Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae) / 生物工程学报

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein / 细胞与分子免疫学杂志

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Preparation and identification of rabbit anti-mouse coiled-coil domain containing 189(Ccdc189)polyclonal antibody / 细胞与分子免疫学杂志

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.

Prediction of epitope region and preparation of mouse polyclonal antibody of human Shisa-like protein 1(SHISAL1) / 细胞与分子免疫学杂志

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.

Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein / 中华预防医学杂志

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.

Specificities and isotypes of erythrocytes autoantibodies in patients with warm autoimmune hemolytic anemia / Especificidades e isotipos de autoanticuerpos eritrocitarios en pacientes con anemia hemolítica autoinmune caliente

Introduction: Autoimmune hemolytic anemia is a rare disorder characterized by hemolysis mediated by autoantibodies directed against red blood cells. The demonstration of antibody specificity is a very difficult procedure since autoantibodies in general are nonspecific of antigens and react with all erythrocytes analyzed. Occasionally, specificity is observed against the Rh system antigens. Objective: To determinate the specificity of erythrocytes autoantibodies in DAT positive autoimmune hemolytic anemia by MAIEA technique. Methods: The specificity and isotype of erythrocyte autoantibodies were determined in the eluate of 109 blood samples from patients with warm autoimmune hemolytic anemia, by means of the MAIEA technique and the use of monoclonal antibodies that recognized 11 blood group systems and the protein CD47. Results: In 100 percent of cases autoantibodies against Rh system antigens were detected; in 24 cases we detected autoantibodies of IgA and IgM isotypes that recognized different antigens that were recognized by IgG isotype autoantibodies. For idiopathic and secondary warm autoimmune hemolytic anemias, predominance was observed against three or more specificities. IgG was detected in 99.09 percent of the eluates, IgA in 35.77 percent and IgM in 16.51 percent. The high degree of hemolysis was related to the presence of several isotype autoantibodies against four or more blood group specificities. Conclusions: The MAIEA technique is a sensitive method that can be used to determine the specificities and isotypes of autoantibodies in patients with warm autoimmune hemolytic anemia.

Introducción: La anemia hemolítica autoinmune es un trastorno poco común, caracterizado por hemólisis mediada por autoanticuerpos dirigidos contra los glóbulos rojos. La demostración de la especificidad de los anticuerpos es un procedimiento muy difícil, ya que los autoanticuerpos en general no son específicos de los antígenos y reaccionan con todos los eritrocitos analizados. Ocasionalmente, se observa especificidad contra los antígenos del sistema Rh. Objetivo: Determinar la especificidad de los autoanticuerpos eritrocitarios en pacientes con anemias hemolíticas autoinmunes PAD positivas con el empleo de la técnica MAIEA Métodos: Se determinó la especificidad e isotipo de los autoanticuerpos eritrocitarios en el eluido de 109 muestras de sangre de pacientes con anemia hemolítica autoinmune caliente, mediante la técnica de MAIEA y el uso de anticuerpos monoclonales que reconocieron 11 sistemas de grupos sanguíneos y la proteína CD47. Resultados: En el ciento por ciento de los casos se detectaron autoanticuerpos contra los antígenos del sistema Rh. En 24 casos se descubrió autoanticuerpos de isotipos IgA e IgM que reconocieron diferentes antígenos que fueron a su vez reconocidos por autoanticuerpos de isotipo IgG. Se observó para las anemias hemolíticas autoinmunes calientes idiopáticas y secundarias; predominio frente a tres o más especificidades. Se detectó IgG en el 99,09 por ciento de los eluidos, IgA en 35,77 por ciento e IgM en 16,51 por ciento. El alto grado de hemólisis se relacionó con la presencia de varios isotipos de autoanticuerpos contra cuatro o más especificidades de grupos sanguíneos. Conclusiones: La técnica MAIEA es un método sensible que puede usarse para determinar las especificidades e isotipos de autoanticuerpos en pacientes con anemia hemolítica autoinmune caliente.

Renal transplant patients with preformed anti-HLA antibodies: early biopsy findings and clinical outcomes / Pacientes transplantados renais com anticorpos anti-HLA pré-formados: achados precoces de biópsia e desfechos clínicos

Abstract Introduction: Renal fibrosis is the end point of a process that begins at transplant, with ischemia reperfusion and early inflammation, and progresses over time with immunological and non-immunological phenomena. Early identification of morphological markers and intervention could improve graft function and survival. Objective: to evaluate the correlation between intensity and specificity of pre-transplant anti-HLA antibodies and kidney allograft pathology in order to identify early risk factors or markers of allograft dysfunction. Methods: A retrospective cohort of kidney transplant recipients with pre-transplant anti-HLA antibodies who underwent graft biopsy within the first two years post-transplant was divided into two groups according to the specificity of anti-HLA antibodies: nonspecific (non-DSA, n = 29) and specific (DSA+, n = 16). Kidney graft pathology, renal function, and proteinuria were analyzed. Results: general characteristics were similar in both groups, except for the higher dose of thymoglobulin in DSA+ group (p < 0.05). The non-DSA group had higher scores for glomerulosclerosis, interstitial inflammation (i) and interstitial fibrosis (ci) (p < 0.05) and higher incidence of cell-mediated acute rejection. No statistical difference in incidence of antibody-mediated rejection, renal function, and proteinuria was observed during follow up. Discussion and conclusions: the difference in inflammation scores and interstitial fibrosis may be associated to the higher incidence of acute cell-mediated rejection and polyomavirus nephropathy in the Non-DSA group. We also should take into account the protective effect of higher doses of thymoglobulin, reducing ischemia reperfusion injury in the DSA+ group. The short follow-up might have been insufficient to detect long-term changes in allograft tissue, renal function, and proteinuria.

Resumo Introdução: A fibrose renal é o desfecho de um processo iniciado no transplante, com reperfusão, isquemia e inflamação precoce, que progride ao longo do tempo com fenômenos imunológicos e não imunológicos. A identificação de marcadores morfológicos e a intervenção precoce poderiam melhorar a função e a sobrevida do enxerto. Objetivo: Avaliar a correlação entre intensidade e especificidade de anticorpos anti-HLA pré-transplante alterações histológicas do enxerto renal, de forma a identificar fatores de risco ou marcadores de disfunção precoces do aloenxerto. Métodos: O presente estudo incluiu uma coorte retrospectiva de receptores de transplante renal sensibilizados com anticorpos anti-HLA no pré-transplante submetidos a biópsia de enxerto nos primeiros dois anos após o transplante. Os grupos foram divididos em função da especificidade dos anticorpos anti-HLA: sem anticorpos doador-específicos (não-DSA, n = 29) e com anticorpos doador-específicos (DSA+, n = 16). Alterações histológicas do enxerto renal, função renal e proteinúria foram analisados. Resultados: Os dois grupos tinham características gerais semelhantes, exceto pela dose mais elevada de timoglobulina administrada nos indivíduos do grupo DSA+ (p < 0,05). O grupo não-DSA teve escores mais elevados de glomeruloesclerose, inflamação intersticial (i) e fibrose intersticial (ci) (p < 0,05), além de maior incidência de rejeição celular aguda (RCA). Não foi observada diferença estatística na incidência de rejeição mediada por anticorpos, função renal ou proteinúria durante o seguimento. Discussão e Conclusões: A diferença nos escores de inflamação e fibrose intersticial pode estar associada à maior incidência de RCA e nefropatia por poliomavírus no grupo não-DSA. Devemos considerar ainda o efeito protetor das doses mais elevadas de timoglobulina na redução da lesão por isquemia-reperfusão no grupo DSA+. O curto período de seguimento pode ter sido insuficiente para detectar alterações de longo prazo no tecido do aloenxerto, função renal e proteinúria.

Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ / 生物工程学报

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.

Preparation of monoclonal antibodies against human interleukin-35 and their application in a quantitative ELISA for interleukin-35 detection / 生物工程学报

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12-200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%-5.6% and 5.6%-7.2%, respectively, and the fortified recovery was 89%-103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.

Preparation of anti-hCG antibody-like molecule by using a RAD peptide display system / 生物工程学报

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.

Alloantibodies to High-Incidence Antigen: Review of Cases and Transfusion Experiences in Korea / 대한수혈학회지

Antibodies to high-incidence red blood cell antigens should be considered if panagglutination reactions are noted in all panel cells, and negative reactions to autologous red blood cells are detected on antibody screening and identification tests. In Korea, most of those antibodies are identified through international reference laboratories. To prevent a hemolytic transfusion reaction, antigen-negative red cells should be provided for those patients who have antibodies to red cell antigens. However, this is nearly impossible when the antibody has specificity to high-incidence red cell antigen. In those cases, transfusion of autologous blood, cryopreserved rare blood and the least incompatible blood components can be considered. In the case of surgery, acute normovolemic hemodilution or intraoperative blood salvage can also be considered. For the patients who have antibodies to high-incidence red cell antigens, it should be discussed to set up a national reference laboratory to quickly identify antibody specificities, and to consider establishing rare blood donor registry and frozen rare blood storage/supply system. This article reviews characteristics of antibodies to high-incidence antigens found in Koreans and also the transfusion experiences of those patients based on literature.

Anticuerpo anti-transglutaminasa tisular en adultos con enfermedad celíaca y su relación con la presencia y duración de la dieta libre de gluten / Anti-transglutaminase antibody in adults with celiac disease and their relation to the presence and duration of gluten-free diet

Introducción: El anticuerpo IgA anti-transglutaminasa tisular 2 (tTG2) es un marcador relevante de la enfermedad celíaca. La utilidad de la determinación de IgA anti-tTG2 está bien establecida para el diagnóstico de la patología, sin embargo su uso para el seguimiento de pacientes con dieta libre de gluten (DLG) no se encuentra del todo esclarecido. Objetivo: Determinar los niveles de IgA anti-tTG2 en pacientes adultos paraguayos con enfermedad celíaca y su relación con la presencia y duración de la DLG. Materiales y métodos: En este estudio observacional descriptivo con componente analítico, transversal, se incluyeron pacientes celíacos adultos, sin (n=23) o con (n=49) DLG. Se determinaron por ELISA los niveles séricos de IgA anti-tTG2. Resultados: Todos (100%) los pacientes celíacos sin DLG presentaron niveles séricos positivos de IgA anti-tTG2. Se observaron niveles séricos de IgA anti-tTG2 significativamente elevados en pacientes celíacos sin DLG en comparación con los niveles en pacientes con DLG. El 35% de los pacientes en tratamiento con DLG (promedio de duración de la dieta = 5,7 años) presentaron niveles positivos (29%) o indeterminados (6%) de IgA anti-tTG2. En relación con la duración de la DLG se observó que al aumentar el tiempo de DLG disminuyen los niveles del auto-anticuerpo (r=-0,2963; p=0,0387). Conclusiones: Los niveles de IgA anti-tTG2 se correlacionaron inversamente con la duración de la DLG. Sin embargo, niveles positivos del anticuerpo persistieron en algunos pacientes, incluso varios años después del inicio de la DLG.

IgA anti-transglutaminase 2 (tTG2) antibody is a relevant marker in celiac disease. The utility of IgA anti-tTG2 determination is well established for the diagnosis, however their use in the follow-up of patients with gluten free diet (GFD) it is not fully established. Objective: To determine IgA anti-tTG2 antibody levels in adult Paraguayan celiac disease patients and its relation to the presence and duration of the GFD. Materials and methods: Adult celiac disease patients without (n=23) or with (n=49) GFD were included in this observational, descriptive, cross-sectional study with analytical component. IgA anti-tTG2 antibody serum levels were analyzed by ELISA. Results: All (100%) celiac disease patients without GFD had positive anti-tTG2 IgA. Serum levels of IgA anti-tTG2 were significantly elevated in celiac disease patients without GFD compared to levels in patients with GFD. 35% of patients treated with GFD (diet average duration = 5.7 years) had positive (29%) or indeterminate (6%) levels of IgA anti-tTG2. In terms of GFD duration we observed that while the GFD period increased, antibody levels decreased (r=0.2963; p=0.0387). Conclusion: IgA anti-tTG2 antibody levels correlated inversely with the GFD duration. However, positive levels of these antibodies persisted in some patients, even several years after the onset of GFD.

Screening of Serum Biomarkers for Distinguishing between Latent and Active Tuberculosis Using Proteome Microarray / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To identify potential serum biomarkers for distinguishing between latent tuberculosis infection (LTBI) and active tuberculosis (TB).</p><p><b>METHODS</b>A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum samples from patients with LTBI and active TB at the systems level. The interaction network and functional classification of differentially expressed antigens were analyzed using STRING 10.0 and the TB database, respectively. Enzyme-linked immunosorbent assays (ELISA) were used to validate candidate antigens further using 279 samples. The diagnostic performances of candidate antigens were evaluated by receiver operating characteristic curve (ROC) analysis. Both antigen combination and logistic regression analysis were used to improve diagnostic ability.</p><p><b>RESULTS</b>Microarray results showed that levels of 152 Mycobacterium tuberculosis (Mtb)-antigen- specific IgG were significantly higher in active TB patients than in LTBI patients (P < 0.05), and these differentially expressed antigens showed stronger associations with each other and were involved in various biological processes. Eleven candidate antigens were further validated using ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031c, Rv1408, and Rv2421c had higher areas under the curve (AUCs) of 0.8520, 0.8152, and 0.7970, respectively. In addition, both antigen combination and logistic regression analysis improved the diagnostic ability.</p><p><b>CONCLUSION</b>Several antigens have the potential to serve as serum biomarkers for discrimination between LTBI and active TB.</p>

Difference in Characteristics in the Formation of Anti-E and Anti-E/-c in Patients with the CDe Phenotype / 대한수혈학회지

BACKGROUND: Anti-E or paired anti-E/-c antibodies can develop in patients with the Rh CDe phenotype. This study examined the differences in transfusion in patients with the CDe phenotype according to formation of anti-E or anti-E/-c antibodies. METHODS: Retrospective reviews were carried out on the results of antibody identification tests performed in 2014. The Rh phenotype and antibody specificity were investigated. The transfusion and medical records of patients with the CDe phenotype were examined. RESULTS: In total, 76 patients were included in the review. Of these 76 patients, 38 (50.0%) were of the CDe phenotype. Anti-E antibodies were the most frequent (60.5%), followed by anti-E/-c antibodies (23.7%). The total transfusion units and platelet transfusion units were significantly higher in patients with anti-E/-c antibodies (P=0.028 and P=0.01, respectively). The distribution of categorized diseases was similar in the patients with the anti-E and anti-E/-c antibodies. A frequency of transfusion episodes greater than or equal to four was higher in patients with hepatobiliary diseases (85.7%). CONCLUSION: In CDe phenotype patients, platelet transfusion was significantly higher in the anti-E/-c positive group than the anti-E positive group, indicating that platelets play a role in red blood cell alloimmunization. Because E is the most immunogenic antigen in Korea, it is important to define the disease group, in which patients with CDe phenotype require a transfusion of E and c-negative blood.

Colangitis biliar primaria. Parte 1. Actualización: generalidades, epidemiología, factores involucrados, fisiopatología y manifestaciones clínicas / Primary biliary cholangitis. Part 1. State of the art, epidemiology, physiopathology and clinical manifestations

La colangitis biliar primaria (CBP), es una colangiopatía crónica caracterizada por la destrucción selectiva de las células epiteliales biliares de conductos hepáticos de pequeño y mediano calibre, que afecta principalmente a mujeres. Los principales síntomas son la fatiga y el prurito, sin embargo, gran porcentaje de los pacientes pueden ser asintomáticos. El diagnóstico se basa en anticuerpos antimitocondriales (AMA) con títulos >1:40, fosfatasa alcalina >1,5 veces del límite superior normal por más de 24 semanas e histología hepática compatible con la patología. Se asocia con múltiples enfermedades principalmente de carácter autoinmune extra hepáticas, enfermedades tiroideas, óseas, entre otras. El tratamiento de primera línea es el ácido ursodesoxicólico (AUDC) que a pesar que no cura la enfermedad, mejora las pruebas del perfil hepático, así como el retraso en la progresión a cirrosis. Actualmente se encuentran en estudio nuevos tratamientos y terapias adyuvantes. El propósito de esta revisión es ofrecer una actualización de este tema que se presenta en los servicios de medicina interna y gastroenterología; para su realización se conformó un equipo interdisciplinario que desarrolló una búsqueda en la base Medline a través de PubMed con las palabras claves correspondientes y se procedió a una lectura crítica y analítica de títulos, resúmenes y textos completos para el filtro, extracción y síntesis de la información encontrada

Primary biliary cholangitis (PBC) is a chronic autoimmune cholangiopathy characterized by a selective destruction of biliary epithelial cells of small and medium caliber hepatic ducts, which mainly affects women. The main symptoms are fatigue and pruritus, however, a large proportion of patients may be asymptomatic. The diagnosis is based on AMA titers >1:40, alkaline phosphatase >1.5 times the upper limit for more than 24 weeks and compatible liver histology. It is associated with multiple autoimmune diseases mainly extrahepatic, thyroid diseases, bone diseases, among others. The first line treatment is ursodeoxycholic acid (UDCA), that improves liver function tests and delay the progression to cirrhosis. Currently, there are new treatments and adjuvant therapies on study. The purpose of this review is to offer an update in this topic, which is very important in gastroenterology and internal medicine. We formed an interdisciplinary team to search in the database Medline thorough PubMed with the key words describe below, we made a critical lecture of the titles and abstracts of each article to write this paper

Polyspecific antibodies without persisting antigen in multiple sclerosis, neurolupus and Guillain-Barré syndrome: immune network connectivity in chronic diseases / Anticorpos poliespecíficos sem antígeno persistente em esclerose múltipla, neurolupus e síndrome de Guillain-Barré: conectividade imunológica em doenças crônicas

ABSTRACT The polyspecific antibody synthesis in multiple sclerosis (MS) gained diagnostic relevance with the frequent combination of measles-, rubella- and varicella zoster antibodies (MRZ-antibody reaction) but their pathophysiological role remains unknown. This review connects the data for intrathecal polyspecific antibody synthesis in MS and neurolupus with observations in the blood of patients with Guillain-Barré syndrome (GBS). Simultaneously increased antibody and autoantibody titers in GBS blood samples indicate that the polyspecific antibodies are based on a general property of an immune network, supported by the deterministic day-to-day concentration variation of antibodies in normal blood. Strongly correlated measles- and rubella- antibody variations point to a particular connectivity between the MRZ antibodies. The immune network, which provides serological memory in the absence of an antigen, implements the continuous change of the MRZ pattern in blood, not followed by the earlier immigrated B cells without corresponding connectivity in the brain. This may explain the different antibody patterns in cerebrospinal fluid, aqueous humor and blood of the individual MS patient. A complexity approach must implement a different view on causation in chronic diseases and causal therapies.

RESUMO A síntese de anticorpos poliespecíficos em esclerose múltipla (EM) ganhou relevância diagnóstica com a combinação frequente de anticorpos contra sarampo, rubéola e varicela-zoster (reação de anticorpos MRZ), mas seu papel fisiopatológico permanece desconhecido. Esta revisão relaciona os dados da síntese intratecal de anticorpos poliespecíficos em EM e Neurolupus com observações no sangue de pacientes com síndrome de Guillain Barré (SGB). Simultaneamente, os títulos aumentados de anticorpos e autoanticorpos em amostras de sangue de SGB indicam que os anticorpos poliespecíficos se baseiam numa propriedade geral de uma rede imunitária, suportada pela variação determinística da concentração diária de anticorpos no sangue normal. As variações fortemente correlacionadas de anticorpos contra sarampo e rubéola apontam para uma conectividade particular entre os anticorpos MRZ. A rede imunitária, que fornece memória sorológica na ausência de um antígeno, implementa a mudança contínua do padrão MRZ no sangue, não seguida pelas células B que imigraram anteriormente sem conectividade no cérebro. Isto pode explicar os diferentes padrões de anticorpos no LCR, humor aquoso e sangue do paciente individual de EM. Uma abordagem complexa deve implementar uma visão diferente sobre a causalidade em doenças crônicas e terapias causais.

False-Positive T-Cell Cytotoxicity Crossmatch Results Due to Autoantibodies in Korean Network for Organ Sharing Crossmatch Tests / 대한이식학회지

BACKGROUND: Basic National Institute of Health (NIH) and sensitive antihuman globulin (AHG) methods are widely used for T-cell complement-dependent cytotoxicity crossmatch (XM) tests. Whereas NIH-negative, AHG-positive (NIH⁻/AHG⁺) results are caused by weak antibodies, NIH⁺/AHG⁻ results are usually due to autoantibodies. We found that solid organ transplantation candidates with NIH⁺/AHG⁻ XM results are repeatedly excluded from allocation of deceased donor organs by the Korean Network for Organ Sharing (KONOS) allocation system. Here, we attempted to demonstrate that these patients do not have donor-specific HLA antibodies (DSAs). METHODS: Sera showing NIH⁺/AHG⁻ results in the analysis of 1,668 KONOS T-cell XM tests were screened for panel reactive antibody (PRA) using a Luminex test. For screen-positive samples, antibody identification was conducted using a Luminex single antigen assay and the presence or absence of class I DSAs was determined. For positive controls, 42 KONOS XM tests showing probable true-positive (NIH⁻/AHG⁺ or NIH⁺/AHG⁺) results were reviewed for PRA results based on electronic medical records and the presence or absence of DSAs was determined. RESULTS: NIH⁺/AHG⁻ results were observed in 1.3% (21/1,668) of KONOS XM tests analyzed. Most of these (18/21, 85.7%) were negative for PRA or DSAs. All probable true-positive cases were either positive for DSAs (24/42, 57.1%) or had high PRA (mean, 92% [range; 42%~100%]), complicating accurate identification of antibody specificities. CONCLUSIONS: NIH⁺/AHG⁻ results are not rare (1.3%) in KONOS XM tests. Most of these results are not due to DSAs, and these patients should not be excluded from organ allocation.

Effect of pre-transplant donor specific antibody on antibody-mediated rejection and graft dysfunction / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of pre-transplant donor specific antibody (DSA) on antibody-mediated rejection (AMR) and function of transplanted kidney.
@*METHODS@#A total of 88 cases of renal transplant recipients were selected. Before surgery, DSA was examined by Luminex liquid phase chip in renal transplant recipients. The recipients were divided into a DSA positive group (n=20) and a DSA negative group (n=68). The follow-up time was 2 years. After the operation, the pathologic morphology of the transplanted kidney was evaluated and classified according to the Banff 2005 standard. The situation for the transplanted kidney was evaluated.
@*RESULTS@#The incidence of AMR in the DSA positive group and negative group was 20% and 1.5%, respectively, with significant difference between them (P0.05).
@*CONCLUSION@#The detection of DSA level before renal transplantation can predict the risk of AMR and the function of transplanted kidney. The MFI intercept point of the highest DSA (MFI>
7909.5) can be used to predict the risk of AMR.

Standardization of Weed Pollen Extracts, Japanese Hop and Mugwort, in Korea

PURPOSE: Japanese hop (Humulus spp.) and mugwort (Artemisia spp.) are notable causes of autumn pollinosis in East Asia. However, Japanese hop and mugwort pollen extracts, which are widely used for the diagnosis, have not been standardized. This study was performed to standardize Japanese hop and mugwort pollen extracts. MATERIALS AND METHODS: Allergen extracts were prepared in a standardized way using locally collected Humulus japonicus and purchased Artemisia vulgaris pollens. The immunoglobulin E (IgE) reactivities of prepared extracts were compared with commercial extracts via IgE immunoblotting and inhibition analyses. Intradermal skin tests were performed to determine the bioequivalent allergy unit (BAU). RESULTS: The IgE reactive components of the extracts via IgE immunoblotting were similar to those of commercial extracts. A 11-kDa allergen showed the strongest IgE reactivity in Japanese hop, as did a 28-kDa allergen in mugwort pollen extracts. Allergenic potencies of the investigatory Japanese hop and mugwort extracts were essentially indistinguishable from the commercial ones. Sums of erythema of 50 mm by the intradermal skin test (SigmaED50) were calculated to be 14.4th and 13.6th three-fold dilutions for Japanese hop and mugwort extracts, respectively. Therefore, the allergenic activity of the prepared extracts was 90827.4 BAU/mg for Japanese hop and 34412 BAU/mg for mugwort. CONCLUSION: We produced Japanese hop and mugwort pollen extracts using a standardized method. Standardized Japanese hop and mugwort pollen extracts will facilitate the production of improved diagnostic and immunotherapeutic reagents.