RSSMembrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells.
Source
Division of Pediatrics, MD Anderson Cancer Center, The University of Texas, Houston, Texas, United States of America.
Abstract
NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy.
- PMID:
- 22279576
- [PubMed - in process]
Ex vivo expansion of human CD8 T cells using autologous CD4 T cell help.
Source
Department of Medical Oncology, Dana-Farber Cancer Institute, Massachusetts, United States of America.
Abstract
BACKGROUND:
Using in vivo mouse models, the mechanisms of CD4(+) T cell help have been intensively investigated. However, a mechanistic analysis of human CD4(+) T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4(+) T cell help of CD8(+) T cell proliferation using a novel in vitro model.
METHODS/PRINCIPAL FINDINGS:
We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3(+) regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4(+) T cells was associated with significantly improved CD8(+) T cell expansion in vitro. The CD4(+) T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4(+) T cell help of CD8(+) T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8(+) T cells.
CONCLUSIONS:
We have developed an in vitro model that advances our understanding of the immunobiology of human CD4(+) T cell help of CD8(+) T cells. Our data suggests that human CD4(+) T cell help can be leveraged to expand CD8(+) T cells in vitro.
- PMID:
- 22279573
- [PubMed - in process]
Circulating Angiopoietin-2 as a Biomarker in ANCA-Associated Vasculitis.
Source
Section of Rheumatology, Department of Medicine, School of Medicine, Vasculitis Center, Boston University, Boston, Massachusetts, United States of America.
Abstract
The endothelial-specific Angiopoietin-Tie2 ligand-receptor system is an important regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to Tie2 receptor renders the endothelial barrier responsive to pro-inflammatory cytokines. We previously showed that circulating Ang-2 correlated with disease severity in a small cohort of critically ill patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. The current study reassessed Ang-2 as a biomarker of disease activity and relapse in AAV. Circulating Ang-2 was measured in 162 patients with severe AAV (BVAS/WG≥3, with or without glomerulonephritis) in a clinical trial. Ang-2 levels during active AAV were compared to levels in the same patients during remission (BVAS/WG = 0). Levels in clinical subsets of AAV were compared, and association with future disease course was assessed. Ang-2 levels were elevated in severe disease (median 3.0 ng/ml, interquartile range 1.9-4.4) compared to healthy controls (1.2, 0.9-1.5). However, they did not reliably decline with successful treatment (median 2.6 ng/ml, interquartile range 1.9-3.8, median change -0.1). Ang-2 correlated weakly with BVAS/WG score (r = 0.17), moderately with markers of systemic inflammation (r = 0.25-0.41), and inversely with renal function (r = -0.36). Levels were higher in patients with glomerulonephritis, but levels adjusted for renal dysfunction were no different in patients with or without glomerulonephritis. Levels were higher in patients with newly diagnosed AAV and lower in patients in whom treatment had recently been started. Ang-2 levels during active disease did not predict response to treatment, and Ang-2 levels in remission did not predict time to flare. Thus, Ang-2 appears to have limited practical value in AAV as a biomarker of disease activity at time of measurement or for predicting future activity.
- PMID:
- 22279570
- [PubMed - in process]
Characterization of an Isotype-Dependent Monoclonal Antibody against Linear Neutralizing Epitope Effective for Prophylaxis of Enterovirus 71 Infection.
Source
Animal Health Biotechnology, Temasek Life Sciences Laboratory, Singapore.
Abstract
BACKGROUND:
Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment.
METHODOLOGY/PRINCIPAL FINDING:
In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16).
CONCLUSION:
We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.
- PMID:
- 22279543
- [PubMed - in process]
The synovium in rheumatoid arthritis.
Source
University of Manitoba, Winnipeg, Manitoba, Canada.
Abstract
Rheumatoid arthritis (RA) is a chronic autoimmune disease targeting multiple joints. The synovium is the primary site of the inflammatory process, which if untreated leads to irreversible damage to the adjacent cartilage and bone. It is now well established that autoantibodies that are characteristic of RA, including rheumatoid factor (RF) and anti-citrulluninated protein antibodies (ACPA), are present before clinical disease onset. Studies in both humans and animal models are beginning to provide new insights into how this asymptomatic autoimmunity evolves into an inflammatory process that is localized in the synovium.Once RA synovitis established, a number of amplification mechanisms serve to sustain the process leading to the persistence of the disease. These mechanisms include engagement of the resident mesenchymal cells and the establishment of ectopic lymphoid structures in the synovium, although the relationship between these lymphoid structures and the presence of RA autoantibodies remains unclear.An enhanced understanding of the mechanisms that initiate and sustain RA synovitis offers unprecedented opportunities for therapeutics, and ultimately prevention strategies.
- PMID:
- 22279509
- [PubMed - in process]
Prevention and Reversal of Antibody Responses Against Factor IX in Gene Therapy for Hemophilia B.
Source
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida Gainesville, FL, USA.
Abstract
Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4(+) T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10 BU within 1 month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specificantibodies to <2 BU, which lasted for >4.5 months. Systemic hF.IX expression increased from undetectable to >200 ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.
- PMID:
- 22279442
- [PubMed - in process]
Safety and tolerability of denosumab for the treatment of postmenopausal osteoporosis.
Source
New Mexico Clinical Research & Osteoporosis Center, Albuquerque, New Mexico, USA.
Abstract
Denosumab is a fully human monoclonal antibody to receptor activator of nuclear factor kappa-B ligand (RANKL), a cytokine member of the tumor necrosis factor family that is the principal regulator of osteoclastic bone resorption. Postmenopausal osteoporosis (PMO) is a systemic skeletal disease associated with high levels of RANKL, resulting in a high rate of bone remodeling and an imbalance of bone resorption over bone formation. By inhibiting RANKL in women with PMO, denosumab reduces the rate of bone remodeling, thereby increasing bone mineral density, improving bone strength, and reducing the risk of fractures. In clinical trials of women with osteoporosis and low bone mineral density, denosumab has been well tolerated, with overall rates of adverse events and serious adverse events in women treated with denosumab similar to those receiving placebo. In the largest clinical trial of denosumab for the treatment of women with PMO, there was a significantly greater incidence of cellulitis reported as a serious adverse event, with no difference in the overall incidence of cellulitis, and a significantly lower incidence of the serious adverse event of concussions with denosumab compared with placebo. The evidence supports a favorable balance of benefits versus risks of denosumab for the treatment of PMO. Assessments of the long-term safety of denosumab are ongoing. Denosumab 60 mg subcutaneously every 6 months is an approved treatment for women with PMO who are at high risk for fracture.
- PMID:
- 22279412
- [PubMed - in process]
ANCA-associated Goodpasture's syndrome in a patient with rheumatoid arthritis on penicillamine.
Source
Division of Nephrology and Renal Transplant Medicine, Medanta Kidney and Urology Institute, Medanta - The Medicity, Gurgaon, Haryana, India.
Abstract
Although penicillamine has been used effectively in the management of a variety of diseases, several adverse reactions have been observed with prolonged administration of this agent. We report a case of Goodpasture's syndrome, as a result of induction of anti-myeloperoxidase antineutrophil cytoplasmic antibodies in a 51 year old man who was being treated with this drug for rheumatoid arthritis. This pulmonary-renal syndrome has been described on rare occasions in patients receiving penicillamine. Treatment with steroids and cyclophosphamide resulted in pulmonary and renal functional recovery.
- PMID:
- 22279343
- [PubMed - in process]
Collagen sheet dressings for cutaneous lesions of toxic epidermal necrolysis.
Source
Department of Plastic Surgery, Sahara Hospital and Ajanta Hospital, Lucknow, Uttar Pradesh, India.
Abstract
Toxic epidermal necrolysis (TEN) is associated with a significant mortality of 30-50% and long-term sequelae. Treatment includes early admission to a burn unit, where management with precise fluid, electrolyte, protein, and energy supplementation, moderate mechanical ventilation, and expert wound care can be provided. Specific treatment with immunosuppressive drugs or immunoglobulins did not show an improved outcome in most studies and remains controversial. We have treated the cutaneous lesions of seven patients of TEN with collagen sheet dressings and have found a significant reduction in morbidity. The sheets are a one-time dressing, easy to apply and they reduce fluid loss, prevent infection, reduce pain, avoid repeated dressings and gradually peal off as the underlying lesions heal.
- PMID:
- 22279282
- [PubMed - in process]
Growth and innate immunity are not limited by selection for high egg testosterone content in Japanese quail.
Source
Department of Animal Physiology and Ethology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic.
Abstract
The effects of maternal androgens on fitness-related traits of offspring are generally assumed to be epigenetic adaptations to the environment that may be encountered by the next generation. Possible constraints of high yolk androgen transfer are still not understood, although a suppressed immune response in offspring is frequently considered. The aim of our study was to examine the innate immune defence in high (HET) and low egg testosterone (LET) lines of Japanese quail, which differ in the hormonal milieu of their eggs, thus providing a good physiological model for the study of androgen-mediated maternal effects. Acute phase response was induced by a lipopolysaccharide injection in 12-day-old quail and plasma corticosterone and the heterophil:lymphocyte ratio were measured at 1 and 3 h post-treatment. Basal levels of non-specific antibodies (IgY) were determined in the circulation. We found that HET quail were heavier than LET quail from the second week of age, indicating enhanced post-hatching growth. At 1 h post-lipopolysaccharide challenge, plasma corticosterone concentrations increased in the HET but not in the LET line. The heterophil:lymphocyte ratio rose in both lines at 3 h post-immune challenge, with a more pronounced response in HET quail. Moreover, HET chicks displayed higher IgY levels than LET chicks, suggesting either enhanced passive immunoprotection or stimulated endogenous antibody production. In conclusion, our data demonstrate that the genetic selection for high egg testosterone content positively influences growth and, simultaneously, does not limit the acute phase response in young quail.
- PMID:
- 22279068
- [PubMed - in process]
Helicobacter pylori Infection Is Associated With an Increased Rate of Diabetes.
Source
Center for Infectious Diseases Epidemiologic Research, Mailman School of Public Health, Columbia University, New York, New York.
Abstract
OBJECTIVEChronic infections could be contributing to the socioeconomic gradient in chronic diseases. Although chronic infections have been associated with increased levels of inflammatory cytokines and cardiovascular disease, there is limited evidence on how infections affect risk of diabetes.RESEARCH DESIGN AND METHODSWe examined the association between serological evidence of chronic viral and bacterial infections and incident diabetes in a prospective cohort of Latino elderly. We analyzed data on 782 individuals aged >60 years and diabetes-free in 1998-1999, whose blood was tested for antibodies to herpes simplex virus 1, varicella virus, cytomegalovirus, Helicobacter pylori, and Toxoplasma gondii and who were followed until June 2008. We used Cox proportional-hazards regression to estimate the relative incidence rate of diabetes by serostatus, with adjustment for age, sex, education, cardiovascular disease, smoking, and cholesterol levels.RESULTSIndividuals seropositive for herpes simplex virus 1, varicella virus, cytomegalovirus, and T. gondii did not show an increased rate of diabetes, whereas those who were seropositive for H. pylori at enrollment were 2.7 times more likely at any given time to develop diabetes than seronegative individuals (hazard ratio 2.69 [95% CI 1.10-6.60]). Controlling for insulin resistance, C-reactive protein and interleukin-6 did not attenuate the effect of H. pylori infection.CONCLUSIONSWe demonstrated for the first time that H. pylori infection leads to an increased rate of incident diabetes in a prospective cohort study. Our findings implicate a potential role for antibiotic and gastrointestinal treatment in preventing diabetes.
- PMID:
- 22279028
- [PubMed - as supplied by publisher]
Apparent Involvement of Plasmin in Early-Stage Follicle Rupture During Ovulation in Medaka.
Abstract
Until recently, the role of the proteolytic system involving serine proteases in follicle rupture during ovulation in mammalian species has been a subject of controversy. We undertook the present study to examine whether proteases play a role in follicle rupture using the teleost medaka (Oryzias latipes) model. Various serine protease inhibitors, including a specific plasmin inhibitor, drastically reduced the rate of ovulation, as assessed by an in vitro ovulation assay, which was established for the fish. Biochemical, molecular biological, and immunological analyses demonstrated that plasminogen/plasmin was present in large follicles destined to ovulate. The active protease, plasmin, was detected in follicles approximately 3 to 7 h before the expected time of ovulation. Specific antibodies against the medaka plasmin light-chain suppressed the ovulation rate of the follicles when antibodies were added to the medium during the period in which active plasmin was generated. This finding was an indication that a plasmin-like protease similar if not identical to plasmin plays a role in follicle rupture during ovulation in the medaka. Our data also indicate that this serine protease participates in the rupture for only a few hours prior to the activation of matrix metalloproteinase (Mmp)-mediated hydrolysis at ovulation. Based on our previous and current data, we propose a follicle rupture model involving two different proteolytic enzyme systems, serine protease and Mmp, in medaka ovulation. The current study is the first to provide evidence of the indispensable role of plasmin or a plasmin-like protease in the ovulation of a non-mammalian vertebrate species.
- PMID:
- 22278979
- [PubMed - as supplied by publisher]
Protease activation and the signal transduction pathway regulating motility in sperm from the water strider Aquarius remigis.
Source
Department of Biology, University of California, Riverside.
Abstract
Many motile processes are regulated such that movement occurs only upon activation of a signaling cascade. Sperm from a variety of species are initially quiescent and must be activated prior to beating. The signaling events leading to the activation and regulation of sperm motility are not well characterized. Mature seminal vesicle sperm from the water strider Aquarius remigis are immotile in vitro, but vigorous motility is activated by trypsin. Trypsin-activated motility was blocked by pretreatment of the sperm with BAPTA-AM to chelate intracellular Ca(2+) and was partially rescued by subsequent addition of A23187 and Ca(2+) . Thapsigargin stimulated motility in the absence of trypsin, suggesting that intracellular Ca(2+) stores are available. In addition, motility could be fully activated by the phosphatase inhibitor calyculin A, suggesting that the immotile state is maintained by an endogenous phosphatase and that kinase activity is required for motility. The MEK1/2 inhibitor U0126 significantly reduced trypsin activated motility, and MPM-2, anantibody which recognizes proline-directed phosphorylation by kinases such as MAPK, recognized components of the water strider sperm flagellum. Antibodies specific for the mouse protease activated receptor PAR2 recognized an antigen on the sperm flagellum. These results suggest that trypsin stimulates a Ca(2+) and MAPK mediated signaling pathway and potentially implicate a PAR2-like protein in regulating motility. © 2012 Wiley Periodicals, Inc.
Copyright © 2012 Wiley Periodicals, Inc.
- PMID:
- 22278949
- [PubMed - as supplied by publisher]
Toll-like receptors 2 and 4 impair insulin-mediated brain activity by interleukin-6 and osteopontin and alter sleep architecture.
Source
*Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology, and Clinical Chemistry.
Abstract
Impaired insulin action in the brain represents an early step in the progression toward type 2 diabetes, and elevated levels of saturated free fatty acids are known to impair insulin action in prediabetic subjects. One potential mediator that links fatty acids to inflammation and insulin resistance is the Toll-like receptor (TLR) family. Therefore, C3H/HeJ/TLR2-KO (TLR2/4-deficient) mice were fed a high-fat diet (HFD), and insulin action in the brain as well as cortical and locomotor activity was analyzed by using telemetric implants. TLR2/4-deficient mice were protected from HFD-induced glucose intolerance and insulin resistance in the brain and displayed an improvement in cortical and locomotor activity that was not observed in C3H/HeJ mice. Sleep recordings revealed a 42% increase in rapid eye movement sleep in the deficient mice during daytime, and these mice spent 41% more time awake during the night period. Treatment of control mice with a neutralizing IL-6 antibody improved insulin action in the brain as well as cortical activity and diminished osteopontin protein to levels of the TLR2/4-deficient mice. Together, our data suggest that the lack of functional TLR2/4 protects mice from a fat-mediated impairment in insulin action, brain activity, locomotion, and sleep architecture by an IL-6/osteopontin-dependent mechanism.-Sartorius, T., Lutz, S. Z., Hoene, M., Waak, J., Peter, A., Weigert, C., Rammensee, H.-G., Kahle, P. J., Häring, H.-U., Hennige, A. M. Toll-like receptors 2 and 4 impair insulin-mediated brain activity by interleukin-6 and osteopontin and alter sleep architecture.
- PMID:
- 22278939
- [PubMed - as supplied by publisher]
Block copolymer arrangement and composition effects on protein conformation using atomic force microscope-based antigen-antibody adhesion.
Source
Nanoprobe Laboratory for Bio- & Nanotechnology and Biomimetics, The Ohio State University, Columbus, Ohio 43210.
Abstract
The conformational changes of fibronectin (FN) deposited on various block copolymers where one block is composed of poly(methyl methacrylate) (PMMA) and the other block is either poly(acrylic acid) (PAA) or poly(2-hydroxyethyl methacrylate) (PHEMA) were investigated using a functionalized atomic force microscope (AFM) tip. The tip was modified with an antibody sensitive to the exposure of the arginine-glycine-aspartic acid (RGD) groups in FN. By studying the adhesive interactions between the antibody and the proteins adsorbed on the block copolymer surface and phase imaging, it was found that the triblock copolymers PAA-b-PMMA-b-PAA and PMMA-b-PHEMA-b-PMMA, which both have large domain sizes, are conducive to the exposure of the FN RGD groups on the surface. On the basis of these results, it is concluded that the surface chemistry as well as the nanomorphology dictated by the block copolymer arrangement could both tune protein conformation and orientation and optimize cell adhesion to the biomaterial surface. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.
Copyright © 2012 Wiley Periodicals, Inc.
- PMID:
- 22278846
- [PubMed - as supplied by publisher]
Development of a human herpesvirus 6 virus species-specific immunoblotting assay.
Source
Department of Pediatrics, Fujita Health University School of Medicine.
Abstract
In order to assess the full spectrum of HHV-6A- and HHV-6B-associated diseases, we sought to develop a HHV-6 virus species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by ORF U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 virus species-specificantibodies were detected by immunoblotting in patient sera. Eighty seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of 12 exanthem subitum convalescent sera reacted exclusively with 101K, while none of 12 acute sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all 5 acute and convalescent sera obtained from transplant recipients reacted exclusively with 101K, two of 6 convalescent sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Thirty-one of 38 sera obtained from healthy adults were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of PBMCs and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed to discriminate between HHV-6A- and HHV-6B-specific antibodies.
- PMID:
- 22278837
- [PubMed - as supplied by publisher]
Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs.
Source
Australian Animal Health Laboratory.
Abstract
This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin which causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, virus was only isolated from affected foetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in six-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of two to three days, virus was shed in nasal and oral secretions, faeces, and urine, typically for less than one week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programs should it ever re-emerge in the pig population.
- PMID:
- 22278823
- [PubMed - as supplied by publisher]
Targeting of Multidrug-Resistant Human Ovarian Carcinoma Cells With Anti-P-Glycoprotein Antibody Conjugates.
Source
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA.
Abstract
A monoclonal antibody (mAb) to P-glycoprotein (Pgp), UIC2, is used as a targeting moiety for N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer/drug [(meso chlorin e(6) mono(N-2-aminoethylamide) (Mce(6) ) or doxorubicin (DOX)] conjugates to investigate their cytotoxicity towards the Pgp-expressing human ovarian carcinoma cell line A2780/AD. The binding, internalization, and subcellular trafficking of a fluorescein labeled UIC2 targeted HPMA copolymer are studied and show localization to the plasma membrane with limited internalization. The specificity of the UIC2-targeted HPMA copolymer/drug conjugates are confirmed using the sensitive cell line A2780 that does not express Pgp.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22278817
- [PubMed - as supplied by publisher]
Allergen Arrays for Antibody Screening and Immune Cell Activation Profiling Generated by Parallel Lipid Dip-Pen Nanolithography.
Source
Karlsruher Institut für Technologie (KIT), Institut für Nanotechnologie (INT), Karlsruhe Nano Micro Facility (KNMF), 76021 Karlsruhe Germany. Sylwia.Sekula-Neuner@kit.edu.
Abstract
Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
- PMID:
- 22278752
- [PubMed - as supplied by publisher]
In vitro determination of anticryptosporidial activity of phytogenic extracts and compounds.
Source
BIOMIN Research Center, Technopark 1, 3430, Tulln, Austria, klaus.teichmann@biomin.net.
Abstract
Cryptosporidiosis caused by Cryptosporidium spp. is an important diarrhoeal disease observed in farm animals and humans, especially in young or immunocompromised individuals. A novel cell culture assay for testing extracts and pure compounds against Cryptosporidium parvum in 96-well microplate format was established and evaluated. It is based on previously described indirect fluorescent antibody techniques and was optimised for higher sample throughput. Rapid assessment of minimal inhibitory concentrations (MICs) was done by checking each well microscopically for the presence or absence of parasite stages. As a novelty, parasite development was quantified by enumeration of clusters of secondary infection (CSI), which typically appeared upon infection with a distinct parasite inoculum after a defined incubation time. Host cell (HCT-8) viability was measured by an integrated non-destructive water-soluble tetrazolium salt assay (WST-1), which facilitated discrimination of antiparasitic activity from possible cytotoxic effects of a test compound against the host cells. Host cell viability was regarded unimpaired when cultures had 75% or more viability when compared to control cultures without test substance. In this study, a maximum density of distinguishable CSI was obtained when cultures were infected with 2.5 × 10(3) oocysts and incubated for 48 h. The applicable inoculum has to be optimised for each batch of oocysts and before each experimental series. Parasite development was inhibited completely by monensin at 134 nM and silymarin at 50 mg/mL. These concentrations were non-toxic to the host cells and comparable to literature data. The percentages of parasite inhibition were determined for monensin and a 50% inhibitory concentration (IC(50)) of 36.6 nM (27.4-45.5) and a 90% inhibitory concentration of 65.9 nM (54.8-90.2) were calculated. The introduced assay is economic because relatively low parasite numbers may be used. If MICs are determined, evaluation is fast, as each well is viewed only briefly under the fluorescence microscope for presence or absence of CSI. Furthermore it is highly critical because only full parasite inhibition is assessed. Counting of CSI is more laborious and time-consuming, but it allows calculation of parasite inhibition rates and parameters like the half maximal inhibitory concentration (IC(50)). This assay shall be used to assess anticryptosporidial activities of various plant waste materials and by-products from the food and the pharmaceutical industries in the course of the EU project SAFEWASTES. Comparison with in vivo models should be performed to further corroborate the results. Automated evaluation by flow cytometry might facilitate higher sample throughput and reduce operator bias.
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