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Gene manipulation and cell vaccines represent innovative strategies to enhance the immunogenicity of cancer cells. We adopted a defective lentivirus derived from the human immunodeficiency virus (HIV)-1 backbone and carrying the enhanced green fluorescent protein (EGFP) gene to transduce primary human acute myelogenous leukemia (AML) and B-precursor acute lymphoblastic leukemia (ALL) cells.AML blasts were maintained with or without cytokines (stem cell factor, FLT3 ligand and interleukin 3) for 48 hours, and successively infected with two spin infection cycles. ALL blasts were cultured on a murine S17 stromal cell line.As regards AML cells, the efficiency of infection at 7 days varied from 8.4 to 37%. As confirmed by cell cycle analysis, cells were, in most of the cases, blocked in different phases of the cycle and did not proliferate during culture: the infection was therefore obtained in the absence of cell proliferation. In contrast, the maintenance of optimal cell viability was of fundamental importance for obtaining good infection levels. As regards ALL blasts, the percentages of infection after 3 days varied from 4.4 to 21%.These preliminary data suggest that gene delivery into primary human AML and B-precursor ALL cells by an HIV-1 derived lentiviral vector could represent a strategy for engineering leukemic cells for use as cancer vaccines.

Recent neuropsychological evidence suggests that acquired brain lesions can, in some instances, abolish the ability to integrate inputs from different sensory modalities, disrupting multisensory perception. We explored the ability to perceive multisensory events, in particular the integrity of audio-visual processing in the temporal domain, in brain-damaged patients with visual field defects (VFD), or with unilateral spatial neglect (USN), by assessing their sensitivity to the 'Sound-Induced Flash Illusion' (SIFI). The study yielded two key findings. Firstly, the 'fission' illusion (namely, seeing multiple flashes when a single flash is paired with multiple sounds) is reduced in both left- and right-brain-damaged patients with VFD, but not in right-brain-damaged patients with left USN. The disruption of the fission illusion is proportional to the extent of the occipital damage. Secondly, a reliable 'fusion' illusion (namely, seeing less flashes when a single sound is paired with multiple flashes) is evoked in USN patients, but neither in VFD patients nor in healthy participants. A control experiment showed that the fusion, but not the fission, illusion is lost in older participants (>50 year-old), as compared with younger healthy participants (

Motor learning interacts with and shapes experience-dependent cerebral plasticity. In stroke patients with paresis of the upper limb, motor recovery was proposed to reflect a process of re-learning the lost/impaired skill, which interacts with rehabilitation. However, to what extent stroke patients with hemiparesis may retain the ability of learning with their affected limb remains an unsolved issue, that was addressed by this study. Nineteen patients, with a cerebrovascular lesion affecting the right or the left hemisphere, underwent an explicit motor learning task (finger tapping task, FTT), which was performed with the paretic hand. Eighteen age-matched healthy participants served as controls. Motor performance was assessed during the learning phase (i.e., online learning), as well as immediately at the end of practice, and after 90 min and 24 h (i.e., retention). Results show that overall, as compared to the control group, stroke patients, regardless of the side (left/right) of the hemispheric lesion, do not show a reliable practice-dependent improvement; consequently, no retention could be detected in the long-term (after 90 min and 24 h). The motor learning impairment was associated with subcortical damage, predominantly affecting the basal ganglia; conversely, it was not associated with age, time elapsed from stroke, severity of upper-limb motor and sensory deficits, and the general neurological condition. This evidence expands our understanding regarding the potential of post-stroke motor recovery through motor practice, suggesting a potential key role of basal ganglia, not only in implicit motor learning as previously pointed out, but also in explicit finger tapping motor tasks.

Unilateral spatial neglect is a neuropsychological syndrome commonly observed after stroke and defined by the inability to attend or respond to contralesional stimuli. Typically, symptoms are assessed using clinical tests that rely upon visual/perceptual abilities. However, neglect may affect high-level representations controlling attention in other modalities as well. Here we developed a novel manual exploration test using a touch screen computer to quantify spatial search behaviour without visual input. Twelve chronic stroke patients with left neglect and 27 patients without neglect (based on clinical tests) completed our task. Four of the 12 "neglect" patients exhibited clear signs of neglect on our task as compared to "non-neglect" patients and healthy controls, and six other patients (from both groups) also demonstrated signs of neglect compared to healthy controls only. While some patients made asymmetrical responses on only one task, generally, patients with the strongest neglect performed poorly on multiple tasks. This suggests that representations associated with different modalities may be affected separately, but that severe forms of neglect are more likely related to damage in a common underlying representation. Our manual exploration task is easy to administer and can be added to standard neglect screenings to better measure symptom severity.

Temporal Binding Window (TBW) represents a reliable index of efficient multisensory integration process, which allows individuals to infer which sensory inputs from different modalities pertain to the same event. TBW alterations have been reported in some neurological and neuropsychiatric disorders and seem to negatively affects cognition and behavior. So far, it is still unknown whether deficits of multisensory integration, as indexed by an abnormal TBW, are present even in Multiple Sclerosis. We addressed this issue by testing 25 participants affected by relapsing–remitting Multiple Sclerosis (RRMS) and 30 age-matched healthy controls. Participants completed a simultaneity judgment task (SJ2) to assess the audio-visual TBW; two unimodal SJ2 versions were used as control tasks. Individuals with RRMS showed an enlarged audio-visual TBW (width range = from − 166 ms to + 198 ms), as compared to healthy controls (width range = − 177/ + 66 ms), thus showing an increased tendency to integrate temporally asynchronous visual and auditory stimuli. Instead, simultaneity perception of unimodal (visual or auditory) events overall did not differ from that of controls. These results provide first evidence of a selective deficit of multisensory integration in individuals affected by RRMS, besides the well-known motor and cognitive impairments. The reduced multisensory temporal acuity is likely caused by a disruption of the neural interplay between different sensory systems caused by multiple sclerosis.

Adaptation to optical prisms displacing the visual scene laterally is a widely investigated instance of visuo-motor plasticity, also because prism adaptation (PA) has been extensively used as a treatment for right-brain-damaged patients suffering from left spatial neglect. The lateral visual displacement brought about by prisms, as indexed by a pointing error in the direction of the displacement, is progressively corrected through repeated pointings: after prism removal, a shift in the direction opposite to the prism-induced deviation occurs in visual, proprioceptive, and visuo-proprioceptive straight-ahead tasks (aftereffects, AEs). The cerebellum and the posterior parietal cortex (PPC) are key components of the bilateral cerebral network subserving the AEs, and the reduction of the pointing error during prism exposure in PA. We report the experimental study of a patient with bilateral occipital and left cerebellar damage, who showed a preserved reduction of the pointing errors to rightward displacing prisms, but not the leftward AEs in the proprioceptive straight-ahead task; instead, visual-proprioceptive and visual AEs were preserved. Anodal transcranial Direct Current Stimulation (tDCS) over the left PPC restored the leftward proprioceptive AEs, and anodal tDCS over the left cerebellum abolished the rightward deviation. Conversely, stimulation over the right PPC or the right cerebellum was ineffective. These results provide novel evidence for neuromodulatory effects of tDCS on defective AEs, through the stimulation over dedicated cortical regions.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Background. Recovery of motor function after stroke may depend on a balance of activity in the neural network involving the affected and the unaffected motor cortices. Objective. To assess whether transcranial direct current stimulation (tDCS) can increase the training-induced recovery of motor functions. Methods. In an exploratory study, 14 patients with chronic stroke and mean Fugl-Meyer Upper Extremity Motor Assessment of 29 (range = 8-50) entered a double-blind sham-controlled study, aimed to investigate neurophysiological and behavioral effects of bihemispheric tDCS (cathodal stimulation of the unaffected motor cortex and anodal stimulation of the affected motor cortex), combined with constraint-induced movement therapy (CIMT). Results. Patients in both groups demonstrated gains on primary outcome measures, that is, Jebsen Taylor Hand Function Test, Handgrip Strength, Motor Activity Log Scale, and Fugl-Meyer Motor Score. Gains were larger in the active tDCS group. Neurophysiological measurements showed a reduction in transcallosal inhibition from the intact to the affected hemisphere and increased corticospinal excitability in the affected hemisphere only in the active tDCS/CIMT group. Such neurophysiological changes correlated with the magnitude of the behavioral gains. Both groups showed a reduction in corticospinal excitability of the unaffected hemisphere. Conclusions. CIMT alone appears effective in modulating local excitability but not in removing the imbalance in transcallosal inhibition. Bihemispheric tDCS may achieve this goal and foster greater functional recovery.

Recent evidence suggests that behavioural gains induced by behavioural training are maximized when combined with techniques of cortical neuromodulation, such as transcranial Direct Current Stimulation (tDCS). Here we address the validity of this appealing approach by investigating the effect of coupling a multisensory visual field exploration training with tDCS of the posterior parietal cortex (PPC). The multisensory visual field exploration training consisted in the practice of visual search through the systematic audio-visual stimulation of the visual field. Neurologically unimpaired participants performed a bimodal exploration training for 30 min, while simultaneously receiving anodal-excitatory PPC tDCS or sham tDCS. In two different experiments, the left and the right hemisphere were stimulated. Outcome measures included visual exploration speed at different time intervals during the training, and the post-training effects on tests assessing visual scanning and visuo-spatial orienting. Results show that PPC tDCS applied to the right, but not to the left, hemisphere increases the training-induced behavioural improvement of visual exploration, as compared to sham tDCS. In addition, right PPC tDCS brings about an improvement of covert visual orienting, in a task different from the visual search practice. In an additional experiment, we confirm that right parietal tDCS by itself, even without the associated training, can lead to enhancement of visual search. Overall, anodal PPC tDCS is a promising technique to enhance visuo-spatial abilities, when combined to a visual field exploration training task. © 2010 Elsevier B.V.

We have investigated the capacity of two human immunodeficiency virus type 1-derived lentivectors, differing in the presence of a 118-bp pol fragment containing the cPPT/CTS element, to transduce human normal primary cells of different hematopoietic lineages. Infection of resting monocytes with a high multiplicity of infection (MOI > 10) revealed that the lentivirus carrying the pol fragment (cPPT) is effective, transducing 75% of cells compared with 36% for the no-cPPT vector. Even at low MOIs (< or =1) the cPPT vector still shows a better transduction efficiency than the no-cPPT vector. Moreover, transduction does not require dendritic cell differentiation. In contrast, infection of nonactivated T lymphocytes showed that both vectors, tested at high MOIs, can transduce a small, although measurable, percentage of cells (up to 10%), which may correspond to G(1a) "activated" cells as detected by simultaneous staining of DNA and RNA, in our cultures in the presence of medium alone. Furthermore, we show that the sole addition of interleukin 2 or interleukin 15 represents a full proliferative signal under our conditions and permits high transduction efficiency (up to 30% with the cPPT vector and 15% with the no-cPPT vector). Still higher transduction of T lymphocytes can be achieved after stimulation with phytohemagglutinin and interleukin 2 (up to 78% with the cPPT vector vs. 42% with the no-cPPT vector). Finally, both viruses do not transduce either resting or proliferating tonsillar B lymphocytes.

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4+ T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8+ T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8+ T cell effectors when used in the inflammatory environment of vaccination. Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1397-7) contains supplementary material, which is available to authorized users.

This study explores whether brain polarization could be effective in modulating multisensory audiovisual interactions in the human brain, as measured by the 'sound-induced flash illusion' (Shams et al., 2000). In different sessions, healthy participants performed the task while receiving anodal, cathodal, or sham tDCS (2 mA, 8 min) to the occipital, temporal, or posterior parietal cortices. We found that up- or down-regulating cortical excitability by tDCS can facilitate or reduce audiovisual illusions, depending on the current polarity, the targeted area, and the illusory percept. Specifically, the perceptual 'fission' of a single flash, due to multiple beeps, was increased after anodal tDCS of the temporal cortex, and decreased after anodal stimulation of the occipital cortex. A reversal of such effects was induced by cathodal tDCS. Conversely, the perceptual 'fusion' of multiple flashes due to a single beep was unaffected by tDCS. This evidence adds novel clues on the cortical substrate of the generation of the sound-flash illusion, and opens new attractive possibilities for modulating multisensory perception in humans: tDCS appears to be an effective tool to modulate the conscious visual experience associated with multisensory interactions, by noninvasively shifting cortical excitability within occipital or temporal areas. (C) 2010 Elsevier Ltd. All rights reserved.

INTRODUCTION Human congenital hypotransferrinemia is a rare disorder characterized by the virtual absence of transferrin in the serum. No information on the causes of the disease is known. MATERIALS AND METHODS Here we describe the identification of a new case, its treatment and the biochemical and genetic defects underlying the disorder. RESULTS At diagnosis the patient had serum Tf levels equal to about 1% of the normal values. The treatment with plasma infusions each month allowed a good erythropoiesis and the prevention of iron overload with no need of red blood cell transfusions or iron chelators. In order to define the genetic basis of the disease, we performed a haplotype analysis of the Tf gene region in the 26 individuals forming the proband's family, and demonstrated that the genetic defect is located in the Tf gene and that it is inherited as a recessive trait. Protein analyses indicate that the proband serum contains two transferrin forms: one of 80 kD analogous to the normal one, and a smaller one of 50 kD, which may arise from a specific degradation or be the gene product of a modified allele. CONCLUSION These data suggest the presence of two Tf alleles carrying genetic defects that cause two distinct abnormalities. One allele causes low expression of an apparently normal protein that probably allowed the survival of the patient in the first years of age. The other allele produces a modified Tf with different biochemical characteristics compared to the normal one.