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.     

Antigen-specific desensitization of patients allergic to penicillin

Three penicillin-allergic patients with life-endangering infections requiring β-lactam antibiotic therapy were desensitized by means of increasing oral then parenteral doses and were treated with full doses of β-lactam agents. Malignant otitis externa caused by Pseudomonas aeruginosa, osteomyelitis caused by Staphylococcus aureus, and bacterial endocarditis caused by an enterococcus were treated with carbenicillin, nafcillin, and benzylpenicillin G, respectively. No acute allergic reactions occurred during desensitization or within 1 wk of the onset of therapy. Immediate wheal and flare skin-test reactions to β-lactam determinants diminished or became negative after the desensitization procedure in each patient. Wheal and flare responses provoked by histamine, compound 48/80, and environmental antigens were not affected by the desensitization procedure or continued β-lactam drug therapy. Mild urticaria appeared after 15 days of penicillin therapy in one patient and after 23 days of carbenicillin therapy in another patient. Skin-test reactions to penicillin reagents had reverted to positive at the time of the urticarial reactions. One patient developed a severe immune hemolytic anemia after 10 days of therapy with nafcillin. The results of this study indicate that acute clinical desensitization of these three penicillin-allergic patients was associated with antigen-specific desensitization of tissue mast cells.     

蝎蜂毒肽对大鼠纤溶系统作用初探

本研究采用大鼠肢体血管灌流和整体给药两种模型，观察蝎蜂毒（ＳＢＰ）对血管理灌流液内纤溶酶原激活物（ＰＡ）活性、血浆优球蛋白纤溶性（ＥＦＡ）和纤溶酶（ＰＬ）活性的影响。结果说明，ＳＢＰ有明显激活纤溶系统作用；其机制可能涉及血管内皮细胞释放ＰＡ活性增加，进一步促使纤溶酶原活化为ＰＬ增多的途径。     

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.     

白细胞介素2和黄芪多糖对小鼠NK细胞作用的细胞化学和超微结构观察

本文报道在白细胞介素2(IL-2)及黄芪多糖(PA)作用下,小鼠NK细胞的细胞化学和超微结构变化。实验表明,NK细胞酸性磷酸酶、酸性醋酸酯酶及氯醋酸AS-D酯酶均为阳性。经IL-2和PA的处理,前两种酶的活性增加,阳性细胞百分率增高,并与NK活性相关。IL-2培养后的NK细胞超微结构表现为胞体变大,表面不规则,胞质丰富,细胞器增多等特点。     

Increased antigen-induced local and systemic mediator release in rhinitis subjects with pulmonary symptoms in the pollen season

In order to delineate parameters that might discriminate between allergic subjects who develop R or R-P symptoms during natural antigen exposure, 26 subjects allergic to grass or ragweed pollen were classified into R or R-P groups, and then the antigen sensitivity and degree of in vivo mediator release were compared. Antigen-skin sensitivity was quantitated by dilutional skin-test titration, and bronchial sensitivity was quantitated by the amount of inhaled antigen required to receive the FEV1 by 20%. Mediator release was determined by measuring the amount of histamine that was released into skin chambers during antigen incubation and the rise in plasma histamine and serum NCA during antigen-induced bronchospasm. Compared to the 13 R subjects, the 13 R-P subjects were: (1) more sensitive to antigen by both skin-test and inhalation challenge, (2) responded to inhalation of antigen with a greater fall in FEV1 and a greater rise in serum NCA and plasma histamine, and (3) released more histamine into skin chambers after antigen incubation. Even when R and R-P subjects were matched by comparing only subjects with equal skin sensitivity to antigen, greater increases in serum NCA and plasma histamine occurred after inhalation of antigen in the R-P subjects. These data are consistent with the hypothesis that allergic rhinitis subjects who develop pulmonary symptoms during natural pollen exposure are more sensitive to antigen and release more mediators in response to antigen administration. It is therefore possible that the degree of mediator release may be an important factor in determining the pattern of clinical responses to antigen exposure.     

Quality validation of platelets obtained from the Haemonetics and Trima Accel automated blood-collection systems

BackgroundPlatelet transfusion is required to treat haemo-oncology or trauma patients. Platelet apheresis (PA) performed with apheresis equipment has increased rapidly in recent years. Leucocyte-reduced platelet apheresis (LRPA) can reduce the risk of platelet refractoriness and febrile nonhemolytic transfusion reactions (FNHTRs) for transfusion. Accordingly, this study aimed to investigate and compare the platelet metabolic and functional responses between PA performed with Haemonetics and LRPA performed with Trima Accel cell separator.MethodsThe qualities of platelets collected through PA and LRPA were evaluated in terms of visual appearance, morphology, platelet-aggregation changes, metabolic activities, and bacterium-screening test during 5-day storage. Statistical analyses included two-sample t-test and generalised estimating equation(GEE) method.ResultsDuring 5-day storage in LRPA, residual leucocytes were all <1.0×10⁶, and the parameters of platelet function were as follows: platelet aggregated to agonists such as adenosine 5′-diphosphate (ADP) and collagen, and the extent of shape change and pO₂ showed no statistically significant difference between PA and LRPA. The hypotonic shock reaction (HSR) on days 0, 1, and 3 were significantly higher in LRPA than in PA (71.78±6.92 vs. 64.10±7.42; P=0.002; 71.53±8.98 vs. 62.96±9.84; P=0.007; 68.05±7.28 vs. 57.76±6.80; P<0.0001, respectively). Values of mean platelet volume (MPV) were statistically larger in PA than in LRPA on days 0, 1, and 3. On day 5, the swirling score was higher in LRPA than in PA. The mean lactate levels had no statistically significant difference between PA and LRPA. Moreover, no growth was observed through bacterium-screening test conducted on 40 samples.ConclusionComparison of LRPA and PA products collected from the Trima Accel and Haemonetics automated blood-collection systems, respectively, revealed that both products possessed good platelet qualities even though additional processes are needed to reduce leucocytes. Furthermore, investigating the outcomes of other apheresis instruments with focus on the safety of donors, products, and recipients is necessary.