Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip.

The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed.

Soluble plantain nonstarch polysaccharides, although increasing caecal load, reduce systemic invasion of Salmonella Gallinarum in the chicken.

UNLABELLED: Soluble plantain (Musa paradisiaca) nonstarch polysaccharides (NSPs) have previously been shown to prevent pathogenic interaction with the intestinal epithelium. Here, we examined whether plantain NSP could prevent the invasion of the intestinal mucosa by Salmonella enterica Gallinarum, a causative agent of fowl typhoid. In vitro assays using B1OXI cells were performed with monolayers pretreated with/without plantain NSP, before inoculation with Salm. Gallinarum 287/91. Chicks were fed from hatch on a pellet diet containing 50 mg day(-1) plantain NSP, followed by oral inoculation with Salm. Gallinarum 287/91 at the age of 6 days. Bacteria were enumerated from the liver, spleen and caecal contents 3 days postinfection. Adhesion and invasion of Salm. Gallinarum to B1OXI cells were inhibited by 10 mg ml(-1) plantain NSP (reduction in invasion 52%; 95% CI 27-77; P < 0.05). In vivo diet supplemented with 50 mg day(-1) plantain NSP reduced the invasion of Salm. Gallinarum in the chick liver (control 4.05 Log10 CFU g(-1) , SE 0.28, vs plantain 2.07 Log10 CFU g(-1) , SE 0.65; P < 0.01) and nonsignificantly in the spleen. Conversely, CFUs were significantly increased in the caeca (control 1.27 Log10 CFU g(-1), SE 0.65, vs plantain 6.04 Log10 CFU g(-1) , SE 0.17; P < 0.001). Plantain NSP feed reduced the systemic infection by Salm. Gallinarum and may have potential in reducing the impact of the disease in avian salmonellosis. The caveat is the increased caecal load of Salm. Gallinarum, although this may reflect both the reduced intestinal invasion and the bacterial multiplication in the caeca. SIGNIFICANCE AND IMPACT OF THE STUDY: Nonstarch polysaccharide (NSP) derived from the plantain (Musa paradisiaca) inhibits interaction with epithelial cells by Salmonella enterica Gallinarum, a causative agent of the disease fowl typhoid. Incorporation of plantain NSP into the poultry feed reduced Salm. Gallinarum levels in the spleen and liver of chicks following experimental infection, although their numbers in the caeca increased. These data demonstrate that alternatives to antimicrobials such as NSP may be useful in the control of invasive salmonellosis in poultry.

Comparative toxinology of Loxosceles reclusa and Corynebacterium pseudotuberculosis.

In contrast to other kinds of phospholipases, phospholipases D that are toxic for humans and animals are not commonly encountered as constituents of venoms or as products of pathogenic microorganisms. Toxic phospholipases D are present, however, in the venom of the brown recluse spider (Loxosceles reclusa) and in supernatants or filtrates of cultures of Corynebacterium pseudotuberculosis. Although the two enzyme toxins are derived from phylogenetically disparate entities, they are similar in molecular weight, charge, substrate specificity, and in several biological activities. They are immunologically distinguishable.

Food additives: Assessing the impact of exposure to permitted emulsifiers on bowel and metabolic health - introducing the FADiets study.

Emulsifiers are common components of processed foods consumed as part of a Western diet. Emerging in vitro cell-line culture, mouse model and human intestinal tissue explant studies have all suggested that very low concentrations of the food emulsifier polysorbate 80 may cause bacterial translocation across the intestinal epithelium, intestinal inflammation and metabolic syndrome. This raises the possibility that dietary emulsifiers might be factors in conditions such as coronary artery disease, type 2 diabetes and Crohn's disease. The potential mechanism behind the observed effects of this emulsifier is uncertain but may be mediated via changes in the gut microbiota or by increased bacterial translocation, or both. It is also unknown whether these effects are generalisable across all emulsifiers and detergents, including perhaps the natural emulsifier lecithin or even conjugated bile acids, particularly if the latter escape reabsorption and pass through to the distal ileum or colon. A major objective of the Medical Research Council (MRC)-funded Mechanistic Nutrition in Health (MECNUT) Emulsifier project is therefore to investigate the underlying mechanisms and effects of a range of synthetic and natural emulsifiers and detergents in vitro and in vivo, and to determine the effects of a commonly consumed emulsifier (soya lecithin) on gut and metabolic health through a controlled dietary intervention study in healthy human volunteers - the FADiets study. This report provides an overview of the relevant literature, discussing the impact of emulsifiers and other additives on intestinal and metabolic health, and gives an overview of the studies being undertaken as part of the MECNUT Emulsifier project.

Phylogeny and natural history of the primate lentiviruses, SIV and HIV.

Studies of primate lentivirus phylogeny over the past decade have established a minimum of five related, but genetically distinct, groups of simian immunodeficiency virus (SIV), each originating from a different African primate species. The hypothesis that HIV-2 (and SIVmac) arose by cross-species transmission from sooty mangabeys (Cercocebus atys has been strengthened by a more detailed characterization of the SIVsm/SIVmac/HIV-2 group of viruses. SIV from all four subspecies of African green monkeys (SIVagm) have been characterized with an apparent chimeric genome structure of SIVagm from West African green monkeys. Although these naturally infected primates remain healthy, cross-species transmission to other primate species may result in immunodeficiency, as caused by SIVsm infection of macaque monkeys (Macaca sp.) and recently, SIVagm infection of pig-tailed macaques (M. nemestrina). Studies of variation within infected individuals have been facilitated by adaptation of the techniques of heteroduplex analysis and single-stranded conformational polymorphism of PCR generated fragments.

Health human resource planning in Barbados and the eastern Caribbean states: a matter of sustainability.

Health and Human Resources (HHR) are very important issues to be considered in healthcare services. While various factors may be of greater significance in one area depending on resources, priorities and stage of economic development, a robust HHR plan is important in all cases. There are many factors such as demographic shifts, changing delivery models, consumer expectations, global shortages and financial restraints that must be considered in proper HHR planning. This manuscript summarizes some of the factors that should be considered and some of the short comings of current HHR planning approaches. Based on our review and experience, we developed a framework for HHR planning and apply the framework to Barbados to try to identify the existing challenges and issues and potential areas for staff and training investments.

Direct demonstration of increased expression of Thomsen-Friedenreich (TF) antigen in colonic adenocarcinoma and ulcerative colitis mucin and its concealment in normal mucin.

Increased binding of the lectin peanut agglutinin is a common feature in epithelial malignancy and hyperplasia. This may have considerable functional importance in the intestine by allowing interaction between the epithelium and mitogenic lectins of dietary or microbial origin. Peanut agglutinin binds the disaccharide Thomsen-Friedenreich (TF, T or core 1) blood group antigen, Gal beta (1-3) GalNAc alpha-, but is not totally specific for this site. Consequently, there has been controversy about the presence of this structure in colon cancer; studies with anti-TF monoclonal antibodies have failed to detect it. We have examined the presence of TF antigen in colonic mucus glycoprotein (mucin) using endo-alpha-N-acetylgalactosaminidase (O-Glycanase), which specifically catalyzes the hydrolysis of TF antigen from glycoconjugates. Samples of adenocarcinoma, inflammatory bowel disease (ulcerative colitis), and normal mucin were treated with O-glycanase, the liberated disaccharide was separated from the glycoprotein and analyzed using dual CarboPac PA-100 column high performance anion-exchange chromatography coupled with pulsed amperometric detection. O-Glycanase treatment released increased amounts of TF antigen from both colonic adenocarcinoma (8.0 +/- 3.9 ng/micrograms protein, n = 11; P < 0.0001 ANOVA) and ulcerative colitis mucin (3.3 +/- 0.3 ng/micrograms protein, n = 5; P = 0.04) compared with mucin samples from histologically normal mucosa distant from carcinoma (1.5 +/- 1.1 ng/micrograms protein, n = 9). However, after mild acid treatment to remove sialic acids and fucose, releasable TF antigen was increased in all nine of these histologically normal mucin samples (5.5 +/- 2.6 ng/micrograms protein, P < 0.0002). We conclude that TF antigen is an oncofetal antigen which is expressed in colon cancer, but is concealed by further glycosylation (sialylation and/or fucosylation) in the normal colonic mucosa.

Technical note: Instantaneous sampling intervals validated from continuous video observation for behavioral recording of feedlot lambs.

When considering methodologies for collecting behavioral data, continuous sampling provides the most complete and accurate data set whereas instantaneous sampling can provide similar results and also increase the efficiency of data collection. However, instantaneous time intervals require validation to ensure accurate estimation of the data. Therefore, the objective of this study was to validate scan sampling intervals for lambs housed in a feedlot environment. Feeding, lying, standing, drinking, locomotion, and oral manipulation were measured on 18 crossbred lambs housed in an indoor feedlot facility for 14 h (0600-2000 h). Data from continuous sampling were compared with data from instantaneous scan sampling intervals of 5, 10, 15, and 20 min using a linear regression analysis. Three criteria determined if a time interval accurately estimated behaviors: 1) ≥ 0.90, 2) slope not statistically different from 1 ( > 0.05), and 3) intercept not statistically different from 0 ( > 0.05). Estimations for lying behavior were accurate up to 20-min intervals, whereas feeding and standing behaviors were accurate only at 5-min intervals (i.e., met all 3 regression criteria). Drinking, locomotion, and oral manipulation demonstrated poor associations () for all tested intervals. The results from this study suggest that a 5-min instantaneous sampling interval will accurately estimate lying, feeding, and standing behaviors for lambs housed in a feedlot, whereas continuous sampling is recommended for the remaining behaviors. This methodology will contribute toward the efficiency, accuracy, and transparency of future behavioral data collection in lamb behavior research.

Molecular cloning of sheep lung dipeptidase: a glycosyl phosphatidylinositol-anchored ectoenzyme that converts leukotriene D4 to leukotriene E4.

Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.

Partial purification and characterization of the antidiuretic hormone-inactivating enzyme from renal plasma membranes.

An antidiuretic hormone-inactivating peptidase located in renal plasma membranes of porcine kidney medulla has been studied. Treatment of antidiuretic hormone (lysine vasopressin) with renal plasma membranes resulted in a progressive loss of biological activity as measured by the rat pressor assay. The reaction of 2,4,6-trinitrobenzenesulfonic acid with released amino groups was employed to follow the peptidase-catalyzed hydrolysis of the hormone. An 83-fold purification of the membrane-bound peptidase was achieved by Lubrol PX solubilization of the membranes followed by DEAE-cellulose, hydroxylopatite, and 8% agarose column chromatography. The molecular weight of the peptidase was 442 000 as determined by 8% agarose gel filtration. An analysis of the antidiuretic hormone hydrolysis products by thin-layer chromatography revealed the presence of trinitrophenyl-glycinamide. The release of glycinamide from the hormone as a function of time was demonstrated. Mg2+ had a slight inhibitory effect and Ca2+ had a strong inhibitory effect on the peptidase activity.

Specificity and inhibition studies of human renal dipeptidase.

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa.

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.

Isolation, sequence and biosynthetic significance of a novel fragment of gastrin-releasing peptide from chicken proventriculus.

The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.

Bioconversion of leukotriene D4 by lung dipeptidase.

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Review article: dietary fibre-microbiota interactions.

BACKGROUND: Application of modern rapid DNA sequencing technology has transformed our understanding of the gut microbiota. Diet, in particular plant-based fibre, appears critical in influencing the composition and metabolic activity of the microbiome, determining levels of short-chain fatty acids (SCFAs) important for intestinal health. AIM: To assess current epidemiological, experimental and clinical evidence of how long-term and short-term alterations in dietary fibre intake impact on the microbiome and metabolome. METHODS: A Medline search including items 'intestinal microbiota', 'nutrition', 'diet', 'dietary fibre', 'SCFAs' and 'prebiotic effect' was performed. RESULTS: Studies found evidence of fibre-influenced differences in the microbiome and metabolome as a consequence of habitual diet, and of long-term or short-term intervention (in both animals and humans). CONCLUSIONS: Agrarian diets high in fruit/legume fibre are associated with greater microbial diversity and a predominance of Prevotella over Bacteroides. 'Western'-style diets, high in fat/sugar, low in fibre, decrease beneficial Firmicutes that metabolise dietary plant-derived polysaccharides to SCFAs and increase mucosa-associated Proteobacteria (including enteric pathogens). Short-term diets can also have major effects, particularly those exclusively animal-based, and those high-protein, low-fermentable carbohydrate/fibre 'weight-loss' diets, increasing the abundance of Bacteroides and lowering Firmicutes, with long-term adherence to such diets likely increasing risk of colonic disease. Interventions to prevent intestinal inflammation may be achieved with fermentable prebiotic fibres that enhance beneficial Bifidobacteria or with soluble fibres that block bacterial-epithelial adherence (contrabiotics). These mechanisms may explain many of the differences in microbiota associated with long-term ingestion of a diet rich in fruit and vegetable fibre.

5-amino salicylic acid absorption and metabolism in ulcerative colitis patients receiving maintenance sulphasalazine, olsalazine or mesalazine.

BACKGROUND: All 5-aminosalicylic acid (5-ASA) preparations are potentially nephrotoxic, but there has been concern that newer delivery systems may increase this risk, either because of altered absorption or altered metabolism. Previous studies of 5-ASA absorption and excretion have usually either been performed in healthy controls or have only examined short-term therapy. 5-ASA and N-acetyl-5-ASA have therefore been measured in blood samples, and N-acetyl-5-ASA in urine samples, from patients with ulcerative colitis on long-term maintenance with different 5-ASA preparations and compared with sensitive markers of renal damage. METHODS: Patients receiving mesalazine (Asacol) (n = 13), sulphasalazine (n = 12) or olsalazine (Dipentum) (n = 8), all at doses within the recommended range were studied. Six-hour and trough serum concentrations of 5-ASA and N-acetyl-5-ASA and 24-h urinary excretion of N-acetyl-5-ASA were measured by high-performance liquid chromatography. RESULTS: Absorption of 5-ASA, assessed as 24-h excretion of N-acetyl-5-ASA expressed as molar % of ingested dose, was greater in patients receiving mesalazine, 23.25 +/- 10.65% (mean +/- s.d.; n = 13), than those receiving sulphasalazine (11.16 +/- 10.52%, n = 12; P = 0.003) or olsalazine (9.70 +/- 3.89%, n = 8; P < 0.002). The ratio of 5-ASA: N-acetyl-5-ASA in the serum 6 h after dose was also greater with mesalazine (1.02 +/- 0.44, mean +/- s.d.) than sulphasalazine (0.54 +/- 0.44, P < 0.02) or olsalazine (0.38 +/- 0.44, P < 0.005). Urinary markers of tubular damage were increased in four of 33 patients, but showed no correlation with concentration of 5-ASA or N-acetyl-5-ASA in serum and N-acetyl-5-ASA in urine, nor with lifetime dose or average daily dose of 5-ASA. CONCLUSIONS: In patients with ulcerative colitis receiving maintenance 5-ASA therapy there was greater absorption and less acetylation of 5-ASA from mesalazine (Asacol) compared with sulphasalazine or olsalazine, but no evidence from this study that this resulted in increased nephrotoxicity.

The mechanism of action of gastrin releasing peptide (GRP) in stimulating avian gastric acid secretion.

The mechanisms of action of gastrin and gastrin releasing peptide (GRP) in stimulating gastric acid secretion were examined in the anaesthetized turkey. Gastrin and GRP produced dose-dependent increases in acid secretion that were inhibited by the gastrin/CCK-B antagonist CI988. The antagonist did not affect the acid secretory responses to histamine or carbachol. A GRP antagonist (M216140) inhibited the acid response to GRP, but not gastrin. The results suggest that in birds, GRP stimulates acid secretion by release of gastrin, which acts in turn on classical gastrin/CCK-B receptors in the proventriculus.

Inhibition of food intake by omeprazole in the chicken.

Chickens treated with the H+/K+ ATPase inhibitor omeprazole, to inhibit gastric acid secretion, failed to gain weight and showed decreased food intake compared with controls. The gastrin antagonist PD134308 reversed the action of omeprazole on food intake. Since exogenous gastrin decreased food intake, and since omeprazole increased plasma gastrin concentrations, the results suggest that elevated plasma gastrin in chicken exerts a satiety effect.

Change in injuries associated with safety belt laws.

Statewide crash data bases from nine states were subjected to time series analyses to detect changes in injuries associated with onset of seat belt laws in the respective states. In each of 18 analyses involving drivers covered by the law observed casualties were below the number forecast on the basis of prior experience and assuming that no law had been enacted. In the case of others, not covered by the law, observed injuries were equally often above or below forecast. Relative to covered drivers not only were the numbers below forecast, but in 12 of the 18 instances there was a statistically significant indication of an abrupt decrease the month the law began.

Evaluating the North Carolina safety belt wearing law.

The North Carolina Seat Belt Law required an evaluation of the effectiveness of the act with a report of the findings to the Legislature three years after the law went into effect. This paper addresses changes in statewide belt usage and in occupant injury associated with that law. Observational data collected bimonthly from a probability sample of 72 sites stratified by geographic region, rural/urban location, road type, and time of day show that belt use rose from a baseline rate of 25% to a warning ticket phase rate of 45%. Belt use then reached 78% upon enforcement and is now nearly 64%. Time series analysis showed that statistically significant reductions in percentages of moderate and serious injuries occurred at the beginning of both the warning ticket and the enforcement phases. Forecasts of injuries and deaths were also developed from the time series models and were compared with observed totals. Warning tickets brought about a modest 5.4% reduction in serious injuries; fatalities among occupants covered by the law showed no change. In contrast, the subsequent enforcement phase saw a reduction of 11.6% in fatalities and 14.6% in serious or worse injuries. This represents an estimated annual savings of 131 lives and over 2,300 serious injuries in North Carolina during the 18 months following onset of enforcement.