Embolization of uterine arteriovenous malformations associated with cyanotic congenital heart disease.

Uterine arteriovenous malformation (AVM) is a rare cause of vaginal bleeding and miscarriage. We report two cases of uterine AVMs in patients with a history of complex congenital heart disease, an association that has not been previously described. Both patients were treated by selective uterine artery embolization, a minimally invasive therapy that has revolutionized the management of uterine AVMs, thus offering an alternative to conventional hysterectomy.

Outcome in AIDS-related systemic non-Hodgkin lymphoma and leptomeningeal disease is not predicted by a CT brain scan.

BACKGROUND AND PURPOSE: AIDS-related systemic non-Hodgkin lymphoma (ARL) remains a significant cause of morbidity and mortality in patients infected with the human immunodeficiency virus (HIV-1), and leptomeningeal disease in this setting has a dismal prognosis. We investigated the utility of brain CT in determining the outcome of leptomeningeal disease, despite MR imaging being the gold standard. MATERIALS AND METHODS: From a cohort of 9621 HIV-1-seropositive individuals, we identified those diagnosed with ARL in the highly active antiretroviral therapy (HAART) era who had both a lumbar puncture and central nervous system imaging using a CT brain scan at the time of initial diagnosis, and we compared survival parameters between those with and without leptomeningeal disease. RESULTS: In a cohort of 82 individuals with ARL treated in the era of HAART, we found that the survival of individuals with leptomeningeal disease defined as the presence of cells in the CSF was worse compared with that of other patients (P = .0026). However, when defined by the presence of abnormal enhancement or parenchymal lesions on a CT scan, the outcome was not significantly different. CONCLUSION: A CT brain scan appears not to offer additional prognostic information following a lumbar puncture in patients with ARL.

Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma.

Survivin is unique for its expression in human malignancies but not in normal adult cells. It has been implicated in sensitisation to chemotherapy and as a prognostic marker in several common cancers. Immunohistochemistry for Survivin, P53 and BCL-2 expression as well as cell proliferative index (Ki-67) and apoptosis index (TUNEL) was conducted on 52 pancreatic and 12 ampullary adenocarcinomas. Survivin was detected in the cytoplasm of carcinoma cells in 46 (88%) of pancreatic tumours. P53 and BCL-2 were detected in 54% and 12% of pancreatic tumours, respectively. Proliferative index was 26.2+/-10.5% and apoptosis index was 1.38+/-0.69%. Prevalence of Survivin expression was significantly higher in P53-positive than in P53-negative cases (P=0.05) but was not associated with BCL-2 expression. Incrementally higher weighted scores of Survivin expression were associated with increased proliferative index (P=0.001). Furthermore, there was linear correlation between increased proliferative index and higher apoptosis index (P<0.001). Surprisingly, higher scores of Survivin expression were associated with increased apoptosis index (P=0.007). Survival characteristics were not influenced by Survivin, P53 or BCL-2 expression, apoptosis index or proliferative index. Ampullary carcinoma showed Survivin expression in 83% of cases. However, unlike pancreatic carcinoma, there was no correlation between Survivin and P53 expression or proliferative index. In conclusion, Survivin is expressed in the majority of pancreatic adenocarcinomas and correlates with both cellular proliferation and apoptosis. Molecular manipulation of Survivin expression may enhance chemotherapy and radiation therapy for pancreatic cancer.

Assessment of chemoreflex sensitivity in free breathing young subjects by correction for respiratory influence.

BACKGROUND: The assessment of autonomic function is an important tool for risk stratification in critically ill patients. Peripheral cardiac chemoreflex sensitivity has been considered a marker for increased risk of sudden cardiac death. In normals, the evaluation of peripheral cardiac chemoreflex sensitivity is performed under controlled breathing conditions during inhalation of hypoxic gas. Since this is poorly tolerated by patients, they are commonly studied under hyperoxic conditions, which are not physiological. METHODS: We studied 20 healthy volunteers who underwent free and controlled breathing of a hypoxic gas mixture (10% O2 in N2) over 5 min. Values of peripheral cardiac chemoreflex sensitivity, corrected for respiratory influence, were compared with the results obtained experimentally under controlled breathing conditions in the same subjects. RESULTS: We found a substantial difference between values obtained during free and controlled breathing (3.64 +/- 0.81 vs. 1.53 +/- 0.32 ms/mmHg, respectively; P < 0.05). After application of a respiratory correction, described and validated in this article, no significant difference was seen for these values (0.89 +/-0.91 vs. 1.53 +/- 0.32 ms/mmHg, P = 0.46). CONCLUSIONS: This approach allows the evaluation of peripheral cardiac chemoreflex sensitivity in free breathing subjects. This correction could improve the assessment of cardiac chemoreflex sensitivity in patients with cardiorespiratory disorders, who find it difficult to control their breathing according to an experimental protocol.

Selective facilitation of the serotonin(1B) receptor causes disorganization of thalamic afferents and barrels in somatosensory cortex of rat.

Alteration of serotonin (5-HT) levels influences developing thalamocortical afferents (TCAs) in primary somatosensory cortex (SI) of rats and mice. The 5-HT(1B) receptor, present on TCAs during the first postnatal week, may be involved in these effects. The present study asked whether administration of 5-nonyloxytriptamine (NNT), a selective 5-HT(1B) receptor agonist, affects TCA organization in rat SI. Littermates were injected five times daily (5x/day), with either 0.1 mg/kg NNT or vehicle from birth to postnatal day 6 (P-6). Animals were killed on P-6, and their brains were processed for high-performance liquid chromatography (HPLC), cytochrome oxidase (CO) histochemistry, cresyl violet, or demonstration of TCAs by placement of 1,1'-dioctadecyl-3,3,3'' 3'-tetra-methylindocarbocyanine perchlorate (Di-I) on thalamocortical radiations. At P-6, NNT treatment decreased 5-HT levels slightly compared with controls, although this difference was not statistically significant. In NNT-treated rats, the Di-I-labeled vibrissae-related pattern showed a range of effects, from fusion of patches related to mystacial vibrissae in treated animals to a less distinct vibrissae-related pattern in SI barrelfield compared with controls. Staining for CO and Nissl stain in layer IV of SI showed a similar range of abnormalities. These results indicate that the agonist action of NNT at the 5-HT(1B) receptor causes TCA disorganization in rat barrel field cortex in the absence of elevated 5-HT.

Nonhomologous bioinjectable materials in urology: 'size matters'?

OBJECTIVE: To assess the factors that influence how particles might become fixed in tissues or migrate from them, by measuring the size of the injectable particles, their susceptibility to phagocytosis and their affinity for fibroblast attachment in culture. MATERIALS AND METHODS: The particle size of three types of particulate unphysiological bioinjectable material, i.e. Urocol (Genesis Medical, Ltd., London), Macroplastiquetrade mark (Uroplasty Ltd., Reading, UK) and Urethrin (Mentor Medical Systems, Wantage, UK) was analysed using phase-contrast light microscopy and confocal microscopy. Human monocytes from peripheral blood were incubated with the three materials in phagocytic studies, where ingestion was determined by confocal microscopy. A fibroblast cell line was used to ascertain the ability of the particles to act as a substrate for cell attachment in culture. RESULTS: The mean (SEM) maximum particle diameters of Macroplastique, Urethrin and Urocol were 209 (5.10) microm, 49 (1.52) microm and 14 (0.39) microm, respectively. Rat peritoneal macrophages and human peripheral blood monocytes commonly ingested Urocol particles; the phagocytosis of Urethrin was rare and that of Macroplastique was not detected. Fibroblasts adhered to Urocol paste and Urethrin particles, but not to Macroplastique. CONCLUSION: Published reports of particle size and phagocytosis are confusing, but a relationship clearly exists. Macroplastique is the largest particle and is least likely to be phagocytosed by human mononuclear phagocytes. Urocol paste is the slowest to dissipate in culture conditions; the flat surfaces of Urethrin, but not Macroplastique, can serve as a substrate for fibroblast anchorage.

Establishment and performance of a magnetic resonance cardiac function clinic.

Our objective was to establish a cardiovascular magnetic resonance (CMR) cardiac function clinic to provide an assessment of cardiac volume, mass, and function in patients with heart failure on the same day as their cardiology outpatient clinic appointment. Sixty-four patients attended the CMR function clinic. The reproducibility, patient acceptability, and time efficiency of the CMR clinic were assessed and compared with radionuclide ventriculography (RNV) and echocardiography (echo). Reports were available in the cardiology outpatient clinic within 2 hr of the CMR appointment time. The reproducibility of volumes, ejection fraction, and mass in this heart failure population was good and comparable with CMR studies in the normal population. CMR was more acceptable to the patients than both RNV and echo (p < 0.05). The total time for CMR was less than that of RNV (42 +/- 4 and 61 +/- 4 min, respectively; p < 0.001) but more than that of echo (echo, 23 +/- 2 min; p < 0.001). Comparison of ejection fractions revealed a correlation between CMR and RNV of 0.7, but Bland-Altman limits of agreement were wide (-10.5% to 18.9%). For CMR versus echo, the correlation was 0.6, and the limits of agreement were wider (-29.9% to 23.3%). The correlation between RNV and echo was 0.2 with wider limits of agreement (-29.8% to 24. 9%). In conclusion, CMR can provide a rapid, reproducible, and patient acceptable assessment of cardiac function in heart failure patients, whereas other methods appear to have a wider variance. The high reproducibility of CMR lends itself to the follow-up of clinical progression and the effect of treatment in patients with heart failure.

Effect of gamma-linolenic acid on cellular uptake of structurally related anthracyclines in human drug sensitive and multidrug resistant bladder and breast cancer cell lines.

This study investigated the effect on drug uptake in multidrug resistant cells by the incorporation of the essential fatty acid gamma-linolenic acid (GLA). The cell lines used were the MCF-7/R resistant human breast cancer and MGH-U1/R bladder cancer. Uptake of drug (doxorubicin, epirubicin, mitoxantrone and idarubicin) after the incorporation of GLA was investigated quantitatively by flow cytometry and qualitatively by confocal microscopy. There was no observable overall increase in drug uptake due to GLA incorporation into the cells as shown by flow cytometry. However, an increase in uptake of the chemotherapeutic agent idarubicin was observed in GLA-treated resistant cells compared with untreated cells using the confocal microscope. This overall increase in cellular drug uptake was not accompanied by a change in cellular drug distribution. Only one drug, mitoxantrone, displayed a change in intracellular drug distribution due to GLA incorporation into MCF-7/R cells. This suggests that essential fatty acid incorporation into the cellular membranes of some resistant cells may cause a shift in the intracellular distribution of certain chemotherapeutic drugs.

Photodynamic therapy of a transplanted pancreatic cancer model using meta-tetrahydroxyphenylchlorin (mTHPC).

Pancreatic cancer is difficult to treat, even for tumours localized to the pancreas. Photodynamic therapy (PDT) is a non-thermal technique for producing localized tissue necrosis with light after prior administration of a photosensitizing drug and it could have a role in the local treatment of these cancers. We studied PDT in a transplanted cancer in the hamster pancreas using the photosensitizer mTHPC (meta-tetrahydroxyphenylchlorin). Fluorescence microscopy showed maximum levels of mTHPC in normal pancreas 2-4 days after sensitization and in tumour at 4-5 days. For PDT, animals were given 0.1 or 0.3 mg kg(-1) mTHPC and the tumour was treated at laparotomy 2 or 4 days later with red light (50 J at 650 nm, continuous or fractionated) delivered via a single fibre touching the tumour surface. The maximum zone of tumour necrosis (seen 3 days after PDT) was 8.7 mm in diameter with continuous irradiation, increasing to 12.4 mm with light fractionation (four equal fractions with 3 min between fractions). The main complication was sealed duodenal perforation, seen in 3 of 16 animals, probably due to inadequate shielding of the duodenum from the light. The duodenal problems seen in hamsters are unlikely to cause trouble in the much thicker human duodenum. PDT tumour necrosis in this animal model has now been shown with a range of photosensitizers, but mTHPC is attractive as it is likely to produce the largest volumes of necrosis around each treatment point with short light exposure times. This technique could have a role in the treatment of localized cancers of the pancreas in patients unsuitable for surgery and can now be considered for preliminary clinical trials.

Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies.

Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management. The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live and in situ by confocal microscopy. The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells. Biopsy material is disaggregated and explanted into culture-grade petri dishes. After incubation for three to seven days plaques of epithelial cells have developed. Classical patterns of sensitive and resistant drug distribution are observed. Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer. Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern. These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.

Localization of anthracycline accumulation in sensitive and resistant urothelial tumor cell lines.

Emergence of resistance to chemotherapy is a serious clinical problem. Our aim was to investigate anthracycline uptake in sensitive and resistant urothelial cancer cell lines and to manipulate uptake by resistant cells. Anthracyclines exhibit natural fluorescence; therefore, the techniques used for detection were high-resolution fluorescence image analysis and confocal microscopy. Different patterns of intracellular drug distribution were observed in the two cell lines. Sensitive cells showed predominantly nuclear drug localization, while in resistant cells there was nuclear and cytoplasmic fluorescence. Manipulation of drug uptake by resistant cells was achieved using an essential fatty acid, gammalinolenic acid, and the calcium channel blocker, verapamil. Both substances increased drug uptake in resistant cells, with verapamil producing patterns of intracellular drug distribution in resistant cells similar to those of sensitive cells.

Enhancement of photodynamic therapy with 5-aminolaevulinic acid-induced porphyrin photosensitisation in normal rat colon by threshold and light fractionation studies.

5-Aminolaevulinic acid (ALA)-induced prophyrin photosensitisation is an attractive option for photodynamic therapy (PDT) since skin photosensitivity is limited to 1-2 days. However, early clinical results on colon tumours using the maximum tolerated oral dose of 60 mg kg-1 showed only superficial necrosis, presumably owing to insufficient intratumoral porphyrin levels, although inadequate light dosimetry may also be a factor. We undertook experiments using ALA, 25-400 mg kg-1 intravenously, to establish the threshold doses required for a PDT effect. Laser light at 630 nm (100 mW, 10-200 J) was delivered to a single site in the colon of photosensitised normal Wistar rats at laparotomy. The animals were killed 3 days later and the area of PDT-induced necrosis measured. No lesion was seen with 25 mg kg-1. The lesion size increased with larger ALA doses and with the light dose but little benefit was seen from increasing the ALA dose above 200 mg kg-1 or the light dose above 100 J. Thus there is a fairly narrow window for optimum doses of drug and light. Further experiments showed that the PDT effect can be markedly enhanced by fractionating the light dose. A series of animals was sensitized with 200 mg kg-1 ALA and then treated with 25 J. With continuous irradiation, the lesion area was 13 mm2, but with a single interruption of 150 s the area rose to 94 mm2 with the same total energy. Results were basically similar for different intervals between fractions (10-900 s) and different numbers of fractions (2-25). This suggests that a single short interruption in the light irradiation may dramatically reduce the net light dose required to achieve extensive necrosis.

Mycoplasma penetrans infection in male homosexuals with AIDS: high seroprevalence and association with Kaposi's sarcoma.

Antibodies to Mycoplasma penetrans were found at an unusually high frequency in male homosexuals with AIDS (55 of 149; 37%) and in human immunodeficiency virus (HIV)-infected asymptomatic homosexuals (13 of 49; 26.5%) but not in intravenous drug users (3 of 308; 1%) and hemophiliacs (1 of 165; 0.6%) with or without HIV-1 infection. Thus, both M. penetrans and Kaposi's sarcoma (KS) occur primarily in male homosexuals and rarely in other groups of patients at high risk of AIDS. Among 414 HIV-1-infected patients, statistical analysis revealed those with M. penetrans antibody were 11.7 times more likely to develop KS. Furthermore, among 198 HIV-infected homosexuals (149 with AIDS and 49 without AIDS), those with KS had M. penetrans-specific antibody at a significantly higher frequency (28 of 47; 59.6%) than did those without KS (27 of 102 with AIDS [26.5%] as well as 13 of 49 without AIDS [26.5%]; odds ratio = 4.1, P < .001). M. penetrans is apparently transmitted sexually through homosexual activity and is epidemiologically linked to formation of KS in homosexual men with AIDS. Parallel tests with M. genitalium revealed no similar link to KS in the same study sample.

Interaction of immune sera with synthetic peptides corresponding to the structural protein region of hepatitis C virus.

Comparison of the deduced amino acid sequence from the structural region of the Hutchinson strain of hepatitis C virus (HCV-H) with four other HCV isolates clearly divides the five isolates into two groups based on sequence homology. The first group includes HCV-H, HCV-1, and HC-J1, while the second includes HCV-J1 and HC-J4. Among the five isolates the first 190 residues (putative nucleocapsid) are highly conserved whereas residues 196-513 exhibit significant diversity and include a hypervariable region encompassing residues 386-404. A series of overlapping decapeptides were synthesized by solid-phase pin technology according to sequence from HCV-H (amino acids 1-513), HC-J4 (amino acids 181-513), and regions from the three other isolates which exhibited sequence variation. A modified ELISA was used to measure immunoreactivity of sera from clinical posttransfusion cases and experimentally infected chimpanzees. Comparison of pre- and postinfection samples revealed 16 clusters of immunoreactive peptides within the structural region, none of which was found in the hypervariable region. Only one cluster (amino acids 73-89) was recognized by all human and chimpanzee sera. Clear variation in the immune response was observed between individuals, although no obvious difference in reactivity between acute and chronic cases was observed. Within individual profiles, the reactivity to each peptide cluster and the total number of reactive clusters increased over time.

A cutaneous B-cell lymphoma of novel immunophenotype.

A case of low-grade B-cell malignant lymphoma with a prolonged restriction to the skin is described. A rearrangement of the immunoglobulin heavy-chain gene was detected using molecular techniques. The histology showed many similarities to primary mucosal lymphomas and this may represent a skin counterpart of these tumours.

Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry.

7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.

Chromatography of beta-adrenergic blocking agents.

The determination of beta-blockers has posed pharmaceutical analysts with a variety of problems arising from the essential characteristics of these compounds as bases and the variability of physicochemical properties of individual drugs. Liquid chromatography has become the favoured method of analysis and to a certain extent there is a standardised approach to analysis based on either solvent or solid-phase extraction and reversed-phase high-performance liquid chromatography coupled to fluorescence detection. The analyst must be aware of interactions occurring during extraction stages. All manipulations should be fully evaluated for individual drugs and metabolites prior to use. Other analytical options are chosen for specific or more demanding applications. The use of unmodified silicas for the liquid chromatography of beta-blockers (and other basic drugs) is an example of a potential alternative mode of chromatography. The stereoselectivity of the pharmacology of beta-blockers has spawned a great deal of literature describing the resolution of enantiomers by chromatographic methods. It is envisaged that this area will achieve greater prominence in the future as drug development pursues optical purity. The demand for the availability of enantiomerically pure pharmaceutical preparations will certainly see developments for preparative-scale separations as well as analytical methods and will surely promote developments in new and established methods of chromatography.

Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors.

In a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl-lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T-cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl-lysine binds to the membrane of heat-inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl-lysine signal, the DNA distribution and surface antigen expression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein.

Changes in antigen expression on human FME melanoma cells after exposure to hypoxia and acidic pH, alone or in combination.

Quantitative changes in the expression of two tumour-associated surface antigens on human FME melanoma cells were studied by flow cytometry after exposure to hypoxia and acidic pH, either alone or in combination. The expression of the p250 antigen recognized by the monoclonal antibody (MAb) 9.2.27 was reduced immediately after exposure to hypoxia. The magnitude and duration of the reduction increased with increasing exposure time. Twelve to 16 hr after the end of a 6-hr exposure to hypoxia the antigen expression reached the normal level, followed by a temporary increase above this level. The p97a antigen recognized by the 4.1 MAb underwent similar changes after exposure to hypoxia for 6 hr. After exposure to hypoxia in acidic environment, the magnitude and duration of the reduction in the expression of the p250 antigen increased with increasing acidity. The enhancement in antigen expression above the normal level was less after hypoxia at acidic pH than after hypoxia at physiological pH. The combined treatment had an additive effect on the expression of the melanoma-associated antigen but did not enhance hypoxia-induced cell killing. The observed changes in antigen expression might be of importance if hypoxic tumour cells are subjected to MAbs conjugated to radioisotopes or cytotoxic agents for diagnostic or therapeutic purposes.

Relationship between changes in antigen expression and protein synthesis in human melanoma cells after hyperthermia and photodynamic treatment.

Hyperthermia and photoactivated hematoporphyrin derivative induce a dose-dependent reduction in the expression of the p250 surface melanoma-associated antigen on the human FME cell line. Expression of this glycoprotein antigen was quantitated by immunofluorescence flow cytometry based on the monoclonal antibody 9.2.27. Decrease in antigen expression was followed by a transient increase above the level for untreated cells, before normalization occurred about one week after treatment. These treatment-induced changes in antigen expression could partly be explained by changes in protein synthesis. This conclusion was based on the following observations: Hyperthermia and photoactivated hematoporphyrin derivative both inhibited protein synthesis. The latter increased again rapidly to rates above normal until antigen expression reached normal level, whereupon the protein synthesis rate decreased to normal. Inhibition of protein synthesis by cycloheximide 1 day after heating, prevented the recovery of antigen expression, demonstrating that protein synthesis is necessary for resumption of normal antigen expression. The changes in both antigen expression and protein synthesis were dose-dependent, and the magnitude and duration of the changes increased with increasing dose. The time courses of the changes in protein synthesis after two different treatments which both inactivated two logs of cells were almost identical, as were the time courses after two lower heat doses inactivating one log of cells. These similarities were reflected in the changes in antigen expression. At the same time as protein synthesis reached its maximum and antigen expression resumed normal level, an increase in the Golgi apparatus was observed ultrastructurally, indicating an increased synthesis rate and transportation of glycoproteins to the cell surface.