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β-thalassemia is a disorder characterized by anemia, ineffective erythropoiesis (IE), and iron overload, whose treatment still requires improvement. The activin receptor-ligand trap Luspatercept, a novel therapeutic option for β-thalassemia, stimulates erythroid differentiation inhibiting the transforming growth factor β pathway. However, its exact mechanism of action and the possible connection with erythropoietin (Epo), the erythropoiesis governing cytokine, remain to be clarified. Moreover, Luspatercept does not correct all the features of the disease, calling for the identification of strategies that enhance its efficacy. Transferrin receptor 2 (TFR2) regulates systemic iron homeostasis in the liver and modulates the response to Epo of erythroid cells, thus balancing red blood cells production with iron availability. Stimulating Epo signaling, hematopoietic Tfr2 deletion ameliorates anemia and IE in Hbb th3/+ thalassemic mice. To investigate whether hematopoietic Tfr2 inactivation improves the efficacy of Luspatercept, we treated Hbb th3/+ mice with or without hematopoietic Tfr2 (Tfr2 BMKO /Hbb th3/+ ) with RAP-536, the murine analog of Luspatercept. As expected, both hematopoietic Tfr2 deletion and RAP-536 significantly ameliorate IE and anemia, and the combined approach has an additive effect. Since RAP-536 has comparable efficacy in both Hbb th3/+ and Tfr2 BMKO /Hbb th3/+ animals, we propose that the drug promotes erythroid differentiation independently of TFR2 and EPO stimulation. Notably, the lack of Tfr2, but not RAP-536, can also attenuate iron-overload and related complications. Overall, our results shed further light on the mechanism of action of Luspatercept and suggest that strategies aimed at inhibiting hematopoietic TFR2 might improve the therapeutic efficacy of activin receptor-ligand traps., (© 2024 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

The NFKBIE gene, which encodes the NF-κB inhibitor IκBε, is mutated in 3-7% of patients with chronic lymphocytic leukemia (CLL). The most recurrent alteration is a 4-bp frameshift deletion associated with NF-κB activation in leukemic B cells and poor clinical outcome. To study the functional consequences of NFKBIE gene inactivation, both in vitro and in vivo, we engineered CLL B cells and CLL-prone mice to stably down-regulate NFKBIE expression and investigated its role in controlling NF-κB activity and disease expansion. We found that IκBε loss leads to NF-κB pathway activation and promotes both migration and proliferation of CLL cells in a dose-dependent manner. Importantly, NFKBIE inactivation was sufficient to induce a more rapid expansion of the CLL clone in lymphoid organs and contributed to the development of an aggressive disease with a shortened survival in both xenografts and genetically modified mice. IκBε deficiency was associated with an alteration of the MAPK pathway, also confirmed by RNA-sequencing in NFKBIE-mutated patient samples, and resistance to the BTK inhibitor ibrutinib. In summary, our work underscores the multimodal relevance of the NF-κB pathway in CLL and paves the way to translate these findings into novel therapeutic options., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Microenvironmental signals strongly influence chronic lymphocytic leukemia (CLL) cells through the activation of distinct membrane receptors, such as B-cell receptors, and inflammatory receptors, such as Toll-like receptors (TLRs). Inflammatory pathways downstream of these receptors lead to NF-κB activation, thus protecting leukemic cells from apoptosis. Dimethyl fumarate (DMF) is an anti-inflammatory and immunoregulatory drug used to treat patients with multiple sclerosis and psoriasis in which it blocks aberrant NF-κB pathways and impacts the NRF2 antioxidant circuit. Our in vitro analysis demonstrated that increasing concentrations of DMF reduce ATP levels and lead to the apoptosis of CLL cells, including cell lines, splenocytes from Eµ-TCL1-transgenic mice, and primary leukemic cells isolated from the peripheral blood of patients. DMF showed a synergistic effect in association with BTK inhibitors in CLL cells. DMF reduced glutathione levels and activated the NRF2 pathway; gene expression analysis suggested that DMF downregulated pathways related to NFKB and inflammation. In primary leukemic cells, DMF disrupted the TLR signaling pathways induced by CpG by reducing the mRNA expression of NFKBIZ, IL6, IL10 and TNFα. Our data suggest that DMF targets a vulnerability of CLL cells linked to their inflammatory pathways, without impacting healthy donor peripheral blood mononuclear cells., (© 2024. The Author(s).)

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose-response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

During melanoma metastasis, tumor cells originating in the skin migrate via lymphatic vessels to the sentinel lymph node (sLN). This process facilitates tumor cell spread across the body. Here, we characterized the innate inflammatory response to melanoma in the metastatic microenvironment of the sLN. We found that macrophages located in the subcapsular sinus (SS) produced protumoral IL1α after recognition of tumoral antigens. Moreover, we confirmed that the elimination of LN macrophages or the administration of an IL1α-specific blocking antibody reduced metastatic spread. To understand the mechanism of action of IL1α in the context of the sLN microenvironment, we applied single-cell RNA sequencing to microdissected metastases obtained from animals treated with the IL1α-specific blocking antibody. Among the different pathways affected, we identified STAT3 as one of the main targets of IL1α signaling in metastatic tumor cells. Moreover, we found that the antitumoral effect of the anti-IL1α was not mediated by lymphocytes because Il1r1 knockout mice did not show significant differences in metastasis growth. Finally, we found a synergistic antimetastatic effect of the combination of IL1α blockade and STAT3 inhibition with stattic, highlighting a new immunotherapy approach to preventing melanoma metastasis., (©2022 The Authors; Published by the American Association for Cancer Research.)

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

Two-photon intravital microscopy (2P-IVM) has become a widely used technique to study cell-to-cell interactions in living organisms. Four-dimensional imaging data obtained via 2P-IVM are classically analyzed by performing automated cell tracking, a procedure that computes the trajectories followed by each cell. However, technical artifacts, such as brightness shifts, the presence of autofluorescent objects, and channel crosstalking, affect the specificity of imaging channels for the cells of interest, thus hampering cell detection. Recently, machine learning has been applied to overcome a variety of obstacles in biomedical imaging. However, existing methods are not tailored for the specific problems of intravital imaging of immune cells. Moreover, results are highly dependent on the quality of the annotations provided by the user. In this study, we developed CANCOL, a tool that facilitates the application of machine learning for automated tracking of immune cells in 2P-IVM. CANCOL guides the user during the annotation of specific objects that are problematic for cell tracking when not properly annotated. Then, it computes a virtual colocalization channel that is specific for the cells of interest. We validated the use of CANCOL on challenging 2P-IVM videos from murine organs, obtaining a significant improvement in the accuracy of automated tracking while reducing the time required for manual track curation., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Chronic lymphocytic leukemia (CLL) is caused by the progressive accumulation of mature CD5+ B cells in secondary lymphoid organs. In vitro data suggest that CD4+ T lymphocytes also sustain survival and proliferation of CLL clones through CD40L/CD40 interactions. In vivo data in animal models are conflicting. To clarify this clinically relevant biological issue, we generated genetically modified Eμ-TCL1 mice lacking CD4+ T cells (TCL1+/+AB0), CD40 (TCL1+/+CD40-/-), or CD8+ T cells (TCL1+/+TAP-/-), and we monitored the appearance and progression of a disease that mimics aggressive human CLL by flow cytometry and immunohistochemical analyses. Findings were confirmed by adoptive transfer of leukemic cells into mice lacking CD4+ T cells or CD40L or mice treated with antibodies depleting CD4 T cells or blocking CD40L/CD40 interactions. CLL clones did not proliferate in mice lacking or depleted of CD4+ T cells, thus confirming that CD4+ T cells are essential for CLL development. By contrast, CD8+ T cells exerted an antitumor activity, as indicated by the accelerated disease progression in TCL1+/+TAP-/- mice. Antigen specificity of CD4+ T cells was marginal for CLL development, because CLL clones efficiently proliferated in transgenic mice whose CD4 T cells had a T-cell receptor with CLL-unrelated specificities. Leukemic clones also proliferated when transferred into wild-type mice treated with monoclonal antibodies blocking CD40 or into CD40L-/- mice, and TCL1+/+CD40-/- mice developed frank CLL. Our data demonstrate that CD8+ T cells restrain CLL progression, whereas CD4+ T cells support the growth of leukemic clones in TCL1 mice through CD40-independent and apparently noncognate mechanisms., (© 2021 by The American Society of Hematology.)

Purpose: In search of novel strategies to improve the outcome of advanced prostate cancer, we considered that prostate cancer cells rearrange iron homeostasis, favoring iron uptake and proliferation. We exploited this adaptation by exposing prostate cancer preclinical models to high-dose iron to induce toxicity and disrupt adaptation to androgen starvation., Experimental Design: We analyzed markers of cell viability and mechanisms underlying iron toxicity in androgen receptor-positive VCaP and LNCaP, castration-resistant DU-145 and PC-3, and murine TRAMP-C2 cells treated with iron and/or the antiandrogen bicalutamide. We validated the results in vivo in VCaP and PC-3 xenografts and in TRAMP-C2 injected mice treated with iron and/or bicalutamide., Results: Iron was toxic for all prostate cancer cells. In particular, VCaP, LNCaP, and TRAMP-C2 were highly iron sensitive. Toxicity was mediated by oxidative stress, which primarily affected lipids, promoting ferroptosis. In highly sensitive cells, iron additionally caused protein damage. High-basal iron content and oxidative status defined high iron sensitivity. Bicalutamide-iron combination exacerbated oxidative damage and cell death, triggering protein oxidation also in poorly iron-sensitive DU-145 and PC-3 cells. In vivo , iron reduced tumor growth in TRAMP-C2 and VCaP mice. In PC-3 xenografts, bicalutamide-iron combination caused protein oxidation and successfully impaired tumor expansion while single compounds were ineffective. Macrophages influenced body iron distribution but did not limit the iron effect on tumor expansion., Conclusions: Our models allow us to dissect the direct iron effect on cancer cells. We demonstrate the proof of principle that iron toxicity inhibits prostate cancer cell proliferation, proposing a novel tool to strengthen antiandrogen treatment efficacy., (©2020 American Association for Cancer Research.)

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

The oxidation of human oxyhemoglobin (HbO 2 ) to methemoglobin (metHb) is an undesirable side effect identified in some promising thiosemicarbazone anti-cancer drugs. This is attributable to oxidation reactions driven by Fe III complexes of these drugs formed in vivo. In this work the Fe III complexes of selected 2-benzoylpyridine thiosemicarbazones (HBpT), 2-acetylpyridine thiosemicarbazones (HApT), and the clinically trialled thiosemicarbazone, Triapine® (3-amino-2-pyridinecarboxaldehyde thiosemicarbazone, H3-AP), have been studied. This was achieved by time-resolved UV-Visible absorption spectroscopy and the sequential oxidation of the α- and β-chains of HbO 2 at distinctly different rates has been identified. A key structural element, namely a terminal -NH 2 group on the thiosemicarbazone moiety, was found to be an important common feature of the most active HbO 2 oxidising complexes that were investigated. Therefore, these studies indicate that an unsubstituted -NH 2 moiety at the terminus of the thiosemicarbazone group should be avoided in the design of future compounds from this class., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bone allograft has become an alternative to autogenous bone due to its decreased operative trauma and the almost unlimited supply of reconstructive material. The aim of the present study was to histologically evaluate the suitability of fresh-frozen bone graft (test group) used in maxillary ridge augmentation, comparing it to autogenous bone (native maxilla: control group). During the re-entry procedures, 9 months after the fresh-frozen allogeneic bone blocks were placed in the atrophic maxillary ridges, bone cores were removed with a trephine bur from test and control treatments in the same patient. Routine histologic processing using hematoxylin and eosin and Picrosirius staining was performed. Mature and immature collagen area and density analysis were carried out for both groups under polarization. The results of Student's t test for paired samples (P > .05) showed no statistically significant difference in mature and immature collagen area or density percentage between test and control groups. Histologically similar bone formation patterns were observed in both groups. We concluded that fresh-frozen bone allograft is a biologically acceptable alternative for augmentation of the deficient alveolar ridge, showing a similar collagen pattern to that of autogenous bone.

Purpose: The present investigation clinically and histologically evaluated the use of fresh-frozen bone in the reconstruction of maxillary alveolar ridges to confirm the effective bone fill and support for the placement of dental implants., Patients and Methods: Fifteen patients who had atrophic maxillary ridge necessitating bone block grafts prior to implant placement were submitted to maxillary reconstructions performed with human block grafts of tibia fresh-frozen chips. Nine months later the re-entry procedures were carried out and at this time a bone core was removed from the grafts for histological analysis., Results: Thirty-four blocks were placed, and the number of blocks each patient received ranged from 1 to 4. During the re-entry procedures, all of the grafts were found to be firm in consistency, well-incorporated, and vascularized. A total of 51 implants were placed over the grafts with a minimum of 40-Newton torque in all cases. None of the implants were lost. The follow-up period ranged from 24 to 35 months. The histological analysis revealed a living bone that showed features characteristic of mature and compact osseous tissue surrounded by marrow spaces., Conclusion: Bone allografts can be successful as graft material for the treatment of maxillary ridge defects. If adequate surgical techniques are adopted, this type of bone graft can be safely used in regions of implant placement as a suitable alternative to autogenous grafts.

Light activation leads to release of NO from a silicate sol-gel material SG-RuNO prepared from the ruthenium complex, [Ru(salen)(OH2)(NO)]+ (salen = N,N'-bis-(salicylidene)ethyl-enediaminato); after photochemical NO photolabilization, SG-RuNO can be regenerated from the spent material via the subsequent reaction with aqueous nitrite.

The complexes [Ru(salen)(NO)Cl] and [Ru(salen)(NO)(H(2)O)](+) were shown to release the nitrosyl ligand as nitric oxide upon exposure to visible light in organic and aqueous solutions respectively, by means of UV-visible, EPR, and FTIR spectroscopies. The former was prepared by a new synthetic route and had its structure determined by single-crystal X-ray diffraction. A crystal of the dichloromethane solvate is orthorhombic, space group Fdd2 (No. 43) and formula C(16)H(14)ClN(3)O(3)Ru.CH(2)Cl(2), with Z = 16 and cell parameters a = 25.489(4), b = 33.435(4), and c = 9.3716(9) A. The electronic absorption spectra of the complexes were calculated using the INDO/S method. The water-soluble complex is a potential drug for antitumoral phototreatment.