Davor Brinc

INTRAVENOUS IMMUNOGLOBULIN AMELIORATES ITP VIA ACTIVATING FC GAMMA RECEPTORS ON DENDRITIC CELLS

Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fcγ receptors (FcγR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 105 IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory FcγRIIB on the initiator cell, although FcγRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating FcγR on dendritic cells.

Closing the Gaps in Pediatric Laboratory Reference Intervals: A CALIPER Database of 40 Biochemical Markers in a Healthy and Multiethnic Population of Children

BACKGROUND Pediatric healthcare is critically dependent on the availability of accurate and precise laboratory biomarkers of pediatric disease, and on the availability of reference intervals to allow appropriate clinical interpretation. The development and growth of children profoundly influence normal circulating concentrations of biochemical markers and thus the respective reference intervals. There are currently substantial gaps in our knowledge of the influences of age, sex, and ethnicity on reference intervals. We report a comprehensive covariate-stratified reference interval database established from a healthy, nonhospitalized, and multiethnic pediatric population. METHODS Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer. RESULTS Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers. CONCLUSIONS The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcgammaRIIB (FcgammaRIIB(-/-) mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcgammaRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

New insight into the mechanism of action of IVIg: the role of dendritic cells.

Summary.  Intravenous immunoglobulin (IVIg) is used to treat an ever‐increasing number of autoimmune diseases. While the exact mechanism of action of IVIg has remained elusive, many theories have been suggested, including mononuclear phagocytic system blockade, autoantibody neutralization by anti‐idiotype antibodies, accelerated pathogenic autoantibody clearance by saturation of the neonatal Fc receptor, cytokine modulation and complement neutralization. More recently, a key role for dendritic cells (DC) in the amelioration of autoimmunity by IVIg has been suggested. Here we will focus on the role that DC may play in IVIg function using data from both mouse and human studies.

Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn

Anti-D is routinely and effectively used to prevent hemolytic disease of the fetus and newborn (HDFN) caused by the antibody response to the D antigen on fetal RBCs. Anti-D is a polyclonal IgG product purified from the plasma of D-alloimmunized individuals. The mechanism of anti-D has not been fully elucidated. Antigenic epitopes are not fully masked by anti-D and are available for immune system recognition. However, a correlation has frequently been observed between anti-D-mediated RBC clearance and prevention of the antibody response, suggesting that anti-D may be able to destroy RBCs without triggering the adaptive immune response. Anti-D-opsonized RBCs may also elicit inhibitory FcgammaRIIB signaling in B cells and prevent B cell activation. The ability of antigen-specific IgG to inhibit antibody responses has also been observed in a variety of animal models immunized with a vast array of different antigens, such as sheep RBCs (SRBC). This effect has been referred to as antibody-mediated immune suppression (AMIS). In animal models, IgG inhibits the antibody response, but the T-cell response and memory may still be intact. IgG does not mask all epitopes, and IgG-mediated RBC clearance or FcgammaRIIB-mediated B-cell inhibition do not appear to mediate the AMIS effect. Instead, IgG appears to selectively disrupt B cell priming, although the exact mechanism remains obscure. While the applicability of animal models of AMIS to understanding the true mechanism of anti-D remains uncertain, the models have nevertheless provided us with insights into the possible IgG effects on the immune response.

Long-term stability of biochemical markers in pediatric serum specimens stored at − 80 °C: A CALIPER Substudy

OBJECTIVES Pediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at -80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions. DESIGN AND METHODS Serum samples collected from children of 0-18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at -80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10-13 month period and each aliquot was subject to one freeze-thaw cycle before analysis. The analysis was performed on VITROS Chemistry System, COBAS INTEGRA 400 Plus and IMMULITE 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time. RESULTS A majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability. CONCLUSIONS The study shows stability for a majority of analytes stored in serum at -80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.

False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125

BACKGROUND By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).

Infection and Cancer: Revaluation of the Hygiene Hypothesis

Several studies have shown that persistent infections and inflammation can favor carcinogenesis. At the same time, certain types of pathogens and antitumor immune responses can decrease the risk of tumorigenesis or lead to cancer regression. Infectious agents and their products can orchestrate a wide range of host immune responses, through which they may positively or negatively modulate cancer development and/or progression. The factors that direct this dichotomous influence of infection-mediated immunity on carcinogenesis are not well understood. Even though not universal, several previous reports have investigated the inverse link of pathogen-induced “benign” inflammation to carcinogenesis and various other pathologies, ranging from autoimmune diseases to allergy and cancer. Several models and ideas are discussed in this review, including the impact of decreased exposure to pathogens, as well as the influence of pathogen load, the timing of infection, and the type of instigated immune response on carcinogenesis. These phenomena should guide future investigations into identifying novel targets within the microbial and host proteome, which will assist in the development of cancer therapeutics and vaccine remedies, analogous to earlier efforts based on helminthic components for the prevention and/or treatment of several pathologies. Clin Cancer Res; 19(11); 2834–41. ©2013 AACR.

IgG-mediated immunosuppression is not dependent on erythrocyte clearance or immunological evasion: implications for the mechanism of action of anti-D in the prevention of haemolytic disease of the newborn?

Haemolytic disease of the newborn (HDN) can be prevented by the passive administration of anti‐D to the mother. The most accepted theory to describe this activity of anti‐D is based upon its ability to clear opsonized erythrocytes before their recognition by the maternal immune system. We examined this hypothesis using a murine model of immunity to foreign erythrocytes. Whereas transfusion of foreign erythrocytes into mice induced immunoglobulin (Ig)M and IgG antibodies specific for the erythrocytes, these humoral immune responses were inhibited when the erythrocytes were opsonized with IgG. To specifically determine if immunological evasion occurs with these opsonized erythrocytes, we examined T‐cell responses from these mice. An erythrocyte‐specific T‐cell response was clearly detected. We then tested whether phagocytosis of opsonized erythrocytes is sufficient to prevent the antibody response. We exposed mononuclear phagocytic cells to sheep red blood cells (SRBC) in vitro and then adoptively transferred the phagocytic cells to recipient mice; opsonized SRBC unexpectedly increased, rather than decreased, the antibody response. These data indicate that removal of opsonized erythrocytes by phagocytic cells does not prevent their immunological recognition and suggest that antigen clearance may not be the predominant mechanism of anti‐erythrocyte action in downregulating the humoral immune response.

Mechanisms of anti-D action in the prevention of hemolytic disease of the fetus and newborn: what can we learn from rodent models?

Purpose of reviewHemolytic disease of the fetus and newborn can be effectively prevented by administration of anti-D to the mother. In this setting, the IgG purified from the plasma of D-alloimmunized donors prevents the maternal immune response to D-positive red blood cells (RBC). Several monoclonal anti-D antibodies have recently been developed for potential use in the setting of hemolytic disease of the fetus and newborn; the functional assays used to assess the potential success of these antibodies have often assumed antigen clearance as the predominant mechanism of anti-D. Unfortunately, the in-vivo success of these monoclonal antibodies has thus far been limited. A similar inhibitory effect of IgG has been observed in animal models with a vast array of different antigens, referred to as antibody-mediated immune suppression (AMIS). Here, studies of AMIS are reviewed and the relevance of these findings for anti-D-mediated immunoprophylaxis is discussed. Recent findingsIn animal models of AMIS, IgG-mediated antigen clearance was not sufficient for prevention of the antibody response to RBC. Furthermore, anti-RBC IgG inhibited B-cell priming to foreign RBC, but failed to prevent a T-cell response and immunological memory. SummaryThe applicability of AMIS models for determining the true mechanism of anti-D, though uncertain, may nevertheless provide knowledge as to potential mechanisms of action of anti-RBC antibodies.