(Strept)avidin-displaying lentiviruses as versatile tools for targeting and dual imaging of gene delivery.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin-biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

Gene expression profiling of malignant mesothelioma cell lines: cDNA array study.

To reveal genes relevant for malignant mesothelioma (MM), we carried out cDNA array experiments on 4 MM cell lines and 2 primary mesothelial cell cultures established from pleural fluid of non-cancer patients. Human cancer gene filters including 588 genes were used for the cDNA array experiments. Our study revealed 26 over-expressed genes that play a role in the regulation of cell fate, cell cycle, cell growth and DNA damage repair and 13 under-expressed genes encoding growth factors, receptors and proteins involved in cell adhesion, motility and invasion to be common to 3 or 4 MM cell lines. We confirmed the cDNA array results using RT-PCR for 5 of the over-expressed genes and for 3 of the under-expressed genes. Our study presents gene expression profiles in MM cell lines and shows the involvement of several genes, such as those encoding JAGGED1, ser/thr protein kinase NIK, Ku80 and cyclin D2, novel in MM.

In vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents.

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2x10(-9), 5x10(-9), 10(-8), 2x10(-8) and 5x10(-8) M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10(-8) M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10(-8) M.

Ultrasonography in the quantification of arterial involvement in Takayasu's arteritis.

OBJECTIVE: To evaluate the accuracy of ultrasonography (US) in the detection and quantification of arterial involvement in Takayasu's arteritis (TA). METHODS: The common carotid and subclavian arteries, and the abdominal aorta of 15 patients with TA were studied by Color Doppler (CD) US and Doppler spectral analysis and compared with angiography. RESULTS: The mean difference (+/-SD) between the percent luminal stenoses measured at angiography and by CD US was 2.0+/-10.3% for the common carotid artery, 4.0+/-23.6% for the subclavian artery and -1.3+/-16.8% for the abdominal aorta. The differences were not statistically significant. However, the agreement of the methods was less than satisfactory as shown by the wide standard deviations. CONCLUSIONS: More efforts are needed to improve the less than optimal agreement of US with angiography regarding the severity of individual stenoses. The technique performs best in the study of carotid arteries.

Effect of weight loss and regional fat distribution on plasma leptin concentration in obese women.

OBJECTIVES: To investigate how circulating leptin concentrations are related to regional fat distribution and whether moderate weight loss alters these relationships. DESIGN: A 6 month, clinical weight reduction trial with measurements before and after weight loss. SUBJECTS: 38 healthy, obese women (age: 44.3+/-9.9 y, BMI: 34.0+/-4.0 kg/m2). MEASUREMENTS: The following measurements were made. 1. indices of obesity and fat distribution: weight, body mass index (BMI), hip circumference (peripheral fat), waist circumference, total body fat (bioelectrical impedance), abdominal fat distribution: visceral fat and abdominal subcutaneous fat (ultrasonography); and 2. Biochemical measurements: plasma leptin and serum insulin. RESULTS: Baseline plasma leptin concentrations were three-fold higher in obese women than in normal weight controls. After weight loss averaging 8.4 kg (9.0%), plasma leptin decreased by a mean of 22.3% (P < 0.001), corresponding to body fat decrease of 16.6% (P < 0.001), abdominal subcutaneous fat decrease of 17.4% (P < 0.001) and visceral fat decrease of 18.7% (P < 0.001). The total amount of body fat correlated with plasma (serum) leptin before (r = 0.64, P < 0.001) and after (r = 0.75, P < 0.001) weight loss. Plasma leptin concentrations expressed per kg of body fat did not change significantly during weight loss. After controlling for body fat, baseline leptin concentrations were significantly associated with hip circumference (r = 0.57, P < 0.001) but not with any indices of abdominal fat distribution. After weight loss the associations became significant for hip and waist circumference as well as for visceral and abdominal subcutaneous fat. Changes in leptin correlated with changes in all indices of obesity except visceral fat. CONCLUSIONS: Plasma leptin concentrations reflect not only total fat mass but also adipose tissue distribution, especially peripheral fat. Plasma leptin values per kilogram of fat mass do not change significantly with modest weight loss.

Prolonged low-back pain in young athletes: a prospective case series study of findings and prognosis.

We investigated the prognosis of low-back pain and the association of clinical symptoms and anatomic findings among young athletes. Consecutive patients, aged between 12 and 18 years, who had low-back pain that had interfered with their training for at least 4 weeks were included in the case series. All the patients participated in a standardized interview and clinical examination, and plain radiographs and magnetic resonance images were also obtained. Most patients also participated in technetium bone scan examination. In 15 out of 19 subjects there were anatomic abnormalities that corresponded with the location and type of clinical symptoms. Twelve subjects had changes in the disk-vertebral end plate complex and eight had a positive bone scan indicative of posterior vertebral arch stress reaction. Six out of eight boys and two out of 11 girls had stress reaction (P = 0.043). Restriction of painful activities was recommended to all subjects, restriction of activities and the use of a dynamic low-back brace for the first 3 months was recommended to patients with posterior vertebral arch stress reaction. The self-reported intensity of low-back pain (scale 0-100) among all the patients was 69 +/- 16 (mean +/- SD) at baseline and 18 +/- 21 at the 1-year follow-up (P < 0.0001). In conclusion, the reasons for prolonged back pain among young athletes are usually established by imaging studies. A knowledge of anatomic abnormalities may help in tailoring training programmes and avoiding the progression of changes during growth. Simple restriction of painful activities usually leads to good recovery.

Cholesterol metabolism and serum and biliary noncholesterol sterols in gallstone patients during simvastatin and ursodeoxycholic acid treatments.

Effects of long-term high-dose ursodeoxycholic acid (UDCA) and simvastatin treatments on cholesterol metabolism and biliary lipid compositions were compared in patients with cholesterol gallstones. Absorption and synthesis of cholesterol, serum and biliary noncholesterol sterols and lipids were determined in 14 patients randomized to UDCA (23-25 mg/kg/d) or simvastatin (40 mg/d) for 1 year. Simvastatin reduced serum low-density lipoprotein cholesterol by 55%, and UDCA, by 9%. Cholesterol absorption was decreased (35%) by UDCA, but nonsignificantly increased by simvastatin (P < .05 for difference of changes caused by the two drugs). Whole-body synthesis and biliary output of cholesterol were both significantly decreased only by UDCA. In addition, UDCA inconsistently increased the proportions of serum and biliary precursor sterols of cholesterol, known to reflect cholesterol synthesis, but did not affect their biliary secretions. Simvastatin, however, dramatically reduced serum and also biliary cholesterol precursor sterol proportions and their biliary secretions and increased proportions of serum and biliary plant sterols and cholestanol, known to reflect cholesterol absorption, but had no effect on their biliary secretion. Only UDCA significantly decreased the molar percentage of cholesterol, the lithogenic index, and the cholesterol/phospholipid (CH/ PL) ratio in bile, whereas both treatments inconsistently decreased the vesicular CH/PL ratio (P < .07 in both groups). It is concluded that both drugs decreased serum cholesterol and inhibited cholesterol synthesis, but had a differing influence on precursor sterols and the absorption of cholesterol. UDCA had more beneficial effects than simvastatin on the antilithogenic properties of bile.

Diagnostic laparoscopy in patients with acute leukemia and suspected hepatic candidiasis.

To assess the value of laparoscopy in the diagnosis of suspected hepatosplenic candidiasis in patients with acute leukemia, a retrospective analysis of 28 laparoscopies was conducted. In all but two cases, imaging of the liver showed focal lesions before laparoscopy. Diagnosis of hepatic candidiasis was established significantly more often when the biopsy was targeted at white nodules (in 12 of 22 laparoscopies) than when targeted randomly or at scars (0 of 6 laparoscopies) (p = 0.017, chi-square test). Yeast was detected more often if the laparoscopy was performed during the three-week period after recovery from neutropenia (in 8 of 12 laparoscopies) than when performed later (in 4 of 16 laparoscopies) (p = 0.028, chi-square test). In addition to the 12 laparoscopically diagnosed patients, eight (29%) patients were diagnosed with disseminated Candida infection by other methods. In another eight (29%) patients the causative agent was not identified. No bleeding or other problems occurred after the laparoscopy. Laparoscopy-guided liver biopsy is most useful if biopsies are targeted to macroscopic lesions and if laparoscopy is performed soon after recovery from neutropenia.

Long-term prognosis of reactive salmonella arthritis.

OBJECTIVES: Reactive joint complications triggered by salmonella gastroenteritis are increasingly reported, but the outcome and long term prognosis of the patients is incompletely known. This study looked at the prognosis of salmonella arthritis in patients hospitalised in 1970-1986. METHODS: Hospital records from two hospitals in southern Finland were screened for patients with the discharge diagnosis of salmonellosis or reactive, postinfectious arthritis or Reiter's disease. For the patients with confirmed diagnosis of reactive salmonella arthritis, data about the acute disease were collected from the hospital records. A follow up study was performed. RESULTS: There were 63 patients (28 women, 35 men, mean age 36.5 years) with salmonella arthritis. Urethritis occurred in 27%, eye inflammation in 13%, and low back pain in 44% of the patients. HLA-B27 was present in 88%. More men than women were HLA-B27 positive. HLA-B27 positive patients had higher erythrocyte sedimentation rate (mean 80.9 v 46.5 mm 1st h, p = 0.0180). Also, extra-articular features and radiological sacroiliitis were seen only in HLA-B27 positive patients. A follow up study was performed on 50 patients mean 11.0 (range 5-22 years) later. Twenty patients had recovered completely. Ten patients had mild joint symptoms, 11 patients had had a new acute transient arthritis, and five acute iritis. Eight patients had developed chronic spondyloarthropathy. Radiological sacroiliitis was seen in six of 44 patients, more frequently in male than in female patients (32% v 0%; p = 0.0289). Recurrent or chronic arthritis, iritis or radiological sacroiliitis developed only in HLA-B27 positive patients. CONCLUSION: Joint symptoms are common after reactive salmonella arthritis. HLA-B27 contributes to the severity of acute disease and to the late prognosis.

Toxicity of ethanol in low concentrations. Experimental evaluation in cell culture.

PURPOSE: To define the threshold ethanol concentration that is toxic to cultured cells. METHODS: Three malignant cell lines and freshly isolated normal rat hepatocytes were exposed to 0-50% (vol.) ethanol (concentrations used were 0, 5, 10, 15, 20, 25, 30, 40 and 50%) on tissue culture plates for 0.25-60 min (exposure times used were 0.25, 5, 10, 20, 30, 40 50 and 60 min). Cytotoxicity was estimated by trypan blue exclusion test and from 3H-thymidine incorporation. RESULTS: All cells were killed by a 15-s exposure to 30-40% ethanol while a concentration as low as 15-20% gave a total response after 5-10-min exposures. After a one-hour exposure of F9 carcinoma cells and hepatocytes, a total or nearly total response was achieved with 10% ethanol. The cytotoxic effect was thus dependent both on the exposure time and on the concentration of ethanol. There were no significant differences in ethanol tolerance among the cell types. CONCLUSION: Ethanol seemed to kill cells in the cell culture effectively in much lower concentrations than those currently used in tumour ablation.

Painful ankle region in rheumatoid arthritis. Analysis of soft-tissue changes with ultrasonography and MR imaging.

PURPOSE: To establish the diagnostic usefulness of ultrasonography (US) and MR imaging in patients with rheumatoid arthritis (RA) suffering from prolonged pain in the ankle region, where plain radiography did not demonstrate any changes. MATERIAL AND METHODS: Seventeen patients were studied with 0.1 T MR imaging and with high-frequency US. Talocrural and subtalar joints (including talonavicular joints), and medial, lateral, and extensor tendons and their synovial sheaths were examined by MR and US. RESULTS: Abnormal findings were found by MR imaging and US in altogether 76% of the patients, by MR alone in 53%, and by US alone in 59%. In 41% of the patients, lesions were demonstrated only by one method. Talocrural and subtalar joints were the most often affected sites (41% each), followed by the peroneus tendon (23%). In the joints, the abnormal findings were interpreted as synovitis; in the tendon areas, mostly as tenosynovitis. MR and US were highly significantly concordant (p < 0.0001), but correlation with clinical features was poor. CONCLUSION: In patients with pain in the ankle region, US and MR imaging can contribute to the diagnosis and localization of the abnormality when the plain radiography is normal. Easily available and inexpensive US can be recommended as the first imaging method after plain radiography. Some divergence seems to exist between US and MR, and in complicated cases both methods are recommended.

Measuring visceral adipose tissue content from contrast enhanced computed tomography.

OBJECTIVE: To study whether visceral adipose tissue (VAT) content can be measured retrospectively from routine contrast enhanced computed tomographic (CT) studies in order to avoid extra radiation exposure and to enable epidemiological quantifications of VAT in various diseases. SUBJECTS AND METHODS: Seven patients were examined by CT and the VAT contents (from -150 HU to -50 HU) were quantified both from their corresponding unenhanced and enhanced images (n = 8-18 per patient). RESULTS: The measured VAT volume in enhanced images was smaller (p = 0.016) than in the corresponding unenhanced images, which is most likely due to the adverse accentuation of partial volume effects. By using our bolus technique, VAT (cm2) in an unenhanced image = 1.11 x VAT in an enhanced image + 2.95, p = 0.0001. Alternatively, rising properly the upper limit for VAT density in an enhanced image compensates the use of i.v. contrast media. CONCLUSION: VAT content may be retrospectively quantified from routine abdominal CT images, but the unfavorable effects of i.v. contrast administration on measurements must be taken into account.

Plasma lathosterol as a screening test for bile acid malabsorption due to ileal resection: correlation with 75SeHCAT test and faecal bile acid excretion.

1. Plasma lathosterol concentration, known to reflect cholesterol and bile acid synthesis, was evaluated as a screening test for bile acid malabsorption, comparing it with faecal bile acid measurements, SeHCAT test and Schilling test in 22 subjects of whom six were healthy controls and 16 had Crohn's disease with ileal resections of varying length. 2. Plasma lathosterols and other non-cholesterol sterols were determined by GLC. Faecal bile acids were measured by GLC, and SeHCAT retention times by gamma camera. The study subjects were divided into two groups according to the degree of bile acid malabsorption: controls (faecal bile acids < 10 mg day-1 kg-1, n = 9) and bile acid malabsorption (faecal bile acids > 10 mg day-1 kg-1, n = 13). 3. Faecal bile acid excretion was 5.9 +/- 1.0 mg day-1 kg-1 in control subjects and 45.7 +/- 6.1 mg day-1 kg-1 in the bile acid malabsorption group. The biological half-life of 75SeHCAT (T1/2) was 95.6 +/- 16.3 h and 14.1 +/- 4.1 h, respectively. Plasma lathosterol levels were significantly elevated in patients with bile acid malabsorption (742 +/- 84 micrograms/ml compared with 400 +/- 59 micrograms/ml in control subjects) and correlated closely with faecal bile acid levels (r = 0.779, P < 0.001), with 75SeHCAT T1/2 (r = -0.524, P < 0.05) and with Schilling test (r = -0.591, P < 0.05). Significant correlations were also obtained for delta 8-cholestenol with faecal bile acids (r = 0.784, P < 0.001) and 75SeHCAT (r = -0.505, P < 0.05). The biological half-life of SeHCAT correlated with faecal bile acid excretion (r = -0.702, P < 0.001). Using mean+2 SD of lathosterol (In micrograms/ml cholesterol) as a cut-off value and 10 mg day-1 kg-1 as the upper limit for faecal bile acid excretion, the test gives 100% sensitivity and 82% specificity for plasma lathosterol determination to detect bile acid malabsorption. 4. The results indicate that both the 75SeHCAT test and plasma lathosterol detect bile acid malabsorption in patients with ileal resections for Crohn's disease. However, plasma lathosterol is a simpler and less expensive method.

Measurement of testicular volume: comparison of 3 different methods.

PURPOSE: We compared 3 commonly used clinical methods to measure testicular volume. MATERIALS AND METHODS: The volumes of 151 testes in 76 adults treated for cryptorchidism during childhood were measured with an ordinary ruler, an orchidometer and ultrasonography. RESULTS: Mean testicular volume was 15 +/- 8 ml. (standard elevation) measured with a ruler, 16 +/- 7 ml. with an orchidometer and 17 +/- 8 ml. with ultrasonography. The correlation among methods was significant. CONCLUSIONS: To measure testicular size use of an ordinary ruler is recommended for clinical purposes because of its simplicity, availability and low cost.

Persistence of enthesopathic changes in patients with spondylarthropathy during a 6-month follow-up.

The persistence of enthesopathic changes was studied by ultrasound (US) in 23 patients with spondylarthropathy during a 6-month prospective trial with sulphasalazine (Salazopyrin). During the follow-up significant improvement was seen in the joint symptoms and in the laboratory variables. By US 78% of the patients had enthesopathy at entry and 74% after 6 months of follow-up. The plantar fascia was the enthesis most frequently affected. Treatment with sulphasalazine had no obvious influence on the persistence of enthesopathy. Enthesopathy is as a rule a constant phenomenon and is probably caused by chronic enthesitis. A parallel resolution of anatomic soft tissue changes and the clinical status was not seen on US. There was also no evidence of a favourable effect of sulphasalazine on enthesopathy in the 6-month follow-up.

Early antibiotic treatment in acute necrotising pancreatitis.

Despite improvements in surgical treatment and intensive care, mortality from severe acute pancreatitis remains high. We have carried out a randomised study of 60 consecutive patients with alcohol-induced necrotising pancreatitis to find out whether early antibiotic treatment can improve outcome. 30 patients were assigned cefuroxime (4.5 g/day intravenously) from admission. In the second group, no antibiotic treatment was given until clinical or microbiologically verified infection or after a secondary rise in C-reactive protein. The inclusion criteria were C-reactive protein concentration above 120 mg/L within 48 h of admission and low enhancement (< 30 Hounsfield units) on contrast-enhanced computed tomography. There were more infectious complications in the non-antibiotic than in the antibiotic group (mean per patient 1.8 vs 1.0, p = 0.01). The most common cause of sepsis was Staphylococcus epidermidis; positive cultures were obtained from pancreatic necrosis or the central venous line in 14 of 18 patients with suspected but blood-culture-negative sepsis. Mortality was higher in the non-antibiotic group (seven vs one in the antibiotic group; p = 0.03). Four of the eight patients who died had cultures from pancreatic necrosis positive for Staph epidermidis. We conclude that cefuroxime given early in necrotising pancreatitis is beneficial and may reduce mortality, probably by decreasing the frequency of sepsis.

Cytogenetic response to asbestos fibers in cultured human primary mesothelial cells from 10 different donors.

The ability of amosite asbestos fibers to induce chromosomal aberrations in human primary mesothelial cells obtained from pleural effusions of 10 noncancerous patients was investigated. The glutathione S-transferase M1 (GSTM1) genotypes of the patients were determined, since the GSTM1 null genotype has been associated with increased susceptibility to lung cancer and chemically induced cytogenetic damage. Four of the patients represented the GSTM1 null genotype, and six the GSTM1 positive genotype. Successful chromosome aberration analyses were obtained from six cases, three of them with the GSTM1 null genotype. The level of aberrant cells in unexposed cultures ranged from 2.0% to 7.5%. Statistically significant increases (2.3-3.0-fold compared to controls) in the number of aberrant cells were observed in two cases only: in one case treated with 1 microgram/cm2 of amosite, and in another treated with 2 micrograms/cm2 of amosite. Cell cultures from four individuals showed minor or no increases in the numbers of aberrant cells in the doses tested (1 and 2 micrograms/cm2). Chromosome breaks were the major type of aberration. The amosite exposed cells with significantly increased aberrations were from patients with GSTM1 positive genotypes. Two cases that showed no cytogenetic response to asbestos fibers were of the GSTM1 null genotype. Thus, our results suggest that the lack of the GSTM1 gene does not render human mesothelial cells more susceptible to chromosomal damage induced by asbestos. GSTM1 null cells appeared, however, to be more sensitive to the growth inhibitory effects of asbestos than did GSTM1 positive cells. Variation in the cytogenetic response of human primary mesothelial cells to asbestos fibers was observed to exist, but the fibers do not appear to be potent inducers of structural chromosomal aberrations in these cells. It remains to be established whether individual sensitivity to asbestos fibers, due to specific genetic traits, exists.