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Purpose: In the present study, we investigated the prognostic value of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 serum levels in patients with metastatic non-small cell lung cancer (NSCLC)., Patients and Methods: From September 1999 to June 2001, pretreatment serum levels of VEGF and MMP-9 were analysed in 194 patients of a randomized phase III trial with enzyme-linked immunoassays., Results: Patients with a VEGF serum level higher than the median serum level (10,995 pg/ml) had a significantly shorter overall survival than those with a lower serum level (P=0.04). The MMP-9 serum level did not correlate with survival. In a multivariate Cox regression analysis, only the pretreatment serum level of VEGF, the Karnofsky performance status, and the presence of bone metastases were identified as independent prognostic factors., Conclusions: The pretreatment VEGF serum level was identified as independent prognostic factor in this study and may help to assess individual risk and treatment profiles in patients with metastatic NSCLC.

Background: Lectins, carbohydrate proteins, bind to glycoconjugates of all mammalian cells, including cancer cells. Aberrant glycosylation, detected by lectin histochemistry, can predict outcome in some tumour entities. One such lectin is aviscumine (recombinant mistletoe lectin). Aviscumine has cytotoxic effects and can therefore be used as anti-tumour therapy., Materials and Methods: Lectin histochemistry with aviscumine was performed on primary tumour sections from resected adenocarcinoma of the lung. Staining results were then correlated with the clinical course of the patients., Results: Most of the adenocarcinomas (92.5%) bound aviscumine. Kaplan-Meier analysis revealed no correlation between aviscumine binding and progression-free survival or overall survival., Conclusion: These results suggest that for the selected group of patients with adenocarcinoma of the lung aviscumine binding activity can not serve as a prognostic factor. More strikingly, however, aviscumine binds to malignant cells in 92.5% of the patients. This is an indicator for the use of aviscumine as a possible target for tumour therapy.

Background: Magic Roundabout (MR; ROBO 4) is a recently discovered gene which encodes a protein that derived its name form the structural homology with the roundabout family of genes. Genes of the roundabout family comprise neuronal-specific cell surface receptors that are involved in axon guidance. MR in contrast showed endothelial specificity in vitro and in vivo using immunohistochemistry and in situ hybridisation. The putative role of MR as an endothelial analogue of Roundabout in angiogenesis makes it an attractive target for anti-angiogenic cancer therapy., Patients and Methods: Using specific antibodies against MR peptide, we screened pretreatment sera from 193 patients with advanced non-small cell lung cancer (NSCLC) for MR-protein levels., Results: Patients with serum levels of MR-protein lower than median (E(450 nm) = 0.652) had a median survival of 41.0 weeks whereas those with a higher serum level had a considerably shorter median survival of 32.4 weeks (P = 0.05). The pretreatment serum level of MR-protein achieved no significance in a univariate Cox regression analysis., Conclusions: This is the first study to present clinical data that link MR expression with outcome in patients with NSCLC, whether the correlation of pretreatment serum levels of MR and survival can be attributed to MR dependent angiogenesis remains to be investigated.

Purpose: To retrospectively analyze the outcomes and benefits from radiation therapy (RT) as a component of multimodal treatment for oligodendroglioma and oligoastrocytoma, assessing local control and survival rates and evaluating prognostic factors., Methods and Materials: We retrospectively reviewed 56 adult patients with supratentorial oligodendroglioma or oligoastrocytoma treated at our institution from January 1990 to December 2003 with fractionated stereotactic RT (FSRT)., Results: Fractionated stereotactic RT was well tolerated in all patients, without side effects. Median survival and progression-free survival calculated from the initiation of radiotherapy were 48 months (range, 2-133 months) and 38 months (range, 2-132 months), respectively. Progression-free survival rates after radiation were 89% at 1 year and 52% at 5 years. Of 26 recurrences, 92% developed in field. With regard to histology, overall survival rates in the World Health Organization (WHO) Grade II group were 89% and 74% at 5 and 10 years, respectively. In patients with WHO Grade III tumors, overall survival rates at 5 and 10 years were 69% and 46%, respectively. No prognosticators could be identified for median survival and progression-free survival after radiotherapy. Median overall survival calculated from primary diagnosis was 77.5 months (range, 3-214 months). The Cox regression multivariate analysis for age and neurologic symptoms showed a significance of p = 0.003 for age and p = 0.037 for the presence of neurologic symptoms on overall survival since primary diagnosis., Conclusions: Commonly, conventional conformal RT is applied in the treatment of brain tumors. In FSRT, the tumor volume can be irradiated with high doses, sparing volume of normal brain tissue. Our data are in accordance with survival times found in the literature. Ninety-two percent of all recurrences occurred within the defined target volume, confirming that reduction of the RT portals by the use of FSRT does not lead to an increased rate of recurrences at the field border or out of field. Fractionated stereotactic RT can therefore be implemented as an effective and safe modality in the therapy of primary oligodendroglioma and oligoastrocytoma.

Background and Purpose: The role of radiochemotherapy in the treatment of primary glioblastoma multiforme is still discussed controversially. To evaluate the feasibility and toxicity of irradiation and concomitant administration of 50 mg/m(2) temozolomide in patients with primary malignant glioma, this phase I/II study was conducted., Patients and Methods: 53 Patients with histologically confirmed WHO grade IV malignant glioma were enrolled into the study. All patients were treated with radiation therapy up to a total dose of 60 Gy using conventional fractionation of 5 x 2.0 Gy/week. Temozolomide was administered orally each therapy day at a dose of 50 mg/m(2)., Results: Prior to radiochemotherapy, complete resection (n = 14), subtotal resection (n = 22) or a biopsy (n = 17) of the tumor was performed. The median time interval between surgery and radiochemotherapy was 21 days. Treatment-related toxicity was very mild. Acute toxicity > grade 2 was observed in one patient who developed grade 4 hemotoxicity. Minor side effects of chemotherapy included nausea and vomiting. No severe late effects were observed. Median progression-free and overall survival were 8 and 19 months, respectively. The overall survival rate was 72% at 1 and 26% at 2 years. Age and extent of surgery significantly influenced survival., Conclusion: The combination of temozolomide plus radiation therapy is feasible and safe in terms of toxicity. Overall survival times were relatively long compared to survival times reported for radiotherapy alone. The application of 50 mg/m(2) of temozolomide can be performed throughout the whole time course without interruption due to side effects and might largely contribute to the prolonged overall survival. Further evaluation is warranted as to which dose of temozolomide is optimal with regard to tumor response and toxicity.

Background: In a phase III study recruiting patients with stage II colon cancer the effect of adjuvant therapy with edrecolomab, a murine monoclonal antibody to the cell-surface glycoprotein 17-1A, was compared to observation alone., Patients and Methods: From January 1997 until July 2000 a total of 377 patients were postoperatively stratified according to tumor stage (T3 vs. T4) and center, and randomly allocated to either treatment with edrecolomab (cohort A, n = 183) or observation (cohort B, n = 194). Patients in cohort A received a total of 900 mg edrecolomab. The study was terminated prematurely because of discontinuation of drug supply in Germany., Results: 305 patients were eligible for the primary endpoint of overall survival and 282 patients for disease-free survival. After a median follow-up of 42 months overall survival and disease-free survival were not significantly different. Toxicity was mild., Conclusions: In the present study, postoperative adjuvant treatment with edrecolomab in patients with resected stage II colon cancer did not improve overall or disease-free survival.

The recent completion of a reference sequence of the human genome now allows a complete characterization of the type II keratin gene domain on chromosome 12q13.13. This, domain, approximately 780 kb in size, is present on nine bacterial artificial chromosome clones sequenced by the Human Genome Sequencing Project. The type II keratin domain contains 27 keratin genes and eight pseudogenes. Twenty-three of these genes and four pseudogenes have been previously reported. This study describes, in addition to the genomic sequencing of the K2p gene and the bioinformatic identification of four keratin pseudogenes, the characterization of cDNA corresponding to three previously undescribed keratin genes K1b, K6l, and Kb20, as well as cDNA sequences for the previously described keratin genes hHb2, hHb4, and K3. Northern analysis of the new keratins K1b, K6l, K5b, and Kb20 using mRNA of major organs as well as of specific epithelial subtypes shows singular expression of these keratins in skin, hair follicles and, for K5b and Kb20, in tongue, respectively. In addition, the obvious discrepancies between the current reference sequence of the human genome and the previously described gene/cDNA sequences for K6c, K6d, K6e, K6f, K6h are investigated, leading to the conclusion that K6c, K6d as well as K6e, K6f are probably polymorphic variants of K6a and K6h, respectively. All 26 human type II keratins found on this domain as well as K18, dtype 1 Keratin, are identified at the genomic and transcriptional level. This appears to be the total complement of functional type II keratins in humans.

Several polymorphisms in DNA repair genes have been reported to be associated with lung cancer risk including XPA (-4G/A), XPD (Lys751Gln and Asp312Asn), XRCC1 (Arg399Gln), APE1 (Asp148Glu) and XRCC3 (Thr241Met). As there is little information on the combined effects of these variants, polymorphisms were analyzed in a case-control study including 463 lung cancer cases [among them 204 adenocarcinoma and 212 squamous cell carcinoma (SCC)] and 460 tumor-free hospital controls. Odds ratios (OR) adjusted for age, gender, smoking and occupational exposure were calculated for the variants alone and combinations thereof. For homozygous individuals carrying the Glu variant of APE1, a protective effect was found (OR = 0.77, CI = 0.51-1.16). Individuals homozygous for the variants XPA (-4A) (OR = 1.53, CI = 0.94-2.5), XPD 751Gln (OR = 1.39, CI = 0.90-2.14) or XRCC3 241Met (OR = 1.29, CI = 0.85-1.98) showed a slightly higher risk for lung cancer overall. In the subgroup of adenocarcinoma cases, adjusted ORs were increased for individuals homozygous for XPA (-4A) (OR = 1.62, CI = 0.91-2.88) and XRCC3 241Met (OR = 1.65; CI = 0.99-2.75). When analyzing the combined effects of variant alleles, 54 patients and controls were identified that were homozygous for two or three of the potential risk alleles [i.e. the variants in nucleotide excision repair, XPA (-4A) and XPD 751Gln, and in homologous recombination, XRCC3-241Met]. ORs were significantly increased when all patients (OR = 2.37; CI = 1.26-4.48), patients with SCC (OR = 2.83; CI = 1.17-6.85) and with adenocarcinoma (OR = 3.05; CI = 1.49-6.23) were analyzed. Combinations of polymorphisms in genes involved in the same repair pathway (XPA + XPD or XRCC1 + APE1) affected lung cancer risk only in patients with SCC. These results indicate that lung cancer risk is only moderately increased by single DNA repair gene variants investigated but it is considerably enhanced by specific combinations of variant alleles. Analyses of additional DNA repair gene interactions in larger population-based studies are warranted for identification of high-risk subjects.

Polymorphisms of genes coding for DNA repair can affect lung cancer risk. A common single nucleotide (-4) G-to-A polymorphism was identified previously in the 5' untranslated region of the XPA gene. In a case-control study in European Caucasians, the influence of this polymorphism on primary lung cancer risk overall and according to histologic subtypes was investigated. Four hundred sixty-three lung cancer cases (including 204 adenocarcinoma and 212 squamous cell carcinoma) and 460 tumor-free hospital controls were investigated using PCR amplification and melting point analysis of sequence-specific hybridization probes. Odds ratios (OR) were calculated by multiple logistic regression analysis adjusting for age, gender, smoking habits, and occupational exposure and showed a slightly enhanced risk for all lung cancer cases as well as for squamous cell carcinoma and adenocarcinoma cases. Gene-environment interactions were analyzed with respect to smoking and occupational exposure. A nearly 3-fold increased risk for adenocarcinoma associated with the XPA AA genotype was observed for occupationally exposed individuals (OR, 2.95; 95% confidence interval, 1.42-6.14) and for heavy smokers (OR, 2.52; 95% confidence interval, 1.17-5.42). No genotype-dependent increase in OR was found for nonexposed individuals or those smoking <20 pack-years. The significant effect of the XPA polymorphism in heavy smokers and occupationally exposed individuals suggests an important gene-environment interaction for the XPA gene. The underlying mechanisms as to why AA homozygotes are predisposed to lung adenocarcinoma and which specific carcinogens are involved remains to be determined.

Background and Purpose: The role of stereotactic radiosurgery (SRS) alone or in combination with whole brain radiotherapy (WBRT) in the treatment of cerebral metastases from breast carcinoma is discussed controversially. To elucidate the role of SRS in this context, a retrospective study evaluating the benefit of SRS and prognostic factors for survival was performed., Patients and Methods: From 1986 to 2003, 62 patients with cerebral metastases from breast cancer were treated for 103 lesions. Ten patients received SRS alone (group 1), 13 patients were treated with WBRT and SRS as a focal boost (group 2), and 39 patients received WBRT and salvage SRS (group 3) for recurrent metastases at a later time point., Results: Survival was increased in patients receiving SRS only compared to WBRT and SRS as a focal boost. Patients < 40 years of age had a favorable outcome (p > 0.04). However, no other prognostic factors could be identified. Overall tolerance of radiation was acceptable. Median local control intervals were 9 months for all patients, 6.5 months in group 1, 4 months in group 2, and 9 months in group 3, respectively. There were no significant intergroup differences., Conclusion: SRS alone is an effective treatment for patients with one to three brain metastases from breast cancer. A randomized trial should be performed to evaluate whether WBRT is a necessary component in the primary treatment of these patients. Salvage SRS is an effective therapy option after WBRT.

Purpose: To evaluate whether cisplatin-based chemotherapy (gemcitabine, vinorelbine, and cisplatin [GVP]) prolongs overall survival in comparison to cisplatin-free chemotherapy (gemcitabine and vinorelbine [GV]) as first-line treatment in patients with advanced non-small-cell lung cancer (NSCLC)., Patients and Methods: Between September 1999 and June 2001, 300 patients with NSCLC stage IIIB with malignant pleural effusion or stage IV disease were randomly assigned to receive GV (gemcitabine 1000 mg/m(2) + vinorelbine 25 mg/m(2) on days 1 and 8 every 3 weeks) or GVP (gemcitabine 1000 mg/m(2) + vinorelbine 25 mg/m(2) on days 1 and 8 + cisplatin 75 mg/m(2) on day 2 every 3 weeks). Primary end point of the study was overall survival., Results: Two hundred eighty-seven patients (GV, 143 patients; GVP, 144 patients) were eligible for analysis. At the time of analysis, April 15, 2002, 209 patients (GV, 103 patients; GVP, 106 patients) of 287 patients had died (73%). No statistically significant difference was observed for overall survival (P =.73; median survival, 35.9 versus 32.4 weeks; 1-year survival rate, 33.6% versus 27.5%) as well as for event-free survival (P =.35; median time-to-event, 19.3 versus 22.3 weeks) between GV and GVP. Two hundred fourteen patients were assessable for best response. The overall response rates were 13.0% for GV versus 28.3% for GVP (P =.004; complete responders, 0% versus 3.8%; partial responders, 13.0% versus 24.5%). Hematologic and nonhematologic toxicity was significantly lower in the GV treatment arm compared with GVP. No statistically significant difference in quality of life was observed., Conclusion: In this phase III study, the cisplatin-based GVP regimen showed no survival benefit as first-line chemotherapy in advanced NSCLC when compared with the cisplatin-free GV regimen, which was substantially better tolerated.

Insulin signaling is enhanced by moderate concentrations of reactive oxygen species (ROS) and suppressed by persistent exposure to ROS. Diabetic patients show abnormally high ROS levels and a decrease in insulin reactivity which is ameliorated by antioxidants, such as N-acetylcysteine (NAC). A similar effect of NAC has not been reported for non-diabetic subjects. We now show that the insulin receptor (IR) kinase is inhibited in cell culture by physiologic concentrations of cysteine. In two double-blind trials involving a total of 140 non-diabetic subjects we found furthermore that NAC increased the HOMA-R index (derived from the fasting insulin and glucose concentrations) in smokers and obese patients, but not in nonobese non-smokers. In obese patients NAC also caused a decrease in glucose tolerance and body fat mass. Simultaneous treatment with creatine, a metabolite utilized by skeletal muscle and brain for the interconversion of ADP and ATP, reversed the NAC-mediated increase in HOMA-R index and the decrease in glucose tolerance without preventing the decrease in body fat. As the obese and hyperlipidemic patients had lower plasma thiol concentrations than the normolipidemic subjects, our results suggest that low thiol levels facilitate the development of obesity. Supplementation of thiols plus creatine may reduce body fat without compromising glucose tolerance.

Genetic polymorphisms of the human CYP3A family affect clinical drug efficacy and may modify cancer risk. CYP3A genes show high sequence similarity that had previously lead to misallocation of CYP3A polymorphisms. Recent studies indicated a high degree of or even complete linkage for certain CYP3A alleles. Reliable LightCycler-based genotyping methods were developed and their degree of linkage in a large Caucasian population (n = 1210) investigated. Strong linkage disequilibrium was confirmed between CYP3A4, CYP3A5, and CYP3AP1 (each at P < 10(-5)). Contrary to some previous results claiming complete linkage between the phenotypically relevant CYP3A5*1 and a variant in a pseudogene promoter region CYP3AP1*1 we found among 428 controls (15 of 66) and 782 lung cancer cases (25 of 115) approximately 22% of CYP3AP1*1 carriers to be homozygous for CYP3A5*3 We conclude that contrary to previous assumptions, the CYP3AP1 genotype is not a reliable predictor for CYP3A5 activity.

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.

Pseudofolliculitis barbae (PFB) is a common hair disorder characterized by a pustular foreign body inflammatory reaction that is induced by ingrown hairs of the facial and submental (barbea) regions after regular shaving. It occurs predominantly in black males, while it is rather rare and usually far less severe in Caucasian males. Black individuals have a higher propensity of developing PFB due to their genetic predisposition for curly hair which inherently possesses a much higher risk of growing back into the skin than straight or wavy hair. The PFB process is, however, not gender dependent nor restricted to the face, but can occur in any skin region once regular shaving, plucking, or other traumatic means of hair removal are instituted. Through a family study and a large-scale investigation of randomly sampled PFB-affected and -unaffected individuals, this study demonstrates that an unusual single-nucleotide polymorphism, which gives rise to a disruptive Ala12Thr substitution in the 1A alpha-helical segment of the companion layer-specific keratin K6hf of the hair follicle, is partially responsible for the phenotypic expression and represents an additional genetic risk factor for PFB.

In discussing design and results of randomized clinical trials, in particular with clinical oncologists, one often encounters the opinion that a phase III trial is a complicated, highly costly, and difficult task. Part of this opinion seems to originate in myths around underlying biostatistical principles such as randomization, sampling and sample size, statistical hypotheses, statistical error probabilities, and statistical power. This work clarifies basic statistical issues of randomized clinical trials and the interpretation of their results. Six issues ('myths') relevant for the design of clinical trials and the interpretation of their results are addressed. They concern choice of study design, choice of participating centers, and recruitment of patients as well as statistical questions of establishing study hypotheses and interpreting p values. These myths are shown to be caused primarily through a misunderstanding of statistical inference and statistical thinking that can be avoided when a rational understanding of statistical principles is translated into a clinical research approach. We also conclude that before clinical evidence is summarized from different studies each study should be examined thoroughly., (Copyright 2003 S. Karger GmbH, Freiburg)

Treatment of hyperlipidemic patients with the thiol compound N-acetylcysteine (NAC) was previously shown to cause a significant dose-related increase in the high-density lipoprotein (HDL)-cholesterol serum level, suggesting the possibility that its disease-related decrease may result from a diminished thiol concentration and/or thiol/disulfide redox status (REDST) in the plasma. We therefore investigated plasma thiol levels and REDST in normo-/hyperlipidemic subjects with and without coronary heart disease (CHD). The thiol level, REDST, and amino acid concentrations in the plasma and intracellular REDST of peripheral blood mononuclear cells (PBMC) have been determined in 62 normo- and hyperlipidemic subjects. Thirty-three of these subjects underwent coronary angiography, because of clinical symptoms of CHD. All groups of hyperlipidemic patients under test and those normolipidemic individuals with documented coronary stenoses showed a marked decrease in plasma thiol concentrations, plasma and intracellular REDST of PBMCs, and a marked increase in plasma taurine levels. Individual plasma thiol concentrations and plasma REDST were strongly negatively correlated with the serum LDL-cholesterol and positively correlated with the serum HDL-cholesterol level. Together with the earlier report about the effect of NAC on the HDL-cholesterol serum level, our findings suggest strongly that lower HDL-cholesterol serum levels may result from a decrease in plasma thiol level and/or REDST possibly through an excessive cysteine catabolism into taurine.

CYP3A isozymes are involved in tobacco carcinogen- and steroid-metabolism, and are expressed in human lung tissue showing interindividual variation in expression and activity. The CYP3A4*1B allele has been associated with a two-fold higher promoter activity and with high-grade prostate cancers. The very frequent intron 3 polymorphism in the CYP3A5 gene (CYP3A5*3) results in decreased CYP3A5 protein levels. A case-control study was conducted in 801 Caucasian lung cancer patients that included 330 adenocarcinomas, 260 squamous cell carcinomas, 171 small cell lung cancers (SCLC) and 432 Caucasian hospital-based controls. CYP3A-genotyping was performed by capillary polymerase chain reaction followed by fluorescence-based melting curve analysis. A significantly increased SCLC risk for CYP3A4*1B allele carriers [odds ratio (OR) 2.25, 95% confidence interval (CI) 1.11-4.55, P=0.02] was found. After dividing cases and controls by gender, an increased lung cancer risk for CYP3A4*1B carriers (OR 3.04, 95% CI 0.94-9.90, P=0.06) for women but not for men (OR 1.00, 95% CI 0.56-1.81) was revealed. Heavier smoking men (> or =20 pack-years) with the CYP3A4*1B allele had a significant OR for lung cancer of 3.42 (95% CI 1.65-7.14, P=0.001) compared to *1A/*1A carriers with lower tobacco exposure (<20 pack-years). For women, the respective OR was 8.00 (95% CI 2.12-30.30, P=0.005). Genotype frequencies were generally in Hardy-Weinberg equilibrium, except for CYP3A5 where a greater than expected number of CYP3A5*1 homozygotes was observed among cases (P=0.006). In addition, we observed linkage disequilibrium of CYP3A4 and CYP3A5 (P<0.00001), but a non-significantly increased lung cancer risk was only found for homozygous CYP3A5*1 allele carriers (OR 5.24, 95% CI 0.85-102.28, P=0.14) but not for heterozygotes. To confirm our observation that the CYP3A4*1B allele increases SCLC risk and modifies the smoking-related lung cancer risk in a gender-specific manner, further studies, including CYP3A haplotype analysis, will be necessary.

Statistical methods for the validation of toxicological in vitro test assays are developed and applied. Validation is performed either in comparison with in vivo assays or in comparison with other in vitro assays of established validity. Biostatistical methods are presented which are of potential use and benefit for the validation of alternative methods for the risk assessment of chemicals, providing at least an equivalent level of protection through in vitro toxicity testing to that obtained through the use of current in vivo methods. Characteristic indices are developed and determined. Qualitative outcomes are characterised by the rates of false-positive and false-negative predictions, sensitivity and specificity, and predictive values. Quantitative outcomes are characterised by regression coefficients derived from predictive models. The receiver operating characteristics (ROC) technique, applicable when a continuum of cut-off values is considered, is discussed in detail, in relation to its use for statistical modelling and statistical inference. The methods presented are examined for their use for the proof of safety and for toxicity detection and testing. We emphasise that the final validation of toxicity testing is human toxicity, and that the in vivo test itself is only a predictor with an inherent uncertainty. Therefore, the validation of the in vitro test has to account for the vagueness and uncertainty of the "gold standard" in vivo test. We address model selection and model validation, and a four-step scheme is proposed for the conduct of validation studies. Gaps and research needs are formulated to improve the validation of alternative methods for in vitro toxicity testing.

Persistent infection with human papillomavirus (HPV), particularly HPV16, represents the prime risk factor in cervical carcinogenesis. HPV variants (e.g., within the E6 gene) together with immunogenetic factors of the host may be responsible either for effective viral clearance, or alternatively, for viral persistence. Peripheral blood from 27 HPV16 positive Swedish women with cervical carcinoma, who had previously been tested for HPV16 E6 variants, was used for human leukocyte antigen (HLA) class I typing. Women with HLA-B*44, HLA-B*51, or HLA-B*57 who were infected with the HPV16 E6 variant L83V had an approximately four- to fivefold increased risk for cancer compared with controls (odds ratio [OR] = 3.5, 95% CI = 1.1-11.1, OR = 4.2, 95% CI = 1.19-14.69, or OR = 4.67, 95% CI = 1.2-18.6, respectively). Epitope predictive algorithm with SYFPEITHI revealed that the variant at amino acid 83 affects the binding affinity in association with HLA-B*44. Interestingly, the HLA-B*15 allele seems protective because it was absent in HPV16 positive cancer. It is concluded that specific HLA class I alleles, combined with certain HPV16 E6 variants, may be crucial for immune surveillance in cervical carcinogenesis. The evaluation of associations of HLA alleles with HPV variants may be helpful in defining prognostic markers and in designing vaccines capable of mediating immune protection against HPV infection.

Aging-related loss of muscle function is a predictor of mortality and a surrogate parameter of the aging process. Its consequences include a high risk for falls, hip fractures, and loss of autonomy. Aging is associated with changes in the oxidant/antioxidant balance including a decrease in plasma thiol (cysteine) concentration. To assess the importance of cysteine, we determined in a double-blind study the effects of N-acetylcysteine on the functional capacity of frail geriatric patients and their response to physical exercise. The subjects on placebo showed only a relatively weak response, and 31% showed even a decrease in more than one parameter during the observation period. Low plasma arginine levels were correlated with a weak overall performance before exercise and a poor response to exercise. N-Acetylcysteine strongly enhanced the increase in knee extensor strength and significantly increased the sum of all strength parameters if adjusted for baseline arginine level as a confounding parameter. N-acetylcysteine had no significant effect on growth hormone and IGF-1 levels but caused a significant decrease in plasma TNF-alpha. These findings may provide a basis for therapeutic intervention and suggest that the loss of function involves limitations in cysteine and one or more other amino acids which may compromise muscular protein synthesis.

MPO participates in the metabolic activation of tobacco carcinogens such as PAHs. A frequent MPO -463 G-->A polymorphism in the promoter region reduces MPO transcription and has been correlated with >4-fold lower benzo[a]pyrene-DNA adduct levels in the skin of coal tar-treated patients. Four of 7 case-control studies found significantly reduced lung cancer risk associated with the A allele. Due to their different etiologies, we examined whether the MPO genotype affects histologic lung cancer types differentially. A case-control study was conducted in 625 ever-smoking lung cancer patients, including 228 adenocarcinomas, 224 SCCs, 135 SCLCs and 340 ever-smoking hospital controls. MPO genotyping was performed by capillary PCR followed by fluorescence-based melting curve analysis. Combining the MPO -463 (G/A+A/A) genotypes, a protective effect approaching significance (OR = 0.75, 95% CI 0.55-1.01) was observed when comparing all lung cancer cases to controls. Among histologic types of lung cancer, a weak protective effect was found for both adenocarcinoma (OR = 0.81, CI 0.55-1.19) and SCC (OR = 0.82, CI 0.56-1.21); a stronger and significant effect was found for SCLC (OR = 0.58, CI 0.36-0.95; p = 0.029). Our results also suggest that the MPO genotype varies among inflammatory nonmalignant lung diseases. In conclusion, our results emphasize the need for a separate analysis of lung cancer histologic types and an adjustment for inflammatory nonmalignant lung diseases in future MPO-related studies. We confirm that the MPO -463 A variant affords a protective effect against lung cancer risk in smokers, which was strongest for SCLC patients., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: To evaluate the prognostic relevance of CEACAM1 and sialyl Lewis X expression in adenocarcinomas of the lung., Patients and Methods: Paraffin wax sections of 93 patients with adenocarcinomas of the lung who underwent surgery between 1990 and 1995 were immunohistochemically investigated using monoclonal anti-CEACAM1 and sialyl Lewis X antibodies. The clinical course of all patients was followed up for a minimum of 5 years., Results: Sixty-one tumors were classified as CEACAM1-positive, and 32 were classified as CEACAM1-negative. Patients with CEACAM1-positive tumors had a significantly poorer overall (P =.00025) and relapse-free (P =.00029) survival than those with CEACAM1-negative tumors. Only three patients did not express the sialyl Lewis X glycotope, whereas 90 tumors (97%) were sialyl Lewis X-positive. In multivariate Cox regression analysis, next to tumor stage and sex, only the expression of CEACAM1 was a significant independent prognostic factor for survival., Conclusion: Expression of CEACAM1 was an independent prognostic factor in our patient population and can be used to stratify patients with adenocarcinomas of the lung into low-risk and high-risk groups. In contrast, the expression of sialyl Lewis X was of no prognostic relevance because it was expressed in 97% of all investigated tumors, and most likely has no influence on the function of CEACAM1 in this tumor entity.

The worldwide incidence of adenocarcinoma of the lung is rising. Unfortunately, no significant prognostic marker beyond the classical TNM staging exists to stratify these patients for appropriate therapy. Because lectins, carbohydrate-binding proteins, have been shown to be useful prognostic markers in several other adenocarcinomas, a panel of lectins [Helix pomatia agglutinin (HPA), Phaseolus vulgaris leukoagglutinin, Ulex europaeus agglutinin, Maackia amurenis agglutinin, Sambucus nigra agglutinin] with different carbohydrate-binding specificities were tested for their prognostic relevance. Paraffin wax sections of 93 patients with adenocarcinomas of the lung who had undergone surgery between 1990 and 1995 were investigated by lectin histochemistry. Lectin-binding data and other known prognostic factors were correlated with survival. In univariate analysis, binding of HPA, Phaseolus vulgaris leukoagglutinin, and Ulex europaeus agglutinin to adenocarcinoma cells were prognostic indicators for overall and relapse-free survival, whereas Maackia amurenis agglutinin and Sambucus nigra agglutinin binding had no prognostic value. However, in a multivariate analysis next to stage and gender only HPA was a significant independent prognostic factor on survival. In conclusion, HPA binding was the primary marker-based predictor of prognosis in our patient population and allows to stratify patients with adenocarcinomas of the lung into a low- and a high-risk group.

Since its introduction by Ostling and Johanson [1984; Biochem Biophys Res Commun 123:291-298] and independent modifications by Singh et al. [1990; Exp Cell Res 175:184-191] and Olive et al. [1988; Radiat Res 112:86-94], the comet assay has been widely used in genetic toxicology, environmental biomonitoring, molecular and human epidemiology, and clinical investigations. There are still several issues to be resolved before the comet assay is accepted as a standard assay for detecting DNA damage and repair in a single cell. One of the major issues is the proper quantification of DNA damage/repair. The aim of this article is to develop a new quantitative measure of DNA damage/repair which is represented in the dose-time-response surface. We propose to use the second derivative (2D) of the dose-time-response surface for measuring DNA repair activity. This approach enables us to represent the DNA repair activity of cells exposed to a DNA-damaging agent with a single number by combining all the information of a dose-time-response experiment. The computation procedure includes the application of linear regression. An SAS/AF-based program, "Comet Assay," was developed for this computation and is freely available on the Internet. We considered the response of each of four DNA damage parameters: tail moment, tail length, tail DNA, and tail inertia for constructing the dose-time-response surface. Using data from 25 patients, we observed that 2Ds based on tail moment and tail DNA were highly correlated and that tail inertia might provide information on a somewhat different aspect of DNA damage/repair., (Copyright 2002 Wiley-Liss, Inc.)

Polymorphic glutathione-S-transferase (GST) genes causing variations in enzyme activity may influence individual susceptibility to lung cancer. In this case-control study (consisting of 389 Caucasian lung cancer patients, including 151 adenocarcinomas (ACs) and 172 squamous cell carcinomas (SCCs), and 353 hospital control subjects without malignant disease, genotype frequencies for GSTM1, GSTM3, GSTP1 and GSTT1 were determined by polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP)-based methods. While adjusted odds ratios (ORs) indicated no significantly increased risk for lung cancer overall due to any single GST genotype, the risk alleles for GSTM1, GSTM3 and GSTP1 conferring reduced enzyme activity were present at higher frequency in SCC than in AC patients. This is consistent with a reduced detoxification of carcinogenic polycyclic aromatic hydrocarbons (PAHs) from cigarette smoke that are more important for the development of SCC than for AC. An explorative data analysis also identified statistically significantly increased ORs for the combinations GSTT1 non-null and GSTP1 GG or AG for lung cancer overall (OR 2.23, CI 1.11-4.45), and for SCC (OR 2.69, CI 1.03-6.99). For lung cancer overall, and especially among SCC patients, the GSTT1 null genotype was underrepresented (SCC 11.2% v. control subjects 19%, P = 0.026, OR 0.57, CI 0.30-1.06). Additionally, in 28 patients with hamartomas, the GSTT1 null genotype was also protective (P = 0.013), while GSTP1 variant allele carriers were overrepresented (OR 2.48, CI 1.06-6.51). In conclusion, GST genotypes may act differently, either by detoxifying harmful tobacco carcinogens and/or by eliminating lung cancer chemopreventive agents. The latter role for GSTT1 would explain the observed lower risk of SCC and hamartoma associated with GSTT1 null. Further confirmatory studies are required.

Background: Postoperative radio-chemotherapy has been established as standard treatment for stage II and III rectal cancer patients in the last decade. To improve the efficacy of this therapy, we decided to evaluate continuous 24-hour infusion of 5-fluorouracil (5-FU) with folinic acid (FA) in combination with local radiation versus standard bolus 5-FU/FA with local radiation in a randomized study. Here we report on the first 28 patients to receive the experimental treatment., Patients and Methods: Patients with stage II and III rectal cancer received weekly 2-hour infusions of FA 500 mg/m2 followed by continuous 24-hour infusions of 5-FU 2,600 mg/m2 postoperatively via a Port-A-Cath system. The first cycle included 8 consecutive weekly administrations, the 1st-4th in full dose, the 5th-8th with 50% reduced dose while local irradiation (45 or 50.4 Gy) was performed. Thereafter, two further chemotherapy cycles (6 weekly administrations, 100% dose) followed., Results: 28 patients received continuous 5-FU/FA treatment, of whom only 21 were evaluable for tolerability. 19 patients (90.4%) completed the first cycle, only 14 patients entered the second treatment cycle. Especially during the combined radiochemotherapy, increased toxicity was observed with grade III/IV diarrhea (n = 2), nausea (n = 1), leukopenia (n = 1), and cardiac toxicity (n = 1)., Conclusion: The high rate of premature treatment dropout indicate that the chosen schedule of weekly high-dose 5-FU/FA continuous infusion and combined postoperative radiotherapy should not be recommended for further use in postoperative adjuvant treatment., (Copyright 2001 S. Karger GmbH, Freiburg)

Seroepidemiological studies have shown previously that cancer patients are less likely to have antibodies against the tumour suppressive adeno-associated virus (AAV) than control groups. To examine the influence of AAV infection on the development of adult T-cell leukaemia lymphoma (ATLL), an endemic disease in Southern Japan that is caused by infection with the human T-cell leukaemia virus type 1 (HTLV-I), the prevalence of serum antibodies to AAV type 2 (AAV-2) was tested in healthy HTLV-I carriers (n = 39) and patients with ATLL (n = 31). The results showed a significant difference in AAV-2 seropositivity between the two groups: Only 29% of the ATLL patients had IgG antibodies against AAV-2, whereas 84.6% of the healthy HTLV-I carriers were seropositive. Analysis of total serum IgG and antibodies against the Epstein-Barr virus (EBV) EBNA1 antigen showed that the lack of AAV antibodies in patients was not due to an ATLL-associated immune deficiency. The lower level of AAV-2 seropositivity in ATLL-patients may indicate that AAV-2 antibody-positive HTLV-I carriers might be less likely to develop ATLL or that loss of AAV-2 antibodies may parallel the development of disease., (Copyright 2001 Wiley-Liss, Inc.)

Purpose: Beside the established maturation of hepatitis B virus (HBV) transcripts at a polyadenylation signal downstream of the HBV x protein open reading frame, maturation at an internal polyadenylation signal has been observed in the chronically infected liver. In the present study, it was the aim to identify the respective circulating full-length and truncated transcripts in plasma/serum of carriers., Experimental Design: Nucleic acids extracted from sera were analyzed using established PCR and reverse transcription-PCR procedures targeted to HBV x protein gene regions. Amplification products were cloned and sequenced., Results: Base substitution patterns were determined, which indicated infection stages advanced to different degrees regardless of the transcript type analyzed. HBV full-length RNA (fRNA) showed a high correlation with hepatitis B e antigen and viral DNA, indicative for a replicative infection. In contrast, truncated RNA (trRNA) appeared to be independent of hepatitis B e antigen and showed only a weak association with circulating viral DNA. No correlation was observed between the levels of trRNA and the apparent liver damage as reflected by alanine transaminase levels. An age-dependent representation of fRNA and trRNA was observed: fRNA decreased progressively to low levels, whereas trRNA remained at comparably high values. trRNA and RNA not polyadenylated at either of the two polyadenylation signals were detected even in the absence of any other conventional HBV seromarker, including viral DNA. This was shown for patients with cryptogenic cirrhosis and hepatitis C virus carriers., Conclusions: The identification of HBV RNA in human serum has a diagnostic potential for apparent and for inapparent infection stages.

The purpose of this phase II study was to investigate the efficacy and safety of gemcitabine plus vinorelbine as first-line chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Eligibility criteria included cytologically or histologically confirmed NSCLC (stage IIIB or IV), no previous chemotherapy, and bidimensionally measurable disease. Patients received 1000 mg/m(2) gemcitabine and 30 mg/m(2) vinorelbine on days 1, 8 and 15 every 4 weeks up to eight courses. From December 1997 to November 1998, 70 patients (59 stage IV and 11 stage IIIB disease), with a median age of 59 years (range 38-74 years) were enrolled. The intent-to-treat response rate was 41% (95% confidence interval (CI) 30-54%) with 1 complete responder (CR) and 28 partial responders (PRs), 15 patients had stable disease (SD) and 26 progressed (PD). Median survival was 8.3 months (95% CI 6.0-9.9 months), median progression-free survival (PFS) was 4.8 months (95% CI 3.9-5.5 months), and 1-year survival rate was 33.5% (95% CI 24.0-46.8%). Patients received a total of 229 cycles. Haematological and non-haematological toxicities were moderate. Transient World Health Organization (WHO)-grade IV leucopenia and thrombocytopenia occurred in 13 (6%) and two (1%) cycles, respectively. The predominant non-haematological toxicity was local reactions of the veins in 19 (27%) patients (WHO-grade II and III). Neurotoxicity was infrequent, non-cumulative, and reversible. The combination of gemcitabine and vinorelbine has demonstrated activity in metastatic NSCLC, with response and survival rates similar to those of cisplatin-based regimens and a more favourable toxicity profile that is well tolerated in an outpatient setting.

Risk factors other than human papillomavirus (HPV) infection per se for cervical cancer development have been investigated recently. It was suggested that HPV 16 E6 variants and the p53 codon 72 arginine polymorphism could be progression markers. Indeed, it has been demonstrated that specific E6 variants and p53 arginine were both enriched in cancer. However, especially with regard to the latter, divergent results have been reported. Our aim was thus to investigate whether p53 arginine is important for cervical carcinogenesis by scaling up samples of the two European cohorts, the initial results of which were reported previously. In addition, we have assessed the occurrence of p53 codon 72 arginine, in combination with specific HPV 16 E6 genotypes. We found p53 arginine to be increased in cancer of both cohorts, consistent with our previous concept. Although specific E6 genotypes increased gradually with the severity of the lesion, p53 arginine was enriched in cancer only. Moreover, the frequency of the arginine allele was similar in groups with different E6 genotypes. It is concluded that p53 arginine is a risk factor for cervical cancer but probably acts independently of E6 variants.

To determine the therapeutic effect of sulfur amino acid supplementation in HIV infection we randomized 40 patients with antiretroviral therapy (ART; study 1) and 29 patients without ART (study 2) to treatment for 7 months with N-acetyl-cysteine or placebo at an individually adjusted dose according to a defined scheme. The main outcome measures were the change in immunological parameters including natural killer (NK) cell and T cell functions and the viral load. Both studies showed consistently that N-acetyl-cysteine causes a marked increase in immunological functions and plasma albumin concentrations. The effect of N-acetyl-cysteine on the viral load, in contrast, was not consistent and may warrant further studies. Our findings suggest that the impairment of immunological functions in HIV+ patients results at least partly from cysteine deficiency. Because immune reconstitution is a widely accepted aim of HIV treatment, N-acetyl-cysteine treatment may be recommended for patients with and without ART. Our previous report on the massive loss of sulfur in HIV-infected subjects and the present demonstration of the immunoreconstituting effect of cysteine supplementation indicate that the HIV-induced cysteine depletion is a novel mechanism by which a virus destroys the immune defense of the host and escapes immune elimination.

Recent cases of fraud in clinical trials have attracted considerable media attention, but relatively little reaction from the biostatistical community. In this paper we argue that biostatisticians should be involved in preventing fraud (as well as unintentional errors), detecting it, and quantifying its impact on the outcome of clinical trials. We use the term 'fraud' specifically to refer to data fabrication (making up data values) and falsification (changing data values). Reported cases of such fraud involve cheating on inclusion criteria so that ineligible patients can enter the trial, and fabricating data so that no requested data are missing. Such types of fraud are partially preventable through a simplification of the eligibility criteria and through a reduction in the amount of data requested. These two measures are feasible and desirable in a surprisingly large number of clinical trials, and neither of them in any way jeopardizes the validity of the trial results. With regards to detection of fraud, a brute force approach has traditionally been used, whereby the participating centres undergo extensive monitoring involving up to 100 per cent verification of their case records. The cost-effectiveness of this approach seems highly debatable, since one could implement quality control through random sampling schemes, as is done in fields other than clinical medicine. Moreover, there are statistical techniques available (but insufficiently used) to detect 'strange' patterns in the data including, but no limited to, techniques for studying outliers, inliers, overdispersion, underdispersion and correlations or lack thereof. These techniques all rest upon the premise that it is quite difficult to invent plausible data, particularly highly dimensional multivariate data. The multicentric nature of clinical trials also offers an opportunity to check the plausibility of the data submitted by one centre by comparing them with the data from all other centres. Finally, with fraud detected, it is essential to quantify its likely impact upon the outcome of the clinical trial. Many instances of fraud in clinical trials, although morally reprehensible, have a negligible impact on the trial's scientific conclusions., (Copyright 1999 John Wiley & Sons, Ltd.)

Methotrexate-albumin conjugate (MTX-HSA) is a novel human albumin-based prodrug conjugate of methotrexate (MTX). A low MTX loading rate provided optimal tumor targeting and therapeutic efficacy during preclinical testing. The objectives of this first Phase I study of MTX-HSA were to determine dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) in a weekly regimen. Seventeen cancer patients who were no longer amenable to standard treatment were enrolled and were evaluable for DLT. Up to eight injections were performed in weekly intervals. Dose escalation was as follows: 20, 40, 50, and then 60 mg/m2 MTX-HSA (based on the amount of MTX bound to albumin). Additional MTX-HSA courses were feasible in case of tumor response. DLT (mainly stomatitis, Common Toxicity Criteria grade 3) occurred, beginning at the 50 mg/m2 dose level after repeated administrations; in one case, thrombocytopenia was dose-limiting. Two events of DLT occurred at the 60 mg/m2 dose level within the first two administrations. Mild anemia, transaminitis, and one case of skin toxicity were found. No significant leukopenia, nausea, renal toxicity, or other toxicities were observed. MTX-HSA was well tolerated. Drug accumulation occurred on the weekly schedule. The half-life of the drug was estimated to be up to 3 weeks. Tumor responses were seen in three patients: (a) a partial response was seen in one patient with renal cell carcinoma (response duration, 30 months, ongoing); (b) a minor response was seen in one patient with pleural mesothelioma (response duration, 31 months, ongoing); and (c) a minor response was seen in one patient with renal cell carcinoma (response duration, 14 months until progression). Poststudy treatment was administered at 2-4-week intervals. No signs of toxicity or drug accumulation were seen. Altered pharmacological properties of MTX-HSA such as plasma half-life, tumor targeting, or intracellular metabolism might have contributed to these responses. The MTD for weekly administration was 4 x 50 mg/m2 MTX-HSA during short-term treatment. A regimen with MTX-HSA injections of 50 mg/m2 every 2 weeks was recommended for a further clinical Phase I study.

Uncertainty in exposure assessment and uncertainty in kinetic models of early effects after exposure to a toxin are addressed in this paper. Sources of uncertainty in the determination of exposure of workers in chemical industry exposed to dioxins are exhibited and a simple kinetic model for biomonitor measurements of the concentrations from occupational exposure is derived. Model uncertainty, and uncertainty in the model parameters of physiologically-based pharmacokinetic models (PBPK models) are addressed when these models are used to estimate the effective dose in risk assessment. Uncertainty in the model parameters originating from the use of different statistical analysis methods is exhibited for Hill type nonlinear kinetics of enzyme induction mediated by a toxin.

RNA from tissue samples of 46 HBsAg seropositive hepatocellular carcinoma (HCC) patients was analysed by an RT/PCR assay which discriminates full-length hepatitis B virus (HBV) RNA polyadenylated at the unique viral poly(A) signal governing replication from truncated HBV RNA polyadenylated at a cryptic poly(A) signal. In the tumor the apparent coexistence was less frequent than in the peritumor while the predominance of one of the two RNAs was more frequent. The mean age of patients with a predominance of truncated RNA in the tumor was 9 years above those patients with a predominance of full length RNA (p < 0.05). An inverse relationship existed between the presence of truncated RNA and the presence of RNA carrying core gene sequences. The results of this study establish truncated RNA as a frequent marker of the chronic infection but leave it open whether it is found preferentially in patients developing HCC or generally in chronically infected individuals.

Feed forward neural networks are compared with standard and new statistical classification procedures for the classification of proteins. We applied logistic regression, an additive model and projection pursuit regression from the methods based on a posterior probabilities; linear, quadratic and a flexible discriminant analysis from the methods based on class conditional probabilities, and the K-nearest-neighbors classification rule. Both, the apparent error rate obtained with the training sample (n = 143) and the test error rate obtained with the test sample (n = 125) and the 10-fold cross validation error were calculated. We conclude that some of the standard statistical methods are potent competitors to the more flexible tools of machine learning.

The elimination of beta-hexachlorocyclohexane (beta-HCH) in humans was investigated in a group of 40 former workers of a lindane-producing plant by analyzing at least 2 blood specimens (3 specimens in 3 workers) from different time points. Assuming a first-order kinetic model for excretion, the median half-life of beta-HCH is 7.2 yr calculated by concentrations in whole blood and 7.6 yr calculated by concentrations in extractable lipids. In univariate analyses an influence of age, percent body fat, and liver disease (additionally in whole blood an influence of contents of extractable lipids) on clearance was observed. All factors show a positive correlation with half-life. According to a multiple regression model, influence of percent body fat calculated according to Deurenberg et al. (1991) is an important covariate in the description of the variations of the clearance rate (calculated on the basis of extractable lipids) of beta-HCH. The data support the assumption of first-order kinetics.

Skeletal muscle catabolism, low plasma glutamine, and high venous glutamate levels are common among patients with cancer or human immunodeficiency virus infection. In addition, a high glycolytic activity is commonly found in muscle tissue of cachectic cancer patients, suggesting insufficient mitochondrial energy metabolism. We therefore investigated (a) whether an "an-aerobic physical exercise" program causes similar changes in plasma amino acid levels, and (b) whether low plasma glutamine or high glutamate levels are risk factors for loss of body cell mass (BCM) in healthy human subjects, i.e., in the absence of a tumor or virus infection. Longitudinal measurements from healthy subjects over longer periods suggest that the age-related loss of BCM occur mainly during episodes with high venous glutamate levels, indicative of decreased muscular transport activity for glutamate. A significant increase in venous glutamate levels from 25 to about 40 microM was seen after a program of "anaerobic physical exercise." This was associated with changes in T lymphocyte numbers. Under these conditions persons with low baseline levels of plasma glutamine, arginine, and cystine levels also showed a loss of BCM. This loss of BCM was correlated not only with the amino acid levels at baseline examination, but also with an increase in plasma glutamine, arginine, and cystine levels during the observation period, suggesting that a loss of BCM in healthy individuals terminates itself by adjusting these amino acids to higher levels that stabilize BCM. To test a possible regulatory role of cysteine in this context we determined the effect of N-acetyl-cysteine on BCM in a group of subjects with relatively low glutamine levels. The placebo group of this study showed a loss of BCM and an increase in body fat, suggesting that body protein had been converted into other forms of chemical energy. The decrease in mean BCM/body fat ratios was prevented by N-acetyl-cysteine, indicating that cysteine indeed plays a regulatory role in the physiological control of BCM.

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.

Human neuroblastoma cells often have deletions of the distal short arm of chromosome 1 (1p). Earlier studies using chromosome analysis had suggested that the 1p deletion is correlated with a poor survival chance for the patient. We have reevaluated this possibility by analyzing 51 neuroblastomas for loss of heterozygosity (LOH) at 1p. We detected LOH in 32% of the cases. LOH did not correlate with the age of the patients at diagnosis or with tumor stage but was correlated significantly with amplification of the MYCN proto-oncogene. Nine of 10 MYCN-amplified tumors had deletions in 1p (P < 0.001). Survival chances of patients with tumors carrying MYCN amplification together with the deletion at 1p were decreased significantly (eight of nine affected patients died) compared with a patient group without any of these aberrations (P < 0.001). However, the deletion of 1p alone without MYCN amplification was not associated with a poor outcome compared with patients who had neither deletion nor amplification (only two of eight affected patients died; P = 0.803). From these data we conclude that 1p deletions are not reliable markers to determine a patient's prognosis. They may, however, identify a subgroup of neuroblastomas in which MYCN is amplified readily, resulting in rapid tumor progression.

Eighty Wistar rats were randomized into two groups. In group 1 vesicosigmoidostomy with proximal colostomy was performed, in group 2, vesicosigmoidostomy. The total tumor incidence did not significantly (group 1 10/40, 25%; group 2 13/40, 32.5%). The tumor spectrum differed, with more adenocarcinomas in group 2 (11/40, 27.5% vs 4/40, 10%; P = 0.047) and urothelial carcinomas only in group 1 (5/40, 2.5%). One hundred and ten other Wistar rats were randomized into three groups. Animals in group A received vesicoileosigmoidostomy, group B, two-step vesicosigmoidostomy with initial separation of urine and the urocolonic anastomosis, group C, vesicosigmoidostomy. Significantly fewer adenocarcinomas were observed in group A (2/40, 5%) than in group B (16/40, 40%; P < 0.002) and group C (9/30, 30%; P < 0.007). These results indicate a similar cancer risk in all continent forms of urinary diversion, at least via colon. Ileal interposition seems to be an effective carcinoma prophylaxis following ureterosigmoidostomy. The proliferative instability at the urointestinal anastomosis is crucial for the pathogenesis and prophylaxis of this form of carcinogenesis, whereas urine seems to play only a minor role.

The DNA excision repair capacity of 23 primary fibroblast lines from patients with dysplastic nevus syndrome was investigated and DNA repair synthesis ("unscheduled DNA synthesis") was determined after UV exposure. Seventeen fibroblast lines from normal donors served as controls. The dose/response experiments included up to ten dose levels and two wavelength ranges: UV-C (using a low-pressure mercury lamp emitting predominantly 254-nm light) and UV-B (artificial "sunlamp" radiation centering around 312-nm light). For each dose level, silver grains over fibroblast nuclei were counted by visual inspection. Twelve cell lines were also evaluated for both UV wavelength ranges using a new semi-automatic image analyzing system. This system included components for rapid sequential identification of both fibroblast nuclei and silver grains sited above them. Silver grains over 100 nuclei were determined for each UV dose level. Dose/response curves were established and analyzed by linear regression. As a quantitative term for assessing DNA excision repair capacity of a cell line we calculated the linear increase (G0) in the number of grains per nucleus, when the UV dose was multiplied by the factor e (i.e. 2.72). The sensitivity of grain detection and resolution of overlapping grains was approximately threefold better in visual than in automatic counting, especially when there were more than 70 grains over nuclei. The time required for visual counting, however, was tenfold that of automatic counting. The variance-weighted mean G0v.w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 79.1 (+/- 1.8- grains/nucleus, that of fibroblast lines from normal donors was 74.2 (+/- 1.7) grains/nucleus. This difference revealed a slightly better repair capability for cell lines from patients but was at the borderline of detection and, therefore, should not be overinterpreted. From the experimental accuracy achieved by determination of the variance-weighted means of the two groups, we would have been able to detect a difference of 7 and more grains [> 2 x (sigma normal+sigma patients)]. The variance-weighted mean G0v.w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 76.4 (+/- 1.4) grains/nucleus, whereas that of fibroblast lines from normal donors was only 66.6 (+/- 1.8) grains/nucleus. This difference was statistically significant and, contrary to expectation, revealed better, not worse post-UV DNA repair capability in cell lines from patients that in those from normal donors.(ABSTRACT TRUNCATED AT 400 WORDS)

HIV-infected individuals and SIV-infected rhesus macaques have, on the average, decreased plasma cysteine and cystine concentrations and decreased intracellular glutathione levels. We show that the cysteine supply and the intracellular glutathione levels have a strong influence on the T cell system. A study of healthy human subjects revealed that persons with intracellular glutathione levels of 20-30 nmol/mg protein had significantly higher numbers of CD4+ T cells than persons with either lower or higher glutathione levels. Persons who moved during a 4-week observation period from the optimal to the suboptimal range (10-20 nmol/mg) experienced, on the average, a 30% decrease in CD4+ T cell numbers. This decrease was prevented by treatment with N-acetyl-cysteine (NAC). NAC caused this relative increase of CD4+ T cell numbers in spite of decreasing glutathione levels and not by increasing the glutathione level. Our studies suggest that the immune system may be exquisitely sensitive not only against a cysteine and glutathione deficiency but also against an excess of cysteine.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to inhibit uroporphyrinogen decarboxylase activity resulting in chronic hepatic porphyria. From a cross-sectional study of 170 workers in chemical industry 68 showed elevated coproporphyrin levels, interpreted as secondary coproporphyrinuria. Three persons suffered from chronic hepatic porphyria in subclinical stages. None of the workers showed an overt porphyria cutanea tarda. A low-grade zinc protoporphyrinemia was observed in three persons. Forty-three of the 170 workers were evaluable for investigating the effect of TCDD on porphyrin levels. No significant correlation was found between TCDD concentration in adipose tissue and the level of uroporphyrin and coproporphyrin. The influence of a chloracne history is described.

200 adolescent (Group I) and 200 adult male Sprague Dawley rats (Group II) were divided into 4 subgroups of 50 animals each. Animals were treated on the 29th (Group I) or 98th day of life (Group II) either with acetone or Amuno carrier in acetone or Amuno on acetone (2.5 mg indomethacin/100 g animal weight in acetone) or with the pure substance indomethacin 2.5 mg/100 g of animal weight in acetone, giving a single application on the shaved dorsal skin. Subsequently the animals remained under observation until their deaths, followed by autopsy and histopathologic examination of several organs. The rats treated with Amuno of indomethacin in the adolescent stage showed lower body weights and a shorter total survival time. The adolescent animals treated with Amuno or indomethacin showed a significantly higher rate of interstitial testicular tumors of the Leydig tumor type, adenomas and adenocarcinomas of the small and large intestine as well as hepatocellular tumors. The total number of neoplasias was higher in the animals treated with Amuno or indomethacin compared to those treated with acetone or carrier with acetone. Application of Amuno or indomethacin also resulted in a higher rate of hyperplasias (sole of foot, lymph nodes, prostate). The impairment of the synthesis of prostaglandins caused by indomethacin apparently results in starting complex pathomechanisms which have effects until the late death of the animals. Adolescent animals were affected more frequently by the application of indomethacin as were already adult animals.

Short-term tests (STTs) for detecting and assessing genotoxic or mutagenic effects have catalyzed the development of biostatistical methods for more than one decade. Most notably, the Ames Salmonella/microsome assay created statistical methodology with a range of applications going beyond genotoxicity. Early approaches with parametric statistical methods appeared to be insufficient and have been replaced by non-parametric ones requiring less restrictive distributional assumptions. There have also been successful attempts to use biomathematical models for establishing dose-response relationships. Overdispersion has been recognized as a major problem for the evaluation of mutagenic count data and methods to cope with it have became available. A theory of generalized linear modelling is emerging to combine dose-response modeling with much less restrictive distributional assumptions, while allowing the inclusion of concomitant factors arising from the experimental conditions. The methodological survey below reviews the present state of this development and is intended to promote further research into biostatistical issues and methods of analysis. Appropriate methods for the design and analysis of STTs are discussed. The progress for the Ames assay was only partially transmitted to the analysis of the large number of other short-term assays. Several such assays are reviewed with respect to their present state of statistical evaluation.

Oral hairy leukoplakia is a lesion on the lateral part of the tongue that contains replicating Epstein-Barr virus (EBV) and presages progression from human immunodeficiency virus (HIV) infection to AIDS. To clarify the role of EBV in the development of the lesions, we used filter in situ DNA hybridization to determine the prevalence of EBV and of human papillomavirus (HPV) in epithelial cells obtained on swabs from the tongue of HIV-infected patients who had hairy leukoplakia, HIV-infected patients who did not have hairy leukoplakia, and healthy uninfected control persons. In samples collected from the 35 uninfected control persons, EBV DNA could not be detected except at low concentrations in three people. In contrast, all but one of the samples from 11 HIV-infected patients who had hairy leukoplakia contained EBV DNA. Of greatest interest, in 19 of 32 HIV-infected patients who had no signs of hairy leukoplakia, EBV DNA was also detected on the epithelium of the tongue. DNA filter in situ hybridization for the detection of HPV serotypes 6, 11, 16, and 18 in all cases yielded negative results. Statistical analysis showed that the presence of EBV DNA was significantly correlated with the clinical status of the HIV-infected persons, as determined by Walter Reed staging classification, whereas hairy leukoplakia was not. It is concluded that detection of EBV DNA in oral epithelium may be an earlier and more powerful predictor of progression to AIDS than is hairy leukoplakia.