Low prevalence of human papillomavirus infection in the healthy oral mucosa of a Northern Italian population.

BACKGROUND: Oral cancer is the sixth most common malignancy in developed countries, representing almost 3% of malignant tumors. Tobacco use and alcohol consumption are well-established risk factors. However, the observation that most patients with oral cancer have not been exposed to these risk factors suggests that additional causes may promote oral carcinogenesis. A link has been suggested between human papillomavirus (HPV) and oral cavity cancer but the significance of HPV contribution to oral carcinogenesis as well as the prevalence of HPV infection in normal oral cavity mucosa remains debated. METHODS: In this study, the prevalence of oral HPV infection was evaluated in 81 randomly selected Northern Italian subjects with clinically normal oral mucosa using a nested PCR on DNA extracted by oral smears. RESULTS AND CONCLUSIONS: No HPV-related lesions were detectable in any of the smears analyzed by cytological approach. nPCR identified HPV DNA in only one (1.2%) of the specimens obtained from clinically healthy oral mucosa and subsequent characterization assigned the positive case to HPV type 90. These data suggest that the incidence of HPV infection in the healthy population might be very low and that other risk factors are likely responsible to promote oral carcinogenesis.

First human case of Usutu virus neuroinvasive infection, Italy, August-September 2009.

We report the first worldwide case of Usutu virus (USUV) neuroinvasive infection in a patient with diffuse large B cell lymphoma who presented with fever and neurological symptoms and was diagnosed with meningoencephalitits. The cerebrospinal fluid was positive for USUV, and USUV was also demonstrated in serum and plasma samples by RT-PCR and sequencing. Partial sequences of the premembrane and NS5 regions of the viral genome were similar to the USUV Vienna and Budapest isolates.

Usutu virus infections in humans: a retrospective analysis in the municipality of Modena, Italy.

OBJECTIVE: To monitor the spread and to evaluate the role for public health of Usutu virus (USUV) in an endemic area of Italy. METHODS: The survey was retrospectively conducted by detecting USUV RNA and USUV antibodies in cerebrospinal fluid and serum samples collected between 2008 and 2011 from 915 patients with or without neurologic impairments in the area of the municipality of Modena, Italy. Organs of birds and pools of mosquitoes were also tested for USUV RNA. Positive samples were partially sequenced and used for phylogenetic analysis. RESULTS: The presence of USUV RNA (1.1%; 95% confidence interval (CI) 0.6-2.0) was significantly (p <0.05) higher than that of West Nile virus (0%; 95% CI 0-0.33). USUV antibody level was 6.57% (95% CI 4.87-8.82), and it was significantly higher (p <0.05) compared to that of West Nile virus (p 2.96, 95% CI 1.89-4.62). Partial genome sequencing of USUV strains detected in humans, birds and mosquitoes revealed high nucleotide sequence identity within them and with the USUV strains isolated in Central Europe. CONCLUSIONS: USUV infection in humans is not a sporadic event in the studied area, and USUV neuroinvasiveness has been confirmed.

Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma.

The development of hepatocellular carcinoma (HCC) in addition to cirrhosis affects males in a significantly higher proportion than females. Liver estrogen receptors increase when HCC develops in males; however, these tumors usually respond poorly to antiestrogens. We have, therefore, hypothesized that, similar to breast cancer, estrogen receptors in males with HCC may be mutated. Variant estrogen receptor transcripts (lacking exon 5 of the hormone binding domain) were investigated by reverse transcription-PCR in 14 patients (7 males and 7 females) with HCC. While females mostly displayed the wild-type transcript (both in peritumoral and in tumor liver tissue), males showed both transcripts in the cirrhotic tissue and almost only the variant in the tumor. As the variant ER transcripts when translated could give rise to truncated receptors still able to constitutively activate transcription, they may be key factors in favoring deregulated proliferation in the male liver.

Type of estrogen receptor determines response to antiestrogen therapy.

Failure of tamoxifen treatment for unresectable hepatocellular carcinomas (HCCs) might be caused by variant estrogen receptors (ERs) in some of these tumors. We therefore planned a study in which antihormonal therapy was done with 80 mg/day tamoxifen or 160 mg/day megestrol according to the presence of wild-type or exon 5-deleted variant ER transcripts. Growth rate (evaluated by MRI) of HCCs characterized by variant ER transcripts was 4 times more rapid than that of HCCs with wild-type ERs. Tumor volume in all patients with wild-type ERs was halved after 9 months of tamoxifen treatment, whereas megestrol in patients with variant ERs only slowed down tumor growth. Choosing antihormonal treatment according to the presence of wild-type or variant ERs in the tumor definitely improves the response rate to tamoxifen; in patients with tumors bearing variant ERs, megestrol causes only a temporary inhibition of tumor growth.

Selection of more pathogenic hepatitis C virus genotype II during long-term follow-up of interferon-treated patients.

The behavior of hepatitis C virus (HCV) infection with regards to type and number of HCV genotypes (tested with genotype-specific nested polymerase chain reaction) was evaluated in 60 patients with anti-HCV-positive chronic active hepatitis without cirrhosis [17 untreated and 43 subjects undergoing single or repeat courses of interferon (IFN) therapy] during a mean follow-up period of 76 +/- 18 months. In untreated patients (2 genotype I, 6 genotype II, 9 mixed infections) 4 out of 9 mixed infections selected for genotype II at the end of follow-up. Of the 43 treated patients 10 were long-term responders with histological remission, 6 were short-term responders, and 22 did not respond. Fifteen of the latter patients received another course of IFN therapy, and only 3 patients responded. Eight of the 10 responders had infection with a single genotype (4 gt I, 3 gt II, 3 gt III). After IFN therapy, all but 2 patients cleared the HCV infection. The responders to the second IFN course (1 gt I, 1 gt II, 1 gt III) remained viremic. Of the short-term responders, 2/6 patients had genotype II and 4 had a mixed infection (3 gt II +/- I and 1 gt II +/- III); gt III became prevalent in the latter in all but one patient. Of the nonresponders 18/24 had more than one genotype, 5 were genotype II at baseline and one had genotype I. At the end of the follow-up period 15/18 with mixed infection had selected for gt II (P < 0.01 vs. untreated patient).(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogens receptors and oxidative damage in the liver.

There is considerable evidence that reactive oxygen species (ROS) have a causative role in chronic hepatic injury and cancer development via direct and indirect mechanisms. Estrogens produce free oxygen radicals through redox cycling and affect cell proliferation, also in the liver. We are presently involved in evaluating the possible relationship between estrogens receptor expression, type of receptor, oxidative DNA damage and c-myc in chronic liver disease. The data on DNA adducts, c-myc mRNA and variant estrogen receptor in patients with HCV- or HBV-related chronic liver disease are suggesting that those positive for variant liver estrogen receptor present higher genomic oxidative damage, as reflected in 8-OHdG levels. We are also observing that patients with chronic hepatitis and cirrhosis, when positive for variant estrogen receptor, present higher c-myc m-RNA expression, a factor reportedly associated with increased genomic instability, augmented cytoproliferation and carcinogenesis. Our own and other author's data are shedding new light on estrogen pathophysiology, liver damage and hepatic cancer.

Preliminary data on the establishment of permanent cell lines continuously producing HIV-1: factors affecting the transmissibility and adaptability of "wild" HIV-1 isolates.

This study examines some technical aspects of the transmission to and permanent adaptability in continuous cell lines of wild HIV-1 isolates. Three cell systems (the lymphocytic cell lines Molt-3 and H-9 and the monocytoid cell line U-937) and two transmission protocols (cell to cell and cell-free) were used. Two different replicative behaviours were observed among isolates efficiently transmitted: a) transmissibility but not adaptability (consisting in a limited length of viral replication); aa) transmissibility and adaptability (consisting in a stable and long term virus production). The second type of replication was confined to viruses from patients with severe immunodeficiency. Technical and viral factors can affect the rate of transmissibility and adaptability: the modality of infection (cell to cell transmission appeared to be the most efficient) and the tropism of the virus (some viruses could infect only one T cell line).

Detection of human T lymphotropic virus type II/b in human immunodeficiency virus type 1-coinfected persons in southeastern Italy.

A preliminary screening of 511 persons at risk for AIDS living in southeastern Italy disclosed 20 cases of seroreactivity to human T lymphotropic viruses (HTLV). To verify and type the HTLV infection among these subjects, confirmatory serologic tests, polymerase chain reaction (PCR), and virus culture were done. No evidence of HTLV-I infection was found. HTLV-II infection was confirmed in 8 cases by HTLV-specific, synthetic peptide EIAs and PCR on uncultured cells; restriction analysis of the PCR-amplified env regions revealed the presence of HTLV-II/b strains in all 8 cases. Four sera were nontypeable by EIA. The finding of such indeterminate reactivities in a geographic area in which HTLV variants were previously described indicates the need for more extensive surveys among the healthy population. HTLV-II was isolated in 5 cases, and virus isolation was mostly dependent on the presence of an actively replicating human immunodeficiency virus type 1 in culture.

Evidence for hepatitis B virus infection in patients with chronic hepatitis C with and without serological markers of hepatitis B.

To assess the influence of HBV infection on anti-HCV-positive chronic liver disease, we performed a prospective case-control study comparing 19 HBsAg-positive, anti-HCV-positive patients with 38 HBsAg-negative, anti-HCV-positive patients, pair-matched for age, sex, and ALT levels. HBV and HCV infections were investigated by standard serology and polymerase chain reaction. HCV RNA was found in all patients with CAH and in 90.0% with cirrhosis (33% HBsAg-positive). HBV DNA sequences were found, in the HBsAg-positive subjects, in 71.4% of CAH and in 83.3% of cirrhotics; in the HBsAg-negative ones, only 10% of CAH but 77.7% of cirrhotics had demonstrable HBV DNA sequences. Consequently, 80.0% of cirrhotics had evidence of both HBV and HCV infection. Conventional serology gives partial information on the true occurrence of HBV infection in HBsAg-negative patients, while PCR defines more accurately the HBV status. When the rate of double infection is defined in this way, it correlates with the presence of cirrhosis.

HIV-1 isolation from small amounts of whole blood: a technical evaluation.

We have evaluated a simple and sensitive culture technique for isolation of Human Immunodeficiency Virus Type-1 (HIV-1) from small amounts of whole blood. Data shown in the paper demonstrate that: 1) cell cultures from small amounts of heparinized whole blood (HWB) allow a high isolation rate in infected subjects at all stages of diseases; 2) among asymptomatic subjects the HIV-1 isolation rate is increased in cell cultures from HWB, with respect to cell cultures from peripheral blood mononuclear cells; 3) cultural results from HWB are not influenced by the presence of detectable serum p24 antigen, but a good correlation was found with the titre of anti p24 antibodies in serum; 4) continuous cell lines (such as Molt-3 cells) instead of peripheral blood mononuclear cells can be used, obtaining good results, for HIV-1 isolation from HWB; 5) frozen samples of HWB can be used in cell cultures for HIV-1 isolation; 6) the type of anticoagulant (Heparin or EDTA) used for the collection of blood does not influence viral replication in cell cultures from whole blood; 7) viral isolation from HWB is highly sensitive; amounts so small as five microliters of whole blood are sufficient, in some cases, to obtain viral replication in cell cultures; 8) the minimal dose of HWB sufficient to infect cell cultures (HWB M.D.I.) varied among different patients. Although this work failed to establish a correlation between this parameter and the clinical and immunological status of patients, it is conceivable that HWB M.D.I. could give information about viral load in blood and have a prognostic significance; 9) the HWB M.D.I. rise in patients treated with Zidovudine, suggesting that this method could be employed in the virological evaluation of trials with antiretroviral drugs.

High doses of alpha-interferon are required in chronic hepatitis due to coinfection with hepatitis B virus and hepatitis C virus: long term results of a prospective randomized trial.

OBJECTIVE: Coinfection with hepatitis B (HBV) and hepatitis C (HCV) viruses is associated with a more severe liver disease, increased frequency in the development of hepatocellular carcinoma, and resistance to interferon (IFN) therapy when performed with the standard dosages used in single infections. In the attempt to verify whether the outcome of IFN therapy in patients with hepatitis B and hepatitis C coinfection can be improved, we have planned a prospective, randomized trial with medium to high dosages of interferon three times a week for 6 months. METHODS: Thirty patients with HBV-HCV coinfection, and chronic hepatitis were randomized to receive either 6 or 9 MU alpha-interferon three times a week for 6 months. Patients were HBsAg positive, anti-HBe positive, HBV DNA negative by dot blot (6/30 positive by polymerase chain reaction), and anti-HCV-positive, HCV RNA positive. Pretreatment and posttreatment liver biopsies were performed. RESULTS: Five patients treated with 9 MU IFN consistently cleared HCV RNA and HBV DNA, whereas none of those treated with 6 MU reacted in a similar fashion (p = 0.045). Responders showed significant improvement of histological activity index in comparison with nonresponders (mean Ishak score pretreatment versus posttreatment p = 0.002). Long term follow-up showed that none of the patients treated with high doses developed cirrhosis whereas 4/14 treated with low doses did develop cirrhosis. CONCLUSION: Even though the percentage was not very high, the sustained response, the striking histological improvement, and the lack of development of cirrhosis achieved in these patients, indicate that with HBV-HCV coinfection, a trial with high doses of interferon is strongly recommended.

Natural history of inoperable hepatocellular carcinoma: estrogen receptors' status in the tumor is the strongest prognostic factor for survival.

Clinical course in hepatocellular carcinoma may be very different. We prospectively evaluated 96 patients with hepatocellular carcinoma unsuitable for radical therapy to investigate factors that could influence survival. Clinical, pathologic, and molecular data of patients were analyzed by univariate and multivariate analysis. The overall actuarial probability of survival at year 1, 2, 3, 4, 5, and 6 was 72%, 41%, 38%, 24%, 20%, and 9%. At univariate analysis, alpha-fetoprotein (AFP) (P =.0082); alkaline phosphatase (P =.0281); bilirubin (P =.0076); etiology (P =.0001); increment of tumor mass at month 3 (P =.0051); type of estrogen receptor (ER) in the tumor (P =.0000); prothrombin time (P =.0003); and portal vein thrombosis (P =.0000) had prognostic significance. At multivariate analysis, only type of ER (P =.0000) and bilirubin (P =.0030) showed independent predictive value for mortality. Survival was significantly longer in patients with wild-type estrogen receptors (P =.0000). Cumulative probability of survival at year 1, 2, 3, 4, 5, and 6 was 94%, 66%, 52%, 43%, 35%, and 18% for wild-type and 51%, 21%, 16%, and 9% for variant estrogen receptors (no patients alive after 4 years). Hepatitis B surface antigen (HBsAg)-positive patients with variant ERs had a median survival of 8 months versus 45 months in anti-hepatitis C virus-positive patients with wild-type ERs (P =.0001). In conclusion, (1) the presence of variant liver ER transcripts in the tumor was the strongest negative predictor of survival in inoperable hepatocellular carcinoma; (2) their presence was associated with spontaneous survival significantly worse than in patients with wild-type estrogen receptors; and (3) HBsAg-positive patients with variant receptors were characterized by the worst survival.

In vitro production of HIV-1 specific antibodies from "at risk" seronegative subjects.

We investigated a group of well characterized seronegative subjects "at risk" for HIV-1 infection including heterosexual partners of HIV-1 infected subjects and intravenous drug abusers. Pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMCs) were studied for their ability to produce antibodies against HIV-1 structural proteins in vitro. Viral activity by means of HIV-1 isolation from PBMCs and presence of serum p24 antigen were also tested. In 7/42 cases (16.6%) HIV-1 immunoreactive specific antibodies were found, mostly directed against the envelope proteins (gp 120/160). Remarkably, none of these in vitro antibody producers yielded HIV-1 isolation in cell cultures or had detectable serum levels of p24 antigen.

Hormonal therapy with megestrol in inoperable hepatocellular carcinoma characterized by variant oestrogen receptors.

Variant liver oestrogen receptor transcripts in hepatocellular carcinoma are associated with aggressive clinical course and unresponsiveness to tamoxifen. To evaluate the impact on survival and on tumour growth of megestrol (progestin drug acting at post-receptorial level) we enrolled 45 patients with HCC characterized by variant liver oestrogen receptors in a prospective, randomized study with megestrol vs. placebo. Presence of variant oestrogen receptors was determined by RT/PCR. 24 patients were randomized to no treatment and 21 to therapy with megestrol 160 mg day(-1). Results were analysed by Kaplan-Meier and Cox methods. Survival of hepatocellular carcinoma characterized by variant oestrogen receptors was extremely poor (median survival 7 months); megestrol significantly improved survival (18 months) (P = 0.0090). Tumour growth at one year was significantly slowed down in megestrol-treated patients (P = 0.0212). Bilirubin levels, presence of portal thrombosis, HBV aetiology and treatment were identified at univariate analysis as factors significantly associated with survival; at multivariate analysis, only megestrol therapy (P = 0.0003), presence of HBV infection (P = 0.0009) and presence of portal vein thrombosis (P = 0.0051) were factors independently related with survival. (1) Megestrol slows down the aggressive tumour growth of patients with hepatocellular carcinoma characterized by variant estrogen receptors and (2) is also able to favourably influence the course of disease, more than doubling median survival.

Long-term follow-up of hepatitis C virus (HCV) infection in liver transplant patients.

Chronic hepatitis represents a frequent event after orthotopic liver transplantation (OLT). To ascertain the influence of HCV infection on the clinical and histological outcome of these patients, we have investigated the long-term outcome of 22 patients with end-stage chronic liver disease undergoing liver transplant focusing the attention on the role of different HCV genotypes in determining recurrence and severity of post-OLT liver disease. For all patients blood samples taken before OLT and 3 months, 1, 2 and 3 years after OLT were tested for antiHCV antibodies by two different enzyme-linked immuno-assays and by recombinant immuno-blot II and for the presence and type of HCV RNA by nested PCR (5' untranslated region and core gene primers). Of the 16 pre-OLT antiHCV-positive patients, 14 (87.5%) had recurrence of HCV infection while 2 cleared HCV. Pre-OLT genotype recurred in 11 of these 14 patients (2 genotype I) 8 genotype II - in 1 case associated with genotype III - and 1 genotype IV). Of the 6 pre-OLT antiHCV-negative patients, only 1 (16.6%) became persistently HCV-infected, with genotypes I and II. The recurrence of genotype II strictly related with development of severe chronic hepatitis while genotype I and IV were associated with milder forms of liver disease and were more easily cleared.

Identification of subjects at risk for colorectal carcinoma through a test based on K-ras determination in the stool.

BACKGROUND & AIMS: The gold standard for screening for colorectal carcinoma is colonoscopy. The aim of this study was to compare endoscopic results with those obtained using the noninvasive screening test of K-ras determination in the stool in a large population of patients undergoing colonoscopy. METHODS: Two hundred thirty consecutive patients were studied by K-ras amplification on stool-derived DNA using polymerase chain reaction and oligomer-specific hybridization. RESULTS: Wild-type K-ras was amplified in 103 of 230 patients (44.8%), the rate of amplification being directly proportional to the presence of an organic disease of the intestine characterized by hyperproliferating mucosa. In 30 of these 103 patients (29.1%), a K-ras mutation was found. Four of 5 with early colorectal carcinoma, all who had K-ras mutations in the tumor, were identified. In first-degree relatives of patients with colorectal carcinoma, all subjects either carrying adenomas > 1 cm in diameter or multiple smaller adenomas were identified. In patients with inflammatory bowel disease, the test identified the only patient with neoplastic transformation. CONCLUSIONS: The sensitivity and specificity of K-ras determination on stool-derived DNA in patients with colorectal carcinoma, in first-degree relatives of patients with colorectal carcinoma, and in patients with inflammatory bowel disease support the opportunity of a large-scale trial to validate its use as a screening test.