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Positive Regulatory Domain (PRDM) gene family members commonly express two main molecular variants, the PR-plus isoform usually acting as tumor suppressor and the PR-minus one functioning as oncogene. Accordingly, PRDM2/RIZ encodes for RIZ1 (PR-plus) and RIZ2 (PR-minus). In human cancers, genetic or epigenetic modifications induce RIZ1 silencing with an expression level imbalance in favor of RIZ2 that could be relevant for tumorigenesis. Additionally, in estrogen target cells and tissues, estradiol increases RIZ2 expression level with concurrent increase of cell proliferation and survival. Several attempts to study RIZ2 function in HeLa or MCF-7 cells by its over-expression were unsuccessful. Thus, we over-expressed RIZ2 in HEK-293 cells, which are both RIZ1 and RIZ2 positive but unresponsive to estrogens. The forced RIZ2 expression increased cell viability and growth, prompted the G2-to-M phase transition and organoids formation. Accordingly, microarray analysis revealed that RIZ2 regulates several genes involved in mitosis. Consistently, RIZ silencing in both estrogen-responsive MCF-7 and –unresponsive MDA-MB-231 cells induced a reduction of cell proliferation and an increase of apoptosis rate. Our findings add novel insights on the putative RIZ2 tumor-promoting functions, although additional attempts are warranted to depict the underlying molecular mechanism.

Il testo Immunobiologia di Janeway è stato pensato per gli studenti dei corsi universitari, ma è così completo da essere adeguato anche come testo di riferimento per specializzandi e per gli stessi medici che praticano l’immunologia. Pur addentrandosi nel mondo della microbiologia, è chiaramente focalizzato sullo studio dell’immunologia e dei suoi principali eventi immunopatologici, quali le reazioni autoimmunitarie, le immunodeficienze, i trapianti e l’immunologia dei tumori. Questa nona edizione è stata completamente riorganizzata ed aggiornata e contiene più di cento immagini nuove. Nell’Appendice I, il Toolbox degli immunologi ha subito una totale rivisitazione, con l’aggiunta di nuove tecniche, tra cui il sistema CRISPR/Cas9 e tecniche di spettrometria di massa e di proteomica. Inoltre è stato creato un elenco di domande che serviranno agli studenti per riflettere attentamente e sintetizzare le conoscenze apprese in ogni capitolo e per preparare al meglio gli esami.

Background: Homozygous mutations in acid-labile subunit (IGFALS) gene result in short stature, very low circulating levels of acid-labile subunit (ALS), insulin growth factor 1 (IGF1) and insulin growth factor binding protein 3 (IGFBP3) and a poor response to growth hormone (GH). The impact of IGFALS mutations heterozygosity on growth is unknown. Patient and Methods: We describe a 10-year-old girl with severe short stature (height -3.2 SDS), heterozygous for a new IGFALS mutation. Results: The girl showed low circulating IGF1, IGFBP3 and ALS levels and normal GH secretion. We found a novel heterozygous frameshift IGFALS mutation (c.1283delA, p.Gln428Argfs*14). Size-exclusion chromatography showed a reduction of the IGF1, IGFBP3 and ALS 150-kDa ternary complex (by about 55%) compared to a control. An IGF-1 generation test, with two different GH dosages, showed a good response in term of increase in IGF1 and in formation of the ternary complex at size-exclusion chromatography. Clinical response after 6 months of therapy with GH was satisfactory (height velocity increased from 3 to 8 cm/year). Conclusion: We suggest that (1) heterozygous IGFALS mutations can be responsible for a subset of patients with severe short stature (below -2.5 SDS), low IGF1 (below -2 SDS) and normal GH secretion, and (2) the identification by IGFALS molecular screening of this subset of patients could help in the administration of the appropriate therapy. Background: Homozygous mutations in acid-labile subunit (IGFALS) gene result in short stature, very low circulating levels of acid-labile subunit (ALS), insulin growth factor 1 (IGF1) and insulin growth factor binding protein 3 (IGFBP3) and a poor response to growth hormone (GH). The impact of IGFALS mutations heterozygosity on growth is unknown. Patient and Methods: We describe a 10-year-old girl with severe short stature (height -3.2 SDS), heterozygous for a new IGFALS mutation. Results: The girl showed low circulating IGF1, IGFBP3 and ALS levels and normal GH secretion. We found a novel heterozygous frameshift IGFALS mutation (c.1283delA, p.Gln428Argfs*14). Size-exclusion chromatography showed a reduction of the IGF1, IGFBP3 and ALS 150-kDa ternary complex (by about 55%) compared to a control. An IGF-1 generation test, with two different GH dosages, showed a good response in term of increase in IGF1 and in formation of the ternary complex at size-exclusion chromatography. Clinical response after 6 months of therapy with GH was satisfactory (height velocity increased from 3 to 8 cm/year). Conclusion: We suggest that (1) heterozygous IGFALS mutations can be responsible for a subset of patients with severe short stature (below -2.5 SDS), low IGF1 (below -2 SDS) and normal GH secretion, and (2) the identification by IGFALS molecular screening of this subset of patients could help in the administration of the appropriate therapy. © 2013 S. Karger AG, Basel.

NTP1 Background: The dynamic intra- and inter-chromosomal links between specific loci contribute to the creation of cell type-specific gene expression profiles and to gene regulation during differentiation processes. Looping is implicated in bringing together far upstream or downstream regions with the gene promoter and body sites, and in establishing contacts between the 5' and 3' ends of genes, since 3' end-processing factors interact with components of the transcriptional machinery. The tumor suppressor PRDM2/RIZ gene plays a role in controlling cellular processes, such as cell cycle progression and regulation of development. The retinoblastoma proteinineracting zinc-finger gene (RIZ) is estrogen responsive and has two alternative promoters, the more downstream of which, promoter 2, is nearby to an EREsequence and is involved in estrogen receptor transcriptional activation. Methods: With the innovative DNA-Picked Chromatin (DPC) assay after timecourse of 17-ßestradiol (E2) induction of MCF-7 breast cancer cells, we highlight preferential interaction between hormone-responsive RIZ promoter and the polyadenylation sites. Gene expression analysis of induced cell RNA was performed with qRT-PCR assay. Results: Within 60’ of E2 treatment of cells, we have observed increased exon segments, exons 9a and 10 (alternative polyA site), linked to isolated promoter 2 and concomitant decrease of exon10 to RIZ promoter 1. The exon 9a shows a low association to RIZ promoter 1 without E2. qRT-PCR also demonstrated increased exon 9a-containing transcripts. Conclusions: The E2 remodels the chromatin architecture of PRDM2/RIZ gene locus to create a loop for the mRNA transcription with polyA-exon 9a, leading to the production of oncogenic variants.

Introduzione- Gli effetti dei glucocorticoidi sull’attività del sistema immunitario e linfocitario sono stati oggetto di studio da più di qualche decennio e sono largamente utilizzati nel trattamento di diverse patologie, anche in cui i processi infiammatori sono causati dall’attività monocitica/linfocitaria con meccanismi di tipo autoimmunitario cellulo-mediati e che causano il danno tessutale permanente (es.tiroidite, ecc.) (1, 4, 5). Seppure appare contraddittorio l’effetto sul sistema immunitario dei corticosteroidi a dosaggi fisiologici (3) rispetto a quelli farmacologici (immunosoppressivi), è noto che i deficit prolungati dell’asse Ipotalamo/ipofisario/corticosurrenalico oltre a produrre effetti endocrino-metabolici possono promuovere le patologie autoimmunitarie e allergiche (1, 4, 5). Dati più recenti indicano che gli effetti degli agonisti dei recettori per i corticosteroidi possono promuovere il differenziamento linfocitario verso il Th2 e T regolatorio e indurre la produzione di citochine immunosoppressive e/o modulatrici, quali IL10, IL4, IL1ra, oltre all'incremento dei fattori cellulari regolatori della pathway di NFkB, come IkBalfa e la deubiquitinasi A20 (3). Tale azione è avvalorata dalla azione degli antagonisti, i quali aumentano la comparsa di patologie autoimmunitarie. La capacità dei recettori specifici di rispondere agli ormoni steroidei, come quella dei recettori nucleari ai propri ligandi (es 1,25-Diidrossicolecalciferolo, acido all-trans-retinoico, PGJ2), potrebbe essere un importante elemento da considerare per un appropriato uso della terapia steroidea immunosoppressiva: la misura della mancata responsività potrebbe spiegare l'eventuale insuccesso della terapia (6) Metodi- Abbiamo analizzato mediante citofluorometria a flusso (FACS BD) le popolazioni linfo-monocitarie da sangue periferico in un gruppo di pazienti (n=8) affetti da patologia tiroidea con esoftalmo sotto trattamento con bolo endovena di cortisonico. Tale trattamento terapeutico consente di valutare la responsività steroidea attraverso il confronto delle popolazioni circolanti nel sangue periferico prima e dopo bolo (circa 3-4 ore). Gli stessi pazienti, sottoposti a sedute settimanali, sono stati osservati per tutto il periodo della terapia (circa 3 mesi). Inoltre, mediante retrotrascriptasi inversa dell’RNA dei PBMC ottenuti dai diversi prelievi e PCR quantitativa, è stata analizzata l’espressione genica dei fattori trascrizionali chiave nel differenziamento delle popolazioni linfocitarie (TBX21, FOXP3, GATA3, RORc) e le loro citochine effettrici (IFNgamma, IL2, IL4, TGFb1, IL10, IL17a, IL6). Risultati- Si osservava in tutti i soggetti una riduzione dei T CD4+ ed un aumento dei NK e NKT nel prelievo post bolo. Dopo il bolo, l’analisi dei T CD4+ CD25+ CD127 low evidenziava una riduzione significativa ed in alcuni pazienti essa diveniva stabile nel tempo. L’analisi dei fattori trascrizionali chiave del differenziamento delle popolazioni (TBX21, FOXP3, GATA3, RORc) evidenziava tali effetti nel corso del trattamento. Conclusione- Ulteriori studi dei cambiamenti stabili nella popolazione linfo-monocitaria, in rapporto ai risultati terapeutici, consentiranno di identificare i parametri vantaggiosi da misurare in corso di trattamento con agonisti corticosteroidei. 1) Glaser, R. and Kiecolt-Glaser, J.K. – Stress-Induced Immune Dysfunction: Implications for Health, Nature Reviews Immunology, 5:10-18, 2005. 2) Mittelstadt PR, Monteiro JP, Ashwell JD. Thymocyte responsiveness to endogenous glucocorticoids is required for immunological fitness. J Clin Invest. 2012 Jun 1. pii: 63067. 3) Rogatsky I, Ivashkiv LB. Glucocorticoid modulation of cytokine signaling. Tissue Antigens. 2006 Jul;68(1):1-12. 4) Webster JI, Stenberg EM Neural immune Interactions. In “Fundamental Immunology” Paul William E; 2008; cap 32:1031-1046 5) Flammer JR, Rogatsky I. Minireview: Glucocorticoids in autoimmunity: unexpected targets and mechanisms. Mol Endocrinol. 2011 Jul;25(7):1075-86. 6) De Iudicibus S, Franca R, Martelossi S, Ventura A, Decorti G. Molecular mechanism of glucocorticoid resistance in inflammatory bowel disease. World J Gastroenterol. 2011 Mar 7;17(9):1095-108.

ST2. PRDM Gene Products in Testicular Germ Cell Tumors E. di Zazzo1, C. Porcile1, C. De Rosa2, A. Marino1, S. Bartollino1, C. Abbondanza2, B. Moncharmont1 1Università degli Studi del Molise, Campobasso, Italy; 2Seconda Università degli studi di Napoli, Naples, Italy Background: Testicular germ cell tumors (TGCT) originate from primordial germ cells blocked at different stages during maturation, reflecting different histological tumor subtypes. A common genetic alteration in TGCT is a deletion of chromosome 1 short arm, where the PRDM2 gene, a member of positive regulatory domain gene family, is located. Moreover recent studies demonstrated that members of PRDM gene family have an essential role in the early stages of testicular development. The aim of this study is to evaluate PRDM gene family members for a possible tumorsuppressor function in TGCT. Methods: PRDM gene expression was assessed by mRNA RT-PCR. Cells were treated with 100 nM 17β-Estradiol (E2), 100 nM DHT or 10 uM RA in serum free medium for 24h. RNA interference was performed using BLOCK-iT™ Pol II miR RNAi system. Proliferation assay was performed with propidium iodide staining and FACS analysis. Results: In GC1 mouse spermatogonial cells treatment with proliferation agents 5α-dihydrotestosterone (DHT) and E2 reduced PRDM2/RIZ1 expression levels whereas PRDM2 total forms showed no variation; the same treatment significantly increased PRDM4 and PRDM10 expression levels. Silencing PRDM2 gene expression by RNA interference increased PRDM10 expression levels and reduced the proliferation rate of spermatogonia. Conclusions: In spermatogonia as in MCF-7 cell line, E2 and DHT regulate PRDM2 gene expression suggesting that PRDM2 gene products could mediate the effect of these agents on cell cycle progression. PRDM4 and PRDM10 are also responsive to steroid hormones and PRDM10 probably cooperates with PRDM2, as demonstrated by the increase of its expression levels after PRDM2 gene silencing.