Peptide Coated Titanium Rods Show Increased Bone Formation in a Rat Femur Model

1998 ◽  
Vol 550 ◽  
Author(s):  
D.M. Ferris ◽  
G.D. Moodie ◽  
P.M. Dimond ◽  
M.G. Ehrlich ◽  
R.F. Valentini
Keyword(s):  
Cell Receptor ◽  
Peptide Sequence ◽  
Pull Out ◽  
Significant Difference ◽  
Short And Long Term ◽  
And Function ◽  
Complex Signaling ◽  
Control Rods

AbstractIn vitro, groups have demonstrated that peptide modified surfaces influence short and long term cell responses like attachment, shape and function via cell receptors known as integrins. These receptors translate information to the nucleus via sets of complex signaling pathways. Little is known about the ability of these surfaces to influence the inherently complex in vivo environment, however. The present study was designed to evaluate the quality and quantity of new bone formed in response to gold coated titanium rods modified with the peptide sequence Arg-Gly-Asp-Cys (RGDC). Quantitative histomorphometric analysis of histologic sections perpendicular to the implant long axis showed a thicker (P < 0.01) shell of new bone formed in response to peptide modified implants (26.2 microns ± 1.9 vs. 20.5 microns ± 2.9) as early as 2 weeks. Mechanical pull-out testing conducted at 4 weeks revealed the average pull-out force of peptide modified rods was 38% greater than gold control rods. The significant difference in the thickness of new bone formed around the implant was maintained at 4 weeks (32.7 microns ± 4.6 vs. 22.6 microns ± 4.0). These results demonstrate the feasibility of developing peptide coated biomaterials designed to elicit a host response at the cell receptor level.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

scholarly journals Ikaros is absolutely required for pre-B cell differentiation by attenuating IL-7 signals

2013 ◽  
Vol 210 (13) ◽  
pp. 2823-2832 ◽  
Author(s):  
Beate Heizmann ◽  
Philippe Kastner ◽  
Susan Chan
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Differentiation ◽  
B Cell Signaling ◽  
And Migration ◽  
And Function

Pre-B cell receptor (pre-BCR) signaling and migration from IL-7–rich environments cooperate to drive pre-B cell differentiation via transcriptional programs that remain unclear. We show that the Ikaros transcription factor is required for the differentiation of large pre-B to small pre-B cells. Mice deleted for Ikaros in pro/pre-B cells show a complete block of differentiation at the fraction C′ stage, and Ikaros-null pre-B cells cannot differentiate upon withdrawal of IL-7 in vitro. Restoration of Ikaros function rescues pre-B cell differentiation in vitro and in vivo and depends on DNA binding. Ikaros is required for the down-regulation of the pre-BCR, Igκ germline transcription, and Ig L chain recombination. Furthermore, Ikaros antagonizes the IL-7–dependent regulation of &gt;3,000 genes, many of which are up- or down-regulated between fractions C′ and D. Affected genes include those important for survival, metabolism, B cell signaling, and function, as well as transcriptional regulators like Ebf1, Pax5, and the Foxo1 family. Our data thus identify Ikaros as a central regulator of IL-7 signaling and pre-B cell development.

Rapid recovery from T lymphopenia by CD28 superagonist therapy

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1764-1770 ◽  
Author(s):  
Karin Elflein ◽  
Marta Rodriguez-Palmero ◽  
Thomas Kerkau ◽  
Thomas Hünig
Keyword(s):  
T Cells ◽  
T Cell ◽  
Opportunistic Infections ◽  
Therapeutic Potential ◽  
Cell Receptor ◽  
Cancer Therapies ◽  
Repertoire Diversity ◽  
And Function

AbstractSlow recovery of T-cell numbers and function contributes to the high incidence of life-threatening infections after cytotoxic cancer therapies. We have tested the therapeutic potential of a novel class of superagonistic CD28–specific antibodies that induce polyclonal T-cell proliferation without T-cell receptor engagement in an experimental rat model of T lymphopenia. We show that in lethally irradiated, bone marrow–reconstituted hosts, CD28 superagonist is able to dramatically accelerate repopulation by a small inoculum of mature, allotype-marked T cells. CD28-driven recovery of CD4 cells was superior to that of CD8 T cells. CD28 superagonist– expanded CD4 T cells had maintained repertoire diversity and were functional both in vitro and in vivo, suggesting that treatment with a human CD28–specific superagonist will protect T-lymphopenic patients from opportunistic infections.

Elucidation of the Roles of Individual Asparagine-Linked Glycans Outside of the B Domain on Factor VIII Secretion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2238-2238 ◽  
Author(s):  
Sundar Rajan Selvaraj ◽  
Hongzhi Miao ◽  
Steven Pipe
Keyword(s):  
Dendritic Cells ◽  
Factor Viii ◽  
Conflicts Of Interest ◽  
Site Directed Mutagenesis ◽  
Significant Difference ◽  
C1 Domain ◽  
Protein Backbones ◽  
And Function

Abstract Abstract 2238 Post-translational modifications play vital roles in the secretion, function, intermolecular interactions and degradation of most secreted and transmembrane proteins. Factor VIII (FVIII) is a heavily glycosylated protein with up to 25 asparagine (Asn)-linked glycans, the bulk of which are present within its B domain. However, deletion of the B domain is not deleterious to FVIII expression and function. In addition, FVIII has several potential Asn-linked glycosylation sequons in its other domains, of which four have been experimentally deduced to be glycosylated: Asn41 and Asn239 in the A1 domain, Asn1810 in the A3 domain and Asn2118 in the C1 domain. Of these, Asn239 and Asn2118 have been determined to comprise complex oligomannose structures. Such complex oligomannose structures have been proposed to play a role in mediating interaction with immunomodulatory cells (i.e. dendritic cells). The present study was aimed at delineating the role(s) of these four Asn-linked glycans in the expression of FVIII in vitro and in vivo and to identify possible bioengineering targets to influence FVIII expression, clearance and processing by immunomodulatory cells. Individual Asn residues were mutated to glutamine (Gln) to create single and multiple glycosylation mutants in both full length (FVIII-WT) and B domain-deleted (BDD)–FVIII, by site-directed mutagenesis. A variant of BDD-FVIII completely devoid of Asn-linked glycans, designated as Degly-BDD-FVIII, was also generated. Transient transfections of the mutants were carried out in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective native FVIII proteins. Antigen and activity assays revealed that the secretion and function of Asn41Gln and Asn1810Gln mutants were only modestly affected (85–90% of WT) but a more significant reduction was observed in the case of Asn239Gln mutant (35–50% of WT). Interestingly, there was no significant difference in secretion or function for Asn2118Gln in either FVIII-WT or BDD-FVIII protein backbones. The double mutants, Asn41/239Gln and Asn239/2118Gln behaved similarly to that of Asn239Gln mutant (30–45% of WT). The triple mutants, Asn41/239/2118Gln and Asn239/1810/2118Gln showed a further decline in secretion (∼30-40% of WT) while Degly-BDD-FVIII demonstrated secretion of only about 15–20% of BDD-FVIII. The FVIII specific activity of each of these glycosylation mutants was similar to the native FVIII proteins. An ELISA-based Von Willebrand Factor (VWF) binding assay revealed no significant differences between immunoaffinity-purified FVIII-WT and Asn2118Gln mutant in their ability to bind VWF. Findings from in vivo expression (via hydrodynamic tail vein injection of plasmid DNA) of these glycosylation mutants in a F8−/− (exon 16 knock-out) hemophilia A mouse model were similar to the in vitro results in the cell lines. Plasma FVIII activity levels were measured 24 hrs post-injection via orbital bleed. While Asn2118Gln (5.2 – 6 U/mL) did not exhibit any difference from BDD-FVIII (4.8 – 5.9 U/ml), Asn239Gln (1.9 – 2.4 U/ml) was expressed at less than 50% of BDD-FVIII levels. The expression of Degly-BDD-FVIII (0.4 – 0.7 U/ml) was further reduced to ∼10% of BDD-FVIII levels. Taken together, these results indicate that of the four Asn-linked glycans, Asn239 was the most crucial for proper secretion of FVIII whereas, Asn2118 did not contribute to the efficiency of FVIII expression. The oligosaccharide structure on Asn239 is positioned at the A1-A2 interface and likely contributes to proper protein folding. However, the sugar moieties on Asn2118 have been shown to be positioned at the A3-C1 domain interface and postulated to participate in packing and stabilization (Shen et al, 2008). This would have suggested that disruption of this residue within the C1 domain might have a deleterious effect on protein secretion or function. Our results with Asn2118Gln in both FVIII-WT and BDD-FVIII protein backbones suggest that this Asn-linked glycosylation can be eliminated without any impact on FVIII expression or function including no impact on FVIII-VWF interaction. This Asn-linked glycan, therefore, could be targeted in bioengineering strategies to determine if eliminating this particular oligomannose structure might impact mannose-receptor mediated uptake of FVIII by dendritic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Effectiveness of Candidate Probiotic Bacteria to Control Vibriosis Disease

2017 ◽  
Vol 5 (2) ◽  
pp. 1
Author(s):  
Mulyati Mulyati ◽  
Suryati Suryati ◽  
Irfani Baga
Keyword(s):  
Survival Rate ◽  
Sea Water ◽  
Challenge Test ◽  
Probiotic Bacteria ◽  
Pathogenicity Test ◽  
Inhibitory Ability ◽  
Significant Difference ◽  
Portunid Crab

The study aims to isolate, characterize, and examine probiotic bacteria's inhibitory ability against Vibrio harveyi bacteria, both in-vitro and in vivo. Methods used in the study consist of 1) An Isolation of Candidate Probiotic Bacteria, 2) An Antagonistic Test of Candidate Probiotic Bacteria in vitro, 3) An Identification of Bacteria, 4) A Pathogenicity Test of Candidate Probiotic Bacteria, 5) An Antagonistic Test of Candidate Probiotic Bacteria against V. harveyi in vivo. According to the isolation of candidate probiotic bacteria, there are 18 isolated candidate probiotic. After being tested for its inhibitory ability in vitro, there are 8 isolates with zone of inhibition as follows: isolate MM 7 from intestine (22 mm), isolate MM 6 from intestine (12 mm), isolate MM 10 from sea water (10 mm), isolate MM 5 from intestine (9 mm), isolate MM 4 from intestine (8 mm), isolate MM 3 from intestine (7 mm), isolate MM 2.2 from intestine (7 mm), isolate MM 2.1 from intestine (7 mm). Eight genera of the candidate probiotic bacteria is derived from Portunid crab, they are Staphylococcus, Streptococcus, bacillus, vibrio, Alcaligenes, Lactobacillus, micrococcus. Before proceeding the V. harveyi bacterial challenge test in vivo, three potential isolates consisting of MM6, MM7 and MM10 as the probiotic bacteria are pathogenicity-tested against V. harveyi. The survival rate of Portunid crab on pathogenicity test using MM6, MM7 and MM10 generates 91.11-100%, while the control generates 100% survival rate. Variance analysis result through post-hoc Tukey's Honest Significant Difference (HSD) test at 95% confidence interval indicates that isolate MM7 and MM10 are significantly able to increase hatchling Portunid crab's survival rate.

scholarly journals Optimizing Manufacturing and Osseointegration of Ti6Al4V Implants through Precision Casting and Calcium and Phosphorus Ion Implantation? In Vivo Results of a Large-Scale Animal Trial

Materials ◽  
2020 ◽  
Vol 13 (7) ◽  
pp. 1670 ◽  
Author(s):  
Wölfle-Roos JV ◽  
Katmer Amet B ◽  
Fiedler J ◽  
Michels H ◽  
Kappelt G ◽  
...  
Keyword(s):  
Ion Implantation ◽  
Large Scale ◽  
In Vivo Studies ◽  
Control Group ◽  
Implant Surface ◽  
Precision Casting ◽  
Pull Out ◽  
Significant Difference ◽  
Calcium And Phosphorus

Background: Uncemented implants are still associated with several major challenges, especially with regard to their manufacturing and their osseointegration. In this study, a novel manufacturing technique—an optimized form of precision casting—and a novel surface modification to promote osseointegration—calcium and phosphorus ion implantation into the implant surface—were tested in vivo. Methods: Cylindrical Ti6Al4V implants were inserted bilaterally into the tibia of 110 rats. We compared two generations of cast Ti6Al4V implants (CAST 1st GEN, n = 22, and CAST 2nd GEN, n = 22) as well as cast 2nd GEN Ti6Al4V implants with calcium (CAST + CA, n = 22) and phosphorus (CAST + P, n = 22) ion implantation to standard machined Ti6Al4V implants (control, n = 22). After 4 and 12 weeks, maximal pull-out force and bone-to-implant contact rate (BIC) were measured and compared between all five groups. Results: There was no significant difference between all five groups after 4 weeks or 12 weeks with regard to pull-out force (p > 0.05, Kruskal Wallis test). Histomorphometric analysis showed no significant difference of BIC after 4 weeks (p > 0.05, Kruskal–Wallis test), whereas there was a trend towards a higher BIC in the CAST + P group (54.8% ± 15.2%), especially compared to the control group (38.6% ± 12.8%) after 12 weeks (p = 0.053, Kruskal–Wallis test). Conclusion: In this study, we found no indication of inferiority of Ti6Al4V implants cast with the optimized centrifugal precision casting technique of the second generation compared to standard Ti6Al4V implants. As the employed manufacturing process holds considerable economic potential, mainly due to a significantly decreased material demand per implant by casting near net-shape instead of milling away most of the starting ingot, its application in manufacturing uncemented implants seems promising. However, no significant advantages of calcium or phosphorus ion implantation could be observed in this study. Due to the promising results of ion implantation in previous in vitro and in vivo studies, further in vivo studies with different ion implantation conditions should be considered.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

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