Pathogens distribution and antimicrobial resistance pattern of blood stream infections in Southern Italian hospital, 2016-2021 surveillance.

Bloodstream infections (BSIs) monitoring and antibiotic susceptibility assumes a priority relevance to guide antibiotic treatment strategies and prevention programs. The study aims to identify the most common causative agents of BSIs, seasonal distribution and variation of antimicrobial susceptibility rates during a 6-year period in a in a Level II EAD Southern Italian Hospital. The study was conducted from 2016 to 2021 at Hospital of National Relevance (AORN) Sant'Anna and San Sebastiano, Caserta, Campania Region in Italy. BSIs Gram positive causative pathogens were S. aureus and Enterococci; Gram negative pathogens were E. coli, K. pneumoniae, P. aeruginosa and A. baumannii. Seasonal distribution showed the main incidence in April-June for Gram positive BSIs pathogens and in July-September months for Gram negative. Antimicrobial susceptibility fluctuations rates from 2016-2018 to 2019-2021 highlighted a significant decrease in S. aureus oxacillin resistance rates. Enterococci incremented resistance was reported for gentamicin. Gram negative pathogens antimicrobial susceptibility revealed decreased carbapenem-resistance rates for K. pneumoniae (-21.5%) and P. aeruginosa (-19.7%). A. baumannii colistin resistance had a significant increase in 2019-2021. K. pneumoniae and E. coli isolates showed decreased trend of extended-spectrum -lactamase-producing (ESBL) and carbapenem-resistant (CRE) resistance profiles. Our finding reflects the success of our Istitution regarding antimicrobial stewardship program and highlights the need to know the trend of antimicrobial resistance characterization focus on local pathogens' profile. In this way, in conjunction with infection control strategies, il could be possible to constantly reduce the spread of Multi Drug Resistant organisms.

Fosfomycin-meropenem synergistic combination against NDM carbapenemase-producing Klebsiella pneumoniae strains.

Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing threat to global public health. Treatment of CPE isolates, like New Delhi metallo-ß-lactamase (NDM), is limited and often necessitates combination therapies. The aim of this study was to evaluate the synergistic meropenem/fosfomycin combination against K.pneumoniae-producing NDM isolates. Fosfomycin/meropenem, fosfomycin/colistin and meropenem/colistin were tested alone and in combination, using e-test and time-kill assay against 20 clinical carbapenemase-producing K. pneumonia (CPKp NDM) isolates collected from September 2022 to December 2022. K. pneumoniae strains were resistant to meropenem, ceftazidime/avibactam and ceftolozano/tazobactam, 75% and 80% of isolates were susceptible for cefiderocol and for colistin respectively. Fosfomycin/meropenem combination was synergic in 95% (n=19) strains. Fosfomycin/colistin and colistin/meropenem combination showed only 10% synergistic combination strains. In 16 isolates (80%) indifference action for fosfomycin/colistin and colistin/meropenem was reported. For 0.8% of CpKP NDM isolates colistin/meropenem and fosfomycin/colistin combinations found to be antagonistic. In this study, time kill assay showed combination therapies action versus K.pneumoniae metallo-b-lactamase producing (NDM) strains and confirmed the synergistic action of fosfomycin/meropenem combination. In vitro synergy testing should be routinely performed in multidrug resistance infections and combo therapies can be used as a possible alternative in targeted patients with the goal of reducing overall antibiotic costs.

Shape-Sensing of Beam Elements Undergoing Material Nonlinearities.

The use of in situ strain measurements to reconstruct the deformed shape of structures is a key technology for real-time monitoring. A particularly promising, versatile and computationally efficient method is the inverse finite element method (iFEM), which can be used to reconstruct the displacement field of beam elements, plate and shell structures from some discrete strain measurements. The iFEM does not require the knowledge of the material properties. Nevertheless, it has always been applied to structures with linear material constitutive behavior. In the present work, advances are proposed to use the method also for concrete structures in civil engineering field such as bridges normally characterized by material nonlinearities due to the behavior of both steel and concrete. The effectiveness of iFEM, for simply supported reinforced concrete beam and continuous beams with load conditions that determine the yielding of reinforcing steel, is studied. In order to assess the influence on displacements and strains reconstructions, different measurement stations and mesh configurations are considered. Hybrid procedures employing iFEM analysis supported by bending moment-curvature relationship are proposed in case of lack of input data in plastic zones. The reliability of the results obtained is tested and commented on to highlight the effectiveness of the approach.

Whole Genome Sequence Dataset of Mycobacterium tuberculosis Strains from Patients of Campania Region.

Tuberculosis (TB) is one of the deadliest infectious disorders in the world. To effectively TB manage, an essential step is to gain insight into the lineage of Mycobacterium tuberculosis (MTB) and the distribution of drug resistance. Although the Campania region is declared a cluster area for the infection, to contribute to the effort to understand TB evolution and transmission, still poorly known, we have generated a dataset of 159 genomes of MTB strains, from Campania region collected during 2018-2021, obtained from the analysis of whole genome sequence. The results show that the most frequent MTB lineage is the 4 according for 129 strains (81.11%). Regarding drug resistance, 139 strains (87.4%) were classified as multi susceptible, while the remaining 20 (12.58%) showed drug resistance. Among the drug-resistance strains, 8 were isoniazid-resistant MTB, 4 multidrug-resistant MTB, while only one was classified as pre-extensively drug-resistant MTB. This dataset expands the existing available knowledge on drug resistance and evolution of MTB, contributing to further TB-related genomics studies to improve the management of this disease.

Acidic mammalian chitinase is not a critical target for allergic airway disease.

The expression of acidic mammalian chitinase (AMCase) is associated with Th2-driven respiratory disorders. To investigate the potentially pathological role of AMCase in allergic airway disease (AAD), we sensitized and challenged mice with ovalbumin or a combination of house dust mite (HDM) plus cockroach allergen. These mice were treated or not treated with small molecule inhibitors of AMCase, which significantly reduced allergen-induced chitinolytic activity in the airways, but exerted no apparent effect on pulmonary inflammation per se. Transgenic and AMCase-deficient mice were also submitted to protocols of allergen sensitization and challenge, yet we found little or no difference in the pattern of AAD between mutant mice and wild-type (WT) control mice. In a separate model, where mice were challenged only with intratracheal instillations of HDM without adjuvant, total bronchoalveolar lavage (BAL) cellularity, inflammatory infiltrates in lung tissues, and lung mechanics remained comparable between AMCase-deficient mice and WT control mice. However BAL neutrophil and lymphocyte counts were significantly increased in AMCase-deficient mice, whereas concentrations in BAL of IL-13 were significantly decreased compared with WT control mice. These results indicate that, although exposure to allergen stimulates the expression of AMCase and increased chitinolytic activity in murine airways, the overexpression or inhibition of AMCase exerts only a subtle impact on AAD. Conversely, the increased numbers of neutrophils and lymphocytes in BAL and the decreased concentrations of IL-13 in AMCase-deficient mice challenged intratracheally with HDM indicate that AMCase contributes to the Th1/Th2 balance in the lungs. This finding may be of particular relevance to patients with asthma and increased airway neutrophilia.

Complement C3a, CpG oligos, and DNA/C3a complex stimulate IFN-α production in a receptor for advanced glycation end product-dependent manner.

The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.

CYPRUS VIRUS INFECTION: A RARE BUT LIFE-THREATENING DIAGNOSIS IN CHILDREN.

A 3.8-year-old Italian girl presenting with high fever and headache developed a decrease in the state of consciousness, apneas, bradycardia thus requiring intensive care and mechanical ventilation. Sandfly fever due to Cyprus virus, previously not reported in a child, outside an endemic area, must be included in the differential diagnosis of acute encephalopathy with apparently no explanation from the usual laboratory work-up.

Profile of Co-Infection Prevalence and Antibiotics Use among COVID-19 Patients.

Bacterial co-infection in COVID-19 patients significantly contributes to the worsening of the prognosis based on morbidity and mortality. Information on the co-infection profile in such patients could help to optimize treatment. The purpose of this study was to describe bacterial co-infections associated with microbiological, clinical, and laboratory data to reduce or avoid a secondary infection. A retrospective cohort study was conducted at Sant'Anna and San Sebastiano Hospital from January 2020 to December 2021. Bacterial co-infection was detected in 14.3% of the COVID-19-positive patients. The laboratory findings on admission showed significant alterations in the median D-dimer, C-reactive protein, interleukin-6, and lactate dehydrogenase values compared to normal values. All inflammatory markers were significantly elevated. The most common pathogens isolated from blood cultures were E. faecalis and S. aureus. Instead, the high prevalence of respiratory tract infections in the COVID-19 patients was caused by P. aeruginosa (41%). In our study, 220 (82.4%) of the COVID-19 patients received antimicrobial treatment. Aminoglycosides and ß-lactams/ß-lactamase inhibitors showed the highest resistance rates. Our results showed that older age, underlying conditions, and abnormal laboratory parameters can be risk factors for co-infection in COVID-19 patients. The antibiotic susceptibility profile of bacterial pathogen infection provides evidence on the importance, for the clinicians, to rationalize and individualize antibiotic usage.

BND-22, a first-in-class humanized ILT2-blocking antibody, promotes antitumor immunity and tumor regression.

BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.

Diagnostic and infection control strategies for Clostridioides difficile infections in a setting of high antimicrobial resistance prevalence.

Clostridioides difficile (CD) is a major nosocomial pathogen and the leading cause of antibiotic-associated diarrhoea. In light of the strong association between antimicrobial use and CD infections (CDI), it may be hypothesised that areas at higher prevalence of antimicrobial resistance, like the region of Campania in southern Italy, could also have a higher rate of CDI. In this multicentre, region-based, prospective study, we analysed such issues, exploiting CDI incidence data collected from local hospitals. In 2016, the Italian National Centre for Disease Control supported a project involving three Italian regions: Friuli Venezia Giulia, Lazio and Campania. In Campania, a network of 49 hospitals willing to participate in the project was created. The project consisted of two phases: a survey on practice patterns concerning CDI and an epidemiological surveillance study. We identified a stringent need to improve awareness about CDI among the regional health-care community, as a widespread lack of surveillance programmes for CDI control was observed (existing in only 40% of participating facilities). Moreover, almost half of the participating hospitals (n=16, 43%) had no standardised procedures or protocols to control and prevent CDI. In the second phase of the study, we collected data of CDI cases during a six-month surveillance programme. In all, 87 CDI cases were observed, for a total of 903,334 patient bed-days and 122,988 admissions. According to the above data, CDI incidence was 0.96 cases/10000 patient bed-days, much lower than expected based on prior studies conducted elsewhere. The results of our study suggest CDI remains a rather neglected clinical issue in Campania. Despite a high burden of antimicrobial resistance and antimicrobial use in our geographic setting, we observed a very low incidence of CDI. Such a low incidence could be explained by underdiagnosis, but could also be related to actual diet, the lower patient age or the specific genetic background. However, further studies are warranted to either confirm or rebut the above hypotheses.

Pan-TGFß inhibition by SAR439459 relieves immunosuppression and improves antitumor efficacy of PD-1 blockade.

TGFß is a pleiotropic cytokine that may have both tumor inhibiting and tumor promoting properties, depending on tissue and cellular context. Emerging data support a role for TGFß in suppression of antitumor immunity. Here we show that SAR439459, a pan-TGFß neutralizing antibody, inhibits all active isoforms of human and murine TGFß, blocks TGFß-mediated pSMAD signaling, and TGFß-mediated suppression of T cells and NK cells. In vitro, SAR439459 synergized with anti-PD1 to enhance T cell responsiveness. In syngeneic tumor models, SAR439459 treatment impaired tumor growth, while the combination of SAR439459 with anti-PD-1 resulted in complete tumor regression and a prolonged antitumor immunity. Mechanistically, we found that TGFß inhibition with PD-1 blockade augmented intratumoral CD8+ T cell proliferation, reduced exhaustion, evoked proinflammatory cytokines, and promoted tumor-specific CD8+ T cell responses. Together, these data support the hypothesis that TGFß neutralization using SAR439459 synergizes with PD-1 blockade to promote antitumor immunity and formed the basis for the ongoing clinical investigation of SAR439459 in patients with cancer (NCT03192345).

Isatuximab Acts Through Fc-Dependent, Independent, and Direct Pathways to Kill Multiple Myeloma Cells.

Isatuximab is a monoclonal antibody targeting the transmembrane receptor and ectoenzyme CD38, a protein highly expressed on hematological malignant cells, including those in multiple myeloma (MM). Upon binding to CD38-expressing MM cells, isatuximab is thought to induce tumor cell killing via fragment crystallizable (Fc)-dependent mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), as well as via direct Fc-independent mechanisms. Here, these mechanisms of action were investigated in MM and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in peripheral blood mononuclear cells derived from healthy donors, and in MM patient-derived samples. Our findings show that isatuximab-mediated cytotoxicity occurred primarily via ADCC and ADCP in MM cell lines and via ADCC and apoptosis in DLBCL cell lines expressing high levels of CD38. We identified the programmed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) pathway and MM cell-secreted transforming growth factor-beta (TGF-ß) as tumor cell-related features that could suppress CD38-mediated ADCC. Furthermore, we established that isatuximab can directly activate natural killer (NK) cells and promote NK cell-mediated cytotoxicity via crosslinking of CD38 and CD16. Finally, isatuximab-induced CDC was observed in cell lines with high CD38 receptor density (>250,000 molecules/cell) and limited expression of inhibitory complement regulatory proteins (CD46, CD55, and CD59; <50,000 molecules/cell). Taken together, our findings highlight mechanistic insights for isatuximab and provide support for a range of combination therapy approaches that could be tested for isatuximab in the future.

Pharmacotherapy of spasticity in children with cerebral palsy.

Spasticity is one of the most common symptoms presented by neurologic patients. Apart from surgical management, drug therapy is an important treatment of children suffering from spasticity. In this review, recent advances in the pharmacologic armamentarium are reported in detail. In particular, there are oral medications (benzodiazepines, baclofen, dantrolene sodium, alpha 2 adrenergic agonists) and parenteral medications (botulinum toxin type A and B, alcohol). Moreover, there is also baclofen that can be administered intrathecally. There are some reports supporting the use of intramuscular alcohol (45% and/or 5-7% phenol) to reduce spasticity without the loss of voluntary movement or loss of sensation. Among these drugs, intrathecal baclofen is one of the most effective substances that can reduce spasticity significantly in the upper and lower extremities. Finally, the effectiveness of therapy with botulinum toxin type A in the management of spasticity is analyzed. Botulinum toxin type A reduces hypertonia in the injected muscles for a period of 2 to 4 months without important side effects. The purpose of this article is to provide an overview of available oral and parenteral drugs for treatment of spasticity in cerebral palsy and to outline indications and contraindications.

Quinine sulfate inhibits invasion of Salmonella typhimurium and Shigella flexneri: a preliminary study.

BACKGROUND: The incidence of typhoid fever and shigellosis parallels that of malaria, so many individuals who are on antimalarial drugs can be found in areas where these diseases are widespread. We investigated the effect of quinine sulfate on the growth and invasion of Salmonella typhimurium and Shigella flexneri M90T to determine whether people on antimalarials can have secondary gain from some protection against typhoid fever and shigellosis. METHODS: The effect of 50 and 100 microM quinine sulfate on the invasive ability of Salmonella typhimurium and Shigella flexneri M90T into human colon adenocarcinoma-2 (Caco-2) cells was studied during the infection period. The invasive efficiency was expressed as the number of viable internalized bacteria by counting the colony-forming units. RESULTS: The invasive ability of Salmonella typhimurium and Shigella flexneri M90T was significantly inhibited by 50 and 100 microM quinine sulfate in a dose-dependent manner (for Salmonella typhimurium) when the drug was added to Caco-2 cell monolayers during the infection period. CONCLUSIONS: Since so many people who are on antimalarial drugs visit and inhabit areas that are endemic to typhoid fever and Shigella infection, a study on the influence of these drugs on the disease is long overdue. Our data indicate that quinine sulfate interferes with the invasion and internalization of Salmonella typhimurium and Shigella flexneri M90T into host cells. Further studies on additional strains/serotypes with other newer antimalarials at various concentrations are needed to verify this effect of quinine sulfate and to draw conclusions on its significance in vivo.

Endocrine and metabolic changes in epileptic patients receiving valproic acid.

It has been well known for many years that valproic acid (VPA) therapy can induce obesity and important endocrine dysfunctions; among these dysfunctions, the most common are hyperandrogenism, menstrual disorders, polycystic ovary syndrome, hyperinsulinism, and changes in LH, FSH, and sexual and thyroid hormones. Moreover, abnormalities in pubertal development and impaired skeletal growth have been reported. The aim of this review is to analyze the main effects of VPA on endocrinological functions in patients with epilepsy in order to understand in depth the pathophysiological mechanisms and, consequently, to improve the care of these patients.

Interferon-gamma cooperates with Helicobacter pylori to induce iNOS-related apoptosis in AGS gastric adenocarcinoma cells.

Helicobacter pylori colonizes the human stomach and causes gastric disease. The resulting gastric damage is a multi-step process involving several molecular factors and different target cells. Th1 cytokines released by neutrophils and lymphoid cells that infiltrate gastric mucosa, nitric oxide production and inducible nitric oxide synthase (iNOS) are associated with immune activation and tissue injury. Many other molecular processes such as apoptosis, as well as angiogenic factors and integrins, are involved in H. pylori pathogenesis. We used cancer gastric cells AGS and MKN as experimental models to evaluate apoptotic rates, iNOS gene expression with and without the presence of interferon-gamma (IFN-gamma), placenta growth factor gene expression and alphav modulation. Our results show that AGS cells stimulated with H. pylori underwent apoptosis. Moreover, the addition of IFN-gamma caused a further increase in iNOS gene expression and in the apoptotic rates. We also found early modulation in PlGF and alphav expression, and noted that p53 and bax gene expression was involved in the apoptotic process. Taken together, these findings demonstrate that H. pylori employs a series of mechanisms to avoid the host defense and cause gastric mucosa damage. One H. pylori pathogenic mechanism for causing gastric damage is the induction of iNOS-dependent apoptosis that is strongly enhanced by IFN-gamma. Thus, data obtained indicate that Th1 cytokines such as IFN-gamma, via modulation of iNOS gene expression, may contribute to an increase in the pathogenicity of H. pylori infections.

Quinine sulfate and bacterial invasion.

BACKGROUND: As many patients who receive antimalarial drugs for treatment of noninfectious, inflammatory diseases are also immunosuppressed and might have a concomitant bacterial infection, we studied the effectiveness of these drugs against bacterial infections, to find out whether they could protect against (and even treat) such conditions and obviate the need for an additional antibiotic drug. METHODS: Effect of QS on bacterial growth: Escherichia coli (E. coli) HB101 pRI203 were cultured overnight at 37 degrees C in TSB and inoculated (approx 1 x 10(7) cells/ml) in MEM in the presence of QS at various concentrations (0, 50 and 100 microM).The effect of QS at concentration of 50 and 100 microM on the entry process of E. coli HB101 pRI203 into HeLa cells was studied under different experimental conditions: 1. QS was incubated with 3 x 10(5) HeLa cells for 60 min at 37 degrees C prior to infection. 2. QS was added to HeLa cell monolayers during the infection period. RESULTS: QS showed no antibacterial activity after 24 h of incubation. The invasive efficiency of the bacteria was significantly inhibited at a dose-dependent manner, when QS was added to HeLa cells for 60 min at 37 degrees C prior to infection (condition 1), and to a lesser extent when added during the period of infection (condition 2). CONCLUSIONS: Although the antimalarials are generally regarded as being inactive against most extracellular bacterial species, our results indicate that QS significantly inhibited the internalization/invasion efficacy of E. coli in the host cells.

Quinine sulfate and HSV replication.

Although antimalarial drugs have been developed primarily to treat malaria, they are also beneficial for many dermatological, immunological, and rheumatological diseases, for which they are mostly used today in the Western world. The aim of the present study was to investigate the effect of quinine sulfate (QS) on the multiplication and adsorption of herpes virus type I (HSV-1). When Vero cells (African green monkey kidney) are infected with HSV-1 in the presence of QS, the viral adsorption is reduced, as demonstrated by a decrease of the number of microscopic plaques of the virus. When the virus-infected Vero cells are incubated in the presence of QS, the multiplication of HSV-1 is also reduced, and the diameter of the plaque are visibly smaller. The practical implications of the antiviral action of antimalarial drugs might be especially important to immunosuppressed patients who receive these drugs for autoimmune collagen-vascular diseases or as additional therapy for AIDS.

Combination of PIM and JAK2 inhibitors synergistically suppresses MPN cell proliferation and overcomes drug resistance.

Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Importantly, overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies.

Identification, characterization and initial hit-to-lead optimization of a series of 4-arylamino-3-pyridinecarbonitrile as protein kinase C theta (PKCtheta) inhibitors.

The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.