Pilot study for cystic fibrosis neonatal screening: the Cuban experience.

Background In Cuba, no screening program for cystic fibrosis (CF) has been implemented yet. The ultramicro enzyme-linked immunosorbent assay (UMELISA)® TIR NEONATAL has been developed for the measurement of immunoreactive trypsin (IRT) in dried blood spots on filter paper. The analytical performance of the kit was evaluated in the national network of laboratories. Methods Newborn dried blood samples (DBS) were evaluated in 16 laboratories. An IRT/IRT/DNA protocol was followed using a cut-off value of 50 ng/mL. The mean, median and percentiles of the distribution were calculated and a two-sample t-test with unequal variance was used for statistical analysis. Influence of perinatal factors on IRT levels was analyzed. Results From January to June 2018, 6470 newborns were studied, obtaining a mean IRT value of 12.09 ng/mL (ranging 0-358 ng/mL) and a median of 8.99 ng/mL. Fifty-two samples (0.78%) were above the cut-off level and 16 samples (0.24%) were elevated in the re-screening process. One of them was confirmed positive by molecular biology (phe508del/c.3120 + 1G > A), constituting the first newborn screened and diagnosed early in Cuba. Second DBS samples were collected on average at 14 days and processed in the laboratory at 16 days of birth. Significant differences were observed (p < 0.05) when evaluating the influence of gender, birth weight (BW) and gestational age (GA) on the IRT values. Lower IRT concentrations were found in samples processed after 10 days of collection. Conclusions The performance of UMELISA® TIR NEONATAL in the laboratories has been satisfactory; hence CF newborn screening (NBS) was extended throughout the country from January 2019.

Biochemical characterization of recombinant Penaeus vannamei trypsinogen.

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.4), which are digestive serine proteases. Despite knowing the properties of trypsins from Pacific white shrimp, Penaeus vannamei, the biochemical properties of shrimp trypsinogens including activation mechanisms and kinetics are unknown, due to difficulties isolating them from natural sources. In the present work, we describe the purification and biochemical characterization of four trypsinogen-like isoforms from recombinant P. vannamei trypsinogen, with a special emphasis on understanding its activation kinetics. The major trypsinogen-like isoform had an apparent molecular mass of 29 kDa. The other three forms of recombinant trypsinogen were: an N-glycosylated form of 32 kDa, a possibly O-glycosylated form of 41 kDa, and a likely double-chain form with a subunit of 23 kDa. The autoactivation profile of three-recombinant trypsinogen-like isoforms showed increased trypsin activity at a rate that was higher than that of bovine trypsinogen. This confirms the hypothesis proposed in the literature of a rapid trypsinogen autoactivation in the absence of aspartates in the activation peptide as it is for P. vannamei trypsinogen.

Ontogeny changes and weaning effects in gene expression patterns of digestive enzymes and regulatory digestive factors in spotted rose snapper (Lutjanus guttatus) larvae.

The study of digestive physiology is an important issue in species that have been introduced in aquaculture like the spotted rose snapper (Lutjanus guttatus). The aims of this study were to describe the expression of digestive enzymes (trypsinogen, chymotrypsinogen, α-amylase, lipoprotein lipase, phospholipase A and pepsinogen) and their relation with orexigenic (neuropeptide Y, NPY) and anorexigenic (cholecystokinin, CCK) factors during the larval development and to evaluate the effect of weaning in their expression. The results showed that the transcripts of all the assayed digestive enzymes, with the exception of pepsinogen, and NPY and CCK were already present in L. guttatus from the hatching stage. The expression of all the enzymes was low during the yolk-sac stage (0-2 days after hatching, DAH), whereas after the onset of exogenous feeding at 2 DAH, their expression increased and fluctuated throughout larval development, which followed a similar pattern as in other marine fish species and reflected changes in different types of food items and the progressive maturation of the digestive system. On the other hand, weaning of L. guttatus larvae from live prey onto a microdiet between 25 and 35 DAH significantly affected the relative expression of most pancreatic digestive enzymes during the first weaning days, whereas chymotrypsinogen 2 and lipoprotein lipase remained stable during this period. At the end of co-feeding, larvae showed similar levels of gene expression regardless of the diet (live prey vs. microdiet), which indicated that larvae of L. guttatus were able to adapt their digestive capacities to the microdiet. In contrast, feeding L. guttatus larvae with live feed or microdiet did not affect the expression of CCK and NPY. The relevance of these findings with regard to current larval rearing procedures of L. guttatus is discussed.

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.

Invertebrate trypsins: a review.

Food protein hydrolysis, a crucial step in digestion, is catalyzed by trypsin enzymes from the digestive apparatus of invertebrates. Trypsin appeared early in evolution and occurs in all phyla and, in the digestive systems of invertebrates, it became the most abundant proteinase. As in vertebrates, invertebrate trypsin is also present in several forms (isoenzymes). Its physiological importance in food protein digestion in several invertebrate species has emerged with compelling evidence; and several other physiological functions, such as regulation of digestive functions, are now settled. Recent advances in the knowledge of invertebrate trypsin synthesis, regulation, genetics, catalytic characteristics; structure, evolution, as well as inhibition, especially in non-Drosophilidae insects and in some crustaceans are reviewed. Most of the existing information is largely based on the use of several tools, including molecular techniques, to answer many still open questions and solve medical, agricultural, and food quality problems.

Pancreatic response of rats fed genetically modified soybean.

Mice fed genetically modified (GM) soybean were not affected in nutritional performance, but pancreatic microscopic features were disturbed. The mechanisms for these contradictory findings are unknown. This study analysed the histology of acinar pancreatic cells and the expression of pancreatitis-associated protein (PAP) and trypsinogen mRNA in rats fed GM soy protein. Two bioassays were run, each one with 34 Wistar rats distributed into two groups fed with non-GM or GM-soy protein (18% protein) for 0, 1, 3, 5, 15 and 30 days. Nutritional evaluation, plasma amylase levels, pancreatic histological analysis and quantification of PAP and trypsinogen mRNAs levels using quantitative real-time RT-PCR were done. No differences in nutritional performance among rats fed non-GM and GM diets were found. The GM, but not the non-GM, diet induced zymogen-granule depletion after 15 days feeding, returning to normal levels after 30 days (P < 0.05). Acinar disorganization started as early as 5 days after initiation of the GM diet and it recovered after 30 days. Levels of PAP mRNA significantly increased in the GM diet between day 1 and day 3 and decreased to the basal level by day 15. Trypsinogen mRNA peaked at two different times; at day 1 and at day 15, decreasing to basal levels after 30 days. Plasma amylase levels remained unchanged at all times. This indicates that GM soy protein intake affected pancreas function, evidenced by the early acute PAP mRNA increased levels and pancreas cellular changes followed by recuperation of acinar cells after 30 days.

CFTR, PRSS1 and SPINK1 mutations in the development of pancreatitis in Brazilian patients.

CONTEXT: Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), in cationic trypsinogen (PRSS1) and in serine protease inhibitor Kazal type 1 (SPINK1) genes have been associated with chronic pancreatitis (alcohol related, idiopathic and hereditary). However, the inheritance pattern is still not clear. PATIENTS: Eighty-two unrelated Brazilian patients with chronic pancreatitis (alcohol-related disease in 64, idiopathic disease in 16, and hereditary disease in 2). Two hundred unrelated individuals with an ethnic distribution comparable to the patients were studied as controls. MAIN OUTCOME MEASURE: Detection of mutations in CFTR, PRSS1, and SPINK1 genes. RESULTS: Mutations in the CFTR gene were found in 8 patients (9.8%) with chronic pancreatitis, 5 of them with idiopathic disease. Interestingly, the only clinical symptom in a male patient in the alcoholic group, who was a compound heterozygote (DeltaF508/R170C) for two CFTR mutations, was pancreatitis without infertility or pulmonary involvement. In the PRSS1 gene, the E79K change in exon 3 was found in one patient (1.2%) with alcohol-related chronic pancreatitis. Four different alterations were identified in the SPINK1 gene. CONCLUSIONS: Mutations in the CFTR gene represent the major cause of idiopathic chronic pancreatitis in Brazilian patients. No mutation was found in the PRSS1 gene among our patients suggesting further genetic heterogeneity for hereditary and idiopathic chronic pancreatitis. Interestingly, the most frequent SPINK1 N34S mutation was not present in patients or controls. Moreover, the -253C allele for the SPINK1 gene was significantly more frequent in patients than controls (P=0.004), suggesting that it might represent a risk factor for the development of pancreatitis in our population.

Serum pancreatic enzymes define the pancreatic phenotype in patients with Shwachman-Diamond syndrome.

OBJECTIVE: To evaluate the role of serum enzymes for defining the pancreatic phenotype in Shwachman-Diamond syndrome (SDS), an inherited multisystem condition. STUDY DESIGN: Serum pancreatic trypsinogen and isoamylase were measured in 164 patients known or presumed to have SDS. The diagnosis was confirmed in 90 patients. Among 74 unconfirmed cases, 35 ("probable SDS") had hematologic dysfunction but lacked documented pancreatic dysfunction, whereas 39 patients ("improbable SDS") lacked both documented pancreatic and hematologic dysfunction. Classification and regression tree (CART) analysis was performed in 90 patients with SDS and 134 control patients to establish a rule for defining the pancreatic phenotype of SDS; the rule was then applied to the patients with unconfirmed diagnosis. RESULTS: In the control patients, serum trypsinogen showed little variation with age, whereas serum isoamylase values rose from birth on, attaining adult values by 3 years. For patients with SDS, serum trypsinogen values were low in young patients and tended to increase with age, whereas serum isoamylase values remained low at all ages. The CART rule combined results from both enzymes and classified the pancreatic phenotype in all but one SDS patient, who was <3 years of age. Excluding patients <3 years of age, CART identified the pancreatic phenotype in 82% and 7% of the "probable SDS" and "improbable SDS" cases, respectively. CONCLUSIONS: Serum pancreatic enzymes are useful for determining the pancreatic phenotype and confirming the diagnosis of SDS.

Classification of chronic pancreatitis including the genetics of trypsin SPINK1 and CFTR

Chronic pancreatitis is not one disease; it is a pathway of injury and fibrosis driven by many factors. The role of genetics is emerging as a major factor, both in terms of understand pathogenesis, and in terms of identifying risk factors and mechanisms. Use of the TIGAR-O classification system, or others will be useful research tools to determine gene-gene, gene-environment, and other interactions that contribute to development of an otherwise mysterious disease

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).

Changes in pancreatic trophism and gene expression during a prolonged fasting period in rats.

To examine the effects of fasting on trophism and gene expression in pancreas, adult male rats were deprived of food from 0-6 d. Total DNA, RNA, and proteins, and specific mRNAs for rat amylase, chymotrypsinogen B, trypsinogen I, proinsulin I, and actin (assessed by employing cloned cDNAs and dot-blot hybridization) were quantitated in pancreas. Body and pancreatic wt diminished progressively to reach 65 and 75% of initial values at the 6th d of fasting. Protein/DNA and total RNA/DNA ratios decreased 2.04 and 2.31-fold, respectively, during 6 d of fasting. The concentration of amylase, chymotrypsinogen B, trypsinogen I, and actin mRNA, expressed as cpm/microgram RNA, decreased significantly throughout the study period, whereas the decrease observed in Proinsulin I mRNA concentration was not significantly different. When mRNA concentrations were refereed to the total content of DNA, however, the decrease was significant for all messengers tested. It is concluded that the prolonged absence of nutrients in the digestive tract exerts negative trophic influence on pancreas and triggers differential changes in pancreatic gene expression. These changes are gradual, asynchronic, and nonparallel.