Faculty Bibliography

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

To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52alpha), and 52beta (amino acids 169-245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received beta-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52alpha or 52beta immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52alpha-immunized mothers, 7% of 86 pups born to 52beta-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52alpha- or 52beta-immunized mice. In the 52alpha group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52beta group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression

Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes

Although a strong clinical association exists between congenital heart block (CHB) and an immune response to SSA/Ro and SSB/La proteins, a causative role of these antibodies in the pathogenesis is just emerging. In a preliminary report, we have demonstrated that IgG fractions isolated from the sera of mothers whose children have CHB are arrhythmogenic in the human fetal heart. To more precisely define the arrhythmogenic effect of anti-SSA/Ro-SSB/La antibodies, we used the readily available rat heart model to record: 1) ECGs from Langendorff beating hearts; 2) action potentials from atrioventricular (AV) nodal preparations; 3) L-type Ca currents, I(Ca) at the whole-cell and single channel levels; and 4) other currents such as the transient outward K+ current, I(to), the inward rectifier K+ current, I(K1), and the Na+ current, I(Na). Perfusion of hearts with purified IgG (800 microg/mL), isolated from the serum of a mother with SSA/Ro and SSB/La antibodies whose child had CHB, resulted in bradycardia associated with 2:1 AV block. Simultaneous action potentials were recorded from dissected atrial and AV nodal areas of the rat heart. Superfusion of these preparations with the same mother's IgG fraction resulted in 2:1 AV block followed by complete inhibition of AV nodal action potential. Because AV nodal electrogenesis is largely dependent on I(Ca), the effect of these antibodies on I(Ca) was subsequently determined. Superfusion of myocytes with whole serum or purified IgG (80 microg/mL) from the same mother consistently inhibited whole cell I(Ca), ensemble average Ba2+ currents (I(Ba)) and open state probability, p(o), without affecting the channel conductance. IgG had no significant effect on I(to), I(K1), or I(Na). Whole sera and IgG fractions from a healthy mother with no detectable anti-SSA/Ro or SSB/La antibodies did not inhibit I(Ca) or I(Ba). These results demonstrate that IgG containing anti-SSA/Ro and -SSB/La antibodies induces complete AV block in beating hearts and in multicellular preparations, thus implicating a preferential interaction of these autoantibodies with Ca channels and/or associated regulatory proteins. This is consistent with the observed inhibition of Ca channels that may be a critical factor contributing to the pathogenesis of CHB

OBJECTIVE: To assess the incidence of Reiter's syndrome aboard The Golden Venture, a ship carrying illegal immigrants from China to the United States. METHODS: After identification of an index case, we conducted telephone interviews with medical staff at immigrant detention centers in Pennsylvania, New York, and Virginia. When a potential case was identified at one facility, we performed a site inspection, reviewing the medical records of all detainees and performing histories and physicals on all those with joint and/or ocular complaints. RESULTS: We identified two patients, both HLA B27 positive, with Reiter's syndrome. The observed incidence (0.87%) approximated the predicted incidence but may have underestimated the actual incidence. We review the history of shipboard Reiter's syndrome, and discuss the pathogenic roles of HLA B27 and particular infectious agents. CONCLUSION: Continued transportation of illegal immigrants from China and other parts of the world is likely to result in occasional clusters of Reiter's syndrome. Physicians treating immigrant populations should remain aware of the possibility of reactive arthritis