Bovine colostral antibodies, purified from cow's milk produced immediately after calving, have enhanced resistance to degradation by intestinal proteases relative to antibodies from human or bovine ...serum, making them of particular interest as orally administered therapeutic agents. However, the basis of this resistance is not well defined. We evaluated the stability of AVX-470, a bovine colostral anti-tumor necrosis factor (TNF) polyclonal antibody used in early clinical studies for treatment of ulcerative colitis, using conditions that mimic the human small intestine. AVX-470 was degraded ∼3 times more slowly than human IgG antibodies or infliximab (a monoclonal mouse-human chimeric IgG). Bovine IgG1 antibodies, the primary component of AVX-470, were slowly cleaved to F(ab′)2 fragments. In contrast, bovine IgG2 and human IgG1 antibodies were cleaved rapidly into Fab and smaller fragments, pointing to specific regions where additional stability might be gained. Infliximab was modified to incorporate the sequences from these regions, including the bovine IgG1 hinge region and a predicted disulfide bonding motif linking the upper hinge region, the CH1 domain, and the light chain. This infliximab-bovine IgG1 chimera (bovinized infliximab) retained the antigen binding and neutralization activity of the WT sequence but was degraded 9-fold more slowly than the unmodified infliximab. This remarkable increase in stability with as few as 18 amino acid substitutions suggests that this bovinization process is a means to enable oral delivery of proven therapeutic antibodies as well as novel antibodies to targets that have been previously inaccessible to therapies delivered by injection.
Recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are associated with alcohol consumption and cigarette smoking. The etiology of RAP and CP is complex, and effects of alcohol and ...smoking may be limited to specific patient subsets. We examined the current prevalence of alcohol use and smoking and their association with RAP and CP in patients evaluated at US referral centers.
The North American Pancreatitis Study 2, a multicenter consortium of 20 US centers, prospectively enrolled 540 patients with CP, 460 patients with RAP, and 695 controls from 2000 to 2006. Using self-reported monthly alcohol consumption during the maximum lifetime drinking period, we classified subjects by drinking status: abstainer, light drinker (< or =0.5 drink per day), moderate drinker (women, >0.5 to 1 drink per day; men, >0.5 to 2 drinks per day), heavy drinker (women, >1 to <5 drinks per day; men, >2 to <5 drinks per day), or very heavy drinker (> or =5 drinks per day for both sexes). Smoking was classified as never, past, or current and was quantified (packs per day and pack-years).
Overall, participants' mean (SD) age was 49.7 (15.4) years; 87.5% were white, and 56.5% were women. Approximately one-fourth of both controls and patients were lifetime abstainers. The prevalence of very heavy drinking among men and women was 38.4% and 11.0% for CP, 16.9% and 5.5% for RAP, and 10.0% and 3.6% for controls. Compared with abstaining and light drinking, very heavy drinking was significantly associated with CP (odds ratio, 3.10; 95% confidence interval, 1.87-5.14) after controlling for age, sex, smoking status, and body mass index. Cigarette smoking was an independent, dose-dependent risk factor for CP and RAP.
Very heavy alcohol consumption and smoking are independent risks for CP. A minority of patients with pancreatitis currently seen at US referral centers report very heavy drinking.
Alcohol has been implicated in the development of chronic pancreatitis (CP) in 60%-90% of patients, although percentages in the United States are unknown. We investigated the epidemiology of ...alcohol-related CP at tertiary US referral centers.
We studied data from CP patients (n = 539) and controls (n = 695) enrolled in the North American Pancreatitis Study-2 from 2000 to 2006 at 20 US referral centers. CP was defined by definitive evidence from imaging or histologic analyses. Subjects and physicians each completed a study questionnaire. Using physician-assigned diagnoses, patients were assigned to an etiology group: alcohol (with/without other diagnoses), nonalcohol (any etiology of CP from other than alcohol), or idiopathic (no etiology identified).
The distribution of patients among etiology groups was: alcohol (44.5%), nonalcohol (26.9%), and idiopathic (28.6%). Physicians identified alcohol as the etiology more frequently in men (59.4% men vs 28.1% women), but nonalcohol (18% men vs 36.7% women) and idiopathic etiologies (22.6% men vs 35.2% women) more often in women (P < .01 for all comparisons). Nonalcohol etiologies were equally divided among obstructive, genetic, and other causes. Compared with controls, patients with idiopathic CP were more likely to have ever smoked (58.6% vs 49.7%, P < .05) or have a history of chronic renal disease or failure (5.2% vs 1.2%, P < .01). In multivariate analyses, smoking (ever, current, and amount) was independently associated with idiopathic CP.
The frequency of alcohol-related CP at tertiary US referral centers is lower than expected. Idiopathic CP and nonalcohol etiologies represent a large subgroup, particularly among women. Smoking is an independent risk factor for idiopathic CP.
To compare patients with chronic pancreatitis (CP) with constant pain patterns to patients with CP with intermittent pain patterns.
This was a prospective cohort study conducted at 20 tertiary ...medical centers in the USA comprising 540 subjects with CP. Patients with CP were asked to identify their pain from five pain patterns (A-E) defined by the temporal nature (intermittent or constant) and the severity of the pain (mild, moderate or severe). Pain pattern types were compared with respect to a variety of demographic, quality of life (QOL) and clinical parameters. Rates of disability were the primary outcome. Secondary outcomes included: use of pain medications, days lost from school or work, hospitalisations (preceding year and lifetime) and QOL as measured using the Short Form-12 (SF-12) questionnaire.
Of the 540 CP patients, 414 patients (77%) self-identified with a particular pain pattern and were analysed. Patients with constant pain, regardless of severity, had higher rates of disability, hospitalisation and pain medication use than patients with intermittent pain. Patients with constant pain had lower QOL (by SF-12) compared with patients who had intermittent pain. Additionally, patients with constant pain were more likely to have alcohol as the aetiology for their pancreatitis. There was no association between the duration of the disease and the quality or severity of the pain.
This is the largest study ever conducted of pain in CP. These findings suggest that the temporal nature of pain is a more important determinant of health-related QOL and healthcare utilisation than pain severity. In contrast to previous studies, the pain associated with CP was not found to change in quality over time. These results have important implications for improving our understanding of the mechanisms underlying pain in CP and for the goals of future treatments and interventions.
Machines of protein destruction—including energy-dependent proteases and disassembly chaperones of the AAA+ ATPase family—function in all kingdoms of life to sculpt the cellular proteome, ensuring ...that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA+ machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.
Background & Aims Idiopathic chronic pancreatitis (ICP) is a complex inflammatory disorder associated with multiple genetic and environmental factors. In individuals without cystic fibrosis (CF), ...variants of CFTR that inhibit bicarbonate conductance but maintain chloride conductance might selectively impair secretion of pancreatic juice, leading to trypsin activation and pancreatitis. We investigated whether sequence variants in the gene encoding the pancreatic secretory trypsin inhibitor SPINK1 further increase the risk of pancreatitis in these patients. Methods We screened patients and controls for variants in SPINK1 associated with risk of chronic pancreatitis and in all 27 exons of CFTR . The final study group included 53 patients with sporadic ICP, 27 probands with familial ICP, 150 unrelated controls, 375 additional controls for limited genotyping. CFTR wild-type and p.R75Q were cloned and expressed in HEK293 cells, and relative conductances of HCO3– and Cl– were measured. Results SPINK1 variants were identified in 36% of subjects and 3% of controls (odds ratio OR, 18.1). One variant of CFTR not associated with CF, p.R75Q, was found in 16% of subjects and 5.3% of controls (OR, 3.4). Coinheritance of CFTR p.R75Q and SPINK1 variants occurred in 8.75% of patients and 0.38% of controls (OR, 25.1). Patch-clamp recordings of cells that expressed CFTR p.R75Q showed normal chloride currents but significantly reduced bicarbonate currents ( P = .0001). Conclusions The CFTR variant p.R75Q causes a selective defect in bicarbonate conductance and increases risk of pancreatitis. Coinheritance of p.R75Q or CF causing CFTR variants with SPINK1 variants significantly increases the risk of ICP.
Abstract Background: Recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are complex syndromes associated with numerous etiologies, clinical variables and complications. We developed the ...North American Pancreatitis Study 2 (NAPS2) to be sufficiently powered to understand the complex environmental, metabolic and genetic mechanisms underlying RAP and CP. Methods: Between August 2000 and September 2006, a consortium of 20 expert academic and private sites prospectively ascertained 1,000 human subjects with RAP or CP, plus 695 controls (spouse, family, friend or unrelated). Standardized questionnaires were completed by both the physicians and study subjects and blood was drawn for genomic DNA and biomarker studies. All data were double-entered into a database and systematically reviewed to minimize errors and include missing data. Results: A total of 1,000 subjects (460 RAP, 540 CP) and 695 controls who completed consentforms and questionnaires and donated blood samples comprised the final dataset. Data were organized according to diagnosis, supporting documentation, etiological classification, clinical signs and symptoms (including pain patterns and duration, and quality of life), past medical history, family history, environmental exposures (including alcohol and tobacco use), medication use and therapeutic interventions. Upon achieving the target enrollment, data were organized and classified to facilitate future analysis. The approaches, rationale and datasets are described, along with final demographic results. Conclusion: The NAPS2 consortium has successfully completed a prospective ascertainment of 1,000 subjects with RAP and CP from the USA. These data will be useful in elucidating the environmental, metabolic and genetic conditions, and to investigate the complex interactions that underlie RAP and CP. study was designed to help determine the prevalence of known genetic variations and autoimmune markers associated with RAP and CP, and to better delineate theinteraction of environmental and genetic factors in the expression of the clinical manifestations of CP and consequential risk of pancreatic cancer. Herein we describe the participating centers, diverse symptomatology, methods used for diagnosis, etiological classification and treatment approaches.
The mechanistic definition of chronic pancreatitis (CP) identifies acute pancreatitis (AP) as a precursor stage. We hypothesized that clinical AP frequently precedes the diagnosis of CP and is ...associated with patient- and disease-related factors. We describe the prevalence, temporal relationship and associations of AP in a well-defined North American cohort.
We evaluated data from 883 patients with CP prospectively enrolled in the North American Pancreatitis Studies across 27 US centers between 2000 and 2014. We determined how often patients had one or more episodes of AP and its occurrence in relationship to the diagnosis of CP. We used multivariable logistic regression to determine associations for prior AP.
There were 624/883 (70.7%) patients with prior AP, among whom 161 (25.8%) had AP within 2 years, 115 (18.4%) within 3–5 years, and 348 (55.8%) >5 years prior to CP diagnosis. Among 504 AP patients with available information, 436 (86.5%) had >1 episode. On multivariable analyses, factors associated with increased odds of having prior AP were a younger age at CP diagnosis, white race, abdominal pain, pseudocyst(s) and pancreatic duct dilatation/stricture, while factors associated with a lower odds of having prior AP were exocrine insufficiency and pancreatic atrophy. When compared with patients with 1 episode, those with >1 AP episode were diagnosed with CP an average of 5 years earlier.
Nearly three-quarters of patients were diagnosed with AP prior to CP diagnosis. Identifying which AP patients are at-risk for future progression to CP may provide opportunities for primary and secondary prevention.
ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six ...potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.
ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP ...modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.
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