Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting.

BACKGROUND: Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. METHODS: The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. RESULTS: Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. CONCLUSIONS: Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.

ATR addiction in multiple myeloma: synthetic lethal approaches exploiting established therapies.

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

Ceruloplasmin oxidized and deamidated by Parkinson's disease cerebrospinal fluid induces epithelial cells proliferation arrest and apoptosis.

In Parkinson's disease, the ferroxidase ceruloplasmin (Cp) is oxidized and deamidated by the pathological cerebrospinal fluid (CSF) environment. These modifications promote the gain of integrin binding properties, fostered by the deamidation of two NGR-motifs present in the Cp sequence that convert into the isoDGR-motif. Through isoDGR/integrin binding, the oxidized/deamidated-Cp (Cp-ox/de) mediates cell adhesion and transduces an intracellular signal in epithelial cells that seems to be addressed to regulate cell cycle, proliferation and cytoskeletal re-arrangement. However, the effect fostered on cells by integrins engagement via Cp-ox/de is not known. We found that in HaCaT epithelial cells, the incubation with Cp-ox/de resulted in proliferation inhibition mediated by isoDGR, cell cycle arrest and apoptosis induction. Similar proliferation inhibition was induced by treatment with purified Cp previously incubated in the CSF from Parkinson's disease patients, but not by Cp incubated in the CSF from healthy subjects. In human primary choroid plexus epithelial cells, a possible in vivo target of Cp-ox/de generated in pathological CSFs, we found that Cp-ox/de mediated cell adhesion via isoDGR/integrins binding and transduced an intracellular signal, which resulted in cell proliferation inhibition. Thus, the generation of Cp-ox/de in pathological CSFs and the consequent apoptosis induction of epithelial cells facing the liquor, might represent a novel mechanism that contributes to neurodegeneration.

Progression of brain white matter hyperintensities in asymptomatic patients with carotid atherosclerotic plaques and no indication for revascularization.

BACKGROUND AND AIMS: Brain white matter hyperintensities (WMHs) have been associated with an increased risk of ischemic stroke and considered as markers of brain ischemia. Progression of WMHs in asymptomatic patients with non-hemodynamically significant carotid plaque could represent a putative marker of plaque vulnerability. We prospectively evaluate progression and determinants of WMHs in this population. METHODS: This prospective study included 51 asymptomatic patients with carotid stenosis <70% that underwent brain magnetic resonance imaging scans at baseline and after a median follow up of 595 days (interquartile range 553-641 days). Patients (mean age of 69 years and 45% females) underwent baseline carotid computed tomography angiography, contrast-enhanced ultrasound for carotid plaque characterization and analysis of subsets of circulating lymphocytes and monocytes by flow cytometry. RESULTS: Seventeen subjects (33.3%) had carotid stenoses of 50-70% (Doppler flow velocity) while the rest had stenoses of <50%. In 25 (49.0%) patients, new WMHs, with 5 new lesions on average and a median volume of 134 mm3, were detected at follow-up. None of the plaque characteristics or of the circulating cellular biomarkers investigated were associated with the global and ipsilateral occurrence of new WMHs whereas, at multivariate analysis, female sex, hypercholesterolemia, and lower glomerular filtration rate (GFR) emerged as independent variables associated with new WMHs. CONCLUSIONS: Half of the patients with carotid plaques of intermediate severity had evidence of WMH progression at follow up. Female gender and systemic factors such as hypercholesterolemia, and lower GFR, but not plaque characteristics or circulating cellular biomarkers, are associated with WMH progression.

Carotid artery plaque uptake of 11C-PK11195 inversely correlates with circulating monocytes and classical CD14++CD16- monocytes expressing HLA-DR.

BACKGROUND: We explored the relation between blood concentrations of monocyte/lymphocyte subsets and carotid artery plaque macrophage content, measured by positron emission tomography (PET) with 11C-PK11195. METHODS AND RESULTS: In 9 patients with carotid plaques we performed 11C-PK11195-PET/computed tomography angiography imaging and measurement of absolute concentrations and frequencies of circulating monocytes and T-cell subsets. Plaque standardized uptake value (SUV) for 11C-PK11195 was negatively correlated with concentrations of total monocytes (r = -0.58, p = 0.05) and CD14++CD16-HLA-DR+ classical subset (r = -0.82, p = 0.005). These correlations hold true also in relation to plaque target to background ratio. No correlation was observed between plaque SUV and CD3+T lymphocytes, CD4+T lymphocytes nor with activated CD3+CD4+T cells expressing HLA-DR. CONCLUSIONS: We first demonstrated a reduction in the absolute concentration of monocytes and particularly in classical monocytes expressing HLA-DR in the presence of an increased uptake of 11C-PK11195 in carotid plaques. The present work, despite being a pilot study comprising only a small number of subjects provides new insights in the search for specific cellular biomarkers with potential diagnostic and prognostic value in patients with a known carotid plaque.

Circulating CD14+ and CD14highCD16- classical monocytes are reduced in patients with signs of plaque neovascularization in the carotid artery.

BACKGROUND AND AIMS: Monocytes are known to play a key role in the initiation and progression of atherosclerosis and contribute to plaque destabilization through the generation of signals that promote inflammation and neoangiogenesis. In humans, studies investigating the features of circulating monocytes in advanced atherosclerotic lesions are lacking. METHODS: Patients (mean age 69 years, 56% males) with intermediate asymptomatic carotid stenosis (40-70% in diameter) were evaluated for maximal stenosis in common carotid artery, carotid bulb and internal carotid artery, overall disease burden as estimated with total plaque area (TPA), greyscale and neovascularization in 244 advanced carotid plaques. Absolute counts of circulating CD14+ monocytes, of classical (CD14highCD16-), intermediate (CD14highCD16+) and non-classical (CD14lowCD16+) monocytes and HLA-DR+ median fluorescence intensity for each subset were evaluated with flow cytometry. RESULTS: No correlation was found between monocytes and overall atherosclerotic burden, nor with high sensitivity C-reactive protein (hsCRP) or interleukin-6 (IL-6). In contrast, plaque signs of neovascularization were associated with significantly lower counts of circulating CD14+ monocytes (297 versus 350 cells/mm3, p = 0.039) and of classical monocytes (255 versus 310 cells/mm3, p = 0.029). CONCLUSIONS: Neovascularized atherosclerotic lesions selectively associate with lower blood levels of CD14+ and CD14highCD16- monocytes independently of systemic inflammatory activity, as indicated by normal hsCRP levels. Whether the reduction of circulating CD14+ and CD14highCD16- monocytes is due to a potential redistribution of these cell types into active lesions remains to be explored.

Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery.

Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34(+) cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

Temporal, quantitative, and functional characteristics of single-KIR-positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation.

In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56(bright)/CD56(dim) NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56(bright), and NKG2A and CD62L up-regulation on CD56(dim), suggest sequential CD56(bright)-to-CD56(dim) NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR(+) NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR(+) NK cells selected from an alloreactive donor.

Frequency and targeted detection of HLA-DPB1 T cell epitope disparities relevant in unrelated hematopoietic stem cell transplantation.

The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by the presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB1 donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions.

Therapeutic and diagnostic applications of minor histocompatibility antigen HA-1 and HA-2 disparities in allogeneic hematopoietic stem cell transplantation: a survey of different populations.

Minor histocompatibility antigens (mHags) HA-1 and HA-2 are encoded by biallelic loci, with immunogenic variants, HA-1H and HA-2V, which induce strong HLA-A2-restricted alloreactive T-cell responses, and nonimmunogenic counterparts, HA-1R and HA-2M, which represent functional null alleles that are poorly presented by HLA class I molecules. HA-1 and HA-2 are potential targets of selective graft-versus-leukemia and graft-versus-tumor reactivity after allogeneic hematopoietic stem cell transplantation (HSCT); however, these applications are restricted to a limited number of patients. Here, we show that a far more frequent application of HA-1 and HA-2 disparity relies on their use as markers for the state of host chimerism after allogeneic HSCT. We have determined allelic frequencies of 29.3% and 70.7% for HA-1H and HA-1R, respectively, and of 83.7% and 16.3% for HA-2V and HA-2M, respectively, in >200 healthy individuals from northern Italy. Similar frequencies were observed in nearly 100 patients affected by hematologic malignancies or solid tumors, thus showing that HA-1 and HA-2 variability are not associated with the presence of cancer. On the basis of these data, we predict that HA-1 and HA-2 can be used in 32.8% and 23.5% of Italian transplant patients, respectively, as markers for the state of host chimerism, whereas exploitation of disparity for these mHags for targeted immunotherapy will be possible in 10.7% and 1.1% of Italian patients, respectively. Retrospective HA-2 typing of bone marrow aspirates obtained from a patient during complete remission or recurrence of acute myeloid leukemia after haploidentical HSCT showed the feasibility of using HA-2 as a surrogate marker for disease monitoring. Because of an apparent north-south gradient for HA-1 allelic frequencies, with higher frequencies for the HA-1H variant reported in white populations from Southern Europe as compared with Northern Europe and North America, the diagnostic applicability of HA-1 disparity will be slightly more frequent in transplant patients from the north. Taken together, our data show that determination of HA-1 and HA-2 variability can be an important parameter for the selection of allogeneic stem cell donors, in particular for patients affected by hematologic malignancies without a tumor-specific molecular marker.

A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation.

The importance of HLA-DPB1 matching for the outcome of allogeneic hematologic stem cell (HSC) transplantation is controversial. We have previously identified HLA-DPB1*0901 as a target of cytotoxic T cells mediating in vivo rejection of an HSC allograft. Here we show that HLA-DPB1*0901 encodes a T-cell epitope shared by a subset of DPB1 alleles that determines nonpermissive mismatches for HSC transplantation. Several T-cell clones obtained from the patient at the time of rejection showed HLA-DP restricted recognition of allogeneic targets expressing HLA-DPB1*0901, *1001, *1701, *0301, *1401, and *4501, but not other alleles. Based on these findings, we developed an algorithm for prediction of nonpermissive HLA-DPB1 mismatches. Retrospective evaluation of 118 transplantations showed that the presence of nonpermissive HLA-DPB1 mismatches was correlated with significantly increased hazards of acute grade II to IV graft-versus-host disease (HR = 1.87, P =.046) and transplantation-related mortality (HR = 2.69, P =.027) but not relapse (HR = 0.98, P =.939), as compared with the permissive group. There was also a marked but statistically not significant increase in the hazards of overall mortality (HR = 1.64, P =.1). These data suggest that biologic characterization of in vivo alloreactivity can be a tool for definition of clinically relevant nonpermissive HLA mismatches for unrelated HSC transplantation.

The neural progenitor-restricted isoform of the MARK4 gene in 19q13.2 is upregulated in human gliomas and overexpressed in a subset of glioblastoma cell lines.

Alterations of 19q13 are frequently observed in glial neoplasms, suggesting that this region harbors at least one gene involved in gliomagenesis. Following our previous studies on structural 19q chromosome rearrangements in gliomas, we have undertaken a detailed FISH analysis of the breakpoints and identified a 19q13.2 intrachromosomal amplification of the MAP/microtubule affinity-regulating kinase 4 (MARK4) gene in three primary glioblastoma cell lines. Recent data suggest that this gene is involved in the Wnt-signaling pathway. We observed that the expression of the alternatively spliced MARK4L isoform is upregulated in both fresh and cultured gliomas and overexpressed in all of the above three glioblastoma cell lines. Interestingly, we also found that MARK4L expression is restricted to undifferentiated neural progenitor cells or proliferating glial precursor cells, whereas its expression is downregulated during glial differentiation. Perturbation of expression using antisense oligonucleotides against MARK4 in glioblastoma cell lines, consistently induced a decreased proliferation of tumor cells. Taken together, these data show that MARK4, which is normally expressed in neural progenitors, is re-expressed in gliomas and may become a key target of intrachromosomal amplification upon 19q rearrangements.