In vivo infectivity of liver extracts after resolution of hepadnaviral infection following therapy associating DNA vaccine and cytokine genes.

DNA-based vaccination appears of promise for chronic hepatitis B immunotherapy, although there is an urgent need to increase its efficacy. In this preclinical study, we evaluated the therapeutic benefit of cytokine (IL-2, IFN-γ) genes co-delivery with DNA vaccine targeting hepadnaviral proteins in the chronic duck hepatitis B virus (DHBV) infection model. Then, we investigated the persistence of replication-competent virus in the livers of apparently resolved animals. DHBV carriers received four injections of plasmids encoding DHBV envelope and core alone or co-delivered with duck IL-2 (DuIL-2) or duck IFN-γ (DuIFN-γ) plasmids. After long-term (8 months) follow-up, viral covalently closed circular (ccc) DNA was analysed in duck necropsy liver samples. Liver homogenates were also tested for in vivo infectivity in neonatal ducklings. Co-delivery of DuIFN-γ resulted in significantly lower mean viremia starting from week 21. Viral cccDNA was undetectable by conventional methods in the livers of 25% and 57% of animals co-immunized with DuIL-2 and DuIFN-γ, respectively. Interestingly, inoculation of liver homogenates from 7 such apparently resolved animals, exhibiting cccDNA undetectable in Southern blotting and DHBV expression undetectable or restricted to few hepatocytes, revealed that three liver homogenates transmitted high-titre viremia (3-5×10(10) vge/mL) to naïve animals. In conclusion, our results indicate that IFN-γ gene co-delivery considerably enhances immunotherapeutic efficacy of DNA vaccine targeting hepadnaviral proteins. Importantly, we also showed that livers exhibiting only minute amounts of hepadnaviral cccDNA could induce extremely high-titre infection, highlighting the caution that should be taken in occult hepatitis B patients to prevent HBV transmission in liver transplantation context.

Contribution of aflatoxin B1 and hepatitis B virus infection in the induction of liver tumors in ducks.

The study of two major risk factors in the development of hepatocellular carcinoma, namely persistent hepatitis virus infection and exposure to dietary aflatoxins, has been hampered by lack of an experimental system. To this end we have used a Pekin duck model to examine the effect of congenital duck hepatitis B virus (DHBV) infection and aflatoxin B1 (AFB1) exposure in the induction and development of liver cancer. AFB1 was administered to DHBV infected or noninfected ducks at two doses (0.08 and 0.02 mg/kg) by i.p. injection once a week from the third month posthatch until they were sacrificed (2.3 years later). Two control groups of ducks not treated with AFB1 (one of which was infected with DHBV) were observed for the same period. Each experimental group included 13-16 ducks. Higher mortality was observed in ducks infected with DHBV and treated with AFB1 compared to noninfected ducks treated with AFB1 and other control ducks. In the groups of noninfected ducks treated with high and low doses of AFB1, liver tumors developed in 3 of 10 and 2 of 10 ducks; in infected ducks treated with the high dose 3 of 6 liver tumors were observed and none in the low dose of AFB1. No liver tumors were observed in the two control groups. Ducks infected with DHBV and treated with AFB1 showed more pronounced periportal inflammatory changes, fibrosis, and focal necrosis compared to other groups. All DHBV carrier ducks showed persistent viremia throughout the observation period. An increase of viral DNA titers in livers and sera of AFB1 treated animals compared to infected controls was frequently observed. No DHBV DNA integration into the host genome was observed, although in one hepatocellular carcinoma from an AFB1 treated duck, an accumulation of viral multimer DNA forms was detected. The metabolism of AFB1 in infected and noninfected duck liver was also examined. The study on the role of DHBV infection and AFB1 in the etiopathogenesis of liver tumors may help to clarify some of the basic mechanisms of carcinogenesis.

Molecular dosimetry of aflatoxin exposure: contribution to understanding the multifactorial etiopathogenesis of primary hepatocellular carcinoma with particular reference to hepatitis B virus.

Aflatoxin exposure and hepatitis B virus infection have been implicated as major risk factors for primary hepatocellular carcinoma (PHC) in high-incidence regions of the world. Investigations using the assay of aflatoxin bound to peripheral blood albumin have shown that exposure can occur throughout the life span of the individual, including during the perinatal period, in high-incidence areas such as The Gambia, Senegal, Kenya, and The People's Republic of China. The possibility of measuring aflatoxin exposure at the individual level permits an investigation of the putative mechanisms of interaction of this carcinogen with HBV in the etiopathogenesis of PHC. Animal models, e.g., Pekin duck and HBV-transgenic mice, have also been used to study these questions, and the available data are reviewed.

Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases.

The main properties of the duck hepatitis B virus (DHBV) DNA polymerase have been studied and compared with those of the human hepatitis B virus (HBV) and of the woodchuck hepatitis virus (WHV) DNA polymerases. All 3 enzymes are active under high salt conditions in the presence of high magnesium concentration. DHBV DNA polymerase was found less sensitive to ethanol and to operate at higher optimal pH than the HBV and WHV DNA polymerases. Like the other two viral endogenous DNA polymerases, the DHBV enzyme was strongly inhibited by phosphonoformic acid but not by aphidicolin, sulfhydryl group blockers or phosphonoacetic acid. Inhibition of DHBV DNA polymerase by the triphosphate derivatives of several nucleoside analogs appeared similar to that reported for HBV or WHV endogenous polymerase. FIACTP was the most, and ACVTP the least effective inhibitor; BVdUTP was of intermediary potency; araCTP and araTTP had a greater inhibitory effect on DHBV DNA polymerase than HBV or WHV DNA polymerase. The similarities in the properties of DHBV and HBV DNA polymerase justify the use of the duck hepatitis B polymerase model for screening and evaluation of potentially active drugs against HBV infection.

Duck hepatitis B virus infection, aflatoxin B1 and liver cancer in ducks.

The association between chronic infection by hepadnaviruses isolated from human (HBV), woodchuck (WHV), ground squirrel (GSHV) and development of hepatocellular carcinoma (HCC) in their respective hosts is well established (reviewed in [11, 15, 17]). By contrast, the association of duck hepatitis B virus (DHBV) infection with HCC is less documented. Pekin ducks congenitally infected with DHBV and followed for several years throughout the world do not develop liver tumors: HCC has been found only in domestic ducks from a single area of China, Qidong. Several factors such as DHBV carrier rate, breed and age of ducks, subtype of DHBV and environmental carcinogens are suspected to contribute to this striking difference between the geographical repartition of liver cancer in DHBV-carrier ducks. In this brief review we will consider successively the role of these different factors in duck liver oncogenesis.

Duck hepatitis B virus (DHBV) as a model for understanding hepadnavirus neutralization.

The role of the immune response to the human hepatitis B virus (HBV) envelope proteins in neutralization of viral infectivity has been well documented. The similarity between HBV, prototype member of the hepadnavirus family, and the closely related duck hepatitis B virus (DHBV) has allowed, use of the latter as a convenient model for the study of molecular mechanisms of HBV replication and neutralization. In this brief review, we will examine the HBV and DHBV envelope proteins and their role as targets for virus neutralization.

Comparative study of DHBV DNA levels and endogenous DNA polymerase activity in naturally infected ducklings in France.

Duck hepatitis B virus (DHBV) was found in the serum of 1-6% of Pekin ducklings originated from French commercial flocks. The viremia was followed in the serum of 5 ducklings over a span of 3 mth by monitoring the levels of DHBV DNA and the endogenous DNA polymerase (DNAp) activity. The DHBV DNA levels in serum were quantified either by the DNA dot hybridization technique including counting of retained radioactivity, or by successive dilutions of each serum sample followed by DNA hybridization. The counting of the retained radioactivity was plotted on a curve and its evolution compared with that of viral DNAp activity. DHBV DNA levels in serum, estimated by both methods paralleled those of the DNAp activity, which peaked at the 4th or 5th week posthatch to decrease and fluctuate thereafter. Occasional discordance between DHBV DNA levels and the endogenous DNAp activity was observed, which could be correlated with the degree of repair of the single stranded gap of serum DHBV DNA. Parallel follow up studies comparing quantitative estimations of serum viral DNA and of DNAp activity, as presented here, may provide some clues for the understanding of the mechanisms involved in the establishment of the HEPA DNA virus carrier state. Such comparative studies may also be crucial for optimal monitoring of antiviral drugs in both human clinical trials and animal experimental studies.

Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS.

Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.

Hepatitis G virus: molecular organization, methods of detection, prevalence, and disease association.

This article reviews data on hepatitis G virus (HGV) prevalence and possible disease associations in various groups of patients. An important fraction of acute or chronic hepatitis cases probably have a viral etiology and are not attributable to known hepatitis viruses. Therefore, researchers continually are looking for new hepatitis viruses. Among the agents found are members of GB hepatitis viruses, including GB-C virus, or HGV. This review presents the history of the discovery of HGV, its molecular biology and some methods of detection; results of clinical and molecular studies of HGV infection also are discussed.

Detection of rabbit haemorrhagic disease virus isolates and sequence comparison of the N-terminus of the capsid protein gene by the polymerase chain reaction.

At present there is no sensitive method for the detection of rabbit haemorrhagic disease virus (RHDV), a calicivirus causing high mortality in rabbit populations. For this purpose a reverse transcriptase polymerase chain reaction (RT-PCR) was established in the N-terminal portion of the RHDV capsid region. The RT-PCR was 10(4)-fold more sensitive than ELISA testing for the detection of the virus and was able to detect as few as 12 copies of template cDNA. By using the RT-PCR test and sequencing, 96.6 to 98.7 per cent homology was demonstrated in the N-terminal portion of the capsid protein of three isolates from geographically and temporally separate outbreaks of viral haemorrhagic disease, indicating that this portion of the RHDV capsid protein is highly conserved.

[Familial hypercholesteremia. Clinico-diagnostic and therapeutic aspects]. / Ipercolesterolemia familiare. Aspetti clinico-diagnostici e terapeutici.

A case of familial hypercholesterolaemia detected in a homozygous subject following the investigation of two large families with a high incidence of the disease, is presented. The genetic background of this form is discussed. Reference is made to the difficulty of identifying heterozygotes and homozygotes and the mode of transmission followed in the two families. The results of 45 months' treatment with cholestiramine and clofibrate and a dietary regimen are described.

Percutaneous ethanol injection of autonomously functioning thyroid nodule.

Percutaneous ethanol injection (PEI) is proposed for treatment of autonomously functioning thyroid nodule, especially for small-mid-sized solitary nodules, for patients refusing the other methods or not proving responsive to radioiodine, or for patients with non toxic nodule. The injection is performed under color Doppler sonography guidance, evaluating the most vascularized areas of the tumor where to primarily inject ethanol solution with 21-22 gauge needles. Serum TSH, FT4, FT3 and TG levels are measured before each treatment session and 3,6,12,24 and 36 months after the end of therapy. Complete remission was achieved by different authors in 86% of cases and the efficacy of response was shown to be inversely proportional to the nodule volume. When TSH remains undetectable, a second cycle of PEI can be performed. If there is complete lack of blood flow signals on color or power Doppler with persisting thyroid hyperfunction, sonographic contrast media can be administered i.v. to assess residual areas of intranodal hypervascularity. In patients with unsuppressed TSH levels before treatment, hormonal changes cannot be used as marker response. Disappearance of nodular hypervascularity at color Doppler sonography and complete normalization of the scintigraphic pattern is usually observed in all cases. PEI is generally well tolerated; no recurrences of the disease and no cases of hypothyroidism have been reported.

[Changes in IGFBP-3 in relation to administration of rGH to deficient children]. / Modificazione dei livelli di IGFBP3 in rapporto alla somministrazione di rGH in bambini deficitari.

Serum IGFBP3 levels were tested in 7 GHD children before the rGH therapy, at the end of the same therapy and 1, 2, 4, and 8 weeks later. The IGFBP3 concentration returned to the pre-therapy levels only 6 weeks after the end of the therapy.

[IGFBP3 in the assessment of growth hormone defects]. / IGFBP3 nella valutazione dei difetti dell'ormone della crescita.

Evaluation of growth hormone dependent IGFBP3, stable in time and with no circadian variations is a useful parameter for diagnosing growth hormone deficit. The IGFBP3 was evaluated using sera, collected and kept at -20 degrees C, from subjects classified according to auxologic characters and response sup. or inf. to 8 ng/ml GH after two stimulation tests and median nocturnal GH sup. or inf. to 3 ng/ml. Two groups were studied: 1) Small stature GHD (growth hormone deficiency): 14 cases; 2) Constitutional small stature (RCC,BSF): 12 cases. A third group composed of 8 normal height, weight and disease-free children formed the control group. The IGFBP3 values were below the 5th percentile in 86% of GHD cases, between the 5th and 95th percentile in 66.6% of constitutionally short stature children and in all normal controls was about the 50th percentile. The IGFBP3 also shows a statistically significant correlation between median nocturnal GH, both in deficient and constitutionally short stature groups (p < 0.01).

Reporte de caso de enfermedad mano-pie-boca con lesiones mucocutáneas que evolucionan a necrosis / A case report of hand, foot, and mouth disease with necrotizing mucocutaneous lesions

La enfermedad de mano-pie-boca es una patología originada en la mayoría de los casos por el virus coxsackie A tipo 16, aunque también puede ser ocasionada por otras cepas de la familia de los coxsackievirus. Dicho virus se propaga principalmente por vía fecal oral y, en menor proporción, por secreciones. Se presenta principalmente en verano, siendo frecuente en niños menores de 10 años. Dentro de dicha enfermedad las lesiones mucocutáneas que evolucionen en necrosis son poco frecuentes, constituyéndose en una complicación severa que requiere hospitalización. En el presente artículo se reporta un caso con diagnóstico de enfermedad mano-pie-boca, que evolucionó hacia lesiones mucocutáneas necróticas, mostrando una respuesta favorable a una terapia de soporte de aciclovir, líquidos y electrolitos.

In most cases, the cause of hand, foot, and mouth disease (HFMD) is coxsackievirus A type 16. The infection can also be caused by other strains of coxsackievirus, spreading mainly by the oral-fecal route, while it is less likely to be transmitted through secretions. HFMD occurs mainly in summer and is more common in children under ten. Skin lesions develop during the disease but rarely become necrotic. When present, they are a severe complication requiring hospitalization. This paper reports the case of a patient with HFMD who developed necrotic mucocutaneous lesions that responded favorably to intravenous acyclovir, fluids, and electrolyte support therapy.

Neurorescue effects and stem properties of chorionic villi and amniotic progenitor cells.

The capability to integrate into degenerative environment, release neurotrophic cytokines, contrast oxidative stress and an inherent differentiation potential towards siteappropriate phenotypes are considered crucial for the use of stem cells in tissue repair and regeneration. Naïve human chorial villi- (hCVCs) and amniotic fluid- (hAFCs) derived cells, whose properties and potentiality have not been extensively investigated, may represent two novel foetal cell sources for stem cell therapy. We previously described that long-term transplantation of hAFCs in the lateral ventricles of wobbler and healthy mice was feasible and safe. In the present study we examine the in vitro intrinsic stem potential of hCVCs and hAFCs for future therapeutic applications in neurodegenerative disorders. Presence of stem lineages was evaluated assessing the expression pattern of relevant candidate markers by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Release of cytokines that may potentialy sustain endogenous neurogenesis and/or activate neuroprotective pathways was quantified by enzyme-linked immunosorbent assays (ELISAs). We also performed an in vitro neurorescue assay, wherein a neuroblastoma cell line damaged by 6-hydroxydopamine (6-OHDA) was treated with hCVC/hAFC-derived conditioned medium (CM). Naïve hCVCs/hAFCs show a neurogenic/angiogenic predisposition. Both cell types express several specific neural stem/progenitor markers, such as nestin and connexin 43, and release significant amounts of brain-derived neurotrophic factor, as well as vascular endothelial growth factor. hCVC and hAFC populations comprise several interesting cell lineages, including mesenchymal stem cells (MSCs) and cells with neural-like phenotypes. Moreover, although CMs obtained from both cell cultures actively sustained metabolic activity in a 6-OHDA-induced Parkinson's disease (PD) cell model, only hCVC-derived CMs significantly reduced neurotoxin-induced apoptosis. In conclusion, this study demonstrates that naïve hAFCs and hCVCs may enhance cell-recovery following neuronal damage through multiple rescue mechanisms, and may provide a suitable means of stem cell therapy for neurodegenerative disorders including PD.

Percutaneous laser ablation of metastatic lymph nodes in the neck from papillary thyroid carcinoma: preliminary results.

CONTEXT: Percutaneous laser ablation (PLA) may be useful in treating patients with metachronous metastatic lymph nodes in the neck. OBJECTIVE: Our objective was to assess PLA as a treatment of difficult-to-treat metachronous cervical lymph node metastases from papillary thyroid carcinoma. DESIGN AND SETTING: We conducted a retrospective analysis of prospectively collected data at a public hospital. PATIENTS: Fifteen patients with previous resection of papillary thyroid carcinoma with elevated serum levels of thyroglobulin (Tg) or anti-Tg antibodies (TgAbs) and 24 metachronous nodal metastases treated between September 2010 and April 2012 were followed with [¹8F]fluorodeoxyglucose (¹8FDG) positron emission tomography (PET)/computed tomography (CT) and contrast-enhanced ultrasound (CEUS). INTERVENTION: Intervention was PLA. OUTCOME MEASURES: Technique feasibility and technical success were evaluated. Tg/TgAb serum levels and ¹8FDG-PET/CT, and CEUS appearance were assessed at 6 and 12 months and compared with baseline. Complications were recorded. RESULTS: PLA was always feasible, and technical success was achieved in all patients. At 6 months, local control was achieved in 11 of 15 patients (73%), with 6 (40%) having serum Tg/TgAb normalized (P = .017 vs baseline). Whereas 20 of 24 (83%) nodes were negative at ¹8FDG-PET/CT and CEUS (P < .001 vs baseline), 4 were ¹8FDG-PET/CT-positive (3 also CEUS-positive). At the 12-month follow-up, local control was achieved in 10 of 14 patients (71.4%). Sixteen of 20 nodes (80%) were negative at ¹8FDG-PET/CT and CEUS (P < .001 vs baseline), 4 were ¹8FDG-PET/CT-positive (2 also CEUS-positive). Four of 10 (40%) patients had normalization of serum Tg/TgAb (P = .098 vs baseline). No major complications occurred. CONCLUSIONS: PLA is potentially feasible, safe, and effective for the treatment of metachronous cervical nodal metastases from papillary thyroid carcinoma. This procedure may reduce or delay a large number of highly invasive repeat neck dissections.

Noninvasive near-infrared live imaging of human adult mesenchymal stem cells transplanted in a rodent model of Parkinson's disease.

BACKGROUND: We have previously shown that human mesenchymal stem cells (hMSCs) can reduce toxin-induced neurodegeneration in a well characterized rodent model of Parkinson's disease. However, the precise mechanisms, optimal cell concentration required for neuroprotection, and detailed cell tracking need to be defined. We exploited a near-infrared imaging platform to perform noninvasive tracing following transplantation of tagged hMSCs in live parkinsonian rats. METHODS: hMSCs were labeled both with a membrane intercalating dye, emitting in the near- infrared 815 nm spectrum, and the nuclear counterstain, Hoechst 33258. Effects of near-infrared dye on cell metabolism and proliferation were extensively evaluated in vitro. Tagged hMSCs were then administered to parkinsonian rats bearing a 6-hydroxydopamine-induced lesion of the nigrostriatal pathway, via two alternative routes, ie, intrastriatal or intranasal, and the cells were tracked in vivo and ex vivo using near-infrared technology. RESULTS: In vitro, NIR815 staining was stable in long-term hMSC cultures and did not interfere with cell metabolism or proliferation. A significant near-infrared signal was detectable in vivo, confined around the injection site for up to 14 days after intrastriatal transplantation. Conversely, following intranasal delivery, a strong near-infrared signal was immediately visible, but rapidly faded and was completely lost within 1 hour. After sacrifice, imaging data were confirmed by presence/absence of the Hoechst signal ex vivo in coronal brain sections. Semiquantitative analysis and precise localization of transplanted hMSCs were further performed ex vivo using near-infrared imaging. CONCLUSION: Near-infrared technology allowed longitudinal detection of fluorescent-tagged cells in living animals giving immediate information on how different delivery routes affect cell distribution in the brain. Near-infrared imaging represents a valuable tool to evaluate multiple outcomes of transplanted cells, including their survival, localization, and migration over time within the host brain. This procedure considerably reduces the number of animal experiments needed, as well as interindividual variability, and may favor the development of efficient therapeutic strategies promptly applicable to patients.

Intracerebroventricular administration of human umbilical cord blood cells delays disease progression in two murine models of motor neuron degeneration.

The lack of effective drug therapies for motor neuron diseases (MND), and in general for all the neurodegenerative disorders, has increased the interest toward the potential use of stem cells. Among the cell therapy approaches so far tested in MND animal models, systemic injection of human cord blood mononuclear cells (HuCB-MNCs) has proven to reproducibly increase, although modestly, the life span of SOD1G93A mice, a model of familial amyotrophic lateral sclerosis (ALS), even if only few transplanted cells were found in the damaged areas. In attempt to improve the potential efficacy of these cells in the central nervous system, we examined the effect and distribution of Hoechst 33258-labeled HuCB-MNCs after a single bilateral intracerberoventricular injection in two models of motor neuron degeneration, the transgenic SOD1G93A and wobbler mice. HuCB-MNCs significantly ameliorated symptoms progression in both mouse models and prolonged survival in SOD1G93A mice. They were localized in the lateral ventricles, even 4 months after administration. However, HuCB-MNCs were not found in the spinal cord ventral horns. This evidence strengthens the hypothesis that the beneficial role of transplanted cells is not due to cell replacement but is rather associated with the production and release of circulating protective factors that may act both at the central and/or peripheral levels. In particular, we show that HuCB-MNCs release a series of cytokines and chemokines with antiinflammatory properties that could be responsible of the functional improvement of mouse models of motor neuron degenerative disorders.

Stem cells in amyotrophic lateral sclerosis: motor neuron protection or replacement?

Given the lack of effective drug treatments for amyotrophic lateral sclerosis (ALS), compelling preclinical data on stem cell research has targeted this disease as a candidate for stem cell treatment. Stem cell transplantation has been effective in several animal models, but the underlying biological pathways of restorative processes are still unresolved. Several mechanisms such as cell fusion, neurotrophic factor release, endogenous stem cell proliferation, and transdifferentiation may explain positive therapeutic results in preclinical animal models, in addition to replacement of lost motor neurons. The clinical target in ALS has shifted from being neuroncentered to focus on the interaction between motor neurons and non-neuronal cells (mainly astroglial or microglial). In fact, one of the fundamental unanswered questions in ALS is whether and how much motor neuron death depends on neighboring cells, and how wildtype non-neuronal cells may protect motor neurons expressing an ALS-causing mutation. Lately, motor neuron replacement has been successfully achieved in animal models with reinnervation of the muscle target. Even if many biological issues need to be solved in preclinical models, preliminary stem cell transplantation trials have been performed in ALS patients with conflicting results. The review discusses relevant topics regarding the application of stem cell research to ALS focusing on their therapeutic relevance and mechanisms of action.