Placental Stem Cells from Domestic Animals: Translational Potential and Clinical Relevance.

The field of regenerative medicine is moving toward clinical practice in veterinary science. In this context, placenta-derived stem cells isolated from domestic animals have covered a dual role, acting both as therapies for patients and as a valuable cell source for translational models. The biological properties of placenta-derived cells, comparable among mammals, make them attractive candidates for therapeutic approaches. In particular, stemness features, low immunogenicity, immunomodulatory activity, multilineage plasticity, and their successful capacity for long-term engraftment in different host tissues after autotransplantation, allo-transplantation, or xenotransplantation have been demonstrated. Their beneficial regenerative effects in domestic animals have been proven using preclinical studies as well as clinical trials starting to define the mechanisms involved. This is, in particular, for amniotic-derived cells that have been thoroughly studied to date. The regenerative role arises from a mutual tissue-specific cell differentiation and from the paracrine secretion of bioactive molecules that ultimately drive crucial repair processes in host tissues (e.g., anti-inflammatory, antifibrotic, angiogenic, and neurogenic factors). The knowledge acquired so far on the mechanisms of placenta-derived stem cells in animal models represent the proof of concept of their successful use in some therapeutic treatments such as for musculoskeletal disorders. In the next future, legislation in veterinary regenerative medicine will be a key element in order to certify those placenta-derived cell-based protocols that have already demonstrated their safety and efficacy using rigorous approaches and to improve the degree of standardization of cell-based treatments among veterinary clinicians.

Dual inhibition of REV-ERBß and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells.

REV-ERBα and REV-ERBß nuclear receptors regulate several physiological processes, including circadian rhythm and metabolism. A previous study reported the REV-ERBα gene to be co-overexpressed with ERBB2 in breast cancer cell lines. Surprisingly, we found that several tumor types, including a number of breast cancer cell lines, predominantly express the REV-ERBß variant. This pattern was independent of ERBB2 and ER status, and opposite to that of non-cancer mammary epithelial HMEC cells, in which REV-ERBα was the major variant. Consistent with this molecular profile, REV-ERB target genes in both circadian and metabolic pathways were derepressed upon silencing of REV-ERBß, but not REV-ERBα. Strikingly, we found that REV-ERBß is a determinant of sensitivity to chloroquine, a clinically relevant lysosomotropic agent that suppresses autophagy. The cytoprotective function of REV-ERBß appears to operate downstream of autophagy blockade. Through compound screening, we identified ARN5187, a novel lysosomotropic REV-ERBß ligand with a dual inhibitory activity toward REV-ERB-mediated transcriptional regulation and autophagy. Remarkably, although ARN5187 and chloroquine share similar lysosomotropic potency and have a similar effect on autophagy inhibition, ARN5187 is significantly more cytotoxic. Collectively, our results reveal that dual inhibition of REV-ERBß and autophagy is an effective strategy for eliciting cytotoxicity in cancer cells. Furthermore, our discovery of a novel inhibitor compound of both REV-ERB and autophagy may provide a scaffold for the discovery of new multifunctional anticancer agents.

Characterization, GFP gene Nucleofection, and allotransplantation in injured tendons of ovine amniotic fluid-derived stem cells.

Amniotic fluid has drawn increasing attention in the recent past as a cost-effective and accessible source of fetal stem cells. Amniotic fluid-derived mesenchymal stem cells (AFMSCs) that display high proliferation rate, large spectrum of differentiation potential, and immunosuppressive features are considered optimal candidates for allogeneic repair of mesenchymal damaged tissues. In this study, ovine AFMSCs (oAFMSCs) isolated from 3-month-old sheep fetuses were characterized for their proliferation rate, specific surface antigen and pluripotency marker expression, genomic stability, and mesenchymal lineage differentiation during their in vitro expansion (12 passages) and after nucleofection. The high proliferation rate of oAFMSCs gradually decreased during the first six subculture passages while the expression of surface molecules (CD29, CD58, CD166) and of pluripotency-associated markers (OCT4, TERT, NANOG, SOX2), the in vitro osteogenic differentiation potential, and a normal karyotype were maintained. Afterwards, oAFMSCs were nucleofected with a selectable plasmid coding for green fluorescent protein (GFP) using two different programs, U23 and C17, previously optimized for human mesenchymal stem cells. Transfection efficiencies were ∼63% and ∼37%, while cell recoveries were ∼10% and ∼22%, respectively. Nucleofected oAFMSCs expressing the GFP transgene conserved their pluripotency marker profile and retained a normal karyotype and the osteogenic differentiation ability. Seven single clones with a GFP expression ranging from 80% to 97% were then isolated and expanded over 1 month, thus providing stably transfected cells with long-term therapeutic potential. The in vivo behavior of GFP-labeled oAFMSCs was tested on a previously validated preclinical model of experimentally induced Achille's tendon defect. The allotransplanted oAFMSCs were able to survive within the host tissue for 1 month enhancing the early phase of tendon healing as indicated by morphological and biomechanical results. Altogether these data suggest that genetically modified oAFMSCs might represent a valuable tool for in vivo preclinical studies in a highly valid translational model.

Ovine amniotic epithelial cells: in vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects.

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Achilles tendon regeneration can be improved by amniotic epithelial cell allotransplantation.

Amniotic epithelial cells (AECs) are ideal seed cells for tissue regeneration, but no research has yet been reported on their tendon regeneration potential. This study investigated the efficiency of AEC allotransplantation for tendon healing, as well as the mechanism involved. To this aim ovine AECs, characterized by specific surface and stemness markers (CD14(-), CD31(-), CD45(-), CD49f, CD29, CD166, OCT4, SOX2, NANOG, TERT), were allotransplanted into experimentally induced tissue defects in sheep Achilles tendon. In situ tissue repair revealed that AEC-treated tendons had much better structural and mechanical recoveries than control ones during the early phase of healing. Immunohistochemical and biochemical analyses indicated that extracellular matrix remodeling was more rapid and that immature collagen fibers were completely replaced by mature ones in 28 days. Moreover, spatial-temporal analysis of cellularity, proliferation index, vascular area, and leukocyte infiltration revealed that AECs induced a specific centripetal healing process that first started in the tissue closer to the healthy portion of the tendons, where AECs rapidly migrated to then progress through the core of the lesion. This peculiar healing evolution could have been induced by the growth factor stimulatory influence (TGF-ß1 and VEGF) and/or by the host progenitor cells recruitment, but also as the consequence of a direct tenogenic AEC differentiation resulting in the regeneration of new tendon matrix. These findings demonstrate that AECs can support tendon regeneration, and their effects may be used to develop future strategies to treat tendon disease characterized by a poor clinical outcome in veterinary medicine.

Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study.

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

Cholinesterase activities and sensitivity to pesticides in different tissues of silver European eel, Anguilla anguilla.

Cholinesterase (ChE) activities were characterized in silver European eel, Anguilla anguilla, grown in the brackish lagoon of Comacchio (Italy). All specimens were harvested at the "lavoriero", a traditional eel trapping weir that captures eels while leaving internal waters at the onset of reproductive migration. To our knowledge, no investigation on ChE was reported in silver eels. Therefore a first characterization of enzyme activity in muscle, brain, liver and plasma of silver eel was carried out, in the presence of different substrates, selective inhibitors, and four pesticides representative of the carbamate and organophosphate classes. Brain and white skeletal muscle showed similar ChE activities, 5- and 10-fold higher than those detected in liver and plasma, respectively. Km values of 0.31 and 0.30 mM, and Vmax values of 40.28 and 35.47 nmol min(-1) mg protein(-1) were obtained in brain and muscle ChE, respectively. Acetycholinesterase was the predominant ChE form in all tissues, as concluded by comparing the effects of BW 284c51, iso-OMPA and eserine. ChE activities in brain and muscle were significantly inhibited by in vitro treatment with pesticides, with the following order of potency: carbofuran>carbaryl>chlorpyrifos≥diazinon.

Experimental study on allografts of amniotic epithelial cells in calcaneal tendon lesions of sheep.

An experimental protocol was designed to study the survival and behaviour of an allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted on three healthy adult sheep. A core lesion was created in both calcaneal tendons under ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml saline. The AECs were obtained from a 60-80-day-old fetus and cultured under standard conditions. After 15 days of collagenase treatment, 2 x 10(6) AECs stained with a vital membrane fluorescent probe (PHK26) were injected under US guidance in 500 microl saline solution into the lesion of one limb. The contralateral untreated limb was used as a control. Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the lesion without any adverse reactions. The damaged tissue of the treated tendons showed a high number of reparative cells in active proliferation that were accumulating collagen within the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized into parallel arrays of fibers oriented along the longitudinal axis of the tendon.

Fusion as the result of sperm-somatic cell interaction.

The research has been designed to investigate whether acrosome-reacted spermatozoa can fuse with somatic cells and to check whether this event may involve the molecular machinery implicated in the sperm-egg fusion. Boar spermatozoa were capacitated in vitro and then treated with A23187 to induce acrosome reaction and activate their fusogenic potential. Reacted spermatozoa, loaded with the membrane-permeant fluorescent dye calcein AM, were incubated with plated granulosa cells or cells derived from stable cell lines: CRFK, VERO, and ESK4. The fusion between spermatozoa and somatic cells was revealed by the diffusion of the fluorescent dye from the sperm to the cell as membrane fusion and cytoplasmic continuity between the two cells were established. The involvement of integrin alpha6 and tetraspanin CD9 in the process of fusion was assessed by carrying out the experiment in the presence of antibodies against these molecules. Moreover, the incidence of fusion displayed by the different cell types used was analyzed in relation to their content in the above molecules assessed by western blot and immunostaining. The role of CD9 was additionally investigated by using CD9-negative cells. The data presented demonstrate that boar spermatozoa can fuse with different somatic cell types derived from different species and the process requires the combined presence of both integrin and tetraspanin molecules on the cell plasma membrane.

The prevalence and clinical implications of c-kit expression in plasma cell myeloma.

AIM: To evaluate the clinical implications of c-kit (CD117) expression in plasma cell myeloma (PCM). METHODS AND RESULTS: We first evaluated the reliability of immunohistochemistry in assessing c-kit expression by comparing the results with those obtained by flow cytometry and gene expression arrays in 22 PCM and in 10 PCM cell lines. Immunohistochemical results showed a perfect concordance with those of flow cytometry; likewise, immunohistochemical and gene expression data were also concordant in all but one PCM and cell lines analysed. Then, we investigated the clinical implications of c-kit immunoreactivity in bone marrow biopsies of 85 PCM patients with a mean follow-up of 41 months. C-kit immunoreactivity was detected in 24 (28.2%) of the 85 cases and it was significantly associated with a high microvessel density, but not with traditional clinicopathological characteristics or with survival. CONCLUSIONS: Our findings suggest that immunohistochemistry is a reliable indicator of c-kit gene expression and reinforce the notion that approximately one-third of PCM express high levels of c-kit. The lack of association with traditional clinicopathological parameters and patient survival suggests that c-kit expression may not be an adjunct in predicting the clinical course of the disease.

Spatio-temporal analysis of vascular endothelial growth factor expression and blood vessel remodelling in pig ovarian follicles during the periovulatory period.

Vascular endothelial growth factor (VEGF) expression pattern and blood vessel remodelling were evaluated during the transition from the preovulatory follicle to the corpus luteum (CL). To this end, prepubertal gilts were treated with equine chorionic gonadotrophin (eCG) to collect preovulatory follicles (60 h after eCG) and with human chorionic gonadotrophin (hCG) to obtain periovulatory follicles 18 h and 36 h later. The VEGF mRNA content was analysed by in situ hybridization, while protein localization in follicular fluid (FF) and in granulosa and theca compartments was evaluated by ELISA, immunohistochemistry or western blot. Blood vessel architecture and vascular area (VA) were investigated using immunohistochemistry for von Willenbrand Factor, a specific endothelial marker. Vascular remodelling was finally tested using Ki-67 immunocytochemistry as a proliferation marker, or alpha-smooth muscle actin (alpha-SMA) as a specific mural cell marker. eCG-treated follicles showed high VEGF levels and two concentric blood vessel networks composed of proliferating endothelial cells without any association with mural components. hCG injection inhibited VEGF synthesis in the granulosa compartment and, as a consequence, the protein fell within the FF. In parallel, endothelial cell proliferation stopped and the VA decreased. Close to ovulation, VEGF production restarted in both follicular compartments and VEGF mRNA content significantly increased in the theca layer. Changes in follicular VEGF secretion were observed; the protein disappeared from FF and was observed in the extracellular matrix. An active angiogenesis characterized the follicle; endothelial cell proliferation was associated with a recruitment of alpha-SMA-positive mural cells. The data presented in this work showed that, in the phases preceding ovulation, a complete vascular remodelling occurs, characterized by both an evident neovascularization and the appearance of blood vessels presenting smooth musculature which could be involved in CL formation after ovulation.

A one-step RT-PCR and a flow cytometry method as two specific tools for direct evaluation of human herpesvirus-6 replication.

In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The first method consisted of a one-step reverse transcription PCR amplifying a part of the late alternatively spliced U100 gene which encode the gp 82-105 viral glycoprotein. Two extraction methods and two RT-PCR kits were evaluated, leading to the selection of TaKaRa mRNA selective PCR kit. The second procedure consisted in a flow cytometry method to analyze the expression of two late viral HHV-6 antigens using 7C7 and 10G6 monoclonal antibodies. Four fixation permeabilization procedures were compared and the preparation of cells with paraformaldehyde (PFA) 4% was found to be optimal. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This kinetic study confirmed that Mabs recognized late antigens and demonstrate that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms are detectable earlier in the cycle.

Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.

B cell-derived chronic lymphocytic leukemia (B-CLL) represents a common malignancy whose cell derivation and pathogenesis are unknown. Recent studies have shown that >50% of CLLs display hypermutated immunoglobulin variable region (IgV) sequences and a more favorable prognosis, suggesting that they may represent a distinct subset of CLLs which have transited through germinal centers (GCs), the physiologic site of IgV hypermutation. To further investigate the phenotype of CLLs, their cellular derivation and their relationship to normal B cells, we have analyzed their gene expression profiles using oligonucleotide-based DNA chip microarrays representative of approximately 12,000 genes. The results show that CLLs display a common and characteristic gene expression profile that is largely independent of their IgV genotype. Nevertheless, a restricted number of genes (<30) have been identified whose differential expression can distinguish IgV mutated versus unmutated cases and identify them in independent panels of cases. Comparison of CLL profiles with those of purified normal B cell subpopulations indicates that the common CLL profile is more related to memory B cells than to those derived from naive B cells, CD5(+) B cells, and GC centroblasts and centrocytes. Finally, this analysis has identified a subset of genes specifically expressed by CLL cells of potential pathogenetic and clinical relevance.

Autoantibodies to human cytosol: a marker of sporadic porphyria cutanea tarda.

The enzymes potentially involved in the pathogenesis of sporadic porphyria cutanea tarda (PCT) reside in liver cytosoles and microsomes. PCT is frequently associated with hepatitis C virus (HCV) infection, which is in turn associated with autoimmune manifestations. To investigate whether autoimmune reactions, possibly triggered by HCV, are involved in the pathogenesis of PCT, we measured by immunoblot autoantibodies to human cytosolic and microsomal liver fractions in 82 patients with PCT (77% with HCV infection), 105 with other liver disorders and 40 healthy subjects. Anti-liver cytosolic antibodies were more frequent in PCT patients (38/82, 46%) than in pathological controls (P < 0.05-P < 0.001) or in healthy subjects (3/40, 8%, P < 0.001). Among PCT patients, anticytosolic antibodies were more frequent in HCV positive (36/63, 57%) than in HCV negative (2/19, 11%, P < 0.05) cases. Reactivity to a 40-kDa cytosolic polypeptide was present in 20 PCT patients (19 HCV positive), being more frequent than in all pathological controls (P < 0.01-P < 0.0001). Histological activity index (P = 0.04) and antibodies to HCV (P = 0.027) - but not HCV RNA - were associated independently with anticytosolic antibodies as assessed by multivariate analysis. In contrast, frequency of antiliver microsomal antibodies was similar in PCT patients (24/82, 29%) and pathological controls (8-26%), being higher in the autoimmune hepatitis control group (23/23, 100%, P < 0.0001). In conclusion, anticytosolic antibodies, particularly to a 40-kDa polypeptide, are frequent in PCT and associated with HCV infection and severity of liver damage.

Genetic analysis of hepatitis A virus outbreak in France confirms the co-circulation of subgenotypes Ia, Ib and reveals a new genetic lineage.

Genetic analysis of selected genome regions of hepatitis A Virus (HAV) suggested that distinct genotype could be defined in different geographic locations. In order to study the degree of genetic variability among HAV isolated during a single epidemic outbreak, sequences from a 148 base pair segment within the VP1 amino terminal region were obtained for eight distinct HAV isolates from an outbreak that occurred in North Bretagne (France). These sequences were compared among themselves and with published sequences from 30 different strains that represented different HAV sub-genotypes that were isolated all over the world. Phylogenetic analysis revealed an extensive genetic heterogeneity among strains belonging to the same outbreak and revealed co-circulation of sub-genotype IA, IB, and the presence of IIIA sub-genotype for the first time in a Mediterranean country.

Follicle activation involves vascular endothelial growth factor production and increased blood vessel extension.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

Mutations in the HFE gene and their interaction with exogenous risk factors in hepatocellular carcinoma.

The possible role of iron in facilitating the development of liver cancer is still debated. The aims of this study were to define the prevalence of the mutations 845G --> A and 187C --> G (C282Y and H63D) in the HFE gene associated with hereditary hemochromatosis in Italian patients with hepatocellular carcinoma occurring in cirrhosis and to analyze the interaction between these mutations and other established risk factors for hepatocellular carcinoma. The HFE gene mutations, performed by polymerase chain reaction, were analyzed in 81 patients (63 males, 18 females) with hepatocellular carcinoma. None of the patients had a phenotype compatible with homozygous hereditary hemochromatosis. Interaction between HFE mutations and exogenous risk factors was analyzed by collecting information on alcohol consumption, hepatitis B and C virus infections, and iron status at the time of diagnosis of chronic liver disease. This analysis was performed only in males to rule out gender influence on patients' iron status by using the case-only approach specifically designed to estimate departure from multiplicative risk ratios under the assumption of independence between genotype and environmental exposure. The prevalence of the C282Y mutation was significantly higher in patients with hepatocellular carcinoma than in normal controls (8.6% vs 1.6%, P < 0.03). At univariate analysis, iron overload was significantly associated with both HFE mutations (P < 0.0001), whereas ongoing hepatitis B virus infection was associated with the C282Y mutation (P < 0.05). By multivariate analysis, a trend for an increased risk of being positive for hepatitis virus markers (OR 2.9, CI 95% 0.9-9.5) and of having been alcohol abusers (OR 3, CI 95% 0.7-14) was observed in patients heterozygous for the HFE mutations. These data indicate that the prevalence of the main mutation associated with hereditary hemochromatosis is significantly higher in cirrhotic Italian patients with hepatocellular carcinoma compared to a normal population and suggest that heterozygotes for HFE mutations exposed to hepatitis virus infections or who had been alcohol abusers could have an increased risk of developing cirrhosis and later liver cancer than people without the mutations exposed to the same risk factors.

Hyperferritinemia, iron overload, and multiple metabolic alterations identify patients at risk for nonalcoholic steatohepatitis.

OBJECTIVE: The aim of this study was to define in patients with hyperferritinemia and normal transferrin saturation the relationships among hyperferritinemia, iron overload, HFE gene mutations, the presence of metabolic alterations, and nonalcoholic steatohepatitis (NASH). METHODS: Forty patients with increased serum ferritin, resistant to dietary restriction and normal transferrin saturation, 90 with ultrasonographic evidence of hepatic steatosis, and 60 obligate heterozygotes for hemochromatosis, all negative for alcohol abuse, hepatitis virus infections, and inflammation were studied. Transferrin saturation, serum ferritin, uric acid, lipids, glucose tolerance, insulin resistance, HFE gene mutations, liver histology, and hepatic iron concentration were analyzed. RESULTS: Of the 40 patients with hyperferritinemia, 29 (72%) had biochemical metabolic abnormalities, 18 of the 26 examined (69%) had insulin resistance, 26 (65%) had the presence of one of the two HFE gene mutations (normal controls, 33 of 128 [26%], p < 0.0001), and all had increased liver iron concentration. Thirty-one patients (77%) had histology compatible with NASH. At univariate analysis, NASH was significantly associated with the presence of metabolic alterations, the C282Y mutation, and severity of fibrosis. At multivariate analysis, NASH was associated with the coexistence of multiple metabolic alterations (odds ratio = 5.2, 95% CI = 0.95-28.7). The risk of having NASH augmented in the presence of higher values of ferritin and liver iron concentration. Among the 90 patients with ultrasonographic evidence of hepatic steatosis, 24 (27%) had increased serum ferritin with normal transferrin saturation, but only six remained hyperferritinemic after dietary restriction. CONCLUSION: Increased ferritin with normal transferrin saturation is frequently found in patients with hepatic steatosis, but it reflects iron overload only in those patients in whom it persists despite an appropriate diet. The simultaneous disorder of iron and glucose and/or lipid metabolism, in most of the cases associated with insulin resistance, is responsible for persistent hyperferritinemia and identifies patients at risk for NASH.

Tumor necrosis factor alpha promoter polymorphisms influence the phenotypic expression of hereditary hemochromatosis.

Severe iron overload usually develops in patients with hereditary hemochromatosis (HHC), but variability in the phenotypic expression of the disease has been reported. This study assessed whether tumor necrosis factor alpha (TNF-alpha) plays a role in phenotypic expression of HHC. Sixty-four patients with HHC and 172 healthy volunteers (controls) were studied. Release of TNF-alpha from stimulated peripheral blood monocytes was measured by enzyme-linked immunosorbent assay, and 308 and 238 TNF-alpha polymorphisms were detected with polymerase chain reaction and restriction fragment-length polymorphism analysis. The relation between TNF-alpha polymorphisms and clinical expression of HHC was evaluated. Patients with HHC released less TNF-alpha than controls, but the difference was significant only in homozygotes for the C282Y mutation. The prevalence of the 308 TNF-alpha polymorphism was similar in patients and controls, whereas the prevalence of the 238 polymorphic allele was significantly lower in patients (3% versus 16%; P =.002). A lower prevalence of cirrhosis was observed in patients with TNF-alpha polymorphism than in those without it (4 of 15 [27%] versus 28 of 49 [57%]), but the difference was not significant (P =.07). In nonhomozygotes for the C282Y mutation, severe liver siderosis was less prevalent in patients with the 308 polymorphism than in those without it (P =.05). Alanine aminotransferase (ALT) values were significantly lower in patients with TNF-alpha polymorphism (P =.006), even when patients with other hepatotoxic factors were excluded. Multivariate analysis showed that TNF-alpha polymorphism was independently associated with ALT values (P =.0008 and P =.045, respectively, in homozygotes and nonhomozygotes for the C282Y mutation) and siderosis in nonhomozygotes (P =.047). Thus, TNF-alpha appears to play a role in HHC by modulating the severity of liver damage. (Blood. 2001;97:3707-3712)