Comparison between semiquantitative and quantitative methods for the assessment of knee synovitis in osteoarthritis using non-enhanced and gadolinium-enhanced MRI.

OBJECTIVE: To compare different semiquantitative and quantitative methods using both non-enhanced and gadolinium-enhanced MRI techniques for the assessment of synovitis in knee osteoarthritis (OA). METHODS: Knees with end-stage clinical OA in patients undergoing total knee replacement surgery were included in this cross-sectional study. MRI was performed on all knees. Standard non-enhanced and gadolinium-enhanced sequences were acquired. Using non-enhanced MRI, we semiquantitatively assessed two features widely used as surrogates for synovitis: effusion-synovitis and Hoffa-synovitis. Using gadolinium-enhanced sequences, we semiquantitatively assessed synovial thickness. We quantitatively evaluated the total synovial volume on the gadolinium-enhanced sequences as well. We assessed the correlations of effusion-synovitis and Hoffa-synovitis with synovial thickness and volume, applying Spearman correlation analysis. The diagnostic performance of both synovitis features on non-enhanced MRI was assessed using synovial thickness on gadolinium-enhanced MRI as the reference. RESULTS: A total of 104 subjects (one knee per subject) were included. Correlations of effusion-synovitis with synovial thickness and volume were r = 0.41 and r = 0.43 (P < .001) r = 0.32 and r = 0.39 (P < .0001). CONCLUSION: Using synovial thickness assessed on gadolinium-enhanced sequences as the reference, effusion-synovitis showed superior correlations and sensitivity. Effusion-synovitis should be preferred over Hoffa-synovitis as a surrogate marker for synovial thickening, in studies of knee OA for which gadolinium-enhanced sequences are not available.

Survival following salvage abdominoperineal resection for persistent and recurrent squamous cell carcinoma of the anus: do these disease categories affect survival?

AIM: This study aimed to investigate the results of salvage abdominoperineal excision (APR) in patients with persistent or recurrent squamous cell carcinoma of the anus (SCCA). METHOD: Patients with anal neoplasia were identified from a prospective database. Patients with invasive SCCA with demonstrated failure of chemoradiation therapy (CRT) who underwent salvage APR for one of three disease categories (persistent, < 6 months post-CRT; early recurrent, 6-24 months post-CRT; late recurrent, > 24 months post-CRT) were included. The primary outcome was overall survival after salvage APR. Tumour size, metastatic lymph nodes (LN), circumferential resection margin positivity (CRM) and neurolymphovascular invasion (NLVI) were correlated with the outcome. RESULTS: Thirty-six patients with a median 3-year overall survival of 46% (median follow-up 24 months) underwent salvage APR due to persistent or recurrent SCCA (14 men, mean age 59 years). Eleven (31%) patients were diagnosed with persistent disease, 17 (47%) with early and 8 (22%) with late recurrence. Two-year overall survival of Stage 0/I/II and III/IV disease was 81.5% and 33.74%, respectively (P = 0.022). Overall disease stage was associated with disease categorization (P = 0.009): patients with persistent disease or early recurrence had a significantly higher disease stage than patients with late recurrence (OR = 20.9 and 17.2). Despite apparently improved survival in patients with late disease recurrence on live table analysis, no significant difference was identified in overall survival when stratified by disease category on log-rank test analysis. CONCLUSION: Persistent and recurrent disease does not show any significant difference in survival, but patients with late recurrence may have a better prognosis.

Smoking initiation among Israeli adolescents: a 24-year time-to-event analysis.

INTRODUCTION: Preventing smoking initiation will protect future generations from smoking-attributable death and disease. This study examines the correlates and patterns of initiation among Israeli youth using time-to-event analysis and other methods. METHODS: Twenty-four consecutive representative samples (1986-2009) of new military recruits (N=50,254) were analyzed. Cox regression and Kaplan-Meier analysis were used to identify factors associated with smoking initiation, and logistic regression was used to identify factors associated with smoking status. RESULTS: The most hazardous age for smoking initiation was seventeen, subsequent to the mean age of smoking initiation (males: 15.7, females: 16.0). Age of initiation and age of greatest hazard for initiation declined among recruits between the years 1986 and 2009. Earlier smoking initiation among boys and girls was significantly associated with low education levels (<12years) (males: HR=2.98, CI: [2.79, 3.18]; females: HR=3.35, CI: [2.96, 3.80]), low paternal education levels, Russian birthplace, and religion. Earlier initiation in boys was associated with high fitness levels and low/medium socio-economic status. Earlier initiation in girls was associated with being Western-born and ever-use of contraception. CONCLUSIONS: Smoking initiation among Israeli youth recruited to the armed forces is associated with individual and family characteristics, particularly low education levels. Time-to-event analysis complements traditional means of understanding smoking initiation by identifying ages at which initiation hazard is high.

Anti-MDA5 positive clinically amyopathic dermatomyositis presenting with severe cardiomyopathy.

BACKGROUND: Anti-MDA5 (Melanoma differentiation-associated gene 5) positive dermatomyositis is a new variant of clinically amyopathic dermatomyositis that presents with characteristic mucocutaneous findings and is associated with a higher risk of developing rapidly progressive interstitial lung disease. Because its presentation differs from that of classical dermatomyositis, this entity can be a diagnostic challenge for the clinician. METHODS & RESULTS: We present the case of a 55-year-old male with a 7-month history of chill sensation, constitutional symptoms and polyarthralgia. Within 3 months, the patient developed progressive heart failure with dyspnoea and orthopnoea, together with characteristic cutaneous lesions. Skin biopsies demonstrated thrombosis of small and medium-sized arteries in the reticular dermis, together with an evolved lobular panniculitis and prominent mucin deposits. CONCLUSIONS: Clinicians should be aware of the characteristic clinical and histopathologic presentation of this variant of dermatomyositis to establish an early diagnosis. Further evidence is needed to clarify the risk of cardiac involvement in this subset of patients.

Effect of rifampin on the pharmacokinetics, safety and tolerability of navitoclax (ABT-263), a dual inhibitor of Bcl-2 and Bcl-XL , in patients with cancer.

WHAT IS KNOWN AND OBJECTIVE: Navitoclax, a first-in-class small molecule Bcl-2 family inhibitor, is metabolized in vitro by the hepatic microsomal cytochrome P450 (CYP) enzymes CYP3A4. Drugs that affect CYP3A4 may therefore have an impact on the pharmacological profile of navitoclax. This study evaluated the effects of co-administration of a potent CYP3A4 inducer rifampin on the pharmacokinetic and safety profiles of navitoclax. METHODS: This open-label, fixed-sequence, 2-period study was performed in twelve subjects with non-haematologic or haematologic malignancy that was relapsed or refractory to standard therapy. A 7-day washout period separated the two treatment periods. On Study Day 1 and Day 8, subjects received a single 250 mg oral dose of navitoclax. Rifampin 600 mg was administrated once daily (QD) on Study Day 4 through Day 10. Blood samples for navitoclax assay were collected prior to dosing (0 h) and at a series of time points through 72 h after dosing on Study Day 1 and Day 8. RESULTS AND DISCUSSION: Co-administration of a single 250 mg dose of navitoclax with 600 mg QD doses of rifampin had a negligible effect on the maximum plasma concentration (Cmax ) of navitoclax [ratio of geometric least square means: 0·84 (90% CI: 0·61-1·16)] but moderately decreased the area under the plasma concentration-time curve (AUC) of navitoclax [ratio of geometric least square means: 0·59 (90% CI: 0·44-0·80)]. Rifampin did not affect the half-life of navitoclax. Co-administration of rifampin did not appear to significantly change the safety profile of navitoclax in the limited number of patients evaluated in this study. WHAT IS NEW AND CONCLUSION: Co-administration navitoclax with rifampin moderately decreased navitoclax AUC, which could be partly due to the induction effect of rifampin on CYP3A4. Further assessment on the mechanism of drug interaction is warranted.

Are postoperative complications more common following colon and rectal surgery in patients with chronic kidney disease?

AIM: According to National Kidney Foundation guidelines, early stages of chronic kidney disease (CKD) can be detected through the estimated glomerular filtration rate (eGFR). We assessed complications following colorectal surgery (CRS) in patients with CKD Stages 3 and 4, as defined by the eGFR. METHOD: Patients with CKD were identified within our database. Patients with an eGFR of 15-59 ml/min (CKD Stages 3 and 4) formed the CKD group and were compared with American Society of Anesthesiology (ASA) score-matched controls with an eGFR of ≥ 60 ml/min. Assessments included demographics, comorbidity, ASA score, operative details and 30-day postoperative outcome. RESULTS: Seventy patients in the CKD group were matched with 70 controls. ASA scores and length of stay did not differ significantly between the groups. CKD patients were older (mean age 76.5 years vs 71.1 years; P < 0.001) and had a lower mean body mass index (24.3 vs 28.2; P < 0.001) compared with controls. Compared with the CKD group, the mean operation time was longer in the control group (181.5 min vs 151.6 min; P = 0.02) and the estimated blood loss was greater (232 ml vs 165 ml; P = 0.004). Postoperative infection was more common in the CKD group (60%vs 40%; P = 0.01). There were no significant differences in reoperation rates, 30-day readmissions or the incidence of acute renal failure (ARF). CONCLUSION: Patients with CKD Stages 3 and 4 had a higher incidence of postoperative infections than matched controls after colorectal surgery. ARF developed in 18.6% of patients. Preoperative optimization should include adequate hydration and assessment of potentially nephrotoxic substances for bowel preparation, preoperative antibiotics and pain control.

Phantom rectum following abdominoperineal excision for rectal neoplasm: appearance and disappearance.

AIM: The sensation that the rectum remains or is functioning after abdominoperineal excision (APE) is called phantom rectum (PR). Its postoperative and long-term morbidity are not well documented. Informed consent may not include the risk and consequences of this condition. We assessed the incidence and morbidity of PR after APE and compared those with vs those without vertical rectus abdominis myocutaneous flaps. METHOD: Patients who underwent APE between 1 January 2004 and 31 December 2008 were identified. Preoperative radiation and operative reconstruction by vertical rectus abdominis myocutaneous (VRAM) flaps were noted. Patients were interviewed by telephone to assess the presence and timing of PR symptoms and their effect on quality of life. RESULTS: Thirty-six of 80 patients who underwent APE were available for follow-up. Twenty-three (64%) described PR symptoms including urgency to evacuate [22 (61%)], sensation of faeces in the rectum [19 (52%)] and sensation of passing flatus [17 (48%)]. Eleven (47%) who had VRAM vs 25 who did not, reported having symptoms of PR at < 3 months after APE. Patients described their symptoms as 'unchanged over time' [20 (56%)], 'gradually decreasing and ultimately disappearing' [13 (35%)] or 'worsening' [3 (9%)]. Preoperative radiation and laparoscopic approach were not associated with PR symptoms. Significantly more patients having a VRAM flap reported early PR symptoms [7/11 (64%) vs 4/25 (16%)] (P = 0.008). CONCLUSION: PR sensations were experienced by 23 (64%) patients who underwent APE for rectal cancer. VRAM reconstruction was associated with early PR presentation. The possibility of PR should be discussed preoperatively in patients undergoing APE for anorectal neoplasm.

Review of postoperative CT and ultrasound for endovascular aneurysm repair using Talent stent graft: can we simplify the surveillance protocol and reduce the number of CT scans?

BACKGROUND: Simplifying a postoperative surveillance protocol for endovascular aneurysm repair (EVAR) requires quality control comparing computerized tomography (CT) and ultrasound (US) results of abdominal aortic aneurysm (AAA) diameter measurements and endoleaks. PURPOSE: To test if US is comparable to CT, then assess a simplified follow-up with our conventional surveillance to assess patient safety. MATERIAL AND METHODS: During 2001-2006, data on 56 patients treated with Talent stent graft were prospectively registered. Median follow-up was 41.5 months (range, 2-94 months), with CT, US, and plain film abdomen X-rays (PFA) at 1, 6, and 12 months, then yearly. Bland-Altman plot was used to assess the agreement between CT and US measuring the AAA diameters and mixed model by the time effect to assess the difference in diameter over time. Sensitivity and specificity for detection of endoleaks by US, with CT as 'gold standard' were calculated. A simplified surveillance protocol with US/PFA at 6 and 8 weeks, CT/US/PFA at 1 year, and yearly US/PFA thereafter, was evaluated. CT was carried out when poor visibility, endoleak detected, AAA diameter increase (≥5 mm) on US or migration (≥10 mm) on PFA. This regime was compared with our conventional follow-up protocol. RESULTS: Diameter measurements on US appear comparable to CT with 91% specificity and 85% sensitivity for endoleaks detected by US. Using the simplified surveillance protocol no endoleaks, migrations, or endotension requiring treatment were overlooked. The simplified protocol generated 53 selective CT scans, avoiding approximately 144 CT scans. If further simplified by omitting the 1-year CT scan, one type II endoleak would be missed with a 1-year delay, eliminating a further 45 CT scans. CONCLUSION: US appears comparable to CT in the follow-up of Talent stent grafts in our institution. The proposed simplified surveillance protocol seems safe and can lead to a significant reduction in the number of CT scans.

Evaluating the needs of risk assessment methods of potentially polluting shipwrecks.

Shipwrecks deteriorate and the probability of a release of oil increases with time on the sea floor. The potential leakage is a risk to the marine environment and may also have social and economic consequences. The purpose of this study was to evaluate existing methods for risk assessment of shipwrecks and suggest a generic risk assessment framework. A risk assessment is necessary for providing decision support on remediation actions and thus enabling an efficient use of available resources. Existing risk assessment methods aimed for assessing shipwrecks were evaluated by comparison to relevant parts of an international standard on risk management. The comparison showed that existing methods lack several key components of risk assessment procedures. None of the evaluated methods provide a comprehensive risk assessment for potentially polluting shipwrecks and few take into account uncertainty and sensitivity. Furthermore, there is a need to develop risk assessment methods considering long-term effects of continuous release of oil into the marine environment. Finally, a generic comprehensive framework for risk assessment of shipwrecks is suggested.

Comparison of PFAS soil remediation alternatives at a civilian airport using cost-benefit analysis.

Contamination of soil and water systems by per- and polyfluoroalkyl substances (PFAS) due to uncontrolled use of aqueous film-forming foams (AFFFs) at firefighting training sites at civilian and military airports is a universal issue and can lead to significant human health and environmental impacts. Remediation of these sites is often complex but necessary to alleviate the PFAS burden and minimise the risks of exposure by eliminating the hotspot/source from which the PFAS spreads. This study presents a probabilistic cost-benefit analysis (CBA) for evaluating PFAS remediation alternatives, which includes monetisation of both direct costs and benefits as well as externalities. The method is applied for a case study to compare five remediation alternatives for managing PFAS contaminated soil at Stockholm Arlanda Airport in Sweden. The social profitability, or the net present value (NPV), of each remediation alternative was calculated in comparison to two reference alternatives - 'total excavation' of the site (Alt 0) or 'do nothing'. Sensitivity analyses and model scenarios were tested to account for uncertainties, including small or large PFAS spreading and simulating different values for the magnitude of annual avoided cost of inaction (i.e., aggregate benefit) from PFAS remediation. In comparison to total excavation, four of the five studied remediation alternatives resulted in a positive mean NPV. Excavation and stabilization/solidification of the hotspot on-site combined with stabilization using activated carbon for the rest of site (Alt 2) had the highest NPV for both spreading scenarios, i.e., Alt 2 was the most socially profitable alternative. Simulations of the annual avoided cost of inaction enabled estimation of the breakeven point at which a remediation alternative becomes socially profitable (NPV > 0) compared to 'do nothing'. Alt 2 had the lowest breakeven point: 7.5 and 5.75 millions of SEK/year for large and small spreading, respectively.

Disseminated tuberculosis, CMV viraemia & haemophagocytic-lymphohistiocystosis syndrome in an adult patient with anti- IFNγ autoantibodies - case report and brief review.

We report a case of an adult female with disseminated tuberculosis, cytomegalovirus viraemia and haemophagocytic-lymphohistiocystosis syndrome associated with neutralizing anti- interferon gamma (IFNγ) autoantibodies demonstrated by absent IFNγ stimulated STAT1 phosphorylation in the presence of patient sera. A brief review of immunodeficiency caused by anti-IFNγ autoantibodies is also described.

A phase I study to determine the safety, pharmacokinetics and pharmacodynamics of a dual VEGFR and FGFR inhibitor, brivanib, in patients with advanced or metastatic solid tumors.

BACKGROUND: This study was designed to determine the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of brivanib in patients with advanced/metastatic solid tumors. PATIENTS AND METHODS: Ninety patients enrolled in this two-part, phase I open-label study of oral brivanib alaninate. The primary objectives of this study were (in part A) dose-limiting toxicity, maximum tolerated dose (MTD) and the lowest biologically active dose level and (in part B) the optimal dose/dose range. The secondary objectives of this study were preliminary evidence of antitumor activity, PK and PD. RESULTS: Across part A (open-label dose escalation and MTD) and part B (open-label dose optimization), 68 patients received brivanib alaninate. Brivanib demonstrated a manageable toxicity profile at doses of 180-800 mg. Most toxic effects were mild. Systemic exposure of the active moiety brivanib increased linearly ≤1000 mg/day. The MTD was 800 mg/day. Forty-four patients were treated at the MTD: 20 with 800 mg continuously, 11 with 800 mg intermittently and 13 with 400 mg b.i.d. doses. Partial responses were confirmed in two patients receiving brivanib ≥600 mg. Dynamic contrast-enhanced magnetic resonance imaging demonstrated statistically significant decreases in parameters reflecting tumor vascularity and permeability after multiple doses in the 800-mg continuous q.d. and 400-mg b.i.d. dose cohorts. CONCLUSION: In patients with advanced/metastatic cancer, brivanib demonstrates promising antiangiogenic and antitumor activity and manageable toxicity at doses ≤800 mg orally q.d., the recommended phase II study dose.

Surgical resection in Crohn's disease: is immunosuppressive medication associated with higher postoperative infection rates?

AIM: The aim of this study was to analyse postoperative infection in patients undergoing surgery for Crohn's disease (CD) according to the use of preoperative immunosuppressants, including infliximab. METHOD: With IRB approval, the records of all patients with CD who underwent abdominal surgery between 2001 and 2008 were reviewed for comorbidity, preoperative medication, type of surgery, stoma construction and postoperative complications. Patients were divided into three categories according to the preoperative medication within 90 days of surgery as follows: infliximab (IFX), other drugs including steroids and/or immunosuppressive agents (OD) and no drugs (ND). RESULTS: Two hundred and twenty-five patients were identified. Preoperative comorbidity, surgical indication and type of surgery were not significantly different among the three groups. Ileocolic resection was the most common procedure [50.8%, IFX group; 61.2%, OD group; 41.3%, ND group (P = 0.09)]. Other procedures included total colectomy (16%), protectomy (15%) and others (18%). Laparoscopic surgery was performed in 47.7%, 45.9% and 29.3% of patients in the IFX, OD and ND groups, respectively (P = 0.04). There were no differences in postoperative rates of infection [pneumonia (P = 0.14), wound infection (P = 0.35), abscess (P = 0.34) or anastomotic leakage (P = 0.44)]. Reoperation was needed in 3.0%, 8.2% and 2.6% of patients in the IFX, OD and ND groups, respectively. Multiple logistic regression found no relationship between infliximab use and infection. CONCLUSION: There was no difference in the rate of postoperative complications among the groups of patients undergoing surgery for CD pretreated with IFX or other immunosuppressive drugs.

Granulomas in Crohn's disease: does progression through the bowel layers affect presentation or predict recurrence?

AIM: The aim of the study was to correlate the presence and pattern of distribution of granulomas in resected specimens to clinical characteristics and outcome in patients undergoing surgery for Crohn's disease. METHOD: Patients with Crohn's disease who underwent surgical resection between 2001 and 2007 were identified. Pathology slides were reviewed for the presence, number and location of granulomas in four representative slides from each specimen. RESULTS: Two-hundred and seven patients were identified. At a mean follow up of 14 months, 39 patients had a recurrence, 22 (57%) of whom underwent reoperation. Ninety-four (45%) patients had granulomas present in the surgical specimen. Patients with granulomas were younger (P<0.001), had a lower preoperative body mass index (P=0.037), were more likely to be female (P=0.017) and were more likely to have extra-intestinal manifestations (P=0.026) or perianal disease (P=0.012). Sites of disease and procedures performed were similar in both groups. Disease recurrence and reoperative rates were similar in both groups, as were length of stay and morbidity rates. The average number of granulomas present in each sampled pathology slide was 7.2, and there was no correlation between number of granulomas and disease severity. No link was found between the depth of involvement of the granulomas and fistulizing or stricturing disease. CONCLUSION: Granulomas were associated with increased extra-intestinal manifestations and perianal disease, a lower body mass index and younger or female patients. There was no correlation between the presence of granulomas and disease progression or recurrence rates during the short follow-up period of this study.

PM-Scl and Th/To in systemic sclerosis: a comparison of different autoantibody assays.

OBJECTIVE: To compare test characteristics of the Euroimmun line blot assay with other assays for two uncommon autoantibody specificities in systemic sclerosis (SSc). METHODS: Patients from the Johns Hopkins Scleroderma Center were assayed routinely using the Euroimmun platform. Patients positive for anti-Th/To (N = 73) and anti-PM-Scl (PM75 and/or PM100; N = 290) by Euroimmun were compared with SSc patients negative for these autoantibodies. For Th/To antibodies, the comparison assay was immunoprecipitation (IP), performed using 4 Th/To complex components: POP1, RPP40, RPP30, and RPP25. For anti-PM-Scl, IPs were performed with PM100 and PM75. Different Euroimmun cut-offs for assigning antibody positive status (≥ 15/+, ≥ 36/++, ≥ 71/+++) were examined. Kappa statistics were calculated to determine agreement between assays. RESULTS: The best performing thresholds for defining anti-PM-Scl positivity were both PM75 and PM100 ≥ 15/+ on Euroimmun, corresponding to a kappa statistic of 0.79, sensitivity 72% and specificity 100%. For anti-Th/To, kappa values were lower for all comparisons (κ < 0.5). Given the high sensitivity of defining anti-Th/To by ≥ 15/+ (91-95%), a potential approach is to use Euroimmun screening (15/+ cut-off), followed by confirmatory IP. CONCLUSION: Given the increasing utilization of Euroimmun and the importance of comparing data across cohorts, continued use of this platform is warranted, acknowledging discordance with IP for some specificities. For these, using a two-step approach (Euroimmun to maximize sensitivity, confirmatory assay to increase specificity) is suggested. KEY POINTS: • For less common SSc autoantibody specificities, some discordances exist between IP and Euroimmun LIA. • The best performing thresholds for defining anti-PM-Scl positivity were both PM75 and PM100 ≥ 15/+ on Euroimmun. • For Th/To, a two-step approach (Euroimmun to maximize sensitivity, confirmatory assay to increase specificity) is suggested.

Novel packages of viral and self-antigens are generated during apoptosis.

Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.

Scleroderma autoantigens are uniquely fragmented by metal-catalyzed oxidation reactions: implications for pathogenesis.

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease.

Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.

Systemic lupus erythematosus is a multisystem autoimmune disease in which the autoantibody response targets a variety of autoantigens of diverse subcellular location. We show here that these autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented endoplasmic reticulum (ER) and ribosomes, as well as the ribonucleoprotein, Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. These autoantigen clusters have in common their proximity to the ER and nuclear membranes, sites of increased generation of reactive oxygen species in apoptotic cells. Oxidative modification at these sites may be a mechanism that unites this diverse group of molecules together as autoantigens.

DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis.

Proteolytic cleavage of key substrates appears to be an important biochemical mechanism underlying the apoptotic process, and the centrality of interleukin 1 beta-converting enzyme (ICE)-like proteases as mediators of apoptosis has been suggested. The identification of the relevant substrates of the ICE protease family during apoptosis therefore constitutes a major challenge. Using human autoantibodies, we demonstrate here that a subset of autoantigens is specifically cleaved early during apoptosis. One of these cleaved molecules is identified as the catalytic subunit of the DNA-dependent protein kinase. The time courses of all proteolytic cleavages are identical and coincide with the onset of morphologic apoptosis. Furthermore, all cleavages share the same inhibition characteristics, which implicate an ICE-like activity(ies). We propose that cleavage of these autoantigens targets these molecules for an autoimmune response by revealing immunocryptic fragments in a proimmune apoptotic setting. Study of the immunogenicity of these fragments may yield insights into the autoimmune targeting of molecules. Moreover, the autoantibodies described will be valuable tools for the elucidation of mechanistically important proteolytic steps along the apoptotic pathway.

Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity.

Systemic autoimmune diseases are a genetically complex, heterogeneous group of disorders in which the immune system targets a diverse but highly specific group of intracellular autoantigens. The molecules targeted are not unified by common structure, function, or distribution in control cells but become clustered and concentrated in surface blebs when cells undergo apoptosis. We show here that the majority of autoantigens targeted across the spectrum of human systemic autoimmune diseases are efficiently cleaved by granzyme B in vitro and during cytotoxic lymphocyte granule-induced death, generating unique fragments not observed during any other form of apoptosis. These molecules are not cleaved by caspase-8, although this protease has a very similar specificity to granzyme B. The granzyme B cleavage sites in autoantigens contain amino acids in the P(2) and P(3) positions that are preferred by granzyme B but are not tolerated by caspase-8. In contrast to autoantigens, nonautoantigens are either not cleaved by granzyme B or are cleaved to generate fragments identical to those formed in other forms of apoptosis. The striking ability of granzyme B to generate unique fragments is therefore an exclusive property of autoantigens and unifies the majority of molecules targeted in this spectrum of diseases. These results focus attention on the role of the cytotoxic lymphocyte granule-induced death pathway in the initiation and propagation of systemic autoimmunity.