Multimodal detection and analysis of microplastics in human thrombi from multiple anatomically distinct sites.

BACKGROUND: Microplastic (MP) pollution has emerged as a significant environmental concern worldwide. While extensive research has focused on their presence in marine organisms and ecosystems, their potential impact on human health, particularly on the circulatory system, remains understudied. This project aimed to identify and quantify the mass concentrations, polymer types, and physical properties of MPs in human thrombi surgically retrieved from both arterial and venous systems at three anatomically distinct sites, namely, cerebral arteries in the brain, coronary arteries in the heart, and deep veins in the lower extremities. Furthermore, this study aimed to investigate the potential association between the levels of MPs and disease severity. METHODS: Thrombus samples were collected from 30 patients who underwent thrombectomy procedures due to ischaemic stroke (IS), myocardial infarction (MI), or deep vein thrombosis (DVT). Pyrolysis-gas chromatography mass spectrometry (Py-GC/MS) was employed to identify and quantify the mass concentrations of the MPs. Laser direct infrared (LDIR) spectroscopy and scanning electron microscopy (SEM) were used to analyse the physical properties of the MPs. Demographic and clinical information were also examined. A rigorous quality control system was used to eliminate potential environmental contamination. FINDINGS: MPs were detected by Py-GC/MS in 80% (24/30) of the thrombi obtained from patients with IS, MI, or DVT, with median concentrations of 61.75 µg/g, 141.80 µg/g, and 69.62 µg/g, respectively. Among the 10 target types of MP polymers, polyamide 66 (PA66), polyvinyl chloride (PVC), and polyethylene (PE) were identified. Further analyses suggested that higher concentrations of MPs may be associated with greater disease severity (adjusted ß = 7.72, 95% CI: 2.01-13.43, p < 0.05). The level of D-dimer in the MP-detected group was significantly higher than that in the MP-undetected group (8.3 ± 1.5 µg/L vs 6.6 ± 0.5 µg/L, p < 0.001). Additionally, LDIR analysis showed that PE was dominant among the 15 types of identified MPs, accounting for 53.6% of all MPs, with a mean diameter of 35.6 µm. The shapes of the polymers detected using LDIR and SEM were found to be heterogeneous. INTERPRETATION: This study presents both qualitative and quantitative evidence of the presence of MPs, and their mass concentrations, polymer types, and physical properties in thrombotic diseases through the use of multimodal detection methods. Higher concentrations of MPs may be associated with increased disease severity. Future research with a larger sample size is urgently needed to identify the sources of exposure and validate the observed trends in the study. FUNDING: This study was funded by the SUMC Scientific Research Initiation Grant (SRIG, No. 009-510858038), Postdoctoral Research Initiation Grant (No. 202205230031-3), and the 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (No. 2020LKSFG02C).

Association Between Temperature During Intensive Care Unit and Mortality in Patients With Acute Respiratory Distress Syndrome.

The relationship between body temperature changes and prognosis in patients with acute respiratory distress syndrome (ARDS) remains inconclusive. Our study aimed to investigate the clinical value of body temperature in the management of ARDS. Data from the Medical Information Mart for Intensive Care III database were collected. Adult patients with ARDS were enrolled and further grouped based on their temperature values in the intensive care unit. Both the maximum (temperaturemax) and minimum (temperaturemin) temperatures were used. The primary outcome was 28-day mortality rate. Polynomial regression, subgroup analysis, and logistic regression analysis were performed in the final analysis. A total of 3922 patients with ARDS were enrolled. There was a U-shaped relationship between 28-day mortality and body temperature. For patients with infection, the elevated temperaturemax (≥37.0°C) was associated with decreased mortality, with an odds ratio ranging from 0.39 to 0.49, using temperaturemax from 36.5°C to 36.9°C as reference. For patients without infection, a similar tendency was observed, but the protective effect was lost at extremely high temperatures (≥38.0°C, p < 0.05). Elevated temperaturemin (≥37.0°C) and decreased temperaturemin (<35.0°C) were associated with increased mortality, using the temperaturemin from 36.0°C to 36.9°C as a reference. Hypothermia was associated with increased mortality in patients with ARDS, while the effect of hyperthermia (≥37.0°C) on the mortality of patients with ARDS was not fully consistent in the infection and noninfection subgroups. Short-term and transient temperatures above 37.0°C would be beneficial to patients with ARDS, but extreme hyperthermia and persistent temperatures above 37.0°C should be avoided.

A Combination of Distinct Vascular Stem/Progenitor Cells for Neovascularization and Ischemic Rescue.

BACKGROUND: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia. METHODS: The ActinCreER/R26VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions. RESULTS: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105highSca1low, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105lowSca1high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice. CONCLUSIONS: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.

NK-like CD8+ γδ T cells are expanded in persistent Mycobacterium tuberculosis infection.

The response of gamma delta (γδ) T cells in the acute versus chronic phases of the same infection is unclear. How γδ T cells function in acute Mycobacterium tuberculosis (Mtb) infection is well characterized, but their response during persistent Mtb infection is not well understood, even though most infections with Mtb manifest as a chronic, clinically asymptomatic state. Here, we analyze peripheral blood γδ T cells from a South African adolescent cohort and show that a unique CD8+ γδ T cell subset with features of "memory inflation" expands in chronic Mtb infection. These cells are hyporesponsive to T cell receptor (TCR)-mediated signaling but, like NK cells, can mount robust CD16-mediated cytotoxic responses. These CD8+ γδ T cells comprise a highly focused TCR repertoire, with clonotypes that are Mycobacterium specific but not phosphoantigen reactive. Using multiparametric single-cell pseudo-time trajectory analysis, we identified the differentiation paths that these CD8+ γδ T cells follow to develop into effectors in this infection state. Last, we found that circulating CD8+ γδ T cells also expand in other chronic inflammatory conditions, including cardiovascular disease and cancer, suggesting that persistent antigenic exposure may drive similar γδ T cell effector programs and differentiation fates.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

BACKGROUND: Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. METHODS: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. RESULTS: In addition to macrophages, we found a high proportion of αß T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αß T cells (CD4

Modest Gains After an 8-Week Exercise Program Correlate With Reductions in Non-traditional Markers of Cardiovascular Risk.

Background: Although engaging in physical exercise has been shown to reduce the incidence of cardiovascular events, the molecular mechanisms by which exercise mediates these benefits remain unclear. Based on epidemiological evidence, reductions in traditional risk factors only accounts for 50% of the protective effects of exercise, leaving the remaining mechanisms unexplained. The objective of this study was to determine whether engaging in a regular exercise program in a real world clinical setting mediates cardiovascular protection via modulation of non-traditional risk factors, such as those involved in coagulation, inflammation and metabolic regulation. Methods and Results: We performed a prospective, cohort study in 52 sedentary patients with cardiovascular disease or cardiovascular risk factors at two tertiary medical centers between January 1, 2016 and December 31, 2019. Prior to and at the completion of an 8-week exercise program, we collected information on traditional cardiovascular risk factors, exercise capacity, and physical activity and performed plasma analysis to measure levels of fibrinolytic, inflammatory and metabolic biomarkers to assess changes in non-traditional cardiovascular risk factors. The median weight change, improvement in physical fitness, and change in physical activity for the entire cohort were: -4.6 pounds (IQR: +2 pounds, -11.8 pounds), 0.37 METs (IQR: -0.076 METs, 1.06 METs), and 252.7 kcals/week (IQR: -119, 921.2 kcals/week). In addition to improvement in blood pressure and cholesterol, patients who lost at least 5 pounds, expended at least 1,000 additional kcals/week, and/or achieved ≥0.5 MET increase in fitness had a significant reduction in plasminogen activator inhibitor-1 [9.07 ng/mL (95% CI: 2.78-15.35 ng/mL); P = 0.026], platelet derived growth factor beta [376.077 pg/mL (95% CI: 44.69-707.46 pg/mL); P = 0.026); and angiopoietin-1 [(1104.11 pg/mL (95% CI: 2.92-2205.30 pg/mL); P = 0.049)]. Conclusion: Modest improvements in physical fitness, physical activity, and/or weight loss through a short-term exercise program was associated with decreased plasma levels of plasminogen activator inhibitor, platelet derived growth factor beta, and angiopoietin, which have been associated with impaired fibrinolysis and inflammation.

Single-Cell Transcriptional Profiling Reveals Sex and Age Diversity of Gene Expression in Mouse Endothelial Cells.

Although it is well-known that sex and age are important factors regulating endothelial cell (EC) function, the impact of sex and age on the gene expression of ECs has not been systematically analyzed at the single cell level. In this study, we performed an integrated characterization of the EC transcriptome of five major organs (e.g., fat, heart-aorta, lung, limb muscle, and kidney) isolated from male and female C57BL/6 mice at 3 and 18 months of age. A total of 590 and 252 differentially expressed genes (DEGS) were identified between females and males in the 3- and 18-month subgroups, respectively. Within the younger and older group, there were 177 vs. 178 DEGS in fat, 305 vs. 469 DEGS in heart/aorta, 22 vs. 37 DEGS in kidney, 26 vs. 439 DEGS in limb muscle, and 880 vs. 274 DEGS in lung. Interestingly, LARS2, a mitochondrial leucyl tRNA synthase, involved in the translation of mitochondrially encoded genes was differentially expressed in all organs in males compared to females in the 3-month group while S100a8 and S100a9, which are calcium binding proteins that are increased in inflammatory and autoimmune states, were upregulated in all organs in males at 18 months. Importantly, findings from RNAseq were confirmed by qPCR and Western blot. Gene enrichment analysis found genes enriched in protein targeting, catabolism, mitochondrial electron transport, IL 1- and IL 2- signaling, and Wnt signaling in males vs. angiogenesis and chemotaxis in females at 3 months. In contrast, ECs from males and females at 18-months had up-regulation in similar pathways involved in inflammation and apoptosis. Taken together, our findings suggest that gene expression is largely similar between males and females in both age groups. Compared to younger mice, however, older mice have increased expression of genes involved in inflammation in endothelial cells, which may contribute to the development of chronic, non-communicable diseases like atherosclerosis, hypertension, and Alzheimer's disease with age.

The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34+ cell erythropoiesis.

As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood-borne infections since donated blood is the only source of RBCs. Although large-scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild-type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of ß-thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood-derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell-cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.

Infectivity and Screening of Anti-piperaquine Genes in Mice Infected with Piperaquine-Sensitive and Piperaquine-Resistant Plasmodium berghei.

BACKGROUND: Piperaquine (PQ) is one of the major components of artemisinin-based combination therapy for malaria. However, the mechanism of PQ resistance has remained unclear. METHODS: In this study, we infected mice with PQ-resistant Plasmodium berghei ANKA strain line (PbPQR) or PQ-sensitive P. berghei ANKA strain line (PbPQS) and their survival rates, parasitemia, and spleen sizes were compared. In addition, we constructed genomic DNA subtractive library of spleens from the infected mice, and screened the potential PQ-resistant related genes from genomic DNA of PbPQR line using the representational difference analysis (RDA) method. Clones of the subtractive library were screened by PCR, and related genes were sequenced and analyzed using BLAST software of NCBI. RESULTS: Compared to PbPQS-infected mice, PbPQR-infected mice survived significantly longer, and had significantly lowered parasitemia rate and significantly increased splenomegaly. Among the total of 502 clones picked, 494 were sequenced and 96 unique PCR fragments were obtained; in which 24 DNA fragments were homologous to chromosomes related to immune function of mice. ORF Finder blasting showed that at the protein level, 26 encoded proteins were homologous to 18 hypothetical PbANKA proteins and 13 encoded proteins were homologous to "ferlin-like protein" family of PbANKA. In addition, there were more immune-related DNA molecules, ubiquitous PbANKA homology at the ORF fragment level, and enriched ferlin-like protein families identified from PbPQR-infected mice than those from PbPQS-infected mice. CONCLUSION: These findings suggest that PbPQR may induce stronger protective immune response than that of PbPQS in infected mice.

Expression of miRNA-206 and miRNA-145 in breast cancer and correlation with prognosis.

Correlation between microRNA (miRNA)-206 and miRNA-145 expression and prognosis in breast cancer was investigated. Breast cancer specimens and paracancerous tissues of 372 breast cancer patients who underwent surgical resection in the First Affiliated Hospital of Shantou University Medical College from September 2010 to September 2014 were included. qRT-PCR was used to detect the expression of miR-206 and miR-145 in breast cancer and paracancerous tissues, and patients were divided into high and low expression groups according to the median expression level to plot survival curve. Expression levels of miR-145 and miR-206 in breast cancer tissues were 2.24±1.23 and 0.76±0.24, respectively. Expression level of miR-145 was significantly lower, while expression level of miR-206 was significantly higher in tumor tissues than in paracancerous tissues (p<0.05). The 3-year survival rates of miR-145 low expression group and miR-206 high expression group were also lower than that of miR-145 high expression group and miR-206 low expression group, respectively (p<0.05). Expression of miR-206 is upregulated and expression of miR-145 is downregulated in breast cancer, which may have an impact on the prognosis of patients. miR-206 and miR-145 may serve as important indicators to predict prognosis of patients with breast cancer in the future.

Commensal microbiota induces colonic barrier structure and functions that contribute to homeostasis.

The intestinal barrier encompasses structural, permeability and immune aspects of the gut mucosa that, when disrupted, may contribute to chronic inflammation. Although gnotobiotic studies have demonstrated the effects of microbiota on mucosal and systemic immunity, as well as intestinal barrier architecture and innate immune characteristics, its impact on barrier function remains unclear. We compared germ-free and conventional mice, as well as mice colonized with human fecal microbiota that were followed for 21 days post-colonization. Colonic barrier structure was investigated by immunohistochemistry, molecular and electron microscopy techniques. Permeability was assessed in colon tissue by Ussing chambers, and by serum LPS and MDP detection using TLR4- and NOD2-NFκB reporter assays. Microbiota profile was determined by Illumina 16S rRNA gene sequencing. Low dose dextran sodium sulfate was administered to assess microbiota-induced barrier changes on resistance to colonic injury. Permeability to paracellular probes and mucus layer structure resembled that of conventional mice by day 7 post-colonization, coinciding with reduced claudin-1 expression and transient IL-18 production by intestinal epithelial cells. These post-colonization adaptations were associated with decreased systemic bacterial antigen exposure and reduced susceptibility to intestinal injury. In conclusion, commensal colonization promotes physiological barrier structural and functional adaptations that contribute to intestinal homeostasis.

Comparative efficacy of individual renin-angiotensin system inhibitors on major renal outcomes in diabetic kidney disease: a network meta-analysis.

Background: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are two drug classes with well-documented renal protective effects. However, whether there is any difference among individual drugs remains unknown. In this study, we aimed to compare the efficacy of individual ACEIs/ARBs on major renal outcomes in adults with diabetic kidney disease (DKD). Methods: We conducted a Bayesian-framework network meta-analysis with a random effects model. We searched PubMed, Embase, Scopus, the Cochrane Central Register of Controlled Trials and ClinicalTrials.gov for clinical trials of ACEIs or ARBs as monotherapy compared with other conventional antihypertensive drugs or placebo. Primary outcomes were end-stage renal disease (ESRD) and albuminuria/proteinuria (including change in albuminuria/proteinuria, progression to macroalbuminuria and remission to normoalbuminuria). Secondary outcome was doubling of serum creatinine levels. We also assessed for hyperkalemia, cough and angioedema/edema. International prospective register of systematic reviews (PROSPERO) registration CRD42016036997. Results: A total of 100 studies with data for 22 365 DKD patients, the majority of whom had type 2 diabetes, were included. Individual ACEIs and ARBs at goal doses showed no significant differences in ESRD and doubling of serum creatinine levels. They also shared similar effects on albuminuria/proteinuria reduction and progression or remission of albuminuria. When combining three outcomes of albuminuria/proteinuria as a single endpoint, most ACEIs/ARBs consistently showed favorable antiproteinuric effect, with little difference in the possibility of being the superior treatment for improving albuminuria/proteinuria. Primary outcomes did not change substantially in meta-regressions and sensitivity analyses. Findings were limited by lack of dose equivalence and paucity of data for some outcomes. Conclusions: Based on the available evidence, individual ACEIs and ARBs at goal doses appeared to have no or little differences in their effect on major renal outcomes.

Bifidobacterium infantis NLS Super Strain Reduces the Expression of α-Defensin-5, a Marker of Innate Immunity, in the Mucosa of Active Celiac Disease Patients.

BACKGROUND: We have previously shown a reduction of gastrointestinal symptoms after the oral administration of Bifidobacterium infantis Natren Life Start super strain (NLS-SS) in untreated celiac disease (CD) patients. The symptomatic improvement was not associated with changes in intestinal permeability or serum levels of cytokines, chemokines, or growth factors. Therefore, we hypothesized that the beneficial symptomatic effect observed previously in patients with CD treated with B. infantis may be related to the modulation of innate immunity. GOALS: To investigate the potential mechanisms of a probiotic B. infantis Natren Life Start super strain on the mucosal expression of innate immune markers in adult patients with active untreated CD compared with those treated with B. infantis×6 weeks and after 1 year of gluten-free diet (GFD). METHODS: Numbers of macrophages and Paneth cells and α-defensin-5 expression were assessed by immunohistochemistry in duodenal biopsies. RESULTS: We showed that GFD decreases duodenal macrophage counts in CD patients more effectively than B. infantis. In contrast, B. infantis decreases Paneth cell counts and expression of α-defensin-5 in CD (P<0.001). CONCLUSIONS: The results identify differential innate immune effects of treatment with B. infantis compared with 1 year of GFD. Further studies are needed to investigate synergistic effects of GFD and B. infantis supplementation in CD.

Altered Esophageal Mucosal Structure in Patients with Celiac Disease.

Background/Aim. Reflux symptoms (RS) are common in patients with celiac disease (CD), a chronic enteropathy that affects primarily the small intestine. We evaluated mucosal integrity and motility of the lower esophagus as mechanisms contributing to RS generation in patients with CD. Methods. We enrolled newly diagnosed CD patients with and without RS, nonceliac patients with classical reflux disease (GERD), and controls (without RS). Endoscopic biopsies from the distal esophagus were assessed for dilated intercellular space (DIS) by light microscopy and electron microscopy. Tight junction (TJ) mRNA proteins expression for zonula occludens-1 (ZO-1) and claudin-2 and claudin-3 (CLDN-2; CLDN-3) was determined using qRT-PCR. Results. DIS scores were higher in patients with active CD than in controls, but similar to GERD patients. The altered DIS was found even in CD patients without RS and normalized after one year of a gluten-free diet. CD patients with and without RS had lower expression of ZO-1 than controls. The expression of CLDN-2 and CLDN-3 was similar in CD and GERD patients. Conclusions. Our study shows that patients with active CD have altered esophageal mucosal integrity, independently of the presence of RS. The altered expression of ZO-1 may underlie loss of TJ integrity in the esophageal mucosa and may contribute to RS generation.

Prevalent drug resistance among oral yeasts from asymptomatic patients in Hainan, China.

The oral cavity is a significant niche of the human microbiome and a gateway for the microbiota in many other human body sites. As a result, understanding the oral microbiota has broad implications for the prevention and management of human infectious diseases. Opportunistic yeast infections are among the most prevalent fungal infections of humans, and most opportunistic yeast pathogens are common residents of the oral mucosa. However, relatively little is known about the drug susceptibility profiles of oral yeasts. Here, we report the species distribution and patterns of antifungal susceptibility profiles among 313 yeasts isolated from the oral cavities of 301 asymptomatic hospitalized patients in Hainan Province in southern China. These yeasts were tested for their susceptibilities to the following five drugs: amphotericin B, fluconazole, itraconazole, ketoconazole, and fluorocytosine. Since none of the sampled hosts had taken any antifungal drugs at least 3 months before samples were taken, we hypothesized that little or no drug resistance should be observed. Contrary to our expectations, our analyses identified that 29 % (91/313) of the isolates were resistant to at least one drug and 14.3 % (45/313) were resistant to two or more of the five common drugs. The potential sources of the observed resistance were discussed.

[Immunological analysis of piperaquine-resistant murine model of Plasmodium berghei ANKA strain].

OBJECTIVE: To analyze the immunological characteristics of murine model of piperaquine sensitive (PQS) line and resistant (PQR) line of Plasmodium berghei (Pb) ANKA strain. METHODS: 64 Kunming mice were divided into three groups, 16 in each of groups A and C, 32 in group B (16 of 32 were used for observing survival days). Each mouse in groups A and B was infected with 1 x 10(7) erythrocytic stage parasites of PbPQS and PbPQR, respectively. Mice in group C were injected with the same volume of normal saline. On days 4, 8, 12 and 16 after inoculation, 4 mice from each group were sacrificed. Blood samples were collected for thin blood smear examination, and parasitemia rate calculated. Spleens were removed and spleen lymphocytes suspension prepared. Spleen lymphocytes were stimulated with ConA, and cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Nitrogen oxide (NO) and IFN-gamma level of spleen cell culture supernatants were detected by the Griess reagent and ELISA methods, respectively. Another 10 mice were each inoculated with 1 x 10(7) parasites of PbPQR line, and the mice were then challenged with lethal PQS line when the parasites turned into blue stained cells. The parasitemia and survival days were recorded. RESULTS: The average survival time of group A was (9.0 +/- 3.0) d, the parasitemia rate was over 50% at 6-12 days post- infection with severe anemia. On 16th day post-infection, no death was recorded in group B with a parasitemia rate of (2666 +/- 254) %. After ConA stimulation, the proliferation of spleen lymphocytes in groups A (0.65 +/- 0.08) and B (0.86 +/- 0.20) at 12 days after infection was significantly higher than that of group C (0.18 +/- 0.03) (P < 00.01). NO level in spleen cell culture supernatant increased with prolonged infection time. On 12th day post-infection, NO level of groups A [(48.80 +/- 3.49) micromol/L] and B [(54.80 +/- 2.17) micromol/L] was higher than that of group C [(7.80 +/- 0.71) micromol/L] (P < 0.01). IFN-gamma concentration in spleen lymphocytes culture supernatant increased with prolonged infection time. The highest IFN-gamma level of group A was (752.20 +/- 39.49) pg/ml on 12th day post-infection, while in group B it was (855.80 +/- 33.65) pg/ml on 8th day after infection, then decreased on 12th day [(620.20 +/- 27.11) pg/ml]. IFN-gamma level showed a significant difference between groups A and B (P < 0.01). In 10 days after challenge, the parasitemia rate in PQR group was up to (2.44 +/- 2.07)%, and gradually disappeared. No parasite was detected on 40th day after challenge and no mice died. CONCLUSION: The proliferation of spleen cells, NO and IFN-gamma levels of spleen lymphocytes culture supernatant in PbANKA strain PQR line are significantly higher than that of PQS line. PbPQR line can induce certain protective immunoreaction.

Increased bacterial translocation in gluten-sensitive mice is independent of small intestinal paracellular permeability defect.

AIM: We investigated whether treatment with gliadin induces a paracellular permeability defect that enhances bacterial translocation to mesenteric lymph nodes (MLN) via resident dendritic cells (DC) expressing TLR-2 or 4 in HCD4/HLA-DQ8 transgenic mice. METHODS: HLA-DQ8 transgenic mice were sensitized and subsequently gavaged with gliadin, in the presence or absence of AT1001 (paracellular permeability inhibitor). Non-sensitized mice were gavaged with indomethacin (permeability inducer) or rice cereal. CD11c and CD103 (DC markers) and TLR-2 and 4 were investigated by immunostaining. Intestinal permeability was assessed by paracellular flux of (51)Cr-EDTA in Ussing chambers. Bacterial translocation to MLN was performed by plate counting on aerobic and anaerobic conditions. RESULTS: In gliadin-treated mice, both (51)Cr-EDTA flux in jejunal mucosa and aerobic and anaerobic bacterial counts in MLN were increased (p < 0.05) compared to indomethacin-treated mice and controls. The inhibitor AT1001 normalized (51)Cr-EDTA flux, but had no effect on bacterial translocation in gliadin-treated mice. In addition, changes in mucosal DC marker distribution such as increased (p < 0.05) trans-epithelial CD103(+) cells and reduction (p < 0.05) of CD11c immunostaining were detected in gliadin-treated mice. Moreover, changes in DC markers and TLR-2 or 4 immunophenotypes were not associated. CONCLUSIONS: Pharmacological restoration of paracellular permeability was not sufficient to prevent bacterial translocation in gluten-sensitive mice. We hypothesize that transcellular mechanisms involving CD103(+)DC and CD11c(+)DC may explain in gluten-sensitive HCD4/HLA-DQ8 transgenic mice the sustained increased bacterial translocation observed in the absence of a significant inflammatory response.

Commensal and probiotic bacteria influence intestinal barrier function and susceptibility to colitis in Nod1-/-; Nod2-/- mice.

BACKGROUND: The intestinal microbiota regulates key host functions. It is unknown whether modulation of the microbiota can affect a genetically determined host phenotype. Polymorphisms in the Nucleotide oligomerization domain (Nod)-like receptor family confer genetic risk for inflammatory bowel disease (IBD). We investigated whether the intestinal microbiota and the probiotic strain Bifidobacterium breve NCC2950 affect intestinal barrier function and responses to intestinal injury in Nod1(-/-); Nod2(-/-) mice. METHODS: Specific pathogen-free (SPF) Nod1(-/-); Nod2(-/-) mice and mice gnotobiotically derived with altered Schaedler flora (ASF) biota were used. SPF Nod1(+/-); Nod2(+/-) littermates (generated by crossing SPF Nod1(-/-); Nod2(-/-) and germ-free C57BL/6 mice) and ASF Nod1(+/-); Nod2(+/-) mice were used as controls. SPF mice were gavaged daily with 10(9) -CFU B. breve for 14 days before colitis induction. Denaturing gradient gel electrophoresis (DGGE) and real-time polymerase chain reaction (PCR) were used to assess microbiota composition. Intestinal permeability was assessed by in vitro and in vivo techniques. Expressions of epithelial apical junction proteins, mucin, and antimicrobial proteins were assessed by quantitative reverse-transcription PCR (qRT-PCR) and immunofluorescence. Responses to intestinal injury were investigated using an acute experimental model of colitis. RESULTS: Under SPF conditions, Nod1(-/-); Nod2(-/-) mice had increased paracellular permeability, decreased E-cadherin, and lower colonic antimicrobial RegIII-γ expression compared to Nod1(+/-); Nod2(+/-) littermate controls. These changes were associated with increased susceptibility to colitis. ASF colonization or B. breve supplementation normalized RegIII-γ expression and decreased susceptibility to dextran sodium sulfate (DSS) colitis in Nod1(-/-); Nod2(-/-) mice. CONCLUSIONS: The intestinal microbiota influences colitis severity in Nod1(-/-); Nod2(-/-) mice. The results suggest that colonization strategies with defined commensals or exogenous specific probiotic therapy may prevent intestinal inflammation in a genetically predisposed host.

Sensitization to gliadin induces moderate enteropathy and insulitis in nonobese diabetic-DQ8 mice.

Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25(+)Foxp3(+) T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4(+) T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4(+) T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25(+)Foxp3(+) T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency.