RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

BACKGROUND: The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. METHODS: After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. RESULTS: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. CONCLUSIONS: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

A molecular circuit linking the BCR to the NAD biosynthetic enzyme NAMPT is an actionable target in Richter syndrome.

ABSTRACT: This works defines, to the best of our knowledge, for the first time a molecular circuit connecting nicotinamide mononucleoside phosphoribosyl transferase (NAMPT) activity to the B-cell receptor (BCR) pathway. Using 4 distinct xenograft models derived from patients with Richter syndrome (RS-PDX), we show that BCR cross-linking results in transcriptional activation of the nicotinamide adenine dinucleotide (NAD) biosynthetic enzyme NAMPT, with increased protein expression, in turn, positively affecting global cellular NAD levels and sirtuins activity. NAMPT blockade, by using the novel OT-82 inhibitor in combination with either BTK or PI3K inhibitors (BTKi or PI3Ki), induces rapid and potent apoptotic responses in all 4 models, independently of their mutational profile and the expression of the other NAD biosynthetic enzymes, including nicotinate phosphoribosyltransferase. The connecting link in the circuit is represented by AKT that is both tyrosine- and serine-phosphorylated by PI3K and deacetylated by sirtuin 1 and 2 to obtain full kinase activation. Acetylation (ie, inhibition) of AKT after OT-82 administration was shown by 2-dimensional gel electrophoresis and immunoprecipitation. Consistently, pharmacological inhibition or silencing of sirtuin 1 and 2 impairs AKT activation and induces apoptosis of RS cells in combination with PI3Ki or BTKi. Lastly, treatment of RS-PDX mice with the combination of PI3Ki and OT-82 results in significant inhibition of tumor growth, with evidence of in vivo activation of apoptosis. Collectively, these data highlight a novel application for NAMPT inhibitors in combination with BTKi or PI3Ki in aggressive lymphomas.

Seroprevalence and Risk Factors Associated with Chlamydia abortus Infection in Sheep and Goats in North-Western Italy.

Chlamydia abortus, although poorly recognized as a human pathogen, is a zoonotic microorganism that can cause many different symptoms in humans, including subclinical infection and fatal illnesses in pregnant women. C. abortus is one of the most common causes of ovine and caprine infectious abortion worldwide, known as the causative agent of the enzootic abortion of ewes (EAE) or ovine enzootic abortion (OEA). To estimate C. abortus seroprevalence and the risk factors related to C. abortus in small ruminants, the sera from 3045 animals (both sheep and goat) belonging to 202 herds were tested and a questionnaire investigating flock management was administered. At the herd level, the true seroprevalence was 56.6% (CI95%: 46.9-66.3%), at sheep-farm and goat-farm level, the true seroprevalence was 71.4% (CI95%: 54.6-88.3%) and 44.8% (CI95%: 41.3-57.0%), respectively. The true seroprevalence was significantly higher among the sheep than the goats. The logistic regression model identified four factors associated with Chlamydia seropositivity: flock size (i.e., farms with >50 heads), contact with cattle, introduction of animals, and Coxiella seropositivity. The study evidenced a high seroprevalence of Chlamydia abortus in small ruminant farms in the Piedmont region. Considering its zoonotic potential and the health consequences in humans, communication to farmers on the importance of vaccination, as well as the sensibilization of farm vets, seem to be strategical.

Meat Juice and Oral Fluid as Alternatives to Serum for Aujeszky Disease Monitoring in Pigs.

Aujeszky Disease Virus (ADV) is a double-stranded DNA virus with a lipoprotein envelope. The natural hosts of the infection are Suidae, but the virus can infect many other mammals. The gold-standard method identified by the WOAH for the diagnosis of Aujeszky disease is the ELISA method. The objective of this study was to compare the performance of meat juice and oral fluid matrices using a commercial ELISA kit designed for serum. A total of 80 blood and oral fluid samples were collected from four pig farms selected for this study. Diaphragm muscle samples of about 100 g and blood samples were collected from 213 animals at the abattoir. These biological matrices were collected from the same animals and tested using a competitive ELISA kit to detect antibodies against ADV. The relative accuracy of the meat juice compared to that of the serum was 96.7% (95% CI: 93.3-98.7%), with 206 correct results out of 213. The relative accuracy of the oral fluid compared to that of the serum was 61.3% (95% CI: 49.7-71.9%), with 58 correct results out of 80. Meat juice has a better combination of sensitivity and specificity than oral fluid. The usage of meat juice in routine diagnostic examinations could be achieved after further investigations to optimize the procedure.

Is the Introduction into a New Environment Stressful for Young Bulls?

Several events in an animal's life are considered stressful. Among them, the most studied and significant are transportation, weaning, and adaptation to climate change. Moreover, other events, such as the separation from the dam, moving from the original farm to another, management practices, such as regrouping with other animals, and new hierarchical conditions, represent routine conditions in the bovine's life, which can influence the animal's homeostasis. The purpose of this study is to evaluate the changes in blood parameters of 45 calves introduced into a new environment from their original farms. Blood samples were collected upon arrival at a genetic center (T1), 7 (T2), 30 (T3), and 120 (T4) days after arrival. Blood count, protein electrophoresis, clinical chemistry, and innate immunity parameters were performed on the samples. Significant alterations in some clinical chemistry parameters were related to liver function in the serum protein and the values of IL-6 and TNF-α; the main cytokines mediating the stress response emerged from the results. The evidence indicates the mild response to adaptation stress by calves raised in close association with people after their introduction into a new environment.

Evaluation of the Immune Response to Transport Stress in the Aosta Valley Breed.

Transportation is a recurring event in a farm animal's life, and it is considered one of the main stressors with possible negative repercussions for both the health and welfare of farm animals. The objective of the present study was to examine the effect of transportation on some blood variables of 45 young bulls moved from their original farms to a livestock collection centre. Transportation took no more than 8 h and was carried out between January and March 2021. Blood samples were taken before transportation (T0), upon arrival at the collection centre (T1), and 7 days after arrival (T2). Samples were processed for blood cell count, clinical chemistry analyses, serum protein electrophoresis, and the evaluation of innate immunity parameters. The results showed a typical stress leukogram with neutrophilia and changes in the neutrophil:lymphocyte ratio. No significant alterations were observed in either serum proteins or pro-inflammatory cytokines. Significant, albeit transient, alterations were observed in some clinical chemistry parameters after transportation, which could be accounted for by stressful conditions such as the transportation itself and handling and mixing with other animals. Our results indicated that the adopted transportation conditions only slightly affected the blood variables under study with no significant impact on animal welfare.

Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy.

BACKGROUND: Hepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits. RESULTS: Twenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM. CONCLUSIONS: The faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

Antagonist of Growth Hormone-Releasing Hormone Potentiates the Antitumor Effect of Pemetrexed and Cisplatin in Pleural Mesothelioma.

Pleural mesothelioma (PM) is an aggressive cancer with poor prognosis and no effective therapies, mainly caused by exposure to asbestos. Antagonists of growth hormone-releasing hormone (GHRH) display strong antitumor effects in many experimental cancers, including lung cancer and mesothelioma. Here, we aimed to determine whether GHRH antagonist MIA-690 potentiates the antitumor effect of cisplatin and pemetrexed in PM. In vitro, MIA-690, in combination with cisplatin and pemetrexed, synergistically reduced cell viability, restrained cell proliferation and enhanced apoptosis, compared with drugs alone. In vivo, the same combination resulted in a strong growth inhibition of MSTO-211H xenografts, decreased tumor cell proliferation and increased apoptosis. Mechanistically, MIA-690, particularly with chemotherapeutic drugs, inhibited proliferative and oncogenic pathways, such as MAPK ERK1/2 and cMyc, and downregulated cyclin D1 and B1 mRNAs. Inflammatory pathways such as NF-kB and STAT3 were also reduced, as well as oxidative, angiogenic and tumorigenic markers (iNOS, COX-2, MMP2, MMP9 and HMGB1) and growth factors (VEGF and IGF-1). Overall, these findings strongly suggest that GHRH antagonists of MIA class, such as MIA-690, could increase the efficacy of standard therapy in PM.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Aryloxyaryl Moiety.

In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia.

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

Modulation of LTCC Pathways by a Melusin Mimetic Increases Ventricular Contractility During LPS-Induced Cardiomyopathy.

AIM: Sepsis-induced cardiomyopathy is commonplace and carries an increased risk of death. Melusin, a cardiac muscle-specific chaperone, exerts cardioprotective function under varied stressful conditions through activation of the AKT pathway. The objective of this study was to determine the role of melusin in the pathogenesis of lipopolysaccharide (LPS)-induced cardiac dysfunction and to explore its signaling pathway for the identification of putative therapeutic targets. METHODS AND RESULTS: Prospective, randomized, controlled experimental study in a research laboratory. Melusin overexpressing (MelOV) and wild-type (MelWT) mice were used. MelOV and MelWT mice were injected intraperitoneally with LPS. Cardiac function was assessed using trans-thoracic echocardiography. Myocardial expression of L-type calcium channel (LTCC), phospho-Akt and phospho-Gsk3-b were also measured. In separate experiments, wild-type mice were treated post-LPS challenge with the allosteric Akt inhibitor Arq092 and a mimetic peptide (R7W-MP) targeting the LTCC. The impact of these therapies on protein-protein interactions, cardiac function, and survival was assessed. MelOV mice had limited derangement in cardiac function after LPS challenge. Protection was associated with higher Akt and Gsk3-b phosphorylation and restored LTCC density. Pharmacological inhibition of Akt activity reversed melusin-dependent cardiac protection. Treatment with R7W-MP preserved cardiac function in wild-type mice after LPS challenge and significantly improved survival. CONCLUSIONS: This study identifies AKT / Melusin as a key pathway for preserving cardiac function following LPS challenge. The cell-permeable mimetic peptide (R7W-MP) represents a putative therapeutic for sepsis-induced cardiomyopathy.

Influence of Anthropic Environmental-Related Factors on Erysipelas in Wild Boar.

Erysipelothrix rhusiopathiae (ER) is an old but still emerging zoonotic infection that is not yet completely understood. ER infects a wide range of species and wild boar is of significant interest because of their similarities to pigs, a known ER reservoir. Moreover, the increase of its densities and the limited data available about ER in this species should be considered. The need is to investigate whether wild boar could represent a risk of erysipelas at the wildlife-domestic-human interface. Here, 1067 sera and 149 tonsils of wild boar from five hunting districts in Northwest Italy were tested using ELISA and bacteriological culture, respectively. Using generalized linear models, we evaluated host and environmental factors influencing ER spread and dynamics. We found an ER seroprevalence of 69.4% among wild boar. Increased human density and pig farm density lead to an increase of ER seropositivity highlighting its association with anthropic environmental-related factors. The high ER percentage of isolation (34.2%) found in healthy wild boar suggests that this species can serve as a healthy carrier. This fact, together with the high seroprevalence, supports a role of wild boar as an ER reservoir. Potential zoonotic and economic risks should be considered in light of these data.

Goat Paratuberculosis: Experimental Model for the Evaluation of Mycobacterium Persistence in Raw Milk Cheese.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic proliferative enteritis found in ruminants, known as paratuberculosis (PTB). The spread of PTB is increasing in countries with advanced animal husbandry practices, leading to significant economic losses. Moreover, a supposed zoonotic role of MAP in Crohn's disease (CD) in humans has been discussed by the scientific community; however, although the association between MAP and CD has generally been accepted, it is still up for debate if MAP is the main cause of CD, a contributing factor, or merely a commensal organism for the development of CD. The aim of this study was to assess the survival of MAP during the entire production process of a traditional Italian goat's raw milk fresh cheese, the "Robiola di Roccaverano", assessing the survival rate and persistence of MAP in the final product. A mix of MAP field isolates from goats of the Roccaverano area and a reference ATCC strain were used to carry out milk in experimental inoculation. Samples of milk, curd and cheese were taken in two consecutive batches of production. Microbiological challenge tests, evaluated by f57-qPCR, showed a significant decrease in MAP charge during the cheesemaking process for both batches, suggesting the productive process has an impact on MAP survival.

Antimicrobial Resistance of Escherichia coli in Dairy Calves: A 15-Year Retrospective Analysis and Comparison of Treated and Untreated Animals.

The health problem of antimicrobial resistance (AMR) involves several species. AMR surveillance is essential to identify its development and design control strategies; however, available data are still limited in some contexts. The AMR profiles of 2612 E. coli strains isolated over a period of 15 years (2002-2016) from calf enteric cases were analyzed to determine the presence of resistance and their temporal dynamics. Furthermore, the AMR profiles and the presence of the major virulence genes of 505 E. coli strains isolated from 1-week- and 2-week-old calves, 406 treated with antimicrobials and 99 untreated, were analyzed and compared to investigate the potential effects of treatment on AMR and strain pathogenicity. Resistance to tetracycline (90.70%) was the most common, followed by resistance to sulfamethoxazole/trimethoprim (77.70%) and flumequine (72.10%). The significantly higher percentage of AMR and virulence gene expression recorded in treated calves, combined with the statistically higher resistance to sulfamethoxazole/trimethoprim in E. coli with K99, corroborates the notion of resistance being induced by the frequent use of antimicrobials, leading to treatments potentially becoming ineffective. The significantly higher resistance to amoxicillin/clavulanic acid, enrofloxacin, and florfenicol in isolates from 1-week-old calves suggests the role of the environment as a source of contamination that should be investigated further.

Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria.

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1-100%] with a specificity of 91.8% (CI 95%, 81.9-97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1-89.2%) with a specificity of 100% (CI 95%, 94.1-100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

The Synergism between DHODH Inhibitors and Dipyridamole Leads to Metabolic Lethality in Acute Myeloid Leukemia.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines; this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Synergistic efficacy of the dual PI3K-δ/γ inhibitor duvelisib with the Bcl-2 inhibitor venetoclax in Richter syndrome PDX models.

A small subset of cases of chronic lymphocytic leukemia undergoes transformation to diffuse large B-cell lymphoma, Richter syndrome (RS), which is associated with a poor prognosis. Conventional chemotherapy results in limited responses, underlining the need for novel therapeutic strategies. Here, we investigate the ex vivo and in vivo efficacy of the dual phosphatidylinositol 3-kinase-δ/γ (PI3K-δ/γ) inhibitor duvelisib (Duv) and the Bcl-2 inhibitor venetoclax (Ven) using 4 different RS patient-derived xenograft (PDX) models. Ex vivo exposure of RS cells to Duv, Ven, or their combination results in variable apoptotic responses, in line with the expression levels of target proteins. Although RS1316, IP867/17, and RS9737 cells express PI3K-δ, PI3K-γ, and Bcl-2 and respond to the drugs, RS1050 cells, expressing very low levels of PI3K-γ and lacking Bcl-2, are fully resistant. Moreover, the combination of these drugs is more effective than each agent alone. When tested in vivo, RS1316 and IP867/17 show the best tumor growth inhibition responses, with the Duv/Ven combination leading to complete remission at the end of treatment. The synergistic effect of Duv and Ven relies on the crosstalk between PI3K and apoptotic pathways occurring at the GSK3ß level. Indeed, inhibition of PI3K signaling by Duv results in GSK3ß activation, leading to ubiquitination and subsequent degradation of both c-Myc and Mcl-1, making RS cells more sensitive to Bcl-2 inhibition by Ven. This work provides, for the first time, a proof of concept of the efficacy of dual targeting of PI3K-δ/γ and Bcl-2 in RS and providing an opening for a Duv/Ven combination for these patients. Clinical studies in aggressive lymphomas, including RS, are under way. This trial was registered at www.clinicaltrials.gov as #NCT03892044.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus and Related Risk Factors in Holdings of Veal Calves in Northwest Italy.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have emerged worldwide as zoonotic pathogens. Data on LA-MRSA in veal calf production in Italy are lacking; the aim of this survey was to fill current knowledge gaps in its prevalence and characteristics. Between February 2012 and January 2013 nasal swabs were taken from 1650 three- to six-month-old veal calves on 55 farms in Piedmont (northwest Italy), including gathering-related epidemiological data. S. aureus were screened for methicillin resistance by phenotypic and molecular (mecA gene detection) methods. MRSA were further genotyped by multilocus sequence typing. About 30% of the herds tested positive for MRSA: three different clonal complexes (CC398, CC97, and CC1) and staphylococcal cassette chromosome mec types (IVa, IVb, and V) were detected. Multilevel logistic regression model indicated poor cleaning, importation from Austria, and animal age as risk factors and coagulase-negative staphylococci colonization as a predictive factor for the occurrence of MRSA. The detection of CCs circulating in pigs and dairy cattle in Italy underscores the ability of the LA-MRSA clones to spread among animal production systems. In addition to maintaining preventive control measures for human health, better cleaning procedures need to be implemented, especially after new calves have been introduced into the herd.