Efficacy assessment of interferon-alpha-engineered mesenchymal stromal cells in a mouse plasmacytoma model

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules, which can inhibit tumor cell proliferation and enhance the anti-tumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-alpha) gene (BM-MSCs/IFN-alpha) to assess in a mouse plasmacytoma model the efficacy of this approach towards neoplastic plasma cells. We found that IFN-alpha can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-alpha significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The anti-tumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-alpha did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-alpha are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs still needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Investigating and Modelling an Engineered Millifluidic In Vitro Oocyte Maturation System Reproducing the Physiological Ovary Environment in the Sheep Model

In conventional assisted reproductive technologies (ARTs), oocytes are in vitro cultured in static conditions. Instead, dynamic systems could better mimic the physiological in vivo environment. In this study, a millifluidic in vitro oocyte maturation (mIVM) system, in a transparent bioreactor integrated with 3D printed supports, was investigated and modeled thanks to computational fluid dynamic (CFD) and oxygen convection-reaction-diffusion (CRD) models. Cumulus-oocyte complexes (COCs) from slaughtered lambs were cultured for 24 h under static (controls) or dynamic IVM in absence (native) or presence of 3D-printed devices with different shapes and assembly modes, with/without alginate filling. Nuclear chromatin configuration, mitochondria distribution patterns, and activity of in vitro matured oocytes were assessed. The native dynamic mIVM significantly reduced the maturation rate compared to the static group (p < 0.001) and metaphase II (MII) oocytes showed impaired mitochondria distribution (p < 0.05) and activity (p < 0.001). When COCs were included in a combination of concave+ring support, particularly with alginate filling, oocyte maturation and mitochondria pattern were preserved, and bioenergetic/oxidative status was improved (p < 0.05) compared to controls. Results were supported by computational models demonstrating that, in mIVM in biocompatible inserts, COCs were protected from shear stresses while ensuring physiological oxygen diffusion replicating the one occurring in vivo from capillaries

Interferon-alpha-engineered Multipotent Mesenchymal Stromal Cells for the treatment of myeloma

Bone marrow mesenchymal stromal cells (BM-MSCs) are nonhematopoietic progenitor cells with multilineage differentiation potential. Exogenously administered BM-MSCs have been shown to survive and proliferate in the presence of malignant cells, becoming stromal cells and supporting tumor growth. Thus, BM-MSCs are attractive candidates to deliver biologically active molecules in the tumor environment in vivo and to enhance specific immune responses. Interferon-a (IFN-a) has been used for years for the maintenance treatment of multiple myeloma (MM), but its administration is limited by the temporary efficacy and the toxicity. We analyzed the in vivo effects of mouse BM-MSCs transduced with IFN-a cDNA in the Sp6 plasmacytoma mouse model. BM-MSCs were transduced with a lentiviral vector containing EGFP cDNA or murine IFN-a cDNA (efficiency = 70%). Two months-old Balb/c mice (Balb/cByJIco, Charles River Italia, Calco, LC, Italy) (H-2d), were injected subcutaneously (s.c.) with the tumorigenic dose of 0.5x106 Sp6 cells (H-2d). The same mice were then weekly injected with 0.5x106 BMMSCs/ IFN-a (1, 4 or 8 doses), in the same anatomical quarter. Some mice was injected s.c. with Sp6 and with BM-MSCs/EGFP s.c. or intravenously (i.v.) to test in vivo homing. Tumor immunohistochemistry was performed with anti-von Willebrand factor, anti- a -smooth muscle actin, anti-CD4, anti-CD8, anti-asialo GM1, anti-CD45, anti-CD90, anti-murine IFN-a. BM-MSCs were capable of homing into Sp6 tumor, forming clusters of cells. Treatment with BM-MSCs/IFN-a resulted in a significant delay in the onset of palpable tumors (event free survival, EFS, of 50% at day +17 for 1 dose, +20 for 4 doses and +64, for 8 doses, whereas Sp6 alone or coinjected with BM-MSCs showed tumor incidence of 100% 10-13 days after injection). Weekly delivery of BMMSCs/ IFN-a induced a significant decrease of kinetics tumor growth and an increment of overall survival (OS) (median OS in controls: 19 days, animals receiving BM-MSCs: 17 days, mice treated with 1 and 4 doses of BM-MSCs/IFN-a: 30-31 days, mice treated with 8 doses:77 days). The antitumor effect is associated with tumor necrosis, reduction in microvessel density, and NK cell infiltration. These findings indicate that transduced BM-MSCs could be useful to deliver anti-cancer molecules in the microenvironment of myeloma and become a promising tool for specific, low-toxic, and long-lasting anti-myeloma therapy

IFN-gamma- Mediated Upmodulation of MHC Class I Expression Associated to a B7-1-Expressing Tumor Cell Vaccine Activates Tumor-Specific Immune Response in a Mouse Model of Prostate Cancer.

Prostatic adenocarcinoma (PCa) carries out a mechanism of immune evasion based on the progressive donwnmodulation of the MHC class I (MHC-I) genes and the lack of the expression of MHC class II genes. The mouse PCa cell lines TRAMP-C1 and TRAMP-C2, derived from the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP), which develops in C57BL/6 male mice transgenic for the SV40 large tumor antigen coding region under the transcriptional control of the prostate-specific rat probasin promoter, represent a suitable animal model to study the influence of MHC-I molecules on protection against tumor development and progression in vivo. In these cell lines, in fact, MHC-I expression decreases after in vitro passaging, a phenotype closely resembling that described in human PCa. TRAMP-C1 and -C2 are tumorigenic when injected in syngeneic C57BL/6 mice. In this study we have analyzed the effect of transfection of TRAMP-C2 with the cDNA of the costimulatory molecule B7-1 (TRAMP-C2/B7 transfectants) on tumor growth in vivo and on tumor-specific immune response. TRAMP-C2/B7 showed impaired growth in vivo, although it did not elicit a protective response against the B7-1 negative TRAMP-C2 parental tumor. Upon increasing MHC-I expression in TRAMP-C2 and TRAMP-C2/B7 cells with IFN-g prior to injection in syngeneic animals, protection against TRAMP-C2 tumor growth rised (tumor incidence: 44%), as well as tumor-specific cytotoxicity of spleen-derived cells. IFN-a increased MHC-I expression in TRAMP-C2 and TRAMP-C2/B7 as well, but it did not inhibit tumor growth (tumor incidence: 100%). Western blot analysis showed that both interferons enhanced TAP-1, TAP-2, LMP-2 and LMP-7 expression, but IFN-g was quantitatively more efficient than IFN-a. These results suggest that IFN-g plays a fundamental role in TRAMP-C2 immunogenicity, by inducing TAP-1, TAP-2, LMP-2 and LMP-7 enough to provide correct production and transport of immunogenic peptides from cytosol to endoplasmic reticulum to be assembled to MHC-I molecules

IFN-gamma-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer

De novo expression of B7-1 impaired tumorigenicity of TRAMP-C2 mouse prostate adenocarcinoma (TRAMP-C2/B7), but it did not elicit a protective response against TRAMP-C2 parental tumor, unless after in vitro treatment with IFN-gamma. TRAMP-C2 cells secrete TGF-beta and show low MHC-I expression. Treatment with IFN-gamma increased MHC-I expression by induction of some APM components and antagonizing the immunosuppressant activity of TGF-beta. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-gamma-mediated fine-tuned modulation of either APM expression or TGF-beta signaling. To explore possible clinical translation, we delivered IFN-gamma to TRAMP-C2 tumor site by means of genetically engineered MSCs secreting IFN-gamma

IFN-gamma- Mediated Upmodulation of MHC Class I Expression Activates Tumor-Specific Immune Response in a Mouse Model of Prostate Cancer

The mouse prostatic adenocarcinoma tumorigenic cell lines TRAMP-C1 and TRAMP-C2 represent a suitable animal model to study the role of major histocompatibility class-I (MHC-I) molecules expression in protection against tumor development and progression in vivo. In these cell lines, MHC-I expression decreases after time of in vitro cell culture, but it can be restored by treatment with IFN-\u3b3. We have transduced TRAMP-C2 cells with the cDNA of the co-stimulatory molecule B7-1. TRAMP-C2/B7 transfectants showed impaired growth in vivo, but they did not elicit a protective response against TRAMP-C2 parental tumor, unless after treatment with IFN-\u3b3 prior to injection. IFN-\u3b3 is an inducer of some components of the MHC-I antigen processing and presentation machinery (APM). IFN-\u3b3 is also an antagonist of the immunosuppressant activity of TGF-\u3b2, largely produced by TRAMP-C2. Thus, immunization with TRAMP-C2/B7 conferred protection against TRAMP-C2-derived tumors in function of the IFN-\u3b3-mediated fine-tuned modulation of either APM expression or TGF-\u3b2 signaling. To explore possible clinical traslation of these results we attempted to deliver IFN-\u3b3 to TRAMP-C2 tumor growth site by means of genetically engineered mesenchymal stem cells (MSCs) secreting IFN-\u3b3. This approach produced results matching those obtained with IFN-\u3b3-treated TRAMP-C2 cells