scholarly journals Healing of sub-critical femoral osteotomies in mice is unaffected by tacrolimus and deletion of recombination activating gene 1

2021 ◽  
Vol 41 ◽  
pp. 345-354
Author(s):  
T-Y Liu ◽  
M Bartnikowski ◽  
AC Wu ◽  
M Veitch ◽  
KA Sokolowski ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Bone Fractures ◽  
Ex Vivo ◽  
Torsional Rigidity ◽  
Bone Volume ◽  
Torsional Stiffness ◽  
Long Bone ◽  
Mouse Strains ◽  
Tissue Volume

Clinical management of delayed healing or non-union of long bone fractures and segmental defects poses a substantial orthopaedic challenge. There are suggestions in the literature that bone healing may be enhanced by inhibiting the activities of T and B lymphocytes, but this remains controversial. To examine this matter in more detail, sub-critical-sized segmental defects were created in the femora of mice and it was assessed whether there might be a benefit from the administration of a Food and Drug Administration (FDA)-approved drug that blocks T cell activation (tacrolimus). Defects were stabilised using an internal plate. In certain groups of animals, 1 mg/kg or 10 mg/kg tacrolimus was delivered locally to the defect site for 3 or 7 d using an implanted osmotic pump with a silicon catheter directing drug delivery into the defect area. Healing was monitored by weekly X-ray and assessed at 12 weeks by mechanical testing, µCT and histology. Radiographic and histological evaluations revealed that 100 % of defects healed well regardless of tacrolimus dosage or duration. A comparison of healed C57BL/6 and Rag1−/− femora by µCT and ex vivo torsion testing showed no differences within mouse strains in terms of bone volume, tissue volume, bone volume/tissue volume ratio, shear modulus, torsional rigidity or torsional stiffness. These data failed to support an important role for tacrolimus in modulating the natural healing of segmental defects under those experimental conditions.

scholarly journals CD14 Expressing Precursors Give Rise to Highly Functional Conventional Dendritic Cells for Use as Dendritic Cell Vaccine

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3818
Author(s):  
Maud Plantinga ◽  
Denise A. M. H. van den Beemt ◽  
Ester Dünnebach ◽  
Stefan Nierkens
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Dendritic Cell Vaccine ◽  
Cell Vaccine ◽  
Activated T Cells

Induction of long-lasting immunity by dendritic cells (DCs) makes them attractive candidates for anti-tumor vaccination. Although DC vaccinations are generally considered safe, clinical responses remain inconsistent in clinical trials. This initiated studies to identify subsets of DCs with superior capabilities to induce effective and memory anti-tumor responses. The use of primary DCs has been suggested to overcome the functional limitations of ex vivo monocyte-derived DCs (moDC). The ontogeny of primary DCs has recently been revised by the introduction of DC3, which phenotypically resembles conventional (c)DC2 as well as moDC. Previously, we developed a protocol to generate cDC2s from cord blood (CB)-derived stem cells via a CD115-expressing precursor. Here, we performed index sorting and single-cell RNA-sequencing to define the heterogeneity of in vitro developed DC precursors and identified CD14+CD115+ expressing cells that develop into CD1c++DCs and the remainder cells brought about CD123+DCs, as well as assessed their potency. The maturation status and T-cell activation potential were assessed using flow cytometry. CD123+DCs were specifically prone to take up antigens but only modestly activated T-cells. In contrast, CD1c++ are highly mature and specialized in both naïve as well as antigen-experienced T-cell activation. These findings show in vitro functional diversity between cord blood stem cell-derived CD123+DC and CD1c++DCs and may advance the efficiency of DC-based vaccines.

A Randomized Phase II Trial of Idiotype Vaccination and Adoptive Autologous T-Cell Transfer in Multiple Myeloma patients

Blood ◽  
2021 ◽  
Author(s):  
Muzaffar H Qazilbash ◽  
Neeraj Y Saini ◽  
Cha Soung-chul ◽  
Zhe Wang ◽  
Edward Stadtmauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase Ii ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Phase Ii Trial ◽  
Ex Vivo ◽  
Vaccine Strategy ◽  
Randomized Phase Ii

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding anti-myeloma idiotype-keyhole limpet hemocyanin (Id-KLH) vaccine to vaccine-specific co-stimulated T cells. In this randomized, phase II trial, eligible patients received either the control (KLH only) or Id-KLH vaccine, an auto-transplant, vaccine-specific co-stimulated T-cells expanded ex-vivo, and two booster doses of the assigned vaccine. In 36 patients (20 in KLH, 16 in Id-KLH) enrolled, no dose-limiting toxicity was seen in either arm. At last evaluation, 6 (30%) and 8 (50%) had achieved complete remission in KLH-only and Id-KLH, respectively (p=0.22) and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; p=0.32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with KLH-only patients, there was a greater change in IR genes in T-cells in Id-KLH patients relative to baseline. Specifically, upregulation of genes associated with activation, induction of effector function, and generation of memory CD8+ T cells after Id-KLH, but not after KLH control vaccination, was observed. Similarly, responding patients across both arms were associated with upregulation of genes associated with T-cell activation. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of Id-KLH patients analyzed. In conclusion, in this combination immunotherapy approach, we observed a significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies.

scholarly journals Mechanical flexural ex vivo study of osteotomized swine femurs stabilized with two types of polyamide 12 rods

2017 ◽  
Vol 47 (8) ◽  
Author(s):  
Juliana Scarpa da Silveira Almeida ◽  
Débora de Oliveira Garcia ◽  
Renato Camargo Bortholin ◽  
Carlos Amaral Razzino ◽  
Cristiane dos Santos Honsho ◽  
...  
Keyword(s):  
Bone Fractures ◽  
Ex Vivo ◽  
Polymeric Materials ◽  
Fracture Site ◽  
Medullary Canal ◽  
Modulus Of Rupture ◽  
Long Bone ◽  
Bone Implant ◽  
Polyamide 12 ◽  
Implant System

ABSTRACT: Long bone fractures are commonly in surgery routine and several bone imobilization techniques are currently available. Technological progress has enabled to use low cost materials in surgical procedures. Thus, the aim of this study was to evaluate the applicability of polyamide 12 rods, solid and hollow in swine femurs, comparing them through flexion strength. This study had as second aim to fix the locking errors, commom place in interlocking nails, once polyamide 12 allows perforation in any direction by orthopaedic screw. Six groups were used: G1 - eight whole swine femurs; G2 - eight whole swine femurs with drilled medullary canal; G3 - two solid polyamide 12 rods; G4 - two hollow polyamide 12 rods; G5 - eight osteotomized drilled swine femurs with a solid polyamide 12 rod implanted in the medullary canal and locked by four 316L stainless steel screws; and G6 - similar to G5 but using hollow rods instead of solid ones. No significant differences were observed for the modulus of rupture between solid and hollow rods, demonstrating that both rods had similar performances. These results led to the speculation that the addition of other polymers to the hollow rods could increase their strength and thus the bone-implant system. Furthermore, the comparison between G1, G5 and G6 could be analyzed using the finite element method in future. New polymeric materials may be developed based on the data from this study, strengthening the bone-implant system and making possible screws to be placed in any direction, nullifying the detrimental forces on the fracture site.

scholarly journals In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

2006 ◽  
Vol 74 (7) ◽  
pp. 3817-3824 ◽  
Author(s):  
Karen L. Wozniak ◽  
Jatin M. Vyas ◽  
Stuart M. Levitz
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

scholarly journals Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

2020 ◽  
Author(s):  
Thomas Vollbrecht ◽  
Aaron O. Angerstein ◽  
Bryson Menke ◽  
Nikesh M. Kumar ◽  
Michelli Faria Oliveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Latent Reservoir ◽  
Antigen Specificity ◽  
Pcr Assay ◽  
Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

scholarly journals GABAA receptor α4-subunit knockout enhances lung inflammation and airway reactivity in a murine asthma model

2017 ◽  
Vol 313 (2) ◽  
pp. L406-L415 ◽  
Author(s):  
Gene T. Yocum ◽  
Damian L. Turner ◽  
Jennifer Danielsson ◽  
Matthew B. Barajas ◽  
Yi Zhang ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Asthma Model

Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate γ-aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4-subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4-subunit global knockout (KO; Gabra40/0) mice. Wild-type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to PBS intranasally 5 days/wk for 3 wk. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation ( P = not significant; n = 4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice ( P < 0.05, n = 8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration ( P < 0.05; n = 4) and increased markers of lung T-cell activation/memory (CD62L low, CD44 high; P < 0.01, n = 4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T-cell receptor as compared with WT CD4+ cells. These data suggest that the GABAAR α4-subunit plays a role in immune cell function during allergic lung sensitization. Thus GABAAR α4-subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.

scholarly journals Flow Cytometric Evaluation of T Cell Activation Markers after Cardiopulmonary Bypass

2014 ◽  
Vol 2014 ◽  
pp. 1-6
Author(s):  
Maja-Theresa Dieterlen ◽  
Hartmuth B. Bittner ◽  
Attila Tarnok ◽  
Jens Garbade ◽  
Stefan Dhein ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Activation Markers ◽  
Increased Risk ◽  
Patients At Risk ◽  
Inflammatory Processes ◽  
Activation Pathways

Background. Cardiopulmonary bypass surgery (CPBS) is associated with an increased risk for infections or with subsequent organ dysfunction. As T cell activation is a central mechanism during inflammatory processes, we developed an assay to evaluate T cell activation pathways in patients undergoing CPBS.Methods. Blood was obtained from eleven patients undergoing CPBS preoperatively, on postoperative day (POD)-3, and on POD-7 and was stimulated with different concentrations of Concanavalin A (ConA). Cyclosporine and sirolimus, inhibiting different pathways of the T cell cycle, were added to blood ex vivo. Expression of T cell activation markers CD25 and CD95 was analyzed by flow cytometry.Results. In untreated blood, expression of CD25 and CD95 significantly increased with higher ConA concentrations(P<0.05)and decreased for all ConA concentrations for both antigens over the study time(P<0.05). Independently from the ConA concentration, inhibition of CD25 and CD95 expression was highest preoperatively for sirolimus and on POD-3 for cyclosporine. At all time points, inhibition of CD25 and CD95 expression was significantly higher after cyclosporine compared to sirolimus treatment(P<0.001).Conclusion. Our results showed that different pathways of T cell activation are impaired after CPBS. Such knowledge may offer the opportunity to identify patients at risk for postoperative complications.

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