Anil D'Souza

46,X,del(X)(q13) Turner's syndrome women with systemic lupus erythematosus in a pedigree multiplex for SLE

Systemic lupus erythematosus (SLE) disproportionately affects women. Recent work demonstrates that men with Klinefelters syndrome (47,XXY men) have a similar risk of developing SLE as do women. We present an unusual African-American family with two SLE-affected individuals in which one of the patients with SLE also has Turners syndrome (46,X,del(X)(q13)). Although not definitive, this family raises interesting questions regarding the function of genes located on the X chromosome in the development of SLE. The paucity of case reports documenting the overlap of SLE with Turners syndrome while there is an association of male SLE with Klinefelters syndrome suggests a lower risk of SLE in women with Turners syndrome. These observations are consistent with a gene dose effect at X with two X chromosomes (46,XX or 47,XXY) conferring higher risk and one X chromosome (46,XY or 45,XO) conferring lower risk of SLE.

Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum.

C1q is of interest in systemic lupus erythematosus (SLE) research due to deficiencies in its activity being associated with the disease. Current published protocols for measuring C1q vary greatly in their results and ease of reproducibility. Due to this, average C1q concentrations have been reported between 56 and 276 microg/mL in non-SLE serum. We present an improved method for quantifying C1q concentrations, which employs a sandwich ELISA. This method has improved precision, cost efficiency, up-scaling, reproducibility, and uses significantly lesser volumes of serum sample when compared to RID and other methods for quantifying C1q. We report an average concentration of 113 +/- 40 microg/mL for C1q in non-SLE serum. The assay designed here will be useful in the high-throughput measurement of serum C1q in SLE cases.

A nonsense mutation of PEPD in four amish children with prolidase deficiency

Encoded by the peptidase D (PEPD) gene located at 19q12‐q13.11, prolidase is a ubiquitous cytosolic enzyme that catalyzes hydrolysis of oligopeptides with a C‐terminal proline or hydroxyproline. We describe here four Amish children with a severe phenotype of prolidase deficiency in the Geauga settlements of Ohio as the first report of prolidase deficiency in the Amish population as well as in the United States. The patients presented with infection, hepatosplenomegaly, or thrombocytopenia, in contrast to most cases previously reported in the literature, presenting with skin ulcers. All four patients had typical facial features, classic skin ulcers, and multisystem involvement. Recurrent infections, asthma‐like chronic reactive airway disease, hyperimmunoglobulins, hepatosplenomegaly with mildly elevated aspartate transaminase (AST), anemia, and thrombocytopenia were common and massive imidodipeptiduria was universal. Prolidase activity in our patients is nearly undetectable. Direct sequencing of PCR‐amplified genomic DNA for all of the exons from the four patients revealed the same homozygous single nucleotide mutation c.793 T > C in exon 11, resulting in a premature stop‐codon at amino acid residue 265 (p.R265X). It is speculated that the severe phenotype in these patients might be associated with the type of the PEPD gene mutation.

Degree of modification of Ro60 by the lipid peroxidation by-product 4-hydroxy-2-nonenal may differentially induce Sjögren syndrome or systemic lupus erythematosus in BALB/c mice

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freunds adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Location of Immunization and Interferon-γ Are Central to Induction of Salivary Gland Dysfunction in Ro60 Peptide Immunized Model of Sjögren's Syndrome

Introduction Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögrens syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved. Methods Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts. Results Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity. Conclusions Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.

Male-only Systemic Lupus

Objective. Systemic lupus erythematosus (SLE) is more common among women than men, a ratio of about 10 to 1. We undertook this study to describe familial male SLE within a large familial SLE cohort. Methods. SLE families (2 or more patients) were identified from the Lupus Multiplex Registry and Repository. Genomic DNA and blood samples were obtained using standard methods. Autoantibodies were determined by multiple methods. Medical records were abstracted for SLE clinical data. Fluorescent in situ hybridization (FISH) was performed with X and Y centromere-specific probes, and a probe specific for the Toll-like receptor 7 gene on the X chromosome. Results. Among 523 SLE families, we found 5 families in which all the SLE patients were male. FISH found no yaa gene equivalent in these families. SLE-unaffected primary female relatives from the 5 families with only-male SLE patients had a statistically increased rate of positive antinuclear antibodies compared to SLE-unaffected female relatives in other families. White men with SLE were 5 times more likely to have an offspring with SLE than White women with SLE, but there was no difference in this likelihood among Black men. Conclusion. Because women in the all-male families had positive antinuclear antibodies, and men are more likely to have children with SLE, these data suggest genetic susceptibility factors that act only in men.

Binding characteristics of progesterone and antiprogestin ZK 98.299 in human endometrial and myometrial cytosol.

The binding of ZK 98.299, a synthetic progesterone antagonist, with human endometrium and myometrium cytosol was studied and compared with that of progesterone. Progesterone showed specific saturable binding to its receptors in both endometrium and myometrium. ZK 98.299 and progesterone were mutually competitive for binding to progesterone receptors; however, the relative binding affinity of ZK 98.299 was 16% that of progesterone. ZK 98.299 exchanged the progesterone-labelled receptor sites. [3H]ZK 98.299 showed specific binding which was linearly related to the cytosol protein concentration. The binding was not saturable at 15 nM of ligand. The binding capacity and binding affinity of ZK 98.299 receptor was less than that of progesterone. Progesterone also partially displaced the binding of [3H]ZK 98.299. This study suggest that ZK 98.299 and progesterone both bind to the same protein. However, whether ZK 98.299 binds to progesterone receptors alone or even to other functionally related sites is not known. It appears that ZK 98.299 when present in higher concentration than progesterone would be an effective receptor ligand.

Interaction of newly synthesized antiprogesterone ZK98299 with progesterone receptor from human myometrium

We have undertaken characterization of binding of the newly synthesized progesterone receptor (PR) antagonist ZK98299 in the cellular fractions of human myometrium. Specific [3H]progesterone and [3H]ZK98299 binding was observed in the cytosol and the nuclear fractions, and could be competitively replaced by either of the steroids in their radioinert form. Although PR occupied by both steroids exhibited nuclear uptake, the extent of nuclear binding was lower with [3H]ZK98299-receptor complexes. The binding of both ligands to PR was a function of the duration of incubation and the protein concentration: it was saturable at 3–6 nM steroids with a dissociation constant of approximately 2 nM. However, the number of ZK98299 binding sites (72 fmoles/mg protein) was lower compared to that of progesterone (322 fmoles/mg protein). The relative binding affinity (RBA) of ZK98299 for the nuclear PR was about 33% that of progesterone. The results of our study suggest that ZK98299 binds to PR in the cytosol and the nuclear fractions. The antiprogestin effects of ZK98299 reported in the literature are PR-mediated and may result from suboptimal nuclear binding/retention of antiprogestin-receptor complexes. Since this study did not involve isolation and study of individual PR isoforms, PR-A and PR-B, the present data should be viewed as representing an average of contributions by the two receptor forms.

Putative sequences on Ro60 three-dimensional structure accessible for 4-hydroxy-2-nonenal (HNE) modification compared to in vitro HNE modification of Ro60 sequences.

We have previously reported accelerated acquisition of new autoreactivity upon immunization with 4-hydroxy-2-nonenal (HNE)-modified Ro60, as well as differential induction of lupus or Sjögrens syndrome by immunization with Ro60 containing varying amounts of HNE. Since the number of HNE molecules on Ro60 appears to be important, we hypothesized that specific sequences on Ro60 are targets for HNE-modification. Using surface plasmon resonance (SPR) we have also shown intramolecular protein-protein interaction between Ro60 and Ro multiple antigenic peptides (MAPs). We also hypothesized that intramolecular protein-protein interaction would be abolished by HNE-modification. To test this hypotheses we investigated (a) the epitopes of Ro60, using 19 Ro MAPs in an in vitro assay (involving HNE-modification of MAPs following immobilization on ELISA plates) to identify targets of HNE modification on Ro60 and (b) the protein-protein interaction between unmodified Ro60 MAPs, immobilized on the sensor surface of BIAcore, and unmodified Ro60 or HNE-modified Ro60 using SPR. New data obtained with SPR strengthens our earlier observation that immunization with HNE-Ro60 induces a stronger response. Unmodified Ro60 bound to several Ro60 MAPs through protein-protein interaction analyzed using SPR. This interaction was totally abrogated using HNE-modified Ro60 suggesting that sequences on Ro had become modified with HNE. When 19 Ro60 MAPs were modified in vitro with HNE, it was found that 10/19 MAPs significantly bound HNE covalently (p<0.001 compared to MAPs binding HNE poorly). The amino acid sequences 126-137, 166-272 and 401-495 on Ro60 were strongly HNE modified. Using computational model system based on the recently published crystal structure for Ro60 enabled us to identify regions on the Ro60 molecule represented by the HNE-modified Ro MAPs, which are part of the exposed tertiary structure of the Ro60 protein.

Antiprogestin ZK-98.299 and progesterone display differential binding characteristics in the human myometrial cytosol

To investigate whether the synthetic progesterone antagonist ZK-98.299 binds to progesterone receptor or also has distinct binding sites, the binding characteristics of ZK-98.299 were compared with those of progesterone in the human myometrial cytosol. [3H]ZK-98.299 and [3H]progesterone showed specific binding in the myometrial cytosol and the binding of each radiolabelled ligand could be displaced with the respective ligand in a dose-response manner. However, while the binding of [3H]progesterone could be completely blocked with progesterone or ZK 98.299, the binding of [3H]ZK-98.299 could not be displaced more than 50%. The non-specific binding of [3H]ZK-98.299 was very high as compared to that of [3H]progesterone. Using [3H]progesterone, the relative binding affinity (RBA) of progesterone was more than that of ZK 98.299, whereas using [3H]ZK-98.299 the RBA of ZK 98.299 exceeded that of progesterone. Treatment of myometrial cytosol with increasing concentrations of -SH-modifying agents (iodoacetamide (IA) 0-10 mM or N-ethylmaleimide (NEM) 0-1000 nM) decreased the binding of progesterone by over 80%, whereas similar treatment did not have appreciable effect on the binding of [3H]ZK-98.299. Although both preformed ligand-receptor complexes were relatively stable in the presence of IA and NEM, the [3H]progesterone-receptor complex was more sensitive as compared to the [3H]ZK-98.299-receptor complex. The addition of 20 mM molybdate in the cytosol had a protective effect against the -SH-modifying agents. [3H]ZK-98.299 and [3H]progesterone-receptor complexes also showed differential stability when incubated at elevated temperatures (25 degrees C and 37 degrees C), [3H]ZK-98.299-binding sites being more thermolabile as compared to [3H]progesterone binding sites. Prior occupation of the receptor by the two ligands gave the complexes the ability to resist an elevated temperature of 25 degrees C. Moreover, molybdate stabilized both the liganded and unoccupied receptors at 25 degrees C. When the ligand-receptor complexes were applied onto a prefocused polyacrylamide gel, the progesterone and ZK-98.299-receptor complexes were resolved and focused at pH 7.2 and 8.4, respectively. The results of this study suggest that although progesterone and ZK-98.299 are mutually competitive for binding to progesterone receptor, ZK-98.299 also has distinct binding sites.