A Pseudo Elevation of CEA Caused by Epidemic Hemorrhagic Fever.

BACKGROUND: Heterophilic antibodies (HA) are one of the main substances that interfere with immunology, especially chemiluminescence immunoassay. Non-specific binding, labeling antibodies, bridging to capture antibodies, or labeling antigens can interfere with the detection process, leading to serious discrepancies between the measured results and clinical manifestations, and even delaying clinical diagnosis and treatment. METHODS: This paper is a case of epidemic hemorrhagic fever causing pseudo CEA elevation caused by heterophagy induced antibodies in the body. RESULTS: The patient's CEA detected on the ABBOTT detection platform was 51.1 ng/mL, and on the ROCHE detection platforms it was 4.66 ng/mL, and treated by PEG precipitation it was 45.2 ng/mL, after diluting the sample the CEA was 50.2 ng/mL, meanwhile the patient's platelets were 96 x 109/L and serum creatinine was 188.4 µmol/L, epidemic hemorrhagic fever IgM antibody was positive. CONCLUSIONS: When the test results do not match clinical symptoms, further confirmation is required through additional testing. Patients who use mouse monoclonal antibody preparations for diagnosis or treatment may have human anti-mouse antibodies in their serum, and the test results may falsely increase or decrease.

Relapse of Nephrotic Syndrome after Adrenocorticotropic Hormone-Induced Remission: Implications of Adrenocorticotropic Hormone Antibodies.

BACKGROUND: Prolonged use of corticosteroids continues to be the mainstay in the management of most proteinuric glomerulopathies, but is limited by extensive side effects. Alternative medications such as adrenocorticotropic hormone (ACTH) have been recently used to treat refractory glomerulopathies and have shown superior outcomes when compared with steroids. However, the clinical responsiveness to ACTH therapy varies considerably with a number of patients exhibiting de novo or acquired resistance. The underlying mechanism remains unknown. METHODS: A patient with steroid-dependent focal segmental glomerulosclerosis (FSGS) developed severe steroid side effects impacting quality of life and was converted to repository porcine ACTH therapy. Immediate response in the form of remission of nephrotic syndrome was noted followed by relapse in 10 weeks. Suspecting the role of some ACTH-antagonizing factors, the patient's serum was examined. RESULTS: Immunoblot-based antibody assay revealed high titers of de novo IgG antibodies in the patient's serum that were reactive to the porcine corticotropin with negligible cross-reactivity to human corticotropin. In vitro, in cultured B16 melanoma cells that express abundant melanocortin receptors, addition of the patient's serum substantially abrogated the porcine corticotropin triggered signaling activity of the melanocortinergic pathway, marked by phosphorylation of glycogen synthase kinase 3ß, thus suggesting a mitigating effect on the biological functionality of porcine corticotropin. CONCLUSION: ACTH is a useful alternative therapeutic modality for refractory proteinuric glomerulopathies like FSGS. However, as quintessential therapeutic biologics, natural ACTH, regardless of purity and origin, is inevitably antigenic and may cause the formation of neutralizing antibodies in some sensitive patients, followed by resistance to ACTH therapy. It is imperative to develop ACTH analogues with less immunogenicity for improving its responsiveness in patients with glomerular diseases.

Xenoantibodies and Complement Activity Determinations by Flow Cytometry in Pig-to-Primate Xenotransplantation.

In pig-to-primate xenotransplantation, flow cytometry assays allow the examination of antibody reactivity to intact antigens in their natural conformation and location on cell membranes. Here we describe in detail the procedures of two flow cytometry assays to measure the antibody-mediated complement-dependent cytotoxicity (CDC) response or serum levels of IgG and IgM xenoantibodies. This information is key for understanding the rejection process of vascularized xenografts and finding strategies to overcome it.

Studying Xenograft Rejection of Bioprosthetic Heart Valves.

Millions of patients with valvular heart disease have benefitted from heart valve replacement since the procedure was first introduced in the 1960s; however, there are still many patients who get early structural valve deterioration (SVD) of their bioprosthetic heart valves (BHV). BHV are porcine, bovine, or equine tissues that have been glutaraldehyde fixed to preserve the tissue and presumably make the tissue immunologically inert. These glutaraldehyde-fixed BHV with anti-calcification treatments last long periods of time in older adults but develop early SVD in younger patients. The consensus at present is that the early SVD in younger patients is due to more "wear and tear" of the valves and higher calcium turnover in younger patients. However, as younger patients likely have a more robust immune system than older adults, there is a new hypothesis that BHV xenografts may undergo xenograft rejection, and this may contribute to the early SVD seen in younger patients.At present, the technology to noninvasively study in vivo whether an implanted BHV in a human patient is undergoing rejection is not available. Thus, a small animal discordant xenotransplant model in young rodents (to match the young patient getting a pig/bovine/equine BHV) was developed to study whether the hypothesis that glutaraldehyde-fixed BHV undergo xenograft rejection had any merit. In this chapter, we describe our model and its merits and the results of our investigations. Our work provides clear evidence of xenograft rejection in glutaraldehyde-fixed tissue, and our small animal model offers an opportunity to study this process in detail.

Interpretation of EBV serology for human body material donors: Is there a need for early antigen IgG and heterophile antibodies testing?

In this report we evaluated a diagnostic algorithm, proposed by the Belgian Superior Health Council, to detect acute and past Epstein-Barr virus (EBV) infections by means of serology in donors of human body material for transplantation. The available EBV serology parameters were tested on eighty serum samples on three random access analysers: Architect i2000 SR, Liasion XL and BioPlex 2200. The EBV sero-status was determined according to the proposed algorithm and results were compared between the different analysers. Seventy one % of the samples gave concordant interpretations on the three analysers. Most of the discordant results were attributable to early antigen (EA) IgG. The knowledge of the EA IgG and heterophile antibodies (HA) IgM status provided only limited added value and was only useful to distinguish between a very early acute infection and false positivity of viral capsid antigen IgM. The diagnostic algorithm proposed by the Belgian Superior Health Council is merely directive and each individual lab remains responsible for the interpretation and implementation of test combinations for the detection of EBV infections. Our study shows the limited added value of testing for EA IgG and HA IgM, based both on clinical and technical performance.

Heterologous Antibodies Adsorption in Xenotransplantation of a Landrace Piglet Kidney Into a Rhesus Monkey.

BACKGROUND: To explore the adsorption of heterologous antibodies in 6 xenotransplants of Landrace piglet kidneys into rhesus monkeys. METHODS: The Landrace piglets and rhesus monkeys were used as donors and recipients, respectively. The donor kidney was the left kidney excised from each Landrace piglet and lavaged with University of Wisconsin solution through the renal artery and vein ex vivo. The renal arteriovenous end of the recipient was preserved. After anastomosis of the renal artery and vein with the arteriovenous end of the recipient for reperfusion, a cross-lymphocyte cytotoxicity test of the heterogeneous kidney was performed. RESULTS: All 6 Landrace piglet kidneys absorbed heterologous antibodies that were pre-existing in the rhesus macaques' kidneys. The cross-lymphocyte toxicity test was performed after the kidney were completely blackened. The cross-lymphocyte toxicity in all each heterogeneous kidney changed from strong positive to weak positive. CONCLUSIONS: Heterologous antibodies were adsorbed in xenotransplants of Landrace piglet kidneys into rhesus monkeys. Xenotransplanted kidney can adsorb heterologous antibodies and consume relevant complements, which is a good model for research of hyperacute rejection in xenotransplantation.

Dose-dependent differential resistance of inbred chicken lines to avian pathogenic Escherichia coli challenge.

Avian pathogenic E. coli (APEC) cause severe respiratory and systemic disease. To address the genetic and immunological basis of resistance, inbred chicken lines were used to establish a model of differential resistance to APEC, using strain O1 of serotype O1:K1:H7. Inbred lines 72, 15I and C.B12 and the outbred line Novogen Brown were inoculated via the airsac with a high dose (107 colony-forming units, CFU) or low dose (105 CFU) of APEC O1. Clinical signs, colibacillosis lesion score and bacterial colonization of tissues after high dose challenge were significantly higher in line 15I and C.B12 birds. The majority of the 15I and C.B12 birds succumbed to the infection by 14 h post-infection, whilst none of the line 72 and the Novogen Brown birds developed clinical signs. No difference was observed after low dose challenge. In a repeat study, inbred lines 72 and 15I were inoculated with low, intermediate or high doses of APEC O1 ranging from 105 to 107 CFU. The colonization of lung was highest in line 15I after high dose challenge and birds developed clinical signs; however, colonization of blood and spleen, clinical signs and lesion score were not different between lines. No difference was observed after intermediate or low dose challenge. Ex vivo, the phagocytic and bactericidal activity of lung leukocytes from line 72 and 15I birds did not differ. Our data suggest that although differential resistance of inbred lines 72, 15I and C.B12 to APEC O1 challenge is apparent, it is dependent on the infectious dose. Research Highlights Lines 15I and C.B12 are more susceptible than line 72 to a high dose of APEC O1. Differential resistance is dose-dependent in lines 15I and 72. Phagocytic and bactericidal activity is similar and dose independent.

The effects of early postoperative immunization with xenogeneic embryo proteins on Lewis lung carcinoma model.

AIM: To investigate the effect of chicken embryo proteins (CEP) as a prototype of xenogeneic vaccine on immune reactions in mice immunized after Lewis lung carcinoma (LLC) surgical removal. MATERIALS AND METHODS: C57Bl male mice were immunized on days 1, 8, and 15 after surgical removal of LLC. The immune response was assessed on days 7, 14, 21 and 28 after tumor resection. Cytotoxic activity of natural killer cells (NK) and cytotoxic T-lymphocytes as well as antibody dependent cellular cytotoxicity was estimated in MTT-assay; specific antibodies were detected in ELISA; lymphocyte proliferation was tested in reaction of in vitro blast transformation. RESULTS: None of the immunized mice developed LLC metastases. Immunization with CEP seems to prevent the potential decrease in NK cell cytotoxic activity and spontaneous blast transformation activity of lymphocytes following the surgically induced stress. Further research on improving immunization schedule and elucidating the mechanisms of NK modulation with CEP is needed.

[False Elevation of Prostate-Specific Antigen Caused by Heterophilic Antibody Interference after Radical Prostatectomy : A Case Report].

We described a 63-year-old man who was diagnosed with clinical T1c prostate cancer, with a Gleason score of 6 (3＋3), and a preoperative prostate-specific antigen (PSA) level of 5. 27 ng/ml. Radical prostatectomy(RP) was performed and final pathologyshowed Gleason score 3＋4, pT2c with negative surgical margin. In spite of suggested surgical radicality, PSA was 3.32, 4.78, 5.93 ng/ml, at 1, 2, and 3 months after RP, respectively. However, radiological investigation revealed no metastasis. Because of this clinical discrepancy, we checked the PSA-α1-antichemotrypsin level and found it to be ≦0.1 ng/ml. From these results, false PSA elevation caused byinterference of positive heterophilic antibodies was suggested and demonstrated byseveral immunoassays.

Xenogeneic cell-based vaccine therapy for colorectal cancer: Safety, association of clinical effects with vaccine-induced immune responses.

An accumulating body of evidence suggests that xenogeneic vaccines can be very effective in breaking the immune tolerance to human tumor-associated antigens (TAAs). We assessed adverse effects, as well as clinical and immune responses induced by a lyophilized xenogeneic polyantigenic vaccine (XPV) prepared from murine melanoma B16 and carcinoma LLC cells in 60 stage IV colorectal cancer patients. Neither grade III/IV toxicities, nor laboratory and clinical signs of systemic severe autoimmune disorders were documented in any XPV-treated patient. Clinical effects of various grades (complete response, partial response and disease stabilization) with duration of no shorter than 6 months was observed in 25 (41.67%) vaccinated patients. The average survival time of the XPV-treated patients was markedly longer than that of the clinically matched control patients (20 vs. 7 months). The overall 3-year survival rate in the XPV-treated and control group was 16.7% (10 patients) and 0%, respectively. Following a course of ten XPV vaccinations, peripheral blood mononuclear cell (PBMC) proliferation assays revealed increased T-cell immune responses to human Caco-2 colon adenocarcinoma-associated antigens. In addition, relative contents of CD25+ FoxP3+regulatory T-cells in patients with proven immunotherapy-mediated clinical effects (responders) were significantly decreased in the blood, which was paralleled by marked increases in serum levels of proinflammatory cytokines, such as interferon-alpha (IFN-α), IFN-É£, and interleukin-8 (IL-8). Serum levels of tumor necrosis factor-alpha (TNF-α), IL-1, IL-4, and IL-6 were not affected in both responder and non-responder patients. In conclusion, this study provides evidence for the safety, clinical feasibility and immunogenicity of xenogeneic composite cell vaccine administration in colorectal cancer patients. This is the first demonstration that clinical effects of such a vaccine are associated with vaccine-induced, proinflammatory immune responses.

Heterophile antibody interference in qualitative urine/serum hCG devices: Case report.

OBJECTIVES: This case report investigates the origin of a false positive result on a serum qualitative human chorionic gonadotropin (hCG) device. PATIENT AND METHODS: A 46-year-old woman diagnosed with chronic myeloid leukemia presented with nausea and vomiting. A qualitative serum hCG test was interpreted as positive; however, a quantitative serum hCG test was negative (<5IU/L). To further investigate this discrepancy, the sample was pretreated with heterophilic blocking reagent (HBR). Additionally, the sample was tested on other qualitative hCG devices composed of antibodies from different animal sources. Blocking reagent from an automated quantitative immunoassay was also tested for its ability to inhibit the heterophile antibody interference. RESULTS: The qualitative test result was negative after pretreatment with heterophilic blocking reagent. Other devices composed of antibodies from different animal sources also demonstrated mixed results with the patient's sample. Blocking reagent obtained from the automated quantitative assay inhibited the heterophile antibody interference in the patient's sample. CONCLUSION: This case demonstrates that positive serum point-of-care hCG results should be interpreted with caution and confirmed with a quantitative serum hCG immunoassay when clinical suspicion is raised.

Repeated spurious elevation of serum prostate-specific antigen values solved by chemiluminescence analysis: A possible interference by heterophilic antibodies.

Heterophilic antibodies are human immunoglobulins directed against various animal antigens. They can produce false-positive results in the analysis of different tumor markers, including prostate-specific antigen. This interference can lead to misdiagnosis, unnecessary tests, and overtreatment in some cases. We present herein the case of a 52-year-old man with repeated spurious elevation of prostate-specific antigen, reaching levels of 108.7 ng/mL, that were suspected to be caused by heterophilic antibodies. The interference was solved by changing the analysis technique. Real values of prostate-specific antigen were less than 1 ng/mL.

Postnatal xenogeneic B-cell tolerance in swine following in utero intraportal antigen exposure.

BACKGROUND: The objective of this study was to investigate the humoral immune response to xenogeneic antigens administered during the fetal state utilizing a baboon-to-pig model. METHODS: Nine fetuses from an alpha-1,3-galactosyltransferase gene knockout (GalT-KO) MGH-miniature swine sow underwent transuterine ultrasound-guided intraportal injection of T-cell depleted baboon bone marrow (B-BM) at mid-gestation. Two juvenile GalT-KO swine undergoing direct B-BM intraportal injection were used as controls. RESULTS: Postnatal humoral tolerance was induced in the long-term surviving piglets as demonstrated by the absence of any antibody response to baboon donor cells. In addition, a second intraportal B-BM administration at 2.5 months post-birth led to no antibody formation despite re-exposure to xenogeneic antigens. This B-cell unresponsiveness was abrogated only when the animal was exposed subcutaneously to third-party xenogeneic and allogeneic antigens, suggesting that the previously achieved humoral non-responsiveness was donor specific. In comparison, the two juvenile GalT-KO control swine demonstrated increasing anti-baboon IgM and IgG levels following intraportal injection. CONCLUSIONS: In summary, xenogeneic B-cell tolerance was induced through in utero intraportal exposure to donor cells and this tolerance persisted following postnatal rechallenge with donor B-BM, but was lost on exposure to third-party antigen, possibly as a result of cross-reactive antibody formation.

Deletion or inhibition of Fc gamma receptor 2B (CD32) prevents FVIII-specific activation of memory B cells in vitro.

Development of inhibitory antibodies against factor VIII (FVIII) is a severe complication of replacement therapy in haemophilia A. Patients with inhibitors are treated with high FVIII doses in the context of immune tolerance therapy (ITT). Data from haemophilia A mouse model suggest that high FVIII concentrations prevent the formation of antibody secreting cells (ASCs) from memory B cells (MBCs) by inducing apoptosis. Fc gamma receptor 2B (CD32) is an important regulator of B cell function, mediating inhibitory signals after cross-linking with the B cell receptor. Here, the role of CD32 in the regulation of FVIII-specific MBCs was investigated using F8-/- and F8-/-CD32-/- knockout mice and monoclonal antibodies (mAbs). The initial immune response was similar between F8-/- and F8-/-CD32-/- mice, including concentration of anti-FVIII antibodies and number of FVIII-specific ASCs in spleen and bone marrow. In contrast, formation of ASCs from MBCs upon rhFVIII re-stimulation in vitro was abolished in F8-/-CD32-/- mice, whereas FVIII/anti-FVIII immune complexes significantly enhanced ASC formation in F8-/- mice. Inhibition of CD32 by mAbs or F(ab)2 fragments prevented ASC formation in a dose-dependent manner. Transfer of B cell-depleted splenocytes using CD45R (B220) depletion from CD32-competent mice did not restore ASC formation in F8-/-CD32-/- cells confirming that CD32 is required on B cells. We conclude that CD32 is a crucial regulator of FVIII-specific B cells and is required for the differentiation of MBCs into ASCs. Inhibition of CD32 could potentially improve the efficacy of FVIII in the context of ITT.

Anti-non-Gal-specific combination treatment with an anti-idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response.

BACKGROUND: B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. METHODS: The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. RESULTS: After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. CONCLUSIONS: This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Production and evaluation of chicken antibodies against a synthetic peptide from glial growth factor / Producción y evaluación de anticuerpos de gallinas contra un péptido sintético del factor de crecimiento glial

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.

Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.

Ischemia-reperfusion injury accelerates human antibody-mediated transplant vasculopathy.

BACKGROUND: The pathogenesis of transplant vasculopathy (TV) is a multifactorial process. We hypothesized that ischemia-reperfusion injury and antibody-mediated damage contribute to the development of TV. METHODS: Human vessels were procured from nine separate donors undergoing cardiac surgery and stored in saline solution on ice until transplantation. BALB/c Rag2IL-2Rγ mice were transplanted with a human vessel graft on day 0. Purified anti-human leukocyte antigen class I antibody (W6/32), isotype control antibody, or saline was injected into recipient mice weekly until day 42, at which point the degree of intimal expansion (IE) of vessels was assessed by histologic analysis. RESULTS: We found that a prolonged cold ischemia time (6-12 hr) alone did not induce IE. In mice that received antibody where vessels were transplanted within 6 hr of procurement, no IE was observed. By contrast, in vessels exposed to more than 6 hr cold ischemia, both W6/32 antibody (30.4%±6.9%) and isotype control antibody (39.5%±6.0%) promoted significant IE (P<0.05 vs. saline [12.4%±1.7%]). Importantly, the isotype control antibody did not cross-react with human tissue. Interestingly, the number of mouse Fc-receptor-positive cells was significantly increased in human vessels exposed to more than 6 hr cold ischemia but only in the presence of antibody (P<0.05). CONCLUSIONS: Antibody, regardless of its specificity, may promote IE in human vessels that are injured through cold ischemia via interaction with Fc-receptor-positive cells. This highlights the importance of controlling the degree of cold ischemia in clinical transplantation in an effort to reduce the risk of TV development.