Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates

Patients with immune complex-(IC) mediated diseases frequently have low levels of CR1 on E. The present study was undertaken to determine the role of circulating IC in causing low E-CR1 levels. E-CR1 were enumerated by measuring the binding of anti-CR1 mAb (E11) and rabbit anti-CR1 antibodies (RbaCR1) to E. In addition, the distribution of CR1 among E was assessed by flow cytometry of E stained with E11 and RbaCR1 and by evaluating the binding of E11-coated fluorescent beads (E11-beads) to E. E11-beads bind to clusters of CR1 on E. Five cynomolgus monkeys (CYN) were preimmunized to bovine gamma-globulin (BGG). E-CR1 changes in these animals were assessed: 1) acutely, during the first 60 min after an infusion of BGG and 2) chronically, during daily administration of BGG infusions over 2 wk. Acutely, there was a decrease in the number of E-CR1 as measured by E11 binding to E (E11/CR1). This decrease was not attributable to occupancy of CR1 by IC because the decrease in E11/CR1 number persisted after the IC had been cleared from E. By comparing the E11/CR1 levels in arterial blood to hepatic vein blood (n = 5), or in pulmonary artery blood (n = 1), we determined that the acute decrease in E11/CR1 number did not occur whereas E circulated through liver, spleen, or lung. The decrease in E11/CR1 number required the binding of IC to E because it did not occur after BGG was infused into nonimmunized CYN (n = 2) or into a preimmunized complement-depleted CYN. The decrease in E11/CR1 number was not due to loss of CR1 from E because E11/CR1 number recovered 24 h after infusion of BGG and in addition, enumeration of E-CR1 with RbaCR1 and E11-beads did not reflect a decrease in E-CR1 number. After several daily BGG infusions there was a persistent decrease in E-CR1 levels and that decrease appeared to be mainly the result of loss of CR1 from E because the decrease was confirmed with all methods of E-CR1 measurement and because E-CR1 levels recovered only slowly after BGG infusions were discontinued. Both in vitro and in vivo IC bound preferentially to subpopulations of E, identified by their ability to bind multiple E11-beads and by their high intensity staining with the anti-CR1 antibodies E11 and RbaCR1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of testicular human chorionic gonadotropin binding and steroidogenesis in adult mice with different forms of hereditary diabetes and obesity

The regulation of testicular hCG binding and steroidogenesis in adult mutant mice with hereditary diabetes and obesity was studied. Low doses of hCG caused no change in hCG binding in obese (ob/ob) mice, whereas, in diabetic (db/db) mice, the increase in binding measured 24 h after hCG administration was not as great as in normal males. Intermediate doses of hCG caused a decrease in hCG binding in obese and normal mice, but not in diabetic animals. However, 72 h after injection of intermediate doses of hCG, a decrease in hCG binding also was observed in diabetic mice. Plasma testosterone was elevated 24 h after hCG injection in all types of mice studied, but the increase in diabetic mice was smaller than in normal animals. However, 72 h after treatment with hCG, plasma testosterone was still elevated in diabetic mice, but not in normal males. In vitro, hCG stimulated testicular testosterone synthesis in all groups of mice, but the observed increase was smaller in diabetic and obese than in normal animals. Plasma LH levels were higher in diabetic than in normal mice, whereas plasma FSH and prolactin levels were lower in obese mice than in normal animals. All parameters (i.e., LH receptors and circulating hormone levels) measured in yellow (Ay/a) mice were similar to those in normal (a/a) mice. The present study indicates that in these models for noninsulin-dependent diabetes, the testicular metabolism of LH receptors and capacity to secrete steroids is altered.     

A new strategy for the decellularization of whole organs by hydrostatic pressure

Tissue engineering has been able to develop novel decellularization-recellularization techniques, which facilitates the research for the generation of functional organs. This is based in the initial obtention of the organ's extracellular matrix (ECM). Therefore, any improvement in the decellularization process would have a positive impact in the results of the recellularization process. Nevertheless, commonly the methods and equipment employed for this process are expensive and thus limit the access of this technique to various research groups globally. To develop a decellularization technique with the exclusive use of hydrostatic pressure of detergent solutions, to have an easily accessible and low-cost technique that meets the basic requirements of acellularity and functionality of the ECM. This experimental study was performed in 10 male Wistar rats, obtaining the liver to carry out serial washes, with 1%, 2%, and 3% Triton X-100 solutions and 0.1% SDS. The washes were performed by using a gravity perfusion system (GPS), which assured us a continuous hydrostatic pressure of 7.5 mmHg. The obtained ECM was processed using stains and immunostaining to determine the residual cell content and preservation of its components. The staining showed a removal of cellular and nuclear components of approximately 97% of the acellular ECM, with an adequate three-dimensional pattern of collagen and proteoglycans. Furthermore, the acellular ECM allowed the viability of a primary hepatocyte culture. The use of the GPS decellularization technique allowed us to obtain an acellular and functional ECM, drastically reducing experimentation costs.     

Interactions between precipitating and nonprecipitating antibodies in the formation of immune complexes

In the present study, we used monoclonal antidinitrophenol (DNP) antibodies to determine certain of the biophysical characteristics of precipitating and nonprecipitating antibodies. In addition, we studied the dynamics of immune complex (IC) formation when precipitating antibodies react with antigen in the presence of nonprecipitating antibodies. The antigen utilized in these studies was DNP-bovine serum albumin. All isolated nonprecipitating anti-DNP antibodies were of the IgG2b isotype, whereas all antibodies with other isotypes (IgG1, IgG3, IgM, IgA and IgE) were precipitating. Nonprecipitating antibodies did not differ significantly from precipitating antibodies in affinity, valence, or isoelectric point. Nonprecipitating antibodies inhibited the formation of precipitable IC between antigen and precipitating antibodies. In addition, preformed IC precipitates were solubilized by nonprecipitating antibodies. The solubilization of IC precipitates was influenced by the isotype of the precipitating antibody and by the antibody:antigen ratio in the IC precipitate. By isokinetic sucrose density centrifugation, we determined that solubilization of IC precipitates by nonprecipitating antibodies was associated with release of free precipitating antibody and formation of soluble IC between the antigen and the nonprecipitating antibody. In conclusion, in this study the nonprecipitating property of mouse anti-DNP antibodies is isotype-specific. Nonprecipitating antibodies compete and displace precipitating antibodies from the antigen, resulting in inhibition of IC precipitation and in IC solubilization. On the basis of the present results, we postulate that antibody-antibody interactions are important determinants of precipitating ability, and that these interactions are a characteristic of antibody isotype.