Incorporation of an interferon-β neutralizing antibody assay into routine clinical practice

2011 ◽  
Vol 17 (11) ◽  
pp. 1333-1340 ◽  
Author(s):  
RA Farrell ◽  
M Espasandin ◽  
N Lakdawala ◽  
PI Creeke ◽  
V Worthington ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Luciferase Assay ◽  
Post Injection ◽  
Post Dose ◽  
Therapeutic Decisions ◽  
Dose Dependent Fashion ◽  
Polymerase Chain ◽  
Dose Dependent

Background: Incorporation of routine clinical testing for neutralizing antibodies (NAbs) to interferon (IFN)-β has remained problematic. With increasing treatment choice for patients, routine NAb testing should be incorporated to aid therapeutic decisions. Objective: We sought to improve interpretation of NAb results by combining the luciferase NAb assay (luciferase gene expression assay under control of interferon-stimulated response element) and in-vivo biomarker (myxovirus A protein, MxA) induction in patients with MS. Methods: Blood samples (serum and PAXGene® for RNA) were obtained pre-injection and 12 hours post-injection of IFN-β from 144 subjects. Sera were tested for NAbs using the luciferase assay. MxA expression was quantified by real-time polymerase chain reaction (PCR). Results: 26% of samples were NAb positive (titre > 20 NU). There was no difference in NAb titres in the pre- or post-dose sera ( p = 0.643). MxA expression was inhibited in a dose-dependent fashion in NAb positive samples. Mean MxA level post-IFN-β: NAb negative 2330 (95% CI 1940–2719), NAb 20–99 NU 1533 (95% CI 741–2324), NAb 100–600 NU 832 (186–1478) and NAb > 600 NU 101 (95% CI 0–224). NAb titre and MxA level correlated strongly: MxA pre- (Spearman r = −0.72, p < 0.0001), MxA post- (Spearman r = −0.79, p < 0.0001) and MxA induction (Spearman r = −0.67, p = 0.0004). Conclusion: A single, 12-hour post-injection sample should be used to test for NAbs using the luciferase assay and IFN-β bioactivity (MxA) in the clinical setting.

scholarly journals Autoinduction of activin genes in early Xenopus embryos

1994 ◽  
Vol 298 (2) ◽  
pp. 275-280 ◽  
Author(s):  
A Suzuki ◽  
T Nagai ◽  
S I Nishimatsu ◽  
H Sugino ◽  
Y Eto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Levels ◽  
Xenopus Embryos ◽  
Treatment Conditions ◽  
Dose Dependent Fashion ◽  
Polymerase Chain ◽  
Stage 1 ◽  
Dose Dependent

Activin exhibits a potent mesoderm inducing activity towards the ectodermal tissue (animal cap) of Xenopus laevis blastulae. Thus in order to investigate the role of activin in morphogenesis of early Xenopus embryos, activation of genes for activin beta A and beta B was examined by the reverse transcription polymerase chain reaction. In vivo, activin beta B mRNA appears to be present in embryonic stage 1 whereas beta A mRNA is undetectable prior to gastrulation. beta B and beta A mRNAs were noted to accumulate after stages 9 and 15 respectively. Activin gene expression in Xenopus animal caps was examined after treatment with various concentrations of activin A. Under these treatment conditions, both activin beta A and beta B mRNAs accumulated in a dose-dependent fashion after 24 h. The same effect was noted for treatment with similar concentrations of activin B. Accumulation of mRNAs was inhibited by the addition of cycloheximide to the culture medium, consistent with the proposition that activin gene expression requires certain protein factors. In total, therefore, these data suggest that an autoinduction mechanism is involved in the regulation of activin mRNA levels in normal Xenopus embryos and that this mechanism may play a pivotal role during early embryonic development.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

scholarly journals Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yongbing Pan ◽  
Jianhui Du ◽  
Jia Liu ◽  
Hai Wu ◽  
Fang Gui ◽  
...  
Keyword(s):  
Phage Display ◽  
Neutralizing Antibodies ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Chain Gene ◽  
Prophylactic Efficacy ◽  
Heavy Chains ◽  
Effective Interventions ◽  
Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

Enhancement of FSH bioactivity in vivo using site-specific antisera

1997 ◽  
Vol 152 (3) ◽  
pp. 355-363 ◽  
Author(s):  
L Ferasin ◽  
G Gabai ◽  
J Beattie ◽  
G Bono ◽  
A T Holder
Keyword(s):  
Biological Activity ◽  
Treatment Period ◽  
Ovarian Function ◽  
Day Treatment ◽  
Site Specific ◽  
Dose Dependent Fashion ◽  
Snell Dwarf ◽  
Fsh Bioactivity ◽  
Dose Dependent

The ability of site-specific antipeptide antisera to enhance the biological activity of ovine FSH (oFSH) in vivo was investigated using hypopituitary Snell dwarf mice. These animals were shown to respond to increasing doses of oFSH (3·3–90 μg/day), administered in two daily injections over a 5-day treatment period, in a highly significant dose-dependent fashion. The responses measured were increases in uterine weight, ovarian weight and the index of keratinisation in vaginal smears. The dose-dependent response to oFSH confirmed the suitability of this animal model for these investigations and suggested the suboptimal dose of oFSH (20 μg/day) for use in enhancement studies. Five peptides derived from the β subunit of bovine FSH (bFSH) (A, residues 33–47; B, 40–51; C, 69–80; D, 83–94; E, 27–39) were used to generate polyclonal antipeptide antisera. Of these peptides, only A and B produced an antiserum (raised in sheep) capable of recognising 125I-bFSH in a liquid phase RIA. Antisera prepared against peptide A or peptide B were found to significantly enhance the biological activity of 20 μg oFSH/day over a 5-day treatment period. The response to antipeptide antisera alone did not differ significantly from that observed in PBS-injected control animals, neither did the response to FSH alone differ from that observed in animals treated with FSH plus preimmune serum. Thus the enhanced responses are dependent upon the presence of FSH plus antipeptide antiserum. Peptides A and B are located in a region thought to be involved in receptor recognition, this may have implications for the mechanism underlying this phenomenon and/or the structure/function relationships of FSH. That FSH-enhancing antisera can be generated by immunisation of animals with peptides A and B suggests that it may be possible to develop these peptides as vaccines capable of increasing reproductive performance, such as ovulation rate. The high degree of sequence homology between ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species. Journal of Endocrinology (1997) 152, 355–363

scholarly journals Kahweol Exerts Skin Moisturizing Activities by Upregulating STAT1 Activity

2021 ◽  
Vol 22 (16) ◽  
pp. 8864
Author(s):  
Hongxi Chen ◽  
Mohammad Amjad Hossain ◽  
Jong-Hoon Kim ◽  
Jae Youl Cho
Keyword(s):  
Proper Time ◽  
Radical Scavenging ◽  
Polymerase Chain Reaction Analysis ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Diphenyltetrazolium Bromide ◽  
Polymerase Chain ◽  
Radical Scavenging Ability ◽  
The Relationship ◽  
Dose Dependent

Kahweol is a diterpene present in coffee. Until now, several studies have shown that kahweol has anti-inflammatory and anti-angiogenic functions. Due to the limited research available about skin protection, this study aims to discern the potential abilities of kahweol and the possible regulation targets. First, the cytotoxicity of kahweol was checked by 3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide assay, while 2,20-azino-bis (3ethylbenzothiazoline-6-sulphonic acid) diammonium salt and 1-diphenyl-2-picryl-hydrazyl were used to examine the radical scavenging ability. Polymerase chain reaction analysis was performed to explore the proper time points and doses affecting skin hydration and barrier-related genes. Luciferase assay and Western blotting were used to explore the possible transcription factors. Finally, fludarabine (a STAT1 inhibitor) was chosen to discern the relationship between skin-moisturizing factors and STAT1. We found that HaCaT cells experienced no toxicity from kahweol, and kahweol displayed moderate radical scavenging ability. Moreover, kahweol increased the outcome of HAS1, HAS2, occludin, and TGM-1 from six hours in a dose-dependent manner as well as the activation of STAT1 from six hours. Additionally, kahweol recovered the suppression of HAS2, STAT1-mediated luciferase activity, and HA secretion, which was all downregulated by fludarabine. In this study, we demonstrated that kahweol promotes skin-moisturizing activities by upregulating STAT1.

scholarly journals The Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody, SC31, isolated from an early convalescent COVID-19 patient, are essential for the optimal therapeutic efficacy of the antibody

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253487
Author(s):  
Conrad E. Z. Chan ◽  
Shirley G. K. Seah ◽  
De Hoe Chye ◽  
Shane Massey ◽  
Maricela Torres ◽  
...  
Keyword(s):  
Therapeutic Efficacy ◽  
Neutralizing Antibodies ◽  
Inflammatory Responses ◽  
Therapeutic Potential ◽  
Rhesus Macaques ◽  
Neutralizing Antibody ◽  
Effector Functions ◽  
Viral Loads ◽  
Therapeutic Doses ◽  
Dose Dependent

Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

scholarly journals Interleukin-6 is a potent thrombopoietic factor in vivo in mice

Blood ◽  
1989 ◽  
Vol 74 (4) ◽  
pp. 1241-1244 ◽  
Author(s):  
T Ishibashi ◽  
H Kimura ◽  
Y Shikama ◽  
T Uchida ◽  
S Kariyone ◽  
...  
Keyword(s):  
Interleukin 6 ◽  
Serum Levels ◽  
In Vivo Studies ◽  
Time Intervals ◽  
Platelet Counts ◽  
Striking Increase ◽  
Biologic Activity ◽  
Dose Dependent Fashion ◽  
Dose Dependent

Abstract To determine the biologic activity of interleukin-6 (IL-6) on megakaryocytopoiesis and thrombocytopoiesis in vivo, the cytokine was administered intraperitoneally to mice every 12 hours at varying doses for five days or for varying time intervals, based on the kinetic analysis of IL-6 serum levels indicating the peak of 40 minutes following injection, with no detection at 150 minutes. A dose-response experiment showed that IL-6 increased platelet counts in a dose- dependent fashion at a plateau stimulation level of 5 micrograms. Administration of 5 micrograms of IL-6 reproducibly elevated platelet counts at five days by approximately 50% to 60% of increase. Moreover, a striking increase in megakaryocytic size in response to IL-6 was elicited by the treatment, but no change in megakaryocyte numbers; whereas IL-6 administration did not expand CFU-MK numbers. The in vivo studies in this manner had negligible effects on other hematologic parameters, with the minor exception of monocyte levels. These data show that IL-6 acts on maturational stages in megakaryocytopoiesis and promotes platelet production in vivo in mice, suggesting that IL-6 functions as thrombopoietin.

scholarly journals METABOLIC ACTIVATION OF 2,6-DIMETHYLANILINE: MUTATIONAL SPECIFICITY IN THE GPT GENE OF AS52 CELLS

2018 ◽  
Vol 11 (9) ◽  
pp. 394
Author(s):  
Seo Hyun Moon ◽  
Min Young Kim
Keyword(s):  
Parent Compound ◽  
Chinese Hamster ◽  
Relative Survival ◽  
Metabolic Activation ◽  
Nested Polymerase Chain Reaction ◽  
Trypan Blue Exclusion ◽  
Polymerase Chain ◽  
Dose Dependent Increase ◽  
Dose Dependent

Objective: The purpose of the current work was to characterize the mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyl transferase (gpt) gene locus were assessed with doses, of which relative survival is 30% or more. Nested polymerase chain reaction-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction on treatment of 2,6-DMA and its metabolites but show a considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethylaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8-fold (13.2×10−5) greater than the spontaneous fraction of 1.62×10−5. Total deletion of the gpt gene sequences was found in 1 (4%) spontaneous and 2 (6%) the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusions: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.

scholarly journals Role of TGF-alpha in the progression of diabetic kidney disease

2017 ◽  
Vol 312 (6) ◽  
pp. F951-F962 ◽  
Author(s):  
Josef G. Heuer ◽  
Shannon M. Harlan ◽  
Derek D. Yang ◽  
Dianna L. Jaqua ◽  
Jeffrey S. Boyles ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Renal Disease ◽  
Neutralizing Antibodies ◽  
Diabetic Kidney Disease ◽  
Transforming Growth Factor ◽  
Renin Angiotensin System ◽  
Neutralizing Antibody ◽  
Diabetic Kidney

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal TGFA mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic 129S6 nephron reduction and Type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

scholarly journals CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1175-1183 ◽  
Author(s):  
Brian Kavanagh ◽  
Shaun O'Brien ◽  
David Lee ◽  
Yafei Hou ◽  
Vivian Weinberg ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cd4 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Effector T Cells ◽  
Activated T Cells ◽  
Ctla4 Blockade ◽  
Dose Dependent Fashion ◽  
Dose Dependent

AbstractCytotoxic T lymphocyte–associated antigen 4 (CTLA4) delivers inhibitory signals to activated T cells. CTLA4 is constitutively expressed on regulatory CD4+ T cells (Tregs), but its role in these cells remains unclear. CTLA4 blockade has been shown to induce antitumor immunity. In this study, we examined the effects of anti-CTLA4 antibody on the endogenous CD4+ T cells in cancer patients. We show that CTLA4 blockade induces an increase not only in the number of activated effector CD4+ T cells, but also in the number of CD4+ FoxP3+ Tregs. Although the effects were dose-dependent, CD4+ FoxP3+ regulatory T cells could be expanded at lower antibody doses. In contrast, expansion of effector T cells was seen only at the highest dose level studied. Moreover, these expanded CD4+ FoxP3+ regulatory T cells are induced to proliferate with treatment and possess suppressor function. Our results demonstrate that treatment with anti-CTLA4 antibody does not deplete human CD4+ FoxP3+ Tregs in vivo, but rather may mediate its effects through the activation of effector T cells. Our results also suggest that CTLA4 may inhibit Treg proliferation similar to its role on effector T cells. This study is registered at http://www.clinicaltrials.gov/ct2/show/NCT00064129, registry number NCT00064129.

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