Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials.

The long-term consequences of adenovirus-mediated conditional cytotoxic gene therapy for gliomas remain uncharacterized. We report here detection of active brain inflammation 3 months after successful inhibition of syngeneic glioma growth. The inflammatory infiltrate consisted of activated macrophages/microglia and astrocytes, and T lymphocytes positive for leucosyalin, CD3 and CD8, and included secondary demyelination. We detected strong widespread herpes simplex virus 1 thymidine kinase immunoreactivity and vector genomes throughout large areas of the brain. Thus, patient evaluation and the design of clinical trials in ongoing and future gene therapy for brain glioblastoma must address not only tumor-killing efficiency, but also long-term active brain inflammation, loss of myelin fibers and persistent transgene expression.

Immunization via hair follicles by topical application of naked DNA to normal skin.

In order to test the immune response generated to small amounts of foreign protein in skin, we applied naked DNA in aqueous solution to untreated normal skin. Topical application of plasmid expression vectors for lacZ and the hepatitis B surface antigen (HBsAg) to intact skin induced antigen-specific immune responses that displayed TH2 features. For HBsAg, specific antibody and cellular responses were induced to the same order of magnitude as those produced by intramuscular injection of the commercially available recombinant HBsAg polypeptide vaccine. Finally, topical gene transfer was dependent on the presence of normal hair follicles.

Subcellular localization of enterokinase (enteropeptidase EC 3.4.21.9) in rat small intestine.

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3% in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (Mr, 2 - 10(6)) and two larger peaks of free enzyme (Mr, 3 - 10(5) and 9 -10). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.

Preparation and evaluation of radioiodinated (iodophenyl)cholines and their morpholinium and piperidinium analogues as myocardial perfusion imaging agents.

A series of nine radioiodinated quaternary ammonium salts related to phenylcholine were synthesized, characterized, and radiolabeled by exchange. These compounds were evaluated as myocardial perfusion imaging agents in mice, pigs, and humans. Mice biodistribution studies showed that five of the nine compounds were taken up in the heart to the same extent as 201Tl+ at 5 min. At 60 min myocardial retention was significantly better than 201Tl+ for six of the compounds. Several of the compounds showed more favorable heart/blood and heart/liver ratios when compared to 201Tl+. Evaluation of three of the more promising compounds in pigs and humans however revealed no selective myocardial uptake.

Routine application of fractionated HMPAO stored at -70 degrees C for WBC scintigraphy.

The radiochemical purity of 99mTc-HMPAO prepared from fractions of reconstituted HMPAO stored at -70 degrees C and its application in the radiolabeling of human granulocytes was investigated. Upon reconstitution of a vial of HMPAO with 1 ml of saline and subsequent freezing at -70 degrees C, small fractions were obtained on each of four consecutive days with the vial being refrozen after each dispensing. Following radiolabeling of the HMPAO fractions with pertechnetate, mean radiochemical purity results met or exceeded manufacturers' specifications for the radiopharmaceutical on each of the four days. Imaging with radiolabeled granulocytes using 99mTc-HMPAO prepared by this technique resulted in high quality clinical studies. These results demonstrate that a vial of HMPAO can be fractionated, after storage at -70 degrees C with no loss of clinical utility for radiolabeling granulocytes and considerable cost savings.

In vivo red blood cell compatibility testing using indium-113m tropolone-labeled red blood cells.

In vivo radionuclide crossmatch is a method for identifying compatible blood for transfusion when allo- or autoantibodies preclude the use of conventional crossmatching techniques. A technique for labeling small volumes of donor red blood cells with [113mIn]tropolone is reported. The use of 113mIn minimizes the accumulation of background radioactivity and the radiation dose especially so when multiple crossmatches are performed. Labeling red cells with [113mIn]tropolone is faster and easier to perform than with other radionuclides. Consistently high labeling efficiencies are obtained and minimal 113mIn activity elutes from the labeled red blood cells. A case study involving 22 crossmatches is presented to demonstrate the technique. The radiation dose equivalent from 113mIn is significantly less than with other radionuclides that may be used to label red cells.

Effect of lesions of the ascending 5-hydroxytryptaminergic pathways on timing behaviour investigated with an interval bisection task.

Seventeen rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei: 12 rats received sham injections. The rats were trained in a series of discrete trials to press lever A following a 2-s presentation of a light stimulus and to press lever B following an 8-s presentation of the same stimulus. Both groups learnt the task rapidly and maintained > 90% accuracy throughout the experiment. When stable performance had been attained, "probe" trials were introduced in which the light was presented for intermediate durations. Both groups showed sigmoid functions relating percent choice of lever B to log stimulus duration. The bisection point (duration corresponding to 50% choice of lever B) was significantly shorter in the lesioned group than in the control group. There was no significant difference between the slopes of the psychophysical functions or the Weber fractions derived for the two groups. The levels of 5-hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, amygdala, nucleus accumbens and hypothalamus were markedly reduced in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected by the lesion. The results confirm the involvement of 5HTergic function in timing behaviour, but suggest that destruction of these pathways does not disrupt the capacity for temporal discrimination.

Effect of lesions of the ascending 5-hydroxytryptaminergic pathways on timing behaviour investigated with the fixed-interval peak procedure.

Twelve rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei; 12 rats received sham lesions. The rats were then trained for 60 sessions under a discrete-trials fixed-interval schedule (peak procedure). In half the trials, a reinforcer became available 40 s after trial onset, and the trial was terminated upon reinforcer delivery; the remaining trials were 120 s in duration, and reinforcement did not occur in these trials. Performance during the 120-s trials was characterized by increasing response rate during the first 40 s of the trial, declining response rate between 40 s and 80 s, and a secondary increase in response rate during the final 40 s of the trial. The lesioned group showed a broader "spread" of the response rate function than the control group (time between attainment of 70% of the peak response rate and subsequent decline of response rate below this level); however, the peak response rate and the time from trial onset until attainment of the peak response rate did not differ significantly between the groups; the spread/peak-time ratio was significantly greater in the lesioned group than in the control group. The levels of 5-hydroxytryptamine (5HT) and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, amygdala, nucleus accumbens and hypothalamus were markedly reduced in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected by the lesion. The results confirm the involvement of 5HTergic function in timing behaviour.

Over-expression of Saccharomyces cerevisiae hsp90 enhances the virulence of this yeast in mice.

Saccharomyces cerevisiae, a yeast of low pathogenic potential, is a rare but well-documented cause of invasive infections in humans. The yeast Candida albicans is a much commoner cause of significant and life-threatening infections. In such infections the heat shock protein hsp90 is an immunodominant antigen associated with protective humoral immunity. In this study it was shown that over-expression of S. cerevisiae hsp90, the amino acid sequence of which shows 84% identity to C. albicans hsp90, significantly increased the virulence of a laboratory strain of S. cerevisiae in mice, both in terms of colony counts in the kidney, liver, and spleen, and in terms of mortality. This is the first direct evidence that hsp90 is a virulence factor.

Automation in the animal house.

Whilst appearing attractive, automation must be appraoched with caution. Much time, effort and money is put into curing 'symptoms' without really defining the problem. Too often organizations launch a programme of automation when what they really need is rationalization. Unless you know precisely how much time and effort is currently involved in an operation, you are in grave danger of making costly mistakes in considering automation. A prerequisite is to make sure that your present operation is as productive as it is capable of being.

Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections.

Temperatures of mice were measured using an infrared high performance non-contact thermometer, after the device had been calibrated using implantable microchips containing temperature transponders. Mice were infected with three species of Candida (isolates) and the resultant disseminated infections monitored. Mouse temperatures could be reliably measured using the infrared device and this measurement caused little distress to the mice. We were further able to demonstrate that mice rarely recovered if their body temperature dropped below 33.3 degrees C (sensitivity 68%, specificity 97%). Adoption of a 33.3 degrees C endpoint in fungal sepsis experiments measured by infrared non-contact thermometer would significantly reduce the suffering in the terminal stages of this type of infection model.

Polymorphonuclear leukocytes labeled with technetium-99m HMPAO. A potential bone marrow imaging agent.

A case of multifocal avascular necrosis of the bone complicating systemic lupus erythematosis is presented in which the extent of involvement was incidentally identified by Tc-99m HMPAO leukocyte imaging. Leukocytes labeled with Tc-99m HMPAO may be an excellent bone marrow imaging agent.

Cellular benign fibrous histiocytoma of the external auditory meatus.

A rare case of cellular benign fibrous histiocytoma (BFH) of the external auditory canal is presented with a discussion of its pathology and management.

Canal wall down techniques for managing cholesteatoma.

Cholesteatoma remains a common otological problem. This series shows that a simple open cavity technique, with meatoplasty, remains a cost-effective option for treatment. Most ears are rendered dry and safe, with cavity problems minimized by careful technique. Subsequent outpatient care is required infrequently and is well tolerated. The hearing is no worse after the operation than before and some ears are suitable for subsequent tympanoplasty.

Efficacy of SCH-56592 in a temporarily neutropenic murine model of invasive aspergillosis with an itraconazole-susceptible and an itraconazole-resistant isolate of Aspergillus fumigatus.

SCH-56592 (SCH) is a novel triazole antifungal agent with excellent in vitro activity against Aspergillus. We compared three doses (5, 10, and 25 mg/kg of body weight) of SCH with itraconazole (ITZ; 25 mg/kg) and amphotericin B (AB; 5 mg/kg) in a temporarily neutropenic murine model of disseminated aspergillosis (lungs and kidneys) against one ITZ-susceptible (AF71) and one ITZ-resistant (AF90) isolate of Aspergillus fumigatus. Treatment started 24 h after infection and lasted for 10 days. Dosing regimens for SCH were once daily for 10 days, those for ITZ were three times daily for 2 days and then twice daily for 3 to 10 days, and those for AB were once daily on days 1, 2, 4, and 7. Both isolates killed 90% of control mice. Kidneys and lungs from survivors were cultured on day 11. Against AF71, all three doses of SCH and ITZ yielded a 90 to 100% survival rate and AB yielded 40% survival (P < or = 0.01 to 0.0001 for all treatment groups compared with the controls). All three doses of SCH were superior to AB in cultures of lung and kidney tissue samples (P < or = 0.01 to 0.0002) and SCH at 25 mg/kg was superior to ITZ in cultures of kidneys (P = 0.01). Against AF90, the highest dose of SCH (25 mg/kg) resulted in a 100% survival rate, compared with 60 and 20% survival rates for the groups treated with SCH at 10 and 5 mg/kg, respectively. Treatment with ITZ yielded no survivors. AB therapy achieved a 50% survival rate. SCH at 25 mg/kg (P < 0.001), SCH at 10 mg/kg (P < or = 0.005), and AB (P < 0.05) were superior to ITZ in cultures of lungs and kidneys. There was a correlation between the MICs of SCH and quantitative organ culture results and between the minimum fungicidal concentration of AB with quantitative organ culture results. SCH appears to be a highly effective anti-Aspergillus compound in this model. There appears to be a degree of cross-resistance between itraconazole and SCH.

The effects of rG-CSF on health-related quality of life in children with congenital agranulocytosis.

A multicentre Phase II clinical trial was recently undertaken to document the value of treatment with recombinant human granulocyte colony-stimulating factor (rG-CSF) in children with congenital agranulocytosis. To assess the impact of such therapy on health-related quality of life (HRQOL), we developed a questionnaire that was administered to the parents of study patients, twice prior to the initiation of treatment, and then monthly thereafter for six months. The questionnaire focused on several aspects of HRQOL that we thought were important in this patient population, including functional status, general health perceptions, activity limitation, disease symptoms, and discomfort associated with therapy. In this paper, we describe the questionnaire that we developed and the process by which it was translated into several languages. We also report on the impact of rG-CSF therapy on HRQOL. A total of 130 questionnaires were administered to the parents of 19 study patients between the ages of four and one-half months and 18 years in 11 study centres in four countries. Although our sample size is small, our findings suggest that treatment with rG-CSF may result in significant improvements in general health perceptions, limitations of daily activities, and symptoms of the disease.

Health-related quality of life in patients with advanced prostate cancer: a multinational perspective.

To explore the value of antiandrogen therapy for advanced prostate cancer, two clinical trials of similar design were recently conducted in six countries throughout Europe. A total of 550 patients with previously untreated metastatic prostate cancer were randomized either to treatment with an antiandrogen or castration. While time to treatment failure, objective tumour response and survival were expected to be similar between study treatments, their effects on health-related quality of life (HRQOL) were expected to differ and were therefore a focus of concern in this trial. To assess these effects, we developed a brief self-administered patient questionnaire covering 10 domains of HRQOL (general health perceptions, pain, emotional well-being, vitality, social functioning, physical capacity, sexual interest, sexual functioning, activity limitation and bed disability), which we translated from English into several other languages. In this paper, we describe the development, content and translation of this survey instrument and report on its reliability and validity in six countries based on data collected for the first 487 patients to complete questionnaires at study entry.

Nasal fracture manipulation: a comparative study of general and local anaesthesia techniques.

Nasal injuries are common conditions treated in either Otolaryngology or Plastic Surgical departments. Manipulation for deformity can be conducted in various ways. The aim of this study is to determine if the anaesthetic technique used for manipulation influences outcomes. Five hundred and fifty-five patients had either local anaesthetic (LA) or general anaesthetic (GA) nasal fracture manipulations in our departments over a 6-year period. Three hundred and twenty-four of these could be contacted and questioned as to subsequent surgical treatments received. Rhinoplasty, septorhinoplasty or septoplasty had been subsequently performed in 3.2% of the GA group and in 17.2% of the LA group (P < 0.0001). We recommend considering this result when treating nasal fractures in conjunction with other important issues of patient preference, financial costs, associated risks, morbidity and facilities available.

Activity of micafungin (FK463) against an itraconazole-resistant strain of Aspergillus fumigatus and a strain of Aspergillus terreus demonstrating in vivo resistance to amphotericin B.

We compared the activity of four doses of micafungin (FK463) with that of amphotericin B, liposomal amphotericin B and itraconazole in a murine model of disseminated aspergillosis. Temporarily neutropenic mice were infected with a lethal dose of either an itraconazole-resistant Aspergillus fumigatus isolate or Aspergillus terreus, a species that is less susceptible to amphotericin B. Treatment was started 24 h after infection and lasted for 7 days. Mice were treated with either amphotericin B (0.5 or 5 mg/kg), liposomal amphotericin (5 or 25 mg/kg), itraconazole (25 or 75 mg/kg) or FK463 (either 1, 2, 5 or 10 mg/kg). Treatment of the A. fumigatus model with either amphotericin B, liposomal amphotericin or FK463 prolonged survival. Doses of FK463 5 and 10 mg/kg had a 100% survival. Treatment of A. terreus infection with either itraconazole or FK463, but not amphotericin B, also prolonged survival. Doses of liposomal amphotericin of 5 and 25 mg/kg were ineffective against A. terreus infection. No treatment regime was able to totally clear the liver or kidneys in either model. The data indicate that FK463 may have a clinical role in the treatment of life-threatening invasive aspergillosis.