Objective. To determine whether immunization of healthy non‐autoimmune mice with 52‐kd SS‐A/Ro induces a secondary antibody response to other components of the 48‐kd SS‐B/La‐60‐kd SS‐A/Ro RNP complex and vice versa, since anti‐52‐kd antibodies have been invariably linked to these antigens in patients with Sjögren's syndrome and in mothers whose children have neonatal lupus. Methods. Female BALB/c mice were immunized with 100 μg of 6 × His recombinant human 48‐kd SS‐B/La, 52‐kd SS‐A/Ro, or 60‐kd SS‐A/Ro proteins, or the 6 × His polypeptide control, each purified by Ni2+ affinity chromatography. Mice subsequently received booster injections with 50 μg of the same antigen every 10–21 days. Immune responses were measured by enzyme‐linked immunosorbent assay (ELISA), immunoblotting of recombinant antigens, and immunoprecipitation of 35S‐methionine‐labeled in vitro translation products. Results. Immunization with 48‐kd SS‐B/La resulted in anti‐48‐kd SS‐B/La antibodies within 45 days, followed 10 days later by a secondary response to 52‐kd SS‐A/Ro, as measured by ELISA. Antibody spreading to 60‐kd SS‐A/Ro was not detected. Immunization with 52‐kd SS‐A/Ro resulted in rapid high‐titer anti‐52‐kd SS‐A/Ro responses within 27 days. Spreading to 48‐kd SS‐B/La occurred in only 1 mouse and 60‐kd SS‐A/Ro was detected in a minority of the mice after prolonged antigen exposure. Immunization with 60‐kd SS‐A/Ro led to anti‐60‐kd SS‐A/Ro responses within 37 days, followed 3 months later by low‐titer anti‐48‐kd SS‐B/La and anti‐52‐kd SS‐A/Ro antibodies. All primary immune responses were confirmed by immunoblotting and immunoprecipitation. While immunoblotting of the recombinant proteins revealed reciprocal intermolecular spreading in the majority of mice, immunoprecipitation clearly demonstrated that predominant spreading was generated after immunization with 48‐kd SS‐B/La, which consistently resulted in antibodies to 52‐kd SS‐A/Ro. Conclusion. The murine responses observed in the present study, demonstrating reciprocal intermolecular spreading to 48‐kd SS‐B/La, 52‐kd SS‐A/Ro, and 60‐kd SS‐A/Ro, support the linkage of 52‐kd SS‐A/Ro with the other proteins, despite their as‐yet‐undetected association in vivo. The marked recruitment of anti‐52‐kd SS‐A/Ro responses elicited by 48‐kd SS‐B/La may provide a lead to exploring the physical interaction, direct or indirect, of 52‐kd SS‐A/Ro with the SS‐A/Ro‐SS‐B/La RNP particle and its presentation to the immune system. These data should facilitate the establishment of a murine model of neonatal lupus.
Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues.
Our data suggest that recurrence is more than anecdotal in patients with bacteremic infections caused by S pneumoniae (2.8%). We believe that recurrence is a warning sign of immunodeficiency. Patients with multiple myeloma, human immunodeficiency virus infection, solid organ tumors, and chronic liver disease with bacteremic pneumococcal infections should be offered antipneumococcal vaccine and other potentially preventive measures, despite doubts about their efficacy. This is justified by the high mortality rate associated with recurrent episodes (47%).
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