From mini-puberty to pre-puberty: early impairment of the hypothalamus-pituitary-gonadal axis with normal testicular function in children with non-mosaic Klinefelter syndrome.

PURPOSE: Klinefelter syndrome (KS) is a genetic disorder caused by the presence of an extra X chromosome in males. The aim of this study was to evaluate the hypothalamic-pituitary-gonadal (HPG) axis and the clinical profile of KS boys from mini-puberty to early childhood. PATIENTS AND METHODS: In this retrospective, cross-sectional, population study, 145 KS boys and 97 controls aged 0-11.9 years were recruited. Serum FSH, LH, testosterone (T), Inhibin B (INHB), sex hormone binding globulin (SHBG) and anti-Müllerian hormone (AMH) were determined. Auxological parameters were assessed. To better represent the hormonal and clinical changes that appear in childhood, the entire population was divided into 3 groups: ≤ 6 months (group 1; mini-puberty); > 6 months and ≤ 8 years (group 2; early childhood); > 8 and ≤ 12 years (group 3; mid childhood). RESULTS: During mini-puberty (group 1), FSH and LH were significantly higher in KS infants than controls (p < 0.05), as were INHB and T (respectively p < 0.0001 and p < 0.005). INHB was also significantly higher in KS than controls in group 2 (p < 0.05). AMH appeared higher in KS than in controls in all groups, but the difference was only statistically significant in group 2 (p < 0.05). No significant differences were found in height, weight, testicular volume, and penile length. CONCLUSIONS: No hormonal signs of tubular or interstitial damage were found in KS infants. The presence of higher levels of gonadotropins, INHB and testosterone during mini-puberty and pre-puberty may be interpreted as an alteration of the HPG axis in KS infants.

Epicardial fat: the role of testosterone and lipid metabolism in a cohort of patients with Klinefelter syndrome.

CONTEXT: Klinefelter syndrome (KS), in which subjects have additional copies of X chromosomes, is the most common male sex chromosome abnormality, with a prevalence of 1 in 660 and an incidence of about 1 in 500-700 newborns. Its sign and symptoms include infertility, generally low testosterone levels, and an increased prevalence of obesity and metabolic syndrome. Epicardial fat thickness (EFT) reflects visceral adiposity rather than general obesity. OBJECTIVE: The aim of this study was to analyze echocardiographic EFT in a cohort of patients with KS in comparison with non-obese and obese euploid controls, and to evaluate its correlation with biochemical parameters. DESIGN, SETTING AND PARTICIPANTS: Two hundred and twenty-one KS patients referred to our Rare Endocrine Diseases clinic and 77 age-matched controls underwent Doppler echocardiography and a full investigation of anthropometric and body composition, Serum levels of total testosterone (T), estradiol (E2), sex hormone binding globulin (SHBG), fasting plasma glucose, insulin, cholesterol and triglycerides were obtained. All participants underwent dual energy X-ray absorptiometry (DEXA) scan to assess truncal body fat (TrBF). MAIN OUTCOME MEASURE: EFT, body composition and metabolic parameters in KS patients and how they are affected by genotype. RESULTS: EFT was greater in KS patients than in healthy non-obese (NOb) controls, but lower than in obese (OB) controls. When KS patients were divided into groups (hypogonadal; eugonadal; receiving testosterone replacement therapy [TRT]), EFT was greater in hypogonadal patients than in NOb controls and eugonadal patients, but showed no difference from the OB controls or TRT patients. Hypogonadal patients showed increased TrBF in comparison with NOb controls and eugonadal and TRT patients, and similar TrBF to OB controls. As expected, there was a strong correlation between BMI and EFT in both KS patients and controls (P < 0.0001). In contrast, there was a strong inverse correlation between testosterone and EFT in the control group, but not in KS patients. EFT was significantly correlated with TrBF in both populations (P < 0.0001). Multivariate analyses showed that the major determinants of both EFT and TrBF were BMI and the presence of KS itself. Testosterone and triglycerides were not included as variables in the models. CONCLUSION: EFT in hypogonadal KS subjects was similar to that of the obese eugonadal controls. Even though there was a direct correlation between BMI and EFT in both populations, the influence of TrBF on EFT was stronger. The presence of the supernumerary X chromosome appeared to be one of the strongest determinants of EFT and TrBF, independent of testosterone levels.

NOTCH1 mutations are associated with high CD49d expression in chronic lymphocytic leukemia: link between the NOTCH1 and the NF-κB pathways.

In chronic lymphocytic leukemia (CLL), stabilizing mutations of NOTCH1, affecting up to 10-15% of cases, have been associated to poor prognosis, disease progression and refractoriness to chemotherapy. NOTCH1 mutations are significantly overrepresented in trisomy 12 CLL, a disease subset frequently expressing CD49d, the α4 chain of the very-late-activation-4 integrin, a well-known key regulator of microenviromental interactions, and negative prognosticator in CLL. In the present study, by analysing a wide cohort of 1180 CLL, we observed a very strong association between the presence of NOTCH1 mutations and the expression of CD49d (P<0.0001), occurring also outside the trisomy 12 CLL subset. Using both the MEC-1 CLL-like cells stably transfected with the NOTCH1 intracellular domain and primary CLL cells bearing a mutated or wild-type NOTCH1 gene configuration, we provide evidence that triggering of the NOTCH1 pathway resulted in a positive CD49d expression regulation, which was driven by a NOTCH1-dependent activation of nuclear factot-κB (NF-κB). Consistently, pharmacological inhibition of the NOTCH1 and/or of the NF-κB pathways resulted in impaired NF-κB nuclear translocation with consequent down-modulation of CD49d expression. Altogether, our data link for the first time NOTCH1 mutations to CD49d expression regulation through the involvement of the NF-κB pathway in CLL.

Fluid intelligence, traits of personality and personality disorders in a cohort of adult KS patients with the classic 47, XXY karyotype.

PURPOSE: Klinefelter's syndrome (KS) is associated with specific neurobehavioral features and personality traits. The aim of our study was to investigate fluid intelligence, personality traits and personality disorders (PD) and possible correlations with testosterone in a cohort of adult KS patients. METHODS: We analyzed 58 adult KS patients with the classic 47, XXY karyotype. The Structured Clinical Interview for axis II disorders was used to assess DSM IV personality disorders. Personality traits were assessed using MMPI-2. Fluid intelligence was tested by using Raven's Standard Progressive Matrices (SPM) Test. Testosterone blood concentration was measured by CMIA. RESULTS: PD prevalence was 31%. Four altered MMPI scales (Social Responsibility, Dominance, Ego Strength and Repression) were found in more than 40% of patients. Overcontrolled hostility and MacAndrew Alcoholism Scale-Revised scales were altered in the PD- group only. Biz-Odd Thinking and Post-Traumatic Stress Disorder scale were associated with the presence of personality disorder. The raw SPM score was 44 ± 10.8 without any significant correlation with testosterone. No significant difference in mean age, SPM raw score and MMPI score was observed between eugonadal, hypogonadal and treated patients. CONCLUSIONS: Most KS patients had average fluid intelligence. PD prevalence was higher than in the general population. Testosterone was not correlated with fluid intelligence, personality traits or PD, but a reduction in marital distress was observed in treated patients. This could suggest that testosterone therapy can improve physical symptoms and this effect could also improve relationship abilities and wellness awareness.

NOTCH1-mutated chronic lymphocytic leukemia cells are characterized by a MYC-related overexpression of nucleophosmin 1 and ribosome-associated components.

In chronic lymphocytic leukemia (CLL), the mechanisms controlling cell growth and proliferation in the presence of NOTCH1 mutations remain largely unexplored. By performing a gene expression profile of NOTCH1-mutated (NOTCH1-mut) versus NOTCH1 wild-type CLL, we identified a gene signature of NOTCH1-mut CLL characterized by the upregulation of genes related to ribosome biogenesis, such as nucleophosmin 1 (NPM1) and ribosomal proteins (RNPs). Activation of NOTCH1 signaling by ethylenediaminetetraacetic acid or by coculture with JAGGED1-expressing stromal cells increased NPM1 expression, and inhibition of NOTCH1 signaling by either NOTCH1-specific small interfering RNA (siRNA) or γ-secretase inhibitor reduced NPM1 expression. Bioinformatic analyses and in vitro activation/inhibition of NOTCH1 signaling suggested a role of MYC as a mediator of NOTCH1 effects over NPM1 and RNP expression in NOTCH1-mut CLL. Chromatin immunoprecipitation experiments performed on NOTCH1 intracellular domain (NICD)-transfected CLL-like cells showed the direct binding of NOTCH1 to the MYC promoter, and transfection with MYC-specific siRNA reduced NPM1 expression. In turn, NPM1 determined a proliferation advantage of CLL-like cells, as demonstrated by NPM1-specific siRNA transfection. In conclusion, NOTCH1 mutations in CLL are associated with the overexpression of MYC and MYC-related genes involved in protein biosynthesis including NPM1, which are allegedly responsible for cell growth and/or proliferation advantages of NOTCH1-mut CLL.

CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia.

CD49d, the alpha-chain of the integrin heterodimer α4ß1, was identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p chromosome involving TP53. In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized. By analyzing a cohort of 778 unselected CLL patients, we assessed the clinical relevance of CD49d as an OS predictor in subgroups defined by mutation/deletion of the TP53, NOTCH1, SF3B1 and BIRC3 genes. In this context, CD49d emerged as an independent predictor of OS in multivariate Cox analysis (Hazard ratio =1.88, P<0.0001). Consistently, high CD49d expression identified CLL subsets with inferior OS in the context of each category of a previously reported hierarchical risk stratification model. Moreover, by evaluating the relative importance of biological prognosticators by random survival forests, CD49d was selected among the top-ranked OS predictor (variable importance =0.0410), along with IGHV mutational status and TP53 abnormalities. These results confirmed CD49d as an independent negative OS prognosticator in CLL also in comprehensive models comprising the novel recurrent mutations. In this context, TP53 disruption and NOTCH1 mutations retained prognostic relevance, in keeping with their roles in CLL cell immuno-chemoresistance.

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation.

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared with controls, NICD-mut transfectants had RBPJ preferentially complexed to NICD and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD-mut transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression.

Fluoxetine increases plasticity and modulates the proteomic profile in the adult mouse visual cortex.

The scarce functional recovery of the adult CNS following injuries or diseases is largely due to its reduced potential for plasticity, the ability to reorganize neural connections as a function of experience. Recently, some new strategies restoring high levels of plasticity in the adult brain have been identified, especially in the paradigmatic model of the visual system. A chronic treatment with the anti-depressant fluoxetine reinstates plasticity in the adult rat primary visual cortex, inducing recovery of vision in amblyopic animals. The molecular mechanisms underlying this effect remain largely unknown. Here, we explored fluoxetine effects on mouse visual cortical plasticity, and exploited a proteomic approach to identify possible candidates mediating the outcome of the antidepressant treatment on adult cortical plasticity. We showed that fluoxetine restores ocular dominance plasticity in the adult mouse visual cortex, and identified 31 differentially expressed protein spots in fluoxetine-treated animals vs. controls. MALDITOF/TOF mass spectrometry identification followed by bioinformatics analysis revealed that these proteins are involved in the control of cytoskeleton organization, endocytosis, molecular transport, intracellular signaling, redox cellular state, metabolism and protein degradation. Altogether, these results indicate a complex effect of fluoxetine on neuronal signaling mechanisms potentially involved in restoring plasticity in the adult brain.

Long term cryopreservation in 5% DMSO maintains unchanged CD34(+) cells viability and allows satisfactory hematological engraftment after peripheral blood stem cell transplantation.

Peripheral blood stem cell cryopreservation is associated with cell damage and decreased viability. We evaluated the impact of up to 10 years of cryopreservation (5% DMSO) on viability of CD34(+) cells utilizing graft samples of consecutive patients (2002-2012) with different malignancies who underwent stem cell collection and transplantation. Viability of CD34(+) cells from oncohaematological patients measured after 5 weeks (97·2 ± 0·6%) or after 9-10 years of cryopreservation (95·9 ± 0·5%) was unaffected. Haemoglobin, granulocyte and platelet recovery after transplantation of long-term cryopreserved grafts occurred within 8-13 days. CD34(+) stem cells can be safely stored up to 9-10 years, without affecting cell viability and clinical effectiveness.

NUP98-HOXA10hd-expanded hematopoietic stem cells efficiently reconstitute bone marrow of mismatched recipients and induce tolerance.

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98- HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

A new freezing and storage procedure improves safety and viability of haematopoietic stem cells and neutrophil engraftment: a single institution experience.

BACKGROUND AND OBJECTIVES: Autologous peripheral blood stem cell transplantation has recently become a standard therapeutic approach to virus-related or infected haematological malignancies. Collection, manipulation, storage and thawing of leukapheresis products in this subset of patients require strict monitoring to prevent infection risk for operators and risk of contamination for other stored bags. MATERIALS AND METHODS: This is a non-randomized retrospective observational study. In the 2000-2002 period, a single bag freezing procedure was used for autologous peripheral blood stem cell transplantation. Bags were stored in tanks containing liquid and gas phase nitrogen. In 2002, the processing procedure was revised, and a second additional safety bag and a new storage tank containing jacketed liquid nitrogen have been used. RESULTS: A total of 524 bags were thawed, of which 121 processed with the single bag method and 403 with the double bag method. Forty-nine and 109 patients were infused respectively. The observed rupture rate with the single bag in liquid and gas phase nitrogen was 17 and 2.5%, respectively, against a rupture rate as little as 0.24% with the new methodology. Viability revealed levels of 84.4% +/- 6.1% and 96.9% +/- 2.4% for the single and double-bag respectively. This statistically significant (P < 0.0001) difference correlated with better neutrophil engraftment. CONCLUSIONS: The new proposed method, based on a double bag and storage freezer without liquid or gas phase nitrogen into a cryogenic chamber, significantly reduces bag rupture and bio-hazard and improves stem cell viability and neutrophil engraftment remarkably.

CD30L up-regulates CD30 and IL-4 expression by T cells.

CD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co-expression of interleukin-4 (IL-4) receptor and with the expansion of specific T-helper 2 (Th2) cell subsets producing IL-4 and expressing CD30. Recombinant CD30L-bearing cells up-regulated the expression of surface CD30 and increased the production of IL-4 and soluble (s) CD30 by co-cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti-CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL-4. Our data indicates a direct role of CD30L(+) neoplastic cells in driving the immune response toward a Th2-polarized non-protective state.

Requirement of the nicotinic acetylcholine receptor beta 2 subunit for the anatomical and functional development of the visual system.

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the beta2 subunit of the neuronal nicotinic receptor, but not in mice lacking the alpha4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, beta2-/- mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the beta2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system.

Integrin pattern and effect on contraction in cultured testicular peritubular myoid cells.

PROBLEM: Neither the integrin pattern nor the biological functions of integrins have been extensively documented in human cultured testicular peritubular myoid cells (TPMC). The integrin pattern and the presence of some proteins of the immunoglobulin superfamily on human TPMC as well as the role of integrins in TPMC contraction were examined. METHOD OF STUDY: Integrin expression was evaluated by immunofluorescence and FACS analysis. To assess the role of integrin in TPMC contraction, human and rat cells were added to a collagen gel system and exposed to contractile stimuli. RESULTS: The immunofluorescence and cytofluorimetric analysis showed that human cultured TPMC express alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, and beta4 integrin subunits, and significant amounts of intercellular adhesion molecule-1 (ICAM-1), whereas they do not present alpha4, beta2, beta7 subunits, nor intercellular adhesion molecule-2 (ICAM-2) and neural cell adhesion molecule (NCAM). The preincubation of human cells with an anti-beta1 mAb and of rat cells with a polyclonal anti-beta1 antibody inhibited TPMC contraction induced by different contractile stimuli. CONCLUSION: Our investigation documented a broad integrin pattern on human cultured TPMC as well as a role for integrins in human and rat TPMC contraction.

From marrow to brain: expression of neuronal phenotypes in adult mice.

After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.

Transcriptional control: rheostat converted to on/off switch.

Individual cells translate concentration gradients of extracellular factors into all-or-none threshold responses leading to discrete patterns of gene expression. Signaling cascades account for some but not all such threshold responses, suggesting the existence of additional mechanisms. Here we show that all-or-none responses can be generated at a transcriptional level. A graded rheostat mechanism obtained when either transactivators or transrepressors are present is converted to an on/off switch when these factors compete for the same DNA regulatory element. Hill coefficients of dose-response curves confirm that the synergistic responses generated by each factor alone are additive, obviating the need for feedback loops. We postulate that regulatory networks of competing transcription factors prevalent in cells and organisms are crucial for establishing true molecular on/off switches.

Normalizing complementary DNA by quantitative reverse transcriptase-polymerase chain reaction of beta2-microglobulin: molecular monitoring of minimal residual disease in acute promyelocytic leukemia.

Reverse transcription (RT)-polymerase chain reaction (PCR) raises unique methodological matters that may hamper the reliability of the procedure, especially when results should direct therapeutic decisions. One of these matters is represented by the RT step. The present study shows that differences in complementary DNA (cDNA) preparations purposely containing increasing amounts of retrotranscribed RNA were not disclosed by nonquantitative RT-PCR by two different housekeeping genes, leading to fictitious results when the expression of a given gene was quantitatively assessed. To overcome this problem, the following are proposed: 1) to evaluate the efficiency of RT step through the quantification, by competitive RT-PCR, of the expression levels of the housekeeping gene beta2-microglobulin (beta2M); 2) to normalize each cDNA preparation to be comprised within 1 standard deviation of the mean value of beta2M absolute level (3.14 +/- 1.14 attomoles/microg RNA) found by analyzing 33 cell lines of hematopoietic origin. To validate this strategy in a clinical setting, serial cDNA samples from patients were checked by conventional and quantitative RT-PCR for beta2M. Again, only a quantitative evaluation of beta2M levels was allowed to unveil significant differences, otherwise undetected, in the efficiency of RT reactions among these cDNA samples. Normalization of samples to obtain cDNA preparations containing comparable beta2M levels, eventually led to an increased sensitivity in the detection of PML-RARalpha fusion transcripts. This approach seems of great value for the monitoring of minimal residual disease in serial patient samples when a tumor-specific marker is available.