Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay

Temperature studies of microtitration plates demonstrate that the use of a common bacteriology incubator for heating the plates can cause a phase lag of over 30 min for the fluid in the wells to reach 37°C from ambient temperature, and that a temperature gradient of as much as 1.6°C can exist between the peripheral and center wells. This gradient is a cause of the ‘rim’ or edge effect noted in enzyme immunoassay using microtitration plates. The problem is corrected by the use of a specially designed forced air microtitration plate incubator.     

Effects of theophylline,terbutaline, and prednisone on antigen-induced bronchospasm and mediator release

Eleven subjects demonstrating clinical, skin, and inhalation sensitivity to grass or ragweed pollen underwent serial inhalation challenges, with and without orally administered theophylline, terbutaline, and prednisone. Comparisons of antigen sensitivity and mediator release were made during these challenges. All three drugs significantly reduced antigen sensitivity (PD₂₀ inhalation units increasing from 670 to ≧ 3,280). Peak plasma histamine levels after antigen challenge decreased from 11.4 ng/ml to ≦ 3.4 ng/ml during all drug administrations. Similarly, the percent increase in serum neutrophil chemotactic activity (NCA) also decreased, from 96% to ≦ 36% during drug administrations. However, even at antigen doses resulting in bronchospasm during drug administration the systemic appearance of NCA and histamine were reduced. We conclude that prednisone, theophylline, and terbutaline significantly reduce antigen-induced bronchospasm and mediator release. The occurrence of bronchospasm despite the inhibition of histamine and NCA suggests either that the local concentration of these mediators are critical or that other mediators produce the bronchospasm observed.     

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.     

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

A hallmark of epithelial cells is polarization, which is achieved by the orchestration of external cues, such as cellular contact, extracellular matrix, signal transduction, growth factors, and spatial organization.¹ Hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell.² The apical poles of adjacent hepatocytes form a continuous network of bile canaliculi into which bile is secreted, whereas the basolateral membrane domain forms the sinusoidal pole, which secretes various components, such as proteins or drugs, into the blood circulation.³ Loss of hepatic polarity has been associated with several cholestatic and developmental disorders, including progressive familial intrahepatic cholestasis (PFIC) and primary sclerosing cholangitis (PSC).^4,5 Although the molecular mechanisms governing hepatocyte polarity have been extensively studied in the in vitro systems, there is still a significant gap in our understanding of how polarity is established within the context of tissue during development or maintained during homeostasis.^6,7 Similarly, the molecular pathways contributing to hepatic polarity are not entirely understood, and a better comprehension of hepatic polarity regulation is thus warranted.Previous studies have confirmed the role of hepatocellular junctions, such as tight and gap junctions, in the maintenance of hepatocyte polarity.^8,9 Studies done in vitro and in vivo have shown that loss of junctional proteins, such as zonula occludens protein (ZO)-1, junctional adhesion molecule-A, and claudins, lead to impairment of polarity and distorted bile canaliculi formation.10, 11, 12, 13 In addition, proteins involved in tight junction assembly, such as liver kinase B1, are also involved in polarity maintenance.¹⁴ Among adherens junction proteins, various in vitro cell culture models have confirmed the role of E-cadherin in the regulation of hepatocyte polarity, possibly through its interaction with β-catenin.^15,16 However, there is a lack of an in vivo model to study the role of adherens junction proteins in hepatocyte polarity and their misexpression contributing to various liver diseases.β-Catenin plays diverse functions in the liver during development, regeneration, zonation, and tumorigenesis.17, 18, 19 The relative contribution of β-catenin as part of the adherens junction is challenging to study because like in other tissues, γ-catenin compensates for the β-catenin loss in the liver.^20,21 To address this redundancy, we previously reported a hepatocyte-specific β-catenin and γ-catenin double-knockout (DKO) mouse model was reported.²² Simultaneous deletion of β-catenin and γ-catenin in mice livers led to cholestasis, partially through the breach of cell-cell junctions. However, more comprehensive understanding of the molecular underpinnings of the phenotype is needed.In the current study, prior preclinical findings of dual β-catenin and γ-catenin loss were extended to a subset of PFIC and PSC patients. In vivo studies using the murine model with hepatocyte-specific dual loss of β-catenin and γ-catenin showed complete loss of hepatocyte polarity compared to the wild-type controls (CONs). Loss of polarity in DKO liver was accompanied by epithelial-mesenchymal transition (EMT), activation of transforming growth factor (TGF)-β signaling, and reduced expression of hepatocyte nuclear factor 4α (HNF4α). Our findings suggest that β-catenin and γ-catenin and in turn adherens junction integrity, are critical for the maintenance of hepatocyte polarity, and any perturbations in this process can contribute to the pathogenesis of cholestatic liver disease.     

Differential effects of interleukin 12 and interleukin 10 on superantigen-induced expression of cutaneous lymphocyte-associated antigen (CLA) and alphaEbeta7 integrin (CD103) by CD8+ T cells

At both cutaneous and mucosal sites, interleukin (IL)-10, IL-12 and transforming growth factor (TGF)-beta are important regulators of chronic inflammatory disease, where cutaneous lymphocyte-associated antigen (CLA) and alphaE integrin (CD103) may be expressed. Stimulation with streptococcal pyrogenic exotoxin C (SpeC) increased the expression of CD103 by CD8+ but not CD4+ T cells. While adding IL-12 augmented the expression of CLA, superantigen-induced expression of CD103 was markedly suppressed by IL-12, which could be reversed by TGF-beta. Antibodies against TGF-beta inhibited, and a combination of anti-TGF-beta and IL-12 completely abrogated the induced CD103 expression. IL-10 strongly decreased the frequency of CLA+ and although not increasing the frequency of CD103+CD8+ T cells, the amount of CD103 expressed per cell was markedly increased. Thus, the expression of CLA and CD103 may be antagonistically regulated by IL-10 and IL-12 and the balance between these cytokines could influence the T cell migration of inflammatory cells into epithelial tissues.     

Role of ubiquitin-mediated proteolysis in the pathogenesis of neurodegenerative disorders

Intraneuronal inclusions containing ubiquitylated filamentous protein aggregates are a common feature of many of the major human neurodegenerative disorders, including Alzheimer's and Parkinson's disease. Loss of function mutations in enzymes of the ubiquitin conjugation/deconjugation pathway are sufficient to cause familial forms of neurodegenerative diseases, suggesting that failure of ubiquitin-mediated proteolysis could also be central to inclusion formation in the more common sporadic cases. Examination of ubiquitin-positive inclusions at the protein level provides evidence of attempted proteasomal proteolysis, however close inspection of the temporal aspects of inclusion formation indicates that ubiquitylation is probably a late event. In this regard, the presence of ubiquitin within inclusions of idiopathic neurodegenerative disorders may indicate not a primary dysfunction of ubiquitin-mediated proteolysis, but rather a secondary, presumably protective cellular response. Within this model, other factors are likely to be initiating in inclusion biogenesis. Consistent with these proposals, non-ubiquitylated forms of the principal ubiquitylated components of Alzheimer's disease neurofibrillary tangles and Parkinson's disease Lewy bodies, tau and alpha-synuclein proteins, respectively, can be degraded by proteasomes in a pathway which does not have an absolute requirement for ubiquitylation. Inhibition of proteasome function in the pathological state, as has been reported in both Alzheimer's and Parkinson's disease, could therefore contribute both to accumulation of non-ubiquitylated forms of aggregation-prone neuronal proteins, as well as impaired clearance of ubiquitylated aggregates.     

^99mTc-DTPA肾动态显像及β2-m水平在评价肾移植受者肾功能中的价值

目的：探讨^99mTc-DTPA肾动态显像半定量参数和血、尿β2-m水平测定对肾移植术后早期并发症诊断与鉴别诊断的价值。方法：28例肾移植病人术后均进行放射性核素^99mTc-DTPA肾动态显像,同时测定移植肾的肾小球滤过率（GFR）、膀胱放射性计数与移植肾放射性计数比值（B/K值）和移植肾放射性1min计数与腹主动脉放射性1min计数比值（K1min/A1min比值）。在进行放射性核素肾动态显像前所有病人均收集其血液和尿液标本,采用放射免疫分析测定血、尿β2-m水平。结果：12例肾功能正常者肾动态显像示肾血流灌注及功能良好,GFR值为（49.1±6.1）ml/min,B/K值均〉3,K1min/A1min比值为8.18±1.41;4例急性排斥反应者肾血流灌注受损程度重于功能相,GFR值为（33.2±5.3）ml/min,B/K值均〈1,K1min/A1min比值为2.59±0.86,β2-m水平以血β2-m升高明显;8例慢性排斥反应者肾血流灌注和功能相均同时受损,GFR值为（19.8±7.5）ml/min,B/K值均〈1,K1min/A1min比值为2.19±0.84,β2-m水平也以血中升高明显;2例肾小管坏死者及2例环孢素A肾中毒者肾血流灌注受损均轻于功能相,GFR值分别为（38.5±4.1）ml/min和（39.4±5.81）ml/min,B/K值均〈1,K1min/A1min比值分别为5.83±0.84和6.01±0.66,β2-m水平以尿中升高显著。结论：放射性核素肾动态显像半定量参数K1min/A1min比值和B/K值,结合肾移植病人术后血、尿β2-m水平联合分析可早期初步鉴别排斥反应的类别,可作为判断移植肾受损程度、原因及预后估测的敏感指标。     

Localization of class i histocompatibility molecule assembly by subfractionation of the early secretory pathway

Class I molecules of the major histocompatibility complex bind peptides derived from cytosolic proteins and display them on the cell surface. This function alerts cytotoxic T cells to the presence of intracellular pathogens. Class I molecule assembly requires the association of the heavy chain with β₂-microglobulin, accompanied by peptide loading via specific transporters. This study localizes where these assembly steps take place, using monoclonal antibodies recognizing class I molecules in different assembly states to analyze subcellular fractions of the early secretory pathway. The distribution of peptide-loaded class I molecules was more localized than the distribution of the total pool of class I molecules in the early secretory pathway. Loaded molecules colocalized with the peptide transporter, free heavy chains, and the chaperone calnexin in high density rough endoplasmic reticulum (RER) membranes. These data suggest that subunit assembly and peptide acquisition occur at the same intracellular site. Class I molecules also localized to less dense subfractions of the early secretory pathway, which contained comparatively less peptide-loaded molecules than the high density RER fractions, at steady state. Following a 15 °C temperature block, class I molecules accumulated in these less dense membrane fractions, indicating that these fractions represent the intermediate compartment where empty class I molecules are trapped in mutant cells. In the presence of cycloheximide, a pool of class I molecules recycling to the RER was detected, suggesting empty molecules recycle to acquire peptide.