Proliferation Of Target Cells Research Articles

Abstract 4017: CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells

Abstract The development of immunotherapies relies on the use of in vitro potency assays, which are crucial for understanding the complex interactions between immune cells (effectors) and cancer cells (targets). Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapeutic approach that exhibits promise; however, varying levels of antigen expression on the surface of tumor cells can influence the efficacy of CAR T cell-mediated killing. To fully understand the impact of target antigen density on CAR T cell activity, we utilized a matrix approach to assess both target cell death, using an impedance-based in vitro potency assay to quantify CAR T cell-mediated cytotoxicity, as well as cytokine release with homogeneous LumitTM immunoassays. SKOV3 (high HER2 expression), A549 (low HER2 expression), or MDA-MB-231 (no HER2 expression) target cells were seeded into a 96-well microplate with embedded electrodes in the substrate that detect the attachment and proliferation of target cells. HER2 CAR T cells were added after 24 hours at effector-to-target (E:T) ratios of 1:5 or 1:1 and target cell cytolysis was recorded continuously by the Maestro Z for 72 hours. Cytolysis of the target cells was calculated by comparing the treated wells to no treatment control wells and full lysis wells (1% TritonX). At 72 hours post CAR T cell addition, complete killing was observed in SKOV3 cells at the 1:1 ratio, while A549 cells exhibited only 80% cytolysis. MDA-MB-231 cells showed 20% cytolysis, likely due to nonspecific killing by non-engineered T cells, as approximately only 78% of the CAR T cell population was CAR positive. Because the cytolysis assay was label-free, cytokine analysis was able to be multiplexed with the same plates. To assess release of pro-inflammatory cytokines, TNF-α and IFN-γ, we collected supernatant post-antigen exposure at 24, 48, and 72 h. At the 1:1 E:T ratio, CAR T cells co-cultured with target cells expressing low (A549) or high (SKOV3) levels of HER2 had the highest TNF-α production at 24 hours, with CAR T cells co-cultured with SKOV3 cells releasing 30.6% more TNF-α when compared to A549 cells. The highest levels of IFN-γ were detected in both A549 and SKOV3 groups at the 1:1 E:T ratio, with the highest levels observed from CAR T cells co-cultured with SKOV3 cells at 72 hours, releasing approximately 1459.6 +/- 357.3 pg/mL of IFN-γ, compared to 1093.8 +/- 387.5 pg/mL IFN-γ released by CAR T cells co-cultured with A549 cells. As expected, CAR T cells co-cultured with MDA-MB-231 cells did not release any detectable TNF-α or IFN-γ at either E:T ratios during the duration of the experiment. Future work will continue to explore multiplexed potency assays for the evaluation of CAR T cell therapies. Citation Format: Sierra Barnes, Denise Sullivan, Dan Lazar, Kim Haupt, Danielle Califano, Stacie Chvatal, Daniel Millard. CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4017.

Abstract 1417: IRF8 driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma

Abstract Glioblastoma (GBM) has an extremely immunosuppressive microenvironment, primarily mediated by the recruitment of immature myeloid cells (IMCs) to the tumor. IMCs suppress T cell function and dampen the anti-tumor response. In this study, we sought to “reprogram” IMCs into antigen-presenting cells (APCs) to enhance anti-tumor T cell responses. To induce IMC differentiation into APCs, we utilized a retroviral replicating vector (RRV) expressing IRF8, a master regulator of type 1 conventional dendritic cell (cDC1) development. We evaluated the impact of IRF8 RRV on in vivo tumor growth kinetics, survival, immunosuppression, and immune microenvironment. Because RRV transduction requires the proliferation of target cells, we assessed the proliferative capacity of myeloid cells in murine and human GBM samples. We utilized the SB28 cell line, a poorly immunogenic murine GBM model. We pre-transduced SB28 cells with IRF8 RRV and implanted them intracranially into syngeneic C57BL/6 mice; controls expressed an empty vector. Intratumoral myeloid cells were proliferative in both murine and human samples. In vivo, tumor and myeloid cells are infected proportionately to their respective levels of proliferation. Mice with IRF8 RRV pre-transduced intracranial tumors had significantly longer survival (median 28 days) compared to controls (median 19 days) (p <0.0001). Additionally, tumor growth was delayed in IRF8 RRV mice. IRF8 RRV mice had significant enrichment of dendritic cells (p = 0.0145), cDC1s (p < 0.0001), T cells (p = 0.0025) and CD8+ T cells (p < 0.0001). Both monocytic and granulocytic IMCs had decreased expression of the immunosuppressive markers Arginase 1 and IDO1. Further, ex vivo co-culture of intra-tumoral myeloid cells with naïve T cells showed a functional decrease in immunosuppression in IRF8 RRV tumors compared to controls (p < 0.0001). To study the mechanism of action, we assessed whether IRF8 transduction of tumor cells alone or both tumor and immune cells, was necessary for the observed survival benefit. To inhibit RRV spread from infected cells to other cells, we treated mice with azidothymidine, a reverse transcription inhibitor. A robust survival benefit and tumor growth inhibition were seen only in mice where the RRV could spread freely (p = 0.0005). This finding shows that both tumor and immune cells must be infected to cause the phenotype and suggests that reprogramming immune cells is required for therapeutic efficacy. Our novel approach used RRV to deliver a myeloid transcriptional regulator in intracerebral GBM. This therapy achieved significant survival benefits and increased intratumoral cytotoxic T cells and APCs, while decreasing myeloid immunosuppression. Further studies will expand upon the mechanism of action and test combination therapies. Citation Format: Megan Montoya, Sara A. Collins, Pavlina Chuntova, Noriyuki Kasahara, Hideho Okada. IRF8 driven reprogramming of the immune microenvironment enhances anti-tumor adaptive immunity and reduces immunosuppression in murine glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1417.

Estrogenic Modulation of Ionic Channels, Pumps and Exchangers in Airway Smooth Muscle.

To preserve ionic homeostasis (primarily Ca2+, K+, Na+, and Cl-), in the airway smooth muscle (ASM) numerous transporters (channels, exchangers, and pumps) regulate the influx and efflux of these ions. Many of intracellular processes depend on continuous ionic permeation, including exocytosis, contraction, metabolism, transcription, fecundation, proliferation, and apoptosis. These mechanisms are precisely regulated, for instance, through hormonal activity. The lipophilic nature of steroidal hormones allows their free transit into the cell where, in most cases, they occupy their cognate receptor to generate genomic actions. In the sense, estrogens can stimulate development, proliferation, migration, and survival of target cells, including in lung physiology. Non-genomic actions on the other hand do not imply estrogen's intracellular receptor occupation, nor do they initiate transcription and are mostly immediate to the stimulus. Among estrogen's non genomic responses regulation of calcium homeostasis and contraction and relaxation processes play paramount roles in ASM. On the other hand, disruption of calcium homeostasis has been closely associated with some ASM pathological mechanism. Thus, this paper intends to summarize the effects of estrogen on ionic handling proteins in ASM. The considerable diversity, range and power of estrogens regulates ionic homeostasis through genomic and non-genomic mechanisms.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

Subcellular elements responsive to the biomechanical activity of triple-negative breast cancer-derived small extracellular vesicles.

Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics.

The HPV16E7 Affibody as a Novel Potential Therapeutic Agent for Treating Cervical Cancer Is Likely Internalized through Dynamin and Caveolin-1 Dependent Endocytosis.

Affibodies targeting intracellular proteins have a great potential to function as ideal therapeutic agents. However, little is known about how the affibodies enter target cells to interact with intracellular target proteins. We have previously developed the HPV16E7 affibody (ZHPV16E7384) for HPV16 positive cervical cancer treatment. Here, we explored the underlying mechanisms of ZHPV16E7384 and found that ZHPV16E7384 significantly inhibited the proliferation of target cells and induced a G1/S phase cell cycle arrest. Furthermore, ZHPV16E7384 treatment resulted in the upregulation of retinoblastoma protein (Rb) and downregulation of phosphorylated Rb (pRb), E2F1, cyclin D1, and CDK4 in the target cells. Moreover, treatment with dynamin or the caveolin-1 inhibitor not only significantly suppressed the internalization of ZHPV16E7384 into target cells but also reversed the regulation of cell cycle factors by ZHPV16E7384. Overall, these results indicate that ZHPV16E7384 was likely internalized specifically into target cells through dynamin- and caveolin-1 mediated endocytosis. ZHPV16E7384 induced the cell cycle arrest in the G1/S phase at least partially by interrupting HPV16E7 binding to and degrading Rb, subsequently leading to the downregulation of E2F1, cyclin D1, CDK4, and pRb, which ultimately inhibited target cell proliferation. These findings provide a rationale of using ZHPV16E7384 to conduct a clinical trial for target therapy in cervical cancer.

Treatment of Acute Wounds With Recombinant Human-Like Collagen and Recombinant Human-Like Fibronectin in C57BL/6 Mice Individually or in Combination.

Wound repair is accomplished by the interaction between the cells involved in the repair and the extracellular matrix (ECM). Collagen is the main component of ECM, which is involved in transduction of signal, transportation of growth factors and cytokines. Fibronectin (FN) is also an important ECM, which participates in the initiation of fibroblast cell (FC) and promotes adhesion, migration, proliferation and differentiation of target cells. Compared with natural protein, the recombinant protein prepared by artificial method has the advantages of poor immunogenicity, wide range of sources, low cost and high activity. In this study, we used recombinant human-like collagen (RHC) and recombinant human-like fibronectin (rhFN) to treat acute wounds in C57BL/6 mice individually or in combination, and explored their effects on wound healing. Our study confirmed that these two recombinant proteins could effectively promote the proliferation, migration and adhesion of FCs. Meanwhile, it could positively regulate the healing speed and quality of acute wounds, re-epithelialization, collagen deposition, inflammation and angiogenesis. Moreover, we proved that the combination of the two was better than the treatment alone. Consequently, it has a good prospect as a new tissue material in the field of skin repair.

Backward bifurcation and stability analysis in a within-host HIV model with both virus-to-cell infection and cell-to-cell transmission, and anti-retroviral therapy

Researches have shown that in addition to direct virus-to-cell infection, viral particles can also be transferred from a productively-infected cell to an uninfected cell through the formation of virological synapses. In order to reduce the viral load in infected individuals, different classes of antiretroviral drugs have been developed, including reverse transcriptase inhibitor (RTI), integrase inhibitor (II), protease inhibitor (PI) and so on. In this paper, we incorporate the mitotic proliferation of target cells which is described by the logistic term, both virus-to-cell infection and cell-to-cell transmission, the intracellular delay and RTI-based therapy into an in-host HIV infection model. Through mathematical analysis, we find that the model undergoes a backward bifurcation when the turn-over rate coefficient of productively-infected cells is smaller than its mitotic proliferation rate coefficient. When the turn-over rate coefficient of productively-infected cells is greater than its mitotic proliferation rate coefficient, the existence of Hopf bifurcation at the chronic-infection equilibrium with and without the intracellular delay is established, respectively. Numerical simulations suggest that the dynamics of the model be sensitive to parameter values and initial conditions, which may be of great significance to control HIV infection. We also show numerical evidence to support the fact that the smaller the therapy efficacy, the higher the viral load in infected individuals.

Dysregulation of LINC00324 associated with methylation facilitates leukemogenesis in de novo acute myeloid leukemia.

LINC00324 was overexpressed and facilitated carcinogenesis in various solid malignant tumors. However, the role of LINC00324 in leukemogenesis remains to be elucidated. The relative expression and unmethylation levels of LINC00324 were detected by real-time quantitative PCR (RT-qPCR) and real-time quantitative methylation-specific PCR (RT-qMSP). Cell proliferation experimental and flow cytometer (FCM) was used to detect the change of proliferation and apoptosis in leukemia cell lines after overexpression of LINC00324. The results showed that the expression of LINC00324 and the methylation level of the promoter region were significantly negatively correlated in AML patients. Moreover, patients with lower LINC00324 expression showed more prolonged overall survival (OS). Remarkably, overexpression of LINC00324 in leukemia cell lines promoted the proliferation of target cells and inhibited their apoptosis. Our findings firstly identified that the hypomethylation of LINC00324 was a common molecular event in de novo AML patients. The abnormally upregulated LINC00324 promotes proliferation and inhibits apoptosis in leukemia cells.

Nanobody-armed T cells endow CAR-T cells with cytotoxicity against lymphoma cells

BackgroundTaking advantage of nanobodies (Nbs) in immunotherapy, we investigated the cytotoxicity of Nb-based chimeric antigen receptor T cells (Nb CAR-T) against lymphoma cells.MethodsCD19 Nb CAR-T, CD20 Nb CAR-T, and Bispecific Nb CAR-T cells were generated by panning anti-human CD19- and CD20-specific nanobody sequences from a natural Nb-expressing phage display library, integrating Nb genes with a lentiviral cassette that included other CAR elements, and finally transducing T cells that were expanded under an optimization system with the above generated CAR lentivirus. Prepared Nb CAR-T cells were cocultured with tumour cell lines or primary tumour cells for 24 h or 5 days to evaluate their biological function.ResultsThe nanobodies that we selected from the natural Nb-expressing phage display library had a high affinity and specificity for CD19 and CD20. CD19 Nb CAR-T, CD20 Nb CAR-T and Bispecific Nb CAR-T cells were successfully constructed, and these Nb CAR-T cells could strongly recognize Burkitt lymphoma cell lines (Raji and Daudi), thereby leading to activation, enhanced proliferation, and specific killing of target cells. Furthermore, similar results were obtained when using patient samples as target cells, with a cytotoxicity of approximately 60%.ConclusionsNanobody-based CAR-T cells can kill both tumour cell lines and patient-derived tumour cells in vitro, and Nb-based CAR-T cells may be a promising therapeutic strategy in future immunotherapy.

Co-administration of platelet-rich plasma and small intestinal submucosa is more beneficial than their individual use in promoting acute skin wound healing.

Acute skin wounds may compromise the skin barrier, posing a risk of infection. Small intestinal submucosa (SIS) is widely used to treat acute and chronic wounds. However, the efficacy of SIS to accelerate wound healing still needs to be improved to meet clinical demands. To tackle this problem, platelet-rich plasma (PRP) is used due to its potency to promote proliferation, migration and adhesion of target cells. In this study, we applied PRP and SIS to skin wounds to explore their effects on wound healing by evaluating re-epithelialization, collagen production, angiogenesis and the inflammatory response. A 1 × 1-cm full-thickness skin defect was established in mice. Sixty mice were divided into four treatment groups: PRP + SIS, PRP, SIS and control. On days 3, 5, 7, 10 and 14 post-surgery, tissue specimens were harvested. Haematoxylin and eosin, Masson's trichrome, immunohistochemical and immunofluorescence double staining were used to visualize epidermal thickness, collagen and vascular regeneration and inflammation. Wound contraction in the PRP and PRP + SIS groups was significantly greater, compared with the other groups, on days 3 and 5 post-surgery. A histological analysis showed higher collagen expression in the PRP and PRP + SIS groups on day 7, which was associated with a thicker epidermal layer on day 14. In addition, immunohistochemical staining showed that CD31-positive blood vessels and vascular endothelial growth factor expression in the PRP + SIS and PRP groups were significantly higher, compared with the control group. Furthermore, immunofluorescence double staining showed that the number of M1 and M2 macrophages in the PRP + SIS and PRP groups was higher, compared with the control and SIS groups alone, on day 3. However, on day 7, the number of M1 macrophages dramatically decreased in the PRP + SIS and PRP groups. The ratio of M2 to M1 macrophages in the PRP + SIS and PRP groups was 3.97 and 2.93 times that of the control group and 4.56 and 3.37 times that of the SIS group, respectively. Co-administration of SIS and PRP has a better effect on promoting angiogenesis, re-epithelialization and collagen regeneration in managing acute wound healing than either agent alone.

The Effect of HRE-Regulated VEGF Expression and Transfection on Neural Stem Cells in Rats.

In this study, we analyzed neural stem cells transfected with the HRE-VEGF gene in groups experiencing different periods of hypoxia. The results of RT-PCR showed that the expression of vascular endothelial growth factor (VEGF) mRNA gradually increased with the prolonged period of hypoxia (p < 0.05). The results from the western-blot test showed that expression of the VEGF protein increased with as the period of hypoxia increased (p < 0.05). The results of MTT combined with Elisa reagent showed that with the prolonged period of hypoxia, the secretion of VEGF protein increased, and that the proliferation of target cells and neural stem cells was better promoted (p < 0.05). These results imply that HRE can safely and effectively regulate VEGF expression. By controlling the period of hypoxia, we can increase the expression level, and limit it in more safe values to avoid the possibility of cancer caused by the over-enhancement of proliferation of target cells due to the overexpression of the VEGF protein.

Three-dimensional mechanical microenvironment enhanced osteogenic activity of mesenchymal stem cells-derived exosomes

Exosomes derived from mesenchymal stem cells (MSCs) through the paracrine pathway have shown great potentials in cell-free therapy for regenerative medicine. Although MSCs have been recently known to respond to the mechanical microenvironment and thus orchestrate their behaviors and functions, the effects of such a biophysical cue on the paracrine pathway of MSCs, especially on the regulation of characteristics and bioactivity of MSC-derived exosomes have rarely been reported. In this study, we used periodontal ligament stem cells (PDLSCs), which were cultured in the three-dimensional(3D) microscale magnetically stretched collagen hydrogels, as the model MSCs to explore the changes in characteristics (e.g., morphology, size distribution, typical surface protein expression, miRNA content, and internalization) and bioactivity of MSC-derived exosomes in response to matrix strain. We found that the levels of 25 miRNAs in exosomes secreted by PDLSCs in the 3D strain microenvironment (SM-Exo) were dramatically different from those obtained from the 3D culture microenvironment (Exo). Next, the bioactivity of SM-Exo was significantly enhanced as reflected by the improved proliferation, migration, and osteogenic differentiation of target cells (e.g., BMSCs). This was further confirmed by in vivo studies, in which PDLSC-derived exosomes obtained from the 3D strain microenvironment showed stronger bioactivity to repair alveolar bone defects in SD rats. This study proposed a novel strategy to promote alveolar bone regeneration via altered PDLSC-derived exosomes in the 3D strain microenvironment, and such an engineered mechanical microenvironment would be an effective tool in other regenerative studies and clinical applications.

New Recombinant Carriers Binding Specifically to the Epidermal Growth Factor Receptor.

New recombinant carriers-modular nanotransporters (MNTs)-with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111In significantly enhanced the cytotoxic effect of 111In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z1907 did not have a similar effect.

HAS3-induced extracellular vesicles from melanoma cells stimulate IHH mediated c-Myc upregulation via the hedgehog signaling pathway in target cells

Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.

Therapeutic mesenchymal stromal stem cells: Isolation, characterization and role in equine regenerative medicine and metabolic disorders.

Mesenchymal stromal cells (MSC) have become a popular treatment modality in equine orthopaedics. Regenerative therapies are especially interesting for pathologies like complicated tendinopathies of the distal limb, osteoarthritis, osteochondritis dissecans (OCD) and more recently metabolic disorders. Main sources for MSC harvesting in the horse are bone marrow, adipose tissue and umbilical cord blood. While the acquisition of umbilical cord blood is fairly easy and non-invasive, extraction of bone marrow and adipose tissue requires more invasive techniques. Characterization of the stem cells as a result of any isolation method, is also a crucial step for the confirmation of the cells' stemness properties; thus, three main characteristics must be fulfilled by these cells, namely: adherence, expression of a series of well-defined differentiation clusters as well as pluripotency. EVs, resulting from the paracrine action of MSCs, also play a key role in the therapeutic mechanisms mediated by stem cells; MSC-EVs are thus largely implicated in the regulation of proliferation, maturation, polarization and migration of various target cells. Evidence that EVs alone represent a complex network 0involving different soluble factors and could then reflect biophysical characteristics of parent cells has fuelled the importance of developing highly specific techniques for their isolation and analysis. All these aspects related to the functional and technical understanding of MSCs will be discussed and summarized in this review.

Downregulation of activin-signaling gene expression in passaged normal human dermal fibroblasts.

Activins are members of the transforming growth factor-β (TGF-β) superfamily and play important roles in proliferation, differentiation, and apoptosis of various target cells. We investigated changes of activin, activin receptor (ActR), and Smad-signaling gene expression with increasing passage number in normal human dermal fibroblasts. The expression of mRNA and protein was measured by reverse transcription-quantitative polymerase chain reaction and immunoblot analysis from passage numbers 5 to 15. Activin A and follistatin transcript levels increased with increasing passage number. ActR types IA, IB, IIA and IIB mRNA levels decreased at high passage number. The levels of Smad2, 3 and 4 protein decreased with increasing passage number, which also attenuated phosphorylation of Smad2 and 3 protein expression. Smad7 was enhanced with increasing passage number. These results suggest that expression of activin-signaling in aging normal human dermal fibroblasts increases activin A and follistatin, whereas ActR-Smad signaling is decreased.

Th1 Cytokine Polymorphism Gene: TNF-Alpha -857C/T Affects the Pathogenesis and Progression of Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is a hematological malignancy characterized by the autonomous growth of immature myeloid cells with impaired differentiation and maturation. Cytokines are low-molecular-weight proteins that play a basic and fundamental role in communication within the immune system. Cytokines induce various effects such as differentiation, proliferation, hematopoiesis, and inflammation of target cells. AML is also closely associated with cytokine networks in terms of proliferation, apoptosis, and differentiation of leukemic cells. Cytokines produced by Th1 involved in cell-mediated immunity are called Th1 cytokines. Th1 cytokine includes TNF-α and IL-2. Several studies have reported that TNF-α is highly expressed in leukemia cells with AML patients. Other studies have also reported that high serum level of TNF-α of AML patients is associated with poor survival outcome. However, the association between Th1 cytokine polymorphisms: TNF-α -857C/T and IL-2-330T/G and the pathogenesis of AML is unclear. Therefore, we investigated the role of these polymorphisms in AML. Materials and Methods: This study included 101 patients with AML [male/female, 56/45; age, 15-86 years; median age, 58 years; MRC classification favorable (n = 38), intermediate (n =56), and adverse (n = 7)] and 202 healthy race-matched controls. All participants provided written informed consent. This study was approved by the Institutional Review Board of Gunma University Hospital. Genotyping was performed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Genotype and allele frequency were compared between patient group and control group by χ2-test. Clinical features were compared using Student's t and χ2 tests. Overall survival (OS) and leukemia free survival (LFS) were calculated using the Kaplan-Meier method. Survival curves were compared using the log-rank test. Analyses were performed using the SPSS software package ver. 25 (IBM, Armonk, NY, USA). P &amp;lt; 0.05 was considered to represent statistical significance. Results: TNF-α -857 C/T nonCC genotype (higher producer type) increases the risk of AML (AML vs. controls = 39.6% vs. 28.2%, OR = 1.67, 95% CI = 1.01-2.75, p = 0.045). Moreover, the frequency of TNF-α -857 C/T T allele (higher producer type) was higher in AML patients compared to controls (AML vs. controls = 24.8% vs. 16.8%, OR = 1.625, 95%CI = 1.078-2.451 p = 0.02). There was no significant difference between AML patients and controls in genotype and allele frequencies of IL-2 -330 T/G. In the analysis of clinical features, the average platelet count was significantly lower in TNF-α -857 C/T TT genotype (higher producer type) (TT vs. nonTT = 2.4±1.4 vs. 4.4±5.9, p &amp;lt; 0.01). TT genotype (higher producer type) was also significantly higher in frequency of MRC classification adverse (TT vs. nonTT = 30.0% vs. 4.4%, p = 0.02) and history of tumor (TT vs. nonTT = 30.0% vs. 6.6%. p =0.04). Moreover, in survival time analysis, patients with TNF-α -857 C/T TT genotype (higher producer type) had significantly shortened OS compared with patients with nonTT genotype (lower producer type) (TT vs. nonTT = 17.2 months vs not reached, p &amp;lt; 0.01). Patients with TT genotype (high producer type) also experienced significantly shortened LFS (TT vs. nonTT = 24.0 months vs not reached, p = 0.04). Furthermore, multivariate analysis of OS revealed TNF-α -857 C/T TT genotype (higher producer type) as an independent prognostic factor (HR = 3.01, 95% CI = 1.04-8.69, p = 0.04), like age and white blood cell count. Conclusion: These results suggest that TNF-α-857 C/T T allele (higher producer type) increases the risk of AML. Furthermore, TNF-α-857 C/T TT genotype (higher producer type) affects the poor prognosis. Therefore, these data suggest the new role of TNF-α polymorphism in AML leukemogenesis. Figure Disclosures Handa: Ono: Research Funding.

Immunization against environmental chemical carcinogens: Pro and contra

The problems of immunoprotection from the environmental chemical carcinogens are discussed. The main experimental argument pro active immunization against carcinogens is a possibility of specific mucosal antibodies (Abs) to inhibit the penetration of carcinogens from environment and to stimulate its excretion with the following decreasing of carcinogen-DNA adducts levels. Hypothesis of cancer immunostimulation after active immunization against carcinogens is based on a high cancer risk in persons with high levels of serum Abs specific to environmental carcinogens coupled with high levels of Abs to endogenous steroids stimulating the proliferation of target cells, for example, Abs to benzo[a]pyrene together with Abs to estradiol. The active immunization could increase the cancer risk much more in those persons. The passive immunization could be an alternative safe approach to avoid this problem.

Intestinal CD4 Depletion in HIV / SIV Infection.

Among the most significant findings in the pathogenesis of HIV infection was the discovery that almost total depletion of intestinal CD4+ T cells occurs rapidly after SIV or HIV infection, regardless of the route of exposure, and long before CD4+ T cell losses occur in blood or lymph nodes. Since these seminal discoveries, we have learned much about mucosal and systemic CD4+ T cells, and found several key differences between the circulating and intestinal CD4+ T cell subsets, both in phenotype, relative proportions, and functional capabilities. Further, specific subsets of CD4+ T cells are selectively targeted and eliminated first, especially cells critically important for initiating primary immune responses, and for maintenance of mucosal integrity (Th1, Th17, and Th22 cells). This simultaneously results in loss of innate immune responses, and loss of mucosal integrity, resulting in mucosal, and systemic immune activation that drives proliferation and activation of new target cells throughout the course of infection. The propensity for the SIV/HIV to infect and efficiently replicate in specific cells also permits viral persistence, as the mucosal and systemic activation that ensues continues to damage mucosal barriers, resulting in continued influx of target cells to maintain viral replication. Finally, infection and elimination of recently activated and proliferating CD4+ T cells, and infection and dysregulation of Tfh and other key CD4+ T cell results in hyperactive, yet non-protective immune responses that support active viral replication and evolution, and thus persistence in host tissue reservoirs, all of which continue to challenge our efforts to design effective vaccine or cure strategies.