Renal Involvement in Primary Sj?gren's Syndrome: A Clinicopathologic Study

Background & objectives: Renal pathology and clinical outcomes in patients with primary Sjögren''s syndrome (pSS) who underwent kidney biopsy (KB) because of renal impairment are reported.Design, setting, participants, & measurements: Twenty-four of 7276 patients with pSS underwent KB over 40 years. Patient cases were reviewed by a renal pathologist, nephrologist, and rheumatologist. Presentation, laboratory findings, renal pathology, initial treatment, and therapeutic response were noted.Results: Seventeen patients (17 of 24; 71%) had acute or chronic tubulointerstitial nephritis (TIN) as the primary lesion, with chronic TIN (11 of 17; 65%) the most common presentation. Two had cryoglobulinemic GN. Two had focal segmental glomerulosclerosis. Twenty patients (83%) were initially treated with corticosteroids. In addition, three received rituximab during follow-up. Sixteen were followed after biopsy for more than 12 mo (median 76 mo; range 17 to 192), and 14 of 16 maintained or improved renal function through follow-up. Of the seven patients presenting in stage IV chronic kidney disease, none progressed to stage V with treatment.Conclusions: This case series supports chronic TIN as the predominant KB finding in patients with renal involvement from pSS and illustrates diverse glomerular lesions. KB should be considered in the clinical evaluation of kidney dysfunction in pSS. Treatment with glucocorticoids or other immunosuppressive agents appears to slow progression of renal disease. Screening for renal involvement in pSS should include urinalysis, serum creatinine, and KB where indicated. KB with characteristic findings (TIN) should be considered as an additional supportive criterion to the classification criteria for pSS because it may affect management and renal outcome.Primary Sjögren''s syndrome (pSS) is a progressive autoimmune disorder involving the exocrine glands (1), typically presenting with keratoconjunctivitis and xerostomia (2). It is characterized pathologically by a predominant lymphocytic infiltrate around epithelial ducts of exocrine glands on salivary gland biopsy (3). Extraglandular manifestations of pSS, once thought to be uncommon, occur in up to 25% of patients. Patients can be afflicted by severe interstitial lung disease (4), cutaneous vasculitis (5), peripheral neuropathy (6), and hematologic complications such as lymphoma (7). They are also at increased risk for celiac sprue (8) and complications from Helicobacter pylori infection (9) such as mucosa-associated lymphatic tissue (MALT)-type lymphoma.Much of our understanding of the clinical presentation of renal involvement in pSS is based on case reports (10–26) and small retrospective cohorts (27–29). Tubulointerstitial nephritis (TIN) remains the most common presentation of renal involvement in pSS and CD4/CD8 T cell subsets are reported to predominate (27,30). This is often characterized by a distal (type I) renal tubular acidosis (RTA) and less commonly proximal (type II) RTA (Fanconi syndrome) (11,31–33). GN is thought to be a rare occurrence, with only case reports available in the literature (10,12–23), and tends to be a late development (34) in the course of the disease.We examined the renal pathologic findings and clinical trends of all patients with pSS who underwent kidney biopsy (KB) at Mayo Clinic since 1967 and assembled a case series of patients with pSS with renal pathologic disease evaluated by renal biopsy at a single center in the United States. This case series aimed to describe the common clinical presentations of renal disease in pSS, the array of pathologic findings of renal involvement in pSS, and trends during follow-up and treatment.     

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion

Yeast vacuole fusion requires 4 SNAREs, 2 SNARE chaperone systems (Sec17p/Sec18p/ATP and the HOPS complex), and 2 phosphoinositides, phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. By reconstituting proteoliposomal fusion with purified components, we now show that phosphoinositides have 4 distinct roles: PI(3)P is recognized by the PX domain of the SNARE Vam7p; PI(3)P enhances the capacity of membrane-bound SNAREs to drive fusion in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P₂ strikingly promotes synergistic SNARE complex remodeling by Sec17p/Sec18p/ATP and HOPS. This ternary synergy of phosphoinositides and 2 SNARE chaperone systems is required for rapid fusion.Intracellular membrane fusion is a conserved reaction, vital for vesicle trafficking, hormone secretion, and neurotransmission. Fusion is regulated by NSF (N-ethylmaleimide-sensitive factor)/Sec18p, αSNAP (soluble NSF attachment protein)/Sec17p, SNAREs (SNAP receptors), Sec1p/Munc18–1p family (SM) proteins, Rab GTPases, and Rab:GTP-binding proteins, termed “Rab effectors” (1–3). Lipids, including phosphoinositides, sterols, diacylglycerol (DAG), and phosphatidic acid (PA), have specific roles in fusion (4–14). Proteins and lipids cooperate for their enrichment in membrane fusion microdomains (6, 8, 15, 16).SNARE proteins are integral or peripheral membrane proteins required for membrane fusion. SNAREs have either a Q or R residue at the center of their SNARE domain and associate in 4-helical QabcR complexes in cis (anchored to one membrane) or in trans (anchored to apposed membranes), where a, b, and c are families of related Q-SNAREs (2, 17, 18). Reconstituted proteoliposomes (RPLs) bearing Q-SNAREs fuse with RPLs bearing an R-SNARE through trans-SNARE-complex assembly (19, 20). This fusion has slow kinetics, requires nonphysiologically high SNARE densities, and causes substantial leakage of luminal contents of the RPLs (21–24).We study membrane fusion with yeast vacuoles (lysosomes). Vacuole fusion (25) requires 3 Q-SNAREs (Vam3p, Vti1p, and Vam7p) and 1R-SNARE (Nyv1p) (26, 27), two SNARE chaperone systems, Sec17p/Sec18p/ATP (28), and the HOPS (homotypic fusion and vacuole protein sorting)/Vps Class C complex (29, 30), the Rab-GTPase Ypt7p (31), and chemically minor but functionally vital “regulatory lipids”: ergosterol (ERG), DAG, PI(3)P, and PI(4,5)P₂ (8). Inactive 4SNARE cis-complexes on isolated organelles are disassembled by Sec17p/Sec18p/ATP (27). The heterohexameric HOPS complex, containing the SM protein Vps33p as a subunit, promotes and proofreads SNARE-complex assembly (32–34). HOPS can physically interact with the Q-SNAREs [Vam7p (35) and Vam3p (36, 37)], 4SNARE cis-complexes (32), GTP-bound Ypt7p (29), and phosphoinositides (35). PI(3)P supports the membrane association of the Qc-SNARE Vam7p, which has no transmembrane domain, through binding its PX domain (38). SNAREs, HOPS, Ypt7p, and regulatory lipids assemble in an interdependent fashion to form a fusion-competent membrane microdomain, the “vertex ring” (8, 16, 39). Trans-SNARE complexes are essential for fusion (26), yet fusion can be accelerated by SNARE-associating factors such as HOPS (14, 35) and by cycles of SNARE complex disassembly and reassembly, termed “remodeling” (40).Membrane fusion has been reconstituted with all purified yeast vacuolar components, including 4SNAREs, vacuolar lipids, 2 SNARE chaperone systems, and phosphoinositides (14). We now show distinct functions of phosphoinositides in RPL fusion: the PX-domain of the SNARE Vam7p recognizes PI(3)P, as reported (38); PI(3)P activates the 3Q-SNAREs to be more fusogenic in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ accelerates fusion by promoting the synergy between Sec17p/Sec18p and HOPS, although this synergy is not a function of the membrane recruitments of these SNARE chaperones. This ternary synergy between phosphoinositides and SNARE chaperones is essential for the assembly and remodeling of SNARE complexes.     

Treatment with IFN-α, -β, or -γ Is Associated with Collapsing Focal Segmental Glomerulosclerosis

Background and objectives: Treatment with IFN is rarely associated with nephrotic syndrome and renal biopsy findings of minimal-change disease or FSGS.Design, setting, participants, & measurements: We report 11 cases of collapsing FSGS that developed during treatment with IFN and improved after discontinuation of therapy.Results: The cohort consists of seven women and four men with a mean age of 48.2 yr. Ten of the 11 patients were black. Six patients were receiving IFN-α for hepatitis C virus infection (n = 5) or malignant melanoma (n = 1), three were receiving IFN-β for multiple sclerosis, and two were treated with IFN-γ for idiopathic pulmonary fibrosis. After a mean and median duration of therapy of 4.0 and 12.6 months, respectively, patients presented with acute renal failure (mean creatinine 3.5 mg/dl) and nephrotic-range proteinuria (mean 24-hour urine protein 9.7 g). Renal biopsy revealed collapsing FSGS with extensive foot process effacement and many endothelial tubuloreticular inclusions. Follow-up was available for 10 patients, all of whom discontinued IFN. At a mean of 23.6 months, nine of 10 patients had improvement in renal function, including one with complete remission and two with partial remission. Among the seven patients with available data, mean proteinuria declined from 9.9 to 3.0 g/d. Four of the seven patients were treated with immunosuppression, and there was no detectable benefit.Conclusions: Collapsing FSGS may occur after treatment with IFN-α, -β, or -γ and is typically accompanied by the ultrastructural finding of endothelial tubuloreticular inclusions. Optimal therapy includes discontinuation of IFN.FSGS is the most common cause of idiopathic nephrotic syndrome in black patients and may be the most frequent cause of nephrotic syndrome in the general population (1–6). The spectrum of FSGS includes primary forms mediated by a putative circulating or permeability factor and a number of secondary forms caused by such diverse insults as hereditable mutations in podocyte genes, drugs, viral infections, and adaptive responses to reduced renal mass or other hemodynamic stress (1). A variety of histologic variants of FSGS have been identified and can be applied to both primary and secondary forms (7–9). Many secondary forms tend to manifest as particular morphologic subtypes (1).The collapsing variant of FSGS is defined by implosive wrinkling and “collapse” of the glomerular basement membrane associated with hypertrophy and hyperplasia of overlying podocytes (10–12). Collapsing FSGS was mainly described in patients with HIV-associated nephropathy (HIVAN) (13) but also was recognized as a variant of idiopathic FSGS (11,12). Both idiopathic collapsing FSGS and HIVAN are most commonly seen in young black patients (8–12,14). Compared with the usual, most common form of FSGS with discrete segmental scars (FSGS not otherwise specified [FSGS NOS]), collapsing FSGS is distinguished by more severe nephrotic syndrome and renal insufficiency at presentation and a more rapid course to renal failure (8–12,14). Central to the morphogenesis of the collapsing variant is podocyte injury that leads to podocyte dedifferentiation, apoptosis, and proliferation, in part through dysregulation of cell cycle–related proteins (15–19). Podocyte precursor cells from the parietal cell layer may contribute to the glomerular epithelial cell proliferation (20).HIVAN is not the only established secondary cause of collapsing FSGS. Collapsing FSGS has been reported in the setting of Parvovirus B19 infection (21) and in patients with hemophagocytic syndrome (with or without underlying lymphoma) (22). Collapsing FSGS also may follow treatment with pamidronate (23), with 15 cases reported in the medical literature (23,24). In contrast, FSGS NOS has been reported to result from treatment with lithium (25), sirolimus (26), and more recently anabolic steroids (27). Although rare cases of collapsing FSGS also have been reported after treatment with IFN-α (28–30), this therapeutic agent is more commonly associated with minimal-change disease (MCD) (31–38) and FSGS NOS (39–47). We report 11 additional cases of collapsing FSGS that developed during treatment with IFN, including six IFN-α (for hepatitis C virus [HCV] infection or melanoma), three IFN-β (for multiple sclerosis [MS]), and two IFN-γ (for idiopathic pulmonary fibrosis).     

Emergence of Gentamicin-Resistant Bacteremia in Hemodialysis Patients Receiving Gentamicin Lock Catheter Prophylaxis

Background and objectives: Antibiotic locks in catheter-dependent chronic hemodialysis patients reduce the rate of catheter-related blood stream infections (CRIs), but there are no data regarding the long-term consequences of this practice.Design, setting, participants, & measurements: Over a 4-year period, from October 1, 2002, to September 30, 2006, we initiated a gentamicin and heparin lock (GHL) protocol in 1410 chronic hemodialysis patients receiving dialysis through a tunneled catheter in eight outpatient units.Results: Within the first year of the GHL protocol, our CRI rate decreased from 17 to 0.83 events per 1000 catheter-days. Beginning 6 months after initiation of the GHL protocol, febrile episodes occurred in 13 patients with coagulase-negative Staphylococcus bacteremia resistant to gentamicin. Over the 4 years of GHL use, an additional 10 patients developed 11 episodes of gentamicin-resistant CRI (including 7 with Enterococcus faecalis), in which there were 4 deaths, 2 cases of septic shock requiring intensive care unit admission, and 4 cases of endocarditis. Because of these events, the GHL protocol was discontinued at the end of 2006.Conclusions: Although the use of GHL effectively lowered the CRI rate in our dialysis population, gentamicin-resistant CRIs emerged within 6 months. Gentamicin-resistant infections are a serious complication of the long-term use of GHLs. Alternative nonantibiotic catheter locks may be preferable to decrease the incidence of CRIs without inducing resistant pathogens.The use of tunneled central venous catheters (TCCs) for vascular access in chronic hemodialysis patients increased from 18% in 1998 to 27% in 2004 (1). Moreover, the current use of TCC can be as high as 30 to 40% in prevalent patients and 74% in incident patients (2). Although providing life-saving therapy to those awaiting maturation or placement of an arteriovenous (AV) fistula or graft, there is a 2- to 3-fold increased risk of death and a 10- to 20-fold higher risk of bacteremia in patients receiving hemodialysis through a TCC compared with a fistula (2–4).The incidence of catheter-related blood stream infections (CRIs) in hemodialysis patients ranges from 2.5 to 5.5 cases per 1000 catheter-days. Ten to 20% of CRIs are associated with metastatic complications such as endocarditis, septic arthritis, and epidural abscess, and they cause considerable financial and physical burdens from catheter loss, repeated access procedures, and hospital admissions (3).Numerous randomized, controlled trials (5–20), as well as meta-analyses of these studies (21–25), have been performed during the past decade to evaluate the benefit of anti-microbial lock solutions in chronic hemodialysis patients with TCCs. These studies have been heterogeneous in nature and used different antibiotics (gentamicin, minocycline, cefazolin, cefotaxime, vancomycin) and nonantibiotic (citrate, taurolidine, EDTA) anti-microbial lock solutions; however, they have all shown a significant decline of 50 to 100% in CRIs compared with standard heparin lock without antibiotics or other sterilizing solutions (3). None of the aforementioned studies have reported anti-microbial resistance or loss of anti-microbial lock solution efficacy, but the longest follow-up period has only been 547 days in one study (13). Therefore, the emergence of bacterial antibiotic resistance from anti-microbial lock solutions and its potential complications remain to be determined.The main objectives of our study were to assess the long-term consequences of a gentamicin and heparin lock (GHL) protocol in maintenance hemodialysis patients using a TCC and to document the associated emergence of gentamicin-resistant bacteremia in these patients.     

Variations in methanobactin structure influences copper utilization by methane-oxidizing bacteria

Methane-oxidizing bacteria are nature’s primary biological mechanism for suppressing atmospheric levels of the second-most important greenhouse gas via methane monooxygenases (MMOs). The copper-containing particulate enzyme is the most widespread and efficient MMO. Under low-copper conditions methane-oxidizing bacteria secrete the small copper-binding peptide methanobactin (mbtin) to acquire copper, but how variations in the structures of mbtins influence copper metabolism and species selection are unknown. Methanobactins have been isolated from Methylocystis strains M and hirsuta CSC1, organisms that can switch to using an iron-containing soluble MMO when copper is limiting, and the nonswitchover Methylocystis rosea. These mbtins are shorter, and have different amino acid compositions, than the characterized mbtin from Methylosinus trichosporium OB3b. A coordinating pyrazinedione ring in the Methylocystis mbtins has little influence on the Cu(I) site structure. The Methylocystis mbtins have a sulfate group that helps stabilize the Cu(I) forms, resulting in affinities of approximately 10²¹ M^-1. The Cu(II) affinities vary over three orders of magnitude with reduction potentials covering approximately 250 mV, which may dictate the mechanism of intracellular copper release. Copper uptake and the switchover from using the iron-containing soluble MMO to the copper-containing particulate enzyme is faster when mediated by the native mbtin, suggesting that the amino acid sequence is important for the interaction of mbtins with receptors. The differences in structures and properties of mbtins, and their influence on copper utilization by methane-oxidizing bacteria, have important implications for the ecology and global function of these environmentally vital organisms.Copper is an essential protein cofactor involved in many important cellular processes (1, 2), and copper-trafficking systems have been extensively studied (1, 3–8). Although copper uptake by eukaryotes is well defined (1, 4, 9), acquisition of this metal by prokaryotes remains poorly understood. Methane-oxidizing bacteria secrete the small copper-binding molecule methanobactin (mbtin) when copper is limiting (10–18), presumably for sequestration of this metal. These organisms have conditionally high requirements for copper (19), primarily for the active site (20) of the particulate methane monooxygenase (pMMO). Almost all known methane-oxidizing bacteria use pMMO for the consumption of methane (19), an important greenhouse gas. A subclass of “switchover” organisms exists that can also produce a less efficient iron-containing soluble MMO (sMMO) under copper-deficient conditions, with pMMO expression up-regulated in response to an increase in the copper-to-cell ratio (15, 21).Methanobactin production has been examined in a number of methane-oxidizing bacteria (22–24), but mbtins from only two organisms have been characterized (13, 18). The mbtin (two forms) from Methylosinus trichosporium OB3b (a switchover organism) is the most extensively studied (13, 15–17, 25–29), and binds a single copper ion coordinated in a distorted tetrahedral arrangement by the nitrogens from two oxazolone rings (29) and the sulfurs from two enethiolate groups. The molecule has a compact arrangement stabilized by a disulfide bridge. The very high affinities for copper that have been determined for the M. trichosporium OB3b molecules are consistent with mbtins playing a role in the acquisition of copper (17). Direct evidence of uptake and cytoplasmic localization has recently been obtained for Cu(I)-mbtin from M. trichosporium OB3b (30). These studies confirm that mbtin is the primary component of an active copper-acquisition system in methane-oxidizing bacteria. Comparisons have been made (13, 15, 16) between mbtins and iron-sequestering siderophores (31, 32), particularly the structurally related pyoverdines. Whereas detailed information is available for siderophore-mediated iron uptake and utilization, almost nothing is known about how mbtins acquire and deliver copper.In this work mbtins have been isolated and characterized from three Methylocystis strains, including switchover and nonswitchover organisms. All of these mbtins have high Cu(I) affinities that are similar to those of the M. trichosporium OB3b mbtins. The N-terminal group present in the Methylocystis mbtins alters the Cu(II) affinity, which will influence acquisition of the metal, and results in different reduction potentials (E_m values) that could dictate the copper release mechanism. Variations in the structures of mbtins affect their ability to provide copper to methane-oxidizing bacteria and to initiate the transition from using sMMO to pMMO in switchover organisms. We have identified features of mbtins that influence how methane-oxidizing bacteria uptake and utilize copper that may influence their capacity to suppress methane in the natural environment.     

Hypophosphatemic Effect of Niacin in Patients without Renal Failure: A Randomized Trial

Background and objectives: Niacin administration lowers the marked hyperphosphatemia that is characteristic of renal failure. We examined whether niacin administration also reduces serum phosphorus concentrations in patients who have dyslipidemia and are free of advanced renal disease.Design, setting, participants, & measurements: We performed a post hoc data analysis of serum phosphorus concentrations that had been determined serially (at baseline and weeks 4, 8, 12, 18, and 24) among 1547 patients who had dyslipidemia and were randomly assigned in a 3:2:1 ratio to treatment with extended release niacin (ERN; 1 g/d for 4 weeks and dose advanced to 2 g/d for 20 weeks) combined with the selective prostaglandin D2 receptor subtype 1 inhibitor laropiprant (L; n = 761), ERN alone (n = 518), or placebo (n = 268).Results: Repeated measures analysis revealed that ERN-L treatment resulted in a net mean (95% confidence interval) serum phosphorus change comparing ERN-L with placebo treatment of −0.13 mmol/L (−0.15 to −0.13 mmol/L; −0.41 mg/dl [−0.46 to −0.37 mg/dl]). These results were consistent across the subgroups defined by estimated GFR of <60 or ≥60 ml/min per 1.73 m², a serum phosphorus of >1.13 mmol/L (3.5 mg/dl) versus ≤1.13 mmol/L (3.5 mg/dl), the presence of clinical diabetes, or concomitant statin use.Conclusions: We have provided definitive evidence that once-daily ERN-L treatment causes a sustained 0.13-mmol/L (0.4-mg/dl) reduction in serum phosphorus concentrations, approximately 10% from baseline, which is unaffected by estimated GFR ranging from 30 to ≥90 ml/min per 1.73 m² (i.e., stages 1 through 3 chronic kidney disease).Abnormalities in calcium-phosphorus homeostasis, including significant elevations in serum phosphorus concentrations, are thought to contribute to arterial stiffening, hypertension, and cardiovascular disease (CVD) risk in patients with advanced chronic kidney disease and ESRD that requires maintenance dialysis (1–6). Observational data from population-based studies suggested that even serum phosphorus concentrations within the normative range are linearly associated with measures of subclinical arteriosclerosis and the development of incident CVD outcomes (7–12). Two cross-sectional studies from patients who underwent cardiac catheterization have further indicated that serum phosphorus concentrations, primarily within the normative range, were directly associated with both the presence and the severity of angiographic coronary artery disease (13,14). Moreover, a graded, independent association between serum phosphorus concentrations (again, within the normative range) and recurrent CVD events was reported among a large clinical trial cohort of patients with a previous myocardial infarction (15).Supplementation of calcium salts, despite their efficacy and tolerability as a phosphorus-lowering treatment in ESRD, may enhance coronary artery and aortic valve calcification (16,17). This observation highlights the need for hyperphosphatemia treatment protocols to balance potential benefits and adverse effects (18–22). Phosphorus-lowering drugs that target other cardiovascular risk factors in chronic kidney disease (CKD), simultaneously, including, for example, dyslipidemia (23), might have additive or synergistic benefits. These findings may also be relevant to populations with less advanced CKD or normal renal function.Preliminary studies suggested that niacin administration (as niacinamide, niceritrol, or nicotinic acid) could be a useful primary or adjunctive treatment for the marked hyperphosphatemia that is characteristic of ESRD (24–30). Several reports from clinical trials of extended-release niacin (ERN) that was given to patients who had dyslipidemia and were free of clinical renal disease and hyperphosphatemia have contained limited additional data noting up to 10% reductions in the serum phosphorus concentrations of actively treated patients (31–34). These repeated clinical observations (24–34) are most plausibly explained by the direct inhibitory effect of niacin compounds on active transport-mediated phosphorus absorption in the mammalian small intestine (35–39).Published studies of patient populations who had dyslipidemia and were receiving ERN that included phosphorus data may have failed to provide information on baseline phosphorus values (33,34), and none (31–34) performed repeated measures analyses to examine the potential effects of niacin treatment on serum phosphorus and calcium concentrations, as well as the calcium-phosphorus products.Focused reexamination of the large, placebo-controlled clinical trial data set assembled by Maccubbin et al. (34) afforded us a unique opportunity to elucidate these and other unresolved issues regarding the impact of niacin given as the fixed-dose combination of ERN and laropiprant (ERN-L), a selective prostaglandin D2 receptor subtype 1 inhibitor that reduces niacin-induced flushing (34) or ERN alone on serum phosphorus and calcium concentrations and calcium-phosphorus products. We further evaluated whether there was evidence for significant effect modification by estimated GFR (eGFR), baseline serum phosphorus concentration, the presence of diabetes, or concurrent hepatic hydroxymethyl glutaryl–CoA reductase inhibitor (statin) use when assessing the potential impact of niacin on these routine clinical measures of calcium-phosphorus homeostasis.     

Outcomes Associated with Serum Calcium Level in Men with Non-Dialysis-Dependent Chronic Kidney Disease

Background and objectives: Elevated serum calcium has been associated with increased mortality in dialysis patients, but it is unclear whether the same is true in non-dialysis-dependent (NDD) chronic kidney disease (CKD). Outcomes associated with low serum calcium are also not well-characterized.Design, setting, participants, & measurements: We examined associations of baseline, time-varying, and time-averaged serum calcium with all-cause mortality in a historic prospective cohort of 1243 men with moderate and advanced NDD CKD by using Cox models.Results: The association of serum calcium with mortality varied according to the applied statistical models. Higher baseline calcium and time-averaged calcium were associated with higher mortality (multivariable adjusted hazard ratio (95% confidence interval): 1.31 (1.13, 1.53); P < 0.001 for a baseline calcium 1 mg/dl higher). However, in time-varying analyses, lower calcium levels were associated with increased mortality.Conclusions: Higher serum calcium is associated with increased long-term mortality (as reflected by the baseline and time-averaged models), and lower serum calcium is associated with increased short-term mortality (as reflected by the time-varying models) in patients with NDD CKD. Clinical trials are warranted to determine whether maintaining normal serum calcium can improve outcomes in these patients.Mineral and bone disorders in chronic kidney disease (CKD) (1) have emerged as novel mortality risk factors in dialysis patients (2–8). Some of these abnormalities (such as serum phosphorus and parathyroid hormone (PTH) levels) have also been implicated in similar ways in patients with non-dialysis-dependent (NDD) CKD (9–12). Serum calcium''s effect on outcomes has been the focus of attention mainly in dialysis patients, where calcium metabolism is significantly distorted (13–19). The use of calcium-containing phosphate binders further complicates the picture because these medications could be involved in the etiology of vascular calcification (20,21), and their roles as therapeutic agents have been intensely debated (22). Supporting the potential role for calcium in cardiovascular disease were epidemiologic studies showing an association between higher calcium and increased mortality (2–8). Some of the same studies have also suggested that extremely low calcium levels may themselves be deleterious (2,3), which has ultimately resulted in recommendations to attain a low-normal serum calcium level in dialysis patients (23). Studies examining the role of calcium in NDD CKD patients are fewer and failed to unequivocally show an association between abnormal calcium levels and vascular calcification (24–27). No study has yet examined the association of calcium levels with mortality in NDD CKD.We examined the association of serum calcium levels with all-cause mortality in a large number of male US veterans with moderate and advanced NDD CKD at a single medical institution.     

Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disease prevalent in several dog breeds. Typically, the initial progressive upper motor neuron spastic and general proprioceptive ataxia in the pelvic limbs occurs at 8 years of age or older. If euthanasia is delayed, the clinical signs will ascend, causing flaccid tetraparesis and other lower motor neuron signs. DNA samples from 38 DM-affected Pembroke Welsh corgi cases and 17 related clinically normal controls were used for genome-wide association mapping, which produced the strongest associations with markers on CFA31 in a region containing the canine SOD1 gene. SOD1 was considered a regional candidate gene because mutations in human SOD1 can cause amyotrophic lateral sclerosis (ALS), an adult-onset fatal paralytic neurodegenerative disease with both upper and lower motor neuron involvement. The resequencing of SOD1 in normal and affected dogs revealed a G to A transition, resulting in an E40K missense mutation. Homozygosity for the A allele was associated with DM in 5 dog breeds: Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, German Shepherd dog, and Chesapeake Bay retriever. Microscopic examination of spinal cords from affected dogs revealed myelin and axon loss affecting the lateral white matter and neuronal cytoplasmic inclusions that bind anti-superoxide dismutase 1 antibodies. These inclusions are similar to those seen in spinal cord sections from ALS patients with SOD1 mutations. Our findings identify canine DM to be the first recognized spontaneously occurring animal model for ALS.Amyotrophic lateral sclerosis (ALS) refers to a heterogeneous group of adult onset human diseases, in which progressive neurodegeneration affecting both the upper and lower motor neuron systems causes advancing weakness and muscle atrophy, and culminates in paralysis and death. Approximately 5 to 10% of ALS cases are familial; the rest appear to be sporadic (1–3). Mutations in SOD1 account for ≈20% of the familial ALS cases and 1 to 5% of the cases of sporadic ALS (1–4); >120 different SOD1 mutations have been identified in ALS patients (http://alsod.iop.kcl.ac.uk/Als/index.aspx). Elucidation of mechanisms underlying ALS has been hampered by a paucity of biological material from affected individuals in early stages of the disease (5). To our knowledge, there are no previous reports of spontaneously occurring animal models of ALS. Thus, ALS research has relied heavily on transgenic rodents expressing mutant human SOD1 (hSOD1^m) to produce a motor neuron disease, which recapitulates many features of ALS (5–7). In contrast, nullizygous SOD1 knockout mice develop normally (8), suggesting that the neurodegeneration in hSOD1^m mice and in ALS patients results from a toxic gain of function (1, 5–8). Although the nature of the toxin is unclear, several experiments suggest that the neurodegeneration occurs because conformational changes in the mutant superoxide dismutase 1 protein (SOD1) alter the biological activity and/or promote the formation of intracellular SOD1 aggregates (1, 4, 9, 10).Canine degenerative myelopathy (DM) has been recognized for >35 years as a spontaneously occurring, adult-onset spinal cord disorder of dogs (11). When pelvic limb hyporeflexia and nerve root involvement were observed, the disease was termed chronic degenerative radiculomyelopathy (12). Initially thought to be specific to German Shepherds, it has also been called German Shepherd dog myelopathy (13). Since these early reports, DM has been diagnosed in several other breeds. The disease is common in certain breeds including the Pembroke Welsh corgi, Boxer, Rhodesian ridgeback, and Chesapeake Bay retriever (14).With DM, there is no sex predilection. Most dogs are at least 8 years old before the onset of clinical signs (11–18). The initial clinical sign is a spastic and general proprioceptive ataxia in the pelvic limbs. At this stage of the disease, the presence of spinal reflexes indicates an upper motor neuron paresis (11). The asymmetric weakness frequently reported at disease onset progresses to paraplegia (11, 12, 14, 16, 18). Hyporeflexia of the myotatic and withdrawal reflexes occur in the latter disease stage (11, 12, 14, 16, 18). The disease duration can exceed 3 years; however, dog owners usually elect euthanasia within a year of diagnosis when their dogs become paraplegic. If the disease is allowed to progress, clinical signs will ascend to affect the thoracic limbs (11, 14, 16). Because various common acquired compressive spinal cord diseases can mimic DM by compromising the upper motor neuron and general proprioceptive pathways, a definitive diagnosis of DM can only be accomplished postmortem by the histopathologic observation of axonal and myelin degeneration, which can occur at all levels of the spinal cord (16–18) and in all spinal cord funiculi, but are consistently most severe in the dorsal portion of the lateral funiculus within the middle to caudal thoracic region (11, 13–18).     

Phase 1 Study of Anti-CTGF Monoclonal Antibody in Patients with Diabetes and Microalbuminuria

Background and objectives: This report summarizes the first phase 1 trial treating patients with microalbuminuric diabetic kidney disease (DKD) using FG-3019, a human monoclonal antibody to connective tissue growth factor (CTGF). CTGF is critically involved in processes of progressive fibrosis, including DKD. This phase 1, open-label, dose-escalation trial evaluated safety, pharmacokinetics, and possible therapeutic effects of FG-3019 on albuminuria, proteinuria, and tubular proteins.Design, setting, participants, and measurements: Microalbuminuric subjects (n = 24) with type 2 (79%) or type 1 (21%) diabetes received 3 or 10 mg/kg FG-3019 dosed intravenously every 14 days for four doses. Albuminuria and safety follow-up were to days 62 and 365, respectively.Results: No infusion was interrupted for symptoms, although 5 of 24 subjects had mild infusion-day adverse events thought to be possibly drug-related. No subject developed anti-FG-3019 antibodies. FG-3019 clearance was lower at 10 mg/kg than at 3 mg/kg, suggesting a saturable elimination pathway. Although this study was not designed for efficacy testing, it was notable that urinary albumin/creatinine ratio (ACR) decreased significantly from mean pretreatment ACR of 48 mg/g to mean post-treatment (day 56) ACR of 20 mg/g (P = 0.027) without evidence for a dose-response relationship.Conclusions: Treatment of microalbuminuric DKD subjects using FG-3019 was well tolerated and associated with a decrease in albuminuria. The data demonstrate a saturable pathway for drug elimination, minimal infusion adverse events, and no significant drug-attributable adverse effects over the year of follow-up. Changes in albuminuria were promising but require validation in a prospective, randomized, blinded study.Patients with diabetic kidney disease (DKD) are at increased risk for cardiovascular complications and early mortality. Those who survive long enough tend to progress to ESRD requiring dialysis or transplantation. Although advances in therapy with angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor type II blockers (ARBs) have attenuated the incident rate of ESRD (1), disease progression remains common (2–4) and diabetes continues to be the leading cause for initiation of dialysis in the United States (1).Connective tissue growth factor (CTGF) is a 349-amino-acid secreted pleiotropic protein belonging to the cysteine-rich CCN (CTGF/Cyr61/Cef10/NOVH) family. Numerous glomerular, tubulointerstitial, and vascular cells types can produce CTGF, and many factors associated with the diabetic condition can stimulate CTGF expression, including hypertension, hyperglycemia, and hyperlipidemia (5–24).CTGF is a critical mediator of extracellular matrix accumulation and coordinates a final common pathway of fibrosis (5,25,26). CTGF has been shown to amplify the fibrogenic activity of TGFβ (27) and IGF-1 (17) and to inhibit the action of antifibrotic and regenerative factors bone morphogenic protein-7 (27,28) and vascular endothelial growth factor (29,30).In type 1 diabetes, plasma and urine CTGF levels correlate with the level of albuminuria and the stage of progressive renal insufficiency (31–34), and the plasma CTGF level is an independent predictor of vascular disease as assessed by intimal medial thickness (35) and of mortality and progression to ESRD (36). In renal biopsy specimens from patients with diabetes, elevated levels of CTGF mRNA are associated with chronic tubulointerstitial damage, albuminuria, and progression of renal insufficiency (37–39).FG-3019 is a recombinant human anti-CTGF monoclonal IgG₁ antibody that has shown activity in rodent models of kidney dysfunction associated with type 1 and 2 diabetes (40–42). Here, we report results of an open-label dose-escalation trial of FG-3019 infusions administered biweekly over 56 days in patients with DKD, the first study designed to evaluate safety and potential therapeutic effect of FG-3019 in this patient population.     

Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197

CRM197 is an enzymatically inactive and nontoxic form of diphtheria toxin that contains a single amino acid substitution (G52E). Being naturally nontoxic, CRM197 is an ideal carrier protein for conjugate vaccines against encapsulated bacteria and is currently used to vaccinate children globally against Haemophilus influenzae, pneumococcus, and meningococcus. To understand the molecular basis for lack of toxicity in CRM197, we determined the crystal structures of the full-length nucleotide-free CRM197 and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA), both at 2.0-Å resolution. The structures show for the first time that the overall fold of CRM197 and DT are nearly identical and that the striking functional difference between the two proteins can be explained by a flexible active-site loop that covers the NAD binding pocket. We present the molecular basis for the increased flexibility of the active-site loop in CRM197 as unveiled by molecular dynamics simulations. These structural insights, combined with surface plasmon resonance, NAD hydrolysis, and differential scanning fluorimetry data, contribute to a comprehensive characterization of the vaccine carrier protein, CRM197.Diphtheria is a contagious respiratory disease that was a major cause of death among children around the world until mass vaccination was introduced in the 1920s. Although diphtheria has now been virtually eliminated in the industrialized world, rare outbreaks still occur worldwide (1, 2). Corynebacterium diphtheriae was shown to be the causative agent of diphtheria by Loeffler in 1885 (3), and Roux and Yersin showed that an extracellular toxin, diphtheria toxin (DT) secreted by C. diphtheriae is responsible for toxicity (4). The formaldehyde-treated detoxified form of DT, diphtheria toxoid, has been successfully used for mass vaccination and is still widely used as a component of combination vaccines (5, 6).A major contribution to the understanding of the mode of action of DT was the discovery of mutated forms in the early 1970s (7). Several phages encoding mutants of DT, named cross-reactive materials (CRMs), were isolated following nitrosoguanidine-based mutagenesis of the phage containing the gene encoding DT. Being naturally nontoxic, CRMs were immediately recognized as having great potential for vaccine development. The most important CRM identified was CRM197, an enzymatically inactive and nontoxic form of DT that contains a single amino acid substitution from Glycine to Glutamate in position 52 (8). Subsequently CRM197 was found to be an ideal carrier for conjugate vaccines against encapsulated bacteria. Here, the carrier protein is covalently linked to poorly immunogenic and T-cell-independent capsular polysaccharides, thus creating T-cell-dependent conjugate antigens that are highly immunogenic in infants (9–11).Vaccines containing CRM197 as a carrier protein have been successfully used to immunize hundreds of millions of children. Such vaccines currently include Menveo®, a recently approved tetravalent conjugate vaccine against serogroups A-C-W135-Y of Neisseria meningitidis, Menjugate® and Meningitec® (against serotype C of N. meningitidis), Vaxem-Hib® and HibTITER® (against Haemophilus influenzae type B, Hib), and the multivalent pneumococcal conjugate Prevnar™ (12).The widespread use of diphtheria toxoid and CRM197 has prompted many investigations of DT and related proteins. Diphtheria toxin is an ADP-ribosylating enzyme that is secreted as a proenzyme of 535 residues and is processed by trypsin-like proteases with release of two fragments (A and B). Fragment A uses NAD as a substrate, catalyzing the cleavage of the N-glycosidic bond between the nicotinamide ring and the N-ribose and mediating the covalent transfer of the ADP-ribose (ADPRT activity) to the modified Histidine 715 (diphthamide) of the elongation factor EF-2. This posttranslational diphthamide modification inactivates EF-2, halting protein synthesis and resulting in cell death. Extensive structural studies elucidated the molecular architecture of DT (13–19). The A fragment of DT (also named C domain) carries the catalytic active site and is the only fragment of the toxin required for the final step of intoxication, while the B fragment carries the R and T domains, which mediate binding to receptors on the host cell surface and promote the pH-dependent transfer of fragment A to the cytoplasm, respectively. An Arginine-rich disulfide-linked loop connects fragment A to fragment B (or domain C to domains TR), and this interchain disulfide bond is the only covalent link between the two fragments after proteolytic cleavage of the chain at position 186.While much progress has been made on the molecular characterization of DT over the last two decades, an understanding of the molecular basis for the lack of toxicity of CRM197 has so far been elusive. Here we present the crystal structures of full-length nucleotide-free (NF)-CRM197, and of CRM197 in complex with the NAD hydrolysis product nicotinamide (NCA). In addition, the differences between CRM197 and DT were elucidated using surface plasmon resonance (SPR), a NAD-glycohydrolase (NADase) activity assay, molecular dynamics (MD) simulations, and differential scanning fluorimetry (DSF).     

Salmonella regulates polyubiquitination and surface expression of MHC class II antigens

Salmonella typhimurium is a facultative pathogen capable of entering and replicating in both professional and non-professional antigen presenting cells. Control of infection requires MHC class II restricted CD4 T-helper cell responses. Here we show that Salmonella infection induced polyubiquitination of HLA-DR, a post-translational modification that led to removal of mature, peptide loaded, αβ dimers from the cell surface. Immature αβIi complexes were unaffected. Surface expression of all class II isotypes, HLA-DP, -DQ, and -DR, was reduced in infected cells, but other cell-surface molecules that traffic through class II peptide loading compartments were unaffected. A Salmonella strain carrying a mutation in ssaV did not induce ubiquitination of class II, implicating Salmonella T3SS-2 effector proteins in the process. T3SS-2 effectors, with established or proposed roles in ubiquitination, were not required for class II down-regulation, suggesting that an additional T3SS-2 effector is involved in regulating MHC class II ubiquitination. Although recognized as a viral immune evasion strategy, here, we demonstrate that bacteria can control surface MHC expression through ubiquitination.Salmonellae are Gram-negative bacteria that cause in a range of animal species. In humans, infection with the serovar Salmonella typhi results in approximately 21 million cases of typhoid fever annually and 200,000 deaths (1). Infection occurs by the oral route and requires virulence factors transported into the host cell by 2 Salmonella-encoded type III secretion systems (T3SS). T3SS-1 is encoded within Salmonella pathogenicity island-1 (SPI-1) and is required for the initial invasion of host cells, while T3SS-2, which is encoded within SPI-2, is required for survival and replication of bacteria within Salmonella-containing vacuoles (SCVs).Infecting bacteria are generally cleared from the circulation but can survive and replicate in cells of the reticuloendothelial system. Clearance requires both CD4⁺ and CD8⁺ T cell-mediated responses, but CD4⁺ responses are of most importance. In mice lacking CD4⁺ T cells, early phases of infection are controlled but ultimately bacterial numbers increase, resulting in death (2). MHC genes have a role in controlling infection and particular haplotypes in humans are associated with either resistance or susceptibility to typhoid fever (3). Bone marrow-derived dendritic cells (DCs) infected with Salmonella show reduced ability to present antigen to T cells (4), providing further evidence that Salmonella interferes with MHC class II antigen presentation.MHC class II molecules play an essential role in adaptive immune responses by presenting peptide antigens to CD4 restricted T cells (5). Class II α and β chains assemble in the endoplasmic reticulum (ER), together with invariant chain (Ii), to form nonomeric complexes (αβIi)₃ (6). These complexes exit the ER and follow the secretory pathway to the trans-Golgi network (TGN). Dileucine based sorting signals, present in Ii, are responsible for targeting into endosomal-lysosomal compartments, either directly from the TGN, or indirectly via the cell surface (7–9). Experiments where clathrin or AP-2 are ablated suggest that a significant proportion of (αβIi)₃ traffics via the plasma membrane (10). Once in the endosomal-lysosomal system, Ii is sequentially degraded leaving a short peptide fragment, CLIP, in the peptide-binding groove (11). HLA-DM and -DO, two class II related molecules, regulate the exchange of CLIP for antigenic peptide (12, 13). The complex nature of the class II antigen-processing pathways provides extensive opportunities for pathogen interference (14).Post-translational regulation of surface class II expression is a key feature of the activation of DCs, the most potent professional antigen presenting cells (APCs) known. Upon sensing invading pathogens, DCs redirect their intracellular pool of MHC class II from late endosomal antigen processing compartments to the cell surface (15, 16). Synchronized relocation of the co-stimulatory molecule B7.2 ensures that the mature DCs exhibit full co-stimulatory capacity (17). Redistribution is controlled through regulated ubiquitination of class II (15, 16).Salmonella interferes with MHC antigen presentation by specifically reducing cell surface HLA-DR expression (18). We set out to determine the molecular mechanism underlying this precise targeting of MHC class II molecules, which are crucial to CD4 T-cell responses.     

N-CDAD in Canada: Results of the Canadian Nosocomial Infection Surveillance Program 1997 N-CDAD Prevalence Surveillance Project

BACKGROUND:

A 1996 preproject survey among Canadian Hospital Epidemiology Committee (CHEC) sites revealed variations in the prevention, detection, management and surveillance of Clostridium difficile-associated diarrhea (CDAD). Facilities wanted to establish national rates of nosocomially acquired CDAD (N-CDAD) to understand the impact of control or prevention measures, and the burden of N-CDAD on health care resources. The CHEC, in collaboration with the Laboratory Centre for Disease Control (Health Canada) and under the Canadian Nosocomial Infection Surveillance Program, undertook a prevalence surveillance project among selected hospitals throughout Canada.

OBJECTIVE:

To establish national prevalence rates of N-CDAD.

METHODS:

For six weeks in 1997, selected CHEC sites tested all diarrheal stools from inpatients for either C difficile toxin or C difficile bacteria with evidence of toxin production. Questionnaires were completed for patients with positive stool assays who met the case definitions.

RESULTS:

Nineteen health care facilities in eight provinces participated in the project. The overall prevalence of N-CDAD was 13.0% (95% CI 9.5% to 16.5%). The mean number of N-CDAD cases were 66.3 cases/100,000 patient days (95% CI 37.5 to 95.1) and 5.9 cases/1000 patient admissions (95% CI 3.4 to 8.4). N-CDAD was found most frequently in older patients and those who had been hospitalized for longer than two weeks in medical or surgical wards.

CONCLUSIONS:

This national prevalence surveillance project, which established N-CDAD rates, is useful as ''benchmark'' data for Canadian health care facilities, and in understanding the patterns and impact of N-CDAD.Key Words: Canada, CDAD, Clostridium difficile-associated diarrhea, Hospital, Nosocomial diarrhea, PrevalenceNosocomial acquisition and transmission of Clostridium difficile are well known (1-4). Despite efforts to control and prevent infections in health care facilities, nosocomially acquired C difficile-associated diarrhea (N-CDAD) persists; some have reported that the number of N-CDAD infections are increasing (5-10). Although the majority of patients remain asymptomatic following acquisition of C difficile (5), it is still the most commonly identified cause of nosocomial diarrhea (5,11,12). While specific antibiotic therapy for C difficile has reduced morbidity and mortality among people with CDAD (13-15), evidence exists that C difficile infection contributes to patient morbidity (7,10) and significantly impacts hospital costs (15-17).Published literature related to the prevalence of CDAD primarily describes periodic outbreaks or endemic situations in health care facilities (7,10,16-19). Because elderly people and those exposed to large amounts of antibiotics have a higher risk of acquiring CDAD, they are commonly surveyed (15,20,21). Specific wards (eg, medical and surgical) where the rates of CDAD are higher are also more frequently studied (2,6,22,23). Multicentre and national surveillance of CDAD in North America and Europe is rare (24-28). In Canada, individual health centres have data on the prevalence and demographics of CDAD cases (6,9,21); however, no national data exist.Many CDAD surveillance studies include community cases (16,17,24-28); however, it is useful to examine specifically N-CDAD cases, because they represent illness that may be prevented by hospital infection prevention and control practices. The primary reservoirs of C difficile in the hospital are humans and the environment (29). Consequently, the nosocomial acquisition of this organism may represent inadequate infection control practices (30). This underscores the importance of instigating measures to monitor the prevalence of N-CDAD, and implementing and assessing the efficacy of any prevention or control practices.There is no Canadian literature that examines the hospital costs of C difficile infections. Worldwide, there are limited data regarding the hospital costs associated with CDAD (16,17). One British study specifically examined the costs of N-CDAD (15). However, all studies suggest that these costs are substantial, which include the expenses of caring for and treating patients with CDAD, combined with the costs associated with C difficile outbreaks (15-17).An N-CDAD prevalence project was undertaken by the Canadian Nosocomial Infection Surveillance Program (CNISP) through participating Canadian health care facilities. CNISP is a collaborative national surveillance program among the Laboratory Centre for Disease Control, Health Canada and the Canadian Hospital Epidemiology Committee (CHEC), a subcommittee of the Canadian Infectious Disease Society. CHEC members participated voluntarily in the CNISP project. The intent of this project was to establish health care facility N-CDAD prevalence rates that could be used as ''benchmark'' data for other Canadian health care facilities, and to assist with the development and evaluation of guidelines that may decrease the incidence and cost of N-CDAD within Canadian health care facilities. The project used standardized case definitions for CDAD and N-CDAD. Non-nominal data were collected and submitted to the Laboratory Centre for Disease Control for compilation, analysis and interpretation. To estimate the burden of N-CDAD on the Canadian health care system, it was necessary to first determine national N-CDAD prevalence rates through a multicentre, geographically diverse surveillance project.     

Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-Å crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, α-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.Glypicans are heparan sulfate proteoglycans (HSPGs) that consist of an approximately 450 amino acid N-terminal protein domain followed by an approximately 100 amino acid stalk region that is attached to the outer cell membrane via a glycosyl-phosphatidylinositol anchor (1). The N-terminal domain of most glypicans is proteolytically processed by a furin-like convertase to produce two chains that remain connected by disulfide bonds (2). This processing appears required for some but not all glypican activity (2, 3). The stalk regions of glypicans are predicted to be largely random coil and typically contain 1–5 heparan sulfate attachment sites (1, 4). Six glypicans are present in humans and mice (glypican-1, -2, -3, -4, -5, and -6); two are present in Drosophila [Dally and Dally-like (Dlp)] (1). Based on sequence similarity, glypicans assort into two subfamilies with glypican-1, -2, -4, -6, and Dlp in one family and glypican-3, -5, and Dally in another (1).Glypicans are active in development in both vertebrates and invertebrates. Loss of Dally in fruit flies results in defects in brain, eye, wings, antennae, and genitalia (5). Loss of glypican-3 in humans is responsible for Simpson–Golabi–Behmel overgrowth syndrome, in which widespread visceral and skeletal abnormalities are present along with a predisposition to tumor formation (6). Loss of glypican-6 has recently been shown to cause omodysplasia, a genetic disorder characterized by variable heart defects, cognitive delay, skeletal and facial abnormalities, and shortness of stature (7). Much of the function of glypicans is attributable to modulation of signaling by several heparin-binding growth factors active during development including members of the fibroblast growth factor, Hedgehog (Hh), Wnt, and transforming growth factor-β families (8–15). Each of these factors functions as a morphogen to elicit distinct concentration-dependent responses within target cells, and glypicans have been shown to be required both for normal response to these factors as well as to establish their proper distribution (9, 10, 12, 16–21). The heparan sulfate attachments of glypicans are clearly important for mediating interactions with these growth factors and downstream signaling components (22, 23), but recent work has demonstrated a role for the N-terminal protein domain, which lacks heparan sulfate modifications, in mediating responsiveness to at least Wnt and Hh signals (23–26).Curiously, glypicans appear able to play both positive and negative roles in mediating Hh signaling. The protein region of Dally-like contributes positively to Drosophila Hh responsiveness, and the developmental defects in omodysplasia, particularly the bone growth defects, are suggestive of a positive role for glypican-6 function in response to Indian hedgehog (7). Notably, glypican-4 and glypican-6 are most similar to Dlp (vs. Dally) and complement Dlp function in a Drosophila cultured cell-based Hh signaling assay (25). In contrast, the protein region of glypican-3, which is more similar to Dally than Dally-like, is a negative regulator of Hh responsiveness in the mouse (24, 25, 27, 28). Based on sequence homology and functional phenotypes, it has thus been speculated that the two major subfamilies of glypicans have evolved opposing activities in Hh signal responsiveness (25).To investigate the molecular basis for glypican function, we have undertaken structural and functional characterization of the N-terminal protein domain of Dlp and report here its 2.4-Å crystal structure. We show that the N-terminal protein domains of glypicans adopt an elongated α-helical structure with no evident homology to any known structure. We have used structure-guided mutagenesis to identify a localized region on the Dlp surface important for the ability of Dlp to mediate Hh signal response. These results are most consistent with Dlp functioning as a binding protein in Hh signaling, but we are unable to detect high-affinity interactions between Dlp and either Hh or an Hh:Ihog complex. These results establish a molecular basis for mapping and comparing functional regions of different glypicans.     

Clinical outcome with oral linezolid and rifampin following recurrent methicillin-resistant Staphylococcus aureus bacteremia despite prolonged vancomycin treatment

Drug-resistant Gram-positive bacteria, especially Staphylococcus aureus, are emerging as the predominant organisms involved in both nosocomial and community-acquired infections. Since the 1980s, vancomycin has been the first-line antibiotic used to treat methicillin- resistant S aureus. However, allergy and intolerance to vancomycin, the increasing number of vancomycin clinical failures and the existence of vancomycin intermediate-susceptible isolates of S aureus suggest that new antibiotics are needed. This paper reports the only known case of a successful clinical outcome with long term oral linezolid and rifampin therapy in the management of recurrent and persistent methicillin-resistant S aureus bacteremia with metastatic infections despite prolonged vancomycin use. More than two years since the initiation of linezolid and rifampin, the study patient has been clinically well with no evidence of adverse drug reactions including cytopenia and hepatic toxicities. Physicians must be aware of the novel developments in antibiotic therapy to treat drug-resistant bacterial infections.Key Words: Linezolid, MRSA bacteremia, Osteomyelitis, VancomycinAccording to the National Nosocomial Infection Surveillance System, Gram-positive pathogens, especially Staphylococcus aureus, are emerging as the predominant organisms involved in both nosocomial and community-acquired infections (1). S aureus is spread via contact and is a common cause of numerous infections including soft tissue infections (cellulitis and impetigo), wound infections, bacteremia with metastatic infections (osteomyelitis, septic arthritis and acute infective endocarditis) and toxic shock syndrome (2-4).Since the introduction of antibiotics in the 1940s, S aureus has been most effective in its ability to develop resistance to antimicrobial agents, thus evoking significant concern in both the public and the health care communities. For example, one year following the introduction of methicillin for the treatment of penicillin-resistant S aureus, the first strain of methicillin-resistant S aureus (MRSA) was identified (5).Over the past two decades, MRSA rates have increased dramatically in both the community and hospital settings, to the point that this pathogen is now endemic in many American centres (5). Methicillin resistance is associated with significant morbidity and mortality. Bacteremia due to MRSA, when compared with methicillin-susceptible S aureus, has a two- to threefold increase in mortality (6-7).Vancomycin was initially introduced in the middle 1960s but gained significant recognition in the early 1980s as the treatment of choice for MRSA (8). While this antibiotic is principally used for the treatment of MRSA infections today, it has a relatively high clinical failure rate (9,10). Various reasons for the decreased clinical efficacy of vancomycin despite the in vitro sensitivity of MRSA to the antibiotic have been suggested. First, vancomycin has decreased bactericidal activity against S aureus (11). Second, its low tissue concentration prevents effective eradication of virulent Gram-positive pathogens (12,13).In July 2002, the first case of infection due to vancomycin-resistant S aureus (VRSA, minimum inhibitory concentration [MIC] greater than 128 µg/mL) related to catheter exit site infection in a Michigan patient was reported (14). There have also been several worldwide reports of glycopeptide-intermediate S aureus (GISA), defined as an MIC of 8 to 16 µg/mL. The first case of GISA was reported in Japan in 1996 (5). Since then, strains of GISA, also resistant to methicillin, were reported from Italy, the United Kingdom, France and the United States (2,15-21). Prolonged intermittent vancomycin use has been associated with the development of GISA and VRSA strains (14,16).Current antimicrobial agents routinely used to treat antibioticresistant Gram-positive organisms, especially MRSA, have limitations in terms of their efficacy. With the emergence of the aforementioned resistant strains, newer antimicrobial agents must be considered.This paper reports the only known case of a successful clinical outcome and no adverse drug reactions with long term linezolid and rifampin therapy in the management of recurrent and persistent MRSA bacteremia with metastatic infections despite prolonged vancomycin use.     

The management of infection and colonization due to methicillin-resistant Staphylococcus aureus: A CIDS/CAMM position paper

Methicillin-resistant Staphylococcus aureus (MRSA) is being seen with greater frequency in most hospitals and other health care facilities across Canada. The organism may cause life-threatening infections and has been associated with institutional outbreaks. Several studies have confirmed that MRSA infection is associated with increased morbidity and mortality compared with infections caused by susceptible strains, even when the presence of comorbidities is accounted for. Treatment of MRSA infection is complicated by the fact that these organisms are resistant to multiple antimicrobial agents, so treatment options are limited. The effectiveness of decolonization therapy (attempting to eradicate MRSA carriage) is also uncertain. This paper reviews the medical management of MRSA infections, discusses the potential role of decolonization and provides an overview of evidence to support recommended infection control practices.Key Words: Methicillin resistance, MRSA, Staphylococcus aureusThe past few decades have witnessed the emergence of methicillin-resistant Staphylococcus aureus (MRSA) as a major hospital-acquired pathogen worldwide (1-4). Although MRSA was first reported in Canada in 1981 (5), MRSA rates in Canadian hospitals have only increased substantially in the last few years. The Canadian Nosocomial Infection Surveillance Program (CNISP) reported that the incidence of MRSA in sentinel hospitals across the country increased from a mean of 0.9 per 100 S aureus isolates in 1995 to 8.2 per 100 isolates in 2001, and from 0.5 cases per 1000 admissions in 1995 to 4.4 per 1000 admissions in 2001 (6,7). Part of this increase may have been related to more frequent screening for MRSA colonization in high risk patients (8). However, a fourfold increase in MRSA infection rates was also observed (from 0.3 infections per 1000 admissions in 1995 to 1.2 infections per 1000 admissions in 2001) (6,7)Although there have been recent reports describing community-onset MRSA in the United States, CNISP data would suggest that MRSA remains predominantly a hospital-acquired pathogen in Canada (6). Nevertheless, it would seem reasonable to expect that an increase in MRSA rates in hospitals will eventually lead to spread of the organism in long term care facilities and the community. In Canada, community-acquired MRSA has been reported most frequently in western Canada, especially among native Aboriginals and intravenous drug users (9,10). Recognized risk factors for MRSA acquisition have included previous hospitalization, admission to an intensive care unit, prolonged hospital stay, proximity to another patient with MRSA, older age, invasive procedures, presence of wounds or skin lesions, and prior antimicrobial therapy (11-15).If MRSA only colonized patients, there would be little reason for concern. However, 20% to 60% of patients identified as being colonized with MRSA in hospital subsequently develop an MRSA infection (12). Using standard criteria for identification of infections, CNISP data indicated that approximately 31% of patients with MRSA in Canadian hospitals were infected (7). In certain high risk populations, staphylococcal infections including bacteremia occur more frequently following colonization with MRSA than after colonization with susceptible strains of S aureus (16). Moreover, MRSA does not merely replace susceptible strains of S aureus as a hospital-acquired pathogen, but rather, it appears to add substantially to the total burden of nosocomial infections (17,18). Although the results are somewhat controversial, several studies have also indicated increased mortality and prolonged hospitalization associated with MRSA infections (19-21). After adjustment for comorbidities, methicillin resistance has been found to be a significant independent risk factor for mortality in bacteremic patients (21-23).Several studies have also documented the economic impact of MRSA in hospitalized patients, demonstrating increased costs associated with managing infections and with the implementation of control measures (19,24,25). The average attributable cost of managing an MRSA infection in a Canadian hospital was estimated to be approximately $14,360, whereas costs associated with managing a patient with MRSA colonization were approximately $1,363 per hospital admission (25).The first strain of S aureus, an MRSA, with reduced susceptibility to vancomycin was reported from Japan in 1996 (26,27). Since then, such strains with vancomycin minimum inhibitory concentrations (MIC) of 8 μg/mL to 16 μg/mL (vancomycin-intermediate S aureus [VISA]) have been reported from several countries in southeast Asia, South America, Europe and the United States (28-30). Of even greater concern has been the recent identification of two infections caused by MRSA with high level resistance to vancomycin (MIC greater than 128 μg/mL; vancomycin-resistant S aureus [VRSA]), mediated by the vanA gene determinant found in vancomycin-resistant enterococci (31,32). These developments have emphasized the need for appropriate use of glycopeptides and other antimicrobial agents in the management of patients with MRSA. This paper reviews options for the treatment of patients with MRSA infection or colonization. The treatment options should be considered appropriate for hospitalized patients as well as for out-patients, and for those residing in long term care facilities.     

CRP Polymorphisms and Progression of Chronic Kidney Disease in African Americans

Background and objectives: Chronic inflammation may play a role in chronic kidney disease (CKD) progression. CRP gene polymorphisms are associated with serum C-reactive protein (CRP) concentrations. It is unknown if CRP polymorphisms are associated with CKD progression or modify the effectiveness of anti-hypertensive therapy in delaying CKD progression.Design, setting, participants, & measurements: We genotyped 642 participants with CKD from the African American Study of Kidney Disease and Hypertension (AASK), selecting five tag polymorphisms: rs2808630, rs1205, rs3093066, rs1417938, and rs3093058. We compared the minor allele frequencies (MAF) of single nucleotide polymorphisms (SNPs) in AASK to MAFs of African Americans from NHANES III. Among AASK participants, we evaluated the association of SNPs with CRP levels and prospectively with a composite: halving the GFR, ESRD, or death.Results: The MAF was higher for the rs2808630_G allele (P = 0.03) and lower for the rs1205_A allele (P = 0.03) in the AASK compared with NHANES III. Among AASK participants, the rs3093058_T allele predicted higher CRP concentrations (P < 0.0001) but not CKD progression. The rs2808630_GG genotype was associated with higher risk of the composite endpoint compared with the AA genotype (P = 0.002). Participants with the rs2808630_GG genotype on angiotensin converting enzyme inhibitors (ACEIs) versus β blockers had increased risk of progression (P = 0.03).Conclusion: CRP SNPs that were associated with higher levels of CRP did not predict CKD progression. The rs2808630_GG genotype was associated with higher risk of CKD progression, and in patients with this genotype, ACEIs did not slow progression.Familial clustering of chronic kidney disease (CKD) and ESRD has been reported in populations throughout the world for most types of nephropathy (1–6). This genetic predisposition to ESRD seems to be strongly associated with race (7,8). Compared with people with no family history of kidney disease, African Americans with a first-degree relative with ESRD have a nine-fold increase in the risk of ESRD compared with a three- to five-fold increase in whites (8). Recently, the candidate gene MYH9 has been identified as associated with nondiabetic ERSD in African Americans, and this association explains some of the disparity in incidence of ESRD observed between whites and African Americans (7,9). However, it is possible that additional genetic variants, such as those related to inflammatory pathways, may also be associated with ESRD.Biomarkers of inflammation, including C-reactive protein (CRP), are increased even in early stages of CKD and have been linked to the risk of CKD progression (10–15). These observations have led to studies examining the genetic basis of inflammation and identification of several candidate genes for ESRD susceptibility (16–19). Recently, several large population-based studies showed that plasma CRP levels are under genetic influence (20–25). Some of these polymorphisms have been consistently associated with CRP levels (higher levels associated with rs3093058_T and lower levels associated with rs1205_A and rs2808630_G) and the risk of cardiovascular events (rs3093058_T) in African Americans (23).CRP gene polymorphisms that affect CRP concentrations may reflect lifetime exposure to CRP more accurately than single time point measurements of serum CRP concentrations. The primary goal of this study was to characterize CRP gene polymorphisms and evaluate their association with CKD progression. We hypothesized that polymorphisms associated with higher levels of CRP would be associated with higher risk of CKD progression. Additionally, we examined whether these polymorphisms modify the renoprotective effects of angiotensin converting enzyme inhibitors (ACEIs), a drug class known to have anti-inflammatory effects (26–28). We hypothesized that patients with polymorphisms associated with higher levels of CRP would benefit most from ACEIs.