Inhaled β2 -agonists and airway responses to allergen

A series of investigations show that the regular use of inhaled β₂ -agonists will increase all aspects of the airway response to allergen. The mechanism of this effect is uncertain; however, it appears to be different from the mechanism that produces tolerance to β₂ -agonist effects. One possibility is that the regular use of β₂ -agonists might induce a mast cell β-receptor dysfunction that might make mast cells more prone to release mediators. As a result β₂ -agonist use plus allergen exposure might cause more mediator release than does allergen exposure alone. The corollary of this is that β₂ -agonist use plus allergen exposure might cause more airway inflammation than does allergen exposure alone. These hypotheses are both testable. I believe that this is a clinically important phenomenon and may well be a major reason for β₂ -agonist–induced worsened asthma control. Further investigations are indicated to identify the mechanism and the clinical relevance of the phenomenon. (J Allergy Clin Immunol 1998;102:S96-9)     

Clearance of p-cresol sulfate and β-2-microglobulin from dialysate by commercially available sorbent technology

Dialysate regeneration by sorbents is an alternative to conventional single-pass dialysis. Little is known about the capacity of sorbents to clear dialysate of “middle molecules” and protein-bound uremic toxins. We studied p-cresol sulfate (PCS) and β-2-microglobulin (β2M) removal from dialysate by a sorbent: 1. PCS (40 mg PCS dissolved in 4 L of fresh dialysate) was recirculated through a sorbent cartridge (SORB Technology, Inc.) for analysis of PCS removal. 2. Spent peritoneal dialysate was recirculated on the “blood” side of a high-flux dialyzer. On the “dialysate” side of the membrane, bicarbonate dialysate was recirculated through a sorbent cartridge. β2M was measured in both streams. Two results are of particular importance for the use of regenerated fluid in chronic dialysis: 1. PCS was virtually completely removed from the dialysate. On average, PCS concentration was reduced to 1.4% of the starting concentration after 60 minutes. PCS extraction across the sorbent was nearly complete at any time. 2. β2M was on average reduced to 14.3% of the starting concentration after 60 minutes. Postsorbent concentrations were consistently below the validated range of the test method. We conclude that PCS and β2M are efficiently removed from the dialysate by commercially available sorbent technology. Spent peritoneal dialysis fluid can be cleared of β2M when circulated against sorbent-regenerated dialysate using a high-flux membrane.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies

The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus—TMN) control wakefulness, and their silencing through activation of GABA_A receptors (GABA_AR) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the β1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA_AR in TMN neurons. In recombinant receptors containing the β3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and β3N265M mice, FDD and propofol, we explored the relative contributions of β1- and β3-containing GABA_AR to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In β3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA_AR by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed β1-type pharmacology, with the same effects achieved by 3 μM propofol and 10 μM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 μM, and in all cells at 10 and 250 μM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1–6 μM) or significantly reduced (12 μM) in β3N265M mice, whereas gating and modulation of GABA_AR by PI24513 as well as modulation by propofol were unchanged. In conclusion, β1-containing (FDD-sensitive) GABA_AR represent the major receptor pool in TMN neurons responding to GABA, while β3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA_AR types.     

Presence of β2-Microglobulin on B- and T-Cells and Lymphocytotoxicity of Anti-β2-Microglobulin Sera

Isolated normal lymphocytes and those of patients with chronic lymphocytic leukemia were shown by immunofluorescence to bear β₂-microglobulin in or on their membranes. Isolated thymocytes displayed similar membrane-associated fluorescence. These findings indicate that both B- and T—cells carry B₂-microglobulin in or on their membranes. Antisera against β₂-microglobulin were found to be cytotoxic in the presence of complement to normal and tissue culture lymphocytes. The presence of β₂-microglobulin found on cells other than B- and T-lymphocytes and the possible function(s) of β₂-microglobulin is discussed.     

Interferon β-1a and β-1b for patients with multiple sclerosis: updates to current knowledge

Introduction: Although multiple sclerosis (MS) remains incurable, interferon beta (IFNβ) has been at the forefront of treatment for many years. Different formulations of IFNβ allow for different levels of exposure: low-dose/frequency with some agents, and high-dose/frequency with others.

Areas covered: This review article discusses existing and emerging efficacy and safety data for IFNβ in MS. Clinical evidence of IFNβ efficacy has been generated and accumulated over many decades. During this time, key clinical trials have demonstrated the benefits of high-dose and/or high-frequency dosing of IFNβ-1a or β-1b, compared with lower levels of exposure, on outcome measures such as relapse rates, disability progression, disease progression and magnetic resonance imaging lesion outcomes.

IFNβ therapy is well tolerated and has one of the best characterized safety profiles of all first line therapies. The overall severity of adverse events (AEs) does not appear to be affected by different IFNβ exposures. Typical AEs that patients may experience with IFNβ are mild, reversible and manageable.

Expert commentary: IFNβ is one of the best characterized treatments for MS, with a large body of clinical and real-world evidence supporting the risk-benefit profile. High-dose/frequency regimens may provide better long-term outcomes.     

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases.     

Expression of β2 integrins and macrophage-associated antigens in meningeal tumours

This study assessed the expression of leukocyte integrins and macrophage-associated antigens in meningiomas. Fourteen benign meningiomas, ten atypical/anaplastic meningiomas, two hemangiopericytomas and one solitary fibrous tumour (SFT) were included. Frozen sections were immunostained using antibodies directed against leukocyte integrins, CD68, CD14, CD2, CD1a, DRC1 and CD34. Their expression was evaluated semi-quantitatively. Ki67 positive cells were counted. Arachnoid membranes served as controls. Arachnoid cells expressed the β2-integrin subunit and KP1. Beta2 was detected in the tumour cells of 14 meningiomas. In nine cases, this was associated with an α-integrin subunit. There was no statistical difference in the expression of β2 between benign and atypical/anaplastic meningiomas. KP1 was constantly expressed by the tumour cells of meningiomas. It was not expressed by other meningeal tumours. CD34 was detected in the fibrous meningiomas, hemangiopericytomas and the SFT. In each tumour, macrophages were more numerous than T lymphocytes. There was no statistical difference in the density of macrophages and T lymphocytes between the benign and atypical/anaplastic meningiomas. There was no correlation between the Ki67 proliferation index and macrophage infiltration. Meningiomas, through the expression of leukocyte antigens, have a very particular phenotype. The expression of β2 integrins could play a role in the attraction of immunocompetent cells in the stroma of meningiomas. Received: 14 April 1999 / Accepted: 15 July 1999     

Polymorphism within the nuclear and 2μm genomes of Saccharomyces cerevisiae

Summary Seven strains of bakers' yeast were obtained as a representative sample of the Spanish baking industry. The nuclear genome was monitored for polymorphism by transverse alternating field electrophoresis (TAFE) and restriction maps of 2 m DNA were produced. All seven strains were uniquely different when evaluated by their total chromosomal lengths whereas only two 2 m variants were defined. There was no apparent correlation between chromosomal and plasmid polymorphism. The extensive chromosomal polymorphism within one 2 m DNA type indicates the rapid and relatively recent evolution of the nuclear genome. The hybrid origin (S. cerevisiae-S.monacensis) of lager yeast was critically evaluated by TAFE analysis of S. cerevisiae and S. carlsbergensis chromosomes. The absence of corresponding S. cerevisiae chromosomes III and XIII in S. carlsbergensis argued against the hybrid origin of lager strains. We discuss limitations of the hybrid origin hypothesis of industrial yeasts and propose that the molecular coevolution observed in 2 m DNA serves as a useful additional mechanism for rationalization of some of the structural polymorphism of the nuclear genome.     

Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.     

Construction and expression of lentiviral vector of rat β-defensin-2 gene

Objective To construct a lentiviral expression vector of rat β-defensin-2(rBD2)gene, and examine its expression by transfected cultured cells,in order to lay the foundation for experiments in vivo.Methods The totaI RNA of rat epithelial ceils was extracted and rBD2 gene was got with PCR amplification.After double-digested and connected the PCR production and lentiviral vector Lentivirus [containing H1 promoter and green fluorescent protein(GFP)],the lentiviral expression vector of rBD2 gene LV-rBD2 was constructed and confirmed by sequencing.The virus-like particles of LV-rBD2 was packed with lentiviral packaging systems and viral titer was determinated by slow-gradient dilution.Expression of rBD2 was tests with RT-PCR and Western Blot after cultured cells had been infected by LV-rBD2.Results The results of gel electrophoresis and DNA sequencing showed that the rBD2 gene was cloned into the lentiviral vector,the sequence is correct.The lentiviral vector particle packaging was complete.the virus titer was adjusted to 1×105 ifu/μl.RT-PCR and Western-blot showed that rBD-2 gene was expressed.Conclusion The lentiviral expression vector of rBD2 gene LV-rBD2 was constructed successful,and could transfecte cells to express rBD2.     

Cooperativity Between Selectins and β2-Integrins Define Neutrophil Capture and Stable Adhesion in Shear Flow

A cooperative, sequential process of molecular recognition governs leukocyte capture, rolling, and arrest on inflamed endothelium. Flowing neutrophils are captured via heterotypic adhesive interactions mediated by endothelial E-selectin, whereas homotypic interactions between neutrophils are mediated by L-selectin. To elucidate how each selectin facilitates the transition to CD18-mediated stable adhesion, E-selectin and L-selectin were expressed at defined site density in a murine pre-B-cell line. Direct observation of two-body collisions revealed that 30% of neutrophil interactions with E-selectin transfectants formed doublets at low shear rate G = 14 s(-1) whereas a threshold shear rate 14 s(-1) < or = G < or = 10 s(-1) was necessary for L-selectin adhesion. Adhesion via L-selectin resisted rupture at high shear stress, while E-selectin tethered doublets remained intact longer once formed. Moreover, higher expression of L-selectin (1100 sites/microm2) than that of E-selectin (220 sites/microm2) was required for comparable heterotypic adhesion efficiency. With a threefold rise in active CD18 upregulated on chemotactically stimulated neutrophils, homotypic adhesion efficiency increased 10-fold compared to less than 5-fold for heterotypic adhesion to selectin transfectants. Co-expression of E-selectin and ICAM-1 boosted adhesion efficiency threefold more than either receptor alone over the range of active CD18 expression. These data are the first to quantify adhesion efficiency mediated by selectin tethering and conformational activation of beta2-integrin in neutrophils in shear flow.     

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and β-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal β-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal β-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized β-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized β-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Intracellular β2-adrenergic receptor signaling specificity in mouse skeletal muscle in response to single-dose β2-agonist clenbuterol treatment and acute exercise

The aim of this study was to clarify the intracellular β₂-adrenergic receptor signaling specificity in mouse slow-twitch soleus and fast-twitch tibialis anterior (TA) muscles, resulting from single-dose β₂-agonist clenbuterol treatment and acute exercise. At 1, 4, and 24 h after single-dose treatment with clenbuterol or after acute running exercise, the soleus and TA muscles were isolated and subjected to analysis. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) increased after single-dose clenbuterol treatment and acute exercise in the soleus muscle but not in the TA muscle. Although there was no change in the phosphorylation of Akt after acute exercise in either muscle, phosphorylation of Akt in the soleus muscle increased after single-dose clenbuterol treatment, whereas that in the TA muscle remained unchanged. These results suggest that p38 MAPK and Akt pathways play a functional role in the adaptation to clenbuterol treatment and exercise, particularly in slow-twitch muscles.     

Fine-tiling array CGH to improve diagnostics for α- and β-thalassemia rearrangements

Implementation of multiplex ligation-dependent probe amplification (MLPA) for thalassemia causing deletions has lead to the detection of new rearrangements. Knowledge of the exact breakpoint sequences should give more insight into the molecular mechanisms underlying these rearrangements, and would facilitate the design of gap-PCRs. We have designed a custom fine-tiling array with oligonucleotides covering the complete globin gene clusters. We hybridized 27 DNA samples containing newly identified deletions and nine positive controls. We designed specific primers to amplify relatively short fragments containing the breakpoint sequence and analyzed these by direct sequencing. Results from nine positive controls showed that array comparative genomic hybridization (aCGH) is suitable to detect small and large rearrangements. We were able to locate all breakpoints to a region of approximately 2 kb. We designed breakpoint primers for 22 cases and amplification was successful in 19 cases. For 12 of these, the exact locations of the breakpoints were determined. Seven of these deletions have not been reported before. aCGH is a valuable tool for high-resolution breakpoint characterization. The combination of MLPA and aCGH has lead to relatively cheap and easy to perform PCR assays, which might be of use for laboratories as an alternative for MLPA in populations where only a limited number of specific deletions occur with high frequency.     

Mechanical properties,electronic structure and bonding of α- and β-tricalcium phosphates with surface characterization

The mechanical properties and electronic structure of α- and β-tricalcium phosphate (TCP) crystals are studied by using two ab initio density functional methods, the Vienna Ab initio Simulation Package (VASP) and the orthogonalized linear combination of atomic orbitals method. Based on the VASP optimized crystal structures, the elastic constants of α- and β-TCP are obtained using an effective stress–strain computational scheme. From the calculated elastic constants, the bulk modulus, shear modulus, Young’s modulus and Poisson’s ratios are obtained. The results show that the mechanical properties of the two crystals are comparable and that α-TCP is somewhat softer than β-TCP. Comparison with experimental extrapolations of the elastic constants shows significant differences, which attest to the difficulty of obtaining single crystal samples. The calculated electronic structure results show that both crystals are large gap insulators with a direct band gap of 4.89 eV for α-TCP and 5.25 eV for β-TCP. Effective charge calculations show that, on average, β-TCP has slightly less charge transfer per Ca than α-TCP. The (0 1 0) ((0 0 1)) surface model for α-TCP (β-TCP) is studied using a supercell slab geometry and fully relaxed to obtain the optimized structures. The estimated surface formation energies are 0.777 and 0.842 J m^?2 for α-TCP and β-TCP, respectively. The electronic structures of the two surface models are compared with the bulk models. Charge density analysis shows that the surfaces of both TCP crystals are positively charged overall owing to the presence of Ca ions near the surfaces.