Structural and functional complexity of the humoral response against the Trypanosoma cruzi ribosomal P2 beta protein in patients with chronic Chagas' heart disease

High levels of antibodies against the C-terminus of the Trypanosoma cruzi TcP2 beta ribosomal protein, defined by the peptide EEEDDDMGFGLFD, named R13, have been measured in sera from patients with chronic Chagas' Heart Disease (cChHD). These antibodies also recognize an epitope on the second extracellular loop of the beta 1-adrenergic receptor, inducing a functional response on cardiomyocytes. The aim of this study was to gain novel insights into the structural basis of this cross-reactivity as well as to evaluate the origin of anti-M2- cholinergic receptor antibodies, which are also commonly found in cChHD patients. To address these questions we immunopurified anti-R13 antibodies and studied the structural requirements of epitope recognition. Results showed that the immunopurified antibodies recognized a conformation of R13 in which the third Glu residue was essential for binding, explaining their low affinity for the mammalian homologue (peptide H13: EESDDDMGFGLFD). Alanine mutation scanning showed individual variations in epitope recognition in each of the studied patients. The importance of a negatively charged residue at position 3 for the recognition of anti-R13 antibodies was further confirmed by competition experiments using a Ser3-phosphorylated H13 analogue, which had 10 times more affinity for the anti-R13 antibody than the native H13 peptide. Moreover, anti-R13 antibodies stimulated either the beta 1-adrenergic or the M2-cholinergic receptor, in strict agreement with the functional properties of the IgG fractions from which they derived, demonstrating that the same parasite antigen may generate antibody specificities with different functional properties. This may be a clue to explain the high variability of electrophysiological disturbances found in cChHD.     

Increased collagen around deformed finger nailfold capillaries in type I diabetes mellitus

Summary Quantitative finger nailfold capillary microscopy was performed in 25 patients with type I diabetes and in 27 healthy control subjects. In the last consecutive 6 patients and 7 controls of these populations, finger nailfold biopsies were taken. Measurements of loop width as an in vivo parameter for deformities of the capillary loops showed significantly higher values in diabetic patients than in controls. Histopathological examination showed markedly and significantly increased deposition of collagen in nailfold dermal papillae of the diabetic patients. The deposition of collagen was positively correlated with the number of capillary endothelial cells in the nailfold dermal papillae and with the size of the papillae in diabetic patients. It is concluded that, in addition to deformity of nailfold capillaries, collagen deposition may also be a sign of metabolic disturbance and perhaps of proliferation of capillary endothelial cells in diabetic microangiopathy.Supported by the Swiss National Fund for Scientific Research, grant number 3.904-0.86     

Comparison of in situ hybridization and immunocytochemistry for the detection of residual beta cells in the pancreas of streptozotocin-treated diabetic rats

The relative efficacy of immunocytochemistry versus in situ hybridization in identifying residual beta cells was studied in rats with streptozotocin-induced diabetes. Consecutive sections of pancreas of streptozotocin-treated diabetic rats and control animals were alternately subjected to in situ hybridization (synthetic oligonucleotides complementary to rat preproinsulin mRNA) and immunocytochemistry (monoclonal antibodies to insulin). The results obtained with both methods were quantitated with the use of computer-assisted image analysis, and the ratio of cells positive by immunocytochemistry to those positive by in situ hybridization was determined. Under normoglycaemic conditions the values obtained by immunocytochemistry correlated well with those obtained by in situ hybridization (immuno/in situ > 95%). In the streptozotocin diabetic animals, however, immunocytochemistry resulted in a distinct underestimation of the number of residual beta cells (immuno/in situ < 80%). This difference was even more striking in small islet cell clusters (<100 m) immuno/in situ 20%). These results suggest that in situ hybridization for prohormone mRNA is the method of choice for the identification of residual or regenerating beta cells with very low insulin content. Caution should be used when interpreting quantitative data in diabetic conditions that are based exclusively on immunocytochemical detection methods.     

Stability of monoclonal antibodies after simulated subcutaneous administration

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.