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Human umbilical vein endothelial cells (HUVEC) propagated in co-culture with fibroblasts form capillary-like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co-cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co-culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary-like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co-culture untreated HUVEC formed capillary-like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel-Palade bodies and transport vesicles. Immuno-EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell-cell contacts, and at the luminal and abluminal side of the capillary-like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor-treated co-cultures. Our results demonstrate that treatment with levamisole or anti-VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP-470 results in formation of short endothelial cell cords. We discuss the implications of these findings.

There have been few studies to date that investigate the effect of race on outcomes related to coronary artery bypass grafting. The objective of the present study was to investigate race as an independent predictor of outcomes among patients undergoing coronary artery bypass graft (CABG). A nested case-control study from a twelve-year hospitalization cohort (N=9671) in which data were collected prospectively was conducted. Cases were African-American patients undergoing CABG (N=644). Controls were randomly selected Caucasian patients undergoing CABG (N=1932). Controls were matched to cases 3:1 on year of surgery. Fifteen preoperative and intraoperative risk factors and 14 outcomes were examined. The 14 outcomes of interest were length of stay, readmission to ICU, total ICU stay, total hours on ventilator post-op, reoperation for bleeding/tamponade, deep sternal wound infection, neurological complications, pneumonia, other pulmonary complications, renal failure, gastrointestinal complications, atrial fibrillation requiring treatment, in-hospital mortality, and intraoperative complications. Regression analysis was used to control for risk factors. Multivariate analysis revealed African-Americans were at greater risk for renal complications (OR 1.88, 95% CI 1.27-2.77), neurological complications (OR 1.34, 95% CI 1.01-1.77), and pulmonary complications (OR 2.11, 95% CI 1.72-2.59). African Americans had a significantly longer hospitalization post-operatively (OR 0.79, 95% CI 0.66-0.96), but were less likely to experience post-operative atrial fibrillation requiring treatment than Caucasians (OR 0.64, 95% CI 0.49-0.84). Even after multiple adjustments, African-Americans undergoing CABG surgery had significantly greater morbidity compared to Caucasian patients.

Background and Aim of Study: The aim of this study was to detect any outcome differences between patients who donated autologous blood versus nondonors undergoing nonemergent cardiac valve surgery. Of further interest was whether autologous donors required less allogeneic blood products overall than patients who did not donate., Methods: We conducted a nested case-control study in which data were collected prospectively on 225 variables. Cases underwent nonemergent, cardiac valve surgery and donated autologous blood products (n = 40). Controls also had nonemergent, cardiac valve surgery but did not donate autologous blood products (n = 120). Cases were matched to controls 1:3 on age (+/-3 years), gender, and New York Heart Association Functional Classification. We controlled for 12 potential confounding variables and examined 17 outcomes of interest. To generate the unadjusted risks of each outcome, chi-square and t-tests were performed comparing cases and controls to each outcome of interest. Then logistic regression analysis investigated the adjusted risk between cases and controls and for the outcomes of interest, each controlling for the potential confounding variables., Results: There were no significant differences between the cases and controls for 11 of the 12 possible confounding variables. Controls had significantly more chronic obstructive pulmonary disorder. There were no significant differences between cases and controls for 13 of the 17 outcomes of interest. Autologous blood donors received more total packed red blood cells (PRBCs) (p = 0.0373) and more total fresh frozen plasma than controls (p = 0.0002). Fewer autologous blood donors required allogeneic packed red blood cell transfusion (p = 0.0134), and the total length of stay was shorter for autologous donors (p = 0.0782)., Conclusion: Four of the 17 outcomes of interest were different for patients who donated autologous blood versus those who did not. Our experience demonstrated that elective cardiac valve surgery can safely reduce (by 18.3%) the need for allogeneic PRBCs by utilizing preoperative autologous blood donation.

The principal cause of a high mortality rate in mesenteric vein thrombosis (MVT) is a delay in diagnosis. Recent data indicate that the mortality rate is decreasing owing to earlier diagnosis and anticoagulation. The authors examined the treatment profile of MVT to see how the increased use of imaging and early anticoagulation has impacted this process. They retrospectively analyzed the treatment paradigm with acute MVT at one institution over a 10-year period. Twenty-three patients were identified. Data were analyzed using chi-squares and Student's t tests. Twenty-three patients (11 men and 12 women with an average age of 51.74 +/-14.8 years) were identified with acute MVT between the years of 1993 and 2003. Five patients had splenic vein thrombosis, 17 had superior mesenteric vein thrombosis, 1 had inferior mesenteric vein thrombosis, and 12 had portal vein thrombosis. Nine patients had combination mesenteric vein segment thrombosis. Thrombolytics were utilized in a total of 6 patients. Four of the 6 patients in whom lytics were utilized had combined mesenteric vein thrombosis; however, these 4 patients did not require surgical intervention. There was no significant difference in length of hospital stay between patients taking lytics versus patients treated with traditional anticoagulation with heparin (p = 0.291). A hypercoagulable state was identified in 66.7% of the patients. Four patients required surgical intervention. The overall mortality rate was 8.7% (2 of 23). The use of thrombolytics was associated with a significant mortality (p = 0.04). The use of antibiotics made no difference in mortality (p = 0.235), nor did antibiotic use influence length of hospitalization (p = 0.192). MVT is relatively rare, and often the delay in diagnosis increases the mortality rate. In the majority of cases prompt anticoagulation will preserve bowel viability and decrease mortality and morbidity rates. The majority of patients do not need surgery. There is a marked increase in mortality rate when these patients progress to surgical intervention. An increased awareness and early diagnosis has led to decreased morbidity and mortality rates.

Background: A study was performed to identify differences between men and women with regard to lung cancer type, stage at diagnosis, and survival in a single hospital system cancer registry., Patients and Methods: A retrospective cohort study was designed based on a study population drawn from the lung cancer tumor registry at a single hospital system composed of 2 independent hospitals in the Midwestern United States. This database included all patients from 1996 to 2002 with known lung cancer or abnormal findings on chest radiography or computed tomography (N=2618). Patients with adenocarcinoma or squamous cell, small-cell, or large-cell carcinoma were included in the study. Data were collected on patient sex, age, cancer type, stage at diagnosis, and survival status., Results: A total of 1216 men and 997 women met inclusion criteria for the study. There was no significant difference in age between sexes at diagnosis. Women were significantly more likely to have adenocarcinoma or small-cell carcinoma but less likely to have squamous cell carcinoma compared with men. There were no significant differences between sexes in the incidence of large-cell carcinoma. No significant differences were found between men and women in terms of cancer stage at diagnosis. There were significant differences in survival between the histologic types at years 3, 4, and 5. Only patients with stage I disease showed a difference between sexes and only for years 2, 3, 4, and 5., Conclusion: Overall differences in lung cancer histology and survival were found between men and women. Because a high mortality rate of lung cancer exists in both sexes, it is important to understand its occurrence and survival rates in both sexes.

Purpose: The goal of this study was to identify and validate novel serum markers of human colorectal cancer as potential candidates for noninvasive detection of early colorectal neoplasm., Experimental Design: Employing two-dimensional gel electrophoresis and mass spectrometry, we analyzed 16 matched colorectal cancer and adjacent normal tissue samples. Proteins found to be elevated in cancer tissue were further validated by generating antibodies which were used for immunoblotting of tissue samples and for the development of highly sensitive immunoassays for assessment of serum samples., Results: In total, 735 different proteins were identified in colon tissue. Strong elevation in colorectal cancer for five proteins was confirmed by immunoblot analysis: transforming growth factor-beta induced protein ig-h3 (betaIG-H3), nicotinamide N-methyltransferase (NNMT), nucleoside diphosphate kinase A (nm23-H1), purine nucleoside phosphorylase (PNPH), and mannose-6-phosphate receptor binding protein 1 (M6P1). Elevated levels of NNMT, which is not predicted to be secreted but is known as a cytoplasmic protein, were found in serum from patients with colorectal cancer. Employing a receiver-operating characteristic curve based on the measurement of 109 patients with colorectal cancer and 317 healthy controls, we obtained an area under the curve of 0.84 for NNMT, which was superior to the established tumor marker carcinoembryogenic antigen with an area under the curve of 0.78., Conclusions: It is proposed that NNMT serum levels may have significance in the early detection and in the management of patients with colorectal cancer.

The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion. For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry. Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections. Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents. We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity. gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA. The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications. Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.

The synthetic anthelmintic compound Levamisole has previously been used in cancer treatment as an adjuvant in combination with 5-fluorouracil. Its mode of action remains unclear, but an immune-stimulatory effect has been suggested. Here, we show that Levamisole inhibits angiogenesis in vitro and tumor growth in vivo. In vitro, Levamisole specifically inhibits proliferation and differentiation of endothelial cells propagated in co-culture with fibroblasts. In vivo, Levamisole inhibits the growth in nude mice of a transplanted human tumor. The use of nude mice as tumor hosts permits the discrimination between the angiogenesis inhibitory effect of Levamisole and its assumed immune-stimulatory effect. Our findings support a possible therapeutic effect of angiogenesis inhibitors in the treatment of cancer and call for further investigations of the mechanism(s) underlying the anti-angiogenic effect of Levamisole.

Objective: The purpose of our study was to assess the feasibility of a closed-chest replacement of the descending thoracic aorta utilizing the daVinci surgical robotic system and to compare hand-sewn running anastomosis to interrupted nitinol clips (Coalescent Surgical)., Methods: Six sheep underwent replacement of the descending aorta using Intuitive's daVinci surgical system. Using the daVinci, the descending aorta was dissected out and individual intercostal arteries were clipped and divided. Following systemic heparinization, the aorta was occluded using percutaneous vascular clamps (Chitwood clamps). The descending aorta was excised and replaced with a woven graft. The proximal and distal anastomoses were varied in each animal between a running 4-0 polypropylene technique and interrupted nitinol clips. Anastomoses were inspected for hemostasis and tested for burst strength., Results: Five of six animals survived the procedure. The average procedure time was 93 minutes. Cross-clamp times range from 55 to 25 minutes (average of 37 minutes). There was no significant difference in time between U-clip anastomoses (17 +/- 4.8 minutes) and sutured anastomoses (10.6 +/- 3.1 minutes). The burst pressure was higher for sutured anastomosis than for U-clips (214.6 +/- 61 and 110 +/- 35, respectively)., Conclusion: Replacement of the descending aorta with a graft is feasible in a closed chest model utilizing Intuitive's daVinci surgical system. While mean burst strengths were higher with a running sutured anastomosis, there was no difference in anastomotic time or ultimate hemostasis between techniques.

Background: As part of the general competencies set forth by the Accreditation Council for Graduate Medical Education (ACGME), residents must have an understanding of systems-based practice, which is demonstrated by an awareness of-and responsiveness to-the comprehensive health care system. Residents must be able to effectively access and use system resources to provide care that is of optimal value. Essential to understanding and implementing systems-based practice is an awareness of how different patient populations perceive health care; one key factor that influences patients' perceptions of health care is their level of education., Methods: We surveyed 2900 adult patients in the Cincinnati, Ohio area and stratified them into 2 groups based on their level of education. Group 1 included patients with some high school education or a high school degree. Group 2 included patients with some college education, an undergraduate degree, or graduate/professional-level coursework. We then compared the groups' perceptions of common health care issues, including physician compensation, patient obligation for medical bills, and increased cost for the freedom to choose a physician., Results: Of 395 respondents, a higher percentage of Group 2 patients, compared with those in Group 1, understood that physicians do not collect 100% of what they bill (p < 0.001) and that businesses do influence the amount a physician is paid by insurance companies (p = 0.009). Conversely, a higher percentage of Group 1 patients thought that physicians are overpaid (p = 0.030) and that they keep a large portion of what they charge (p < 0.001). Further, fewer Group 1 respondents felt obligated to pay medical bills not covered by insurance (p = 0.002); they also were less willing to pay more for better medical care (p = 0.002) or for the freedom to choose a physician (p = 0.015)., Conclusions: This study indicates that patients with a lower level of education believe that physicians are overpaid and that they keep a large portion of insurance reimbursement. These findings may explain why fewer patients in this group feel that they are obligated to pay medical bills not covered by insurance and why they are less likely to pay more for better medical care or for the freedom to choose a physician.

Objective: To assess if coronary artery bypass grafting (CABG) patients with dialysis-dependent end stage renal disease (ESRD) experience greater intraoperative and postoperative morbidity and mortality compared to CABG patients without ESRD., Methods: We conducted a nested case-control study from an 8-year hospitalization cohort in which data were collected prospectively. Inclusion criteria included CABG surgery and age greater than 18 years. Cases were patients with dialysis dependent ESRD (N = 28) and controls were patients without ESRD (N = 84). Cases were matched to controls 1:3 on age, gender, tobacco history, and New York Heart Association Functional Class. The outcomes of interest were mortality, intensive care unit length of stay, total length of hospitalization, time on the ventilator, wound complications, pulmonary complications, neurological complications, gastrointestinal complications, arrhythmia, and intraoperative complications. Using logistic regression we controlled for 13 potential confounding variables., Results: There were no significant differences between the groups with the exception of total length of hospitalization. Patients with dialysis-dependent ESRD had a significantly longer total hospitalization stay (21%) than patients without ESRD. There were no significant differences for the remaining nine outcomes of interest, including perioperative morbidity or mortality., Conclusion: Intraoperative and postoperative morbidity and mortality for CABG were not increased for patients with dialysis-dependent ESRD compared to patients without ESRD. However, patients on dialysis undergoing CABG experienced a greater length of hospitalization than patients undergoing CABG who were not on dialysis.

Background: The aim of this study was to assess risk factors and outcomes of gastrointestinal (GI) complications in patients undergoing coronary artery bypass surgery (CABG)., Study Design: We conducted a nested case-control study from a 9-year hospitalization cohort (n = 7,345) in which data were collected prospectively. Patients developed GI complications (n = 66) and controls did not (n = 330). Cases were matched to controls 1:5 on type of surgery. We examined 16 risk factors and 14 outcomes., Results: Five risk factors proved significant in predicting GI complications. Patients were more likely to be older than age 70, to be on dialysis, to have left ventricular hypertrophy, and to be on anticoagulants; the procedure was also more likely to be urgent. There was no significant difference between the cases and controls for the remaining 11 risk factors. We also computed correlation coefficients among the significant variables; using regression analysis, we found that patients undergoing CABG had a threefold increase in the risk of GI complications if they were older than age 70 (odds ratio [OR] 1.06, 95% CI 1.03 to 0.97), if they were on dialysis (OR 1.87, 95% CI 1.98 to 1.22), and if their procedure was urgent (OR 1.91, 95% CI 1.07 to 3.4). Eleven outcomes proved significant. Patients with GI complications ran a greater risk of mortality; required more additional procedures; suffered arrhythmia that required treatment; and were more likely to have neurologic, pulmonary, renal, and sternal wound complications. They also had greater length of hospitalization, intensive care unit length of hospitalization, ventilator time, and postoperative creatine phosphokinase levels., Conclusions: In patients undergoing CABG surgery, urgency of the procedure, age greater than 70 years, and dialysis all significantly increased the risk of a GI complication. Patients with GI complications also experienced more negative outcomes.

Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.

Since solid tumours and metastases depend on adequate blood supply, much research is focused on inhibition of angiogenesis. Unfortunately, most known angiogenesis inhibitors have serious side effects when used as therapeutic agents in man. It is therefore important to develop methods to identify well-tolerated and efficient angiogenesis inhibitors. As a method for identification of new angiogenesis inhibitors we have further developed the procedure described by Bishop et al. (Angiogenesis 1999;3:335-44) to a quantitative ELISA-based fibroblast and endothelial cell co-culture angiogenesis assay. In each well of a 96-microwell plate, human umbilical vein endothelial cells (HUVEC) are seeded onto normal human dermal fibroblasts (NHDF) and propagated in co-culture for 72 h with or without a potential angiogenesis inhibitor. The effect on total cell proliferation is evaluated by quantitative immunochemical measurement of DNA, and on endothelial tube formation by quantification of CD 31, von Willebrand factor, and collagen IV. After ELISA reading, the morphology of the tubular structures formed by HUVEC is visualised with BCIP/NBT, permitting a quantitative result and a qualitative evaluation of cell morphology from the same well. We have used the assay to demonstrate the effect of well-known angiogenesis inhibitors on HUVEC tube formation.

It must be assumed that all tumor cells produce proteins which do not belong to a normal cell. These are called tumor-associated or tumor-specific antigens. In the classic immune surveillance theory it is believed that the cellular immune defense (the T-cell system) continuously discovers and eliminates newly arisen tumor cells which express such tumor-specific antigens. Since then it has been shown that one of the preconditions for the T-cell system to be able to recognize antigens is that they are presented by MHC class I histocompatibility antigens. There is a continual processing and presentation of all intracellular proteins in a cell. Thus, a tumor cell which produces an abnormal protein will also present this and thereby expose itself to being killed by cytotoxic T cells. The antigens are presented in the form of short peptides (8-9 aminoacids), which arise as a result of controlled degradation of the original proteins. The peptides thus formed are transported by specialised molecules in the so-called endogenous antigen processing and presentation pathway, and are eventually bound to and presented by MHC class I molecules. It has been shown that many tumors express less MHC class I on their surface compared to the normal tissue from which they have arisen, and also that patients with reduced immune function have an increased incidence of certain forms of cancer. It is therefore widely believed that a low MHC class I level contributes to the ability of tumor cells to avoid the T-cell-mediated immune defense. The aim of the present research project was to confirm the existence of a T-cell-mediated immune selection in primary tumors. Another of its goals was to elucidate the extent to which tumor cells with low MHC class I expression showed poor ability to present antigen, and whether the reason for this could be found in one or more of the molecular systems which participate in antigen processing and presentation. By using the chemical carcinogen 3-methylcholanthrene a total of 144 tumors were induced in immunologically normal and T-cell defective mice, respectively. It was assumed that tumors induced in normal mice would be immune selected, whilst this would not be the case for tumors from T-cell defective mice. This enabled us to work with a tumor-material where the two populations only differed in that the one part had undergone selection by a T-cell system and the other had not. Tumor induction time turned out to be shorter in immune defective than in normal mice, and the tumor frequency was higher, which might be due to the fact that in normal mice tumor growth was inhibited and in certain cases stopped by the T cells. On transplantation of the uncloned cell lines which were established from the primary tumors to immunologically normal congenic recipients, we were able to show that most of the tumors which originated from mice with a functional T-cell system, and which must therefore be assumed to have undergone selection in the primary tumor host, were not immunogenic and were therefore accepted. On the other hand, most tumors which originated from T-cell-defective mice were rejected as a sign that immunogenic tumor cells, assumed to have expressed tumor antigen, had not been eliminated in the primary tumor host. Still, we found that the ability of tumor cells to induce an immune response on transplantation was not reflected in their MHC class I expression. Both tumor lines from immunodeficient and normal mice had highly varying MHC class I levels, and contrary to expectations the highest levels were seen in tumor lines from immunologically normal mice. At the same time we found that the expression levels for the three different MHC class I molecules were the same in the individual tumor lines, which might indicate that the three genes are syn-regulated. The MHC class I mRNA content in tumors from normal mice was generally concordant with the surface level of MHC protein. Among the tumor lines from immunodeficient mice, on the other hand, we found several where there was no such agreement, which was taken to indicate that tumor cells with deviant MHC class I gene transcription had not been eliminated, in contrast to in the immunocompetent tumor hosts. The ability of tumor cells to present antigen was investigated by infecting cells with virus and thereafter assessing their ability to function as target cells for virus-specific T cells in a cytotoxic test. Their ability to do this varied considerably, but showed a correlation with their MHC class I expression. Among the transplanted tumor lines that were not able to present viral antigen, the majority were accepted, while most of the tumor lines which were rejected on transplantation possessed the ability to present virus. Closer analysis of the composition of proteasomes, heat shock protein content and TAP molecule function, which are all involved in the antigen processing system, did not immediately reveal any defects. Treatment with interferon gamma, which is known to upregulate the transcription of MHC class I and a number of other proteins which are involved in antigen presentation, showed that by far the majority of the tumor lines were able to respond normally. This was also true for the tumor lines which had deviant MHC class I gene transcription and the cells which showed poor ability to present viral antigen. We did find, however, three cell lines which did not respond to interferon gamma, and they all had defective interferon gamma-signaling, not because they did not express the interferon-receptor on the surface, but possibly on account of their lacking phosphorylation of an intracellular signal molecule, Stat1.

Protein tyrosine phosphatase-like IA-2 autoantigen is one of the major targets of humoral autoimmunity in patients with insulin-dependant diabetes mellitus (IDDM). In an effort to define the epitopes recognized by autoantibodies against IA-2, we generated five human mAbs (hAbs) from peripheral B lymphocytes isolated from patients most of whom had been recently diagnosed for IDDM. Determination and fine mapping of the critical regions for autoantibody binding was performed by RIA using mutant and chimeric constructs of IA-2- and IA-2beta-regions. Four of the five IgG autoantibodies recognized distinct epitopes within the protein tyrosine phosphatase (PTP)-like domain of IA-2. The minimal region required for binding by three of the PTP-like domain-specific hAbs could be located to aa 777-979. Two of these hAbs cross-reacted with the related IA-2beta PTP-like domain (IA-2beta aa 741-1033). A further PTP-like domain specific hAb required the entire PTP-like domain (aa 687-979) for binding, but critical amino acids clustered in the N-terminal region 687-777. An additional epitope could be localized within the juxtamembrane domain (aa 603-779). In competition experiments, the epitope recognized by one of the hAbs was shown to be targeted by 10 of 14 anti-IA-2-positive sera. Nucleotide sequence analysis of this hAb revealed that it used a V(H) germline gene (DP-71) preferably expressed in autoantibodies associated with IDDM. The presence of somatic mutations in both heavy and light chain genes and the high affinity or this Ab suggest that the immune response to IA-2 is Ag driven.

An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell-competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell-mediated lysis.

The idea of immunological surveillance against cancer has existed for nearly 100 years but as no conclusive evidence has yet been published the importance of the cellular immune defense in the detection and removal of incipient or existing tumors is still a hotly debated subject. However, in order to select a relevant immunotherapeutic strategy in the treatment of cancer, a fundamental understanding of the basic immunologic conditions under which a tumor develops and exists is a prerequisite. Therefore, a murine model was set up that we hoped would enable us to confirm or reject the theory of immunological surveillance. A large panel of methylcholanthrene induced tumors was established in T-cell immunodeficient nude mice and congenic normal mice to study the influence of the immune system on developing tumors. As nude mice developed tumors fastest and with the highest incidence, we concluded that in this model the immune system constituted a 'tumor-suppressive factor' delaying and sometimes abrogating tumor growth, i.e. performing immune surveillance. Immunogenicity of the tumors was assessed by transplantation back to normal histocompatible mice. Tumors originating from the immunodeficient nude mice turned out to be far more immunogenic than tumors from normal mice, resulting in a high rejection rate. CD8+ cytotoxic T cells were found to be indispensable for this rejection, leading to the conclusion that the cytotoxic T cells perform immune selection in normal mice, eliminating immunogenic tumor cell variants in the incipient tumor. In this review, we discuss the difficulties facing immunotherapy when conclusions are drawn from the presented observations and hypotheses.

Nucleotide sequence and biochemical analysis of D-beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M(r) of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature.

The risk and clearance of GB virus type C (GBV-C)/hepatitis G virus (HGV) infection was investigated in a cohort of homosexual men (n=180; median follow-up time, 7 years). The interaction between GBV-C/HGV RNA and antibodies against the E2 region of the virus, and the clinical impact of chronic GBV-C/HGV infection were studied. GBV-C/HGV RNA was detected by RT-PCR, and E2 antibodies were assessed by an immunoassay. At baseline, 63% of the participants had evidence of previous or current GBV-C/HGV infection. The GBV-C/HGV incidence rate was 2 per 100 person-years (95% confidence interval 0. 9-3.8) and was similar to the HIV incidence. The incidence of GBV-C/HGV infection was significantly higher in those reporting unprotected anal intercourse (3.6 per 100 person-years compared to 0 in the group without such sexual contacts). The occurrence of E2 antibodies was strongly associated with GBV-C/HGV RNA clearance. A loss of E2 antibodies was observed at a rate of 1.5 per 100 person-years. It was higher among HIV-infected individuals. Chronic GBV-C/HGV infection was not associated with clinical or biochemical evidence of liver disease.

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine/threonine phosphatases in STAT3 signaling in human antigen-specific CD4(+) T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1/PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine/threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogen-activated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.

An experiment was set up to investigate the relationship, if any, between cell surface MHC class I expression and the growth rate for skin tumors induced by two different UV radiation regimens in hairless mice. Two groups of 20 hairless mice were each irradiated with either a UVA radiation source (2 SED per session) or broad-spectrum UV radiation (UVB) (8.1 SED per session) 5 days a week during the entire experiment. In the UVA group, 17 out of 20 animals developed tumors, and 10 of these grew to a diameter of > or = 5 mm. In the UVB group, 19 out of 20 animals developed tumors, and 15 of these grew to a diameter of > or = 5 mm. The tumor induction time, i.e. the time from the start of UV treatment to tumor appearance, was found to be significantly longer (p<0.01) in the UVA than in the UVB group. This is in accordance with previous findings. Of the 25 tumors growing to a diameter of > or = 5 mm, 11 were established as cultured cell lines (4 UVA and 7 UVB tumors). These uncloned cell lines were analyzed for surface expression of major histocompatibility complex class I by FACS analysis. There was a clear correlation between high MHC class I expression and slow growth of the individual tumors (p<0.05). This suggests a role for the MHC class I governed, i.e. cytotoxic T-cell-mediated, reactions in deciding the fate of UV-induced skin cancers. No correlation was found between MHC class I expression and tumor induction time.

The clinical significance and course of acute hepatitis G virus (HGV) infection were studied by measuring HGV RNA and antibody to HGV envelope protein E2 (HGV-E2 antibody). A total of 59 patients with transfusion-associated non-A, non-B hepatitis, who were followed-up for more than 1 year, were selected retrospectively. HGV RNA was measured by reverse transcriptase (RT) and nested polymerase chain reaction (PCR) was performed, using primer sets, in the 5'-non-coding region of the HGV genome. HGV-E2 antibody was measured by enzyme-linked immunosorbent assay (ELISA) using recombinant E2 protein. Of the 59 patients, 51 (86%) were infected with hepatitis C virus (HCV) and 12 (20%) were infected with HGV; 11 of the 12 with HGV infection were also infected with HCV. HGV viraemia was cleared during the follow-up period in seven of the 12 patients with HGV infection. All these seven patients seroconverted for HGV-E2 antibody just before or just after the clearance of HGV viraemia. In contrast, all five patients without clearance of HGV viraemia were negative for HGV-E2 antibody (P = 0.0013). Of seven patients with continuous HGV viraemia at 1 year from the onset of acute hepatitis, four with HCV RNA showed chronic elevation of alanine aminotransferase (ALT) but three without HCV RNA did not. The severity of acute hepatitis was similar between patients with both HGV and HCV infections and in those with HCV infection alone. The majority of patients with HGV infection cleared the virus during long-term follow-up. Appearance of HGV-E2 antibody was associated with the clearance of HGV viraemia. An abnormal ALT level was noted to depend on HCV infection but not on HGV infection in both the acute and chronic phases of transfusion-associated hepatitis.

In order to elucidate the structure and morphology of hepatitis G virus (HGV), a recently isolated flavivirus, we generated a panel of eight monoclonal antibodies (MAbs) against the putative second envelope protein (E2) following DNA immunization. The MAbs were shown to be specific for four different epitopes on recombinant E2. MAb Mc6 was the only antibody able to detect the linear epitope LTGGFYEPL. In addition, Mc6 was able to immunoprecipitate viral particles in human blood samples as detected by reverse transcription-PCR amplification of HGV RNA. This precipitation could be competed by addition of saturating amounts of the linear peptide or abolished by addition of Nonidet P-40. We conclude that, albeit lacking the N-terminal sequence of a functional core protein, HGV builds classical viral particles displaying E2 envelope protein on their outer surfaces.

Using confocal laser scanning microscopy we studied sections of the T24B, a human bladder carcinoma, grown in C.B.-17 scid/scid or NMRI nu/nu mice in order to examine the relationship between tumor tissue and tumor vessels. Tumor cells were labelled with FITC-anti-cytokeratin and blood vessel endothelia with Cy3-labelled BS-I lectin. In contrast to our expectation, no major leaks in the endothelial lining of blood vessels were observed. We are looking for a suitable marker for mouse lymphatics in order to investigate their possible role.

We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5' noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (P < 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (P < 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (P < 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.

The second envelope protein (E2) of the hepatitis G virus (HGV) was expressed in Chinese hamster ovary (CHO) cells and showed a molecular weight of approximately 60 to 70 kd, with 15 to 25 kd of the size contributed by N-linked glycosylation. An enzyme-linked immunosorbent assay (ELISA) using HGV-E2 was developed to test for antibodies to this protein (anti-E2) in human sera. High sensitivity was achieved by developing monoclonal antibodies (mAbs) to HGV-E2, which were used as capture antibodies in the ELISA. Our studies revealed that 16% of healthy Spanish blood donors were exposed to HGV, indicating that additional routes of viral transmission besides parenteral exposure might exist. An even higher prevalence of exposure to HGV (52%-73%) was found in several groups at risk of parenteral exposure to infectious agents, i.e., intravenous drug users, transfusion history, hemophiliacs, and hepatitis C virus (HCV)-positive patients. Most anti-E2-positive patients were HGV-RNA-negative and vice versa, indicating an inverse correlation of these two viral markers. A panel of 16 posttransfusion patients followed for up to 16 years revealed that patients who develop an anti-E2 response become HGV-RNA-negative, while patients who do not develop anti-E2 are persistently infected. Immunity to HGV seems to be long-lasting, because circulating antibody to E2 could still be detected 14 years after seroconversion. Sequence comparisons showed that E2 is highly conserved among isolates collected worldwide, indicating that immune escape variants are not common in HGV infections. This reflects on a molecular level why HGV infections usually are cleared spontaneously by the host. However, possible mechanisms of HGV persistence, as found in some patients, remain to be elucidated.

Seventy-eight uncloned tumour cell lines, each established from a primary sarcoma induced with methyl-cholanthrene in immunocompetent nu/+ BALB/c and C.B.-17 mice or in immunodeficient nu/nu BALB/c and severe combined immunodeficient (SCID) mice, were examined for sensitivity to interferon-gamma (IFN-gamma) as measured by tumour cell augmentation of major histocompatibility complex (MHC) class I expression. The tumour cells were cultured with IFN-gamma and their expression of Kd, Dd and Ld was measured by fluorescence-activated cell sorter analysis. All but three of the 78 tumour lines up-regulated Kd, Dd and Ld to a variable degree in response to IFN-gamma, indicating that IFN-gamma resistance is not a common property of these sarcomas. The tumour cell lines varied greatly in their MHC class I expression before as well as after IFN-gamma stimulation. There was a tendency towards a higher MHC expression after IFN-gamma stimulation in tumour lines from immunocompetent mice compared to immunodeficient mice, but no common maximum MHC class I expression level was found for the 78 tumour cell lines. Three of the tumour lines, all from immunodeficient mice, completely failed to respond to IFN-gamma by up-regulating MHC class I expression. The same three also displayed absence of IFN-gamma-induced Stat1 beta tyrosine phosphorylation and low Stat1 alpha tyrosine phosphorylation, indicating a defect in the signal transduction pathway affecting phosphorylation of Stat1. These findings strongly suggest a link between defects in Stat1 phosphorylation and the failure to up-regulate MHC class I. In all tumour lines tested, the Stat1 Western blotting revealed a 78 kDa protein (p78) not previously described.

Background: Hepatitis G virus (HGV/GBV-C) RNA indicating current infection has been frequently isolated from the sera of transplant recipients and other multitransfused individuals. Lifetime exposure to the virus, however, is unknown. We carried out a study to determine the prevalence and risk factors of HGV antibodies and of HGV RNA among renal transplant recipients, and to investigate possible associations between HGV RNA and immunosuppressive treatment., Methods: HGV RNA was detected by reverse transcriptase-polymerase chain reaction, and HGV antibodies (anti-E2) by a newly developed immunoassay. To assess risk factors for HGV exposure, univariate and multivariate analysis was performed., Results: Of the 221 patients, 14% were HGV RNA positive and 40% had HGV antibodies. Both HGV RNA and anti-HGV were present in only two individuals. Thus, the overall HGV exposure prevalence was 53%. It increased significantly with the number of blood transfusions. In logistic regression, the adjusted HGV exposure prevalence odds ratio was 5.7 (95% confidence interval [CI]: 2.2-15) among patients with > or =10 transfusions (baseline: no transfusions). Other independent risk factors were a longer duration of hemodialysis and a longer time interval since transplantation. HGV viremia was not associated with the type of immunosuppressive treatment. Alanine aminotransferase levels were not significantly increased among HGV RNA-positive patients., Conclusions: Much higher proportions of renal transplant recipients were exposed to HGV than is suggested by HGV RNA detection alone. The majority of infected individuals apparently eliminate the virus over time. Contaminated blood transfusions have to be regarded as a main risk factor for HGV infection.

In a previous study, we demonstrated that eight sarcomas induced by chemical carcinogenesis in nude mice were rejected by syngeneic immunocompetent recipients at a much higher rate than eight sarcomas induced with the same method in syngeneic immmunocompetent mice. In the present study, we investigated these 16 sarcomas for structural and quantitative aberrations in components of the MHC class I-restricted antigen-processing and -presentation pathway. Considerable discrepancies between mRNA levels and cell surface protein expression of MHC class I (Kd, Dd and Ld) molecules were observed almost exclusively in the tumours derived from nude mice. Several of the nude mouse-derived tumours also displayed incongruent levels of heavy chain mRNA and beta2-microglobulin mRNA. These findings are taken as indications of abnormal regulation of gene transcription in nude mouse tumours, and if this abnormal regulation extends to the entire genome, it may explain the pronounced immunogenicity of these tumours. Proteasome composition, heat shock protein expression, TAP-molecule inducibility and intercellular adhesion molecule-1 expression were investigated in the same tumours. We found no indications of structural defects or quantitative differences in these molecules between the two groups of tumours.

With the aim of studying possible T-cell mediated selection of the cells in growing tumours, 108 mice of the C.B-17 strain, either immunocompetent C.B-17 mice or histocompatible immunodeficient C.B-17 severe combined immune deficiency (scid) were treated with 3-methylcholanthrene (MCA) in two different dosages. A total of 51 tumours were obtained, 44 of which were established as uncloned tumour cell lines, and used for further study. Tumour incidence correlated with carcinogen-dosage in that more tumours developed in groups treated with a high MCA dose than in groups treated with a low MCA dose, but not with immune status of the tumour host. No significant difference in the level of MHC class I molecule expression was found between the two groups of tumours. The rate of rejection after transplantation to syngeneic immunocompetent hosts was significantly higher for the scid tumours than for the non-scid tumours. The authors suggest that this reflects an immunoselection performed by T cells in the immunocompetent host in which the tumour originated, which has eliminated highly immunogenic tumour cells, leaving non-immunogenic tumour cells to grow.

Background: A flavivirus designated hepatitis G virus (HGV) has been isolated from the serum of patients with non-A-E hepatitis. Hitherto, the presence of HGV RNA in serum has been detected with the reverse transcription-polymerase chain reaction (RT-PCR) amplification method. We have now developed an immunoassay for antibodies against an HGV protein., Methods: Recombinant HGV envelope protein E2 was used as antigen in an ELISA. 80 blood donors, 99 intravenous-drug users, and 11 patients with acute post-transfusion hepatitis were tested for antibodies to E2. The HGV-RNA status was assessed by RT-PCR., Findings: Anti-E2 seroprevalence was 9% among the blood donors and 41% among the drug users; HGV-RNA prevalence was 2.5% and 38%, respectively. Whereas anti-E2 prevalence increased with the duration of drug use, HGV-RNA prevalence declined in parallel. In each group, the presence of anti-E2 and HGV RNA was almost mutually exclusive: none of the blood donors and only 4% of the drug users were positive for both markers at the same time. Of the 11 post-transfusion patients--who were all HGV-RNA positive and anti-E2 negative at the onset of disease--four developed antibodies to E2 during the following year, and two of the four subsequently became HGV-RNA negative., Interpretation: We conclude that a humoral immune response to E2 is associated with loss of detectable HGV viraemia. Thus, E2-specific antibodies might serve as a useful marker for diagnosing recovery from HGV infections. The immunoassay we describe should facilitate investigation of suspected infections and may be helpful in the elucidation of the clinical significance of HGV.

A panel of sarcomas induced with 3-methylcholanthrene in normal and immunodeficient mice was studied for their capacity to present antigen by the endogenous, MHC class I restricted pathway. Lymphocytic choriomeningitis virus was used to infect cultured tumour cells, and the infected cells were tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.-17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule Ld of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation to immunocompetent, histocompatible recipients.

The recently identified hepatitis G virus (HGV) is parenterally transmitted; the impact of sexual transmission is unknown. Moreover, it is unclear what proportion of HGV-infected persons may develop persistent viremia. Sera from injecting drug users (IDUs), non-drug-injecting homosexual and bisexual men with high levels of sexual risk behavior, and blood donors were tested for HGV RNA and hepatitis C virus (HCV) RNA by reverse transcriptase-polymerase chain reaction and for antibodies to human immunodeficiency virus, hepatitis B virus, and HCV. HGV RNA was detected in 33% of IDUs (n = 130), 11% of homosexual and bisexual men (n = 101), and 2% of blood donors (n = 90). HGV RNA seroprevalence significantly decreased with increasing time since first drug injection, whereas the seroprevalences of both HCV RNA and anti-HCV antibody increased. Thus, a high proportion of HGV-infected persons may clear the virus and develop protective antibodies. The relatively high HGV RNA prevalence among non-drug-injecting homosexual and bisexual men indicates that sexual contact may be another important route of HGV transmission.

Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T-cell-mediated defense mechanisms may confer upon the bearer a lower resistance to 3-methylcholanthrene and a different MHC profile of the ensuing tumor.

To detect possible differences in immunogenicity between tumors induced in T cell-deficient mice and phenotypically normal congenic mice, 16 sarcomas, 8 having developed in nude BALB/c mice and 8 having developed in congenic normal (nu/+) mice, were transplanted to normal BALB/c recipients and the rates of rejection or acceptance were registered. The 16 tumors were chosen randomly from a panel of 39 sarcomas induced with 0.5% or 0.1% 3-methylcholanthrene and maintained as cell lines in culture. Out of the tumors originating from nude mice, 66% were rejected by the normal BALB/c recipients, while only 30% of the tumors originating from normal mice were rejected. Tumors with short induction times from normal mice were more readily accepted than tumors with long induction times. Tumors originating from nude mice had significantly longer mean latency times after transplantation to both normal and nude recipients than tumors originating from normal mice. Contrary to what has been reported by others, there was no correlation between the rejection rates of the individual tumors and their Kd, Dd or Ld major histocompatibility complex (MHC) class I surface expression as measured by flow cytometric analysis of cultured tumor cells. The Kd, Dd and Ld proteins of the transplanted tumor lines were analyzed by isoelectric focusing for the occurrence of mutations resulting in altered charge of the MHC protein. No such mutations were found, ruling out MHC mutations of that kind as the source of immunogenicity in the cell lines used in these experiments. Our results suggest the existence of a T cell-mediated selection in the original tumor cell mass of tumors induced in normal mice, adapting the tumor to growth in a host with a functional T cell system, but apparently there is no connection between this loss of immunogenicity and loss of MHC class I expression.

Omp alpha is an outer-membrane protein that spans the periplasmic space of the hyperthermophilic eubacterium Thermotoga maritima. The molecule contains a globular head with an apparent diameter of 8 nm and a rod-shaped tail of 40 nm length. The sequence of the globular domain is homologous to a conserved region of cell wall-bound proteins and probably attaches Omp alpha to the peptidoglycan. The sequence of the rod domain resembles that of coiled coil proteins and ends in a transmembrane segment that anchors Omp alpha to the outer membrane. We have analysed Omp alpha by scanning transmission electron microscopy (STEM) and by statistical sequence analysis methods. The Omp alpha rod is a tetramer with an unusual periodicity of hydrophobic residues close to 3.6 that differs from the 3.5 periodicity of canonical coiled coils. This is due to periodic omissions of three residues in the heptad repeat pattern ("stutters") whose effect is to locally distort the packing of hydrophobic layers in the core of the coiled coil. Residues in position alpha are shifted to occupy a position halfway between positions alpha and d (x layers) and residues in positions d and e are shifted so that both participate in core packing interactions (da layers). Such distorted layers are frequently found in helical bundles and are characteristic of helices that do not undergo supercoiling. The only homo-oligomeric coiled coil of known structure which contains x and da layers is the three-stranded coiled coil of influenza haemagglutinin. Using geometric constraints derived from this structure, we have built a model for the Omp alpha rod in which the helices have a crossing angle of less than 15 degrees and maintain a residual degree of supercoiling with a pitch of approximately 40 nm. Our analysis of distorted layers in the hydrophobic core of coiled coils and helical bundles shows that stutters must not be viewed as discontinuities but rather as a departure from the canonical "knobs-into-holes" packing that allows helices to interact at a low angle without supercoiling. Although stutters have been considered to weaken helical interactions, their occurrence in a rigid, highly thermostable coiled coil indicates that this may not be generally true. Our analysis also indicates that skips and stutters are two different conventions for describing the same underlying structural feature.

The hormone binding site of rat and human natriuretic peptide clearance receptor (NPR-C), a single transmembrane receptor, has been further refined by mutagenesis. In addition to residue 188 (rat Ala, human Ile), which completely inverts the pharmacology of the rat and human receptors [Engel et al. (1994) J. Biol. Chem. 269, 17005-17008], we report a second key residue at position 205 (rat Tyr, human Asn) which modulates affinity to a limited number of ligands. Orthologous mutation of both residues results in tighter binding for human and weaker binding for rat NPR-C. The ligand binding fold of the receptor is formed by at least the first half of the extracellular domain containing two intramolecular disulfide loops, with the two affinity-modulating residues 188 and 205 in the second loop.

This study compared the efficacy of inhaled salmeterol (SM) 50 micrograms twice a day with SM 100 micrograms once a day (at night) and placebo in patients with poorly controlled nocturnal asthma despite treatment with corticosteroids, oral long-acting bronchodilators, or both. This was a two-center, double-blind, randomized, crossover study with three treatment periods, each of 3 weeks' duration. The first treatment period was preceded by two 1-week run-in periods, and the last treatment period was followed by a 2-week follow-up period. In the three treatment periods, patients received either SM 50 micrograms twice a day, SM 100 micrograms nightly, or placebo, in randomized order. Salbutamol metered-dose inhaler was given as relief medication. Of the 41 randomized patients, 38 were evaluable for more than one treatment period. Efficacy and safety were determined by daily record card data: morning and evening peak expiratory flow rates (PEFR), daytime and nighttime asthma symptom scores, and rescue salbutamol use. At clinic visits, FEV1 and FVC were measured, and the physicians' and the patients' respective assessments of the study mediation were noted. The study showed that the two doses, SM 50 micrograms twice a day and SM 100 micrograms nightly, were equally effective in controlling nocturnal asthma symptoms and were significantly better than placebo. SM was well tolerated, and no unexpected problems were revealed. The adverse events reported during this study were related either to the patient's underlying disease or to an intercurrent respiratory infection.

The natriuretic peptide receptor-C (NPR-C) constitutes greater than 95% of the natriuretic peptide binding sites in vivo. This cell surface glycoprotein is a disulfide-linked homodimer with a subunit molecular weight of 68,000. Two sources and types of ANP affinity-purified human NPR-C were used to map disulfide linkages and glycosylation sites of this receptor by mass spectrometry: the extracellular domain obtained by papain cleavage of a receptor-IgG fusion protein expressed in Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data describe the intra- and intermolecular linkages in NPR-C, providing a model for the homologous guanylyl cyclase receptors, NPR-A and NPR-B; both of the cyclase receptors likely contain the first amino-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.

This study compared the efficacy and tolerability of sodium cromoglycate (SC) and beclomethasone dipropionate (BDP) in adult patients with bronchial asthma inadequately treated with bronchodilators alone. The study was a double-blind, randomized, double-dummy, parallel-group study. Patients with mild to moderate symptomatic asthma, inadequately treated with bronchodilators only, were, after a 2-week run-in (base-line) period, randomized to 8 weeks of treatment with either SC 10 mg four times daily or BDP 100 micrograms four times daily. Salbutamol metered-dose inhaler was given as relief medication. A total of 37 patients were randomized for treatment, 19 patients in the SC group and 18 patients in the BD group. Efficacy and safety were determined by daily record card data: morning and evening peak-expiratory-flow rates (PEFR), daytime and nighttime asthma symptom scores, and rescue salbutamol use. At clinic visits, FEV1 and FVC were measured, as were the physician's and the patient's assessment of the medication at the end of the study. The safety and tolerability of the trial medication were assessed by monitoring adverse events throughout the study. A clinically and statistically significant improvement of the asthma in FEV1, symptom scores, rescue medication, and global opinion of efficacy was observed, and both groups provided equivalent efficacy. The morning PEFR as well as the evening PEFR for both groups improved, but was statistically significant only for the BDP group (M-PEFR). Both drugs were well tolerated with only a few minor adverse events. This trial shows that SC and BDP are equally effective anti-inflammatory treatments for mild to moderate bronchial asthma in adults.

We report the cloning and expressing of rat natriuretic peptide receptor-C (NPR-C), which binds naturally occurring and synthetic ligands with higher affinity than human NPR-C. Using rat/human hybrids and site-directed mutagenesis, we identified residue 188, Ala for rat and Ile for human, which modulates hormone binding. Orthologous mutagenesis at position 188 for either rNPR-C or hNPR-C results in a complete reversal of the pharmacology. To our knowledge, this is the first example of a single transmembrane domain receptor for which a single residue dictates the ligand binding properties; previous examples are limited to seven transmembrane receptors.

Many human tumors express low amounts of HLA class I molecules relative to the normal cells from which they are derived. From experimental work it is clear that the malignant behavior of a tumor cell may depend on its MHC class I expression. Therefore, it is of obvious interest to study the HLA class I expression of human tumors in their various stages. We have studied the HLA class I expression by the cells in premalignant epithelial lesions and invasive carcinoma of the bladder and uterine cervix using immunoperoxidase staining for beta 2-microglobulin of paraffin-embedded tissue. We here assume that beta 2-microglobulin expression by malignant and premalignant cells equals HLA class I expression. Thirty-two of the 36 invasive tumors expressed less overall beta 2-microglobulin than cells from the normal epithelium. In contrast, approximately two-thirds of 34 premalignant bladder epithelia and 47 premalignant cervix epithelia displayed higher overall beta 2-microglobulin expression than the normal epithelium. Thus, a systematic large-scale elimination of HLA class I high-expressing tumor cell variants may take place only after the tumor penetrates the basement membrane.

The effect of inhaled levomepromazine (Nozinan, Veractil) on bronchial responsiveness to inhaled histamine was investigated in asthmatics. In a double blind, randomized controlled study, 12 asthmatics (FEV1% pred 52-96%, and PC20 histamine 1.01 mg/ml (geometric mean)) were challenged before and after inhalation of levomepromazine in three different doses. Before and after each inhalation of levomepromazine, PC20, FEV1, the continuous reaction time (CRT) and the subjective sedation score (VAS) were determined. A dose-dependent increase in PC20 was observed after inhalation of levomepromazine. PC20 was increased by up to 4.02 two-fold concentration differences (doubling), i.e. up to a 38-fold increase from the basic values. Inhalation of the two higher doses of levomepromazine had a small sedative effect evaluated from an increase in CRT and the VAS-score and corresponding to the plasma concentrations. We conclude that inhaled levomepromazine has a dose-dependent protective effect on histamine-induced bronchial hyperresponsiveness in asthmatics and that inhalation of levomepromazine was well tolerated. The mechanism by which levomepromazine acts on histamine-induced bronchial hyperresponsiveness is not known but it could be partly explained by the antihistaminic effect. In this respect levomepromazine bears comparison with the most potent second generation antihistamines. The plasma concentrations of levomepromazine measured corresponded to those seen after oral intake of 5-10 mg levomepromazine.

We have discovered a new oligomeric protein component associated with the outer membrane of the ancestral eubacterium Thermotoga maritima. In electron micrographs, the protein, Omp alpha, appears as a rod-shaped spacer that spans the periplasm, connecting the outer membrane to the inner cell body. Purification, biochemical characterization and sequencing of Omp alpha suggest that it is a homodimer composed of two subunits of 380 amino acids with a calculated M(r) of 43,000 and a pI of 4.54. The sequence of the omp alpha gene indicates a tripartite organization of the protein with a globular NH2-terminal domain of 64 residues followed by a putative coiled-coil segment of 300 residues and a COOH-terminal, membrane-spanning segment. The predicted length of the coiled-coil segment (45 nm) correlates closely with the spacing between the inner and outer membranes. Despite sequence similarity to a large number of coiled-coil proteins and high scores in a coiled-coil prediction algorithm, the sequence of the central rod-shaped domain of Omp alpha does not have the typical 3.5 periodicity of coiled-coil proteins but rather has a periodicity of 3.58 residues. Such a periodicity was also found in the central domain of staphylococcal M protein and beta-giardin and might be indicative of a subclass of fibrous proteins with packing interactions that are distinct from the ones seen in other two-stranded coiled-coils.

Tumors were induced in athymic, T-cell-deficient nude mice and in syngeneic normal haired mice by treatment with low doses of 3-methylcholantrene (MCA). The tumors were studied for tumor cell expression of MHC class I molecules and for immunogenicity by transplantation to syngeneic haired recipients. Ten tumors were obtained by the MCA treatment, six from nude and four from haired mice. They were all fibrosarcomas as judged from their microscopic appearance. Five of the "nude" tumors expressed measurable amounts of MHC class I molecules and two of them expressed high amounts. Both were immunogenic in the sense that they evoked a cytotoxic T-cell response in transplanted haired recipients. Only one of the four "haired" tumors expressed measurable amounts of MHC class I, and none of them were immunogenic. These findings support the concept that some tumors are immunoselected at an early point of time in their existence in a host with a normal immune system and that this results in an elimination of tumor cell variants which are highly immunogenic for the T-cell system, leaving the low or non-immunogenic variants. These take over and grow and kill their host. The results suggest that tumor cell variants expressing high amounts of MHC class I are important targets in the immunoselection in hosts with a normal immune system.

The activity of the inhibitor of activated Stuart factor (anti-Xa) was evaluated together with the activity of the antiactivator of plasminogen (antiplasminogen) and the amount and nature of the fibrinogen/fibrin degradation products (FDP), as indicators of the existence of the risk for hypercoagulability in patients with acute and recent myocardial infarction (AMI and RMI, respectively). The activity of anti-Xa was diminished in the AMI group with a p less than 0.001. The antiplasminogen activity was increased in both groups with a p less than 0.001; on the other hand, the amount of FDP was increased in the AMI patients and they were of the 'early' type, while in the RMI group the increase was not as marked, but the FDP were of the 'late' type. In both groups the euglobulin lysis time was very prolonged, while the plasminogen did not vary significantly. The tests described appear to be valuable tools for studying the status of the cardiovascular system during cardiac rehabilitation.