Monoclonal IgG in MGUS and multiple myeloma targets infectious pathogens.

Subsets of mature B cell neoplasms are linked to infection with intracellular pathogens such as Epstein-Barr virus (EBV), hepatitis C virus (HCV), or Helicobacter pylori. However, the association between infection and the immunoglobulin-secreting (Ig-secreting) B proliferative disorders remains largely unresolved. We investigated whether the monoclonal IgG (mc IgG) produced by patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM) targets infectious pathogens. Antigen specificity of purified mc IgG from a large patient cohort (n = 244) was determined using a multiplex infectious-antigen array (MIAA), which screens for reactivity to purified antigens or lysates from 9 pathogens. Purified mc IgG from 23.4% of patients (57 of 244) specifically recognized 1 pathogen in the MIAA. EBV was the most frequent target (15.6%), with 36 of 38 mc IgGs recognizing EBV nuclear antigen-1 (EBNA-1). MM patients with EBNA-1-specific mc IgG (14.0%) showed substantially greater bone marrow plasma cell infiltration and higher ß2-microglobulin and inflammation/infection-linked cytokine levels compared with other smoldering myeloma/MM patients. Five other pathogens were the targets of mc IgG: herpes virus simplex-1 (2.9%), varicella zoster virus (1.6%), cytomegalovirus (0.8%), hepatitis C virus (1.2%), and H. pylori (1.2%). We conclude that a dysregulated immune response to infection may underlie disease onset and/or progression of MGUS and MM for subsets of patients.

Serum hemorphin-7 levels are decreased in obesity.

OBJECTIVE: Hemorphin peptides exhibit biological activities that interfere with the endorphin system, the inflammatory response, and blood-pressure control. VV-hemorphin-7 and LVV-hemorphin-7 peptides exert a hypotensive effect, in particular, by inhibiting the renin-angiotensin system. Furthermore, levels of circulating hemorphin-7 peptides have been found to be decreased in diseases such as type 1 and type 2 diabetes. DESIGN AND METHODS: Because type 2 diabetes and obesity share common features, such as insulin resistance, microinflammation, high glomerular-filtration rate (GFR), and cardiovascular risk, we evaluated serum VV-hemorphin-7 like immunoreactivity (VVH7-i.r.) levels, using an enzyme-linked immunosorbent assay method, on a group of 54 obese subjects without diabetes or hypertension, compared with a group of 33 healthy normal-weight subjects. RESULTS: Circulating VVH7-i.r. levels were significantly decreased in the obese group compared with the control group (1.98 ± 0.19 vs. 4.86 ± 0.54 µmol/l, respectively, P < 0.01), and a significant negative correlation between VVH7-i.r. and diastolic blood pressure (DBP) was found in obese patients (r = -0.35, P = 0.011). There was no significant correlation between VVH7-i.r. level and insulin resistance, metabolic syndrome, or GFR. CONCLUSIONS: The decreased serum hemorphin-7 found in obese subjects, as in diabetes, may contribute to the development of hypertension and to the cardiovascular risk associated with these metabolic diseases.

Multiplexed infectious protein microarray immunoassay suitable for the study of the specificity of monoclonal immunoglobulins.

Enzyme-linked immunosorbent assays (ELISAs) used to detect antibodies specific for common infectious agents such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), Toxoplasma gondii (T. gondii), and hepatitis C virus (HCV) are time-consuming and require large volumes of samples, which restrict their use. We propose a new assay based on a multiplexed infectious protein (MIP) microarray combining different epitopes representative of the four germs. Antigens and lysates were printed on nitrocellulose slides to constitute the microarray. First, the microarray was incubated with human serum samples. Then, the suitability of the microarray for analysis of the specificity of purified monoclonal immunoglobulin (mc Ig) was assessed using serum and mc Ig of HCV-positive patients. Bound human immunoglobulin G (IgG) was detected using fluorescently labeled secondary antibodies, and the signals were quantified. Results obtained in serum samples with the new MIP microarray immunoassay were compared with ELISAs; we observed concordances of 95% for EBV, 93% for CMV, 91% for T. gondii, and 100% for HCV. Regarding purified mc Ig of HCV-positive patients, 3 of 3 recognized antigens printed on the microarray. Hence, the novel EBV/CMV/T. gondii/HCV MIP microarray allows simultaneous diagnosis of polyclonal and monoclonal immune response to infectious diseases using very small volume samples.

Proteolytic degradation by cathepsin D of glycated hemoglobin from diabetes patients gives rise to hemorphin-7 peptides.

Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients (n=8) and normal healthy control subjects (n=2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients.

Hemorphin 7 reflects hemoglobin proteolysis in abdominal aortic aneurysm.

OBJECTIVE: In human abdominal aortic aneurysm, the accumulation of blood-derived cells and proteases within the mural thrombus plays a pivotal role in the evolution toward vessel wall rupture. We sought to identify peptides released from abdominal aortic aneurysm specimens, characterized by an intraluminal thrombus. METHODS AND RESULTS: Intraluminal thrombus samples were analyzed by differential proteomics, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. A 1309-Da peptide was detected in larger amounts in the newly formed luminal thrombus layer relative to older layers. It was identified as being LVVYPWTQRF (known as LVV-Hemorphin 7), a peptide generated from hemoglobin by cathepsin D. By immunohistochemical analysis, we showed that Hemorphin 7 (H7) colocalizes with cathepsin D and cathepsin G in the luminal layer of the intraluminal thrombus. In vitro, cathepsin G was able to generate H7 peptides at pH 7.4, whereas cathepsin D was only active in acidic conditions. Finally, H7 peptides were shown to be increased 3- to 4-fold in sera of abdominal aortic aneurysm patients relative to controls, and their levels were positively correlated with the volume of the thrombus. CONCLUSIONS: Our results suggest that circulating H7 peptides may reflect proteolysis of hemoglobin in the aneurysmal intraluminal thrombus and may be used as a biological marker of pathological vascular remodeling.