Heterogeneity Of Antibodies Research Articles

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Introduction: Common variable immune deficiency (CVID) is the most common form of primary immunodeficiency (prevalence 1: 10,000 – 50,000). CVID is a heterogeneous antibody deficiency syndrome characterized by hypogammaglobulinemia and immune dysregulation. Pulmonary manifestations are most common,

Long‐term anti‐FVIII antibody response in Bethesda‐negative haemophilia A patients receiving continuous replacement therapy

It has previously been shown that patients with haemophilia A may develop non-neutralizing anti-factor VIII (FVIII) antibodies (NNA) that escape detection by the Bethesda assay, but are detected using immune-based assays. We and others found NNAs to be directed not only towards non-functional parts of the protein, but towards all regions of the FVIII protein. We also showed a heterogeneous antibody response towards different FVIII products. However, the clinical relevance and the natural history of NNA remain unclear. Therefore, we followed a cohort of unrelated subjects with haemophilia A for 4years with the goal of exploring the long-term development of NNA using an enzyme-linked immunosorbent assay (ELISA). Ten of 78 subjects (12·8%) exhibited an immune response that was transient and heterogeneous, and none of the subjects developed an FVIII inhibitor. The result of the ELISA was examined in relation to clinical variables and no significant associations between a positive ELISA and age, F8 mutation, port-à-cath implantation and HCV infection were shown. Interestingly, patients with NNA had significantly fewer bleeding episodes (P=0·048) compared with NNA-negative subjects. The results indicate that the immune response to FVIII products within an individual may vary over time. However, the clinical impact of NNA remains unclear.

Variabler Immundefekt

Common variable immunodeficiency (CVID) represents the most common clinically relevant form of primary immunodeficiency. This heterogeneous antibody deficiency syndrome is characterized not only by susceptibility to bacterial respiratory tract infections but displays additional signs of immune dysregulation, such as autoimmunity, chronic inflammation and lymphoproliferation in more than 30 % of the patients. Due to poor awareness the diagnosis is often delayed by 4-6 years. A close collaboration in patient care with a center specialized in primary immunodeficiency is recommended. Regular follow-up visits include assessment of adequate immunoglobulin replacement therapy and screening for manifestation of secondary complications. Regular substitution with intravenous or subcutaneous immunoglobulins has more or less normalized life expectancy of patients with isolated susceptibility to bacterial infections. Therefore, the current core task in the management of CVID patients is the elaboration of more effective and safer forms of prophylaxis and treatment of sequelae of immune dysregulation in the lungs, intestines and liver of affected patients.

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

BackgroundInfluenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.ResultsBoth H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.ConclusionOnly in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.

From Phenomenology to Mechanism: Resistance to Annexin A5 Anticoagulant Activity Identifies a Subset of Patients with Antiphospholipid Antibodies and Thrombosis

Abstract 631 Background:The antiphospholipid (aPL) syndrome (APS) is a diagnostic category that is defined by non-mechanistic empirically-derived assays - the lupus anticoagulant (LA) assay and aPL-antibody immunoassays for anticardiolipin and anti-ß2 glycoprotein-I antibodies. APS patients have heterogeneous antibodies and it is likely that several mechanisms are involved in the disease process. Annexin A5 (AnxA5) is a potent anticoagulant protein that forms 2-dimensional crystals over membranes that express phosphatidyl serine and shields the phospholipid from availability for phospholipid-depended coagulation reactions. We previously demonstrated that aPL antibodies can disrupt this crystallization and accelerate coagulation reactions. We therefore investigated whether disruption of AnxA5 anticoagulant activity might mark patients with histories for aPL antibody associated thrombosis. Design:We investigated samples from 966 adult patients from a large urban academic medical center with the following categories of histories: thrombosis, including venous thromboembolism (VTE) and stroke (n=433); pregnancy complications attributable to placental insufficiency (n=218); autoimmune disease (n=279); cancer (n=63); renal disease (n=101); liver disease (n=51); HIV (n=23); and free of known disease (n=140) (total >966 because of concurrent diagnosis). An assay that detects annexin A5 resistance (A5R) was performed on all samples. Results were expressed as “% AnxA5 anticoagulant activity”. A quintile analysis was performed to obtain cutoffs for A5R values. Values among groups were compared using student t-test, one way ANOVA and odds ratio analysis. Results:aPL-antibody positive patients with thrombosis (VTE and/or stroke) have significantly lower mean A5R values (194±57, n=76) than aPL-antibody negative patients with a thrombotic event (238±41, n=175; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 54% (41/76) of patients in the aPL-antibody positive and thrombosis group had A5R values within the lowest quintile of results (<199%), in contrast to 15% (26/175) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and thrombosis (OR: 13.7, p<0.001). aPL-antibody positive patients with pregnancy complications (PCs) have significantly lower A5R values (218±45, n=111) than aPL-negative patients with pregnancy complications (248±43, n=107; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 28% (31/111) of patients in the aPL-antibody positive and pregnancy complication group had A5R values within the lowest quintile of results (<199%), in contrast to 8% (9/107) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and pregnancy complications (OR: 4.6, p<0.001). There were no significant differences in AnxA5 anticoagulant activity in patients with cancer, HIV, and renal disease, however there were significantly lower A5R values in patients with autoimmune disease and liver disease. The latter appeared to correlate with severity of liver disease. Patients with triple aPL-antibody positivity or greater had significantly lower mean A5R values (155±48%, n=23, p<0.001), than patients with double positivity (193±55%, n=44, p<0.01), or single positivity (214±47, n=111, p<0.05). All aPL-positive groups had significantly lower A5R values than patients who were negative for aPL antibodies (240±43%, p<0.001). [Display omitted] Conclusion:A subset of ∼50% of aPL-antibody positive patients with thrombosis and ∼30% with pregnancy complications showed evidence for this mechanism - resistance to annexin A5 anticoagulant activity. The identification of specific mechanisms for aPL-antibody associated thrombosis and pregnancy complications offers the possibilities of improving diagnosis and developing targeted treatments for this enigmatic disorder. Disclosures:No relevant conflicts of interest to declare.

Hashimoto’s encephalopathy cases: Chinese experience

BackgroundHashimoto’s encephalopathy is a poorly understood syndrome consisting of heterogeneous neurological symptoms and high serum antithyroid antibody titers, typically responding to steroids. More clinical series studies are required to characterize the clinical, laboratory and imaging features, and outcomes, especially in the Chinese population.MethodsWe analyzed the clinical, laboratory, and imaging features and outcomes of thirteen consecutive patients with Hashimoto’s encephalopathy diagnosed in Xuan Wu Hospital, Beijing from 2005 to 2010 retrospectively.ResultsCognitive impairment (84.6%) and psychiatric symptoms (38.5%) were the most frequent symptoms. Seizures (30.8%) and myoclonus (7.7%) were less common than previously described. Three (23.1%) patients showed abnormal signals in hippocampus or temporal lobe, which were believed related to their memory disorders or seizures. MRI changes showed resolution paralleling clinical improvement in one patient. Among eight patients who received steroid therapy, five patients recovered, one patient improved with residual deficits, and two patients relapsed or had no effect. Among five non-steroid treated patients, three patients experienced stable remission with antiepileptic drugs or general neurotrophic therapy, and two patients experienced continuous deterioration.ConclusionsMost patients with Hashimoto’s encephalopathy showed good response to steroids. Some patients improved without steroid therapy. Considering its reversible course, we recommend that Hashimoto’s encephalopathy should always be in the differential diagnosis while evaluating disorders of the central nervous system.

Pulsed high intensity focused ultrasound increases penetration and therapeutic efficacy of monoclonal antibodies in murine xenograft tumors

The success of radioimmunotherapy for solid tumors remains elusive due to poor biodistribution and insufficient tumor accumulation, in part, due to the unique tumor microenvironment resulting in heterogeneous tumor antibody distribution. Pulsed high intensity focused ultrasound (pulsed-HIFU) has previously been shown to increase the accumulation of 111In labeled B3 antibody (recognizes Lewisy antigen). The objective of this study was to investigate the tumor penetration and therapeutic efficacy of pulsed-HIFU exposures combined with 90Y labeled B3 mAb in an A431 solid tumor model. The ability of pulsed-HIFU (1MHz, spatial averaged temporal peak intensity=2685Wcm−2; pulse repetition frequency=1Hz; duty cycle=5%) to improve the tumor penetration and therapeutic efficacy of 90Y labeled B3 mAb (90Y-B3) was evaluated in Ley-positive A431 tumors. Antibody penetration from the tumor surface and blood vessel surface was evaluated with fluorescently labeled B3, epi-fluorescent microscopy, and custom image analysis. Tumor size was monitored to determine treatment efficacy, indicated by survival, following various treatments with pulsed-HIFU and/or 90Y-B3. The pulsed-HIFU exposures did not affect the vascular parameters including microvascular density, vascular size, and vascular architecture; although 1.6-fold more antibody was delivered to the solid tumors when combined with pulsed-HIFU. The distribution and penetration of the antibodies were significantly improved (p-value<0.05) when combined with pulsed-HIFU, only in the tumor periphery. Pretreatment with pulsed-HIFU significantly improved (p-value<0.05) survival over control treatments.

Aspects immunologiques du syndrome des antiphospholipides

Antiphospholipid antibodies constitute a group of heterogeneous antibodies, which mainly recognize complexes made of proteins and anionic phospholipids. The nature of these complexes is currently better defined, as well as known, the structure of the antiphospholipid antibodies owing to the analysis of the monoclonal forms of these antibodies which were also studied both in terms of their precise specificities and cross-reactivity. However, the origin of these autoantibodies is not clearly understood, as well as the possible link between antiphospholipid antibodies present in healthy individuals, and those observed in autoimmune diseases. Only a fraction of antiphospholipid antibodies are pathogenic and directly responsible for the clinical manifestations of the antiphospholipid syndrome, but there is, to date, no biological test able to accurately detect pathogenic antiphospholipid antibodies. The diverse mechanisms which link these autoantibodies to the occurrence of symptoms, mainly during obstetrical complications, are better understood, and suggest new therapeutic avenues.

Analysis of bulk and virus-specific CD8+ T cells reveals advanced differentiation of CD8+ T cells in patients with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous antibody deficiency syndrome with alterations in T cell regulation and function in a subgroup of patients. We assessed phenotype and function of bulk and virus-specific CD8+ T cells of a cohort of 34 HLA-A2+ CVID patients by pentamer technology and flow cytometry in relationship to their immunological and clinical phenotypes. Bulk CD8+ T cells displayed a shift toward a more antigen experienced and activated differentiation state. The advanced differentiation pattern was mainly found in a subgroup of CVID patients with lymphadenopathy and granulomatous disease. This effect existed independently of the patients' CMV status even so CMV-associated immunosenescence was more evident in CVID patients than in CMV-positive immunocompetent controls. As the phenotype and function of virus-specific CD8+ T cells were normal in CVID the induction of antiviral immunity by prophylactic immunization appears to be a logical and desirable aim for this group of patients.

Pathophysiology of β2-glycoprotein I in antiphospholipid syndrome

Since beta(2)-glycoprotein I (beta(2)GPI) was described as the major antigenic target for antiphospholipid antibodies, many studies have focused their attention to the physiological role of beta(2)GPI and anti-beta(2)GPI antibodies on autoimmune-mediated thrombosis. Studies reporting the physiological role of beta(2)GPI have been numerous, but the exact mechanism of action(s) has yet to be completely determined. beta(2)GPI's epitopes for anti-beta(2)GPI autoantibodies have been characterized, however, not all of the heterogeneous anti-beta(2)GPI antibodies are pathogenic. The pathophysiologic role of beta(2)GPI has been reported in the fields of coagulation, fibrinolysis, angiogenesis, and atherosclerosis. Our understanding of the impact of beta(2)GPI, its metabolites and autoantibodies to beta(2)GPI on these physiological functions may contribute to the development of better therapeutic strategies to treat and prevent autoimmune-mediated atherothrombotic vascular disease.

In-solution virus capture assay helps deconstruct heterogeneous antibody recognition of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) on whole virions is heterogeneous, so molecular analysis of Env with monoclonal antibodies (MAbs) is challenging. Virus capture assays (VCAs) involving immobilized MAbs are typically used, but these assays suffer from immobilization artifacts and do not provide binding constants. Furthermore, we show here that certain HIV-1 neutralizing MAbs, including 2G12, 4E10, 2F5, Z13e1, and D5, will capture virion particles completely devoid of Env. We modified the VCA such that MAbs and virions are incubated in solution, and unbound MAbs are removed prior to the capture step. This modification nearly eliminated evidence of Env-independent binding by MAbs to virions and allowed determination of apparent affinity constants in solution. Three important qualitative observations were further revealed. First, neutralizing MAbs 2F5, 4E10, and Z13e1 against the membrane-proximal external region (MPER) of HIV-1 gp41 were found to capture virions efficiently only if a significant amount of uncleaved gp160 or synthetic MPER peptide was present. Second, we show how non-native forms of Env vary by Env genotype and that Env from HIV-1(JR-FL) is more homogeneously trimeric than that from HIV-1(JR-CSF). Third, we determined that Env containing all or parts of gp41, including uncleaved gp160, binds spontaneously to free virions. This exogenous Env is an indiscriminate molecular "bridge" between Env-specific Ab and virions and can affect VCA analyses, particularly using pseudotyped virions. Heterogeneity in Env from endogenous and exogenous sources might also subvert humoral immunity to HIV-1, so in-solution VCAs may help to dissect this heterogeneity for vaccine design purposes.

Monitoring the Structural Changes of Conjugated Antibodies by High-Resolution Electron Microscopy and Individual-Particle Electron Tomography

Peptides have promise as potent and selective drug candidates but have not succeeded primarily because of poor pharmacokinetics. The fusion of these peptides to our scaffold antibody has produced molecules, CovX-Bodies, which protect the peptide from renal elimination and/or enzymatic degradation. However, it is possible that the fusion process may in itself affect the intrinsic properties of the antibody scaffold (eg. effector function). Changes in function may relate to changes in structure. However, the structural study of highly dynamic and structurally heterogenous antibodies by X-ray crystallization and NMR is extremely difficult. These studies were designed to examine structural changes that may have occurred during the conjugation process.Here, we studied the structural changes of antibodies with the high-resolution electron microscopy and individual-particle electron tomography (IPET, for details, see the abstract presented by Lei Zhang and Gang Ren). We found 1) the average angle between the Fab regions was 55 degrees +-15 in unconjugated antibodies, but 40 degrees +-10 in unconjugated antibodies and 2) the average sizes of the Fc domain were similar regardless of conjugation. However, 3) the Fc domain shape differed significantly depending on conjugation. The Fc domains of conjugated antibodies were significantly elongated (>30%). Using IPET, we reconstructed a dozen of three-dimensional density maps of individual conjugated and unconjugated antibodies. Comparing the maps, it showed that all three domains of conjugated antibodies were elongated after fusing with the drug. This example demonstrates that this method could be a useful tool for monitoring the structural changes of antibodies.

Frequency of Treg Cells Is Reduced in CVID Patients with Autoimmunity and Splenomegaly and Is Associated with Expanded CD21lo B Lymphocytes

Common variable immunodeficiency is a heterogeneous antibody deficiency syndrome with autoimmune and inflammatory complications in a significant proportion of patients. The study was designed to evaluate the role of T regulatory (Treg) cells in common variable immunodeficiency (CVID) patients with autoimmunity. The number and frequency of Treg cells (CD4(+), CD25(hi), Foxp3(+)) were evaluated in patients and controls, and Foxp3 expression in different subgroups of CVID patients with common clinical manifestations was compared. CVID patients had significantly fewer Treg cells than controls, and low frequency of Treg cells was associated with expansion of CD21(lo) B cells in patients. Patients with autoimmunity had significantly reduced frequency but normal numbers of regulatory T cells, whilst patients with splenomegaly had significant reduction in frequency and number of regulatory T cells. Foxp3 is useful on its own or as an adjunct to classify CVID patients although the possibility of reduction in Treg cells as a secondary phenomenon cannot be excluded.

Pathophysiology of Anti-PL Syndrome in Thrombosis.

Abstract SCI-45The antiphospholipid (aPL) syndrome (APS) is an enigmatic autoimmune thrombotic disorder that was initially identified through astute clinical observations and the development of a quantitative test for aPL antibodies. The condition is marked by 2 types of assays: 1) immunoassays that were derived from the “biologic false positive syphilis” test and 2) coagulation assays that detect “lupus anticoagulants” (LAs), which are inhibitors of phospholipid-dependent coagulation reactions. The most recent consensus investigational criteria for diagnosing APS require that patients have evidence for thrombosis and/or pregnancy complications attributable to placental insufficiency and also laboratory evidence for persistent (aPL) antibodies, detected by high levels of IgG or IgM antibodies against cardiolipin and β2-glycoprotein I (β2GPI) and/or through abnormal LA assays. The thrombotic disorder requires long term anticoagulant treatment, which is accompanied by the risk of bleeding complications. Several mechanisms have been postulated and can be classified as involving: 1) aPL antibody-mediated inhibition of endogenous anticoagulant mechanisms; 2) aPL antibody-triggered signaling events on target cells (vascular endothelial cells, monocytes, platelets and trophoblasts) that promote proadhesive and prothrombotic phenotypes; and 3) aPL antibody-mediated complement activation. At present, it appears that the APS diagnostic entity actually includes several distinct subsets that reflect the actions of heterogeneous antibodies directed against different epitopes of phospholipid-binding proteins which then may yield different clinical sequelae. We have accumulated significant data indicating that a major one of these mechanisms involves aPL antibody-mediated disruption of annexin A5 (AnxA5) activity. AnxA5 is a potent anticoagulant protein whose anticoagulant properties are a consequence of its high affinity for anionic phospholipid. The protein forms 2-dimensional crystals on phospholipid surfaces that shield the phospholipids from availability for coagulation reactions. AnxA5 appears to play a thrombomodulatory role on the surfaces of cells lining the placental and systemic vasculatures. It is highly expressed on the apical membranes of placental syncytiotrophoblasts, the location where maternal blood interfaces with fetal cells. aPL antibody-antigen complexes disrupt the ordered crystallization of AnxA5, displace the protein from phospholipid membrane surfaces and thereby accelerate coagulation reactions. This effect of the antibodies has been demonstrated on artificial bilayers, on cultured trophoblasts and on endothelial cells and platelets. This disruption has been appears to be a consequence of aPL antibodies that recognize a specific epitope within domain I of β2GPI, the key antigen recognized by aPL antibodies. Based upon these data, we developed a novel clinical assay “the AnxA5 resistance (A5R) test” to identify patients who have antibodies that interfere with the anticoagulant activity of AnxA5. Initial studies indicate that a large proportion of APS patients have evidence for A5R. It therefore appears possible that measurement for A5R may provide a mechanistic assay for APS. We are also developing treatments to target this mechanism and protect AnxA5 from antibody-mediated disruption which may open novel nonanticoagulant approaches to treating APS. DisclosuresOff Label Use: The presentation will include research on in vitro effects of hydroxychloroquine; the drug is not FDA approved for the treatment of patients with antiphospholipid syndrome who do not also have concurrent systemic lupus erythematosus or rheumatoid arthritis.

Possible Involvement of Chemokine‐induced Platelet Activation in Thrombophilic Diathesis of Antiphospholipid Syndrome

Among the heterogeneous antiphospholipid antibodies, many studies suggest that those directed to beta2-glycoprotein I (beta2GPI) are the major pathogenic antibodies in antiphospholipid syndrome (APS). They have been shown to activate the coagulation pathway via several mechanisms, activate platelets via thrombin formation, and suppress fibrinolysis. Additionally, we propose another possible mechanism that involves certain chemokines and results in platelet activation. This hypothesis is based on the observations that anti-beta2GPI antibodies stimulated monocytes to secrete inflammatory cytokines such as IL-1beta and TNF-alpha, which in turn stimulated vascular endothelial cells to express chemokines such as CX3CL1 and CCL5. CX3CL1 increased the ability of normal platelets to adhere to collagen at a high shear rate, while CCL5 induced platelet aggregation. Expression of tissue factor, IL-1beta, and TNF-alpha by monocytes stimulated with anti-beta2GPI antibodies, as well as CX3CL1 and CCL5 by vascular endothelial cells stimulated with IL-1beta or TNF-alpha were all suppressed by the NF-kappaB-specific inhibitor DHMEQ. These results suggest that the NF-kappaB pathway may be a potential therapeutic target relating to both the coagulation pathway and platelet activity.

Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in HIV-1 elite controllers

To determine the spectrum of antiviral antibodies in HIV-1-infected individuals in whom viral replication is spontaneously undetectable, termed HIV controllers (HICs). Multicenter French trial ANRS EP36 studying the viral control in HICs. Neutralizing Antibody (nAb) activities (neutralization assay, competition with broadly reactive monoclonal antibodies, and reactivity against the viral MPER gp41 region), FcgammaR-mediated antiviral activities, antibody-dependent cell cytotoxicity (ADCC), as well as autoantibody levels, were quantified in plasma from 22 controllers and from viremic individuals. The levels of these different antibody responses and HIV-specific CD8 T cell responses quantified by enzyme-linked immunosorbent spot (ELISPOT) IFNgamma assay were compared in each controller. The levels of antibody against the gp120 CD4 binding site, gp41, as well as Env epitopes near to the sites bound by broadly nAbs 2F5 and 1b12 were not different between HICs and viremic individuals. We did not find significant autoantibody levels in HICs. The magnitude and breadth of nAbs were heterogeneous in HICs but lower than in viremic individuals. The levels of nAbs using FcgammaR-mediated assay inhibition were similar in both groups. Regardless of the type of antibody tested, there was no correlation with HIV-specific CD8 T cell responses. ADCC was detectable in all controllers tested and was significantly higher than in viremic individuals (P < 0.0002). There was no single anti-HIV-1 antibody specificity that was a clear correlate of immunity in controllers. Rather, for most antibody types, controllers had the same or lower levels of nAbs than viremic individuals, with the possible exception of ADCC antibodies.

A rapid immunochromatographic assay for the detection of Mycobacterium tuberculosis antigens in pulmonary samples from HIV seropositive patients and its comparison with conventional methods

As tuberculosis generates a highly heterogeneous antibody repertoire, its diagnosis requires tests based on cocktails of antigens. We describe a new, rapid method called rapid immunochromatographic assay (RICA) for cocktail-based diagnosis, which can detect Mycobacterial antigens in sputum specimens. Six antigenic fractions of pathogenic Mycobacterium tuberculosis were used in combination as the capture antigens in the control line of the flow-through assay. Antigen detection of 200 sputum samples from HIV seropositive patients by RICA assay gave a sensitivity of 97.9%, specificity of 99.0%, positive predictive value of 98.9%, negative predictive value of 98.0%, false positive rate of 0.9%, false negative rate of 2.0%, prevalence rate of 49%, likelihood ratio for positive results 97 and likelihood ratio for negative results 0.02. The combination of RICA and AFB staining gave a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, negative predictive value of 100%, false positive rate of 0%, false negative rate of 0%, likelihood ratio for negative results 0. The assay was simple, rapid and economical for the detection of M. tuberculosis infection and suitable for large scale screening of samples in endemic areas without any sophisticated equipment. The results of the assay proved to be superior to conventional methods and combined with clinical data, could form the basis for starting an earlier course of treatment.

Improvement in soluble expression levels of a diabody by exchanging expression vectors

Accumulating evidence suggests that bispecific antibody fragments (BsAbs) seem set to join monoclonal antibodies as powerful therapeutic and diagnostic agents, particularly for targeting cancer. One type of recombinant BsAbs are diabodies, which are constructed from heterogeneous single-chain antibodies and can increase antigen-specific cytotoxicity in T cells by cross-linking tumor antigens with T cell associated antigens. Some diabodies, however, cannot be solubly expressed in sufficient quantities, which limits their use in clinical therapeutics. Previously we constructed an anti-CD20 × CD3 diabody, which effectively directs the lytic potential of cytolytic T cells toward CD20 + malignant B cells and shows marked antitumor efficacy in vivo. Here, to increase the amount of soluble product for clinical trials, we used an alternative expression vector under the T7 promoter but retained the stII signal sequences to ensure that the expressed protein is secreted to the periplasm of Escherichia coli. We achieved a periplasmic, soluble product by optimizing the conditions for induction with a yield of 8–9 mg/L after affinity chromatography purification. This is nearly a five times greater yield than obtained with the previous vector. The diabodies generated from this modified vector retain dual binding specificity for both CD20-positive and CD3-positive cell lines. Taken together, these results suggest that changing expression vectors may be an alternative strategy to accomplish high-level expression of active BsAb proteins from E. coli.

Flowcytometric phenotyping of common variable immunodeficiency

Common variable immunodeficiency (CVID) comprises heterogeneous antibody deficiency disorders. To classify this heterogeneous syndrome, clinical as well as immunologic parameters have been combined. Flowcytometric analysis of circulating T and B lymphocyte subpopulations has become an important tool in this endeavor of disease classification. Multicolor flowcytometric analysis of circulating lymphocytes. The flowcytometric analysis of B and T cell subpopulations in the blood of CVID patients has contributed significantly to the identification of separate groups within the CVID population. In addition, the flowcytometric analysis of the inducible costimulator on activated T cells, CD19 and BAFF-R on B cells are valid screening methods for three of the four known genetic defects associated with CVID. Only TACI deficiency can not be sufficiently detected by flowcytometric measures. Flowcytometric classification of patients with CVID has become a standard procedure during the diagnostic work up. This should be performed according to common guidelines to guarantee world wide comparability between different immunodeficiency centers.

GAD Autoantibody Affinity and Epitope Specificity Identify Distinct Immunization Profiles in Children at Risk for Type 1 Diabetes

Autoantibodies to insulin and GAD are features of preclinical type 1 diabetes in children. For insulin autoantibodies, the antibody affinity and epitope specificity predict which children progress to diabetes. We asked whether autoantibodies to GAD (GADAs) are heterogeneous in affinity and epitope recognition and whether diabetes-related GADA are restricted to high-affinity responses. GADA affinity was measured by competitive binding experiments with [(125)I]-labeled and -unlabeled recombinant human GAD65 in the first GADA-positive sample from 95 children with a type 1 diabetes family history who were prospectively followed from birth and in follow-up samples from 65 of these children. At first GADA appearance, affinity ranged from 10(7) to 10(10) l/mol. Affinity was higher in multiple islet autoantibody-positive children (P < 0.0001) and in HLA DR3-positive children (P = 0.006). GADA affinities were >10(9) l/mol in 52 of 53 multiple autoantibody-positive children. In contrast, children who were single GADA positive often had lower affinity GADA and/or GADA with specificities that were restricted to minor NH(2)-terminal GAD65 epitopes. At follow-up, affinity increased from low to high in 3 of 65 children. All 24 children who developed diabetes had high-affinity GADAs before diabetes onset. Children develop discrete, heterogeneous antibody responses to GAD that could arise from distinct immunization events, only some of which are diabetes relevant. Subtyping the GADA responses using affinity measurement will improve type 1 diabetes risk assessment.