The FDA approved PI3K inhibitor GDC-0941 enhances in vitro the anti-neoplastic efficacy of Axitinib against c-myc-amplified high-risk medulloblastoma.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/ß in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi-kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti-neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c-myc-amplified Non-WNT/Non-SHH and SHH-TP53-mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/ß and c-kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi-modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC-0941 enhances the anti-neoplastic efficacy of Axitinib against c-myc-amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.

Brentuximab vedotin exerts profound antiproliferative and pro-apoptotic efficacy in CD30-positive as well as cocultured CD30-negative germ cell tumour cell lines.

Prognosis in patients suffering from high-risk, refractory and relapsed germ cell tumours (GCT) often comprising of CD30-positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody-drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30-expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30-negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS- and flow cytometry-based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT-PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30-positive GCT27 EC line exerting marked time-dependent antiproliferative and pro-apoptotic activity. CD30-negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose-dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30-negative JAR cocultured with CD30-positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30-negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro-apoptotic activity against both CD30-positive as well as CD30-negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high-risk GCT of heterogeneous CD30 positivity.

Threshold and Target for Blood Pressure Lowering in the Elderly.

PURPOSE OF REVIEW: Detection of elevated blood pressure values in elderly patients represents a common clinical condition associated with an increased cardiovascular risk. This has been shown to be the case in both systodiastolic and isolated systolic hypertension as well. However, despite the evidence of the benefits of the blood pressure lowering intervention in terms of reduction of cardiovascular morbidity and mortality, at least two issues related to antihypertensive drug treatment in aged individuals are still undefined: (1) the blood pressure threshold at which antihypertensive drug should be initiated and (2) the blood pressure goals of the therapeutic intervention. RECENT FINDINGS: The present paper will critically review the evidence available so far on these two issues as well as the position of current guidelines and consensus statements. Emphasis will be given to the analysis of the new data of the Systolic Blood Pressure Interventional Trial (SPRINT), which have recently demonstrated the benefits, even in individuals aged more than 75 years, of a tight blood pressure reduction to systolic blood pressure to 120 mmHg or less. The potential limitations of the trial will be also critically addressed and the expectations of ongoing clinical studies investigating the issue in elderly patients properly emphasized. Although of interest, the results of the SPRINT trial encompass a number of limitations which limit their applicability to the general elderly hypertensive population. A prudent approach will be to adopt in clinical practice the less intensive and more conservative targets recommended by current guidelines.

Anti-CD3ε mAb improves thymic architecture and prevents autoimmune manifestations in a mouse model of Omenn syndrome: therapeutic implications.

Omenn syndrome (OS) is an atypical primary immunodeficiency characterized by severe autoimmunity because of activated T cells infiltrating target organs. The impaired recombinase activity in OS severely affects expression of the pre-T-cell receptor complex in immature thymocytes, which is crucial for an efficient development of the thymic epithelial component. Anti-CD3ε monoclonal antibody (mAb) treatment in RAG2(-/-) mice was previously shown to mimic pre-TCR signaling promoting thymic expansion. Here we show the effect of anti-CD3ε mAb administration in the RAG2(R229Q) mouse model, which closely recapitulates human OS. These animals, in spite of the inability to induce the autoimmune regulator, displayed a significant amelioration in thymic epithelial compartment and an important reduction of peripheral T-cell activation and tissue infiltration. Furthermore, by injecting a high number of RAG2(R229Q) progenitors into RAG2(-/-) animals previously conditioned with anti-CD3ε mAb, we detected autoimmune regulator expression together with the absence of peripheral immunopathology. These observations indicate that improving epithelial thymic function might ameliorate the detrimental behavior of the cell-autonomous RAG defect. Our data provide important therapeutic proof of concept for future clinical applications of anti-CD3ε mAb treatment in severe combined immunodeficiency forms characterized by poor thymus function and autoimmunity.

Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients.

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8(+) T(N) cells (CD4(-)CD62L(+)CD45RA(+)) and CD8(+) T(CM) (CD4(-)CD62L(+)CD45RA(-)) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T(N) and T(CM) we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF.

CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8+ T-cells. Two major MDSC subsets were recently shown to play an equal role in MDSC-induced immune dysfunctions: monocytic- and granulocytic-like. We isolated three fractions of MDSC, i.e. CD11b+/Gr-1high, CD11b+/Gr-1int, and CD11b+/Gr-1low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b+/Gr-1int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8+ T-cell activation, while CD11b+/Gr-1high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b+/Gr-1int but not CD11b+/Gr-1high cells were also immunosuppressive in vivo following adoptive transfer. CD11b+/Gr-1low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM-CSF was necessary to induce preferential expansion of both CD11b+/Gr-1int and CD11b+/Gr-1low subsets in the spleen of tumor-bearing mice and mediate tumor-induced tolerance whereas G-CSF, which preferentially expanded CD11b+/Gr-1high cells, did not create such immunosuppressive environment. GM-CSF also acted on granulocyte-macrophage progenitors in the bone marrow inducing local expansion of CD11b+/Gr-1low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor-released GM-CSF.

A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome.

Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.

Targeting EpCAM by a Bispecific Trifunctional Antibody Exerts Profound Cytotoxic Efficacy in Germ Cell Tumor Cell Lines.

Outcome in high-risk patients with refractory or relapsed germ cell tumours (GCT) remains poor. Novel strategies enhancing therapeutic efficacy whilst limiting therapeutic burden are warranted, yet immunotherapy approaches geared towards activating endogenous antitumor responses have not been successful thus far. Redirection of cytotoxic effector cells by bispecific antibodies represents a promising approach in this setting. We demonstrate that the Epithelial Cell Adhesion Molecule (EpCAM) is broadly expressed in GCT cell lines of different histologic origin including seminoma, choriocarcinoma (CHC), and embryonal carcinoma (EC). In these GCT lines of variable EpCAM surface expression, targeting T cells by the prototypic bispecific EpCAM/CD3-antibody (bAb) Catumaxomab together with natural killer (NK) cell engagement via the Fc domain promotes profound cytotoxicity across a broad range of antibody dilutions. In contrast, tumor cell lysis mediated by either immune cell subset alone is influenced by surface density of the target antigen. In the CHC line JAR, NK cell-dependent cytotoxicity dominates, which may be attributed to differential surface expression of immunomodulatory proteins such as MHC-I, CD24, and Fas receptors on CHC and EC. In view of redirecting T cell therapy mediated by bispecific antibodies, such differences in GCT immunophenotype potentially favoring immune escape are worth further investigation.

The hidden hypothesis: A disseminated tuberculosis case.

CASE PRESENTATION: 77-year-old former smoker admitted because of fatigue and abdominal distention. Past medical history positive for two previous hospitalizations for pericardial and pleural effusions (no diagnosis achieved). At admission erythrocyte sedimentation rate was 122mm per hour. Baseline investigations revealed ascitic, pleural and pericardial effusion. Effusions were tapped: neoplastic cells and acid-fast bacilli (AFB) were not identified, aerobic and mycobacterial culture resulted negative. QuantiFERON TB-Gold test was negative. Total body PET-CT and autoimmunity panel were negative. A neoplastic process was considered the most likely explanation. Before signing off the patient to comfort care, a reassessment was performed and an exposure to tuberculosis during childhood was documented. Because of constrictive pericarditis, pericardiectomy was performed: histologic examination showed chronic pericardial inflammation without granulomas, but Ziehl-Neelsen stain identified AFB and PCR was positive for Mycobacterium tuberculosis complex. Patient was started on anti-TB therapy with resolution of the effusions in the following months. Genes associated with defects in innate immunity were sequences and dentritic cells were studied, but no alterations were identified. DISCUSSION: A Bayesian approach to clinical decision making should be recommended. Interpretation of diagnostic tests should take into account the imperfect diagnostic performance of the majority of these tests. Further studies to investigate genetic susceptibility to tuberculosis are needed.

Clinical and prognostic value of hypertensive cardiac damage in the PAMELA Study.

Because subclinical alterations in cardiovascular structure reflect cumulative damage induced by risk factors and represent an intermediate stage between risk factor exposure and cardiovascular events, this damage is regarded as a marker of increased cardiovascular risk in different clinical settings, including the general population. The Pressioni Monitorate e Loro Associazioni (PAMELA) is an originally designed research study aimed at assessing the normal values and prognostic significance of ambulatory and home blood pressure in a representative sample of the Northern Italian general population. Because the study protocol included the collection of electrocardiographic (ECG) and echocardiographic (ECHO) data, the prevalence and clinical correlates, as well as the prognostic value of subclinical cardiac alterations, have been extensively investigated. This article is a review of the findings of the PAMELA study regarding the clinical aspects and prognostic significance of cardiac abnormal phenotypes such as left ventricular hypertrophy, left atrial dilatation and aortic root dilation.

Differential effects of enalapril-felodipine versus enalapril-lercanidipine combination drug treatment on sympathetic nerve traffic and metabolic profile in obesity-related hypertension.

Scanty information is available on the effects of combination drug treatment based on an ACE inhibitor and a calcium channel blocker on the neurometabolic alterations characterizing obesity-related hypertension (OHT). After 2-week run-in with enalapril (20 mg), 36 OHTs were randomized according to a double-blind crossover design to a combination therapy with either lercanidipine 10 mg (L) or felodipine extended release 5 mg (F), each lasting 8 weeks. Measurements included clinic and ambulatory blood pressure (BP) and heart rate, homeostasis model assessment index, plasma norepinephrine, and muscle sympathetic nerve activity. Patients with uncontrolled BP were then uptitrated to 20 mg/d (L) and 10 mg/d (F) combined with enalapril 20 mg, respectively, for further 8 weeks. For similar BP reductions, enalapril-lercanidipine (EL) caused norepinephrine and MSNA increases significantly less pronounced than those seen with enalapril-felodipine, the lesser sympathoexcitation observed with EL being coupled with a significant improvement in homeostasis model assessment index. This was the case also when L and F were uptitrated in the combination. In OHT, at variance from enalapril-felodipine, EL combination is almost entirely devoid of any major sympathoexcitatory effect and is associated with an improvement in insulin sensitivity.

Selective preservation of bone marrow mature recirculating but not marginal zone B cells in murine models of chronic inflammation.

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4(+) naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation.

Impact of different definitions of the metabolic syndrome on the prevalence of organ damage, cardiometabolic risk and cardiovascular events.

OBJECTIVES: We compared definitions of metabolic syndrome performed by ATPIII [the National Cholesterol Education Program Adult Treatment Panel III; three criteria of the following: systolic blood pressure >or=130 mmHg and/or diastolic blood pressure >or=85 mmHg, fasting serum glucose >or=110 mg/dl, high-density lipoprotein plasma cholesterol or=150 mg/dl, waist circumference >or=102 cm (men) or 88 cm (women)], AHA (the American Heart Association; same cut-off of ATPIII except serum glucose >or= 100 mg/dl) and IDF [the International Diabetes Federation; mandatory criteria of visceral obesity with reduced cut-off of 94 cm (men) or 80 cm (women), and at least two criteria with the same cut-off as in AHA] for their impact on metabolic syndrome prevalence, cardiac organ damage, long-term risk of cardiovascular events and death for any cause and risk of developing diabetes mellitus, in-office and out-of-office hypertension and left ventricular hypertrophy (LVH). METHODS: In 2051 participants, we measured office, home and ambulatory blood pressure as well as metabolic, anthropometric and echocardiographic variables. Measurements were performed between 1990 and 1992 and repeated 10 years later. Information on long-term incidence of cardiovascular events and all-cause deaths was also collected. RESULTS: Prevalence of metabolic syndrome was significantly greater when using the AHA and IDF as compared to the ATPIII definition. Prevalence of LVH was higher in participants with than without metabolic syndrome and similar for the three definitions. Over 12-year follow-up, there were 179 cardiovascular events and 233 deaths for any cause. The risk of cardiovascular events and death was markedly greater for participants with as compared with those without metabolic syndrome, regardless of the definition of metabolic syndrome. This was the case also for the risk of new-onset diabetes mellitus, office, home and ambulatory hypertension and new-onset LVH. CONCLUSION: Risks of fatal and nonfatal cardiovascular events, diabetes mellitus, hypertension and LVH were similar for the three definitions of metabolic syndrome. However, the AHA and IDF definitions are more sensitive than that of ATPIII in identifying metabolic syndrome condition.

Purinergic control of T cell activation by ATP released through pannexin-1 hemichannels.

T cell receptor (TCR) stimulation results in the influx of Ca(2+), which is buffered by mitochondria and promotes adenosine triphosphate (ATP) synthesis. We found that ATP released from activated T cells through pannexin-1 hemichannels activated purinergic P2X receptors (P2XRs) to sustain mitogen-activated protein kinase (MAPK) signaling. P2XR antagonists, such as oxidized ATP (oATP), blunted MAPK activation in stimulated T cells, but did not affect the nuclear translocation of the transcription factor nuclear factor of activated T cells, thus promoting T cell anergy. In vivo administration of oATP blocked the onset of diabetes mediated by anti-islet TCR transgenic T cells and impaired the development of colitogenic T cells in inflammatory bowel disease. Thus, pharmacological inhibition of ATP release and signaling could be beneficial in treating T cell-mediated inflammatory diseases.

Tumors hamper the immunogenic competence of CD4+ T cell-directed dendritic cell vaccination.

Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44(high)CD62L(low), IL-2(+), IFN-gamma(+)) and central memory-like lymphocytes (CD44(high)CD62L(high), only IL-2(+)) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.

The immunogenicity of dendritic cell-based vaccines is not hampered by doxorubicin and melphalan administration.

Immunization of cancer patients is most effective in tumor-free conditions or in the presence of minimal residual disease. In the attempt to develop new strategies able to control tumor recurrence while allowing the development of protective immunity, we have investigated the immunogenic potential of two distinct vaccine formulations when provided alone or upon single and repeated treatment with chemotherapeutics drugs. Vaccine-induced T cell responses were first investigated by tracing Ag-specific T cell responses in mice bearing detectable frequencies of Ag-specific TCR transgenic CD4 and CD8 T cells. These studies indicated that immunization with peptide-pulsed dendritic cells and soluble Ag plus adjuvant elicited a comparable expansion and differentiation of CD4 and CD8 effector cells in the peripheral lymphoid tissues when provided alone or shortly after Doxorubicin or Melphalan administration. We also analyzed the potency of the combined vaccination in transgenic adenocarcinoma mouse prostate mice, which develop spontaneous prostate cancer. Dendritic cell-based vaccination elicited potent tumor-specific cytotoxic responses in mice bearing prostate intraepithelial neoplasia both in the absence and in the presence of Doxorubicin. Together our results indicate that Doxorubicin- or Melphalan-based chemotherapy and Ag-specific vaccination can be combined for adjuvant treatments of cancer patients.