The cobas p 630 instrument: a dedicated pre-analytic solution to optimize COBAS® AmpliPrep/COBAS® TaqMan® system workflow and turn-around-time.

The cobas p 630, a fully automated pre-analytical instrument for primary tube handling recently introduced to complete the Cobas(®) TaqMan systems portfolio, was evaluated in conjunction with: the COBAS(®) AmpliPrep/COBAS(®) TaqMan HBV Test, v2.0, COBAS(®) AmpliPrep/COBAS(®) TaqMan HCV Test, v1.0 and COBAS(®) AmpliPrep/COBAS(®) TaqMan HIV Test, v2.0. The instrument performance in transferring samples from primary to secondary tubes, its impact in improving COBAS(®) AmpliPrep/COBAS(®) TaqMan workflow and hands-on reduction and the risk of possible cross-contamination were assessed. Samples from 42 HBsAg positive, 42 HCV and 42 HIV antibody (Ab) positive patients as well as 21 healthy blood donors were processed with or without automated primary tubes. HIV, HCV and HBsAg positive samples showed a correlation index of 0.999, 0.987 and of 0.994, respectively. To assess for cross-contamination, high titer HBV DNA positive samples, HCV RNA and HIV RNA positive samples were distributed in the cobas p 630 in alternate tube positions, adjacent to negative control samples within the same rack. None of the healthy donor samples showed any reactivity. Based on these results, the cobas p 630 can improve workflow and sample tracing in laboratories performing molecular tests, and reduce turnaround time, errors, and risks.

Aurora B: a new prognostic marker and therapeutic target in cancer.

Aurora B is a serine-threonine kinase belonging to the highly conserved Aurora family of mitotic kinases. Aurora B is a chromosomal passenger protein involved in chromosome segregation, spindle-checkpoint, and cytokinesis. Alteration of each of these steps could induce aneuploidy, one of main features, and driving force of cancer progression. The overexpression of Aurora B has been observed in several tumor types, and has been linked with a poor prognosis of cancer patients. In this review we will focus on the role of Aurora B in cancer development, its role as a prognostic marker, and the clinical outcome of recently developed Aurora(s) inhibitors.

Long-term antibody persistence and immune memory to hepatitis B virus in adult celiac patients vaccinated as adolescents.

Aim of this study was to investigate the anti-HBs antibody persistence and immune memory to hepatitis B virus in adult celiacs vaccinated as adolescents and the effect of a booster administration in non-protected individuals. Eleven years after primary vaccination, the proportion of vaccinees with titres ≥ 10 mIU/ml and antibody geometric mean concentrations (GMCs) were lower among celiac patients than among controls (68.6% vs 91.7%, p<0.01; GMCs 29.38 mIU/ml vs 250.6 mIU/ml, p<0.001). Participants with anti-HBs below 10 mIU/ml received a booster dose and were retested 2 weeks later to assess the anamnestic response. Post-booster anti-HBs levels were still <10 mIU/ml in 71.4% celiacs and 25% controls (p<0.01). Our findings indicate that the prevalence of seroprotective levels of anti-HBs detected eleven years after primary immunization as well as the frequency of response to a booster dose of vaccine are lower in celiac patients compared to healthy controls.

Immunocompromised patients with HBsAg a determinant mutants: comparison of HBsAg diagnostic assays.

Hepatitis B surface antigen (HBsAg) is considered the best marker for the diagnosis of hepatitis B virus (HBV) infection. Mutations of the s gene involving amino acid substitutions within the a determinant could affect the sensitivity of diagnostic tests. In the present study, HBsAg mutants were detected in 3 immunocompromised patients, previously found to be HBsAg negative and anti-HBs positive. All patients had high levels of HBV-DNA, whereas HBsAg tests gave discordant results. Immunosuppression can cause viral reactivation of occult HBV infection in these patients and favour the selection of HBsAg a determinant mutants.

Prolonged persistence of lamivudine-resistant mutant and emergence of new lamivudine-resistant mutants two years after lamivudine withdrawal in HBsAg-positive chronic hepatitis patient: a case report.

Lamivudine can select resistant hepatitis B virus (HBV) tyrosine-methionine-aspartate-aspartate aminoacid motif (YMDD) mutants, which usually disappear in few months after lamivudine withdrawal. We report an unusual case of a male adult patient who showed a prolonged persistence of the M204I mutation up to 24 months after lamivudine withdrawal followed by the emergence of new distinct YMDD mutants (namely M204V, V207L). Only 42 months after lamivudine withdrawal wild-type YMDD motif became dominant over the YMDD mutants. To our knowledge, a so prolonged persistence of a YMDD mutant and also the emergence of new YMDD mutants many months after drug withdrawal are unusual.

Severe reactivation of hepatitis B virus infection in a patient with hairy cell leukemia: Should lamivudine prophylaxis be recommended to HBsAg-negative, anti-HBc-positive patients?

The reverse seroconversion to hepatitis B virus infection has been sporadically described in onco-haematological patients receiving cytotoxic therapy or allogeneic bone marrow transplantation and can be associated with the development of acute icteric hepatitis. We present a male HBsAg-negative, anti-HBc-positive patient with Hairy Cell Leukemia who developed acute B hepatitis more than 1 year after the last course of 2-CdA and 6 months after splenectomy, while the patient was receiving therapy with alphaIFNr. The acute B hepatitis promptly responded to lamivudine therapy followed by viral clearance.

Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas.

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.

Traumatic brain edema in diffuse and focal injury: cellular or vasogenic?

The objective of this study was to confirm the nature of the edema, cellular or vasogenic, in traumatic brain injury in head-injured patients using magnetic resonance imaging techniques. Diffusion-weighted imaging methods were quantified by calculating the apparent diffusion coefficients (ADC). Brain water and cerebral blood flow (CBF) were also measured using magnetic resonance and stable Xenon CT techniques. After obtaining informed consent, 45 severely injured patients rated 8 or less on Glasgow Coma Scale (32 diffuse injury, 13 focal injury) and 8 normal volunteers were entered into the study. We observed that in regions of edema, the ADC was reduced, signifying a predominantly cellular edema. The ADC values in diffuse injured patients without swelling were close to normal and averaged 0.89 +/- 0.08. This was not surprising, as ICP values for these patients were low. In contrast, in patients with significant brain swelling ADC values were reduced and averaged 0.74 +/- 0.05 (p < 0.0001), consistent with a predominantly cellular edema. We also found that the CBF in these regions was well above ischemic threshold at time of study. Taking these findings in concert, it is concluded that the predominant form of edema responsible for brain swelling and raised ICP is cellular in nature.

Continuous cerebral compliance monitoring in severe head injury: its relationship with intracranial pressure and cerebral perfusion pressure.

BACKGROUND: Cerebral compliance expresses the capability to buffer an intracranial volume increase while avoiding a rise in intracranial pressure (ICP). The autoregulatory response to Cerebral Perfusion Pressure (CPP) variation influences cerebral blood volume which is an important determinant of compliance. The direction of compliance change in relation to CPP variation is still under debate. The aim of the study was to investigate the relationship between CPP and compliance in traumatic brain injured (TBI) patients by a new method for continuous monitoring of intracranial compliance as used in neuro-intensive care (NICU). METHOD: Three European NICU's standardised collection of CPP, compliance and ICP data to a joint database. Data were analyzed using an unpaired student t-test and a multi-level statistical model. RESULTS: For each variable 108,263 minutes of data were recorded from 21 TBI patients (19 patients GCS/=20 mmHg in 20% and CPP<60 mmHg for 10.7% of the time. Compliance was lower (0.51+/-0.34 ml/mmHg) at ICP>/=20 than at ICP<20 mmHg (0.73+/-0.37 ml/mmHg) (p<0.0001). Compliance was significantly lower at CPP<60 than at CPP>/=60 mmHg: 0.56+/-0.36 and 0.70+/-0.37 ml/mmHg respectively (p<0.0001). The CPP - compliance relationship was different when ICP was above 20 mmHg compared with below 20 mmHg. At ICP<20 mmHg compliance rose as CPP rose. At ICP>/=20 mmHg, the relation curve was convexly shaped. At low CPP, the compliance was between 0.20 and 0.30 ml/mmHg. As the CPP reach 80 mmHg average compliance was 0.55 ml/mmHg., but compliance fell to 0.40 ml/mmHg when CPP was 100 mmHg. CONCLUSIONS: Low CPP levels are confirmed to be detrimental for intracranial compliance. Moreover, when ICP was pathological, indicating unstable intracranial equilibrium, a high CPP level was also associated with a low volume-buffering capacity.

Three-phase sequential combined treatment with lamivudine and interferon in young patients with chronic hepatitis B.

Alpha-interferon (IFN) or lamivudine monotherapy are ineffective in treating chronic HBeAg positive patients with high viral load and low alanine aminotransferase (ALT) levels. We investigated whether priming lamivudine treatment might enhance the antiviral and immunostimulant action of lamivudine/IFN combination in young tolerant patients. Eleven chronic HBeAg positive patients received: 100 mg/day lamivudine for 3 months followed by IFN 5 MU/m2/tiw with lamivudine 100 mg/day for 6 months and then lamivudine alone 100 mg/day for 9 months. Quantitative hepatitis B virus (HBV)-DNA was evaluated during treatment and core-promoter, precore and polymerase HBV mutants were detected by direct sequencing at the end of therapy. Serum HBV-DNA levels dropped during lamivudine monotherapy and in combination with IFN. After IFN withdrawal, viraemia transiently increased to high levels in five of 11 (45%) patients who showed rt M204V/I lamivudine mutant resistant. Two patients cleared HBeAg without anti-HBe seroconversion. One patient presented core-promoter (A1762T/G1764A) and precore stop codon mutations. Hence, three-phase sequential combined lamivudine/IFN treatment reduced HBV-DNA serum level, but did not lead to HBeAg and HBV-DNA clearance in these highly viraemic, normal ALT patients. Lamivudine/IFN combination did not prevent the emergence of YMDD lamivudine resistance. New schedules of antiviral treatments must be evaluated in this population at risk of disease progression.

Aurora B expression in normal testis and seminomas.

Aurora/Ipl1-related kinases are a conserved family of proteins that have multiple functions during mitotic progression. High levels of Aurora kinases are characteristic of rapidly dividing cells and tumours. Aurora B encodes a protein that associates with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. In this study the expression and the localisation of Aurora B throughout germinal epithelial progression in normal testis and its neoplastic counterpart were analysed. Immunocytochemistry and RT-PCR analysis of mouse germinal epithelium cells showed the presence of Aurora B in spermatogonia and occasionally in spermatocytes. Western blot analysis revealed the typical Aurora B isoform ( approximately 41 kDa) in the same cellular types. A similar distribution was observed in human testis by immunohistochemistry. Moreover, the distribution and the expression of Aurora B were investigated in neoplasms derived from germ cells. Surgical samples of seminomas were analysed, and a high percentage of Aurora B positive cells (51%) was detected; the expression of Aurora B was significantly related to the MIB-1 proliferation marker (R=0.816). The data presented here demonstrate that Aurora B expression occurs in spermatogonial division. Furthermore, our results indicate that the expression of Aurora B is a consistent feature of human seminomas.

Efficacy and limitations of preemptive therapy against cytomegalovirus infections in heart transplant patients.

OBJECTIVES: Cytomegalovirus (CMV) disease often represents a serious complication that promotes opportunistic infections in heart transplant recipients. In this study we evaluated the impact of preemptive gancylovir therapy, guided by pp65 antigenemia on the morbidity associated with viral reactivation. PATIENTS AND METHODS: We have performed a CMV infection surveillance program since March 1999, with antigenemia pp65 determinations weekly for the first 2 months biweekly in the third months, and monthly to the sixth month. Patients with pp65 antigenemia value >/= 10 positive cells per 2 x 10(5) polymorphonuclear cells (PMN) were treated with intravenous gancyclovir followed by 1 month of oral gancyclovir. RESULTS: Among the 107 patients who underwent the virological monitoring, 80 were pp65 antigenemia-positive with preemptive therapy administered in 48 cases. Five patients displayed symptomatic CMV disease (4.7% vs 18% rate in the period of 1988 to 1998 before the introduction of virologic monitoring; P <.01). We observed only one case of gancyclovir-resistant pneumonia which was successfully treated with foscarnet. CMV recurrence in 10 patients required a second cycle of gancyclovir treatment. Our experience included 13 opportunistic infections (12.7%) with 11 antigenemia-positive. CONCLUSIONS: Preemptive therapy drastically reduces the incidence of CMV disease and the associated morbidity. Compared to universal prophylaxis, this approach may avoid unnecessary pharmacologic treatment in more than 50% of transplant recipients. Indeed, preemptive therapy does not fully prevent CMV disease, because it may manifest at the first antigenemia determination, and furthermore may select gancyclovir-resistant strains.

Treatment of fever in neurosurgical patients.

Even moderate temperature elevations soon acute cerebral damage may markedly worsen initial brain injury. These effects may justify aggressive antipyretic treatment in neurosurgical intensive care unit (NICU). On the basis of a literature survey, it is observed that fever is extraordinarily common in the neurosurgical intensive care unit during the acute phase of subarachnoid hemorrhage, stroke, and traumatic brain injury. Several clinical studies also suggest worsened neurologic outcome in patients who are febrile compared to those who are not. Pyrexia is more frequent in infected than noninfected patients. Infections (mainly in the respiratory tract) are usually diagnosed in the majority of febrile NICU patients. Laboratory investigations are quite clear regarding the adverse effects of fever in terms not only of functional outcomes, but also histological and neurochemical injury. Even though fever may cause diagnostic confusion (central fever vs infectious), the potentially devastating effects of pyrexia in patients with cerebral diseases may proceed to treat in any case. An attempt to correct fever appears warranted in all patients with acute cerebral damage in order to obtain a better functional recovery and to limit maximally any further insult to the brain. Some of the more common and innovative methods to control body temperature in order to mitigate the detrimental effects of pyrexia following acute neurological injury are explored. Maintenance of normothermia appears to be a desirable therapeutic goal in managing the patients with damaged or at-risk brain tissue. However, it has not been established conclusively that the benefits of antipyretic therapy outweigh its risks and that despite a sound physiologic argument for controlling fever in the brain-injured patient, there is no evidence that doing so will improve their outcome.

Continuous cerebral compliance monitoring in severe head injury: its relationship with intracranial pressure and cerebral perfusion pressure.

Cerebral Compliance describes the ability of cranial content to accommodate volume variations. Intracranial vascular compartment is thought to be one of the most important determinants of Compliance. Cerebral perfusion pressure (CPP) has a significant influence upon the calibre of cerebral vessels and consequently, upon blood volume. This study was designed to investigate the influence of CPP on intracranial volumes balance, described by cerebral compliance, in severe traumatic brain injured patients (TBI). Nine TBIs were studied. The Spiegelberg ventricular catheter continuously measured ICP and Compliance. Compliance, CPP and ICP were digitally collected for a total of 737 hours of monitoring (44239 total data). Compliance was lower at CPP < 60 than at CPP > or = 60 (0.51 +/- 0.3 versus 0.64 +/- 0.3 ml/mmHg). The ICP level influenced the relation between CPP and Compliance. At ICP < 20 (LICP; 80.3% of data) Compliance and CPP were not significantly related. At ICP > or = 20 mmHg (HICP; 19.7% of data), Compliance varied with changes in CPP. When CPP < 60 mmHg, Compliance showed a trend to decrease as CPP decreased (R2 = 0.85). At CPP > or = 60 mmHg Compliance decreased with CPP (R2 = 0.83). In the range of low CPP vasoparalysis is impending. However, when ICP is pathological, at high CPP our results may express vasodilatation instead of expected vasoconstriction from normal autoregulation.

[Role of assisted breathing in severe traumatic brain injury]. / La ventilazione assistita nel trauma cranico grave.

BACKGROUND: Based on available data, there is no definite clinical research describing option, timing and effects of assisted as opposed to controlled ventilation to successfully treat acute severely brain-injured patients. This study demonstrates pressure support ventilation as a possible alternative to controlled ventilation in the acute phase of brain injury. We illustrated which factors influenced the shift from total (CPPV) to partial ventilatory support (PS-SIGH) and the consequences of assisted ventilation on cerebral hemodynamics. METHODS: a) EXPERIMENTAL DESIGN: Retrospective, cohort study. b) SETTING: Adult intensive care unit of a university hospital. c) Patients population: Forty-two severe head-trauma victims (GCS

Control by the endogenous cannabinoid system of ras oncogene-dependent tumor growth.

We investigated the effect of 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA), a stable analog of the endocannabinoid anandamide, on a rat thyroid epithelial cell line (FRTL-5) transformed by the K-ras oncogene, and on epithelial tumors derived from these cells. Met-F-AEA effect in vivo was evaluated in a nude mouse xenograft model, where K-ras-transformed (KiMol) cells were implanted subcutaneously. Met-F-AEA (0.5 mg/kg/dose) induced a drastic reduction in tumor volume. This effect was inhibited by the CB1 receptor antagonist SR141716A (0.7 mg/kg/dose) and was accompanied by a strong reduction of K-ras activity. Accordingly, KiMol cells and tumors express CB1 receptors. Met-F-AEA inhibited (IC50 ~5 mM) the proliferation in vitro and the transition to the S phase of KiMol cells and it reduced K-ras activity; these effects were antagonized by SR141716A. Met-F-AEA cytostatic action was significantly smaller in nontransformed FRTL-5 cells than in KiMol cells. Met-F-AEA treatment exerted opposite effects on the expression of CB1 receptors in KiMol and FRTL-5 cells, with a strong up-regulation in the former case and a suppression in nontransformed cells. The data suggest that: 1) Met-F-AEA inhibits ras oncogene-dependent tumor growth in vivo through CB1 cannabinoid receptors; and 2) responsiveness of FRTL-5 cells to endocannabinoids depends on whether or not they are transformed by K-ras.

Human papilloma virus 16 E7 oncogene does not cooperate with RET/PTC 3 oncogene in the neoplastic transformation of thyroid cells in transgenic mice.

We have previously reported that the thyroid-targeted expression of the RET/PTC3 oncogene (Tg-RET/PTC3) in transgenic mice induces follicular hyperplasia with papillary architecture, resulting in a modest increase of the thyroid gland volume, followed by the appearance of papillary carcinomas in approximately 1-year-old animals. In order to analyze the genetic alterations that may cooperate with RET/PTC3 in the development or progression of thyroid tumors, we interbred Tg-RET/PTC3 mice with Tg-E7 transgenic mice, which express the E7 oncogene of the human papilloma virus 16 in thyroid cells. Tg-E7 mice develop large colloid goiters with small papillae and well-differentiated thyroid carcinomas in older animals. Here we show that thyroid lesions in Tg-RET/PTC3-Tg-E7 double transgenics were morphologically different from those occurring in Tg-RET/PTC3 mice, while they were virtually indistinguishable from those occurring in Tg-E7 mice. In addition, the coexpression of RET/PTC3 and E7 oncogenes neither enhanced the malignant phenotype nor reduced the latency period of thyroid lesions with respect to parental transgenic lines. We conclude that the coexpression of RET/PTC3 and E7 lacks any cooperative effect in the neoplastic transformation of thyroid cells and that the E7-induced thyroid phenotype is dominant with respect to the RET/PTC3 one.

HMGI-C gene expression is not required for in vivo thyroid cell transformation.

We have previously demonstrated that HMGI proteins are required for the transformation of rat thyroid cells by v-mos and v-ras-Ki oncogenes. To determine whether HMGI proteins are also required for in vivo thyroid carcinogenesis, mice carrying a disrupted HMGI-C gene (pygmy mice) were either treated with radioactive iodine or crossed with transgenic mice carrying the E7 papilloma virus oncogene under the transcriptional control of thyroglobulin gene promoter. The pygmy mice developed thyroid carcinomas with the same frequency as occurred in wild-type mice without significant macroscopic and microscopic differences. Therefore, these results indicate that HMGI-C gene expression is not required in in vivo thyroid cell malignant transformation.

HCV and GBV-c/HGV infection in HIV positive patients in southern Italy.

Flaviviridae-hepatitis C virus (HCV) and GB virus C/hepatitis G virus (GBV-C/HGV)--and human immunodeficiency virus (HIV) frequently show similar modes of transmission. HCV and GBV-C/HGV infection was assessed in 134 consecutive patients with evidence of HIV infection, living in Campania, Italy. Data obtained from this cohort were compared with those obtained from 252 age- and sex-matched HCV infected patients without evidence of HIV infection (HCV control group). Following enzymatic immunoassays, samples were tested for the presence of HCV-RNA by RT-PCR. The HCV-RNA positive sera were genotyped by LiPA procedure. The prevalence of HCV infection in HIV patients was 19.40% and the largest group of HIV-HCV co-infected patients (84.62%) was represented by intravenous drug users (IVDU). The distribution of HCV genotypes in HIV-HCV patients was different, compared to that observed in HCV control group. HCV genotypes la (50%) and 3a (23.08%) were more frequently detected in HIV HCV patients, compared to HCV control group (5.16 and 5.56% for la and 3a, respectively). Conversely, HCV genotypes lb (55.70%) and 2a/2c (30.26%) were more represented in HCV control group, compared to HIV-HCV patients (15.38 and 0% for lb and 2a/2c, respectively). GBV-C/HGV seroprevalence was 41.04% in HIV patients and 6.54% in healthy control individuals. Differently from HCV, GBV-C/HGV infection did not correlate to a preferential risk behaviour in the HIV cohort. Comparative analysis of HCV and GBV-C/HGV infection indicates that the use of injecting drugs might play a key role in the epidemiology of HCV and, in particular, of la and 3a HCV genotypes, in HIV patients.