Alfredo Procino

HOX gene network is involved in the transcriptional regulation of in vivo human adipogenesis

Adipogenesis is regulated by the sequential activation of a series of transcription factors: the C/EBP proteins of type β and δ trigger the process while PPARγ and C/EBPα induce the differentiation from pre‐adipocyte to adipocyte, followed by adipo‐specific gene expression. A number of observations suggest the involvement of genes controlling embryonal development in adipogenesis. In human thyroid follicular carcinoma, it has been recently identified an oncogenetic fusion protein resulting from the interaction between the isoform PPARγ1 of PPARγ and the homeoprotein encoded by the PAX‐8 gene. Recent observations have pointed out that gene expression associated with adipocyte differentiation in vivo and in vitro, although partially overlapping, is actually different. HOX genes make up a network of transcription factors (homeoproteins) controlling embryonal development as well as crucial functions of adult eukaryotic cells. The molecular organization of this network of 39 genes appears to be unique in the genome and probably acts regulating phenotypic cell identity. In the present study we have analyzed the expression of the complete HOX gene network, in vivo, in different deposits of human white adipose tissue and in embryonal brown adipose tissues. Most of the genes in the HOX network are active in white as well as brown adipose tissue. Furthermore HOX genes display a deposit‐specific expression in white adipose tissue. Moreover, expression of the paralogous group 4 genes (HOX A4, HOX B4, HOX C4, and HOX D4), together with that of isolated genes in the network, appears to discriminate between white and brown adipose tissue. This data allows us to postulate the involvement of the HOX network in transcriptional regulation of human adipogenesis and to hypothesize on the molecular mechanisms that could be implicated.

Immunologic evaluation during the first year of life of infants born to cyclosporine-treated female kidney transplant recipients : Analysis of lymphocyte subpopulations and immunoglobulin serum levels

BACKGROUND In rodents, CsA has been shown to affect T-cell development, giving rise to an abnormal production of mature T cells and the absence of many T-cell subsets as well as to autoimmunity. Surprisingly, only a few studies investigated the effect of the immunosuppressive drug on the immune system of the human fetus. METHODS We examined six infants born to female kidney transplant recipients who had received cyclosporine and methylprednisolone throughout their pregnancies. Peripheral blood was obtained 1 day and 2, 4, 6, and 12 months after birth, and two-color flow cytometric immunophenotyping of lymphocytes was performed. RESULTS Total T cells, as well as CD4+ and CD8+ T cells, were low at birth, but normalized thereafter. Among T-cell activation markers, the expression of CD25, the alpha chain of the interleukin-2 receptor, was below the normal range or low range throughout the study period, and HLA-DR expression was extremely low at birth and failed to increase up to 12 months. The number of total B cells was lower than normal at birth, but steeply increased over time. In contrast, B-cell subset bearing CD5 antigen was severely depleted throughout the first year of life. Total IgG concentration was significantly lower than in controls at 2 months, mainly because of subnormal levels of IgG1 and IgG3 subclasses, which remained in the low range up to 6 months. Finally, infants showed normal numbers of true natural killer (NK) cells (CD3-CD16+CD56+), whereas the expression of CD57 antigen, defining non-MHC-restricted cytotoxic lymphocytes, was barely detectable at birth and failed to increase over time, in both CD8+ and CD8- subsets. Of note, none of the infants had clinical evidence of an immunodeficient state. CONCLUSIONS continuous exposure to CsA in utero seemingly impairs T-, B-, and NK-cell development and/or maturation, and most of its effects are still apparent at 1 year, which might suggest that conventional vaccinations should be delayed in these infants.

Adult human neural crest-derived cells for articular cartilage repair.

HOX-negative, differentiated neural crest–derived adult cells from the nasal septum display self-renewal capacity and environmental plasticity and are compatible for articular cartilage repair. Cells from Nose Repair Tissue in Joint Cartilage repair remains a yet unmet clinical need, with few viable cell therapy options available. Taking cells from the knee or ankle to repair worn cartilage requires additional surgery and, in turn, pain and healing for the patient. As such, a new, accessible cell source would greatly benefit these patients. Here, Pelttari and colleagues looked up the nose for cells that may have the capacity to regenerate cartilage. Nasal septum cells arise from the neuroectoderm—the tissue that gives rise to the nervous system—and are better at repairing tissues than their mesoderm counterparts. These regenerative capabilities have been attributed to a lack of homeobox (HOX) gene expression. The authors therefore investigated whether nasal chondrocytes (HOX-negative, neuroectoderm origin) were compatible with an articular cartilage environment, like the knee joint (HOX-positive, mesoderm origin). The authors discovered that adult human nasal chondrocytes were able to self-renew and also, to their surprise, adopt a HOX-positive profile upon implantation into a mesoderm environment; in goats, this led to repair of experimental articular cartilage defects. In an ongoing clinical trial, human nasal chondrocytes have been shown to be safe once transplanted, suggesting translation of this new, easy-to-access cell source for repairing damaged joints. In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile—a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC–based engineered tissues for the treatment of traumatic articular cartilage lesions.

Interleukin-6 release from peripheral mononuclear cells is associated to disease activity and treatment response in patients with lupus nephritis.

Sir, We read with great interest the article by De La Torre, et al. recently published in Lupus. The authors, through clinical and flow cytometric evaluations, demonstrate that there is an increased expression of interleukin-6 (IL-6) agonistic receptor gp130 on peripheral lymphocytes, drawn from patients with systemic lupus erythematosus (SLE). In addition, they interestingly report a significant association between gp130 expression and disease activity score, suggesting that gp130 could provide a useful tool for monitoring patients with SLE. In agreement with these findings, we report the results of a small longitudinal study, which we performed in patients with lupus nephritis, in order to evaluate the relationship among IL-6 release from peripheral blood mononuclear cells (PBMCs), proteinuria and disease activity. In this study, we demonstrate a strong correlation between IL-6 spontaneous release and both systemic and renal disease activity. Moreover, we supply evidence that an effective treatment results in decreasing clinical disease activity and IL-6 production by PBMCs. Fourteen patients (36.5 10.7 year, 12 women) affected by SLE, as defined by American Rheumatism Association criteria, with renal involvement (histological findings:WHO class III for six patients, class IV for five patients and class V for three patients) were enrolled. These patients underwent induction therapy with intravenousmethylprednisolone, followed by oral prednisone, and 6-monthly boluses of intravenous cyclophosphamide. After the induction, patients started the maintenance therapy with oral prednisone combined with either cyclosporine A (eight patients) or mycophenolate mofetil (six patients). A 12-month follow-up was performed. SLE disease activity index 2000 (SLEDAI-2K) was calculated every 3 months, as previously reported. Similarly, urine protein/creatinine (P/C) ratio and creatinine clearance on 24-h urine collection were calculated. IL-6 spontaneous production by PBMCs was evaluated before the initiation of the induction treatment (basal condition) and after 12 months. PBMCs were isolated from blood samples by lymphoprep gradient density centrifugation and were cultured for 24 h. At the end of the incubation period, cell-free supernatants were collected, and IL-6 concentration was evaluated by ELISA, as described elsewhere. IL-6 release by cultured PBMCs was significantly higher in basal condition when compared with posttreatment values (443.0 84.1 vs. 27.4 17.8 pg/mL; P< 0.001) (Figure 1A), whereas there was no significant difference in IL-6 levels between patients treated with mycophenolate or cyclosporine (20.2 10.5 vs. 32.7 20.9 pg/mL, ns). At 12month check-up, patients showed a significant decrease of both proteinuria and SLEDAI-2K, when compared with basal values (P/C ratio 1.76 1.25 vs. 0.69 0.38, P< 0.05, and 8.4 2.2 vs. 4.9 2.4, P< 0.01, respectively). Creatinine clearance did not significantly change (basal value 81.4 18.8 vs. 12-month value 81.3 17.2). IL-6 production resulted significantly associated to both SLEDAI-2K and P/C ratio (rs1⁄4 0.4180, P1⁄4 0.0002 and rs1⁄4 0.2670, P1⁄4 0.0049, respectively) (Figure 1B). However, this association could be due, at least in part, to the fact that an effective treatment may independently decrease both SLEDAI and IL-6 levels, acting as a confounding factor. High serum and urinary IL-6 levels have already been reported in lupus nephritis, but the relationship between IL-6 production and disease activity is still debated. Showing a close correlation between IL-6 spontaneous release and SLE activity, our data, in addition to those reported by De La Torre, could contribute to better define the important role of IL-6 activation in lupus nephritis. Longitudinal and bigger studies are required to confirm these findings and to discriminate the effects of different treatments on inflammatory status in patients with lupus nephritis.

Serum fetuin a in hemodialysis: a link between derangement of calcium-phosphorus homeostasis and progression of atherosclerosis?

BACKGROUND Fetuin A, a circulating inhibitor of ectopic calcification, is downregulated in hemodialysis and has been shown to predict cardiovascular mortality in this setting. The association of altered calcium-phosphorus with serum fetuin A levels is still a matter of debate. Although carotid intima-media thickness (cIMT) is a strong predictor of major cardiovascular events, its association with serum fetuin A levels is poorly defined. STUDY DESIGN Cohort study. PARTICIPANTS & SETTINGS 174 uremic patients on long-term hemodialysis therapy enrolled in 4 university hospitals. PREDICTORS Serum fetuin A levels at the beginning of the study (T0) and after 12 months (T12). OUTCOMES Progression of atherosclerosis assessed by means of cIMT measurements at 24 months (T24); cardiovascular morbidity and mortality at 36 months. RESULTS Serum fetuin A concentrations at T0 and T12 were 282.3 +/- 79.4 and 290.0 +/- 92.2 microg/mL, respectively. Mean T0 and T24 cIMT values were 1.02 +/- 0.2 and 1.06 +/- 0.2 mm, respectively (P < 0.001). Fatal and nonfatal cardiovascular disease occurred in 36 and 86 patients by 36 months, respectively. In multivariate logistic regression, higher calcium-phosphorus product was associated with lower serum fetuin A level (odds ratio, 0.96; 95% confidence interval [CI], 0.93 to 1.00; P = 0.02). Multiple regression analysis showed that T0 serum fetuin A level was associated with T24 cIMT (P = 0.01) after adjustments for age, cholesterol level, high-sensitivity C-reactive protein level, previous cardiovascular events, and T0 cIMT. In a multivariate Cox regression analysis, cardiovascular mortality was independently associated with a 1-tertile lower T0 serum fetuin A level, and a 1-tertile higher T0 cIMT value was independently associated with greater cardiovascular mortality (hazard ratio, 0.45; 95% CI, 0.15 to 0.65; P = 0.007 and hazard ratio, 10.00; 95% CI, 3.16 to 31.73; P < 0.001, respectively) after adjustment for age and previous cardiovascular events. LIMITATION Length of follow-up. CONCLUSION Calcium-phosphorus product in hemodialysis patients inversely correlated with serum fetuin A level, which, in turn, was associated inversely with progression of atherosclerotic lesions and cardiovascular mortality in this study population.

The HOX genes network in uro-genital cancers: mechanisms and potential therapeutic implications.

Genito-urinary malignancies (prostate, bladder, renal and testicular cancers) rank high among human tumors with an incidence that varies with age and organ involvement. Prostate cancer is the most commonly detected male cancer followed by bladder and kidney cancers, less frequent in women. Testicular cancer, although rare, is the most frequent cancer in males under 35. The majority of oncogenic and tumor suppressor signaling pathways involved with urogenital cancers converge on sets of transcription factors that ultimately control gene expression resulting in tumor formation and metastatic progression. The activity of these transcription factors is modulated by multiple mechanisms spanning from transcriptional regulation, deregulation of the splicing, maturation, export and location of mRNAs, protein synthesis and post-translational modifications. The recent involvement of the epigenitic mechanisms in the generation and the evolution of cancer has produced a great deal of interest. This is related to the possibility that revealing these mechanisms able to regulate the cell memory program (the gene systems polycomb, trithorax and HOX) may generate important biological and therapeutic achievements. The HOX gene network is the only physically and functionally identifiable transcription factor network located in the human genome controlling crucial cellular processes. Here we describe the implication of the HOX genes in the urogenital embryonic development and cancers. We further highlight the mechanisms uncovered along these processes and involving the HOX genes. Finally, we foresee the specific targeting of HOX genes and in general the cell memory gene program in the therapeutic setting of urogenital malignancies due to their upstream location in these stepwise cell processes and their early deregulation in cancer evolution.

Immunomodulatory Effect of Continuous Venovenous Hemofiltration during Sepsis: Preliminary Data

Introduction. Severe sepsis and septic shock are the primary causes of multiple organ dysfunction syndrome (MODS), which is the most frequent cause of death in intensive care unit patients. Many pro- and anti-inflammatory mediators, such as interleukin-6 (IL-6), play a strategic role in septic syndrome. Continuous renal replacement therapy (CRRT) removes in a nonselective way pro- and anti-inflammatory mediators. Objective. To investigate the effects of continuous venovenous hemofiltration (CVVH) as an immunomodulatory treatment of sepsis in a prospective clinical study. Methods. High flux hemofiltration (Qf = 60 ml/Kg/hr) was performed for 72 hr in thirteen critically ill patients suffering from severe sepsis or septic shock with acute renal failure (ARF). IL-6 gene expression was measured by real-time PCR analysis on RNA extracted from peripheral blood mononuclear cell before beginning of treatment (T0) and after 12, 24, 48, and 72 hours (T1–4). Results. Real-time PCR analysis demonstrated in twelve patients IL-6 mRNA reduction after 12 hours of treatment and a progressive increase after 24, 48, and 72 hours. Conclusions. We suggest that an immunomodulatory effect might exist during CVVH performed in critically ill patients with severe sepsis and septic shock. Our data show that the transcriptional activity of IL-6 increases during CVVH.

The HOX genes network in metabolic diseases.

Fat distribution is associated with metabolic risk. Differences in cellular characteristics and metabolic functions of these depots have been described, but the molecular mechanisms involved are not understood. The pathogenesis and pathophysiology of metabolic disease can be better understood by studying the molecular mechanisms that control the development and function of adipose tissue (adipogenesis). Homeobox genes are transcription factors that act during normal development and contain the homeobox, a 183bp DNA sequence coding for a 61 amino acid domain defined as homeodomain (HD). Class 1 homeobox genes (Hox genes) have a critical role in controlling positional information and tissue patterning during development. The expression of the whole HOX gene network in different deposits of normal adult human white adipose tissue (intraperitoneal, extra‐peritoneal and dermis) indicate a marked expression in adipose tissue. Furthermore, this expression seems to vary in different bodily deposits of white adipose tissue and between white and brown adipose tissue. The purpose of this mini‐review is to discuss the role of HOX genes in metabolic diseases.

Expression of lumbosacral HOX genes, crucial in kidney organogenesis, is systematically deregulated in clear cell kidney cancers.

Homeobox-containing genes are involved in different stages of kidney organogenesis, from the early events in intermediate mesoderm to terminal differentiation of glomerular and tubular epithelia. The HOX genes show a unique genomic network organization and regulate normal development. The targeted disruption of paralogous group 11 HOX genes (HOX A11, HOX C11 and HOX D11) results in a complete loss of metanephric kidney induction. Despite a large amount of data are related to the early events in the kidney development, not much is known about HOX genes in advanced kidney organogenesis and carcinogenesis. Here, we compare the expression of the whole HOX gene network in late-stage human foetal kidney development with the same patterns detected in 25 pairs of normal clear cell renal carcinomas (RCCs) and 15 isolated RCC biopsy samples. In the majority of RCCs tested, HOX C11 is upregulated, whereas HOX D11, after an early involvement becomes active again at the 23rd week of the foetal kidney development, is always expressed in normal adult kidneys and is deregulated, together with HOX A11 and lumbosacral locus D HOX genes. Thus, through its function of regulating phenotype cell identity, the HOX network plays an important role in kidney carcinogenesis. Lumbosacral HOX genes are involved in the molecular alterations associated with clear cell kidney cancers and represent, through their deregulation, a molecular mark of tubular epithelial dedifferentiation occurring along tumour evolution, with the restoration of genetic programs associated with kidney organogenesis. The deregulation of lumbosacral HOX genes in RCCs supports (i) the consideration of the HOX gene transcriptome as the potential prognostic tool in kidney carcinogenesis and (ii) the possibility to foresee clinical trials with the purpose of targeting these genes to achieve a therapeutic effect in RCC patients.

A translational approach to micro-inflammation in end-stage renal disease: molecular effects of low levels of interleukin-6

Inflammation plays a key role in the progression of cardiovascular disease, the leading cause of mortality in ESRD (end-stage renal disease). Over recent years, inflammation has been greatly reduced with treatment, but mortality remains high. The aim of the present study was to assess whether low (<2 pg/ml) circulating levels of IL-6 (interleukin-6) are necessary and sufficient to activate the transcription factor STAT3 (signal transducer and activator of transcription 3) in human hepatocytes, and if this micro-inflammatory state was associated with changes in gene expression of some acute-phase proteins involved in cardiovascular mortality in ESRD. Human hepatocytes were treated for 24 h in the presence and absence of serum fractions from ESRD patients and healthy subjects with different concentrations of IL-6. The specific role of the cytokine was also evaluated by cell experiments with serum containing blocked IL-6. Furthermore, a comparison of the effects of IL-6 from patient serum and rIL-6 (recombinant IL-6) at increasing concentrations was performed. Confocal microscopy and Western blotting demonstrated that STAT3 activation was associated with IL-6 cell-membrane-bound receptor overexpression only in hepatocytes cultured with 1.8 pg/ml serum IL-6. A linear activation of STAT3 and IL-6 receptor expression was also observed after incubation with rIL-6. Treatment of hepatocytes with 1.8 pg/ml serum IL-6 was also associated with a 31.6-fold up-regulation of hepcidin gene expression and a 8.9-fold down-regulation of fetuin-A gene expression. In conclusion, these results demonstrated that low (<2 pg/ml) circulating levels of IL-6, as present in non-inflamed ESRD patients, are sufficient to activate some inflammatory pathways and can differentially regulate hepcidin and fetuin-A gene expression.