CD99 Expression and Prognostic Impact in Glioblastoma: A Single-Center Cohort Study.

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan-Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

Targeting SARS-CoV-2 by synthetic dual-acting thiol compounds that inhibit Spike/ACE2 interaction and viral protein production.

The SARS-CoV-2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin-converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS-CoV-2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell-free system as demonstrated by enzyme-linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS-CoV-2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti-SARS-CoV-2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.

A checklist for assessing the methodological quality of concurrent tES-fMRI studies (ContES checklist): a consensus study and statement.

Low-intensity transcranial electrical stimulation (tES), including alternating or direct current stimulation, applies weak electrical stimulation to modulate the activity of brain circuits. Integration of tES with concurrent functional MRI (fMRI) allows for the mapping of neural activity during neuromodulation, supporting causal studies of both brain function and tES effects. Methodological aspects of tES-fMRI studies underpin the results, and reporting them in appropriate detail is required for reproducibility and interpretability. Despite the growing number of published reports, there are no consensus-based checklists for disclosing methodological details of concurrent tES-fMRI studies. The objective of this work was to develop a consensus-based checklist of reporting standards for concurrent tES-fMRI studies to support methodological rigor, transparency and reproducibility (ContES checklist). A two-phase Delphi consensus process was conducted by a steering committee (SC) of 13 members and 49 expert panelists through the International Network of the tES-fMRI Consortium. The process began with a circulation of a preliminary checklist of essential items and additional recommendations, developed by the SC on the basis of a systematic review of 57 concurrent tES-fMRI studies. Contributors were then invited to suggest revisions or additions to the initial checklist. After the revision phase, contributors rated the importance of the 17 essential items and 42 additional recommendations in the final checklist. The state of methodological transparency within the 57 reviewed concurrent tES-fMRI studies was then assessed by using the checklist. Experts refined the checklist through the revision and rating phases, leading to a checklist with three categories of essential items and additional recommendations: (i) technological factors, (ii) safety and noise tests and (iii) methodological factors. The level of reporting of checklist items varied among the 57 concurrent tES-fMRI papers, ranging from 24% to 76%. On average, 53% of checklist items were reported in a given article. In conclusion, use of the ContES checklist is expected to enhance the methodological reporting quality of future concurrent tES-fMRI studies and increase methodological transparency and reproducibility.

Characterization of FLT3-ITDmut acute myeloid leukemia: molecular profiling of leukemic precursor cells.

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/-,CD25-,CD99low/-) (p = 0.0005) and mononuclear cells (MNCs) (p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

Stairways to the brain: Transcutaneous spinal direct current stimulation (tsDCS) modulates a cerebellar-cortical network enhancing verb recovery.

It has long been assumed that the language function is hierarchically organized into specific cortical areas. Here, for the first time, we present direct evidence that the spinal cord takes part in language processing. In a randomized-double blind design, sixteen aphasics underwent a language treatment combined with transcutaneous spinal direct current stimulation (tsDCS). During the treatment, each subject received tsDCS (20 min, 2 mA) over the thoracic vertebrae (IX-X vertebrae) in two different conditions: (1) anodal, and (2) sham while performing a verb naming task. Each experimental condition was run in five consecutive daily sessions over two weeks. Before and after each condition, all patients underwent a resting state functional magnetic resonance imaging (rs-fMRI). After anodal tsDCS, significant functional connectivity changes were found in a cerebellar-cortical network recruiting regions such as the left cerebellum, the right parietal and premotor cortex known to be also involved in action-related verb processing. Indeed, this increase of connectivity significantly correlated with the greatest amount of improvement found in verb naming. In line with our experimental data, we also found a greater improvement after anodal tsDCS also on untreated items of the language test but only on tasks which required the use of verbs, such as verb naming and picture description. No significant changes were found in noun naming. Thus, this evidence emphasizes, for the first time, that the neural response due to tsDCS combined with language treatment changes during the course of recovery by enhancing activity into cortical regions which influence verb processing.

High-Definition Transcranial Direct Current Stimulation Improves Verb Recovery in Aphasic Patients Depending on Current Intensity.

High-definition transcranial direct current stimulation (HD-tDCS) is a variant of tDCS, which produces more focal stimulation, delimiting brain current flow to a defined region compared to conventional tDCS. To date, only one study has been conducted to investigate HD-tDCS effects on language recovery in aphasia. Here, we aimed to assess the effects of cathodal HD-tDCS on verb naming by comparing two current intensities: 1 vs 2 mA. In a double-blinded cross over study, two groups of 10 aphasic individuals were submitted to active cathodal HD-tDCS and sham stimulation over the right homolog of Broca's area, while performing a verb naming task. Indeed, we reasoned that, by applying inhibitory current over the right Broca's area, we would decrease the inhibitory impact from the right hemisphere to the left perilesional cortex, thus boosting language recovery. The groups differed in the intensity of the active stimulation (1 mA or 2 mA). In both groups, each condition was carried out in five consecutive daily sessions with one week of interval between the two experimental conditions. A significant improvement in verb naming was found only after cathodal HD-tDCS at 2 mA, which endured one week after the end of treatment. The improvement was not observed on the group receiving cathodal HD-tDCS at 1 mA. Our findings showed that HD-tDCS applied to the right intact hemisphere are efficacious for language recovery. These results indicate that HD-tDCS represents a promising new technique for language rehabilitation. However, systematic determination of stimulation intensity appears to be crucial for obtaining relevant effects.

Transcranial direct current stimulation (tDCS) facilitates verb learning by altering effective connectivity in the healthy brain.

Recent studies have shown that the left inferior frontal gyrus (IFG) plays a key role in language learning. Facilitatory stimulation over this region by means of anodal transcranial direct current stimulation (tDCS) can modulate linguistic abilities in healthy individuals and improve language performance in patients with post-stroke aphasia. Neuroimaging studies in healthy participants have suggested that anodal tDCS decreases task-related activity at the stimulated site when applied during different language tasks, and changes resting-state connectivity in a larger network of areas associated with language processing. However, to date, the neural correlates of the potential beneficial effects of tDCS on verb learning remain unclear. The current study investigated how anodal tDCS during verb learning modulates task-related activity and effective connectivity in the healthy language network. To this end, we combined a verb learning paradigm during functional neuroimaging with simultaneous tDCS over the left IFG in healthy human volunteers. We found that, relative to sham stimulation, anodal tDCS significantly decreased task-related activity at the stimulated left IFG and in the right homologue. Effective connectivity analysis showed that anodal tDCS significantly decreased task-related functional coupling between the left IFG and the right insula. Importantly, the individual decrease in connectivity was significantly correlated with the individual behavioural improvement during anodal tDCS. These results demonstrate, for the first time, that the behavioural improvements induced by anodal tDCS might be related to an overall decrease in processing effort both with respect to task-related activity and effective connectivity within a large language network.

Transcranial Cerebellar Direct Current Stimulation Enhances Verb Generation but Not Verb Naming in Poststroke Aphasia.

Although the role of the cerebellum in motor function is well recognized, its involvement in the lexical domain remains to be further elucidated. Indeed, it has not yet been clarified whether the cerebellum is a language structure per se or whether it contributes to language processing when other cognitive components (e.g., cognitive effort, working memory) are required by the language task. Neuromodulation studies on healthy participants have suggested that cerebellar transcranial direct current stimulation (tDCS) is a valuable tool to modulate cognitive functions. However, so far, only a single case study has investigated whether cerebellar stimulation enhances language recovery in aphasic individuals. In a randomized, crossover, double-blind design, we explored the effect of cerebellar tDCS coupled with language treatment for verb improvement in 12 aphasic individuals. Each participant received cerebellar tDCS (20 min, 2 mA) in four experimental conditions: (1) right cathodal and (2) sham stimulation during a verb generation task and (3) right cathodal and (4) sham stimulation during a verb naming task. Each experimental condition was run in five consecutive daily sessions over 4 weeks. At the end of treatment, a significant improvement was found after cathodal stimulation only in the verb generation task. No significant differences were present for verb naming among the two conditions. We hypothesize that cerebellar tDCS is a viable tool for recovery from aphasia but only when the language task, such as verb generation, also demands the activation of nonlinguistic strategies.

Moving Beyond the Brain: Transcutaneous Spinal Direct Current Stimulation in Post-Stroke Aphasia.

Over the last 20 years, major advances in cognitive neuroscience have clearly shown that the language function is not restricted into the classical language areas but it involves brain regions, which had never previously considered. Indeed, recent lines of evidence have suggested that the processing of words associated to motor schemata, such as action verbs, modulates the activity of the sensorimotor cortex, which, in turn, facilitates its retrieval. To date, no studies have investigated whether the spinal cord, which is functionally connected to the sensorimotor system, might also work as an auxiliary support for language processing. We explored the combined effect of transcutaneous spinal direct current stimulation (tsDCS) and language treatment in a randomized double-blind design for the recovery of verbs and nouns in 14 chronic aphasics. During each treatment, each subject received tsDCS (20 min, 2 mA) over the thoracic vertebrae (10th vertebra) in three different conditions: (1) anodic, (2) cathodic and (3) sham, while performing a verb and noun naming tasks. Each experimental condition was run in five consecutive daily sessions over 3 weeks. Overall, a significant greater improvement in verb naming was found during the anodic condition with respect to the other two conditions, which persisted at 1 week after the end of the treatment. No significant differences were present for noun naming among the three conditions. The hypothesis is advanced that anodic tsDCS might have influenced activity along the ascending somatosensory pathways, ultimately eliciting neurophysiological changes into the sensorimotor areas which, in turn, supported the retrieval of verbs. These results further support the evidence that action words, due to their sensorimotor semantic properties, are partly represented into the sensorimotor cortex. Moreover, they also document, for the first time, that tsDCS enhances verb recovery in chronic aphasia and it may represent a promising new tool for language treatment.

Differential effects of bihemispheric and unihemispheric transcranial direct current stimulation in young and elderly adults in verbal learning.

For the past few years, the potential of transcranial direct current stimulation (tDCS) for the treatment of several pathologies has been investigated. In the language domain, several studies, in healthy and brain-damaged populations, have already shown that tDCS is effective in enhancing naming, repetition and semantic word generation. In those studies, different tDCS electrode configurations have been tested, however, a direct comparison between different montages in verbal learning has never been conducted. In this study, we aimed to explore the impact of bihemispheric and unihemispheric tDCS on verbal learning task performance in two groups (young vs. elderly). Fifteen healthy volunteers participated per group. Each participant received three stimulation conditions: unihemispheric anodal tDCS over the left temporal area, bihemispheric tDCS over the left (anodal) and right (cathodal) temporal areas and a sham condition. During active stimulation, tDCS (20min, 2mA) was applied while each participant learned twenty pseudowords (arbitrarily assigned to corresponding pictures). No significant differences were found between the three conditions for the young group with regard to accuracy and vocal reaction times. In contrast, in the elderly group, real stimulation improved performance compared to sham but bihemispheric tDCS was more efficient than unilateral stimulation. These results suggest that bihemispheric stimulation is more effective in improving language learning but this effect is age-dependent. The hypothesis is advanced that cortical changes in the course of aging might differentially impact on tDCS efficacy on behavioral performance. These data may also have implications for treatment of stroke patients with language impairment.

Right sensory-motor functional networks subserve action observation therapy in aphasia.

Recent studies have shown that the systematic and repetitive observation of actions belonging to the experiential human motor repertoire without verbal facilitation enhances the recovery of verbs in non fluent aphasia. However, it is still an open question whether this approach extends its efficacy also on discourse productivity by improving the retrieval of other linguistic units (i.e. nouns, sentences, content words). Moreover, nothing is known regarding the neural substrates which support the language recovery process due to action observation treatment.In the present study, ten non fluent aphasics were presented with two videoclips (real everyday life context vs. familiar pantomimed context), each video for six consecutive weeks (Monday to Friday, weekend off). During the treatment, they were asked to observe each video and to describe it without verbal facilitation from the therapist. In all patients, language measures were collected before and at the end of treatment. Before and after each treatment condition (real vs. pantomimed context), each subject underwent a resting state fMRI. After the treatment, significant changes in functional connectivity were found in right sensory-motor networks which were accompanied by a significant improvement for the different linguistic units in the real context condition. On the contrary, the language recovery obtained in the pantomimed context did not match any functional modification. The evidence for a recruitment of the sensory-motor cortices during the observation of actions embedded in real context suggests to potentially enhance language recovery in non fluent aphasia through a simulation process related to the sensory-motor properties of actions.

Bilateral Transcranial Direct Current Stimulation Language Treatment Enhances Functional Connectivity in the Left Hemisphere: Preliminary Data from Aphasia.

Several studies have already shown that transcranial direct current stimulation (tDCS) is a useful tool for enhancing recovery in aphasia. However, no reports to date have investigated functional connectivity changes on cortical activity because of tDCS language treatment. Here, nine aphasic persons with articulatory disorders underwent an intensive language therapy in two different conditions: bilateral anodic stimulation over the left Broca's area and cathodic contralesional stimulation over the right homologue of Broca's area and a sham condition. The language treatment lasted 3 weeks (Monday to Friday, 15 sessions). In all patients, language measures were collected before (T0) and at the end of treatment (T15). Before and after each treatment condition (real vs. sham), each participant underwent a resting-state fMRI study. Results showed that, after real stimulation, patients exhibited the greatest recovery not only in terms of better accuracy in articulating the treated stimuli but also for untreated items on different tasks of the language test. Moreover, although after the sham condition connectivity changes were confined to the right brain hemisphere, real stimulation yielded to stronger functional connectivity increase in the left hemisphere. In conclusion, our data provide converging evidence from behavioral and functional imaging data that bilateral tDCS determines functional connectivity changes within the lesioned hemisphere, enhancing the language recovery process in stroke patients.

Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing's sarcoma.

Ewing's sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and "up-scalable" process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells.

The human antibody fragment DIATHIS1 specific for CEACAM1 enhances natural killer cell cytotoxicity against melanoma cell lines in vitro.

Several lines of evidence show that de novo expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is strongly associated with reduced disease-free survival of patients affected by metastatic melanoma. Previously published investigations report that homophilic interactions between CEACAM1 expressed on natural killer (NK) cells and tumors inhibit the NK cell-mediated killing independently of major histocompatibility complex class I recognition. This biological property can be physiologically relevant in metastatic melanoma because of the increased CEACAM1 expression observed on NK cells from some patients. Moreover, this inhibitory mechanism in many cases might hinder the efficacy of immunotherapeutic treatments of CEACAM1 malignancies because of tumor evasion by activated effector cells. In the present study, we designed an in vitro experimental model showing that the human single-chain variable fragment (scFv) DIATHIS1 specific for CEACAM1 is able to enhance the lytic machinery of NK cells against CEACAM1 melanoma cells. The coincubation of the scFv DIATHIS1 with CEACAM1 melanoma cells and NK-92 cell line significantly increases the cell-mediated cytotoxicity. Moreover, pretreatment of melanoma cells with scFv DIATHIS1 promotes the activation and the degranulation capacity of in vitro-expanded NK cells from healthy donors. It is interesting to note that the melanoma cell line MelC and the primary melanoma cells STA that respond better to DIATHIS1 treatment, express higher relative levels of CEACAM1-3L and CEACAM1-3S splice variants isoforms compared with Mel501 cells that are less responsive to DIATHIS1-induced NK cell-mediated cytotoxicity. Taken together, our results suggest that the fully human antibody fragment DIATHIS1 originated by biopanning approach from a phage antibody library may represent a relevant biotechnological platform to design and develop completely human antimelanoma therapeutics of biological origin.

CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin.

PURPOSE: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. EXPERIMENTAL DESIGN: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. RESULTS: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. CONCLUSIONS: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death.

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems.

Bihemispheric tDCS enhances language recovery but does not alter BDNF levels in chronic aphasic patients.

PURPOSE: Several studies have shown that transcranial direct current stimulation (tDCS) is a useful tool to enhance language recovery in aphasia. It has also been suggested that modulation of the neurotrophin brain-derived neurotrophic factor (BDNF) might be part of the mechanisms involved in tDCS effects on synaptic connectivity. However, all language studies have previously investigated the effects using unihemispheric stimulation. The purpose of the present study is to investigate the role of bihemispheric tDCS on language recovery and BDNF serum levels. METHODS: Seven aphasic persons underwent an intensive language therapy in two different conditions: real bihemispheric stimulation over the left and right Broca's areas and a sham condition. RESULTS: After the stimulation, patients exibited a significant recovery in three language tasks (picture description, noun and verb naming) compared to the sham condition which persisted in the follow-up session. No significant differences were found in BDNF serum levels after tDCS stimulation and in the follow-up session. However, a significant positive correlation was present for the real stimulation condition between percent changes in BDNF levels and in the verb naming task. CONCLUSIONS: The data suggest that this novel approach may potentiate the recovery of language in chronic aphasia. They also emphasize the importance to further investigate the role of possible biomarkers associated with tDCS treatment response in language recovery.