Outcomes of Cystic Fibrosis Screening-Positive Infants With Inconclusive Diagnosis at School Age.

BACKGROUND AND OBJECTIVES: Cystic fibrosis (CF) screen-positive infants with an inconclusive diagnosis (CFSPID) are infants in whom sweat testing and genetic analysis does not resolve a CF diagnosis. Lack of knowledge about the health outcome of these children who require clinical follow-up challenges effective consultation. Early predictive biomarkers to delineate the CF risk would allow a more targeted approach to these children. METHODS: Prospective, longitudinal, multicenter, Canada-wide cohort study of CF positive-screened newborns with 1 to 2 cystic fibrosis transmembrane conductance regulator gene variants, of which at least 1 is not known to be CF-causing and/or a sweat chloride between 30 and 59 mmol/L. These were monitored for conversion to a CF diagnosis, pulmonary, and nutritional outcomes. RESULTS: The mean observation period was 7.7 (95% confidence interval 7.1 to 8.4) years. A CF diagnosis was established for 24 of the 115 children with CFSPID (21%) either because of reinterpretation of the cystic fibrosis transmembrane conductance regulator genotype or because of increase in sweat chloride concentration ≥60 mmol/L. An initial sweat chloride of ≥40 mmol/l predicted conversion to CF on the basis of sweat testing. The 91 remaining children with CFSPID were pancreatic sufficient and showed normal growth until school age. Pulmonary function as well as lung clearance index in a subgroup of children with CFSPID were similar to that of healthy controls. CONCLUSIONS: Children with CFSPID have good nutritional and pulmonary outcomes at school age, but rates of reclassifying the diagnosis are high. The initial sweat chloride test can be used as a biomarker to predict the risk for CF in CFSPID.

Immunoreactive Trypsinogen and Free Carnitine Changes on Newborn Screening after Birth in Patients Who Develop Type 1 Diabetes.

Are free carnitine concentrations on newborn screening (NBS) 48-72 h after birth lower in patients who develop type 1 diabetes than in controls? A retrospective case-control study of patients with type 1 diabetes was conducted. NBS results of patients from a Sydney hospital were compared against matched controls from the same hospital (1:5). Multiple imputation was performed for estimating missing data (gestational age) using gender and birthweight. Conditional logistic regression was used to control for confounding and to generate parameter estimates (α = 0.05). The Hommel approach was used for post-hoc analyses. Results are reported as medians and interquartile ranges. A total of 159 patients were eligible (80 females). Antibodies were detectable in 86. Median age at diagnosis was 8 years. Free carnitine concentrations were lower in patients than controls (25.50 µmol/L;18.98-33.61 vs. 27.26; 21.22-34.86 respectively) (p = 0.018). Immunoreactive trypsinogen was higher in this group (20.24 µg/L;16.15-29-52 vs. 18.71; 13.96-26.92) (p = 0.045), which did not persist in the post-hoc analysis. Carnitine levels are lower and immunoreactive trypsinogen might be higher, within 2-3 days of birth and years before development of type 1 diabetes as compared to controls, although the differences were well within reference ranges and provide insight into the pathogenesis into neonatal onset of type 1 diabetes development rather than use as a diagnostic tool. Given trypsinogen's use for evaluation of new-onset type 1 diabetes, larger studies are warranted.

Development of mutant human immunoreactive trypsinogen 1 (IRT1) and mutant human immunoreactive trypsinogen 2 (IRT2) for use in immunoassays.

Immunoreactive Trypsinogen (IRT) is a protein-based pancreatic proenzyme that has an important role in protein digestion in humans. In human body, once IRT present in the small intestine, the proteolytic cleavage activates trypsinogen into trypsin. When IRT is in the active form, it is capable to cleave antibodies, other proteins and even itself while it is desired to use in immunoassays. According to the literature, there are three important IRT isoforms called Immunoreactive Trypsinogen 1 (IRT1), Immunoreactive Trypsinogen 2 (IRT2), and Immunoreactive Trypsinogen 3 (IRT3). However, trypsinogen 1 (cationic trypsinogen, IRT1) and trypsinogen 2 (anionic trypsinogen, IRT2) are the major isoforms in human pancreatic juice and used in the diagnosis of cystic fibrosis (CF). In this study, it is aimed to restrain its proteolytic activity with K23D mutation, which changes lysine (K) residue at the 23rd position to aspartic acid (D). Because we wanted to produce a hassle-free human recombinant immune reactive trypsinogen proenzyme which has similar antigenic properties with the native form. It is also aimed that the mutant IRTs do not exhibit proteolytic activity for the development of durable detection kits with a longer shelf life for both two isoforms. The innovation was actualized in order to use IRTs as a standard antigen in Immunoassays such as ELISA kits. The gene was synthesized as mutated and expressed in P. pastoris X-33 strain. The loss of proteolytic activity has been proven with the BAEE test. Antigenic properties of K23D IRTs and the effect of proteolytic inactivation on their performance in immunoassays were assessed with ELISA and Western Blot. In ELISA results K23D mutated IRTs showed higher signals than Wild-Type forms.

Transgenic Expression of PRSS1R122H Sensitizes Mice to Pancreatitis.

BACKGROUND & AIMS: Mutations in the trypsinogen gene (PRSS1) cause human hereditary pancreatitis. However, it is not clear how mutant forms of PRSS1 contribute to disease development. We studied the effects of expressing mutant forms of human PRSS1 in mice. METHODS: We expressed forms of PRSS1 with and without the mutation encoding R122H (PRSS1R122H) specifically in pancreatic acinar cells under control of a full-length pancreatic elastase gene promoter. Mice that did not express these transgenes were used as controls. Mice were given injections of caerulein to induce acute pancreatitis or injections of lipopolysaccharide to induce chronic pancreatitis. Other groups of mice were fed ethanol or placed on a high-fat diet to induce pancreatitis. Pancreata were collected and analyzed by histology, immunoblots, real-time polymerase chain reaction, and immunohistochemistry. Trypsin enzymatic activity and chymotrypsin enzymatic activity were measured in pancreatic homogenates. Blood was collected and serum amylase activity was measured. RESULTS: Pancreata from mice expressing transgenes encoding PRSS1 or PRSS1R122H had focal areas of inflammation; these lesions were more prominent in mice that express PRSS1R122H. Pancreata from mice that express PRSS1 or PRSS1R122H had increased levels of heat shock protein 70 and nuclear factor (erythroid-derived 2)-like 2, and reduced levels of chymotrypsin C compared with control mice. Increased expression of PRSS1 or PRSS1R122H increased focal damage in pancreatic tissues and increased the severity of acute pancreatitis after caerulein injection. Administration of lipopolysaccharide exacerbated inflammation in mice that express PRSS1R122H compared to mice that express PRSS1 or control mice. Mice that express PRSS1R122H developed more severe pancreatitis after ethanol feeding or a high-fat diet than mice that express PRSS1 or control mice. Pancreata from mice that express PRSS1R122H had more DNA damage, apoptosis, and collagen deposition and increased trypsin activity and infiltration by inflammatory cells than mice that express PRSS1 or control mice. CONCLUSIONS: Expression of a transgene encoding PRSS1R122H in mice promoted inflammation and increased the severity of pancreatitis compared with mice that express PRSS1 or control mice. These mice might be used as a model for human hereditary pancreatitis and can be studied to determine mechanisms of induction of pancreatitis by lipopolysaccharide, ethanol, or a high-fat diet.

Generation and characterization of murine monoclonal antibodies against immunoreactive trypsinogen for newborn screening of cystic fibrosis.

Cystic fibrosis (CF) is a multisystem disorder that reduces quality of life and survival in affected individuals. In newborns, the release of pancreatic enzymes into the blood raises the levels of immunoreactive trypsinogen (IRT), the main marker for CF screening, which is detected in dried blood samples on filter paper by immunoenzymatic assays. In Cuba, CF has an estimated incidence of 1/9862 live births and should be included in the national basic newborn screening (NBS) panel given its benefits in terms of nutrition, lung function and survival. The Immunoassay Center develops and produces diagnostic kits allowing the establishment of large-scale NBS programs for inherited metabolic disorders in Cuba and other Latin American countries. IRT-specific monoclonal antibodies (MAbs) obtained at the Immunoassay Center are essential for developing an affordable immunoassay for IRT to support CF NBS in our low-income country. An immunization scheme with trypsinogen-1 originated two IgG1-producing murine hybridomas. 4C9C9 and 4C9E11 MAbs recognized different determinants on both trypsin-1 and trypsin-2 molecules. Both antibodies identified conformational epitopes on the molecule of trypsin-1 and of its zymogen. As 4C9E11 MAb cross-reacted with proteins structurally and functionally related to trypsinogen, it was used as revealing antibody in a sandwich-type UMELISA® assay for IRT determination with 4C9C9 MAb for capture. This combination, aside from detecting several commercially available trypsins, adequately quantified IRT from dried blood samples on filter paper of newborns. The evaluation of the assay's accuracy yielded percentage recoveries ranging 93.3-109.2% for commercial controls. The properties of the studied MAbs demonstrate their suitability for being used in a sandwich-type UMELISA® assay for the CF NBS in Cuba.

A product of immunoreactive trypsinogen and pancreatitis-associated protein as second-tier strategy in cystic fibrosis newborn screening.

BACKGROUND: In cystic fibrosis newborn screening (CFNBS), immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) can be used as screening parameters. We evaluated the IRT×PAP product as second-tier parameter in CFNBS in newborns with elevated IRT. METHODS: Data on 410,111 screened newborns including 78 patients with classical cystic fibrosis (CF) from two European centers were retrospectively analyzed by discrimination analysis to identify a screening protocol with optimal cutoffs. We also studied differences in PAP measurement methods and the association of IRT and PAP with age. RESULTS: PAP values differed systematically between fluorometric and photometric assays. The IRT×PAP product showed better discrimination for classical CF than PAP only as second-tier screening parameter (p<0.001). In CF patients, IRT decreased while PAP values remained high over years. In newborns without CF, IRT decreased after birth over weeks while PAP increased within days. CONCLUSIONS: The IRT×PAP product performs well as second-tier cutoff parameter for CFNBS. Screening quality parameters depend on the analytic method and on age at blood collection.

Assessing the improvements in the newborn screening strategy for cystic fibrosis in the Balearic Islands.

OBJECTIVES: Newborn screening strategies for cystic fibrosis (CF) are run worldwide, and aim at the early detection of the disorder to significantly improve the quality of life. Elevated levels of immunoreactive trypsinogen (IRT) represent a high likelihood for the screened child to be affected with CF. However, the specificity of IRT is low. The objective of this study was to assess the screening program in the Balearic Islands during the past 14 years. DESIGN & METHODS: We evaluated all results of the screening program after 14 years, by considering all changes in the protocol and assessing the number of positive samples, the mutations detected, the number of sweat tests performed, the incidence of CF and the presence of false-negative cases. RESULTS: Despite a great variability among the different Balearic Islands, the global incidence of CF was 1:6059 for the 14 years assessed. The incidence in the smaller islands is about 5 times higher than in Majorca (1:2376 versus 1:10,613). After different changes in the protocol, an IRT cut-off value of 60 ng/mL was established. The two most common mutations are ΔF508 and G542X, in accordance with other geographical regions. CONCLUSIONS: The changes in the protocol helped reduce the number of sweat tests performed without any increase in the false-negative rate.

Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: challenges in newborn dried blood spot screening.

On May 23-24, 2011, a workshop entitled "Immunoreactive Trypsinogen (IRT) as a Biomarker for Cystic Fibrosis: Technical Issues and Challenges" was held in Annapolis, Maryland. The two-day workshop was co-hosted by the National Newborn Screening and Genetics Resource Center, Austin, Texas, and the Association of Public Health Laboratories, Silver Spring, Maryland, in collaboration with the Health Resources and Services Administration and the Centers for Disease Control and Prevention. Participants included nearly 40 representatives from U.S. state public health and commercial laboratories performing newborn dried blood spot screening tests for cystic fibrosis (CF), the federal government, academic research institutions, and commercial vendors of products used in newborn screening. Representatives from selected European CF newborn screening programs were also present. The workshop focused on identifying key IRT testing issues and mechanisms for achieving their resolution and laboratory harmonization in order to reduce, or eliminate completely, the late identified CF cases following a negative newborn screen. Informative findings are reported, their impacts on improving IRT screening are described, and their implications are discussed.

Serologic issues in IgG4-related systemic disease and autoimmune pancreatitis.

PURPOSE OF REVIEW: IgG4-related systemic disease (ISD) is a recently recognized syndrome affecting multiple organs. Autoimmune pancreatitis (AIP) is the pancreatic manifestation of ISD and mimics pancreatic cancer. Current data show frequent association with serum IgG4 elevation and other serologic abnormalities. Here we explore the diagnostic and possible prognostic utility and pathogenetic implications of serologic abnormalities in ISD. RECENT FINDINGS: Serum IgG4 elevations (>140 mg/dl) are seen in 70-80% of AIP patients and also in 5% of normal population and 10% of pancreatic cancer making it an unsuitable single marker for diagnosis. However, when combined with other features of AIP, it can be of great diagnostic value though its utility in monitoring of therapy or as a marker or predictor of relapse is limited. Several other antibodies have been identified in AIP against pancreas-specific antigens like trypsinogens I and II, pancreatic secretory trypsin inhibitor (PSTI) and plasminogen binding protein (PBP) and other nonpancreas-specific antigens. Anti-PBP antibodies appear to have potential diagnostic utility but require further validation. SUMMARY: No single serologic marker is diagnostic of ISD. Serum IgG4 elevation has convincing diagnostic utility when combined with other disease features although its value in disease monitoring may be limited.

Newborn screening for cystic fibrosis by use of a multiplex immunoassay.

BACKGROUND: Since its beginnings, newborn screening for cystic fibrosis (CF) using an assay for immunoreactive trypsinogen (IRT) has been plagued by a high rate of false-positive results (screen positive, diagnosis negative), despite attempts to reduce this rate by use of altered cutoffs and second-tier DNA testing. IRT exists as 2 isoforms: IRT1 and IRT2, with IRT2 being more closely aligned with pancreatic disease, including CF. Assay standardization between programs is a continuing problem because the IRT assays currently in use variously recognize either 1 or both isoforms. Here we report the development of a multiplexed assay for both forms of IRT simultaneously. METHODS: Using 2 different Luminex bead sets, we developed assays for each IRT isoform separately and then combined them. Using the sum of IRT1 and IRT2 values (IRT1+IRT2), we compared the results with a CF kit currently in use. RESULTS: In a sample set consisting of 16 cases confirmed positive for CF, we established a cutoff at >97 microg/L total IRT. Seven of 8 carriers with 1 CF mutation screen-positive by the standard method were also screen-positive by IRT1+IRT2. Of 32 cases screen-positive by standard IRT, 11 were screen-negative by IRT1+IRT2. None of these 11 cases had CF mutations identified by the screening program. CONCLUSIONS: These data indicate that the multiplex method with specificity for 2 isoforms of IRT has performance comparable to that of a standard IRT method and the advantage of improved standardization by detection of the 2 isoforms.

Trypsin is a multifunctional factor in spermatogenesis.

Trypsin is well known as a pancreatic enzyme that is typically secreted into the intestine to digest proteins. We show in our current study, however, that trypsin is also a key factor in the control of spermatogenesis. A progestin in teleost fish, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), is an essential component of the spermatogenesis pathway, particularly during the initiation of the first meiotic division. In the course of our investigations into the mechanisms underlying progestin-stimulated spermatogenesis, we identified that eel trypsinogen is upregulated in eel testis by DHP treatment. Trypsinogen is expressed in the Sertoli cells surrounding spermatogonia and in the membranes of spermatids and spermatozoa. Using an in vitro eel testicular culture system, we further analyzed the roles of trypsin in spermatogenesis. The inhibition of trypsin using specific antibodies or serine protease inhibitors was found to compromise DHP-induced spermatogenesis. A low dose of trypsin induces DNA synthesis and the expression of Spo11, a molecular marker of meiosis, in germ cells. By comparison, a higher dose of trypsin partially induced spermiogenesis. Furthermore, trypsin was detectable in the membranes of the spermatozoa and found to be associated with fertilization in fish. Our results thus demonstrate that trypsin and/or a trypsin-like protease is an essential and multifunctional factor in spermatogenesis.

A new cystic fibrosis newborn screening algorithm: IRT/IRT1 upward arrow/DNA.

OBJECTIVE: To evaluate an immunoreactive trypsinogen (IRT) IRT/IRT1 upward arrow/DNA algorithm, aimed at improving sensitivity while decreasing cystic fibrosis (CF) carrier identification. STUDY DESIGN: New technologies allow the measurement of the second IRT level solely in infants with an elevated first IRT level. Specimens with an elevated second IRT level undergo mutation analysis. We tested the projected efficacy with retrospective data from Colorado. RESULTS: All known infants with CF would have been identified with our proposed IRT cutoff points, and 3 would have been missed with our mutation panel. Two of 3 missed cases would have been identified by using a failsafe method (IRT >99.9th percentile), yielding a sensitivity rate of 99.7% (95% CI, 98.4-99.9). Estimated reduction in carrier detection was 80% compared with IRT/DNA. CONCLUSION: IRT/IRT1 upward arrow/DNA appears to improve cystic fibrosis newborn screen sensitivity while decreasing carrier identification, providing an alternative to IRT/IRT in states that obtain 2 blood spots.

Risk factors of developing asthma in children with recurrent wheezing in the first three years of life / Factores de riesgo para el desarrollo de asma en niños con dificultad respiratoria recurrente en los tres primeros años de vida

Introduction: recurrent wheezing is a common problem during the first years of life, but it is still difficult to identify which of these children may develop asthma in the future. Objectives: To study risk factors of developing asthma in a group of patients with frequent wheezing during the first three years of life. Material and methods: A prospective study was performed of a group of 60 patients, aged below three, referred to our Hospital for recurrent wheezing. Age, sex, parental and personal history of atopy, clinical features, laboratory tests, evolution and response to treatment were analyzed. Results: 60 patients were enrolled in study. Most of children were boys and have had the first episode of wheezing after the 6 months of life. 63 % had personal history of atopy and 55 % parental history of allergy. The group of atopic children had more wheezing exacerbations and worse evolution than the group of non atopic. They also had more treatment necessities. Conclusions: The identification of young children at high risk of developing asthma could permit an early intervention before irreversible changes in the airway appeared

Introducción: Las sibilancias recurrentes son un problema frecuente en los primeros años de vida. Sin embargo, es aún dificultoso identificar cuáles de estos niños van a desarrollar asma en el futuro. Objetivos: estudiar cuáles son los factores de riesgo de desarrollar asma en un grupo de pacientes con broncoespasmo de repetición durante los primeros tres años de vida. Material y métodos: Estudio prospectivo de 60 pacientes con edad igual o inferior a los 3 años, remitidos a nuestro centro por crisis de broncoespasmo repetidas. Se recoge información acerca de: edad, sexo, antecedentes familiares y personales de atopia, exploración física y pruebas alergológicas. Asimismo, evolución clínica y respuesta al tratamiento. Resultados: De los 60 pacientes implicados en el estudio, la mayor parte eran varones y presentaron el primer episodio de broncoespasmo antes de los 6 meses de vida. El 63% tenían antecedentes personales de atopia y el 55% antecedentes familiares de alergia. Los pacientes atópicos presentaron más crisis de sibilancias y peor evolución a corto plazo que los no atópicos. Asimismo, requirieron más tratamiento de mantenimiento. Conclusiones: La identificación precoz de los niños con elevado riesgo de desarrollar asma permitiría una intervención temprana antes de que se desarrollaran cambios irreversibles en la vía aérea

One-step immunochromatography assay for detection of high-level canine serum trypsin-like immunoreactivity.

We developed a one-step immunochromatography assay kit to measure high levels of canine trypsin-like immunoreactivity (cTLI) for bedside estimation of canine pancreatitis. The serum cTLI level can be determined within 10 min by visual comparison of color strengths in the test and reference zones. The serum cTLI levels determined by this method correlate well with canine TLI-ELISA and can be classified into 3 categories: cTLI levels higher than 60 ng/ml were considered positive; 20-60 ng/ml, weakly positive; and less than 20 ng/ml, negative. Twelve dogs suspected of pancreatitis were examined using this method; 4 dogs were positive, 2 were weakly positive, and 6 were negative. This test can detect a high level of serum cTLI and a positive result in the TLIH test will provide critical information for evaluation of pancreatitis in dogs.

Development and characterization of dried blood spot materials for the measurement of immunoreactive trypsinogen.

OBJECTIVES: In response to increasing numbers of states in the US that test newborn babies for cystic fibrosis (CF), the Newborn Screening Quality Assurance Programme initiated a pilot proficiency testing programme for immunoreactive trypsinogen (IRT), the biomarker for CF. Dried blood spot specimens (DBS) were used to evaluate the performance of laboratories that screen babies for CF. METHODS: DBS were prepared from human whole blood enriched with physiologically relevant levels of IRT. Various methods of making IRT-enriched DBS were used to optimize the recovery and stability of the biomarker, including preparation of DBS from either intact or lysed red blood cells, varying the timing of IRT addition to blood before dispensing onto filter paper, adding a protease inhibitor cocktail, and treating serum with charcoal before IRT enrichment. The recovery and stability of IRT in DBS were assessed. Newborn screening laboratories were offered the opportunity to test blind-coded DBS in the pilot PT programme. RESULTS: IRT was stable in the filter paper matrix when stored for one year at either -20 degrees C or 4 degrees C. Fifty percent more IRT was recovered from DBS prepared with lysed red blood cells where the IRT was added to blood just before dispensing; however, protease inhibitors did not improve IRT recovery. CONCLUSIONS: IRT in the DBS matrix is stable and can be shipped worldwide under ambient conditions. Optimal IRT recovery was achieved by adjustment of DBS production practices. Laboratories receiving specimens accurately measured IRT by a variety of commercial and in-house methods.

Cystic fibrosis carriers have higher neonatal immunoreactive trypsinogen values than non-carriers.

Following cystic fibrosis (CF) neonatal screening implementation, a high frequency of heterozygotes has been reported among neonates with elevated immunoreactive trypsinogen (IRT) and normal sweat chloride levels. We studied the relationship between normal IRT values and CF heterozygosity: 10,000 neonates were screened for CF by IRT measurement and tested for 40 CF mutations; the 294 carriers detected were coupled with newborns negative to the same genetic testing, and the two groups' IRT levels compared. Heterozygotes had higher IRT levels than their controls (mean 35.32 vs. 27.58 microg/L, P<0.001). Even within normal trypsinogen range, the probability of being a CF carrier increases with neonatal IRT concentration.

Experimentally induced Vogt-Koyanagi-Harada disease in two Akita dogs.

We have investigated whether a Vogt-Koyanagi-Harada (VKH)-like disease can be induced in Akita dogs by immunizing them with tyrosinase related protein 1 (TRP1), and compared the alterations induced to those of Akita dogs with a spontaneously occurring disease that resembles human VKH disease. Two Akita dogs were immunized with a peptide mixture of human TRP1. The changes in the eyes were followed by slit-lamp biomicroscopy, ophthalmoscopy, and fluorescein angiography (FA). The eyes, skin, and brains were studied by standard histological methods at about 20 months after the first immunization in one dog (dog 1), and at 3 weeks after the second immunization in the second dog (dog 2). Both dogs developed chorioretinal disease 3-4 weeks after the first immunization. Many inflammatory cells infiltrated into the anterior chamber and anterior vitreous. The fundus showed geographic, multifocal exudative retinal detachments. Multifocal leakages of fluorescein were detected from the choroid. Histologically, exudative retinal detachment was present, and inflammatory cells were seen in the subretinal space in the eyes of dog 2 taken three weeks after the second immunization. The choroid was thickened by the infiltration of inflammatory cells in some lesions. Dalen-Fuchs nodules were seen in the eye of dog 2. Depigmentation, pigment dispersion, and infiltration of many inflammatory cells around hair follicles and vessels were seen in the skin taken three weeks post-immunization. The clinical course and changes in the eyes and skin were very similar to those seen in the Akita dogs with spontaneously occurring VKH disease. We concluded that a VKH-like disease had been induced in these dogs, and this supports the tentative conclusion that the spontaneously occurring chorioretinal disease in Akita dogs is VKH disease.