The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

Therapeutic Effect of an Antibody-Derived Peptide in a Galleria mellonella Model of Systemic Candidiasis.

The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans. A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

Candida isolates causing candidemia show different degrees of virulence in Galleria mellonella.

We aim to assess intra- and interspecies differences in the virulence of Candida spp. strains causing candidemia using the invertebrate Galleria mellonella model. We studied 739 Candida spp. isolates (C. albicans [n = 373], C. parapsilosis [n = 203], C. glabrata [n = 92], C. tropicalis [n = 53], and C. krusei [n = 18]) collected from patients with candidemia admitted to Gregorio Marañon Hospital (Madrid, Spain). Species-specific infecting inocula (yeast cells/larva) were adjusted (5 × 105 [C. albicans, and C. tropicalis], 2 × 106-5 × 106 [C. parapsilosis, C. glabrata, and C. krusei]) and used to infect 10 larvae per isolate; percentage of survival and median survival per isolate were calculated. According to the interquartile range of the median survival, isolates with a median survival under P25 were classified as of high-virulence and isolates with a median survival over P75 as of low virulence. The median survival of larvae infected with different species was variable: C. albicans (n = 2 days, IQR <1-3 days), C. tropicalis (n = 2 days, IQR 1.5-4 days), C. parapsilosis (n = 2 days, IQR 2-3.5 days), C. glabrata (n = 3 days, IQR 2-3 days), and C. krusei (n = 7 days, 6.5->8 days) (P < .001). Differences in virulence among species were validated by histological examination (day +1 post-infection) in the larvae infected by the isolates of each virulence category and species. Virulence-related gene expression in C. albicans isolates did not reach statistical significance. We report species-specific virulence patterns of Candida spp. and show that isolates within a given species have different degrees of virulence in the animal model.

EPIRETINAL FIBROSIS REMOVAL IN AN ARGUS II-IMPLANTED EYE: Histological Characteristics and Functional Results.

PURPOSE: 1) To investigate morphologic and histochemical characteristics of an epiretinal fibrosis removed in an Argus II-implanted eye; 2) to evaluate the Argus II function before and after the fibrosis removal, and 3) to compare morphologic and functional data. METHODS: Fibrosis, which developed between the Argus II prosthesis and the retina two years after implant, was surgically removed. Its morphologic and histochemical characteristics were evaluated both in light and transmission electron microscopy, with special stains and immunohistochemistry. The Argus II function was evaluated during the follow-up before surgical removal and 1 month later. RESULTS: Fibrosis was successfully removed. It was composed of a fibrotic tissue with spindle cells arranged in nodular aggregates with a symmetric distribution, mixed with an inflammatory infiltrate. Extra- and intracellular, irregular, small iron particles were found and confirmed ultrastructural characterization with degenerative cellular changes. The repositioned Argus II restored, and its function was partially nearly to normal values 1 month after surgery. CONCLUSION: Fibrosis can develop between the Argus II and the retina with increasing reduced function. Morphologic characteristics of the removed fibrosis suggested a pathogenesis based on an inflammatory process involved in a foreign body reaction with progressing connective tissue deposition leading to sclerosis. Adequate clinical follow-up is critical to successful removal of the fibrosis with reactivation of the Argus II function.

Therapeutic Effect of Iron Citrate in Blocking Calcium Deposition in High Pi-Calcified VSMC: Role of Autophagy and Apoptosis.

In chronic kidney disease (CKD), the first cause of mortality is cardiovascular disease induced mainly by vascular calcification (VC). Recently, iron-based phosphate binders have been proposed in advanced CKD to treat hyperphosphatemia. We studied the effect of iron citrate (iron) on the progression of calcification in high-phosphate (Pi) calcified VSMC. Iron arrested further calcification when added on days 7-15 in the presence of high Pi (1.30 ± 0.03 vs 0.61 ± 0.02; OD/mg protein; day 15; Pi vs Pi + Fe, p < 0.01). We next investigated apoptosis and autophagy. Adding iron to high-Pi-treated VSMC, on days 7-11, decreased apoptotic cell number (17.3 ± 2.6 vs 11.6 ± 1.6; Annexin V; % positive cells; day 11; Pi vs Pi + Fe; p < 0.05). The result was confirmed thorough analysis of apoptotic nuclei both in VSMCs and aortic rings treated on days 7-15 (3.8 ± 0.2 vs 2.3 ± 0.3 and 4.0 ± 0.3 vs 2.2 ± 0.2; apoptotic nuclei; arbitrary score; day 15; Pi vs Pi + Fe; VSMCs and aortic rings; p < 0.05). Studying the prosurvival axis GAS6/AXL, we found that iron treatment on days 9-14 counteracted protein high-Pi-stimulated down-regulation and induced its de novo synthesis. Moreover, iron added on days 9-15 potentiated autophagy, as detected by an increased number of autophagosomes with damaged mitochondria and an increase in autophagic flux. Highlighting the effect of iron on apoptosis, we demonstrated its action in blocking the H2O2-induced increase in calcification added both before high Pi treatment and when the calcification was already exacerbated. In conclusion, we demonstrate that iron arrests further high Pi-induced calcium deposition through an anti-apoptotic action and the induction of autophagy on established calcified VSMC.

Inhibition of ceramide de novo synthesis by myriocin produces the double effect of reducing pathological inflammation and exerting antifungal activity against A. fumigatus airways infection.

BACKGROUND: Fungal infections develop in pulmonary chronic inflammatory diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF). The available antifungal drugs may fail to eradicate fungal pathogens, that can invade the lungs and vessels and spread by systemic circulation taking advantage of defective lung immunity. An increased rate of sphingolipid de novo synthesis, leading to ceramide accumulation, was demonstrated in CF and COPD inflamed lungs. The inhibitor of sphingolipid synthesis myriocin reduces inflammation and ameliorates the response against bacterial airway infection in CF mice. Myriocin also inhibits sphingolipid synthesis in fungi and exerts a powerful fungistatic effect. METHODS: We treated Aspergillus fumigatus infected airway epithelial cells with myriocin and we administered myriocin-loaded nanocarriers to A. fumigatus infected mice lung. RESULTS: We demonstrate here that de novo synthesized ceramide mediates the inflammatory response induced by A. fumigatus infection in airway epithelia. CF epithelial cells are chronically inflamed and defective in killing internalized conidia. Myriocin treatment reduced ceramide increase and inflammatory mediator release whereas it upregulated HO1 and NOD2, allowing the recovery of a functional killing of conidia in these cells. Myriocin-loaded nanocarriers, intratracheally administered to mice, significantly reduced both the inflammatory response induced by A. fumigatus pulmonary challenge and fungal lung invasion. CONCLUSIONS: We conclude that inhibition of sphingolipid synthesis can be envisaged as a dual anti-inflammatory and anti-fungal therapy in patients suffering from chronic lung inflammation with compromised immunity. GENERAL SIGNIFICANCE: Myriocin represents a powerful agent for inflammatory diseases and fungal infection.

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model.

Both neuronal acetylcholine and nonneuronal acetylcholine have been demonstrated to modulate inflammatory responses. Studies investigating the role of acetylcholine in the pathogenesis of bacterial infections have revealed contradictory findings with regard to disease outcome. At present, the role of acetylcholine in the pathogenesis of fungal infections is unknown. Therefore, the aim of this study was to determine whether acetylcholine plays a role in fungal biofilm formation and the pathogenesis of Candida albicans infection. The effect of acetylcholine on C. albicans biofilm formation and metabolism in vitro was assessed using a crystal violet assay and phenotypic microarray analysis. Its effect on the outcome of a C. albicans infection, fungal burden, and biofilm formation were investigated in vivo using a Galleria mellonella infection model. In addition, its effect on modulation of host immunity to C. albicans infection was also determined in vivo using hemocyte counts, cytospin analysis, larval histology, lysozyme assays, hemolytic assays, and real-time PCR. Acetylcholine was shown to have the ability to inhibit C. albicans biofilm formation in vitro and in vivo. In addition, acetylcholine protected G. mellonella larvae from C. albicans infection mortality. The in vivo protection occurred through acetylcholine enhancing the function of hemocytes while at the same time inhibiting C. albicans biofilm formation. Furthermore, acetylcholine also inhibited inflammation-induced damage to internal organs. This is the first demonstration of a role for acetylcholine in protection against fungal infections, in addition to being the first report that this molecule can inhibit C. albicans biofilm formation. Therefore, acetylcholine has the capacity to modulate complex host-fungal interactions and plays a role in dictating the pathogenesis of fungal infections.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity.

BACKGROUND: Candida albicans infections have become increasingly recognised as being biofilm related. Recent studies have shown that there is a relationship between biofilm formation and poor clinical outcomes in patients infected with biofilm proficient strains. Here we have investigated a panel of clinical isolates in an attempt to evaluate their phenotypic and transcriptional properties in an attempt to differentiate and define levels of biofilm formation. RESULTS: Biofilm formation was shown to be heterogeneous; with isolates being defined as either high or low biofilm formers (LBF and HBF) based on different biomass quantification. These categories could also be differentiated using a cell surface hydrophobicity assay with 24 h biofilms. HBF isolates were more resistance to amphotericin B (AMB) treatment than LBF, but not voriconazole (VRZ). In a Galleria mellonella model of infection HBF mortality was significantly increased in comparison to LBF. Histological analysis of the HBF showed hyphal elements intertwined indicative of the biofilm phenotype. Transcriptional analysis of 23 genes implicated in biofilm formation showed no significant differential expression profiles between LBF and HBF, except for Cdr1 at 4 and 24 h. Cluster analysis showed similar patterns of expression for different functional classes of genes, though correlation analysis of the 4 h biofilms with overall biomass at 24 h showed that 7 genes were correlated with high levels of biofilm, including Als3, Eap1, Cph1, Sap5, Plb1, Cdr1 and Zap1. CONCLUSIONS: Our findings show that biofilm formation is variable amongst C. albicans isolates, and categorising isolates depending on this can be used to predict how pathogenic the isolate will behave clinically. We have shown that looking at individual genes in less informative than looking at multiple genes when trying to categorise isolates at LBF or HBF. These findings are important when developing biofilm-specific diagnostics as these could be used to predict how best to treat patients infected with C. albicans. Further studies are required to evaluate this clinically.

Canine glioblastoma-derived extracellular vesicles as precise carriers for glioblastoma imaging: Targeting across the blood-brain barrier.

The treatment of glioblastoma (GBM) faces significant challenges due to the difficulty of delivering drugs through the blood-brain barrier (BBB). Extracellular vesicles (EVs) have emerged as potential carriers for targeted drug delivery to brain tumors. However, their use and distribution in the presence of an intact BBB and their ability to target GBM tissue are still under investigation. This study explored the use of EVs for GBM targeting across the BBB. Canine plasma EVs from healthy dogs and dogs with glioma were isolated, characterized, and loaded with diagnostic agents. Biodistribution studies were conducted in healthy murine models and a novel intranasal model that preserved BBB integrity while initiating early-stage GBM growth. This model assessed EVs' potential for delivering the contrast agent gadoteric acid to intracranial tumors. Imaging techniques, such as bioluminescence and MRI, confirmed EVs' targeting and delivery capabilities thus revealing a selective accumulation of canine glioma-derived EVs in brain tissue under physiological conditions. In the model of brain tumor, MRI experiments demonstrated the ability of EVs to accumulate gadoteric acid within GBM to enhance contrast of the tumoral mass, even when BBB integrity is maintained. This study underscores the potential of EVs derived from glioma for the targeted delivery of drugs to glioblastoma. EVs from dogs with glioma showed capacity to traverse the BBB and selectively accumulate within the brain tumor. Overall, this research represents a foundation for the application of autologous EVs to precision glioblastoma treatment, addressing the challenge of BBB penetration and targeting specificity in brain cancer therapy.

Ceramide present in cholangiocarcinoma-derived extracellular vesicle induces a pro-inflammatory state in monocytes.

Cholangiocarcinoma (CCA) is a rare cancer characterized by a global increasing incidence. Extracellular vesicles (EV) contribute to many of the hallmarks of cancer through transfer of their cargo molecules. The sphingolipid (SPL) profile of intrahepatic CCA (iCCA)-derived EVs was characterized by liquid chromatography-tandem mass spectrometry analysis. The effect of iCCA-derived EVs as mediators of inflammation was assessed on monocytes by flow cytometry. iCCA-derived EVs showed downregulation of all SPL species. Of note, poorly-differentiated iCCA-derived EVs showed a higher ceramide and dihydroceramide content compared with moderately-differentiated iCCA-derived EVs. Of note, higher dihydroceramide content was associated with vascular invasion. Cancer-derived EVs induced the release of pro-inflammatory cytokines in monocytes. Inhibition of synthesis of ceramide with Myriocin, a specific inhibitor of the serine palmitoyl transferase, reduced the pro-inflammatory activity of iCCA-derived EVs, demonstrating a role for ceramide as mediator of inflammation in iCCA. In conclusion, iCCA-derived EVs may promote iCCA progression by exporting the excess of pro-apoptotic and pro-inflammatory ceramides.

MICA/B-targeted antibody promotes NK cell-driven tumor immunity in patients with intrahepatic cholangiocarcinoma.

The major histocompatibility complex-class I chain related proteins A and B (MICA/B) is upregulated because of cellular stress and MICA/B shedding by cancer cells causes escape from NKG2D recognition favoring the emergence of cancers. Cholangiocarcinoma (CCA) is a relatively rare, though increasingly prevalent, primary liver cancer characterized by a late clinical presentation and a dismal prognosis. We explored the NKG2D-MICA/B axis in NK cells from 41 patients with intrahepatic cholangiocarcinoma (iCCA). The MICA/B-specific 7C6 mAb was used for ex vivo antibody-dependent cytotoxicity (ADCC) experiments using circulating, non tumor liver- and tumor-infiltrating NK cells against the HuCCT-1 cell line and patient-derived primary iCCA cells as targets. MICA/B were more expressed in iCCA than in non-tumoral tissue, MICA transcription being higher in moderately-differentiated compared with poorly-differentiated cancer. Serum MICA was elevated in iCCA patients in line with higher expression of ADAM10 and ADAM17 that are responsible for proteolytic release of MICA/B from tumor. Addition of 7C6 significantly boosted peripheral, liver- and tumor-infiltrating-NK cell degranulation and IFNγ production toward MICA/B-expressing established cell lines and autologous iCCA patient target cells. Our data show that anti-MICA/B drives NK cell anti-tumor activity, and provide preclinical evidence in support of 7C6 as a potential immunotherapeutic tool for iCCA.

Schwann cell hamartoma: case report.

BACKGROUND: Colorectal polyps of mesenchymal origin represent a small percentage of gastrointestinal (GI) lesions. Nevertheless, they are encountered with increasing frequency since the widespread adoption of colonoscopy screening. CASE PRESENTATION: We report a case of a small colonic polyp that presented as intramucosal diffuse spindle cell proliferation with a benign cytological appearance, strong and diffuse immunoreactivity for S-100 protein, and pure Schwann cell phenotype. Careful morphological, immunohistochemical and clinical evaluation emphasize the differences from other stromal colonic lesions and distinguish it from schwannoma, a circumscribed benign nerve sheath tumor that rarely arises in the GI tract. CONCLUSION: As recently proposed, this lesion was finally described as mucosal Schwann cell hamartoma.

Endothelial-Mesenchymal Transition in COVID-19 lung lesions.

Sars-Cov-2 infection is still a healthcare emergency and acute respiratory distress failure with Diffuse Alveolar Damage (DAD) features is the main causes of patients' death. Pathogenic mechanisms of the disease are not clear yet, but new insights are necessary to improve therapeutic management, to prevent fatal irreversible multi-organ damage and to adequately follow up those patients who survive. Here we investigated, by histochemistry and immunohistochemistry, a wide number of mapped lung specimens taken from whole body autopsies of 7 patients dead of COVID-19 disease. Our data confirm morphological data of other authors, and enlarge recent reports of the literature suggesting that Endothelial-Mesenchymal Transition might be central to COVID-19 lung fibrosing lesions. Furthermore, based upon recent acquisition of new roles in immunity and vascular pathology of the CD31 molecule, we hypothesize that this molecule might be important in the development and treatment of COVID-19 pulmonary lesions. These preliminary findings need further investigations to shed light on the complexity of Sars-Cov-2 disease.

CT colonography followed by elective surgery in patients with acute diverticulitis: a radiological-pathological correlation study.

PURPOSE: To perform a radiologic-pathologic correlation analysis of sigmoid colon in patients undergoing pre-operative CT Colonography (CTC) after an episode of acute diverticulitis (AD). METHODS: Fifty-nine consecutive patients (31/28 M/F; 58 ± 13 years) underwent CTC 55 ± 18 days after AD, 8 ± 4 weeks before surgery. Thirty-seven patients (63%) underwent conventional abdominal CT at time of AD. An experienced blinded radiologist retrospectively analyzed all images: disease severity was graded according to the Ambrosetti classification on conventional CT and according to the diverticular disease severity score (DDSS) on CTC. A GI pathologist performed a dedicated analysis, evaluating the presence of acute and chronic inflammation, and fibrosis, using 0-3 point scale for each variable. RESULTS: Of 59 patients, 41 (69%) had at least one previous AD episode; twenty-six patients (44%) had a complicated AD. DDSS was mild-moderate in 34/59 (58%), and severe in 25/59 (42%). All patients had chronic inflammation, while 90% had low-to-severe fibrosis. Patients with moderate/severe fibrosis were older than those with no/mild fibrosis (61 ± 13 versus 54 ± 13). We found a significant correlation between DDSS and chronic inflammation (p = 0.004), as well as DDSS and fibrosis (p = 0.005). Furthermore, fibrosis was correlated with complicated acute diverticulitis (p = 0.0.27), and with age (p = 0.067). At multivariate analysis, complicated diverticulitis was the best predictor of fibrosis (odds ratio 4.4). Patient age and DDSS were other independent predictors. CONCLUSION: DDSS-based assessment on preoperative CTC was a good predictor of chronic colonic inflammation and fibrosis. In addition, the presence of complicated diverticulitis on CT during the acute episode was most predictive of fibrosis.

Brainstem neuropathology in two cases of COVID-19: SARS-CoV-2 trafficking between brain and lung.

INTRODUCTION: SARS-CoV-2 might spread through the nervous system, reaching respiratory centers in the brainstem. Because we recently reported neurophysiological brainstem reflex abnormalities in COVID-19 patients, we here neuropathologically assessed structural brainstem damage in two COVID-19 patients. MATERIALS AND METHODS: We assessed neuropathological features in two patients who died of COVID-19 and in two COVID-19 negative patients as controls. Neuronal damage and corpora amylacea (CA) numbers /mm2 were histopathologically assessed. Other features studied were the immunohistochemical expression of the SARS-CoV-2 nucleoprotein (NP) and the Iba-1 antigen for glial activation. RESULTS: Autopsies showed normal gross brainstem anatomy. Histopathological examination demonstrated increased neuronal and CA damage in Covid-19 patients' medulla oblongata. Immunohistochemistry disclosed SARS-CoV-2 NP in brainstem neurons and glial cells, and in cranial nerves. Glial elements also exhibited a widespread increase in Iba-1 expression. Sars-Co-V2 was immunohistochemically detected in the vagus nerve fibers. DISCUSSION: Neuropathologic evidence showing SARS-CoV-2 in the brainstem and medullary damage in the area of respiratory centers strongly suggests that the pathophysiology of COVID-19-related respiratory failure includes a neurogenic component. Sars-Co-V2 detection in the vagus nerve, argues for viral trafficking between brainstem and lung.

Identification of potential therapeutic targets in malignant mesothelioma using cell-cycle gene expression analysis.

Cell-cycle defects are responsible for cancer onset and growth. We studied the expression profile of 60 genes involved in cell cycle in a series of malignant mesotheliomas (MMs), normal pleural tissues, and MM cell cultures using a quantitative polymerase chain reaction-based, low-density array. Nine genes were significantly deregulated in MMs compared with normal controls. Seven genes were overexpressed in MMs, including the following: CDKN2C, cdc6, cyclin H, cyclin B1, CDC2, FoxM1, and Chk1, whereas Ube1L and cyclin D2 were underexpressed. Chk1 is a principal mediator of cell-cycle checkpoints in response to genotoxic stress. We confirmed the overexpression of Chk1 in an independent set of 87 MMs by immunohistochemistry using tissue microarrays. To determine whether Chk1 down-regulation would affect cell-cycle control and cell survival, we transfected either control or Chk1 siRNA into two mesothelioma cell lines and a nontumorigenic (Met5a) cell line. Results showed that Chk1 knockdown increased the apoptotic fraction of MM cells and induced an S phase block in Met5a cells. Furthermore, Chk1 silencing sensitized p53-null MM cells to both an S phase block and apoptosis in the presence of doxorubicin. Our results indicate that cell-cycle gene expression analysis by quantitative polymerase chain reaction can identify potential targets for novel therapies. Chk1 knockdown could provide a novel therapeutic approach to arrest cell-cycle progression in MM cells, thus increasing the rate of cell death.

Unexpected thymoma in a challenging case of hyperparathyroidism.

We report the case of a woman with primary hyperparathyroidism suspected of mediastinal ectopic parathyroid adenoma revealed to be a thymoma. Our aim was to focus on some possible criticisms in distinguishing between ectopic parathyroid and thymus.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1-2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines' ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients' BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Evidence of SARS-CoV-2 Transcriptional Activity in Cardiomyocytes of COVID-19 Patients without Clinical Signs of Cardiac Involvement.

AIMS: A considerable proportion of patients affected by coronavirus respiratory disease (COVID-19) develop cardiac injury. The viral impact in cardiomyocytes deserves, however, further investigations, especially in asymptomatic patients. METHODS: We investigated for SARS-CoV-2 presence and activity in heart tissues of six consecutive COVID-19 patients deceased from respiratory failure showing no signs of cardiac involvement and with no history of heart disease. Cardiac autopsy samples were collected within 2 h after death, and then analysed by digital PCR, Western blot, immunohistochemistry, immunofluorescence, RNAScope, and transmission electron microscopy assays. RESULTS: The presence of SARS-CoV-2 into cardiomyocytes was invariably detected in all assays. A variable pattern of cardiomyocyte injury was observed, spanning from absence of cell death and subcellular alterations hallmarks, to intracellular oedema and sarcomere ruptures. In addition, we found active viral transcription in cardiomyocytes, by detecting both sense and antisense SARS-CoV-2 spike RNA. CONCLUSIONS: In this autopsy analysis of patients with no clinical signs of cardiac involvement, the presence of SARS-CoV-2 in cardiomyocytes has been detected, determining variable patterns of intracellular damage. These findings suggest the need for cardiologic surveillance in surviving COVID-19 patients not displaying a cardiac phenotype.