Multiplicity Results for a Perturbed Elliptic Neumann Problem

The existence of three solutions for elliptic Neumann problems with a perturbed nonlinear term depending on two real parameters is investigated. Our approach is based on variational methods

Nonlinear elliptic equations involving the p-Laplacian with mixed Dirichlet-Neumann boundary conditions

In this paper, a nonlinear differential problem involving the $p$-Laplacian operator with mixed boundary conditions is investigated. In particular, the existence of three non-zero solutions is established by requiring suitable behavior on the nonlinearity. Concrete examples illustrate the abstract results

One-dimensional nonlinear boundary value problems with variable exponent

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Interleukin-21 induces the differentiation of human umbilical cord blood CD34-lineage- cells into pseudomature lytic NK cells

<p>Abstract</p> <p>Background</p> <p>Umbilical cord blood (UCB) is enriched with transplantable CD34⁺cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34^-lineage^-cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34^-lineage^-cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34^-lineage^-cells.</p> <p>Results</p> <p>CD34^-lineage^-cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α, and cytolytic activity against NK-sensitive tumour cell targets. CD34^-lineage^-cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34^-lineage^-cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)^-CD56⁺CD16^-/+phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-γ, GM-CSF and CCL3/MIP-1α, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.</p> <p>Conclusion</p> <p>This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.</p

Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies

<p>Abstract</p> <p>Background</p> <p>Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the <it>in vivo </it>and ex vivo release of immune modulating cytokines remain unexplored.</p> <p>Methods</p> <p>Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. <it>In vitro</it>-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.</p> <p>Results</p> <p>Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4⁺FoxP3⁺<it>bona fide </it>Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered <it>in vitro</it> monocytic<it/> secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated <it>in vitro </it>after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.</p> <p>Conclusions</p> <p>Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.</p

Thymoglobulin, interferon-γ and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures

<p>Abstract</p> <p>Background</p> <p>Cytokine-induced killer (CIK) cells are typically differentiated <it>in vitro </it>with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol.</p> <p>Methods</p> <p>Peripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of <it>bona fide </it>regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the <it>in vitro </it>cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells.</p> <p>Results</p> <p>CIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells.</p> <p>Conclusions</p> <p>TG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.</p

Identification of the hemangioblast in postnatal life

AbstractPostnatal CD34+ cells expressing vascular endothelial growth factor receptor 2 (KDR) generate hematopoietic or endothelial progeny in different in vitro and in vivo assays. Hypothetically, CD34+KDR+ cells may comprise hemangioblasts bipotent for both lineages. This hypothesis is consistent with 2 series of experiments. In the first series, in clonogenic culture permissive for hematopoietic and endothelial cell growth, CD34+KDR+ cells generate large hemato-endothelial (Hem-End) colonies (5% of seeded cells), whereas CD34+KDR− cells do not. Limiting-dilution analysis indicates that Hem-End colonies are clonally generated by single hemangioblasts. Sibling cells generated by a hemangioblast, replated in unicellular culture, produce either hematopoietic or Hem-End colonies, depending on the specific culture conditions. Identification of endothelial cells was based on the expression of VE-cadherin and endothelial markers and with lack of CD45 and hematopoietic molecules, as evaluated by immunofluorescence, immunocytochemistry, and reverse transcription–polymerase chain reaction. Furthermore, endothelial cells were functionally identified using low-density lipoprotein (LDL) uptake and tube-formation assays. In the second series, to evaluate the self-renewal capacity of hemangioblasts, single CD34+KDR+ cells were grown in 3-month extended long-term culture (ELTC) through 3 serial culture rounds—that is, blast cells generated in unicellular ELTC were reseeded for a subsequent round of unicellular ELTC. After 9 months, 10% blasts from tertiary ELTC functioned as hemangioblasts and generated macroscopic Hem-End colonies in clonogenic culture. These studies identified postnatal hemangioblasts in a CD34+KDR+ cell subset, endowed with long-term proliferative potential and bilineage differentiation capacity. Although exceedingly rare, hemangioblasts may represent the lifetime source/reservoir for primitive hematopoietic and endothelial progenitors

Tracing the footprints of SARS-CoV-2 in oceanic waters

The detection of SARS-CoV-2 in water environments has predominantly focused on wastewater, neglecting its presence in oceanic waters. This study aimed to fill this knowledge gap by investigating the occurrence of SARS-CoV-2 in remote sea and oceanic waters, at large distances from the coastline. Forty-three 500-liter samples were collected between May 2022 and January 2023 from the Atlantic Ocean, the Mediterranean Sea, the Arctic region, the Persian Gulf and the Red Sea. Using molecular detection methods including real-time RT-qPCR and nested PCR followed by sequencing, we successfully detected SARS-CoV-2 RNA in 7 of the 43 marine water samples (16.3&nbsp;%), and specifically in samples taken from the Atlantic Ocean and the Mediterranean Sea. The estimated concentrations of SARS-CoV-2&nbsp;genome copies in the positive samples ranged from 6 to 470 per 100&nbsp;l. The presence of mutations characteristic of the Omicron variant was identified in these samples by amplicon sequencing. These findings provide evidence of the unforeseen presence of SARS-CoV-2 in marine waters even at distances of miles from the coastline and in open ocean waters. It is important to consider that these findings only display the occurrence of SARS-CoV-2 RNA, and further investigations are required to assess if infectious virus can be present in the marine environment

A novel prostate cell type-specific gene signature to interrogate prostate tumor differentiation status and monitor therapeutic response (running title: Phenotypic classification of prostate tumors)

In this study, we extracted prostate cell-specific gene sets (metagenes) to define the epithelial differentiation status of prostate cancers and, using a deconvolution-based strategy, interrogated thousands of primary and metastatic tumors in public gene profiling datasets. We identified a subgroup of primary prostate tumors with low luminal epithelial enrichment (LumElow). LumElow tumors were associated with higher Gleason score and mutational burden, reduced relapse-free and overall survival, and were more likely to progress to castration-resistant prostate cancer (CRPC). Using discriminant function analysis, we generate a predictive 10-gene classifier for clinical implementation. This mini-classifier predicted with high accuracy the luminal status in both primary tumors and CRPCs. Immunohistochemistry for COL4A1, a low- luminal marker, sustained the association of attenuated luminal phenotype with metastatic disease. We found also an association of LumE score with tumor phenotype in genetically engineered mouse models (GEMMs) of prostate cancer. Notably, the metagene approach led to the discovery of drugs that could revert the low luminal status in prostate cell lines and mouse models. This study describes a novel tool to dissect the intrinsic heterogeneity of prostate tumors and provide predictive information on clinical outcome and treatment response in experimental and clinical samples

Quantitative Microbial Risk Assessment as support for bathing waters profiling

Profiling bathing waters supported by Quantitative Microbial Risk Assessment (QMRA) is key to the WHO's recommendations for the 2020/2021 revision of the European Bathing Water Directive. We developed an areaspecific QMRA model on four pathogens, using fecal indicator concentrations (E. coil, enterococci) for calculating pathogen loads. The predominance of illness was found to be attributable to Human Adenovirus, followed by Salmonella, Vibrio, and Norovirus. Overall, the cumulative illness risk showed a median of around 1 case/10000 exposures. The risk estimates were strongly influenced by the indicators that were used, suggesting the need for a more detailed investigation of the different sources of fecal contamination. Area-specific threshold values for fecal indicators were estimated on a risk-basis by modelling the cumulative risk against E. coll. and enterococci concentrations. To improve bathing waters assessment, we suggest considering source apportionment locally estimating of pathogen/indicator ratios, and calculating site-specific indicators thresholds based on risk assessment