Lactobacillus acidophilus Induces a Strain-specific and Toll-Like Receptor 2–Dependent Enhancement of Intestinal Epithelial Tight Junction Barrier and Protection Against Intestinal Inflammation

Defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease. To date, no effective therapies that specifically target the intestinal TJ barrier are available. The purpose of this study was to identify probiotic bacterial species or strains that induce a rapid and sustained enhancement of intestinal TJ barrier and protect against the development of intestinal inflammation by targeting the TJ barrier. After high-throughput screening of >20 Lactobacillus and other probiotic bacterial species or strains, a specific strain of Lactobacillus acidophilus, referred to as LA1, uniquely produced a marked enhancement of the intestinal TJ barrier. LA1 attached to the apical membrane surface of intestinal epithelial cells in a Toll-like receptor (TLR)-2–dependent manner and caused a rapid increase in enterocyte TLR-2 membrane expression and TLR-2/TLR-1 and TLR-2/TLR-6 hetero-complex–dependent enhancement in intestinal TJ barrier function. Oral administration of LA1 caused a rapid enhancement in mouse intestinal TJ barrier, protected against a dextran sodium sulfate (DSS) increase in intestinal permeability, and prevented the DSS-induced colitis in a TLR-2– and intestinal TJ barrier–dependent manner. In conclusion, we report for the first time that a specific strain of LA causes a strain-specific enhancement of intestinal TJ barrier through a novel mechanism that involves the TLR-2 receptor complex and protects against the DSS-induced colitis by targeting the intestinal TJ barrier.

Intestinal epithelial tight junctions (TJs) are the apical-most junctional complexes and act as a functional and structural barrier against the paracellular permeation of harmful luminal antigens, which promote intestinal inflammation.¹ The increased intestinal permeability caused by defective intestinal epithelial TJ barrier or a leaky gut is an important pathogenic factor that contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) and other inflammatory conditions of the gut, including necrotizing enterocolitis and celiac disease.^2,3 Clinical studies in patients with IBD have found that a persistent increase in intestinal permeability after clinical remission is predictive of poor clinical outcome and early recurrence of the disease, whereas normalization of intestinal permeability correlates with a sustained long-term clinical remission.4, 5, 6 Accumulating evidence has found that a defective intestinal TJ barrier plays an important role in exacerbation and prolongation of intestinal inflammation in IBD. Currently, no effective therapies that specifically target the tightening of the intestinal TJ barrier are available.Intestinal microbiota play an important role in modulating the immune system and in the pathogenesis of intestinal inflammation.⁷ Patients with IBD have bacterial dysbiosis in the gut, characterized by a decrease in bacterial diversity and an aberrant increase in some commensal bacteria, which are an important factor in the pathogenesis of intestinal inflammation.^8,9 Normal microbial flora of the gastrointestinal tract consists both of bacteria that are known to have beneficial effects (probiotic bacteria) on intestinal homeostasis and bacteria that could potentially have detrimental effects on gut health (pathogenic bacteria).¹⁰ The modulation of intestinal microflora affects the physiologic and pathologic states in humans and animals. For example, fecal transplantation from healthy, unaffected individuals to patients with refractory Clostridium difficile colitis is curative in up to 94% of the treated patients, and transfer of stool microbiome from obese mice induces obesity in previous lean mice, whereas transfer of microbiome from lean mice preserves the lean phenotype.11, 12, 13 The beneficial effects of gut microbiota are host and bacterial species-specific.¹⁴ Although multiple studies indicate that some commensal bacteria play a beneficial role in gut homeostasis by preserving or promoting the intestinal barrier function, because of conflicting reports, it remains unclear which probiotic species cause a persistent predictable enhancement in the TJ barrier and could be used to treat intestinal inflammation by targeting the TJ barrier. For example, some studies suggest that Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus cause a modest enhancement in the intestinal epithelial TJ barrier, whereas others have found minimal or no effect of these probiotic species on the intestinal TJ barrier.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 The major aim the current study was to perform a high-throughput screening of Lactobacillus and other bacterial species to identify probiotic species that induce a rapid, predictable, and marked increase in the intestinal epithelial TJ barrier and protect against the development of intestinal inflammation by preserving the intestinal TJ barrier.In the studies described herein, most of the probiotic species tested (>20 species or strains) had a modest or minimal effect on intestinal TJ barrier function. L. acidophilus uniquely caused a rapid and marked increase in intestinal TJ barrier function. Further analysis indicated that the effect of L. acidophilus was strain-specific, limited to a specific strain of L. acidophilus, and did not extend to other L. acidophilus strains. The L. acidophilus enhancement of the intestinal TJ barrier was mediated by live bacterial-enterocyte interaction that involved Toll-like receptor (TLR)-2 heterodimeric complexes on the apical membrane surface of intestinal epithelial cells. Our animal studies also found that L. acidophilus causes a marked enhancement in mouse intestinal barrier function and protects against the dextran sodium sulfate (DSS)–induced colitis by preserving and augmenting the mouse intestinal barrier function in a strain-specific manner.     

A NEW METHOD FOR DETECTING EYE MOVEMENT IN SLEEP

A new method of detecting eye movements (EMs) during sleep is described. The method consists of an electromechanical measurement using micro-miniaturized silver cup electrodes. These electrodes, when placed on the eyelid, produce electro-oculographic (EOG) recordings similar to the usual electrical method. The eyelid method offers the advantage of a relatively “clean” recording showing only EMs and movement artifact, with no intermingling of EEG and EOG. Furthermore, the method is at least one and one half times as sensitive as the usual EOG technique. In addition to these two special advantages, it also offers the features of conventional methods, convenience of DC coupling, independence from signal converters, ease of analyzing EM directionality, and durability despite the small size of the electrodes. Fabrication of the electrodes, recording configurations, and simultaneous comparisons to both the usual EOG technique and to a strain gauge method are described.     

C to T transition at the first nucleotide of codon 63 of the β-globin gene corresponding to hemoglobin M-Saskatoon in an Indonesian boy

Summary Hemoglobin (Hb) M-Saskatoon, a variant of methemoglobin, is characterized by mild hemolysis. It is caused by the substitution of a histidine by a tyrosine at the 63rd amino acid residue of the -globin chain. Amplification and sequence analysis of genomic -globin DNA from an Indonesian boy diagnosed as having the more severe disease thalasemia demonstrated the presence of a C to T transition at nucleotide 473 in one of the two -blogin genes resulting in a histidine to tyrosine substitution at 63rd residue. This amino acid change matched with that reported in Hb M-Saskatoon. This nucleotide change abolished a recognition site for the restriction endonucleaseNlaIII.NalIII digestion of the corresponding -globin DNA amplified from the patient's parents indicated that the mutation was inherited through from his mother. This result shows that the world-wide distribution of Hb M-Saskatoon extends to Indonesia, where it was not previously identified. Possible causes of the unusually severe symptoms observed in the case are discussed.     

Nasal congestion as a side effect of cromolyn sodium

Cromolyn sodium, a recently introduced antiasthmatic medication, is known occasionally to produce maculopapular and urticarial rashes, and increased bronchospasm. A patient is reported with a previously unpublished side effect of this medication, i.e., severe nasal congestion.     

一起戊型肝炎暴发的血清抗体比较研究

目的评价戊型肝炎(戊肝)抗体E2-IgM(抗-HEV E2-IgM)酶联免疫试剂(捕获法)在反映戊肝流行特征和对戊肝早期诊断中的作用.方法在首发病例26 d后对某单位戊肝暴发人群和相邻单位对照人群进行血清抗-HEV E2-IgM、IgG检测;部分人群进行美国Genelabs抗-HEV IgM、IgG的平行检测.结果抗-HEV E2-IgM试剂捕获法在对照人群中仅检出1例阳性(0.11%),且阳性和阴性之间可明显区分,其特异度为99.89%;在暴发流行人群中检出145例阳性(8.66%),显著高于对照人群(P＜0.001);暴发人群中戊肝患者的血清学动态变化为抗-HEV E2-IgM在暴露时间为30～60d时保持较高吸光度(A值),随着时间的延长A值呈明显下降趋势;抗-HEV E2-IgM阳性在该组人群中与性别和年龄无关;在暴发人群抗-HEV E2-IgM(+)的115例患者中,Genelabs抗-HEV IgG检测出88份阳性,检出率为76.52%,漏检27例,漏检率为23.48%.对照人群随机抽样179例患者中有20例Genelabs抗-HEVI IgG(+),阳性率为11.17%.在110份抗-HEV E2-IgM(+)的血样中,Genelabs抗-HEVIgM只检测出76份,检出率为69.09%;有16例患者Genelabs抗-HEV IgG、IgM均未能检出;Genelabs抗-HEV IgG和IgM的不一致率为25.45%.结论抗-HEV E2-IgM具有99%左右的特异度,与在正常人群中检出较高阳性率的Genelabs抗-HEV IgG试剂相比,减少了假阳性;抗-HEV E2-IgM与Genelabs抗-HEV IgM、IgG相比,对急性戊肝感染诊断的灵敏度提高了25%～30%,减少了漏诊;抗-HEV E2-IgM试剂盒操作简单、快速,本底较好,不存在酶结合物不足的问题.     

CⅡTA M1-RNA对MHCⅡ类分子表达的抑制作用

目的探讨MHCⅡ类分子转录激活因子（CⅡTA）的M1-RNA对细胞表面MHCⅡ类分子表达的抑制。方法M1-RNA是核糖核酸酶P的催化活性单位，设计并克隆针对CⅡTA第452、629位点的M1-RNA（分别为M1-452-GS、M1-629-GS）及其相应的CⅡTA靶基因，分别插入pUC19、pGEM-7zf（＋）载体，进行细胞外切割活性筛选。将细胞外切割作甩明显的M1-629-GS亚克隆入psNAV载体（psNAV-M1-629-GS，pA629）并稳定转染ECV304细胞株，流式细胞术检测经典的MHCⅡ（HLA-DR、-DP、-DQ）类抗原表达，RT-PCR检测CⅡTA的mRNA水平。结果pA629阳性ECV304细胞株与对照组比较，HLA-DR、-DP抗原表达分别降低了89．21％及92．31％；同时CⅡTA的mRNA含量降低（P〈0．05）。结论CⅡTA的M1-RNA（M1-629-GS）降低了自身mRNA含量，从而阻止其调控的MHCⅡ类分子的表达。     

A phage display system for detection of T cell receptor-antigen interactions

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCRα chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCRα chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the α chains were derived. Cross-specificity analysis using two antigen-binding TCRα chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCRα-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

Nonrandom chromosomal changes in untreated retinoblastomas

The karotypic patterns of 15 retinoblastomas were examined. Five tumors were found to have two distinct stem lines and, therefore, the chromosomal patterns of 20 tumor cell lines are reported. Three nonrandom chromosomal changes, namely, a loss of a chromosome #13, the presence of an i(6p), or a trisomy of 1q were observed. The potential importance of these chromosomal changes in tumor development is discussed, particularly the loss of a chromosome #13 or the gain of an i(6p). At least one of the three chromosomal changes was found in 75% of the tumor lines analyzed.     

Induction of specific anti-actin antibodies and their measurement by ELISA technique.

Using the ELISA technique we have been able to quantify antibodies directed against actin and to follow the kinetics of antibody production. Specific anti-actin antisera have been raised in rabbits by immunization with chemically modified white muscle rabbit actin. Two or three dinitrophenyl groups linked per actin molecule were sufficient to break natural tolerance, while linkage of three phosphorylcholine groups to actin was not.