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Obejctives Serosurveys can be used to monitor COVID-19 seroprevalence and conduct surveillance. Dried blood spot (DBS), used increasingly as a valuable sample to assay severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies (Ab), has several advantages, particularly in infants, due to the limited amount of blood required and its utility in testing a large number of samples in a limited time-frame. We evaluated SARS-CoV-2 IgG Ab prevalence in newborn DBS in the Trentino region of Italy, during the time period January 2020 – December 2021. Methods Anti-SARS-CoV-2 IgG levels were determined in DBS by means of Anti-SARS-CoV-2 QuantiVac IgG ELISA assay (Euroimmun, Lubeck, Germany). Results Analyses included 2,400 DBS from newborns (54% M, 46% F), samples being collected 2–3 days after birth. The first DBS that tested positive for anti-SARS-CoV-2 IgG antibodies was found in March 2020 and, up to May 2020, only 4 positive results were detected overall. Starting from June 2020, the positivity thresholds increased according to the epidemiological waves of the COVID-19 pandemic in Italy, with a robust increment in the winters of 2020 and 2021. The percentage of positive DBS rose from 0 to 6% to 10–47%, in 2020 and 2021, respectively. Conclusions This study demonstrates DBS is a suitable tool for both epidemiological purposes and surveillance in the SARS-CoV-2 pandemic, particularly in newborns and pregnant women, saving blood waste and sparing patients any discomfort.

Objectives The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10, and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release. Methods Whole blood (Li–He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li–He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China). Results Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and the time between the last vaccination or positivity. Conclusions Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific T cells, especially when rapid analyses are required, such as for organ transplantation.

Performance of two disposable devices for identifying subjects with low anti-SARS-CoV-2 protection was compared with that of automated enzyme-linked immunosorbent (ELISA) and chemiluminescent (CLIA) assay.In July 2021, 123 healthcare workers (HCW), twice vaccinated by BNT162b2/Comirnaty mRNA (BioNTech-Pfizer), underwent Ab iRapid COVID-19 Quant "Neutralizing" Self-test (iRapid Self-test) and "Neutralizing" Professional-use (iRapid pro) (DIESSE, Diagnostica Senese, Siena, Italy). Simultaneously, serum Ab were determined by Maglumi 2000 plus (anti S-RBD CLIA assay, Snibe Diagnostics, Shenzhen, China) and SARS-CoV-2 "Neutralizing" Ab Chorus ELISA (DIESSE, Siena, Italy). Results were evaluated against two "protective-thresholds", 90 kBAU/L and 506 kBAU/L.HCW mean age, 46.2 (±12.6) years; 26 (20.5%), males, 101 (79.5%), females. The mean time interval (and standard deviation) between the first vaccine dose and Ab determination was 129.5 (±36.4) days and was neither gender (p = 0.879) nor age (p = 0.341) related. With Maglumi, 114 (89.7%) and 43 (33.8%) HCW presented Ab ≥ 90 kBAU/L and Ab ≥ 506 kBAU/L, respectively; with Chorus, 96 (75.6%) presented Ab values ≥506 kBAU/L. CLIA and ELISA agreement was 56.7%. At 90 kBAU/L, iRapid self-test and Pro sensitivities were 98.2% (95% CI: 92.7-99.8), specificity 69.2% (95% CI: 38.6-90.9%) and 76.9% (46.2-95%), respectively. At 506 kBAU/L, iRapid sensitivities were 58.1-91.6%, and specificities, 89-96.6%. On evaluating Ab at4 and ≥4 months, protective titers had decreased.iRapid semi-quantitative devices had very good overall agreements of 95.1% and 95.9% for detecting individuals with low anti-SARS-CoV-2 protection.

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca

Calcium ion (Ca

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.

The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD(+)-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

Abstract 1457 Acute lymphoblastic leukemia (ALL) is the most frequent form of childhood cancer. Although therapy efforts have achieved cure rates of above 80%, about every fifth patient encounters relapse, which is associated with poor outcome. Risk stratification based on prognostic factors is critical for selecting appropriate treatment intensity to improve therapy efficacy and reduce toxicity. However, in spite of new strategies for risk stratification, the majority of relapsed patients are initially stratified into non-high risk groups pointing out the need to identify new prognostic markers that might refer to novel therapeutic targets. We have recently shown in a NOD/SCID/huALL mouse xenotranplant model that early relapse of pediatric B-cell precursor (BCP-) ALL patients is reflected by rapid engraftment (time to leukemia/ TTLshort) of primary ALL cells. In this study we aimed to identify differently expressed and activated proteins in xenograft ALL of distinct engraftment subgroups employing a Reverse Phase Protein Array (RPPA) strategy. Levels of total or activated proteins were analyzed in patient derived xenograft BCP-ALL samples characterized with respect to their NOD/SCID engraftment phenotype (TTLshort n= 7, TTLlong n= 9). The proteome of each sample was immobilized on nitrocellulose coated glass slides and overall protein expression or phosphorylation of 51 key signaling molecules was detected by incubation with the corresponding specific antibodies. Protein expression and/ or activation was quantified and compared between the different engraftment subgroups (TTLshort/ early relapse versus TTLlong/ good prognosis). RPPA results were validated by Western Blot analyses. The association of NOD/SCID/huALL engraftment and clinical patient outcome was analyzed and revealed a significantly inferior survival of patients with the TTLshort in contrast to the TTLlong phenotype also in this subgroup of patients (Kaplan Meier, log rank, P=.003). Upon comparison of the protein expression data (Shrinkage t-test, P CYCLIN B is a positive regulator of the cell cycle and highly expressed in the G2/M phase. Consistent with its pro-proliferative function, CYCLIN B was identified to be up-regulated in leukemia samples with rapid NOD/SCID/huALL growth (TTLshort/ early relapse phenotype) (P=.013). Interestingly, the level of CYCLIN B protein expression correlates negatively to TTL (Spearman, rs=-.516, P=.041), suggesting a direct association of leukemia cell cycle progression and leukemia engraftment in the NOD/SCID/huALL xenograft model. Beta-CATENIN, a molecule involved in WNT-signaling and cell adhesion but also associated with apoptosis inhibition and cell growth in other hematological malignancies, was found to be over-expressed in TTLshort (P=.006). Furthermore, beta-CATENIN protein levels were reversely correlated to TTL (Spearman, rs=-.557, P=.025). Additionally, ANNEXIN I, involved in the regulation of inflammation and apoptosis and reported to be up-regulated in hairy cell leukemia, showed high protein levels in TTLshort (P=.033). Protein-kinase C alpha was previously reported to induce apoptosis in an ALL- cell line and showed increased activation in non-relapsing pediatric T-ALL patient samples. In line with this, we found higher levels of PKC alpha activation in the TTLlong -group (P= 8.5e–9), indicating an association with good prognosis. Taken together, this study identified differentially expressed proteins in prognostic subgroups of BCP-ALL patients with distinct clinical outcomes, which can be further evaluated as new prognostic markers and therapeutic targets. Disclosures: Debatin: Apogenix GmbH: Patents & Royalties, Research Funding.

Abstract 744 Background: T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL pediatric patients remain at high risk for early relapse. It is therefore very important to identify new prognostic biomarkers and to develop new therapeutic approaches for these patients. More specific and less toxic therapies might be developed through the identification of molecular targets, such as aberrantly activated phosphoproteins, that offer the possibility to use specific kinase inhibitors. In this research we first explored the phosphoproteomic profile of pediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), and we further investigated the origin and the functional significance of Protein Kinase C alpha (PKCα) downregulation in patients that are liable to relapse. Methods: We analyzed 98 pediatric T-ALL patients at diagnosis using RPPA. This innovative approach allows to profile phosphorylation modifications and to characterize the activation state of cellular protein networks. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with one of 53 different antibodies included in the study. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features through statistical analyses. Gene expression analysis was performed by means of HG-U133 Plus 2.0 array. Permission for this study was obtained following the tenets of the Declaration of Helsinki. A set of commercially available human T-ALL cell lines was studied by Western Blot and cells were treated with Ro-32-0432, a commercial PKC inhibitor. Results: The phosphoproteomic profile of T-ALL patients was compared between patients subgroups at diagnosis. Comparison between relapsed vs non relapsed patients samples revealed the downregulation of PKCα(S657) in relapsed patients (t test with BH multiplicity corrections p=0.02) and Relapse Free Survival analysis through Kaplan-Meier estimates showed that patients with low PKCα(S657) have a high probability to relapse. Notably, 54.5% of patients with low PKCα(S657) relapsed whereas no relapses were observed among patients with high PKCα activity. Multivariate analysis showed no correlation between PKCα(S657) levels and other parameters currently used for clinical characterization of T-ALL patients. Differences in PKCα(S657) between relapsed and non relapsed patients were confirmed by Western Blot in two independent sets of T-ALL patients. Analysis of PKCα total protein form revealed concordance between total protein expression and PKCα(S657) levels. Furthermore, gene expression analysis revealed a significant difference in PKCα gene expression levels, comparable with levels of protein expression among T-ALL patients studied by RPPA. Gene expression data were validated by Sybr Green Real-Time Quantitative PCR. Moreover, gene pathway analysis in a larger cohort of T-ALL pediatric patients, divided by high and low PKCα mRNA levels, revealed the phosphatidylinositol signalling pathway as the most important deregulated pathway. Of note, this pathway is involved in the production of DAG and Ca2+, the PKCα cofactors that play an essential role in mediating its translocation from the cytosol to the plasmatic membrane and subsequent activation. PKCα and PKCα(S657) were then analysed by Western Blot in five T leukemic cell lines (DND41, P12 ICHIKAWA, MOLT3, TALL1 and CEM). These cell lines were treated with a PKCα inhibitor and PKCα inhibition effects were studied by the means of MTT assay and Trypan Blue cell count. An increase in cell proliferation was observed in each treated cell line 9 hours after PKCα inhibition. Conclusions: PKCα is differentially expressed in T-ALL pediatric patients. Low levels of PKCα(S657) are associated with a higher probability to relapse and downregulation or absence of PCKα seems to be related to a more aggressive T-ALL phenotype. Ongoing studies are needed to better define its role as a new prognostic marker. Disclosures: No relevant conflicts of interest to declare.

Osteoblast suppression is the hallmark of Multiple Myeloma (MM) osteolytic bone lesions mainly due to the capacity of MM cells to inhibit the osteogenic differentiation of bone marrow (BM) mesenchymal cells (MSC). Many evidences suggest that Wnt signaling is critically involved in the regulation of osteoblast formation. Recently, in murine osteoprogenitor cells and in MM mouse models it has been shown that activation of canonical Wnt pathway stimulate osteoblast formation and blunts MM-induced bone destruction. In this study we have investigated whether modulation of both canonical and noncanonical Wnt signaling pathway may affect osteogenic differentiation of human MSC and counterbalance the suppressive effect of MM cells. First we checked the potential expression of Wnt activators and inhibitors by human MSC and osteoprogenitor cells (PreOB) by gene arrays. We found that both cells expressed the activator of non-canonical Wnt pathways Wnt5a but lack of express the main activators of canonical Wnt signaling as Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptor FZD2 and FZD5 was also detected in both cells as well as that of FZD3, FZD6 and FDZ7 and the Wnt canonical co-receptors LRP5 and LRP6. On the other hand we found that both inhibitors of canonical and non-canonical Wnt pathways DKK-1 and sFRP-1 were expressed by MSC. Secondly, activation of either canonical or non-canonical Wnt signaling pathway by Wnt3a and Wnt5a treatment respectively was performed in human MSC to evaluate the effect on osteogenic differentiation and the expression of osteoblast related markers (Collagen I, Osteocalcin and Alkaline Phosphatase). We found that Wnt5a treatment but not Wnt3a significantly increased the early osteogenic differentiation and the expression of alkaline phosphatase in MSC. Consistently in a co-culture system with MM cells Wnt5a treatment blunted, at least in part, the inhibitory effect of MM cells on alkaline phosphatase expression by MSC and PreOB. To go further inside, we evaluated in both primary human BM MSC and the human MSC cell line HS-5 the effect of either the activation of non-canonical Wnt signaling by Wnt5a overexpression using a lentivirus vector or the Wnt5a suppression using siRNA. Wnt5a over-expression in MSC induced the activation of Wnt/Ca++ non-canonical pathway as demonstrated by the increase of Wnt5a secretion and phospho-PKC expression detected by westernblot analysis. Consequently to non-canonical Wnt signal activation we found a significant increase of alkaline phosphatase expression by MSC cells as well as of their osteogenic differentiation. Interestingly, analyzing the gene expression profile by microarray, we found that Wnt5a overexpression in MSC also affects the expression of chemokines, inflammatory cytokines and pro-angiogenic molecules. In conclusion our data indicate that activation of non-canonical Wnt signal pathway may represent a potential target in MM microenvironment to counterbalance the inhibitory effect of MM cells on osteogenic differentiation of human MSC.

Hypoxia is a common feature of solid tumors associated to angiogenesis and malignant phenotype. Tumor adaptation to hypoxia is mainly due to the hypoxia-inducible factor (HIF)-1α, a key transcription factor that regulates angiogenesis and tumor progression. As known multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasmacells into a hypoxic microenvironment as the bone marrow (BM) that critically supports their growth and survival. However the effect of hypoxia on MM cells and the role of HIF-1α in MM-induced angiogenesis actually are not known. Recently we have demonstrated that the tumor suppressor gene ING4 may exert an anti-angiogenic effect through the inhibition of HIF-1α activity in MM cells in hypoxic condition suggesting a role of HIF-1α in MM-induced angiogenic switch. To go further insight this issue, in this study, first we checked the level of BM oxygen tension in a cohort of MM patients (n°=25) at the diagnosis as compared to healthy donors and MGUS subjects. The mean pO2 ± SD was 52.3±9 mmHg (p=NS) in MM patients similar to that observed in the controls, confirming that MM cells are exposed in vivo to hypoxic microenvironment. Thereafter HIF-1α protein expression by MM cells was checked by immunohistochemistry on bone biopsies showing the presence of HIF-1α stabilization at nuclear level in malignant plasmacells as well as in BM stromal cells (BMSC) into the BM. Consequently the effect of hypoxia and HIF-1α in both MM and BMSC cells was checked. Human myeloma cell lines (JJN3 and RPMI-8226) and BMSC were transfected with a pool of siRNA anti-HIF-1α to knockout HIF-1α and then exposed to low oxygen tension. A gene expression profiling evaluation was performed by microarray analysis using Gene Chips U133plus 2.0 (Affymetrix). Data were then validated by real time PCR. We found that hypoxia significantly upregulated the expression of the pro-angiogenic molecules in both MM and BMSC cells including Vascular Endothelial Growth Factor (VEGF), Osteopontin (OPN) and Interleukin-8 (IL-8) blunted by siRNA anti-HIF-1α. Genes belonging to glycolysis and HIF-1α regulating signal pathways were found to be also regulated by HIF-1α in MM cells in hypoxic condition. These observations were confirmed in purified CD138+ MM cells (n°=11) exposed to hypoxia that induced a significant up-regulation of the pro-angiogenic molecules and the modulation of glycolysis and ubiquitin mediated proteolysis signal pathways. Finally, the potential expression and role of HIF-1α in MM cells was also investigated in normoxic condition. Whereas the presence of HIF-1α mRNA was observed in all HMCLs and primary MM cells tested, HIF-1a protein stabilization and activity was observed at nuclear level in 2 out of 6 HMCLs and in about 38% of MM patients evaluated suggesting that a hypoxia independent stabilization of HIF-1α may occur in MM cells. Consistently, in normoxic condition, HIF-1α knock out by siRNA significantly affected in HMCLs either pro-angiogenic molecules as VEGF or several genes belonging to cell cycle regulation. In conclusion our data underline the role of hypoxia in the regulation of the angiogenic signature of MM cells and the BM microenvironment and suggest that HIF-1α could be a potential target in MM.