Diversity of T-cell antigen receptor variable genes used by mycosis fungoides cells.

The expression of T-cell antigen receptor beta-chain variable genes (V beta) was evaluated in 28 cases of mycosis fungoides. A novel polymerase chain reaction (PCR) technique was used to associate expression of particular V beta genes with monoclonal T-cell populations. In addition, the same biopsies used for PCR analysis were also examined for reactivity with a panel of seven monoclonal antibodies that specifically recognized V beta proteins from four different families. Only three cases clearly stained with the antibodies, a result consistent with a diverse set of V beta genes being used. This was confirmed by PCR analysis, which indicated that V beta genes from many different families were expressed by these tumors. Preferential use of the V beta 8 family, which had been previously use of the V beta 8 family, which had been previously reported for this disease, was not evident among the cases analyzed.     

Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Galactose metabolism in mice with galactose-1-phosphate uridyltransferase deficiency: sucklings and 7-week-old animals fed a high-galactose diet

Mice deficient in galactose-1-phosphate uridyltransferase (GALT) demonstrate abnormal galactose metabolism but no obvious clinical phenotype. To further dissect the pathways of galactose metabolism in these animals, galactose oxidation and metabolite levels were studied in 16-day-old sucklings and the effect of a 4 week prior exposure to a 40% glucose or 40% galactose diet was determined in 7-week-old mice. Suckling GALT-deficient (G/G) mice slowly oxidized [1-14C]galactose to 14CO2, 4.0% of the dose when fed and 7.9% when fasted compared to normal animals 38.3 and 36.4% in 4 h, respectively. Plasma of G/G sucklings contained 11.1 mM galactose and erythrocyte galactose 1-phosphate levels were 28.2 and 31.9 mg/dl packed cells. Galactose, galactitol, galactonate, and galactose 1-phosphate were found in G/G suckling mouse tissues. The tissue galactose concentrations were 10% or less of that in plasma, suggesting that there was limited cellular entry of galactose. In 7-week-old fasted mice with 4 weeks prior exposure to glucose or galactose-containing diet, 4-h oxidation was 12.9 and 15.0% of the administered radiolabeled galactose, respectively. Normal animals oxidized 33.9 and 37.9% of the dose when fed the same diets, respectively. The ability of G/G mice to oxidize galactose in the absence of GALT activity suggests the presence of alternate metabolic pathways for galactose disposition. G/G mice fed the galactose-free 40% glucose diet had erythrocyte galactose 1-phosphate levels ranging from 6.4 to 17.7 mg/dl packed cells and detectable galactose and galactose metabolites in tissues, suggesting that these animals endogenously produced galactose. The plasma of 40% galactose-fed G/G mice contained 9.1 mM galactose with red blood cell galactose 1-phosphate averaging 43.6 mg/dl. Tissues of these animals also contained high levels of galactose and galactose 1-phosphate. Liver contained over 4 micromol/g galactonate but little galactitol. Despite the elevated galactose and galactose 1-phosphate, the animals tolerated the high-galactose diet and were indistinguishable from normal animals, exhibiting no manifestations of galactose toxicity seen in human GALT-deficient galactosemia. The data suggest that high galactose 1-phosphate levels do not cause galactose toxicity and that high galactitol in combination with galactose 1-phosphate may be a prerequisite. Absence of GALT appears necessary but insufficient to produce human galactosemic phenotype.     

p53, Ki-67, and serum alpha feto-protein as predictors of hepatocellular carcinoma recurrence in liver transplant patients.

Patients with hepatocellular carcinoma who undergo orthotopic liver transplantation (OLT) are at risk for post-transplant tumor recurrence. The aim of this study was to evaluate whether expression of p53 and Ki-67 in hepatocellular carcinoma lesions present in explanted liver tissue was associated with time to tumor recurrence after OLT. Subjects consisted of 20 consecutive patients who underwent OLT and were found to have hepatocellular carcinoma in the liver explant. Immunostaining for p53 and Ki-67 was performed by standard methods. The presence of nuclear immunostaining in >10% of the tumor tissue was considered positive. Time to recurrence of hepatocellular carcinoma after OLT was compared between patients with positive and negative immunostaining by the log rank test. Multivariate analysis was performed using a Cox regression model to control for potentially confounding clinical factors. Time to post-transplant hepatocellular carcinoma recurrence was significantly more rapid in p53+ (P=0.0007) and Ki-67+ cases (P=0.001). These associations remained significant in multivariate analysis. Furthermore, time to recurrent hepatocellular carcinoma was significantly shorter in patients with a serum alpha feto-protein (AFP) level >or=100 ng/ml at time of diagnosis, compared to those with an AFP level <100 ng/ml (P=0.003). In conclusion, expression of p53 and Ki-67 in hepatocellular carcinoma lesions, and a serum AFP level >or=100 ng/ml were associated with more rapid recurrence of hepatocellular carcinoma after OLT. Identification of patients at risk for early post-transplant recurrence could be used to guide surveillance and adjuvant treatment strategies.     

Examination of the Short-Term Efficacy of a Parent-Based Intervention to Prevent Skin Cancer

The research evaluated an intervention strategy designed to prevent skin cancer in young adolescents. The intervention used parents as change agents to effectively communicate the risks of skin cancer and encourage their children to avoid high-risk sun-related behaviors while increasing positive sun-safe behaviors. Three hundred and forty parents in two regions of the United States were educated about the dangers of risky sun behaviors and how to convey information about skin cancer prevention to their children. Parents were then encouraged to talk with their children about these issues over a 1-month period prior to the onset of summer. Following this time period, children whose parents received and implemented the intervention materials were compared with a control sample of 129 children. These two groups were matched on age, gender, and school on number of sunburns and sunburn severity, attitudes and beliefs, and sunbathing behavior. Children in the treatment condition differed significantly from controls in the predicted directions on all outcome variables. The findings are discussed in terms of reducing skin cancer risk behaviors of children via parent-based intervention approaches.     

Roxithromycin, Clarithromycin, and Azithromycin Attenuate the Injurious Effects of Bioactive Phospholipids on Human Respiratory Epithelium in Vitro

The effects of the bioactive phospholipids (PL), platelet-activating factor (PAF), lyso-PAF, and lysophosphatidylcholine (LPC) on the beat frequency and structural integrity of human ciliated respiratory epithelium were studied in vitro, in the presence or absence of polymorphonuclear leukocytes (PMNL), the antimicrobial agents, roxithromycin, clarithromycin, and azithromycin and the antioxidative enzymes catalase and superoxide dismutase (SOD). All three PL caused dose-dependent slowing of ciliary beat frequency (CBF) and epithelial damage (ED) at concentrations 1 g/ml, which were unaffected by inclusion of the antimicrobial agents and antioxidative enzymes. When epithelial strips were exposed to the combination of PMNL and PL, there was significant potentiation of ciliary dysfunction and ED, which was ameliorated by pretreatment of the PMNL with the antimicrobial agents or by inclusion of catalase, but not SOD. These results demonstrate that LPC, PAF, and lyso-PAF cause epithelial damage by direct mechanisms which are oxidant-independent, as well as by indirect mechanisms involving phagocyte-derived reactive oxidants. Macrolides and azalide antimicrobial agents may have beneficial effects on airway inflammation in asthma and microbial infections by protecting ciliated epithelium against oxidative damage inflicted by PL-sensitized phagocytes.     

Fluid and solid mechanical implications of vascular stenting

Vascular stents have emerged as an effective treatment for occlusive vascular disease. Despite their success and widespread use, outcomes for patients receiving stents are still hampered by thrombosis and restensosis. As arteries attempt to adapt to the mechanical changes created by stents, they may in fact create a new flow-limiting situation similar to that which they were intended to correct. In vitro fluid mechanics and solid mechanics studies of stented vessels have revealed important information about how stents alter the mechanical environment in the arteries into which they are placed. Adverse nonlaminar flow patterns have been demonstrated as well as remarkably high stress concentrations in the vessel wall. In vivo studies of stented vessels have also shown a strong relationship between stent design and their dynamic performance within arteries. Alterations in pressure and flow pulses distal to the stent have been observed, as well as regional changes in vascular compliance. Considering the influence of flow and stress on the vascular response and the suboptimal clinical outcomes associated with stenting, knowledge gained from stent/artery mechanics studies should play an increasingly important role in improving the long-term patency of these devices. © 2002 Biomedical Engineering Society. PAC2002: 8719Rr, 8780-y, 8719Uv