BackgroundThe CD47/SIRPα axis mediates a ‘don’t eat me’ signal exploited by tumor cells to escape macrophage-mediated immune surveillance. Anti-CD47 therapies have shown promising clinical results in ...solid and hematological malignancies; however, efficacy is hindered by systemic toxicity. We hypothesized that local delivery of a therapeutic, able to interfere with the CD47/SIRPα axis within an oncolytic viral chassis, would induce high payload expression paired with oncolytic activity and low systemic exposure, ultimately resulting in improved tumor control.MethodsAlpha-201-macro1 is a first-in-class, replication-defective oncolytic virus encoding a protein payload able to interfere with the CD47/SIRPα axis. Disruption of the interaction between CD47 and SIRPα was confirmed in vitro using a plate-based CD47 displacement assay. Bioactivity was tested in an ex vivo phagocytosis assay using Raji cells and differentiated M1 macrophages treated with conditioned medium obtained from tumor cells infected with Alpha-201-macro1 or control vectors. Phagocytosis was measured by flow cytometry. Anti-tumor efficacy of Alpha-201-macro1, delivered intratumorally at 3x107 PFU, was assessed in A549 tumor-bearing BALB/c-Nude mice (N=8 per group) and compared to anti-CD47 antibody therapy (clone: B6H12; intraperitoneal, 10 mg/kg). Treatments were administered on day 1, 4, 7, 10, and 13 after randomization. All values are reported as mean±SEM.ResultsConditioned media from Alpha-201-macro1 infected cells selectively disrupted binding of SIRPα to CD47 in a payload- (and not vector-) dependent manner. Additionally, conditioned medium from Alpha-201-macro1 infected cells resulted in a dose-dependent increase in macrophage-mediated target cell phagocytosis (3 PFU/cell: 4.16±0.17%, 10 PFU/cell: 17.8±2.55%) that was greater than the effect of the anti-CD47 antibody (6.86±1.68%, p=0.017 compared to 10 PFU/cell Alpha-201-macro1). Pretreatment with conditioned medium from Alpha-201 vectors encoding irrelevant payloads did not significantly affect macrophage phagocytosis, demonstrating the specificity of the effect of the therapeutic payload. In vivo, treatment with Alpha-201-macro1 resulted in tumor growth inhibition compared to the vehicle control group (217.1±17.4 mm3 vs. 312.7±30.5 mm3, p=0.023). This effect was dependent on payload expression, as a control vector did not show statistically significant tumor activity. There was a trend towards greater efficacy with local Alpha-201-macro1 delivery compared to systemic anti-CD47 antibody (217.1±17.4 mm3 vs. 248.7±27.9 mm3, p=0.38).ConclusionsAlpha-201-macro1 enhances macrophage phagocytosis ex vivo and exerts anti-tumor efficacy in vivo, effects which exceed those of a systemically administered anti-CD47 antibody. Further in vivo studies of Alpha-201-macro1 and modified, multi-payload versions of this vector, in combination with immune checkpoint inhibitors, are ongoing.Ethics ApprovalAll procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC).
CD248 (Endosialin) is a type 1 membrane protein involved in developmental and pathological angiogenesis through its expression on pericytes and regulation of PDGFRβ signalling. Here we explore the ...function of CD248 in skeletal muscle angiogenesis. Two distinct forms of capillary growth (splitting and sprouting) can be induced separately by increasing microcirculatory shear stress (chronic vasodilator treatment) or by inducing functional overload (extirpation of a synergistic muscle). We show that CD248 is present on pericytes in muscle and that CD248-/- mice have a specific defect in capillary sprouting. In contrast, splitting angiogenesis is independent of CD248 expression. Endothelial cells respond to pro-sprouting angiogenic stimulus by up-regulating gene expression for HIF1α, angiopoietin 2 and its receptor TEK, PDGF-B and its receptor PDGFRβ; this response did not occur following a pro-splitting angiogenic stimulus. In wildtype mice, defective sprouting angiogenesis could be mimicked by blocking PDGFRβ signalling using the tyrosine kinase inhibitor Imatinib mesylate. We conclude that CD248 is required for PDGFRβ-dependant capillary sprouting but not splitting angiogenesis, and identify a new role for CD248 expressed on pericytes in the early stages of physiological angiogenesis during muscle remodelling.
The roe deer is a seasonally breeding species with a reproductive cycle regulated by endogenous rhythms and photoperiod-sensitivity. Sexually mature bucks show hormonal and testicular activation ...during the reproductive season, with a peak in the rut period, and following gradual involution. Hair is a good matrix for non-invasive endocrinological analyses that provide long-term information without being influenced by the hormones' pulsating release patterns in blood. The aim of the work was to quantify hair concentrations of testosterone and cortisol in wild roe deer bucks hunted during the pre- and post-rut period, using a radioimmunoassay methodology, and to look for differences between the two periods. The secondary objective was the evaluation of possible correlations of such hair concentrations with blood and morphometric parameters of the testes. Both hormones showed statistical differences, with opposing trends, when comparing the two periods: testosterone increased while cortisol decreased. The correlation analysis was in agreement with existing literature regarding metabolism/actions of these hormones and testicular morphometric parameters. This study represents the first report of the use of radioimmunoassay techniques to quantify testosterone and cortisol in roe deer hair, and may provide interesting insights into their reproductive physiology.
When it comes to neuroscience, pigs represent an important animal model due to their resemblance with humans' brains for several patterns including anatomy and developmental stages. Cerebrospinal ...fluid (CSF) is a relatively easy-to-collect specimen that can provide important information about neurological health and function, proving its importance as both a diagnostic and biomedical monitoring tool. Consequently, it would be of high scientific interest and value to obtain more standard physiological information regarding its composition and dynamics for both swine pathology and the refinement of experimental protocols. Recently, proton nuclear magnetic resonance (1H NMR) spectroscopy has been applied in order to analyze the metabolomic profile of this biological fluid, and results showed the technique to be highly reproducible and reliable. The aim of the present study was to investigate in both qualitative and quantitative manner the composition of Cerebrospinal Fluid harvested form healthy newborn (5 days old-P5) and young (30-P30 and 50-P50 days old) piglets using 1H NMR Spectroscopy, and to analyze any possible difference in metabolites concentration between age groups, related to age and Blood-Brain-Barrier maturation. On each of the analyzed samples, 30 molecules could be observed above their limit of quantification, accounting for 95-98% of the total area of the spectra. The concentrations of adenine, tyrosine, leucine, valine, 3-hydroxyvalerate, 3-methyl-2-oxovalerate were found to decrease between P05 and P50, while the concentrations of glutamine, creatinine, methanol, trimethylamine and myo-inositol were found to increase. The P05-P30 comparison was also significant for glutamine, creatinine, adenine, tyrosine, leucine, valine, 3-hydroxyisovalerate, 3-methyl-2-oxovalerate, while for the P30-P50 comparison we found significant differences for glutamine, myo-inositol, leucine and trimethylamine. None of these molecules showed at P30 concentrations outside the P05 -P50 range.
Aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) has been used to describe the histological lesion associated with metal-on-metal (M-M) bearings. We tested the hypothesis that the ...lymphoid aggregates, associated with ALVAL lesions resemble tertiary lymphoid organs (TLOs). Histopathological changes were examined in the periprosthetic tissue of 62 M-M hip replacements requiring revision surgery, with particular emphasis on the characteristics and pattern of the lymphocytic infiltrate. Immunofluorescence and immunohistochemistry were used to study the classical features of TLOs in cases where large organized lymphoid follicles were present. Synchrotron X-ray fluorescence (XRF) measurements were undertaken to detect localisation of implant derived ions/particles within the samples. Based on type of lymphocytic infiltrates, three different categories were recognised; diffuse aggregates (51%), T cell aggregates (20%), and organised lymphoid aggregates (29%). Further investigation of tissues with organised lymphoid aggregates showed that these tissues recapitulate many of the features of TLOs with T cells and B cells organised into discrete areas, the presence of follicular dendritic cells, acquisition of high endothelial venule like phenotype by blood vessels, expression of lymphoid chemokines and the presence of plasma cells. Co-localisation of implant-derived metals with lymphoid aggregates was observed. These findings suggest that in addition to the well described general foreign body reaction mediated by macrophages and a T cell mediated type IV hypersensitivity response, an under-recognized immunological reaction to metal wear debris involving B cells and the formation of tertiary lymphoid organs occurs in a distinct subset of patients with M-M implants.
Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the ...characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41
Lyve1
and CD41
Lyve1
cells in the LNs. CD41
Lyve1
cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41
cells in human LNs as well. We demonstrated that murine CD41
cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41
Lyve1
cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41
Lyve1
cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41
cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41
cells in the function of LNs.
Acoustic radiation forced impulse (ARFI) is an integrated ultrasound method, measuring stiffness by point shear wave elastography. To evaluate the diagnostic performance of the ARFI of the liver and ...the spleen, combined with spleen dimension and platelet count, in predicting high-risk esophageal varices (HRVs) in cirrhotic patients, a prospective and cross-sectional study was conducted between February 2017 and February 2021. The following ratio scores were calculated based on ARFI measurements: ALSDP (ARFI Liver-Spleen Diameter-to-Platelet Ratio Score), ASSDP (ARFI Spleen-Spleen Diameter-to-Platelet Ratio Score), ASSAP (ARFI Spleen-Spleen Area-to-Platelet Ratio Score), and ALSAP (ARFI Liver-Spleen Area-to-Platelet Ratio Score). In 100 enrolled subjects, spleen ARFI, ASSDP, and ASSAP were significantly associated with HRVs in the prospective short- and long-term follow-ups and in the cross-sectional study (
< 0.05), while ALSDP and ALSAP were associated with HRVs only in the prospective long-term follow-up and cross-sectional study (
< 0.05). ASSAP was the best ARFI ratio score for HRVs at the long-term follow-up value of area under curve (AUC) = 0.88, although all the ARFI ratio scores performed better than individual liver and spleen ARFI (AUC > 0.7). In our study, ARFI ratio scores can predict, in well-compensated cirrhotic patients, the risk of developing HVRs in short- and long-term periods.
Einkorn wheat (
) is characterized by high content of proteins, bioactive compounds, such as polyunsaturated fatty acids, fructans, tocols, carotenoids, alkylresorcinols, and phytosterols, and lower
...-,
-amylase and lipoxygenase activities compared to polyploid wheat. These features make einkorn flour a good candidate to provide healthier foods. In the present study, we investigated the effects of einkorn bread (EB) on the intestinal physiology and metabolism of the pig model by characterizing the glycemic and insulinemic response, and the microbiota and metabolome profiles. Sixteen commercial hybrid pigs were enrolled in the study; four pigs were used to characterize postprandial glycemic and insulinemic responses and twelve pigs underwent a 30-day dietary intervention to assess microbiota and metabolome changes after EB or standard wheat bread (WB) consumption. The postprandial insulin rise after an EB meal was characterized by a lower absolute level, and, as also observed for glucose, by a biphasic shape in contrast to that in response to a WB meal. The consumption of EB led to enrichment in short-chain fatty acid producers (e.g.,
,
and
) in the gut microbiota and to higher metabolic diversity with lower content of succinate, probably related to improved absorption and therefore promoting intestinal gluconeogenesis. The observed changes, at both a compositional and metabolic scale, strongly suggest that EB consumption may support a health-promoting configuration of the intestinal ecosystem.
BackgroundEffective therapies for PDAC are urgently needed. CAN-2409 is a replication-defective adenovirus encoding the HSV-thymidine kinase gene, administered intratumorally in combination with a ...prodrug (valacyclovir or acyclovir). Cells transduced with CAN-2409 activate prodrug, resulting in immunogenic cell death and release of tumor neoantigens within the inflammatory tumor microenvironment. Together, this results in in situ vaccination against the patient’s tumor. A phase 1 clinical trial in PDAC previously showed marked infiltration by CD8+ T cells and initial evidence of clinical activity after administration of CAN-2409+prodrug.MethodsPaTK02 is an open-label phase 2 clinical trial evaluating safety and efficacy of CAN-2409+prodrug combined with standard of care (SOC) chemoradiation (CR) and surgery for borderline resectable (BR) or locally advanced (LA) PDAC. Three courses of CAN-2409+prodrug were administered after induction chemotherapy, during CR, and during surgery. Primary endpoints include resection rate and overall survival (OS) rate (24 months). CA19–9 longitudinal changes were reported for treatment arm (n=9) and controls (n=9). Immune profiling of serial samples for 4 test arm patients and 3 controls was performed using OLINK.Results19 patients were enrolled by data cutoff (01MAY2023): 10 in test arm (7 BR, 3 LA) and 9 in control arm (6 BR, 3 LA). 3 BR patients and 1 LA patient were successfully resected in both test and control arms. Median OS was not reached in BR patients receiving CAN-2409 compared to 12.5 mo amongst controls; OS24 was estimated as 62.5% in CAN-2409 vs. 16.7% in control patients (figure 1). Mortality was comparable by arm in LA patients. No dose-limiting toxicities were reported, including no cases of pancreatitis. Most adverse events were grade 1 and 2; common related events were nausea and fatigue (n=4 each). CAN-2409 treatment was associated with lower CA19–9 trajectory in post-treatment samples (figure 2). We observed, in test arm patients, an increase in levels of soluble granzymes B and H (respectively, 21% and 14% increase vs. 0% and 8% decrease in control arm). IL-10 increased by 11% for test arm patients and decreased by 5% in control patients, while IFNγ increased by 19% in test patients, but only 8% in control patients.ConclusionsIn patients undergoing SOC treatment for PDAC, treatment with CAN-2409+prodrug was well tolerated and associated with increased OS in BR patients. CAN-2409+prodrug reduced CA19–9 levels, suggesting an effect on tumor volume consistent with CAN-2409’s mechanism of action. Biomarker analysis demonstrated systemic activation of the immune response.Abstract 653 Figure 1OS in borderline resectable PDAC by treatment arm, time from enrollmentAbstract 653 Figure 2CA 19–9 trajectory by treatment arm
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