and specificity were 29% (95% CI = 20% - 38%) and 89% (95% CI = 83% - 95%), respectively. The positive and negative predictive values were 75% (95% CI = 66.4% - 83.6%) and 53% (95% CI = 46% - 60%), respectively. CONCLUSION: Agreement between malaria parasitaemia using microscopy and mRDT positivity increased with increase in the parasite density. The mRDT might be negative when malaria parasite density using microscopy is low.

Optimization of pH Elution Conditions in Immune Complexome Analysis for Comprehensive Identification of Immune Complex Antigens.

The identification of antigens incorporated into immune complexes (IC-antigens) is important for studying the pathophysiology of immunological diseases. Immune complexome analysis identifies IC-antigens by analyzing ICs collected from biological fluids by IC-capturing beads. In this study, we optimized the method to improve its comprehensiveness while maintaining selectivity for IC-antigens by comparing the number of identified peptides (model IC experiment) or proteins (human pooled serum) eluted from Protein G beads using different pH solutions (pH 2.0 - 11.0).

Centrosome amplification is a frequent event in circulating tumor cells from subjects with metastatic breast cancer.

Centrosome amplification (CA) is a common phenomenon in cancer, promotes genomic stability and cancer evolution, and has been reported to promote metastasis. CA promotes a stochastic gain/loss of chromosomes during cell division, known as chromosomal instability (CIN). However, it is unclear whether CA is present in circulating tumor cells (CTCs), the seeds for metastasis. Here, we surveyed CA in CTCs from human subjects with metastatic breast cancer. CTCs were captured by CD45 exclusion and selection of EpCAM-positive cells using an exclusion-based sample preparation technology platform known as VERSA (versatile exclusion-based rare sample analysis). Centriole amplification (centrin foci> 4) is the definitive assay for CA. However, determination of centrin foci is technically challenging and incompatible with automated analysis. To test if the more technically accessible centrosome marker pericentrin could serve as a surrogate for centriole amplification in CTCs, cells were stained with pericentrin and centrin antibodies to evaluate CA. This assay was first validated using breast cancer cell lines and a nontransformed epithelial cell line model of inducible CA, then translated to CTCs. Pericentrin area and pericentrin area x intensity correlate well with centrin foci, validating pericentrin as a surrogate marker of CA. CA is found in CTCs from 75% of subjects, with variability in the percentage and extent of CA in individual circulating cells in a given subject, similar to the variability previously seen in primary tumors and cell lines. In summary, we created, validated, and implemented a novel method to assess CA in CTCs from subjects with metastatic breast cancer. Such an assay will be useful for longitudinal monitoring of CA in cancer patients and in prospective clinical trials for assessing the impact of CA on response to therapy.

Prevalencia del antígeno D débil y su clasificación fenotípica en donantes voluntarios de sangre / Prevalence of "weak" D antigen and its phenotypic classification in voluntary blood donours / Prevalência do antígeno D "fraco" e sua classificação fenotípica em doadores voluntários de sangue

En el campo de la medicina transfusional la correcta identificación de los fenotipos del sistema Rh y en especial del antígeno D debe ser de manera inequívoca por su relevancia clínica. El antígeno D tiene variantes denominadas D parcial, D débil y DEL, las que se producen por mutaciones de los alelos RHD/RHCE o por una supresión en la expresión fenotípica. Se trató de un estudio descriptivo, retrospectivo de corte transversal en el que se realizó una revisión de registros primarios durante el período 2011-2014 validados de acuerdo con el protocolo de Hernández-Sampieri R. Se utilizó estadística descriptiva mediante la aplicación del software informático SPSS versión 22.0 y se estableció la relación entre variables independientes a través del análisis estadístico de Chi-cuadrado. Se determinó una prevalencia de donantes RhD negativos de 1,8 a 2,5% y RhD débil de 1,79 a 2,28%. La fenotipificación serológica permitió identificar que los tipos 2 y 5 eran los más frecuentes. También se estableció la existencia de aloinmunización por anti-D, anti-C y anti-E. Se estableció de esta manera la existencia de D débil y una importante aloinmunización en la población de donantes de sangre tipificados como D negativo y D débil, por lo que se recomienda implementar un algoritmo de identificación del antígeno D en servicios de medicina transfusional.

In the field of transfusion medicine, the correct identification of the phenotypes of the Rh system and especially of the D antigen must be unequivocal for clinical relevance. The D antigen has variants called partial D, weak D and DEL. These are produced by mutations of the RHD/RHCE alleles or a suppression in phenotypic expression. The objective of this study was to establish the frequency of weak D antigen in the population of blood donours from 17 Ecuadorian states and their phenotypic combinations. It was a descriptive, retrospective cross-sectional study performed during the 2011-2014 period and validated with primary records in accordance with the Hernández-Sampieri R protocol. A descriptive statistics through the application of SPSS computer software version 22.0 was used and the relationship between independent variables through the Chi-square statistic method was established. A prevalence of RhD negative donours from 1.8 to 2.5% and weak D 1.79 to 2.28% was observed The serological phenotyping made it possible to identify that type 2 and 5 were the most frequent. The presence of alloimmunization by anti-D, anti-C and anti-E was also established. Besides, the presence of weak D types and significant alloimmunization in the donour population of blood typed as D negative and weak was established, so it is recommended to implement an algorithm for the identification of D antigen in transfusional medicine services.

No campo da medicina transfusional, a correta identificação dos fenótipos do sistema Rh e especialmente do antígeno D deve ser inequívoca devido a sua relevância clínica. O antígeno D tem variantes chamadas de D parcial, D fraca e DEL, as quais são produzidos por mutações dos alelos RHD/RHCE ou por uma supressão na expressão fenotípica. O objetivo deste estudo foi estabelecer a frequência do antígeno D fraco em uma população de doadores de sangue de 17 províncias equatorianas e suas combinações fenotípicas. Foi uma estudo descritivo, retrospectivo de corte transversal em que se realizou uma revisão dos registros primários validados de acordo com o Protocolo Hernández-Sampieri R durante o período 2011-2014. Utilizou-se estatísticas descritivas através da aplicação do software informático SPSS versão 22.0 e a relação entre variáveis independentes através da análise estatística de qui-quadrado. Foi determinada uma prevalência de doadores RhD negativos de 1,8 a 2,5% e RhD fraco de 1,79 a 2,28%. A genotipagem serológica permitiu identificar que os tipos 2 e 5 são os mais frequente. A existência de alo imunização por anti-D, anti-C e anti-E também foi estabelecida. A existência de D fraco e uma alo imunização significativa na população de doadores de sangue tipificados como D negativo e fraco, por isso é recomendado implementar um algoritmo de identificação do antígeno D em serviços de medicina transfusional.

Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA.

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

Analysis of Phenotype and Genotypein an Inherited Coagulation Factor VII Deficiency Pedigree.

BACKGROUND: To identify potential mutations and analyze the phenotype in an inherited factor VII (FVII) deficiency pedigree. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, FVII activity (FVII:C), FVII antigen (FVII:Ag) and other coagulant parameters of the proband and family members were measured. Calibrated automated thrombin generation measurements were used to detect coagulation status. All the exons, exonintron boundaries and 5', 3' untranslated sequences of the F7 gene in the proband and other family members were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing. The detected mutations were confirmed by sequencing the other strand and analyzed by PIC software. RESULTS: The PT in the proband was prolonged and further study showed that the FVII:C and FVII:Ag were below the normal range, 3% and 38%, respectively. Double heterozygous mutations in the proband were identified: an A to G mutation at position 11,459 in exon 8, resulting in a p.Lys341Glu substitution, and a 2bp deletion (nt 27 del CT) mutation in exon 1a, leading to a frameshift and the creation of a premature stop codon. Both endogenous thrombin potential (ETP) and peak height in the proband were declined. Her father, brother, daughter, son, and niece were heterozygous for the nt 27 del CT mutation, while her mother, little brother, little niece, and little nephew were heterozygous for the Lys341Glu mutation, and their ETP and peak height obtained more than half normal ETP and peak height, respectively. Model analysis indicated that the Lys341Glu mutation will interrupt the electrovalent bond between 341Lys and 289Asp. CONCLUSIONS: Compound heterozygous mutations (p.Lys341Glu and nt27 del CT) which were responsible for the bleeding tendency were found in a pedigree of inherited FVII deficiency. The Lys341Glu mutation is firstly reported and nt27 del CT may be one of the mutational spots in Chinese population.

Antigen array for serological diagnosis and novel allergen identification in severe equine asthma.

Severe equine asthma (sEA), which closely resembles human asthma, is a debilitating and performance-limiting allergic respiratory disorder which affects 14% of horses in the Northern Hemisphere and is associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental proteins. A comprehensive microarray platform was developed to enable the simultaneous detection of allergen-specific equine IgE in serum against a wide range of putative allergenic proteins. The microarray revealed a plethora of novel pollen, bacteria, mould and arthropod proteins significant in the aetiology of sEA. Moreover, the analyses revealed an association between sEA-affected horses and IgE antibodies specific for proteins derived from latex, which has traditionally been ubiquitous to the horse's environment in the form of riding surfaces and race tracks. Further work is required to establish the involvement of latex proteins in sEA as a potential risk factor. This work demonstrates a novel and rapid approach to sEA diagnosis, providing a platform for tailored management and the development of allergen-specific immunotherapy.

Plasma phenotypes of protein S Lys196Glu and protein C Lys193del variants prevalent among young Japanese women.

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.

Plasma thrombin-activatable fibrinolysis inhibitor (TAFI) antigen levels in acromegaly patients in remission

Background/aim: Acromegaly is associated with increased morbidity andmortality, mostly due to cardiovascular complications.Plasma thrombin-activatable fibrinolysis inhibitor (TAFI) antigen levels are associated with coagulation/fibrinolysis and inflammation. Plasma TAFI may play a role in arterial thrombosis in cardiovascular diseases. In this study, it was aimed to evaluate the thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and homocysteine levels in patients with acromegaly and healthy control subjects. Materials and methods: Plasma TAFI antigen and homocysteine levels in 29 consecutive patients with acromegaly and 26 age-matched healthy control subjects were measured. All patients included in the study were in remission. The TAFIa/ai antigen in the plasma samples was measured using a commercially available ELISA kit. Results: Routine biochemical parameters, fasting blood glucose, prolactin, thyroid stimulating hormone, total-cholesterol, low density lipoprotein cholesterol, triglyceride, and homocysteine levels were similar in the 2 groups (P > 0.05), whereas the plasma TAFI antigen levels were significantly elevated in the acromegalic patients (154.7 ± 94.0%) when compared with the control subjects (107.2 ± 61.6%) (P = 0.033). No significant correlation was identified by Pearson's correlation test between the plasma TAFI antigen and homocysteine levels (r = 0.320, P = 0.250). Conclusion: A significant alteration in the plasma TAFI antigen levels was detected in acromegaly. Increased plasma TAFI antigen levels might aggravate prothrombotic and thrombotic events in patients with acromegaly.

Bivalent property of intra-platelet VWF in liver regeneration and HCC recurrence: A prospective multicenter study.

BACKGROUND AND AIMS: A striking difference has been observed in structure and functional properties between plasma and platelet von Willebrand factor (VWF). While the existing evidence has revealed a clinical relevance of plasma VWF-Ag in liver regeneration (LR) and different cancers, this study was designed to explore the properties of intra-platelet (IP) and serum VWF-Ag in patients with hepatocellular carcinoma (HCC) undergoing partial hepatectomy. METHODS: A total of 40 patients undergoing partial hepatectomy were prospectively recruited from 3 institutions. VWF-Ag concentrations were evaluated mainly in serum and platelet extracts. Patients were followed-up for postoperative liver dysfunction and HCC recurrence. RESULTS: We observed a post-resection increase in the concentration of VWF-Ag in serum and platelet. Patients with postoperative liver dysfunction had substantially reduced serum and IP VWF-Ag concentrations. After a 2-year follow-up, patients with higher post-resection serum and IP VWF-Ag concentrations were found to develop early HCC recurrence. Likewise, IP VWF-Ag was able to independently predict post-resection early HCC recurrence. CONCLUSION: This multicenter, prospective, pilot study demonstrates a bivalent property of IP VWF in LR and oncological outcome; low preoperative VWF appeared to have a negative association on post-resection liver dysfunction, whereas, patients with higher post-resection VWF-Ag concentrations were found to have early HCC recurrence.