IL28B polymorphisms, IP-10 and viral load predict virological response to therapy in chronic hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma and the identification of the predictors of response to antiviral therapy is an important clinical issue. AIM: To determine the independent contribution of factors including IL28B polymorphisms, IFN-gamma inducible protein-10 (IP-10) levels and the homeostasis model assessment of insulin resistance (HOMA-IR) score in predicting response to therapy in chronic hepatitis C (CHC). METHODS: Multivariate analysis of factors predicting rapid (RVR) and sustained (SVR) virological response in 280 consecutive, treatment-naive CHC patients treated with peginterferon alpha and ribavirin in a prospective multicentre study. RESULTS: Independent predictors of RVR were HCV RNA <400 000 IU/mL (OR 11.37; 95% CI 3.03-42.6), rs12980275 AA (OR 7.09; 1.97-25.56) and IP-10 (OR 0.04; 0.003-0.56) in HCV genotype 1 patients and lower baseline γ-glutamyl-transferase levels (OR=0.02; 0.0009-0.31) in HCV genotype 3 patients. Independent predictors of SVR were rs12980275 AA (OR 9.68; 3.44-27.18), age <40 years (OR=4.79; 1.50-15.34) and HCV RNA <400 000 IU/mL (OR 2.74; 1.03-7.27) in HCV genotype 1 patients and rs12980275 AA (OR=6.26; 1.98-19.74) and age <40 years (OR 5.37; 1.54-18.75) in the 88 HCV genotype 1 patients without a RVR. RVR was by itself predictive of SVR in HCV genotype 1 patients (OR 33.0; 4.06-268.32) and the only independent predictor of SVR in HCV genotype 2 (OR 9.0, 1.72-46.99) or genotype 3 patients (OR 7.8, 1.43-42.67). CONCLUSIONS: In HCV genotype 1 patients, IL28B polymorphisms, HCV RNA load and IP-10 independently predict RVR. The combination of IL28B polymorphisms, HCV RNA level and age may yield more accurate pre-treatment prediction of SVR. HOMA-IR score is not associated with viral response.

Long-term outcome of chronic hepatitis B in Caucasian patients: mortality after 25 years.

OBJECTIVE: To assess risk factors for liver-related death, we re-evaluated, after a median follow-up of 25 years, a cohort of 70 Caucasian patients with hepatitis B e antigen (HBeAg) positive chronic hepatitis (CH) at presentation. METHODS: Follow-up studies included clinical and ultrasound examinations, biochemical and virological tests, and cause of death. RESULTS: Sixty-one (87%) patients underwent spontaneous HBeAg seroconversion. During a median period of 22.8 years after HBeAg seroclearance, 40 (66%) patients became inactive carriers, whereas the remaining 21 (34%) showed alanine aminotransferase elevation: one (1%) had HBeAg reversion, nine (15%) detectable serum HBV DNA but were negative for HBeAg, eight (13%) concurrent virus(es) infection and three (5%) concurrent non-alcoholic fatty liver disease. Liver-related death occurred in 11 (15.7%) patients, caused by hepatocellular carcinoma in five and liver failure in six. The 25-year survival probability was 40% in patients persistently HBeAg positive, 50% in patients with HBeAg negative CH or HBeAg reversion and 95% in inactive carriers. Older age, male sex, cirrhosis at entry and absence of sustained remission predicted liver-related death independently. The adjusted hazard ratios (95% CI) for liver related death were 33 (3.01-363) for persistently HBeAg positive patients and 38.73 (4.65-322) for those with HBeAg negative CH or HBeAg reversion relative to inactive carriers. CONCLUSION: Most patients with HBeAg seroconversion became inactive carriers with very good prognosis. The risk of liver-related mortality in Caucasian adults with CH is strongly related with sustained disease activity and ongoing high level of HBV replication independently of HBeAg status.

Evidence of Neospora caninum DNA in wild rodents.

Seventy-five house mice (Mus musculus), 103 rats (Rattus norvegicus) and 55 field mice (Apodemus sylvaticus) from North-West Italy were PCR analysed for Neospora caninum infection. Brain, kidney and muscle tissues collected from the above mentioned animals were tested by PCR using Np6 and Np21 primers. The brain tissue from 2 house mice and 2 rats, the kidney from 4 rats, 1 house mouse and 1 field mouse and muscle from 10 rats, 8 house mice and 1 field mouse were tested positive for N. caninum. Sequencing showed a 96-97% identity of PCR products with N. caninum NC1 sequence. Our findings support previous report on house mouse and rat, and for the first time, provides the evidence of the infection also in field mice. Based on our data, it could be hypothesized that mice can act as a reservoir of N. caninum, and they can play a role in maintaining/spreading N. caninum infection also in the sylvatic cycle. The possibility that dogs could be infected by eating infected house mice suggests new opportunities for N. caninum prophylaxis and control.

Seroprevalence of antibodies to Neospora caninum in urban and rural dogs in north-west Italy.

Sera were collected from 490 dogs from north-west Italy. One hundred and eighty-eight dogs were urban, while 302 dogs were rural. Among the latter, 190 were shepherd dogs and 112 were cattle farm dogs. Sera were tested for the presence of antibodies against Neospora caninum using the Neospora agglutination test. Seroprevalence at 1/40, 1/80, 1/160 dilution titres was significantly higher in rural (36.4%, 19.5%, 9.9% respectively) than in urban dogs (20.2%, 10.6%, 4.8% respectively). Seroprevalence did not differ significantly in males and females. In shepherd dogs, prevalence increased according to dogs' age, thus suggesting a post-natal exposure by horizontal transmission. The observed higher seroprevalence in rural dogs suggests the importance of lifestyle and alimentary habits (i.e. aborted foetuses, placentas and small mammals) in the acquisition of N. caninum infection. Our results confirm that dogs are exposed to N. caninum and play an important role in the epidemiology of N. caninum.

Insight into molecular changes of the FIX protein in a series of Italian patients with haemophilia B.

Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X-linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause-effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software-constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease.

Antibodies to Neospora caninum in stray cats from north Italy.

Antibodies to Neospora caninum were determined in serum samples from 282 stray cats coming from four colonies near Turin (north-west Italy). Sera were tested using a Neospora Agglutination Test (NAT). Seroprevalence was 24.8% at 1:80, 12.8% at 1:160 and 5.3% at 1:320 dilution. Seroprevalence in females and males, in different colonies and in different age classes, did not differ significantly. Our results confirm that domestic cats are exposed to N. caninum and the observed seroprevalences suggest that risk of exposure is high and N. caninum should be considered in differential diagnosis in cats with neurological signs.

Urinary nitrite/nitrate concentrations and total antioxidant capacity in patients with chronic hepatitis C in therapy with interferon and ribavirin.

In this study we tried to elucidate the role of nitric oxide (NO) in chronic hepatitis C in relation to antioxidant status, since the mechanisms by which hepatitis C virus (HCV) causes cell damage and the factors underlying its resistance to therapy are not well understood. Before and after one and six months of therapy with alpha-interferon and ribavirin, we measured nitrite/nitrate urinary levels, total antioxidant capacity and selenium serum concentrations in 14 patients with chronic hepatitis C and in 9 healthy subjects. Before therapy, mean urinary nitrite/nitrate levels of patients were not different from those of healthy subjects, but after a 6-month treatment with alpha-interferon and ribavirin, these NO metabolites were higher in virologically negative patients (responders). Moreover, while no changes in selenium were observed in all patients, total antioxidant capacity was significantly higher in non-responders and well correlated with hyperuricemia (due to cell damage) observed in these subjects. Instead, uric acid decreased as free molecule in serum in responders, while we found the excretion of high NO levels as nitrite/nitrate. Our data allow us to hypothesize a role for NO as predictive of the success of therapy, since nitrite/nitrate increase in the urine of some patients precedes disappearance of the virus observed at the end of therapy.

Germ-line origin of intron 1 inversion in two haemophilia A families.

Factor VIII gene inversion of intron 1 has recently been reported to be the mutation responsible for haemophilia A in about 5% of severe cases. In our series of patients, which is made up of 77 Italian cases negative for intron 22 inversion, the mutation was found in three sporadic and in one familial patients, with an overall frequency of 5.2%. The carrier status of the patients' female relatives was assessed by mutation analysis and showed that only two-thirds of cases could be considered truly sporadic. The germ-line origin of the mutation was investigated in the two sporadic families by haplotype analysis on genomic DNA of the patients' maternal grandparents. These studies indicated that both mutation events had occurred in the germ cell lines of the patients' healthy grandfather, suggesting that, as already demonstrated for the inversion of intron 22, the male germ cell line is more susceptible to the intrachromosome recombination which leads to the inversion of intron 1.

Flow cytometric and functional characterization of AC133+ cells from human umbilical cord blood.

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte-macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62.3%, and median AC133+ population purity was 97.9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

Congenital hypoplastic anaemia in a patient with a new multiple congenital anomalies-mental retardation syndrome.

We report on a girl with congenital hypoplastic anaemia, "coarse" face, generalized hypertrichosis with scalp hypotrichosis, short fifth finger, hypoplastic toenails, and mental retardation. A sister of the proposita, who died at the age of 1 year, had severe congenital anaemia, hypoplastic fingernails, low birth weight, failure to thrive, and repeated upper respiratory tract infections. Based on family history, we suspect that hypoplastic anaemia and the same multiple congenital anomalies-mental retardation syndrome (MCA/MR) were also present in this sister. To the best of our knowledge, this patient represents the first report of congenital hypoplastic anaemia and such a complex MCA/MR syndrome, probably inherited as an autosomal recessive trait.

Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells.

BACKGROUND AND OBJECTIVE: The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors. DESIGN AND METHODS: Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS: The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. INTERPRETATION AND CONCLUSIONS: These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.

Detection of megakaryocyte colonies in plasma clot cultures by immunoenzymatic staining.

In vitro induced megakaryocytic differentiation/maturation of megakayocyte (meg) progenitors represents an important tool for investigating cytokine-induced in vitro thrombocytopoiesis. We have developed an assay which allows the in situ study of human meg progenitor-derived colonies, cultured on a plasma clot in the presence of cytokines. Plates were immunostained by using an anti-alpha IIb beta 3 monoclonal antibody and an alkaline phosphatase-labeled secondary antibody. alpha IIb beta 3-bearing cells were stained an intense red and were clearly differentiated from the negative cells. Processed plates were stable for some weeks at 4 degrees C. The described procedure is easy to perform and allowed us to enumerate the meg colonies and assess colony morphology and cell ploidy.

Collection of peripheral blood hematopoietic progenitors (PBHP) from patients with severe aplastic anemia (SAA) after prolonged administration of granulocyte colony-stimulating factor.

The aim of this study was to test whether prolonged administration of granulocyte colony-stimulating factor (G-CSF) would allow the collection by leukapheresis of PBHP in patients with SAA. For this purpose, nine SAA patients, 7 to 46 years old, six of whom were enrolled at diagnosis of their disease and three after previous immunosuppression had failed, were treated with antilymphocyte globulin (ALG) (day 1 to 5), cyclosporin A (5 mg/kg/d orally) (day 6 to 90) and G-CSF 5 micrograms/kg/d (day 6 to 90). A total of 40 leukaphereses were performed, (range 2 to 7 per patient), between days +10 and +168 from G-CSF treatment. White blood cell count at the time of harvest ranged from 1.2 to 18.1 x 10(9)/L. Results can be summarized as follows: the median number of cells collected per patient was 5.0 x 10(8)/kg (range 2.6 to 18.7), the median number of CD34+ cells was 1.8 x 10(6)/kg (range 0.27 to 3.8) and the median number of colony-forming units granulocyte-macrophage (CFU-GM) was 3.9 x 10(4)/kg (range 0 to 39). Twenty leukaphereses performed between days +33 and +77 of G-CSF treatment grew granulocyte macrophages and erythroid colonies in vitro. No colony growth was obtained from 20 leukaphereses performed before day +33 or after day +80. In six patients the total number of CFU-GM recovered were in the range described for autologous peripheral blood stem cell grafts. (2.6 to 39 x 10(4)/kg). In conclusion, this study suggests that circulating hematopoietic progenitors can be recovered after ALG priming and after at least 1 month of G-CSF treatment in a proportion of patients with SAA. Whether these cells will be suitable for autologous transplantation remains to be determined.

Acute lymphoblastic leukemia in Noonan syndrome: report of two cases.

Only two cases of Noonan Syndrome (NS) associated with tumor have previously been reported. The authors describe two new cases of NS with acute lymphoblastic leukemia (ALL), which were part of a series of 370 consecutive ALL untreated patients.

Chronic childhood neutropenia: studies on the in vitro clonogenicity of bone marrow myeloid progenitors.

Bone marrow mononuclear cells (MNC) from 6 pediatric patients with chronic neutropenia were tested for myeloid colony formation upon stimulation with the supernatant of the 5637 cell line or with recombinant granulocyte-macrophage colony-stimulating factor or interleukin 3. Heterogeneous patterns of response of myeloid progenitors were observed in the individual patients, with no colony growth in 2 cases and abnormalities of colony formation or composition in two additional cases. Morphologic and surface marker analyses showed that the bone marrow of some patients contained an excess of lymphocytes with an altered subset distribution. In order to investigate whether or not there was a relationship between the latter abnormality and the observed clonogenic defects, marrow MNC were tested for myeloid colony formation before and after lymphocyte depletion. No evidence for a cell-mediated suppression of colony growth was obtained; likewise, patient sera failed to inhibit colony formation by normal bone marrow myeloid progenitors. Taken together, these data make it unlikely that, in our cases, immunologic mechanisms were responsible for the pathogenesis of chronic neutropenia.