MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

Activation of the mitochondrial caspase pathway and subsequent calpain activation in monkey RPE cells cultured under zinc depletion.

PURPOSE: Decreased zinc levels in the macula are reported in patients with age-related macular degeneration, and the zinc chelator N,N,N',N'-tetrakis (2- pyridylmethyl) ethylenediamine) (TPEN) causes death of human retinal pigment epithelial (RPE) cells. The purpose of the present study was to investigate signal transduction pathways during cell death initiated by TPEN, using monkey RPE cells. METHODS: RPE cells were cultured with TPEN. Activation of calpains and caspases, and proteolysis of their substrates were detected by immunoblotting. Incubation of calpain inhibitor SNJ-1945 or caspase inhibitor z-VAD-fmk was used to confirm activation of specific proteases. RESULTS: TPEN caused a time-dependent decrease in viable RPE cells. Cell death was accompanied by activation of calpain-1, caspase-9, and caspase-3. SNJ-1945 inhibited calpain activation and slightly inhibited caspase-9 activation. z-VAD-fmk inhibited caspases and calpain-1 activation. TPEN did not activate caspase-12. CONCLUSIONS: Relative zinc deficiency in RPE cells causes activation of cytosolic calpain and mitochondrial caspase pathways without ER stress.

Prospective, long-term study of fat-soluble vitamin status in children with cystic fibrosis identified by newborn screen.

OBJECTIVE: To prospectively evaluate the biochemical status of vitamins A, D, and E in children with cystic fibrosis (CF). SUBJECTS: A total of 127 infants identified by the Colorado CF newborn screening program. DESIGN: Vitamin status (serum retinol, 25-hydroxy vitamin D, ratio of alpha-tocopherol/total lipids) and serum albumin were assessed at diagnosis (4 to 8 weeks), ages 6 months, 12 months, and yearly thereafter, to age 10 years. RESULTS: Deficiency of 1 or more vitamins was present in 44 (45.8%) of 96 patients at age 4 to 8 weeks as follows: vitamin A 29.0%, vitamin D 22.5%, and vitamin E 22.8%. Of these patients with initial deficiency, the percent that was deficient at 1 or more subsequent time points, despite supplementation, was vitamin A 11.1%, vitamin D 12.5%, and vitamin E 57.1%. Of the initial patients with vitamin sufficiency, the percent who became deficient at any time during the 10-year period was as follows: vitamin A 4.5%, vitamin D 14.4%, and vitamin E 11.8%. The percent of patients deficient for 1 or more vitamins ranged from 4% to 45% for any given year. CONCLUSIONS: Despite supplementation with standard multivitamins and pancreatic enzymes, the sporadic occurrence of fat-soluble vitamin deficiency and persistent deficiency is relatively common. Frequent and serial monitoring of the serum concentrations of these vitamins is therefore essential in children with CF.

Infants and young children with cystic fibrosis have high levels of serum sialyl Lewis(a) antigen.

Previous studies suggest that serum levels of the mucin-associated sialyl Lewis(a) (Le(a)) antigen (NeuAC alpha 2-3 Gal beta 1-3 [Fuc alpha 1-4]GlcNAc beta 1...) correlate with the pulmonary status of cystic fibrosis (CF) patients who have Le(a) or Lewis(b) blood types and can form the antigen. However, there is little information on serum sialyl Le(a) antigen levels in CF patients or normal children younger than 9 y. We measured serum antigen levels using the MAb 19-9 in normal term neonates, CF infants and young children, and infants and children who had bronchopulmonary dysplasia or asthma. The mean serum sialyl Le(a) antigen level of the CF patients was 46.7 U/mL, significantly above (p < 0.01) the mean levels of the three other groups. The mean serum sialyl Le(a) levels of the three non-CF groups were not significantly different from one another or from published normal values. We conclude that serum sialyl Le(a) antigen levels are elevated early in CF, but are normal in asthma and bronchopulmonary dysplasia patients.

Clinical evaluation of the macroduct sweat collection system and conductivity analyzer in the diagnosis of cystic fibrosis.

The purposes of this study were to compare sweat tests used in diagnosing cystic fibrosis (CF), as performed with the Macroduct collection system, with those utilizing the more laborious quantitative pilocarpine iontophoresis test (QPIT), and to ascertain the efficacy of the Sweat-Chek conductivity analyzer in eliminating some possibly unnecessary chloride analyses. A Macroduct sweat test was performed on one arm and a QPIT on the other on 1090 patients, 93 of whom had CF. Of these, 514 patients (43 with CF) also had a conductivity determination on the Macroduct sweat sample. All subjects were referred to the laboratory of one of us (K.B.H.) for sweat testing. Of the QPIT samples, 0.7% were inadequate, as were 6.1% of those from the Macroduct system. When sodium and chloride concentrations from the two tests were compared, the standard errors of the estimate were 3.90 and 3.85, respectively. Agreement within 8 mEq/L could then be expected with 95% confidence limits. With use of the Sweat-Chek analyzer, no patient with CF was found to have a conductivity of less than 90 mmol/L, whereas 430 (91%) of the non-CF subjects had a conductivity of less than 50 mmol/L. None of those 430 subjects had a sweat chloride value > 32 mmol/L. We conclude that the Macroduct collection system provides results equally as satisfactory as those provided by the QPIT and that the Sweat-Chek analyzer frequently eliminates the necessity of measuring chloride concentrations.

Pancreatic insufficiency, growth, and nutrition in infants identified by newborn screening as having cystic fibrosis.

To evaluate the impact of early pancreatic insufficiency on growth and nutritional status in cystic fibrosis, we studied 49 infants identified by a newborn screening program. Pancreatic insufficiency, determined by increased 72-hour fecal fat excretion, was present in 59% (23/39) of infants at diagnosis (7.0 +/- 0.8 weeks; mean +/- SEM). Before initiation of pancreatic enzyme replacement, growth and nutritional status of pancreatic-insufficient (n = 16) and pancreatic-sufficient (n = 13) infants were compared. Pancreatic-insufficient infants gained less weight from birth to diagnosis (13.4 +/- 3.4 vs 22.3 +/- 4.0 gm/day; p = 0.05), had decreased triceps skin-fold thicknesses (4.5 +/- 0.3 vs 6.1 +/- 0.4 mm; p less than 0.005), and had lower blood urea nitrogen (3.07 +/- 0.42 vs 4.62 +/- 0.65 mg/dl; p = 0.02) and albumin (2.99 +/- 0.14 vs 3.54 +/- 0.14 gm/dl; p less than 0.01) levels despite higher gross calorie (154 +/- 8 vs 116 +/- 13 kcal/kg per day; p less than 0.01) and protein intakes (2.81 +/- 0.21 vs 2.14 +/- 0.33 gm/kg per day; p = 0.03). Fecal nitrogen loss was correlated with fat loss (r = 0.79; p less than 0.001). Fat malabsorption was present in 79% (30/38) and 92% (33/36) of infants tested at 6 months and 12 months of age, respectively, indicating that pancreatic insufficiency persists and increases in frequency throughout infancy. We conclude that pancreatic insufficiency is prevalent in young infants with cystic fibrosis and has a significant impact on growth and nutrition.

Efficacy of statewide neonatal screening for cystic fibrosis by assay of trypsinogen concentrations.

BACKGROUND: To evaluate the feasibility and efficacy of measuring immunoreactive trypsinogen in blood to screen for cystic fibrosis, we performed this test in 279,399 newborns in Colorado from 1982 to 1987. METHODS: Immunoreactive trypsinogen was measured in dried blood spots when the infants were 1 to 4 days old; if the level was elevated (greater than or equal to 140 micrograms per liter), the measurement was repeated (mean age, 38 days); if the level was again elevated, sweat testing was performed (mean age, 49 days). For the second test, two cutoff levels (120 and 80 micrograms per liter) were evaluated. RESULTS: We found an incidence of cystic fibrosis of 1 in 3827 (0.26 per 1000), with 3.2 newborns per 1000 requiring repeat measurement. When adjusted for race and compliance with testing, the incidence among the white infants (1 in 2521) was close to the expected incidence. The false positive rate with the initial cutoff level (92.2 percent) was similar to the rate found in neonatal screening programs for other diseases. False negative results occurred because of laboratory error or changes in procedure (three infants) and trypsinogen concentrations lower than the initial cutoff level (three infants) or lower than the remeasurement cutoff level of 120 micrograms per liter (one infant). Sweat tests were negative in 168 infants with an elevated initial trypsinogen level but a level below 80 micrograms per liter on remeasurement, confirming the value of 80 micrograms per liter as an appropriate cutoff for repeat-test results. Overall, 95.2 percent of the infants with cystic fibrosis (95 percent confidence interval, 85 to 99 percent) who did not have meconium ileus could be identified with the use of a trypsinogen cutoff level of 140 micrograms per liter on initial testing and 80 micrograms per liter on repeat testing. CONCLUSIONS: Statewide screening for cystic fibrosis based on measurements of immunoreactive trypsinogen in dried blood spots is feasible and can be implemented with acceptable rates of repeat testing and false positive and false negative results.

Early bacteriologic, immunologic, and clinical courses of young infants with cystic fibrosis identified by neonatal screening.

To understand better the events associated with the initiation of lung disease in young children with cystic fibrosis (CF), we prospectively performed a longitudinal study examining the early bacteriologic, immunologic, and clinical courses of 42 children with CF diagnosed after identification by neonatal screening. Serial evaluations included history and physical examination, chest radiographs, throat cultures for bacteria, and determinations of serum immunoglobulin levels and circulating immune complexes. At a mean follow-up age of 27 months, 19% of the children had serial throat cultures positive for Pseudomonas aeruginosa; the first positive culture was found at a mean age of 21 months. In three infants the initial P. aeruginosa isolates were mucoid. As determined by typing with a DNA probe, serial P. aeruginosa isolates from each patient were identical over time but were genetically distinct from isolates recovered from other patients. Of 11 infants with P. aeruginosa, nine (82%) had previous isolates of Staphylococcus aureus or Haemophilus influenzae; all had received prior antibiotic therapy. In comparison with other infants with CF, children with P. aeruginosa grown on serial throat cultures more frequently had daily cough (p less than 0.01), lower chest radiograph scores (p less than 0.05), and elevated levels of circulating immune complexes (p less than 0.01). None of the study infants had persistent hypogammaglobulinemia or hypergammaglobulinemia. We conclude that (1) S. aureus and H. influenzae remain the isolates most frequently recovered from infants with CF; (2) initial recovery of P. aeruginosa by throat culture is often preceded by the onset of chronic respiratory signs; (3) elevations of circulating immune complexes can occur early, often after the initial recovery of P. aeruginosa; and (4) early P. aeruginosa isolates are genetically distinct, demonstrating the lack of cross-colonization in this newborn population.

The rise in the total iron-binding capacity after iron overdose.

STUDY OBJECTIVES: To determine if and to what extent the total iron-binding capacity (TIBC) would increase following an iron overload, and to identify specific iron-binding proteins that might be responsible for the increased TIBC. DESIGN: A prospective laboratory investigation. SETTING: A certified regional poison control center. PARTICIPANTS: Six healthy adult male volunteers. MEASUREMENTS AND MAIN RESULTS: All volunteers ingested 20 mg/kg of elemental iron. Blood samples were drawn at hourly intervals for eight hours and analyzed for serum iron, TIBC, transferrin, ferritin, lactoferrin, glucose, bicarbonate, and WBC. Within two hours of ingestion, subjects developed symptoms of toxicity, including nausea, lightheadedness, vomiting, severe crampy abdominal pain, and voluminous diarrhea. The TIBC was statistically significantly increased at all points measured from one to six hours. Despite rising above 300 micrograms/dL in five of six subjects, the serum iron never exceeded the TIBC in any subject. Transferrin and ferritin did not increase to account for the increased TIBC. The lactoferrin levels did increase, but they did not correlate with significant increases in the TIBC. CONCLUSIONS: Twenty mg/kg of elemental iron caused clinical toxicity in this study, and after iron overload the colorimetric TIBC increased by unknown mechanisms.

Early respiratory course in infants with cystic fibrosis: relevance to newborn screening.

Respiratory morbidity and mortality during infancy are important problems in the care of CF patients whether they are diagnosed conventionally or through newborn screening. Although the mechanisms of lung disease in CF remain to be elucidated, two potential pathophysiologic mechanisms--viral infection and undernutrition--can be associated with respiratory morbidity in infancy. Colonization of some infants with Pseudomonas and the presence of early mucus casts and cytokines in bronchoalveolar lavage suggest that pathophysiologic processes that are important in later life may begin in infancy. The early respiratory abnormalities, morbidity and mortality seen in CF indicate the need for future investigations of the respiratory course and interventional trials in infancy.

Fat-soluble vitamins in infants identified by cystic fibrosis newborn screening.

Fat-soluble vitamin status was assessed in 36 infants diagnosed with cystic fibrosis by newborn screening in the Colorado Program. At the time of diagnosis of cystic fibrosis, 36% of infants were hypoalbuminemic, 21% had vitamin A deficiency, 35% had vitamin D deficiency, and 38% had vitamin E deficiency. None had vitamin K deficiency. Supplementation with pancreatic enzymes, a multiple vitamin preparation, and additional vitamin E was associated with normalization of serum albumin, retinol, and 25-hydroxyvitamin D and negative PIVKA testing at age 6 and 12 months. Several patients remained vitamin E deficient, but this was felt to be due to poor compliance. Biochemical evidence of fat-soluble vitamin deficiency is common before age 3 months in infants with CF and responds to supplementation in the first year of life.

Fat-soluble-vitamin status during the first year of life in infants with cystic fibrosis identified by screening of newborns.

We investigated the fat-soluble-vitamin status during the first year of life in 36 infants with cystic fibrosis (CF) consecutively identified by screening of newborns. At initial evaluation (at age 51.0 +/- 26.7 d) 36% of patients were hypoalbuminemic, 21% had low serum retinol, 35% had low serum 25-hydroxyvitamin D. 38% had low serum alpha-tocopherol and low ratios of serum vitamin E to total lipids, and none had elevated protein in vitamin K absence (PIVKA). Hypoalbuminemia was more common in breast-fed than in formula-fed infants. Seventy-two-hour fecal fat excretion correlated inversely with serum alpha-tocopherol. Treatment with oral pancreatic enzyme supplements, a multiple vitamin, and additional vitamin E was associated with normalization of serum albumin, retinol, and 25-hydroxyvitamin D and negative PIVKA at age 6 and 12 mo. Approximately 10% of patients remained vitamin E deficient. Biochemical evidence of fat-soluble-vitamin deficiencies is common before age 3 mo in patients with CF and, except for vitamin E, these deficiencies corrected with standard therapy.

Bilirubin binding capacity and free bilirubin concentration: fluorescence quenching compared with peroxidase oxidation and sephadex column elution techniques.

Fluorescence quenching was compared with the techniques of peroxidase oxidation and Sephadex column elution for determining free bilirubin concentrations and the capacity of albumin to bind bilirubin and assessed as to its suitability for use as a routine method in a clinical laboratory. The poor reproducibility of the fluorescence quenching method made it unacceptable and peroxidase oxidation was found to be the most satisfactory technique. The Sephadex column elution technique did not measure free bilirubin concentration but gave a good estimate of binding capacity. This method is, however, limited by the sample size required for performing the determinations.

A solution to the problem of bilirubin interference with the kinetic Jaffé method for serum creatinine.

We measured bilirubin interference with the kinetic Jaffé method for serum creatinine. Both pooled sera with added bilirubin and icteric patients' sera were used and results with of which gave more nearly "true" values.

Drugs and children: methods for therapeutic monitoring.

In this era of polypharmacy, the incidence of adverse reactions due to drug therapy has increased alarmingly since the precise effects on the metabolism of a drug given in combination with other drugs can never be predicted with certainty. Inadequate therapy due to insufficient medication or to factors which diminish absorption or enhance metabolism may be equally undesirable. The consequences to patients in terms of increased morbidity and financial cost of prolonged hospitalization may be considerable. For pediatric patients, particularly in the newborn period, these hazards may be much more dangerous. There is a need for more investigation into the validity of procedures in current use for the determination of drug levels in biologic fluids and into the interpretation of the values they produce. In addition clinical chemists and clinical pharmacologists are faced with the challenge of defining those drugs for which blood level information would be advantageous and developing rapid, sensitive, and accurate assays which can be performed by the routine clinical laboratory. The day may be not too far away when a major proportion of the workload of the clinical laboratory consists of assays primarily designed as an aid to therapy rather than diagnosis.