Inhibition of Ryanodine Receptor 1 Reduces Endoplasmic Reticulum (ER) Stress and Promotes ER Protein Degradation in Cyclic Nucleotide-Gated Channel Deficiency.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in cone phototransduction. Mutations in genes encoding the channel subunits CNGA3 and CNGB3 account for about 80% of all cases of achromatopsia and are associated with progressive cone dystrophies. CNG channel deficiency leads to cellular/endoplasmic reticulum (ER) calcium dysregulation and ER stress-associated cone apoptosis. This work investigated the role of the ER calcium channel ryanodine receptor 1 (Ryr1) in ER stress and cone degeneration in CNG channel deficiency. The AAV-mediated CRISPR/SaCas9 genome editing was used to knock down Ryr1 specifically in cones. CNG channel-deficient mice displayed improved cone survival after subretinal injection of AAV2-SaCas9/gRNA-Ryr1, manifested as increased expression levels of cone proteins M-opsin, S-opsin, and cone arrestin. Knockdown of Ryr1 also led to reduced ER stress and increased expression levels of the ER-associated degradation proteins. This work demonstrates a role of Ryr1 in ER stress and cone degeneration in CNG channel deficiency, and supports strategies targeting ER calcium regulation for cone preservation.

Slowing Progression of Cardiovascular Calcification With SNF472 in Patients on Hemodialysis: Results of a Randomized Phase 2b Study.

BACKGROUND: The high cardiovascular morbidity and mortality in patients with end-stage kidney disease could be partially caused by extensive cardiovascular calcification. SNF472, intravenous myo-inositol hexaphosphate, selectively inhibits the formation and growth of hydroxyapatite. METHODS: This double-blind, placebo-controlled phase 2b trial compared progression of coronary artery calcium volume score and other measurements of cardiovascular calcification by computed tomography scan during 52 weeks of treatment with SNF472 or placebo, in addition to standard therapy, in adult patients with end-stage kidney disease receiving hemodialysis. Patients were randomized 1:1:1 to SNF472 300 mg (n=92), SNF472 600 mg (n=91), or placebo (n=91) by infusion in the hemodialysis lines thrice weekly during hemodialysis sessions. The primary end point was change in log coronary artery calcium volume score from baseline to week 52. The primary efficacy analysis combined the SNF472 treatment groups and included all patients who received at least 1 dose of SNF472 or placebo and had an evaluable computed tomography scan after randomization. RESULTS: The mean change in coronary artery calcium volume score was 11% (95% CI, 7-15) for the combined SNF472 dose group and 20% (95% CI, 14-26) for the placebo group (P=0.016). SNF472 compared with placebo attenuated progression of calcium volume score in the aortic valve (14% [95% CI, 5-24] versus 98% [95% CI, 77-123]; P<0.001) but not in the thoracic aorta (23% [95% CI, 16-30] versus 28% [95% CI, 19-38]; P=0.40). Death occurred in 7 patients (4%) who received SNF472 and 5 patients (6%) who received placebo. At least 1 treatment-emergent adverse event occurred in 86%, 92%, and 87% of patients treated with SNF472 300 mg, SNF472 600 mg, and placebo, respectively. Most adverse events were mild. Adverse events resulted in discontinuation of SNF472 300 mg, SNF472 600 mg, and placebo for 14%, 29%, and 20% of patients, respectively. CONCLUSIONS: Compared with placebo, SNF472 significantly attenuated the progression of coronary artery calcium and aortic valve calcification in patients with end-stage kidney disease receiving hemodialysis in addition to standard care. Future studies are needed to determine the effects of SNF472 on cardiovascular events. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02966028.

Myxoma virus M013 protein antagonizes NF-κB and inflammasome pathways via distinct structural motifs.

Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.

IL-33 Drives Eosinophil Infiltration and Pathogenic Type 2 Helper T-Cell Immune Responses Leading to Chronic Experimental Ileitis.

Although a clear association has been established between IL-33 and inflammatory bowel disease, mechanistic studies to date, primarily using acute murine models of colitis, have yielded contradicting results, demonstrating both pathogenic and protective roles. We used a well-characterized, spontaneous model of inflammatory bowel disease [ie, SAMP1/YitFc (SAMP) mice] to investigate the role of IL-33 during chronic intestinal inflammation. Our results showed marked eosinophil infiltration into the gut mucosa with increased levels of eotaxins and type 2 helper T-cell (Th2) cytokines as disease progressed and became more severe, which could be reversed upon either eosinophil depletion or blockade of IL-33 signaling. Exogenous IL-33 administration recapitulated these effects in ilea of uninflamed (parental) control AKR/J mice. Human data supported these findings, showing colocalization and up-regulation of IL-33 and eosinophils in the colonic mucosa of inflammatory bowel disease patients versus noninflamed controls. Finally, colonization of commensal flora by fecal material transplantation into germ-free SAMP and the presence of the gut microbiome induced IL-33, subsequent eosinophil infiltration, and mounting of Th2 immune responses, leading to exacerbation of chronic intestinal inflammation characteristic of SAMP mice. These data demonstrate a pathogenic role for IL-33-mediated eosinophilia and activation of Th2 immunity in chronic intestinal inflammation that is dependent on the gut microbiome. Targeting IL-33 may represent a novel therapeutic approach to treat patients with inflammatory bowel disease.

Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis.

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation after conventional anti-TNF therapy in Crohn's disease and ulcerative colitis (UC) patients and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, whereas elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium, and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, whereas stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6, and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Association of Suppressive Myeloid Cell Enrichment with Aggressive Oropharynx Squamous Cell Carcinoma.

BACKGROUND: While immune-cell infiltrated tumors, such as human papillomavirus positive (HPV+) ororpharyngeal squamous cell carcinomas (OPSCC) have been associated with an improved clinical prognosis, there is evidence to suggest that OPSCCs are also subjected to increased immunoregulatory influence. The objective of this study was to assess whether patients with clinically aggressive OPSCC have a distinct immunosuppressive immune signature in the primary tumor. METHODS: This retrospective case-control study analyzed 37 pre-treatment tissue samples from HPV+ and HPV-negative OPSCC patients treated at a single institution. The cases were patients with known disease recurrence and the controls were patients without disease recurrence. An mRNA-expression immune-pathway profiling was performed, and correlated to clinical outcomes. The TCGA head and neck cancer database was utilized to make comparisons with the institutional cohort. RESULTS: In our cohort, HPV-negative and HPV+ patients with known disease recurrence both had significantly increased suppressive monoctyte/macrophage and granulocyte cell-expression-profile enrichment. Similar findings were found in the TCGA cohort when comparing HPV-negative to positive patients. CONCLUSIONS: our study demonstrates that patients with recurrent HPV+ OPSCC had suppressive monocyte/macrophage and granulocyte immune-cell enrichment, similar to those seen in the more aggressive HPV-negative OPSCC.

Spontaneous, immune-mediated gastric inflammation in SAMP1/YitFc mice, a model of Crohn's-like gastritis.

BACKGROUND & AIMS: Crohn's disease (CD) can develop in any region of the gastrointestinal tract, including the stomach. The etiology and pathogenesis of Crohn's gastritis are poorly understood, treatment approaches are limited, and there are not many suitable animal models for study. We characterized the features and mechanisms of chronic gastritis in SAMP1/YitFc (SAMP) mice, a spontaneous model of CD-like ileitis, along with possible therapeutic approaches. METHODS: Stomachs from specific pathogen-free and germ-free SAMP and AKR mice (controls) were evaluated histologically; the presence of Helicobacter spp was tested in fecal pellets by polymerase chain reaction analysis. In vivo gastric permeability was quantified by fractional excretion of sucrose, and epithelial tight junction protein expression was measured by quantitative reverse-transcription polymerase chain reaction analysis. The effects of a proton pump inhibitor (PPI) or corticosteroids were measured, and the ability of pathogenic immune cells to mediate gastritis was assessed in adoptive transfer experiments. RESULTS: SAMP mice developed Helicobacter-negative gastritis, characterized by aggregates of mononuclear cells, diffuse accumulation of neutrophils, and disruption of epithelial architecture; SAMP mice also had increased gastric permeability compared with controls, without alterations in expression of tight junction proteins. The gastritis and associated permeability defect observed in SAMP mice were independent of bacterial colonization and reduced by administration of corticosteroids but not a PPI. CD4(+) T cells isolated from draining mesenteric lymph nodes of SAMP mice were sufficient to induce gastritis in recipient SCID mice. CONCLUSIONS: In SAMP mice, gastritis develops spontaneously and has many features of CD-like ileitis. These mice are a useful model to study Helicobacter-negative, immune-mediated Crohn's gastritis.

Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1).

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

Trial design and baseline characteristics of CaLIPSO: a randomized, double-blind placebo-controlled trial of SNF472 in patients receiving haemodialysis with cardiovascular calcification.

BACKGROUND: The objective of CaLIPSO, a Phase 2b, randomized, double-blind, placebo-controlled clinical trial, is to test the hypothesis that myo-inositol hexaphosphate (SNF472) attenuates the progression of cardiovascular calcification in patients receiving maintenance haemodialysis. Here we report the trial design and baseline characteristics of trial participants. METHODS: Adult patients on maintenance haemodialysis (≥6 months) with an Agatston coronary artery calcium score, as measured by a multidetector computed tomography scanner, of 100-3500 U were enrolled. Patients were stratified by Agatston score (100-<400, 400-1000 or >1000 U) and randomized in a 1:1:1 ratio to receive placebo, SNF472 300 mg or SNF472 600 mg administered intravenously three times weekly during each haemodialysis session. RESULTS: Overall, 274 patients were randomized. The mean age of trial participants was 63.6 (standard deviation 8.9) years and 39% were women. The coronary artery, aorta and aortic valve median (25th-75th percentile) Agatston scores at baseline were 730 U (315-1435), 1728 U (625-4978) and 103 U (31-262), respectively, and the median (25th-75th percentile) calcium volume scores at baseline were 666 (310-1234), 1418 (536-4052) and 107 (38-278), respectively. Older age and diabetes mellitus were associated with higher calcium scores at baseline. CONCLUSIONS: The CaLIPSO trial enrolled patients on haemodialysis with pre-existent cardiovascular calcification to test the hypothesis that SNF472 attenuates its progression in the coronary arteries, aorta and aortic valve.

Effects of Myo-inositol Hexaphosphate (SNF472) on Bone Mineral Density in Patients Receiving Hemodialysis: An Analysis of the Randomized, Placebo-Controlled CaLIPSO Study.

BACKGROUND AND OBJECTIVES: In the CaLIPSO study, intravenous administration of SNF472 (300 or 600 mg) during hemodialysis significantly attenuated progression of coronary artery and aortic valve calcification. SNF472 selectively inhibits formation of hydroxyapatite, the final step in cardiovascular calcification. Because bone mineral is predominantly hydroxyapatite, we assessed changes in bone mineral density in CaLIPSO. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Patients with coronary artery calcification at screening (Agatston score of 100-3500 U) were randomized 1:1:1 to receive placebo, 300 mg SNF472, or 600 mg SNF472 as an intravenous infusion during hemodialysis three times weekly for 52 weeks. Dual-energy x-ray absorptiometry (DXA) scans were obtained at baseline (screening) and end of treatment, and between-group changes from baseline were compared using analysis of covariance. RESULTS: Among 274 randomized patients, 202 had evaluable DXA scans at baseline and postrandomization (the DXA-modified intention-to-treat population). Mean (95% confidence interval) changes in total-hip bone mineral density from baseline to week 52 were -1.5% (-2.7% to -0.3%), -1.5% (-2.7% to -0.4%), and -2.5% (-3.8% to -1.2%) in the placebo, 300 mg SNF472, and 600 mg SNF472 groups, respectively. Mean (95% confidence interval) changes in femoral-neck bone mineral density from baseline to week 52 were -0.3% (-1.6% to 1.0%), -1.0% (-2.3% to 0.2%), and -2.6% (-4.0% to -1.3%), respectively. Regression analyses showed no correlation between change in coronary artery calcium volume and change in bone mineral density at either location. Changes in serum alkaline phosphatase, calcium, magnesium, phosphate, and intact parathyroid hormone levels were similar across treatment groups. Clinical fracture events were reported for four of 90, three of 92, and six of 91 patients in the placebo, 300 mg SNF472, and 600 mg SNF472 groups, respectively. CONCLUSIONS: Bone mineral density decreased modestly in all groups over 1 year. In the 600 mg SNF472 group, the reduction appeared more pronounced. Reported fractures were infrequent in all groups. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Effect of SNF472 on Progression of Cardiovascular Calcification in End-Stage-Renal-Disease (ESRD) Patients on Hemodialysis (HD), NCT02966028.

A novel assay to measure calcification propensity: from laboratory to humans.

Cardiovascular calcification (CVC) contributes to morbidity and mortality in patients undergoing dialysis. We examined the pharmacodynamic effects of SNF472, a calcification inhibitor, on plasma calcium phosphate crystallization using spectrometric measurements, and its correlations with effects on CVC in rats or humans. Rats (N = 38) injected with vitamin D (days 1-3) to induce CVC were infused with saline or SNF472 (days 1-12). Inhibition of CVC was 50-65% with SNF472 3 mg/kg and ~ 80% with SNF472 10 or 30 mg/kg. SNF472 dose-dependently inhibited calcium phosphate crystallization, which correlated with inhibition of CVC (r = 0.628, P = 0.005). In patients with calciphylaxis (N = 14), infusion of SNF472 (~ 7 mg/kg) during hemodialysis for 12 weeks inhibited calcium phosphate crystallization by nearly 70%. In patients with CVC (N = 274), infusion of SNF472 during hemodialysis for 52 weeks inhibited calcium phosphate crystallization (placebo: 15%; 300 mg: 61%; 600 mg: 75%), which correlated with inhibition of CVC (r = 0.401, P = 0.003). These findings show a direct correlation between inhibition of calcium phosphate crystallization in plasma and inhibition of CVC both in a rat model and in humans, supporting the use of the pharmacodynamic assay in clinical trials as a potentially predictive tool to evaluate the activity of calcification inhibitors.

Effects of SNF472, a Novel Inhibitor of Hydroxyapatite Crystallization in Patients Receiving Hemodialysis - Subgroup Analyses of the CALIPSO Trial.

INTRODUCTION: Coronary artery calcium (CAC) is highly prevalent and linked with poor outcomes in patients receiving maintenance hemodialysis, and its reduction may improve patient prognosis. SNF472, a selective inhibitor of hydroxyapatite crystallization, slows CAC progression in patients receiving maintenance hemodialysis. In this analysis, we assessed the efficacy of SNF472 in prespecified patient subgroups. METHODS: In a randomized clinical trial SNF472 300 mg, SNF472 600 mg, or placebo were infused thrice weekly in 91, 92, and 91 patients receiving maintenance hemodialysis and with CAC at baseline, respectively. In prespecified subanalyses, the percent change in CAC volume score (CACvs) from baseline to week 52 in modified intention-to-treat (mITT) and per-protocol (PP) populations was calculated in the following subgroups: age, sex, diabetes mellitus, dialysis vintage, prior atherosclerotic cardiovascular disease, baseline use of non-calcium and calcium-based phosphate binders, calcimimetics, activated vitamin D, warfarin, and statins. RESULTS: In the main trial, SNF472 significantly reduced CACvs progression compared with placebo (11% versus 20% mITT analyses; P = 0.016; 8% vs. 24% PP analyses; P < 0.001). Treatment differences for CACvs progression were similar across all subgroups, and all interaction P values were non-significant in mITT and PP analyses. CONCLUSIONS: SNF472 treatment for 52 weeks reduced CACvs progression compared with placebo in a broad range of patients receiving maintenance hemodialysis. Future studies will determine the impact of SNF472 on cardiovascular events in this population.

Mechanism of action of SNF472, a novel calcification inhibitor to treat vascular calcification and calciphylaxis.

BACKGROUND AND PURPOSE: No therapy is approved for vascular calcification or calcific uraemic arteriolopathy (calciphylaxis), which increases mortality and morbidity in patients undergoing dialysis. Deposition of hydroxyapatite (HAP) crystals in arterial walls is the common pathophysiologic mechanism. The mechanism of action of SNF472 to reduce HAP deposition in arterial walls was investigated. EXPERIMENTAL APPROACH: We examined SNF472 binding features (affinity, release kinetics and antagonism type) for HAP crystals in vitro, inhibition of calcification in excised vascular smooth muscle cells from rats and bone parameters in osteoblasts from dogs and rats. KEY RESULTS: SNF472 bound to HAP with affinity (KD ) of 1-10 µM and saturated HAP at 7.6 µM. SNF472 binding was fast (80% within 5 min) and insurmountable. SNF472 inhibited HAP crystal formation from 3.8 µM, with complete inhibition at 30.4 µM. SNF472 chelated free calcium with an EC50 of 539 µM. Chelation of free calcium was imperceptible for SNF472 1-10 µM in physiological calcium concentrations. The lowest concentration tested in vascular smooth muscle cells, 1 µM inhibited calcification by 67%. SNF472 showed no deleterious effects on bone mineralization in dogs or in rat osteoblasts. CONCLUSION AND IMPLICATIONS: These experiments show that SNF472 binds to HAP and inhibits further HAP crystallization. The EC50 for chelation of free calcium is 50-fold greater than a maximally effective SNF472 dose, supporting the selectivity of SNF472 for HAP. These findings indicate that SNF472 may have a future role in the treatment of vascular calcification and calcific uraemic arteriolopathy in patients undergoing dialysis.

Improvement in wound healing, pain, and quality of life after 12 weeks of SNF472 treatment: a phase 2 open-label study of patients with calciphylaxis.

BACKGROUND: Calciphylaxis in end-stage renal disease is characterized by painful necrotic skin ulcers and high mortality. There are no approved therapies. SNF472, an intravenous formulation of myo-inositol hexaphosphate, inhibits the formation and growth of hydroxyapatite crystals, the final common pathway in the pathogenesis of vascular calcification. METHODS: In this open-label, single-arm study, calciphylaxis patients on thrice-weekly hemodialysis and standard care, received intravenous SNF472 3 times per week for 12 weeks. The primary endpoint was wound healing assessed using the quantitative Bates-Jensen Wound Assessment Tool (BWAT). Pain visual analog scale (VAS), quality of life (wound-QoL), and qualitative wound image review were secondary endpoints. Quantitative changes from baseline were analyzed by paired t-tests using multiple imputation to account for missing observations. RESULTS: Fourteen patients received SNF472. Improvements from baseline to week 12 were observed for mean BWAT score (- 8.1; P < 0.001), pain VAS (- 23.6 mm; P = 0.015) and wound-QoL global score (- 0.90; P = 0.003). Of the 9 patients with ulcerated lesions at baseline who completed treatment, wound image review showed improvement for 7. SNF472 was well tolerated with no serious treatment-related adverse events. The most common adverse events were infections which occur frequently in patients on hemodialysis. None of these were considered as treatment-related. CONCLUSIONS: SNF472 was well-tolerated and improvements from baseline to week 12 in wound healing, pain, and quality of life were observed. A randomized, double-blind, placebo-controlled trial is planned to evaluate SNF472 in patients with calciphylaxis.

Drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death.

BACKGROUND: In November 2001, the Food and Drug Administration (FDA) approved drotrecogin alfa (activated) (DrotAA) for adults who had severe sepsis and a high risk of death. The FDA required a study to evaluate the efficacy of DrotAA for adults who had severe sepsis and a low risk of death. METHODS: We randomly assigned adult patients with severe sepsis and a low risk of death (defined by an Acute Physiology and Chronic Health Evaluation [APACHE II] score <25 or single-organ failure) to receive an intravenous infusion of placebo or DrotAA (24 microg per kilogram of body weight per hour) for 96 hours in a double-blind, placebo-controlled, multicenter trial. The prospectively defined primary end point was death from any cause and was assessed 28 days after the start of the infusion. In-hospital mortality within 90 days after the start of the infusion was measured, and safety information was collected. RESULTS: Enrollment in the trial was terminated early because of a low likelihood of meeting the prospectively defined objective of demonstrating a significant reduction in the 28-day mortality rate with the use of DrotAA. The study enrolled 2640 patients and collected data on 2613 (1297 in the placebo group and 1316 in the DrotAA group) at the 28-day follow-up. There were no statistically significant differences between the placebo group and the DrotAA group in 28-day mortality (17.0 percent in the placebo group vs. 18.5 percent in the DrotAA group; P=0.34; relative risk, 1.08; 95 percent confidence interval, 0.92 to 1.28) or in in-hospital mortality (20.5 percent vs. 20.6 percent; P=0.98; relative risk, 1.00; 95 percent confidence interval, 0.86 to 1.16). The rate of serious bleeding was greater in the DrotAA group than in the placebo group during both the infusion (2.4 percent vs. 1.2 percent, P=0.02) and the 28-day study period (3.9 percent vs. 2.2 percent, P=0.01). CONCLUSIONS: The absence of a beneficial treatment effect, coupled with an increased incidence of serious bleeding complications, indicates that DrotAA should not be used in patients with severe sepsis who are at low risk for death, such as those with single-organ failure or an APACHE II score less than 25.

Use of an integrated clinical trial database to evaluate the effect of timing of drotrecogin alfa (activated) treatment in severe sepsis.

INTRODUCTION: Several studies have indicated that early identification and treatment of patients with severe sepsis using standard supportive care improves outcomes. Earlier treatment with drotrecogin alfa (activated) (DrotAA) may also improve outcomes in severe sepsis. Using a recently constructed integrated severe sepsis database, our objectives in this study were to describe the influence of baseline clinical characteristics on timing of DrotAA treatment in patients with severe sepsis, to evaluate the efficacy of DrotAA with respect to timing of administration, and to examine the association between early intervention with DrotAA and patient outcomes, using adjustments for imbalances. METHODS: The database comprises data from 4,459 patients with severe sepsis (DrotAA, n = 3,228; placebo, n = 1,231) included in five clinical trials conducted in tertiary care institutions in 28 countries. Placebo data came only from randomized trials, whereas data for the DrotAA group came from randomized (PROWESS) and open-label/observational (ENHANCE) trials. RESULTS: Increased time-to-treatment with DrotAA was significantly associated with more organ dysfunction, greater need of mechanical ventilation, vasopressor use, or recent surgery. Earlier treatment was associated with higher baseline Acute Physiology and Chronic Health Evaluation (APACHE II) scores. Adjusted and unadjusted survival analyses suggested that compared with placebo, DrotAA treatment provided a potential survival benefit, regardless of time to treatment. Survival curves of DrotAA patients treated early compared with those treated late began to separate at 14 days. By 28 days, patients treated earlier had higher survival than those treated later (76.4% versus 73.5%, p = 0.03). Sepsis-induced multiorgan dysfunction was the most common cause of death followed by refractory shock and respiratory failure. Modeling of the treatment effect, as a function of time to treatment, suggested increased benefit with earlier treatment. CONCLUSION: Using an integrated database of five severe sepsis trials and appropriate statistical adjustments to reduce sources of potential bias, earlier treatment with DrotAA seemed to be associated with a lower risk-adjusted mortality than later treatment. These data suggest that earlier treatment with DrotAA may provide most benefit for appropriate patients.

Racial differences in outcome and treatment effect in congestive heart failure.

BACKGROUND: In congestive heart failure (CHF), it is unknown whether race affects mortality and whether the effect of treatments differs by race. METHODS: This study was a post hoc analysis of data from the DIG study that evaluated the effect of digoxin on morbidity and mortality in CHF. RESULTS: Investigators followed 897 black and 6660 white participants for a mean of 37 months. Compared with whites, blacks were younger (60 +/- 13 vs 65 +/- 11 years). Total mortality was 34.2% in blacks and 33.6% in whites; hospitalization for worsening CHF occurred in 39% of blacks and 28% of whites. Cox regressions with race as the only covariate showed no effect of race on risk for death (relative risk = 1.04, 95% CI 0.93-1.18, P = .49) but an increase in CHF hospitalization in blacks (relative risk = 1.52, 95% CI 1.35-1.70, P = .0001). Multivariate Cox regression showed no difference by race in risk for death or death/hospitalization for CHF and no difference in the effect of digoxin on either end point. CONCLUSION: Race is not an independent predictor of mortality in CHF. The effect of digoxin on morbidity and mortality in CHF does not differ in blacks and whites.

Lack of evidence for qualitative treatment by disease severity interactions in clinical studies of severe sepsis.

INTRODUCTION: The design of clinical trials of interventions aimed at reducing mortality in patients with severe sepsis assumes that the relative treatment effect of the intervention is independent of the patients' risk for death. We reviewed published data from phase III clinical studies of severe sepsis to determine whether a relationship exists between risk for death and the relative benefit of the investigational agent. Such an interaction might warrant a change in the assumptions that underlie current trial designs. METHODS: We conducted a systematic review of published phase III, randomized, placebo-controlled trials in adult patients with sepsis, severe sepsis, or septic shock up to November 2004. All studies enrolled patients with known or suspected infection, evidence of a systemic response to the infection, and one or more organ dysfunctions resulting from the systemic response. RESULTS: Twenty-two publications, investigating 17 molecular entities, fulfilled criteria for phase III or equivalent studies aimed at reducing mortality in adult patients with severe sepsis or septic shock. Three studies achieved the prospectively defined primary end-point of a statistically significant reduction in 28-day all-cause mortality. The control group mortality rates for these studies were 31%, 43% and 61%, indicating that the beneficial effects of adjunct therapies could be demonstrated over a wide range of illness severity. Analysis of subgroup data from failed studies provided no evidence that the efficacy of the therapeutics being investigated varied by baseline placebo mortality rates. Among all studies, interventions with anticoagulant activity or anti-inflammatory activity did not appear to be harmful in patients with evidence of less coagulopathy or less inflammation. CONCLUSION: Our review of published clinical data does not support the hypothesis that mortality risk of the population studied alters the relative treatment effect associated with anti-inflammatory or other agents used to treat severe sepsis. Clinical studies in severe sepsis should continue to enroll patients over a wide range of disease severity, as long as patients enrolled have evidence of sepsis-induced organ dysfunction(s), patients are at an appreciable risk for death (e.g. as evidenced by admission to an intensive care unit), and the potential for benefit outweighs the potential for harm.

Pneumocystis carinii pneumonia after renal transplantation.

Being immuno-suppressed, renal allograft recipients are at increased risk of contracting various infectious complications. Pneumocystis carinii pneumonia (PCP) is one of the important opportunistic infection causing high morbidity and mortality in these patients. Majority of studies has reported the occurrence of PCP during 6 months to one year after renal transplantation. This communication describes occurrence of PCP in five renal allograft recipients 10 weeks to 72 months after transplantation. In view of elusive presentation, strong clinical and radiological suspicion followed by direct demonstration of the organisms is essential for early diagnosis and prompt treatment. These observations also indicate that PCP is an emerging opportunistic infection in immuno-compromised patients in tropical countries.