Publications by authors named "Giulia Scalia"

23 Publications

  • Page 1 of 1

Ocrelizumab depletes T-lymphocytes more than rituximab in multiple sclerosis.

Mult Scler Relat Disord 2021 Apr 28;49:102802. Epub 2021 Jan 28.

Multiple Sclerosis Clinical Care and Research Centre, Department of Neuroscience, Reproductive Sciences and Odontostomatology, University of Naples Federico II, Naples, Italy. Electronic address:

Background: We aim to directly compare changes in lymphocyte subpopulations between chimeric (rituximab) and humanised (ocrelizumab) anti-CD20 antibodies in multiple sclerosis (MS).

Methods: In this retrospective analysis of prospectively collected data, we included 88 patients with MS, treated with rituximab (n=50) or ocrelizumab (n=38). We used flow cytometry in the peripheral blood to count total lymphocytes and lymphocytes expressing different phenotypic markers (CD4, CD8, CD19, CD20, CD4/CD8 ratio), before treatment and after 1, 3 and 6 months.

Results: On linear mixed effect regression models, after 1, 3 and 6 months, patients treated with rituximab and with ocrelizumab were similar in total lymphocyte count, CD19 lymphocytes, CD20 lymphocytes and CD4/CD8 ratio. However, patients treated with ocrelizumab presented with lower CD4 T lymphocytes and CD8 T lymphocytes after 1, 3 and 6 months (all p<0.05). No between-treatment difference in EDSS progression was found.

Discussion: B-cell levels in the peripheral blood were equally decreased by rituximab and ocrelizumab. On the contrary, CD4 and CD8 T lymphocyte reduction was more pronounced in ocrelizumab, when compared with rituximab, suggesting a broader immunomodulatory effect for the humanised antibody to be confirmed and correlated with clinical efficacy in the long term.
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http://dx.doi.org/10.1016/j.msard.2021.102802DOI Listing
April 2021

Immunocytometric analysis of COVID patients: A contribution to personalized therapy?

Life Sci 2020 Nov 29;261:118355. Epub 2020 Aug 29.

Dipartimento di Malattie Infettive e Emergenze Infettive, Divisione di Malattie Infettive Respiratorie, Ospedale Cotugno, AORN dei Colli, Naples, Italy.

Aims: This study aims to cast light on immunocytometric alterations in COVID-19, a potentially fatal viral infection with heterogeneous clinical expression and a not completely defined pathophysiology.

Methods: We studied 35 COVID patients at hospital admission testing by cytofluorimetry a large panel of lymphocyte subpopulations and serum tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-17A and the soluble receptor of IL-17A (IL-17RA).

Key Findings: At hospital admission, total lymphocytes and most T and B subpopulations were reduced in 50-80% of patients, with close relationship to disease severity. While activated T helper 1 (TH1) and TH17 cells resulted normal or higher. Serum IL-6 was increased in all patients, while TNF-α and IL-17A were higher in advanced stages. A patient subset with low severity had very high IL-17RA levels. Tocilizumab treatment caused an increase of IL-17A in 3/6 patients and a reduction in 3 others, while the lymphocyte number increased in 3 patients and did not change in the others.

Significance: Cytofluorimetry revealed a functional exhaustion of most lymphocyte populations in COVID patients not involving activated TH1 and TH17. Consequently, there was a relevant cytokines production that contributes to impair the respiratory inflammation. The increase of TH17 and IL-17 in a subset of cases and the evidence of a significant increase of IL-17RA (that prevents the interaction of IL-17 with the cell receptor) in patients with low severity suggest that some patients could benefit from monoclonal antibodies treatment targeting IL-17 pathway. Immunocytofluorimetric markers may contribute to a personalized therapy in COVID patients.
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http://dx.doi.org/10.1016/j.lfs.2020.118355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456265PMC
November 2020

Clinical Phenotype, Immunological Abnormalities, and Genomic Findings in Patients with DiGeorge Spectrum Phenotype without 22q11.2 Deletion.

J Allergy Clin Immunol Pract 2020 10 12;8(9):3112-3120. Epub 2020 Jul 12.

Department of Translational Medical Sciences, Pediatrics Section, Federico II University of Naples, Naples, Italy. Electronic address:

Background: The phenotype of early embryonic fourth branchial arch defects encompasses a wide spectrum of clinical conditions including DiGeorge syndrome (DGS), velocardiofacial syndrome, and conotruncal anomaly face syndrome. The majority of the patients have a 22q11.2 deletion. However, in 6% to 17% of patients, the identification of a genetic cause remains unknown through fluorescence in situ hybridization. In these patients, the clinical features and the immunological abnormalities are not well defined.

Objective: To describe the main genomic abnormalities, clinical features, and immunological abnormalities of a cohort of patients resembling the 22q11.2 deletion phenotype in the absence of 22q11.2 locus alterations.

Methods: Eleven patients from unrelated nonconsanguineous families with suspected 22q11.2 deletion syndrome (22q11.2DS) according to Tobias criteria were enrolled. Array-comparative genomic hybridization was performed in 10 patients. A phenotypic and immunological assessment was performed in all patients.

Results: The majority of patients had a phenotype overlapping with 22q11.2DS and immunological abnormalities suggestive of abnormalities in T-cell development, being severe in 2 of them. Most subjects suffered from recurrent infections. Clinically overt autoimmune manifestations were identified in 2 (18%) subjects. New pathogenic or likely pathogenic genomic regions associated with 22q11.2DS features were identified.

Conclusion: Patients with a DGS-like phenotype share the same features of the classical 22q11.2DS associated with other rare genomic alterations. Severe forms of immunodeficiency may also be observed in this group.
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http://dx.doi.org/10.1016/j.jaip.2020.06.051DOI Listing
October 2020

In Ataxia-Telangiectasia, Oral Betamethasone Administration Ameliorates Lymphocytes Functionality through Modulation of the IL-7/IL-7Rα Axis Paralleling the Neurological Behavior: A Comparative Report of Two Cases.

Immunol Invest 2021 Feb 13;50(2-3):295-303. Epub 2020 May 13.

Department of Translational Medical Sciences-Section of Pediatrics, Federico II University , Naples, Italy.

Ataxia-Telangiectasia (A-T) is characterized by cerebellar neurodegeneration and immunodeficiency. Recent studies suggest that very low glucocorticoids (GCs) doses may help improve A-T neurological phenotype in some patients. Interestingly, in GCs studies an unexpected improvement of lymphocytes proliferation in some A-T patients has been observed. GCs are able to upregulate IL-7 Rα expression and rescue it from the recycling. In this study, we compared several immunological functions, including PBMC proliferative responses, cell activation events and IL-7/IL-7 Rα axis functionality, with the neurological behavior during an GCs treatment between the most Responder patient to GC and the Non-Responder at all. During GC treatment, we observed an increase of lymphocyte proliferation upon stimulation with PHA or IL-7 only in the Responder. This finding paralleled the increase in the surface expression of IL-7 R and up-regulation of the CD69 T-cell activation marker. Internalization and recycling of IL-7 R occurred properly only in the Responder. Microarray analysis revealed a remarkable difference in the DE-genes levels among Responder and Non-Responder, mostly concerning miRNAs and Multiple Complex families. Our findings suggest that the improvement of lymphocyte functionality, which correlates to the neurological behavior, is mediated through an effect of GCs on the IL-7/IL-7 Rα axis.
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http://dx.doi.org/10.1080/08820139.2020.1761379DOI Listing
February 2021

Clinical, Immunological, and Functional Characterization of Six Patients with Very High IgM Levels.

J Clin Med 2020 Mar 17;9(3). Epub 2020 Mar 17.

Department of Translational Medical Sciences-Section of Pediatrics, Federico II University, 80131 Naples, Italy.

Very high IgM levels represent the hallmark of hyper IgM (HIGM) syndromes, a group of primary immunodeficiencies (PIDs) characterized by susceptibility to infections and malignancies. Other PIDs not fulfilling the diagnostic criteria for HIGM syndromes can also be characterized by high IgM levels and susceptibility to malignancies. The aim of this study is to characterize clinical phenotype, immune impairment, and pathogenic mechanism in six patients with very high IgM levels in whom classical HIGM syndromes were ruled out. The immunological analysis included extended B-cell immunophenotyping, evaluation of class switch recombination and somatic hypermutation, and next generation sequencing (NGS). Recurrent or severe infections and chronic lung changes at the diagnosis were reported in five out of six and two out of six patients, respectively. Five out of six patients showed signs of lymphoproliferation and four patients developed malignancies. Four patients showed impaired B-cell homeostasis. Class switch recombination was functional in vivo in all patients. NGS revealed, in one case, a pathogenic mutation in . In a second case, the gene, implicated in B- and T-cell development, survival, and activity was identified as a potential candidate gene. Independent of the genetic basis, very high IgM levels represent a risk factor for the development of recurrent infections leading to chronic lung changes, lymphoproliferation, and high risk of malignancies.
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http://dx.doi.org/10.3390/jcm9030818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141334PMC
March 2020

Imbalance Between Interleukin-1β and Interleukin-1 Receptor Antagonist in Epicardial Adipose Tissue Is Associated With Non ST-Segment Elevation Acute Coronary Syndrome.

Front Physiol 2020 5;11:42. Epub 2020 Feb 5.

Department of Translational Medical Science, University of Naples Federico II, Naples, Italy.

Introduction: Interleukin-1beta (IL-1β) is crucially involved in the pathogenesis of coronary atherosclerotic diseases (CAD) and its inhibition has proven cardiovascular benefits. Epicardial adipose tissue (EAT) is a local source of inflammatory mediators which may negatively affect the surrounding coronary arteries. In the present study, we explored the relationship between serum and EAT levels of IL-1β and IL-1 receptor antagonist (IL-1ra) in patients with chronic coronary syndrome (CCS) and recent acute coronary syndrome (ACS).

Methods: We obtained EAT biopsies in 54 CCS (Group 1) and 33 ACS (Group 2) patients undergoing coronary artery bypass grafting. Serum and EAT levels of IL-1β and IL-1ra were measured in all patients. An immunophenotypic study was carried out on EAT biopsies and the CD86 events were studied as markers of M1 macrophages.

Results: Circulating levels of IL-1β were significantly higher in the overall CAD population compared to a control group [7.64 pg/ml (6.86; 8.57) vs. 1.89 pg/ml (1.81; 2.29); < 0.001]. In contrast, no differences were observed for serum IL-1ra levels between CAD and controls. Comparable levels of serum IL-1β were found between Groups 1 and 2 [7.6 pg/ml (6.9; 8.7) vs. 7.9 pg/ml (7.2; 8.6); = 0.618]. In contrast, significantly lower levels of serum IL-1ra were found in Group 2 compared to Group 1 [274 pg/ml (220; 577) vs. 603 pg/ml (334; 1022); = 0.035]. No differences of EAT levels of IL-1β were found between Group 2 and Group 1 [3.4 pg/ml (2.3; 8.4) vs. 2.4 pg/ml (1.9; 8.0); = 0.176]. In contrast, significantly lower EAT levels of IL-1ra were found in Group 2 compared to Group 1 [101 pg/ml (40; 577) vs. 1344 pg/ml (155; 5327); = 0.002]. No correlation was found between EAT levels of IL-1β and CD86 and CD64 events.

Conclusion: The present study explores the levels of IL-1β and IL-1ra in the serum and in EAT of CCS and ACS patients. ACS seems to be associated to a loss of the counter-regulatory activity of IL-1ra against the pro-inflammatory effects related to IL-1β activation.
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http://dx.doi.org/10.3389/fphys.2020.00042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012938PMC
February 2020

Incidence and Predictive Risk Factors of Infective Events in Patients With Multiple Sclerosis Treated With Agents Targeting CD20 and CD52 Surface Antigens.

Open Forum Infect Dis 2019 Nov 21;6(11):ofz445. Epub 2019 Oct 21.

Department of Clinical Medicine and Surgery, Section of Infectious Diseases, University of Naples Federico II, Naples, Italy.

Objective: Monoclonal antibodies (MAbs) directed against the CD20 and CD52 antigens are used increasingly in patients with multiple sclerosis (MS). Several life-threatening opportunistic infections have been reported in postmarketing case series. The aim of this study was to investigate the incidence of infections and associated prognostic factors during the first year of treatment in patients receiving anti-CD20 (ocrelizumab or rituximab) or anti-CD52 MAbs (alemtuzumab).

Methods: A retrospective study was conducted in patients with MS referring to the Neurodegenerative Diseases Center at the University of Naples Federico II who received MAbs between November 2015 and June 2018.

Results: A total of 163 patients were enrolled. Approximately 40% of patients experienced lymphocytopenia during treatment. Eighty-six infective events were reported in 67 patients (41%). Bacterial infections were significantly more frequent with anti-CD20, whereas viral infections prevailed with alemtuzumab. Cytomegalovirus reactivation rates were significantly higher in the alemtuzumab group than in patients on anti-CD20 (51% vs 6%, < .001). The overall annualized infection rate was 1.1 per patient-year, higher in patients on anti-CD52 versus those on anti-CD20 regimens (1.5 vs 0.8 per patient-year). Alemtuzumab treatment, prior exposure to ≥2 MS drugs, and iatrogenic immune impairment significantly and independently predicted an infection event (adjusted hazard ratio [aHR], 2.7; = .013; aHR, 1.7; = .052; and aHR, 2.9; = .004; respectively).

Conclusions: Given their considerable infection risk, MS patients receiving MAbs should undergo timely follow up and tailored preventive interventions. Anti-CD52-based treatment, prior exposure to MS drugs, and on-treatment immune impairment are significant predictive factors of infection and their evaluation could help clinicians to stratify a patient's risk of infection.
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http://dx.doi.org/10.1093/ofid/ofz445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837838PMC
November 2019

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis.

Am J Hum Genet 2019 09 22;105(3):549-561. Epub 2019 Aug 22.

Division of Allergy and Immunology, Department of Pediatrics, University of Utah, Salt Lake City, UT 84112, USA.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4 but lower than normal CD8 cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1 (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.
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http://dx.doi.org/10.1016/j.ajhg.2019.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731368PMC
September 2019

Ibrutinib as salvage therapy in mantle cell lymphoma with central nervous system involvement in a pretreated unfit patient.

Leuk Lymphoma 2018 07 11;59(7):1734-1737. Epub 2017 Oct 11.

a Department of Clinical Medicine and Surgery , Federico II University Medical School , Naples , Italy.

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http://dx.doi.org/10.1080/10428194.2017.1387910DOI Listing
July 2018

CD4/CD8 ratio during natalizumab treatment in multiple sclerosis patients.

J Neuroimmunol 2017 08 12;309:47-50. Epub 2017 May 12.

Department of Neurosciences, Reproductive Science and Odontostomatology, Federico II University, Naples, Italy. Electronic address:

Although improved, the risk of progressive multifocal leukoencephalopathy is a constant threat for patient affected by multiple sclerosis treated with natalizumab. We performed a 24months longitudinal study aimed to evaluate the total WBC and lymphocytes subsets modifications and their correlations with anti-JCV antibody index after 1 and 2years of natalizumab treatment. Natalizumab induced an increase of WBC, total and C19+ lymphocytes together with a decrease of CD3+, CD4+ T lymphocytes and CD4/CD8 ratio, which was positively related to anti-JCV antibodies index at month 0, 12 and 24. Our study confirms that lymphocytes subsets are modified under NAT therapy. Implications of lymphocyte subsets alterations in the pathogenesis of PML are under analyses.
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http://dx.doi.org/10.1016/j.jneuroim.2017.05.006DOI Listing
August 2017

DiGeorge-like syndrome in a child with a 3p12.3 deletion involving MIR4273 gene born to a mother with gestational diabetes mellitus.

Am J Med Genet A 2017 Jul 24;173(7):1913-1918. Epub 2017 Apr 24.

Department of Translational Medical Sciences, Pediatric section, Federico II University, Naples, Italy.

Chromosome 22q11.2 deletion is the most common chromosomal alteration associated with DiGeorge syndrome (DGS), even though this is not the only underlying cause of DGS. In rare patients, mutations in a single gene, TBX1, have been described resulting in a DGS phenotype. Recently, it has been reported that at least part of the TBX1 mutant phenotype is due to excessive bone morphogenetic proteins (BMP) signaling. Evidence suggests that miRNA may modulate the expression of critical T-box transcriptional regulators during midface development and Bmp-signaling. We report on a 7-year-old Caucasian male born to a mother affected with gestational diabetes (GDM) who had a 371Kb-interstitial deletion of 3p12.3 identified by array CGH, involving the ZNF717, MIR1243, and 4273 genes. The child presented with a DiGeorge anomaly (DGA) associated with unilateral renal agenesis and language delay. The immunological evaluation revealed a severe reduction and impairment of T lymphocytes. FISH analysis and TBX1 sequencing were negative. Among the miRNA-4273 predicted target genes, we found BMP3, which is involved in several steps of embryogenesis including kidney and lung organogenesis and in insulin gene expression. Since, DGA is not commonly found in newborns of diabetic mothers, we hypothesize that the pathogenesis of DGA associated with GDM is multifactorial, involving both genetic and/or epigenetic cofactors.
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http://dx.doi.org/10.1002/ajmg.a.38242DOI Listing
July 2017

Lipopolysaccharide-Elicited TSLPR Expression Enriches a Functionally Discrete Subset of Human CD14 CD1c Monocytes.

J Immunol 2017 05 24;198(9):3426-3435. Epub 2017 Mar 24.

Department of Translational Medical Sciences, School of Medicine, University of Naples Federico II, 80131 Naples, Italy;

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express and mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14 monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14 CD16 monocytes, TSLPR monocytes (TSLPR mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., , , , ). Strikingly, TSLPR mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14 CD1c cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of and mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14 CD16 monocytes and prompt further ontogenetic and functional analysis of CD14 CD1c and LPS-activated CD14 CD1c TSLPR mono.
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http://dx.doi.org/10.4049/jimmunol.1601497DOI Listing
May 2017

Enhancement of 5-FU sensitivity by the proapoptotic rpL3 gene in p53 null colon cancer cells through combined polymer nanoparticles.

Oncotarget 2016 Nov;7(48):79670-79687

Laboratory of Biochemistry, Department of Pharmacy, University of Napoli Federico II, 80131 Napoli, Italy.

Colon cancer is one of the leading causes of cancer-related death worldwide and the therapy with 5-fluorouracil (5-FU) is mainly limited due to resistance. Recently, we have demonstrated that nucleolar stress upon 5-FU treatment leads to the activation of ribosome-free rpL3 (L3) as proapoptotic factor. In this study, we analyzed L3 expression profile in colon cancer tissues and demonstrated that L3 mRNA amount decreased with malignant progression and the intensity of its expression was inversely related to tumor grade and Bcl-2/Bax ratio. With the aim to develop a combined therapy of 5-FU plus plasmid encoding L3 (pL3), we firstly assessed the potentiation of the cytotoxic effect of 5-FU on colon cancer cells by L3. Next, 10 μM 5-FU and 2 μg of pL3 were encapsulated in biocompatible nanoparticles (NPs) chemically conjugated with HA to achieve active tumor-targeting ability in CD44 overexpressing cancer cells. We showed the specific intracellular accumulation of NPs in cells and a sustained release for 5-FU and L3. Analysis of cytotoxicity and apoptotic induction potential of combined NPs clearly showed that the 5-FU plus L3 were more effective in inducing apoptosis than 5-FU or L3 alone. Furthermore, we show that the cancer-specific chemosensitizer effect of combined NPs may be dependent on L3 ability to affect 5-FU efflux by controlling P-gp (P-glycoprotein) expression. These results led us to propose a novel combined therapy with the use of 5-FU plus L3 in order to establish individualized therapy by examining L3 profiles in tumors to yield a better clinical outcomes.
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http://dx.doi.org/10.18632/oncotarget.13216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346744PMC
November 2016

Reciprocal interplay between thyroid hormone and microRNA-21 regulates hedgehog pathway-driven skin tumorigenesis.

J Clin Invest 2016 06 9;126(6):2308-20. Epub 2016 May 9.

The thyroid hormone-inactivating (TH-inactivating) enzyme type 3 iodothyronine deiodinase (D3) is an oncofetal protein that is rarely expressed in adult life but has been shown to be reactivated in the context of proliferation and neoplasms. D3 terminates TH action within the tumor microenvironment, thereby enhancing cancer cell proliferation. However, the pathological role of D3 and the contribution of TH metabolism in cancer have yet to be fully explored. Here, we describe a reciprocal regulation between TH action and the cancer-associated microRNA-21 (miR21) in basal cell carcinoma (BCC) skin tumors. We found that, besides being negatively regulated by TH at the transcriptional level, miR21 attenuates the TH signal by increasing D3 levels. The ability of miR21 to positively regulate D3 was mediated by the tumor suppressor gene GRHL3, a hitherto unrecognized D3 transcriptional inhibitor. Finally, in a BCC mouse model, keratinocyte-specific D3 depletion markedly reduced tumor growth. Together, our results establish TH action as a critical hub of multiple oncogenic pathways and provide functional and mechanistic evidence of the involvement of TH metabolism in BCC tumorigenesis. Moreover, our results identify a miR21/GRHL3/D3 axis that reduces TH in the tumor microenvironment and has potential to be targeted as a therapeutic approach to BCC.
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http://dx.doi.org/10.1172/JCI84465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887175PMC
June 2016

Molecular evidence for a thymus-independent partial T cell development in a FOXN1-/- athymic human fetus.

PLoS One 2013 9;8(12):e81786. Epub 2013 Dec 9.

Department of Translational Medical Sciences, Pediatric Section, "Federico II" University, Naples, Italy.

The thymus is the primary organ able to support T cell ontogeny, abrogated in FOXN1(-/-) human athymia. Although evidence indicates that in animal models T lymphocytes may differentiate at extrathymic sites, whether this process is really thymus-independent has still to be clarified. In an athymic FOXN1(-/-) fetus, in which we previously described a total blockage of CD4(+) and partial blockage of CD8(+) cell development, we investigated whether intestine could play a role as extrathymic site of T-lymphopoiesis in humans. We document the presence of few extrathymically developed T lymphocytes and the presence in the intestine of CD3(+) and CD8(+), but not of CD4(+) cells, a few of them exhibiting a CD45RA(+) naïve phenotype. The expression of CD3εεpTα, RAG1 and RAG2 transcripts in the intestine and TCR gene rearrangement was also documented, thus indicating that in humans the partial T cell ontogeny occurring at extrathymic sites is a thymus- and FOXN1-independent process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081786PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3857207PMC
September 2014

Human skin-derived keratinocytes and fibroblasts co-cultured on 3D poly ε-caprolactone scaffold support in vitro HSC differentiation into T-lineage committed cells.

Int Immunol 2013 Dec 13;25(12):703-14. Epub 2013 Sep 13.

Department of Translational Medical Sciences, 'Federico II' University, Naples 80131, Italy.

In humans, the thymus is the primary lymphoid organ able to support the development of T cells through its three-dimensional (3D) organization of the thymic stromal cells. Since a remarkable number of similarities are shared between the thymic epithelial cells (TECs) and skin-derived keratinocytes and fibroblasts, in this study we used human keratinocytes seeded with fibroblasts on the 3D poly ε-caprolactone scaffold to evaluate their ability to replace TECs in supporting T-cell differentiation from human haematopoietic stem cells (HSCs). We observed that in the multicellular biocomposite, early thymocytes expressing CD7(+)CD1a(+), peculiar markers of an initial T-cell commitment, were de novo generated. Molecular studies of genes selectively expressed during T-cell development revealed that TAL1 was down-regulated and Spi-B was up-regulated in the cell suspension, consistently with a T-cell lineage commitment. Moreover, PTCRA and RAG2 expression was detected, indicative of a recombinant activity, required for the generation of a T-cell receptor repertoire. Our results indicate that in the multicellular biocomposite, containing skin-derived elements in the absence of thymic stroma, HSCs do start differentiating toward a T-cell lineage commitment. In conclusion, the construct described in this study exerts some properties of a lymphoid organoid, suitable for future clinical applications in cell-based therapies.
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http://dx.doi.org/10.1093/intimm/dxt035DOI Listing
December 2013

Gliadin does not induce mucosal inflammation or basophil activation in patients with nonceliac gluten sensitivity.

Clin Gastroenterol Hepatol 2013 Oct 30;11(10):1294-1299.e1. Epub 2013 Apr 30.

Gastrointestinal Unit, Department of Medicine and Surgery, University of Salerno, Salerno, Italy.

Background & Aims: Nonceliac gluten-sensitive (NCGS) patients report intestinal and extra-intestinal symptoms shortly after ingesting gluten; these symptoms disappear on gluten-free diets, although these patients have no serologic markers of celiac disease or intestinal damage. In fact, there is no evidence for mucosal or serologic modifications in those individuals. We investigated immunologic responses of duodenal mucosa samples and peripheral blood basophils, isolated from NCGS patients, after exposure to gliadin.

Methods: Participants underwent a complete clinical evaluation to exclude celiac disease while on a gluten-containing diet, a skin prick test to exclude wheat allergy, and upper endoscopy (n = 119) at 2 tertiary medical centers in Italy. Patients were considered to have NCGS based on their symptoms and the current definition of the disorder. Subjects were assigned to the following groups: patients with celiac disease on gluten-free diets (n = 34), untreated patients with celiac disease (n = 35), patients with NCGS (n = 16), or controls (n = 34). Duodenal biopsy samples collected during endoscopy were incubated with gliadin peptides, and levels of inflammatory markers were assessed. Peripheral blood basophils were extracted and incubated with gliadin peptides or a mix of wheat proteins; activation was assessed based on levels of CD203c, CD63, and CD45.

Results: Duodenal mucosa samples collected from 69 patients with celiac disease showed markers of inflammation after incubation with gliadin. Some, but not all, markers of inflammation were detected weakly in biopsy samples from 3 controls and 3 NCGS patients (P = .00 for all markers). There were no significant increases in the levels of CD63 and CD203c in NCGS patients.

Conclusions: Unlike the duodenal mucosa from patients with celiac disease, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin. The in vitro gliadin challenge therefore should not be used to diagnose NCGS.
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http://dx.doi.org/10.1016/j.cgh.2013.04.022DOI Listing
October 2013

ImageStream promyelocytic leukemia protein immunolocalization: in search of promyelocytic leukemia cells.

Cytometry A 2012 Mar 20;81(3):232-7. Epub 2012 Jan 20.

CEINGE-Biotecnologie Avanzate, Naples, Italy.

Acute promyelocytic leukemia (APL) is a hematological emergency in which a rapid diagnosis is essential for early administration of appropriate therapy, including all-trans retinoic acid before the onset of fatal coagulopathy. Currently, the following methodologies are widely used for rapid initial diagnosis of APL: 1) identification of hypergranular leukemic promyelocytes by using classical morphology; 2) identification of cells with diffuse promyelocytic leukemia (PML) protein distribution by immunofluorescence microscopy; 3) evidence of aberrant promyelocyte surface immunophenotype by conventional flow cytometry (FCM). Here, we show a method for immunofluorescent detection of PML localization using ImageStream FCM. This technique provides objective per-cell quantitative image analysis for statistically large sample sizes, enabling precise and operator-independent PML pattern recognition even in electronic and real dilution experiments up to 10% of APL cellular presence. Therefore, we evidence that this method could be helpful for rapid and objective initial diagnosis and the prompt initiation of APL treatment.
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http://dx.doi.org/10.1002/cyto.a.22013DOI Listing
March 2012

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies.

BMC Physiol 2008 May 29;8:13. Epub 2008 May 29.

Servizio di Immunoematologia e Medicina Trasfusionale, Ospedale A. Cardarelli, Napoli, Italy.

Background: Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients.

Results: In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion: Our study, comparing surface antigen expression of ALDH+/CD34+, ALDH-/CD34+ and ALDH+/CD34- progenitor cell subsets in human bone marrow, clearly indicated that ALDH+CD34- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.
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http://dx.doi.org/10.1186/1472-6793-8-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426712PMC
May 2008

Complete remission induced by G-CSF in a patient with acute myeloid leukemia with t(8;21)(q22;q22).

Hematol J 2003 ;4(3):218-21

Division of Hematology, Cardarelli General Hospital, Via Niccoló Piccinni 6, Naples 80128, Italy.

We describe a case of acute myeloid leukemia (AML) with t(8;21) in which complete remission (CR) was obtained with G-CSF given at 10 microg/kg in the absence of concomitant cytotoxic chemotherapy. CR was achieved following 2 weeks of therapy and confirmed by investigating minimal residual disease by four-color flow cytometry analysis. During treatment with G-CSF, maturing cells with cytoplasmic Auer Rods were observed in the peripheral blood, suggesting a differentiation effect. This case adds further evidence for a specific role of G-CSF in the treatment of AML with t(8;21), namely in patients who are not eligible for aggressive chemotherapy.
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http://dx.doi.org/10.1038/sj.thj.6200225DOI Listing
August 2003

Secretory phospholipases A2 activate selective functions in human eosinophils.

J Immunol 2003 Mar;170(6):3279-88

Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy.

Secretory phospholipases A(2) (sPLA(2)s) are released in large amounts in the blood of patients with systemic inflammatory diseases and accumulate at sites of chronic inflammation, such as the airways of patients with bronchial asthma. Blood eosinophils or eosinophils recruited in inflammatory areas therefore can be exposed in vivo to high concentrations of sPLA(2). We have examined the effects of two structurally different sPLA(2)s (group IA and group IIA) on several functions of eosinophils isolated from normal donors and patients with hypereosinophilia. Both group IA and IIA sPLA(2) induced a concentration-dependent release of beta-glucuronidase, IL-6, and IL-8. Release of the two cytokines was associated with the accumulation of their specific mRNA. In addition, sPLA(2)s induced the surface expression of CD44 and CD69, two major activation markers of eosinophils. In contrast, none of the sPLA(2)s examined induced the production of IL-5, the de novo synthesis of leukotriene C(4) and platelet-activating factor, or the generation of superoxide anion from human eosinophils. Incubation of eosinophils with the major enzymatic products of the sPLA(2)s (arachidonic acid, lysophosphatidylcholine, or lysophosphatidic acid) did not reproduce any of the enzymes' effects. In addition, inactivation of sPLA(2) enzymatic activity by bromophenacyl bromide did not influence the release of beta-glucuronidase or of cytokines. Stimulation of eosinophils by sPLA(2)s was associated with activation of extracellular signal-regulated kinases 1/2. These results indicate that sPLA(2)s selectively activate certain proinflammatory and immunoregulatory functions of human eosinophils through mechanism(s) independent from enzymatic activity and from the generation of arachidonic acid.
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http://dx.doi.org/10.4049/jimmunol.170.6.3279DOI Listing
March 2003