Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis

OBJECTIVE: The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHODS: The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. RESULTS: CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. CONCLUSION: These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.     

Amiloride delays the ischemia-induced rise in cytosolic free calcium

An increase in cytosolic free calcium (Cai) has been shown to occur early during ischemia in perfused rat, ferret, and rabbit hearts. It has been proposed that this increase in Cai may occur as a result of exchange of Nai for Cao, which occurs as a result of an increase in Nai arising from exchange of Nao for H+i. The latter exchange is stimulated by the intracellular acidification that occurs during ischemia. To test this hypothesis, we examined Cai, Nai, ATP, and pHi during ischemia in rats in the presence and absence of 1 mM amiloride, a Na-H exchange inhibitor. Cai was measured using 19F nuclear magnetic resonance (NMR) of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA)-loaded rat hearts. Nai was measured using 23Na NMR, and the shift reagent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetramethylenephosph onate (Tm[DOTP]-5) was used to separate Nai and Nao. ATP and pH were determined from 31P NMR measurements. During 20 minutes of ischemia, amiloride did not significantly alter the ATP decline but did significantly attenuate the rise in Nai and Cai. After 20 minutes of ischemia, time-averaged Cai was 1.0 +/- 0.2 microM (mean +/- SEM) in amiloride-treated hearts compared with 2.3 +/- 0.9 microM in nontreated hearts. After 20 minutes of ischemia, Nai in the untreated heart was threefold greater than control, whereas in the amiloride-treated heart, Nai was not significantly different from control. These data are consistent with the involvement of Na-Ca exchange in the rise in Cai during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA–RON2 pair

Plasmodium falciparum and Toxoplasma gondii are widely studied parasites in phylum Apicomplexa and the etiological agents of severe human malaria and toxoplasmosis, respectively. These intracellular pathogens have evolved a sophisticated invasion strategy that relies on delivery of proteins into the host cell, where parasite-derived rhoptry neck protein 2 (RON2) family members localize to the host outer membrane and serve as ligands for apical membrane antigen (AMA) family surface proteins displayed on the parasite. Recently, we showed that T. gondii harbors a novel AMA designated as TgAMA4 that shows extreme sequence divergence from all characterized AMA family members. Here we show that sporozoite-expressed TgAMA4 clusters in a distinct phylogenetic clade with Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) proteins and forms a high-affinity, functional complex with its coevolved partner, TgRON2_L1. High-resolution crystal structures of TgAMA4 in the apo and TgRON2_L1-bound forms complemented with alanine scanning mutagenesis data reveal an unexpected architecture and assembly mechanism relative to previously characterized AMA–RON2 complexes. Principally, TgAMA4 lacks both a deep surface groove and a key surface loop that have been established to govern RON2 ligand binding selectivity in other AMAs. Our study reveals a previously underappreciated level of molecular diversity at the parasite–host-cell interface and offers intriguing insight into the adaptation strategies underlying sporozoite invasion. Moreover, our data offer the potential for improved design of neutralizing therapeutics targeting a broad range of AMA–RON2 pairs and apicomplexan invasive stages.Phylum Apicomplexa comprises >5,000 parasitic protozoan species, many of which cause devastating diseases on a global scale. Two of the most prevalent species are Toxoplasma gondii and Plasmodium falciparum, the causative agents of toxoplasmosis and severe human malaria, respectively (1, 2). The obligate intracellular apicomplexan parasites lead complex and diverse lifestyles that require invasion of many different cell types. Despite this diversity of target host cells, most apicomplexans maintain a generally conserved mechanism for active invasion (3). The parasite initially glides over the surface of a host cell and then reorients to place its apical end in close contact to the host-cell membrane. After this initial attachment, a circumferential ring of adhesion (termed the moving or tight junction) is formed, through which the parasite actively propels itself while concurrently depressing the host-cell membrane to create a nascent protective vacuole (4).Formation of the moving junction relies on a pair of highly conserved parasite proteins: rhoptry neck protein 2 (RON2) and apical membrane antigen 1 (AMA1). Initially, parasites discharge RON2 into the host cell membrane where an extracellular portion (domain 3; D3) serves as a ligand for AMA1 displayed on the parasite surface (5–8). Intriguingly, recent studies have shown that the AMA1–RON2 complex is an attractive target for therapeutic intervention (9–12). The importance of the AMA1–RON2 pairing is also reflected in the observation that many apicomplexan parasites encode functional paralogs that are generally expressed in a stage-specific manner (13–15). We recently showed that, in addition to AMA1 and RON2, T. gondii harbors three additional AMA paralogs and two additional RON2 paralogs (14, 15): TgAMA2 forms a functional invasion complex with TgRON2 (15), TgAMA3 (also annotated as SporoAMA1) selectively coordinates TgRON2_L2 (14), and TgAMA4 binds TgRON2_L1 (15). Despite substantial sequence divergence, structural characterization of all AMA–RON2D3 complexes solved to date [TgAMA1–TgRON2D3 (16), PfAMA1–PfRON2D3 (17), and TgAMA3–TgRON2_L2D3 (14)] reveal a largely conserved architecture and binding paradigm. Intriguingly, however, sequence analysis indicates that TgAMA4 and TgRON2_L1 are likely to adopt substantially divergent structures with an atypical assembly mechanism.To investigate the functional implications of the AMA4–RON2_L1 complex in T. gondii, we first established that TgAMA4 is part of a highly divergent AMA clade that includes the functionally important malaria vaccine candidate Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) (18–20) and that TgRON2_L1 displays a similar divergence consistent with coevolution of receptor and ligand. We then show that TgAMA4 and TgRON2_L1 form a high-affinity binary complex and probe its overall architecture and underlying mechanism of assembly using crystal structures of TgAMA4 in the apo and TgRON2_L1D3 bound forms. Finally, we show proof of principle that TgAMA4 and TgRON2_L1 form a functional pairing capable of supporting host-cell invasion. Collectively, our study reveals exceptional molecular diversity at the parasite–host-cell interface that we discuss in the context of the unique invasion barriers encountered by the sporozoite.     

Clinical and pathologic spectrum of 46,XY gonadal dysgenesis: its relevance to the understanding of sex differentiation

The condition termed "46,XY gonadal dysgenesis" is characterized by a 46,XY karyotype and incomplete testicular determination. It is likely the result of a mutation in the gene for the testicular determination factor or in another gene involved in the early stages of testicular differentiation. In view of the present interest in the identification of gene(s) initiating the differentiation of the embryonic gonads into testes, we have reviewed the phenotype of 15 patients with 46,XY gonadal dysgenesis to use this information for future molecular studies. Seven patients presented a complete form, 46,XY pure gonadal dysgenesis, including streak gonads, normal Müllerian structures, and normal female external genitalia. The structure of the streak gonads in these patients presented some variation. Eight patients presented an incomplete form, 46,XY partial gonadal dysgenesis, with ambiguous external genitalia and partial development of Müllerian and Wolffian structures. Among them, 3 had bilateral dysgenetic testes, and 4 had a streak gonad on one side with a contralateral dysgenetic testis. The streak gonads showed ovarian stroma with occasional primitive sex cords devoid of germ cells. However, a primordial follicle was observed in 1 streak gonad. The dysgenetic testes showed disorganized seminiferous tubules and ovarian stroma. In some patients, the ovarian stroma was intermixed with testicular tissue, while in others, distinct ovarian and testicular portions were present. In 1 patient, the dysgenetic testis contained a focus of well-differentiated ovarian tissue with primordial follicles. Our observations support the hypothesis that streak gonads in 46,XY pure gonadal dysgenesis arise from fetal ovaries and that dysgenetic testes in the partial form in 46,XY partial gonadal dysgenesis develop from ovotestis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combination chemotherapy (M-2) protocol (BCNU, cyclophosphamide, vincristine, melphalan, and prednisone) for Waldenstrom macroglobulinemia: preliminary report

Fourteen consecutively referred, symptomatic patients with Waldenstrom's macroglobulinemia (ages 52-87 yr) have been treated with the 5-drug M-2 protocol (BCNU, cyclophosphamide, vincristine, melphalan, and prednisone). Three patients were previously treated and 11 patients were untreated. The majority of patients were symptomatic from hyperviscosity. All patients have responded to therapy. Two patients have achieved complete remissions and 12 patients partial remissions to date. None of the patients with symptomatic hyperviscosity has required plasmapheresis since therapy with the M-2 has been initiated. Lymphadenopathy, hepatosplenomegaly, and anemia have also responded to treatment. Follow-up data are limited, with survival from initiation of therapy with the M-2 ranging from 2+ to 35% mo (median 17+ mo) 2+-40+ mo from time of diagnosis). Combination chemotherapy for Waldenstrom's macroglobulinemia with the M-2 protocol appears to increase the response rate in patients with symptomatic disease. Further survival analysis will be carried out.     

Deficiency of Fibroblast Growth Factor-Inducible 14 (Fn14) Preserves the Filtration Barrier and Ameliorates Lupus Nephritis

TNF ligand superfamily member 12, also known as TNF-related weak inducer of apoptosis (TWEAK), acts through its receptor, fibroblast growth factor-inducible 14 (Fn14), to mediate several key pathologic processes involved in tissue injury relating to lupus nephritis. To explore the potential for renal protection in lupus nephritis by targeting this pathway, we introduced the Fn14 null allele into the MRL-lpr/lpr lupus mouse strain. At 26–38 weeks of age, female Fn14-knockout MRL-lpr/lpr mice had significantly lower levels of proteinuria compared with female wild-type MRL-lpr/lpr mice. Furthermore, Fn14-knockout mice had significantly improved renal histopathology accompanied by attenuated glomerular and tubulointerstitial inflammation. There was a significant reduction in glomerular Ig deposition in Fn14-knockout mice, despite no detectable differences in either serum levels of antibodies or splenic immune cell subsets. Notably, we found that the Fn14-knockout mice displayed substantial preservation of podocytes in glomeruli and that TWEAK signaling directly damaged barrier function and increased filtration through podocyte and glomerular endothelial cell monolayers. Our results show that deficiency of the Fn14 receptor significantly improves renal disease in a spontaneous lupus nephritis model through prevention of the direct injurious effects of TWEAK on the filtration barrier and/or modulation of cytokine production by resident kidney cells. Thus, blocking the TWEAK/Fn14 axis may be a novel therapeutic intervention in immune-mediated proliferative GN.