scholarly journals IgG1 Thioether Bond Formation in Vivo

2013 ◽  
Vol 288 (23) ◽  
pp. 16371-16382 ◽  
Author(s):  
Qingchun Zhang ◽  
Matthew R. Schenauer ◽  
John D. McCarter ◽  
Gregory C. Flynn
Keyword(s):  
Light Chain ◽  
Reaction Rate ◽  
Human Subjects ◽  
Light Chains ◽  
Chain Reaction ◽  
Healthy Human ◽  
Conversion Rates ◽  
Healthy Human Subjects

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.

scholarly journals A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects

2015 ◽  
Vol 4 ◽  
Author(s):  
P. Ramnani ◽  
A. Costabile ◽  
A. G. R. Bustillo ◽  
G. R. Gibson
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Human Subjects ◽  
Secretory Iga ◽  
Healthy Human ◽  
Double Blind ◽  
Bacterial Populations ◽  
Healthy Human Subjects ◽  
Gradient Gel Electrophoresis

AbstractThis placebo-controlled, randomised, double-blind, cross-over human feeding study aimed to determine the prebiotic effect of agave fructans. A total of thirty-eight volunteers completed this trial. The treatment consisted of 3 weeks' supplementation with 5 g/d of prebiotic agave fructan (Predilife) or equivalent placebo (maltodextrin), followed by a 2-week washout period following which subjects were crossed over to alternate the treatment arm for 3 weeks followed by a 2-week washout. Faecal samples were collected at baseline, on the last day of treatment (days 22 and 58) and washout (days 36 and 72), respectively. Changes in faecal bacterial populations, SCFA and secretory IgA were assessed using fluorescentin situhybridisation, GC and ELISA, respectively. Bowel movements, stool consistencies, abdominal comfort and mood changes were evaluated by a recorded daily questionnaire. In parallel, the effect of agave fructans on different regions of the colon using a three-stage continuous culture simulator was studied. Predilife significantly increased faecal bifidobacteria (log109·6 (sd0·4)) and lactobacilli (log107·7 (sd0·8)) compared with placebo (log109·2 (sd0·4);P = 0·00) (log107·4 (sd0·7);P= 0·000), respectively. No change was observed for other bacterial groups tested, SCFA, secretory IgA, and PGE2concentrations between the treatment and placebo. Denaturing gradient gel electrophoresis analysis indicated that bacterial communities were randomly dispersed and no significant differences were observed between Predilife and placebo treatments. Thein vitromodels showed similar increases in bifidobacterial and lactobacilli populations to that observed with thein vivotrial. To conclude, agave fructans are well tolerated in healthy human subjects and increased bifidobacteria and lactobacilli numbersin vitroandin vivobut did not influence other products of fermentation.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

scholarly journals Morphometric changes in the human pulmonary acinus during inflation

2012 ◽  
Vol 112 (6) ◽  
pp. 937-943 ◽  
Author(s):  
A. J. Hajari ◽  
D. A. Yablonskiy ◽  
A. L. Sukstanskii ◽  
J. D. Quirk ◽  
M. S. Conradi ◽  
...  
Keyword(s):  
Human Subjects ◽  
Alveolar Volume ◽  
Healthy Human ◽  
Lung Inflation ◽  
Pulmonary Acinus ◽  
Anisotropic Expansion ◽  
Healthy Human Subjects ◽  
Alveolar Duct ◽  
Gas Volume

Despite decades of research into the mechanisms of lung inflation and deflation, there is little consensus about whether lung inflation occurs due to the recruitment of new alveoli or by changes in the size and/or shape of alveoli and alveolar ducts. In this study we use in vivo 3He lung morphometry via MRI to measure the average alveolar depth and alveolar duct radius at three levels of inspiration in five healthy human subjects and calculate the average alveolar volume, surface area, and the total number of alveoli at each level of inflation. Our results indicate that during a 143 ± 18% increase in lung gas volume, the average alveolar depth decreases 21 ±5%, the average alveolar duct radius increases 7 ± 3%, and the total number of alveoli increases by 96 ± 9% (results are means ± SD between subjects; P < 0.001, P < 0.01, and P < 0.00001, respectively, via paired t-tests). Thus our results indicate that in healthy human subjects the lung inflates primarily by alveolar recruitment and, to a lesser extent, by anisotropic expansion of alveolar ducts.

scholarly journals Activation of Coagulation by Administration of Recombinant Factor VIIa Elicits Interleukin 6 (IL-6) and IL-8 Release in Healthy Human Subjects

2003 ◽  
Vol 10 (3) ◽  
pp. 495-497 ◽  
Author(s):  
Evert de Jonge ◽  
Philip W. Friederich ◽  
George P. Vlasuk ◽  
William E. Rote ◽  
Margaretha B. Vroom ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Interleukin 6 ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Human Subjects ◽  
In Vitro Experiments ◽  
Healthy Human ◽  
Healthy Human Subjects ◽  
Proinflammatory Responses

ABSTRACT The activation of coagulation has been shown to contribute to proinflammatory responses in animal and in vitro experiments. Here we report that the activation of coagulation in healthy human subjects by the administration of recombinant factor VIIa also elicits a small but significant increase in the concentrations of interleukin 6 (IL-6) and IL-8 in plasma. This increase was absent when the subjects were pretreated with recombinant nematode anticoagulant protein c2, the inhibitor of tissue factor-factor VIIa.

scholarly journals The effect of marine oil-derived n-3 fatty acids on transepithelial calcium transport in Caco-2 cell models of healthy and inflamed intestines

2007 ◽  
Vol 97 (2) ◽  
pp. 281-288 ◽  
Author(s):  
Jennifer Gilman ◽  
Kevin D. Cashman
Keyword(s):  
Fatty Acids ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Human Subjects ◽  
Healthy Human ◽  
Marine Oil ◽  
Ca Transport ◽  
Tnf Α

Marine oil-derived n-3 fatty acids have been shown to stimulate intestinal Ca absorption in animal studies, but the effects of such fatty acids on Ca absorption in human subjects are relatively unknown. In particular, n-3 fatty acids may be of therapeutic value for some Crohn's disease patients who experience Ca malabsorption. Therefore, the aim of the present study was to investigate the effect of 20 : 5n-3 and 22 : 6n-3 on transepithelial Ca transport across monolayers of healthy Caco-2 cells as well as of TNF-α-treated Caco-2 cells (an in vitro model of Crohn's disease). Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers, which were treated with 80 μm-20 : 5n-3, 80 μm-22 : 6n-3, or 40 μm-20 : 5n-3+40 μm-22 : 6n-3 for 6 or 8 d, with or without co-treatment with TNF-α (10 ng/ml) (n 11–15 monolayers per treatment). On day 16, transepithelial and transcellular transport of 45Ca and fluorescein transport (a marker of paracellular diffusion) were measured. Treatment of healthy and inflamed Caco-2 cells with 20 : 5n-3, 22 : 6n-3 and both fatty acids combined for 8 d significantly (P < 0·005–0·01) increased total transepithelial Ca transport compared with that in control, effects which were mediated by an enhanced rate of transcellular Ca transport. The effects of n-3 fatty acids on Ca absorption after 6 d were less clear-cut. In conclusion, the present in vitro findings highlight the need to investigate the effect of marine oil-based n-3 fatty acids on Ca absorption in vivo in studies of healthy human subjects as well as of Crohn's disease patients.

scholarly journals Secreted microbial metabolites modulate gut immunity and inflammatory tone

2019 ◽  
Author(s):  
Rabina Giri ◽  
Emily C. Hoedt ◽  
Khushi Shamsunnahar ◽  
Michael A. McGuckin ◽  
Mark Morrison ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Inflammatory Responses ◽  
Human Subjects ◽  
Intestinal Immunity ◽  
Healthy Human ◽  
Peripheral Blood Mononuclear ◽  
Healthy Human Subjects ◽  
Gut Immunity ◽  
Inflammatory Bowel

AbstractEvidence is emerging that microbiome–immune system crosstalk regulates the tenor of host intestinal immunity and predisposition to inflammatory bowel disease (IBD). We identified five NF-κB suppressive strains affiliated with Clostridium clusters IV, XIVa and XV that independently suppressed secretion of the chemokine IL-8 by peripheral blood mononuclear cells and gut epithelial organoids from healthy human subjects, as well as patients with the predominant IBD subtypes, Crohn’s disease and ulcerative colitis. The NF-κB suppressive Clostridium bolteae AHG0001, but not C. bolteae BAA-613, suppressed cytokine-driven inflammatory responses and endoplasmic reticulum stress in gut epithelial organoids derived from Winnie mice that develop spontaneous colitis. This predicted in vivo responses thereby validating a precision medicine approach to treat Winnie colitis and suggesting the microbiome may function as an extrinsic regulator of host immunity. Finally, we identified a novel molecule associated with NF-κB suppression indicating gut bacteria could be harnessed to develop new therapeutics.

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