Escherichia coli Nissle 1917 for probiotic use in piglets: evidence for intestinal colonization.

AIMS: This study was prompted to investigate the intestinal localization and colonization of orally administered Escherichia coli Nissle 1917 (EcN) in piglets. METHODS AND RESULTS: EcN was fed to ten EcN-negative piglets (3 months) over seven consecutive days. Faecal samples were collected repeatedly and tested for EcN-DNA by a combined culture/PCR assay and for viable EcN by culture methods, respectively. EcN-DNA was detectable in faeces of all piglets within the first 24 h after it was added to the feed. After the administration of EcN had been stopped, the presence of EcN-DNA in faecal samples indicated that all piglets shedded EcN with their faeces intermittently through up to 33 days. In addition, E. coli strains indistinguishable from EcN by all markers tested (rdar colony morphotype, multiplex PCR and GEI II-PCR analyses, XbaI-pattern, K5 phage susceptibility) were isolated from faecal samples and from mucosal swabs taken at euthanasia at the end of the experiment. CONCLUSIONS: EcN colonizes the intestine and persists in conventionally reared piglets for at least 4 weeks upon oral administration. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study have implications for efficacy and safety assessments of EcN as a probiotic strain for use in pigs.

Hemoglobin A2 levels in healthy persons, sickle cell disease, sickle cell trait, and beta-thalassemia by capillary isoelectric focusing.

Simultaneously measuring major and minor hemoglobin (Hb) variants by capillary isoelective focusing, we obtained HbA2 intervals in healthy volunteers (n = 412) (reference value) and patients with HbS or beta-thalassemia. We classified normal HbA2 reference intervals into three age groups: 5 months or younger (1.2% +/- 1.5%), 6 months to 1 year (2.2% +/- 0.9%), and 1 year or older (2.4% +/- 0.9%). These intervals were comparable to those used with other methods. Patients 1 year of age or older with HbS had significantly higher HbA2 levels (sickle cell trait, 2.9% +/- 0.9%; sickle cell anemia, 2.8% +/- 1.0%; P < .05). Although reference HbA2 intervals overlapped those in patients with HbS, no overlap in HbA2 levels was noted between these groups and patients with beta-thalassemia (observed range, 4.3% to 7.5%). The higher than normal HbA2 interval in patients with HbS must be considered before a diagnosis of sickle cell trait or sickle cell disease with beta-thalassemia is made.

Predictors of glycemic control in children with type 1 diabetes: the importance of race.

Diabetes is a common cause of kidney failure and blindness among young adults, particularly of African-American descent. Since glycemic control is a predictor of diabetes complications, we evaluated the impact of multiple factors including a special multidisciplinary management program on glycosylated hemoglobin in children with Type 1 diabetes. Data was collected from pediatric diabetes clinics in New Orleans, LA and Baltimore, MD. In New Orleans, hemoglobin A(1c) was higher in African-American patients 12. 5+/-3.3% (n=71) vs. 10.7+/-2.1% (n=80) in Caucasian children, p<0. 0001. Longer duration of diabetes was also associated with higher hemoglobin A(1c) in both races. The effect of race on hemoglobin A(1c) was independent of the influence of sex, insurance status, body mass index (BMI) z-score, and number of clinic visits. Covariate analysis with mean blood glucose levels indicated that higher hemoglobin A(1c) was attributable to higher mean blood glucose levels in African-American children. From the Baltimore data, a multidisciplinary intervention program led to improved total glycosylated hemoglobin for Caucasian patients but not for African-American children. Poorer glycemic control of African-American children is likely to predispose them to a higher likelihood of developing microvascular complications as they mature. Standard hospital-based multidisciplinary programming for diabetes management may have limited effectiveness in improving glycemic control of African-American children with diabetes. Innovative intervention programs are needed for these high-risk patients.

Laboratory diagnosis of structural hemoglobinopathies and thalassemias by capillary isoelectric focusing.

Structural hemoglobinopathies and thalassemias are congenital hemoglobin (Hb) disorders that cause anemia, morbidity, and mortality resulting from abnormal Hb function. Structurally different normal Hb variants include HbA(2), HbF (fetal hemoglobin), and HbA (adult hemoglobin). Each is a protein tetramer consisting of two α-globins, and either two δ-, γ-, or ß-globins, respectively. Fetal Hb (α(2)γ(2)) predominates in neonates (60-95% of total Hb), but declines to <1% in older children and adults. HbA(2) (α(2)δ(2)) is a minor constituent with apparently normal function that is not detected in neonates, but increases to about 2-3% of HbA (α(2)ß(2)) in older children and adults. Mutations in the genes that regulate the structure and synthesis of α-, ß-, γ- and δ-globins produce abnormal and often dysfunctional Hb variants (structural hemoglobinopathy), or cause decreased synthesis of normal Hb variants (thalassemia) (1,2). DNA mutations that cause structural hemoglobinopathies are most commonly diagnosed by indirect assays that identify abnormal gene products (i.e., Hb variants) rather than abnormal genes. Over 600 abnormal structural Hb variants have been reported (3), most of which (95%) differ from normal HbA by replacement of a single amino acid (2). Although some structural mutations are benign, many (50% of ß-variants and 20% of α-variants) alter Hb solubility, stability, or oxygen affinity in ways that adversely affect Hb function. In contrast, thalassemia syndromes are caused either by deletions of entire genes or by mutations that affect the production or processing of normal globin mRNAs.

Quantification of human cytomegalovirus by competitive PCR and capillary electrophoresis.

Human cytomegalovirus (CMV) is a ubiquitous pathogen found in 40-100% of adults, and in about 1% of live births in the United States (1). It is the most common fetal and perinatal infectious organism; approx 10% of infected neonates are born with symptomatic congenital CMV disease, which is the most common cause of mental retardation and childhood deafness. CMV is a significant pathogen in immunocompromised individuals, including organ transplant recipients (2-4), and in acquired immune deficiency syndrome (AIDS) patients (5,6). Infection is characterized by latency, chronic infection, and reactivation, a progression similar to that observed in other members of the herpesvirus family. Because CMV infection is usually controlled by the host cellular immune system, primary infections can occur without obvious symptoms, and progress to latency may go unnoticed. Latent infection may persist throughout life, but primary or reactivated infection, coupled with impaired host immune response, can rapidly produce symptomatic CMV disease.

Zinc, copper, and metallothionein metabolism after jejunoileal bypass surgery or small bowel resection in rats.

Liver dysfunction is a frequent complication of jejunoileal bypass (JIB) surgery, a procedure commonly used until recently to treat morbid obesity. It has been suggested that liver failure in JIB patients is due to bacterial overgrowth and translocation from the bypassed intestine. Because invading microorganisms cause hepatic inflammation these experiments evaluated zinc, copper, and metallothionein (MT) in two experimental rat models of intestinal surgery to determine whether their distribution in plasma and tissues was similar to the highly characteristic pattern observed during an inflammatory response. In the JIB rat model 90 per cent of the small intestine was isolated from the flow of digesta but remained viable in the abdominal cavity. In the small bowel resection (SBR) model 90 per cent of the small intestine was removed and the remaining intestine was resected. Data collected 21 days after surgery showed decreased growth rate and plasma zinc in the SBR and JIB rats that was significantly improved by supplemental zinc. All other measures of zinc, copper, and MT metabolism in the SBR rats were similar to those of controls. In JIB rats, however, liver copper, MT protein, and MT mRNA were significantly elevated, and a high proportion of the intracellular zinc and copper was associated with MT. The pattern of zinc, copper, and MT distribution in systemic circulation and liver of JIB rats suggests hepatic inflammation superimposed on low zinc and copper status. Lack of a similar response in the SBR rats confirms the involvement of the bypassed intestinal segment and supports the hypothesis that bacterial overgrowth and translocation are responsible for liver inflammation and dysfunction in JIB patients.

Effect of transfusion on hemoglobin variants in preterm infants.

OBJECTIVE: To determine the effect of transfusion on hemoglobin (Hb) variants in very low birth weight infants and to correlate these changes with parameters measured in routine complete blood counts. METHODS: Hb variants were measured by capillary isoelectric focusing on 126 specimens from 25 very low birth weight infants during their hospital course. These results were compared with transfusion frequency, mean corpuscular volume (MCV), and red cell distribution width. RESULTS: Mean initial Hb F level before transfusion was 87.1 +/- 5.1%, and a single 15 ml/kg packed red blood cell transfusion decreased the mean Hb F level to 54.0 +/- 4.7%. With frequent transfusions in the first month of life, there was progressive decline in Hb F content such that Hb F made up < 15% of the total Hb after five transfusions. There was a linear correlation between Hb F content and MCV, with a correlation coefficient (r) of 0.77 and a p value of < 0.0001. In most instances, when the MCV fell below 100 fl, the Hb F content was < 50%; when the MCV fell below 95 fl, the Hb F content was < 25%. There was a nonlinear correlation between Hb F content and red cell distribution width. CONCLUSION: Transfusion in very low birth weight infants results in a rapid transition from Hb F to Hb A predominance. This transition is marked by a reduction in MCV that allows for prediction of the Hb F content.

Rapid determination of egg weight and specific gravity using a computerized data collection system.

A rapid data-collection system for determining egg weight and specific gravity is described. The method links a microcomputer and an electronic balance to facilitate specific gravity determination by the displaced water (CADW) or Archimedes' method. Measurements obtained by this technique were compared with those determined by the saline flotation method. Repeatability measures showed both methods to be relatively precise. The CADW method was faster and reduced opportunities for clerical error.

Analysis of hemoglobin variants by capillary isoelectric focusing.

Clinical laboratory evaluation of congenital hemoglobin disorders requires both the accurate identification of major and minor hemoglobin variants, and precise quantitation of these variants over a wide range of concentrations. Capillary isoelectric focusing is an automated, microanalytical technique that produces diagnostic information comparable to that obtained from multiple conventional assays now used by most hospital laboratories. This report describes the advantages of capillary isoelectric focusing for the routine primary assessment of hemoglobinopathies and thalassemias. The use of capillary isoelectric focusing for the identification of unusual hemoglobinopathies, including an extremely rare doubly heterozygous disorder (hemoglobin C/E disease), is described. Application of the technique for rapid (< 4 minutes) neonatal hemoglobinopathy screening, and for the analysis of Hb A1c to monitor glycemic control in diabetic subjects is also discussed. In an increasingly competitive and cost-conscious clinical diagnostic market, capillary isoelectric focusing is a rapid, specific, precise, and low-cost method for comprehensive primary analysis of hemoglobin variants.

Appropriateness of the Zucker Diabetic Fatty rat as a model for diabetic microvascular late complications.

Male obese Zucker Diabetic Fatty (ZDF) rats develop type 2 diabetes around eight weeks of age, and are widely used as a model for human diabetes and its complications. The objective of the study was to test whether the complications manifested in the kidney and nerves of ZDF rats really correspond to human diabetic complications in their being related to the hyperglycaemic state. Four groups of ZDF rats were used. One lean (Fa/?) and one obese (fa/fa) untreated group served as non-diabetic and diabetic controls. In two further groups of obese (fa/fa) rats, diabetes was prevented by pioglitazone or delayed by food restriction. All rats were monitored up to 35 weeks of age with respect to their blood glucose, HbA1c and insulin levels, their kidney function (urinary glucose excretion, renal glucose filtration, glomerular filtration rate, albumin/creatinine ratio), and their nerve function (tactile and thermal sensory threshold and nerve conduction velocity). Pioglitazone prevented the development of diabetes, while food restriction delayed its onset for 8-10 weeks. Accordingly, kidney function parameters were similar to lean non-diabetic rats in pioglitazone-treated rats and significantly improved in food-restricted rats compared with obese controls. Kidney histology paralleled the functional results. By contrast, nerve functional evaluations did not mirror the differing blood glucose levels. We conclude that the ZDF rat is a good model for diabetic nephropathy, while alterations in nerve functions were not diabetes-related.

Cysteine-rich intestinal protein binds zinc during transmucosal zinc transport.

The mechanism of zinc absorption has not been delineated, but kinetic studies show that both passive and carrier-mediated processes are involved. We have identified a low molecular mass zinc-binding protein in the soluble fraction of rat intestinal mucosa that could function as an intracellular zinc carrier. The protein was not detected in liver or pancreas, suggesting a role specific to the intestine. The protein binds zinc during transmucosal zinc transport and shows signs of saturation at higher luminal zinc concentrations, characteristics consistent with a role in carrier-mediated zinc absorption. Microsequence analysis of the protein purified by gel-filtration HPLC and SDS/PAGE showed complete identity within the first 41 N-terminal amino acids with the deduced protein sequence of cysteine-rich intestinal protein [Birkenmeier, E. H. & Gordon, J. I. (1986) Proc. Natl. Acad. Sci. USA 83, 2516-2520]. These investigators showed that the gene for this protein is developmentally regulated in neonates during the suckling period, conserved in many vertebrate species, and predominantly expressed in the small intestine. Cysteine-rich intestinal protein contains a recently identified conserved sequence of histidine and cysteine residues, the LIM motif, which our results suggest confers metal-binding properties that are important for zinc transport and/or functions of this micronutrient.

Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants.

Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification.

Quantification of hemoglobin variants by capillary isoelectric focusing.

Capillary isoelectric focusing (cIEF) was used to identify and quantify major and minor hemoglobin (Hb) variants. Whole blood (approximately 10 microL required) hemolysate was analyzed with a commercial instrument equipped with a 50 microns (i.d.) x 27 cm coated capillary filled with 20 g/L ampholytes (pH 6-8) in 4 g/L methylcellulose (MC). Cathode and anode solutions were 20 mol/L NaOH and 100 mol/L H3PO4 in MC, respectively. Samples (approximately 40 nL) were applied via autosampler by low-pressure injection, focused for 3 min at 30 kV, and mobilized by simultaneous voltage and low pressure past the detector, where absorbance at 415 nm was analyzed by an automated data acquisition system. Blood from subjects with sickle cell trait, Hb S/C disease, and various beta-thalassemias were analyzed by cIEF in < 15 min. cIEF was used to separate Hb S from Hb D-Los Angeles. Assay precision determined with commercial controls gave CV < 2% for Hb A and S, and 1-11% for minor Hb variants A2, F, and A1c. Results obtained by cIEF for patients' samples agreed well with values determined by conventional assays (r2 > 0.95). The results demonstrate that cIEF is a rapid, sensitive, high-resolution automated method for routine quantitative clinical analysis of Hb variants.

Effect of EDTA and zinc-methionine complex on zinc absorption by rat intestine.

The effects of zinc chelators on 65Zn uptake, absorption and tissue distribution were determined in rats using ligated duodenal loops. 65Zn was supplied as Zn-methionine complex, ZnCl2, ZnCl2 + L-methionine or ZnCl2 + EDTA. The effect of EDTA was also determined in the presence of phytic acid. Absorption of 65Zn was markedly reduced in rats given Zn-methionine complex or ZnCl2 + EDTA. The 65Zn level in tissues (liver, bone, muscle, skin, kidney, and thymus) of rats given ZnCl2 + EDTA was also significantly reduced compared to that of rats given ZnCl2. Reduced absorption due to phytic acid was not improved by EDTA, although EDTA increased mucosal 65Zn retention. High performance gel filtration chromatography showed six 65Zn-binding peaks in the mucosal cytosol of rats given ZnCl2. A seventh peak attributable to Zn-EDTA was observed in cytosol from rats given ZnCl2 + EDTA. A comparable peak in plasma was not observed. Both EDTA and Zn-methionine complex reduced 65Zn-binding to a low-molecular-weight component of mucosal cytosol that was not metallothionein. The results suggest that Zn-EDTA is transported intact from the lumen into mucosal cells but not across the basolateral membrane. The adverse effects of EDTA and Zn-methionine complex on zinc absorption were associated with reduced 65Zn-binding to a component of mucosal cytosol that may be involved in zinc absorption.

Cysteine-rich intestinal protein and intestinal metallothionein: an inverse relationship as a conceptual model for zinc absorption in rats.

Dietary zinc may regulate zinc absorption in part via the inhibitory effect of intestinal metallothionein, but the mechanism is unknown. We recently showed that cysteine-rich intestinal protein (CRIP) binds zinc during transmucosal zinc transport, and that CRIP may function as an intracellular zinc carrier. The present experiments examine the interaction of CRIP and metallothionein with zinc to evaluate their potential roles in the mechanism of zinc absorption. Intestinal metallothionein concentrations were lower and zinc absorption rates from isolated intestinal loops were higher in rats fed a low zinc diet compared with those fed a high zinc diet or given parenteral zinc to induce metallothionein synthesis. Zinc status did not affect the apparent CRIP concentration, but markedly altered the distribution of 65Zn in intestinal cytosol as determined by gel filtration HPLC. More 65Zn was associated with CRIP (40 vs. 14%) and less was bound to metallothionein (4 vs. 52-59%) in rats fed the low zinc diet compared with rats of high zinc status. Luminal zinc concentration also affected the distribution of 65Zn in the cytosol. CRIP bound progressively less (from 42 to 25%) of the 65Zn taken up from the lumen as the luminal zinc concentration was increased from 5 to 300 mumol/L. Collectively these data suggest that CRIP is a saturable, intracellular zinc transport protein, and that metallothionein inhibits zinc absorption by binding zinc in competition with CRIP. A hypothetical model for the mechanism of transcellular zinc absorption involving metallothionein and CRIP is presented and discussed.

Intestinal metallothionein gene expression and zinc absorption in rats are zinc-responsive but refractory to dexamethasone and interleukin 1 alpha.

The effects of dexamethasone and interleukin 1 alpha on intestinal metallothionein gene expression and zinc absorption were studied. Rats given parenteral zinc served as positive controls. A single intraperitoneal or intravenous dose of dexamethasone, interleukin 1 alpha or zinc markedly increased liver metallothionein synthesis 3-9 h after injection. Intestinal metallothionein mRNA and metallothionein protein were not affected by dexamethasone or interleukin 1 alpha, but were markedly increased by parenteral zinc. Absorption of 65Zn from isolated duodenal segments was inversely related to intestinal metallothionein concentration in rats given zinc, but was not affected by dexamethasone or interleukin 1 alpha. Plasma zinc concentrations decreased in rats given dexamethasone or interleukin 1 alpha and increased in those given zinc, but they were not related to 65Zn absorption. Similarly, multiple intraperitoneal administration of either dexamethasone or interleukin 1 alpha, or oral administration of dexamethasone, for 7 d markedly increased liver metallothionein synthesis but did not affect intestinal metallothionein concentration or 65Zn absorption. These results suggest that intestinal metallothionein gene expression and 65Zn absorption are refractory to glucocorticoid hormone and interleukin 1 alpha.

Role of metallothionein and cysteine-rich intestinal protein in the regulation of zinc absorption by diabetic rats.

Hyperzincuria and low Zn absorption in diabetic animals and humans have prompted speculation that diabetics are more susceptible to Zn deficiency. There is little information, however, describing the effects of diabetes on the biochemical mechanisms of intestinal Zn transport. We evaluated Zn absorption in streptozotocin-induced diabetic rats based on a model of Zn transport in which cysteine-rich intestinal protein serves as an intracellular carrier that is inhibited by metallothionein (MT). Apparent absorption and retention of Zn and Cu in rats fed a purified diet were measured in a balance study 15-17 d after induction of diabetes. The rate of 65Zn absorption from isolated intestinal segments, molecular distribution of 65Zn in mucosal cytosol, and tissue MT levels were measured on d 20-22. Food consumption, and thus Zn and Cu intake, by diabetic rats was twice that of controls. Although fractional absorption (percent) of Zn and Cu was lower in the diabetic rats, net absorption (micrograms/100 g body weight/d) was higher. The higher net absorption in the diabetic group was offset, however, by higher urinary excretion, so that Zn and Cu retention was similar in both groups of animals. Low fractional absorption is attributable to the down-regulation of intestinal Zn transport, as indicated by the lower rate of 65Zn absorption from isolated intestinal segments in the diabetic rats. Down-regulation of intestinal transport is in turn attributable to higher concentrations of intestinal MT, which resulted in more 65Zn in the mucosal cytosol bound to MT, an inhibitor of Zn transport, and less to cysteine-rich intestinal protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary isoelectric focusing of hemoglobin variants in the pediatric clinical laboratory.

We have used capillary isoelectric focusing (cIEF) to diagnose and monitor hemoglobinopathies in children for over three years. This report describes a major revision of our original method (Clin. Chem. 1994, 40, 2288-2295) that improves the analysis of hemoglobin (Hb) variants by cIEF, especially capillary performance and quantitation precision for minor variants. The revised method uses mixed ampholytes (2% pH 6-8:3-10; 10:1), lower viscosity methylcellulose (0.375%) solution, between-sample capillary conditioning with methanol, and hemolysates prepared from red blood cells (RBC) instead of whole blood. Collectively, these changes prolonged capillary life by minimizing capillary exposure to NaOH, standardized the sample matrix, and improved the precision of peak autointegration. The between-run quantitation imprecision (% relative standard deviation) of the revised method was 0.1-3.5% for all diagnostically important major and minor Hb variants present at normal or abnormal levels. The results show the use of the revised method for (i) posttranslationally modified Hb present at low concentrations in normal blood, (ii) Hb oxidation products produced by improper sample storage, (iii) differential diagnosis of S/beta + thalassemia, G-Philadelphia trait, S/C-Harlem disease, and Hb H disease, (iv) sensitive detection of minor variants like Hb A2' as indicators of an alpha globin mutation, and (v) neonatal screening using dried blood collected on filter paper. The results show that high-efficiency separation and precise quantitation of Hb variants over a wide range of concentrations makes cIEF a comprehensive assay that can be used without adjunct analyses for the automated primary evaluation of hemoglobinopathies and thalassemias.

Expression of cysteine-rich intestinal protein in rat intestine and transfected cells is not zinc dependent.

The cysteine-rich intestinal protein (CRIP) is a member of a superfamily of proteins containing the LIM motif (a double zinc finger) that has been shown to bind zinc. The role of zinc in the regulation of CRIP was examined in adult rats, cultured intestinal epithelial cells and in a transient transfection system. When adult male rats were fed diets with various amounts of zinc, the amount of ileal CRIP mRNA was only 19% lower in rats fed a zinc-deficient diet (1 mg Zn/kg) and was not different in the zinc-supplemented group (180 mg Zn/kg) compared with the zinc-adequate group (30 mg Zn/kg). In contrast, metallothionein mRNA levels were 76% lower and 80% greater than control levels in the zinc-deficient and zinc-supplemented groups, respectively. Using the chloramphenicol acetyltransferase (CAT) reporter gene, 5'-deletion products of the CRIP genomic promoter were tested for basal and zinc-induced CAT activity in transiently transfected IEC-6 cells. Treatment of the cells with zinc did not alter CAT activity of any construct. These results suggest that CRIP is not directly regulated by zinc in the intestine of rats.

Up-regulation of CD40 ligand and induction of a Th2 response in children immunized with pneumococcal polysaccharide vaccines.

We wished to determine whether pneumococcal polysaccharide antigens induce mRNA expression of CD40 ligand (CD40L) and Th1 or Th2 cytokines in unimmunized individuals in vitro and whether immunization with the 23-valent pneumococcal polysaccharide vaccine induces changes in CD40L and cytokine mRNA expression. Children with recurrent respiratory infections were studied before and 4 to 6 weeks after receiving the pneumococcal vaccine. One patient who failed to respond to the polysaccharide vaccine subsequently received a single dose of the experimental 7-valent pneumococcal conjugate vaccine. Unimmunized healthy adults were included as controls. Quantification of mRNA expression of CD40L, interleukin-4 (IL-4), IL-12p40, and gamma interferon (IFN-gamma) was performed by reverse transcription-PCR and enzyme-linked immunosorbent assay (ELISA)-PCR with resting and stimulated peripheral blood mononuclear cells. Serum immunoglobulin G (IgG) anti pneumococcal antibody levels were measured by ELISA. The results showed a significant increase in the expression of mRNAs for CD40L and IL-4, but not IL-12p40 or IFN-gamma, in stimulated cultures from unimmunized individuals. CD40L and IL-4 mRNA expression was significantly higher in postimmunization than in preimmunization samples stimulated with the individual pneumococcal serotypes. These results suggest that pneumococcal polysaccharide antigens specifically up-regulate CD40L expression and induce a Th2 response in vitro which parallels the increase in IgG antipneumococcal antibody levels in serum.