The fracture strength and the failure mode of lithium disilicate or resin nano ceramics as a crown, overlay, or endocrown restoration on endodontically treated teeth.

INTRODUCTION: Different materials and restorative concepts have been proposed over the years to restore endodontically treated teeth (ETT). Monolithic ceramic and composite restoration can be lute to the tooh, without the use of a post. However, little is known how the material stiffness and presence of a composite core will affect the survival and failure mode. The objective of this in-vitro study was to evaluate the fracture strength and failure mode of endodontically treated molars, restored with ceramic or hybrid composite monolithic restoration, in the presence of absence of a composite core. MATERIALS AND METHODS: Sixty depulped molars were restored with a lithium-disilicate (e.max CAD) or hybrid composite (Cerasmart) restoration. Both materials were used in a monolithic approach, but with 3 different designs: (a) monolithic endocrown, (b) crown with a separate composite core, and (c) overlay without core buildup or pulpal extension. Ten sound teeth were used as control group. All groups were thermocycled (10,000 cycles), subsequently loaded in a chewing simulator (100,000 cycles) and finally loaded until fracture. RESULTS: Peak fracture loads and failure modes were registered. No significant differences were seen between the groups in terms of fracture load. Failure modes were statistically significantly different among groups with significant correlation between restoration type and material. (p < 0.001 and p = 0.033, respectively). No group presented significantly higher fracture resistance. Although ceramic crowns and overlays presented the highest repairability, all restored ETT were within the range of the intact tooth' fracture strength. CONCLUSION: No restoration presented significant different fracture loads. However, the type of restoration and material choice were correlated to the fracture mode.

Orthogonal proteomics methods warrant the development of Duchenne muscular dystrophy biomarkers.

BACKGROUND: Molecular components in blood, such as proteins, are used as biomarkers to detect or predict disease states, guide clinical interventions and aid in the development of therapies. While multiplexing proteomics methods promote discovery of such biomarkers, their translation to clinical use is difficult due to the lack of substantial evidence regarding their reliability as quantifiable indicators of disease state or outcome. To overcome this challenge, a novel orthogonal strategy was developed and used to assess the reliability of biomarkers and analytically corroborate already identified serum biomarkers for Duchenne muscular dystrophy (DMD). DMD is a monogenic incurable disease characterized by progressive muscle damage that currently lacks reliable and specific disease monitoring tools. METHODS: Two technological platforms are used to detect and quantify the biomarkers in 72 longitudinally collected serum samples from DMD patients at 3 to 5 timepoints. Quantification of the biomarkers is achieved by detection of the same biomarker fragment either through interaction with validated antibodies in immuno-assays or through quantification of peptides by Parallel Reaction Monitoring Mass Spectrometry assay (PRM-MS). RESULTS: Five, out of ten biomarkers previously identified by affinity-based proteomics methods, were confirmed to be associated with DMD using the mass spectrometry-based method. Two biomarkers, carbonic anhydrase III and lactate dehydrogenase B, were quantified with two independent methods, sandwich immunoassays and PRM-MS, with Pearson correlations of 0.92 and 0.946 respectively. The median concentrations of CA3 and LDHB in DMD patients was elevated in comparison to those in healthy individuals by 35- and 3-fold, respectively. Levels of CA3 vary between 10.26 and 0.36 ng/ml in DMD patients whereas those of LDHB vary between 15.1 and 0.8 ng/ml. CONCLUSIONS: These results demonstrate that orthogonal assays can be used to assess the analytical reliability of biomarker quantification assays, providing a means to facilitate the translation of biomarkers to clinical practice. This strategy also warrants the development of the most relevant biomarkers, markers that can be reliably quantified with different proteomics methods.

A Proof of Principle Proteomic Study Detects Dystrophin in Human Plasma: Implications in DMD Diagnosis and Clinical Monitoring.

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD.

Experiences of Stress - A Focus Group Interview Study Among Swedish Adolescents During the COVID-19 Pandemic.

Adolescence can be a stressful period in life. The period contains challenges associated with the transition from childhood to adulthood (body changes, changes in interpersonal relationships, and identity changes). The aim was to investigate experiences of stress among adolescents in addition to stress related to the COVID-19 pandemic. Focus group interviews (n = 8) were conducted with girls (n = 22) and boys (n = 19) aged 13-15 in southern Sweden. The transcribed interviews were analyzed with qualitative content analysis. Analysis of the collected material resulted in two categories with four sub-categories each of which highlights adolescents' experiences of stress. The results show that adolescents' have a variety of experiences of stress, i.e., what they mean are the sources of stress and how stress is manifested. The adolescents experienced how stress was manifested both physically and emotionally. This affected both their sleep and performance. The adolescents reflected on both positive and negative manifestations of stress.

Rimeporide as a first- in-class NHE-1 inhibitor: Results of a phase Ib trial in young patients with Duchenne Muscular Dystrophy.

Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies.

Fracture strength of monolithic all-ceramic crowns made of high translucent yttrium oxide-stabilized zirconium dioxide compared to porcelain-veneered crowns and lithium disilicate crowns.

OBJECTIVE: The aim of the study was to provide data on the fracture strength of monolithic high translucent Y-TZP crowns and porcelain-veneered high translucent Y-TZP crown cores and to compare that data with the fracture strength of porcelain-veneered Y-TZP crown cores and monolithic lithium disilicate glass-ceramic crowns. MATERIALS AND METHODS: Sixty standardized crowns divided into six groups (n = 10) were fabricated: monolithic high translucent Y-TZP crowns, brand A, monolithic high translucent Y-TZP crowns, brand B, veneered high translucent Y-TZP crown cores, brand A, veneered high translucent Y-TZP crown cores, brand B, heat-pressed monolithic lithium disilicate crowns and veneered Y-TZP crown cores. All crowns were thermocycled, cemented onto dies, cyclically pre-loaded and finally loaded to fracture. RESULTS: The monolithic Y-TZP groups showed significantly higher fracture strength (2795 N and 3038 N) compared to all other groups. The fracture strength in the veneered Y-TZP group (2229 N) was significantly higher than the monolithic lithium disilicate group (1856 N) and the veneered high translucent Y-TZP groups (1480 N and 1808 N). CONCLUSIONS: The fracture strength of monolithic high translucent Y-TZP crowns is considerably higher than that of porcelain-veneered Y-TZP crown cores, porcelain-veneered high translucent Y-TZP crown cores and monolithic lithium disilicate crowns. The fracture strength of a crown made of monolithic high translucent Y-TZP is, with a large safety margin, sufficient for clinical use for the majority of patients. Porcelain-veneered Y-TZP crown cores show higher fracture resistance than monolithic lithium disilicate crowns.

Creating Designer Engineered Extracellular Vesicles for Diverse Ligand Display, Target Recognition, and Controlled Protein Loading and Delivery.

Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag-fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein-engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles.

Bond strength between titanium and polymer-based materials adhesively cemented.

The aim was to evaluate the bond strength between titanium and polymer-based materials for prosthetic restorations, cemented with different adhesive cement systems. Eight groups with 13 specimens in each group were included. Each specimen consisted of two parts: a cylinder of titanium resembling a titanium base, and a cylinder of one of two polymer-based materials Micro Filled Hybrid (MFH) or Telio CAD and cemented with one of four adhesive cement systems, namely Multilink Hybrid Abutment, Panavia V5, RelyX Ultimate and G-Cem LinkAce. The titanium was sandblasted with 50 µm Al2O3 and treated according to each cement manufacturer's recommendations. The polymer-based materials were pre-treated according to the manufacturer's instructions including sandblasting for MFH. After cementation, the groups were water stored for one day before thermocycling: 5000 cycles in 5-55 °C. A shear bond strength test was performed (crosshead speed 0.5 mm/min) and data was analysed with one-way ANOVA, Tukey's test. Telio CAD cemented with Panavia V5 and G-Cem LinkAce showed significantly lower bond strength compared to all other groups, due to spontaneous debonding. The highest numerical bond strength was found in the group of MFH cemented with RelyX Ultimate or with G-Cem LinkAce. Generally, the Telio CAD groups showed lower bond strength values than the MFH groups. The conclusions are that pre-treatment methods and choice of cement system are of importance for polymer-based materials for prosthetic restorations. The bond strength is adequate for provisional cementation irrespective of cement system when pre-treating by sandblasting, but cement dependent without sandblasting.

Accuracy of 3D printed polymers intended for models and surgical guides printed with two different 3D printers.

The purpose of the study was to evaluate the accuracy: trueness and precision of photopolymers used for dental models and surgical guides printed with two different digital light processing (DLP) printers. Forty specimens of four materials; E-dentstone®, E-shell®, NextDent™ Model, NextDent™ SG, and two designs; models A and B (n=5), were manufactured (DDDP, EvoDent). Trueness was evaluated by comparing values for 26 parameters with the CAD models' reference values and precision through standard deviation. The trueness and precision were higher for linear than for angle parameters. X- and Y-axes showed higher trueness than Z-axis and model B higher trueness than model A. The conclusions are; the accuracy is dependent on the design of the object. The linear precision appears to be high. The highest trueness was observed for a surgical guide polymer (NextDent™ SG). The definition of clinically relevant accuracy and acceptable production tolerance should be evaluated in future studies.

Circadian Genes as Exploratory Biomarkers in DMD: Results From Both the mdx Mouse Model and Patients.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients' plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Longitudinal serum biomarker screening identifies malate dehydrogenase 2 as candidate prognostic biomarker for Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal disease for which no cure is available. Clinical trials have shown to be largely underpowered due to inter-individual variability and noisy outcome measures. The availability of biomarkers able to anticipate clinical benefit is highly needed to improve clinical trial design and facilitate drug development. METHODS: In this study, we aimed to appraise the value of protein biomarkers to predict prognosis and monitor disease progression or treatment outcome in patients affected by DMD. We collected clinical data and 303 blood samples from 157 DMD patients in three clinical centres; 78 patients contributed multiple blood samples over time, with a median follow-up time of 2 years. We employed linear mixed models to identify biomarkers that are associated with disease progression, wheelchair dependency, and treatment with corticosteroids and performed survival analysis to find biomarkers whose levels are associated with time to loss of ambulation. RESULTS: Our analysis led to the identification of 21 proteins whose levels significantly decrease with age and nine proteins whose levels significantly increase. Seven of these proteins are also differentially expressed in non-ambulant patients, and three proteins are differentially expressed in patients treated with glucocorticosteroids. Treatment with corticosteroids was found to partly counteract the effect of disease progression on two biomarkers, namely, malate dehydrogenase 2 (MDH2, P = 0.0003) and ankyrin repeat domain 2 (P = 0.0005); however, patients treated with corticosteroids experienced a further reduction on collagen 1 serum levels (P = 0.0003), especially following administration of deflazacort. A time to event analysis allowed to further support the use of MDH2 as a prognostic biomarker as it was associated with an increased risk of wheelchair dependence (P = 0.0003). The obtained data support the prospective evaluation of the identified biomarkers in natural history and clinical trials as exploratory biomarkers. CONCLUSIONS: We identified a number of serum biomarkers associated with disease progression, loss of ambulation, and treatment with corticosteroids. The identified biomarkers are promising candidate prognostic and surrogate biomarkers, which may support drug developers if confirmed in prospective studies. The serum levels of MDH2 are of particular interest, as they correlate with disease stage and response to treatment with corticosteroids, and are also associated with the risk of wheelchair dependency and pulmonary function.

Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Decreased IgA antibody production in the stomach of gastric adenocarcinoma patients.

Gastric adenocarcinoma is closely associated with Helicobacter pylori infection. It is also much more frequent in patients with common variable immunodeficiency or selective IgA-deficiency than in the general population. To investigate a possible link between local antibody production and gastric tumors, we studied gastric B cell infiltration and local IgA production in patients with H. pylori induced gastric adenocarcinomas. These studies showed that total and H. pylori-specific IgA antibody levels were substantially lower in gastric tissue from the cancer patients compared to those from asymptomatic H. pylori carriers. However, serum IgA levels were similar in the cancer patients and asymptomatic carriers. As could be expected, H. pylori infected asymptomatic carriers had considerably increased IgA antibody levels compared to uninfected subjects. We conclude that patients suffering from gastric adenocarcinoma have a dramatically decreased local IgA production in the stomach compared to asymptomatic H. pylori infected individuals.

The effects of dual PPARα/γ agonism compared with ACE inhibition in the BTBRob/ob mouse model of diabetes and diabetic nephropathy.

The leptin-deficient BTBRob/ob mouse develops progressive albuminuria and morphological lesions similar to human diabetic nephropathy (DN), although whether glomerular hyperfiltration, a recognized feature of early DN that may contribute to renal injury, also occurs in this model is not known. Leptin replacement has been shown to reverse the signs of renal injury in this model, but in contrast, the expected renoprotection by angiotensin-converting enzyme (ACE) inhibition in BTBRob/ob mice seems to be limited. Therefore, to investigate the potential renal benefits of improved metabolic control in this model, we studied the effect of treatment with the dual peroxisome proliferator-activated receptor (PPAR) α/γ agonist AZD6610 and compared it with the ACE inhibitor enalapril. AZD6610 lowered plasma glucose and triglyceride concentrations and increased liver size, but had no significant effect in reducing albuminuria, whereas enalapril did have an effect. Nephrin and WT1 mRNA expression decreased in the kidneys of BTBRob/ob mice, consistent with podocyte injury and loss, but was unaffected by either drug treatment: at the protein level, both nephrin and WT1-positive cells per glomerulus were decreased. Mesangial matrix expansion was reduced in AZD6610-treated mice. GFR, measured by creatinine clearance, was increased in BTBRob/ob mice, but unaffected by either treatment. Unexpectedly, enalapril-treated mice showed intrarenal arteriolar vascular remodeling with concentric thickening of vessel walls. In summary, we found that the BTBRob/ob mouse model shows some similarities to the early changes seen in human DN, but that ACE inhibition or PPARα/γ agonism afforded limited or no kidney protection.

Retinoid-related orphan receptor γ (RORγ) adult induced knockout mice develop lymphoblastic lymphoma.

RORγ is a nuclear hormone receptor which controls polarization of naive CD4+ T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma.

The discovery of a selective and potent A2a agonist with extended lung retention.

Although the anti-inflammatory role of the A2a receptor is well established, controversy remains with regard to the therapeutic value for A2a agonists in treatment of inflammatory lung diseases, also as a result of unwanted A2a-mediated cardiovascular effects. In this paper, we describe the discovery and characterization of a new, potent and selective A2a agonist (compound 2) with prolonged lung retention and limited systemic exposure following local administration. To support the lead optimization chemistry program with compound selection and profiling, multiple in vitro and in vivo assays were used, characterizing compound properties, pharmacodynamics (PD), and drug concentrations. Particularly, pharmacokinetic-PD modeling was applied to quantify the effects on the cardiovascular system, and an investigative toxicology study in rats was performed to explore potential myocardial toxicities. Compound 2, in comparison to a reference A2a agonist, UK-432,097, demonstrated higher solubility, lower lipophilicity, lower plasma protein binding, high rat lung retention (28% remaining after 24 h), and was efficacious in a lung inflammatory rat model following intratracheal dosing. Despite these properties, compound 2 did not provide a sufficient therapeutic index, that is, separation of local anti-inflammatory efficacy in the lung from systemic side effects in the cardiovascular system. The plasma concentration that resulted in induction of hypotension (half maximal effective concentration; EC50 0.5 nmol/L) correlated to the in vitro A2a potency (rIC50 0.6 nmol/L). Histopathological lesions in the heart were observed at a dose level which is threefold above the efficacious dose level in the inflammatory rat lung model. In conclusion, compound 2 is a highly potent and selective A2a agonist with significant lung retention after intratracheal administration. Despite its local anti-inflammatory efficacy in rat lung, small margins to the cardiovascular effects suggested limited therapeutic value of this compound for treatment of inflammatory lung disease by the inhaled route.

Chinese nurses' ethical concerns in a neurological ward.

Our aim was to describe Chinese nurses' experiences of workplace distress and ethical dilemmas on a neurological ward. Qualitative interviews were performed with 20 nurses. On using latent content analysis, themes emerged in four content areas: ethical dilemmas, workplace distress, quality of nursing and managing distress. The ethical dilemmas were: (1) conflicting views on optimal treatment and nursing; (2) treatment choice meeting with financial constraints; and (3) misalignment of nursing responsibilities, competence and available resources. The patients' relatives lacked respect for the nurses' skills. Other dilemmas could be traced to the transition from a planned to a market economy, resulting in an excessive workload and treatment withdrawal for financial reasons. Lack of resources was perceived as an obstacle to proper patient care in addition to hospital organization, decreasing the quality of nursing, and increasing moral and workplace distress. The nurses managed mainly by striving for competence, which gave them hope for the future.

Oral immunization with a Salmonella enterica serovar typhi vaccine induces specific circulating mucosa-homing CD4(+) and CD8(+) T cells in humans.

The kinetics and homing characteristics of T-cell responses in humans after mucosal immunizations have not been well characterized. Therefore, we have investigated the magnitude and duration of such responses as well as the homing receptor expression of antigen-specific peripheral blood T cells by using an oral model vaccine, i.e., the live, attenuated Salmonella enterica serovar Typhi vaccine (Ty21a). Eight volunteers were each given three doses of the vaccine 2 days apart, and blood samples, from which CD4(+) and CD8(+) T cells were selected by the use of magnetic beads, were collected before vaccination and at regular intervals thereafter. To purify the potentially antigen-specific gut-homing T cells, CD45RA(-) integrin beta(7)(+) cells were further sorted by flow cytometry. The sorted cells were then stimulated in vitro with the serovar Typhi vaccine strain, and the proliferation of cells and the cytokine production were measured. Following vaccination, there was a large increase in both the proliferation of and the gamma interferon (IFN-gamma) production by blood T cells stimulated with the vaccine strain. The responses were seen among both CD4(+) and CD8(+) T cells, although the CD8(+) cells produced the largest amounts of IFN-gamma. Peak responses were seen 7 to 14 days after the onset of vaccination. Furthermore, most of the IFN-gamma produced by both CD4(+) and CD8(+) cells emanated from cells with the potential to home to mucosal tissues, as the integrin beta(7)-expressing memory T cells produced around 10-fold more IFN-gamma than the remaining populations. In conclusion, we demonstrate that oral vaccination with a live oral bacterial vaccine induces antigen-specific CD4(+) and CD8(+) memory T cells, almost all of which express the gut-homing integrin beta(7).