Comparison of normal piglet bladder damage after PDT with oral or intravesical administration of ALA.

5-Aminolaevulinic-acid (ALA) can be used as an alternative drug in photodynamic therapy of the bladder, since the selective formation of protoporphyrin IX (PpIX) in the tumour and the virtual absence of induced skin photosensitivity are theoretically advantageous for clinical use. A preclinical study was performed, using an in vivo normal piglet bladder model, in order to determine the maximum drug and light doses for reversible tissue damage. Various ALA doses were administered either orally or instilled in the bladder and different radiant exposures were applied. Bladder biopsies were taken at regular intervals and tissue damage was investigated histologically. After oral ALA-administration the PpIX concentration was determined in plasma, erythrocytes and various tissues. In the case of oral administration, reversible bladder damage was observed using 60-75 mg/kg ALA combined with a radiant exposure of 100 J/cm(2) (direct radiant exposure plus scattered 632 nm light) 5-7 h later. For an oral ALA dose of up to 150 mg/kg, the maximum PpIX concentration is reached at approximately 5 h following administration and in neither skin nor bladder tissue is PpIX present at 10-11 h after administration. This ALA dose combined with a radiant exposure of 200 J/cm(2) produces irreversible bladder damage (extensive necrosis and ulceration). In the case of intravesical instillation for 4-4.75 h, an ALA dose of 2.5 g in 50 ml phosphate buffered saline and a radiant exposure of 100 J/cm(2) are still too high to obtain reversible tissue damage; at this dose one of the 13 pigs developed a shrunken bladder with a fibrotic, thickened bladder wall. These drug and light combinations reported above should be regarded as upper limits in pigs and can serve as an indication for the toxicity of the treatment in a clinical setting.

Negative sural nerve biopsy in neurolymphomatosis.

Patients with non-Hodgkin's lymphoma occasionally develop widespread invasion of peripheral nerves by tumor cells or neurolymphomatosis (NL). Clinically this usually results in asymmetrical, progressive, and painful polyneuropathy. Diagnosis rests on the identification of tumor cells in peripheral nerves. To avoid false-negative biopsy findings in patients with malignant lymphomatous infiltration of peripheral nerves it has been recommended to biopsy clinically involved nerves. We present two patients with histologically confirmed NL in whom sural the nerve biopsy finding was negative despite clinical and neurophysiological evidence of involvement of the sural nerve a. The clinical features of NL are reviewed. Some patients with neurolyphomatosis have only focal or proximal involvement of nerves, requiring the biopsy of an affected part of these nerves. Magnetic resonance imaging may be useful in identifying affected nerves.

An analogue laser optical disc in comparison with cinefilm for visual analysis of coronary narrowings before and after coronary angioplasty.

This study evaluated an analogue laser optical disc (MVP) as an alternative for cinefilm angiography in the visual analysis of coronary angiograms. Visual analysis was performed independently by 5 observers using cinefilm and MVP before and after PTCA (194 coronary lesions in 88 patients) and the outcomes were compared with QCA. The mean percentage diameter stenosis on cinefilm and MVP yielded similar results compared to QCA. Regression analysis showed a good correlation between the mean cinefilm and MVP values per diameter stenosis (p < 0.001). Bland-Altman plots confirmed these findings. Qualitative analysis for detection of coronary dissections after PTCA showed an incidence of 31.3% (cinefilm) and 21.8% (MVP) (p < 0.05). The results of this study indicate that the visual analysis of the coronary angiograms using the analogue laser optical disc (MVP) yields similar results compared to the cinefilm concerning coronary lesion severity, although there is an underestimation of coronary dissections.

The prognostic significance of expression of the multidrug resistance-associated protein (MRP) in primary breast cancer.

In the present study, we determined the frequency and intensity of MRP protein expression by monoclonal antibody immunohistochemistry in a series of 259 resected invasive primary breast carcinomas, and we evaluated MRP immunoreactivity in relation to patient and tumour characteristics, relapse-free (RFS) and overall survival (OS). The immunostaining was graded on a semiquantitative scale that ranged from (-) to ( ). Overall, 34% of the tumours were positive for anti-MRP antibody: 19% showed weak cytoplasmic staining (+), 14% had clear cytoplasmic staining (++) and only 1% of the tumours had a strong cytoplasmic as well as membranous staining ( ). MRP expression was not related to patient's age, menopausal status, tumour size, differentiation grade, oestrogen and progesterone receptor level or lymph node involvement. In an exploratory univariate analysis of all patients, only primary tumour size and number of lymph nodes involved were significantly associated with shortened RFS (P < 0.001 and P < 0.001 respectively) and OS (P = 0.02 and P < 0.001 respectively). In Cox univariate analysis for RFS in subgroups of patients stratified by menopausal status, tumour size, nodal status, adjuvant systemic therapy and oestrogen and progesterone receptor status, MRP expression was associated with increased risk for failure in patients with small tumours (T1), in node-negative patients and in node-positive patients who received adjuvant systemic chemotherapy with cyclophosphamide, methotrexate and 5-fluorouracil (CMF); the relative hazard rate (RHR) for relapse was increased in the presence of MRP, with RHR values with 95% confidence limits (CL) of 2.8 (1.2-6.9), 2.1 (1.0-4.2) and 2.8 (0.8-9.9) respectively. In analysis for OS, expression of MRP was also associated with increased risk for failure in patients with small tumours (T1) [RHR (95% CL) 2.3 (0.9-6.0)] and in node-positive patients who received adjuvant systemic chemotherapy with CMF [RHR (95% CL) 3.7 (0.8-17.1)] but not in node-negative patients [RHR (95% CL) 1.1 (0.4-2.6)]. In conclusion, our results show that MRP is frequently overexpressed in primary breast cancer and suggest that MRP expression might be of prognostic significance in the subgroups of patients with the more favourable prognosis, i.e. patients with small tumours and node-negative patients, as well as in the setting of adjuvant systemic chemotherapy. In primary breast cancer, MRP might be related to altered cell biological behaviour, including a more aggressive phenotype, and resistance to adjuvant systemic chemotherapy.

Inhibition and stimulation of LFA-1 and Mac-1 functions by antibodies against murine CD18. Evidence that the LFA-1 binding sites for ICAM-1, -2, and -3 are distinct.

The murine CD18 monoclonal antibody (mAb) M18/2 was reported to inhibit lymphoma metastasis [Zahalka, M. A. et al. (1993) J. Immunol. 150, 4466]. To identify the pathways potentially blocked, we studied the effects of M18/2 compared with two new mAb against murine CD18, GAME-46, and -245. Whereas the GAME mAb blocked most Mac-1-mediated interactions, M18/2 had no effect, or even stimulated. The same was true for adhesion of LFA-1 to ICAM-1. To test effects on interactions with different ICAMs, we used L cells transfected with human ICAM-1, -2, and -3. As previously described, mouse LFA-1 does not bind to human ICAM-1 but we show here that mouse LFA-1 does bind to human ICAM-2 and -3. Again, the GAME mAb blocked completely, but M18/2 did not. These results indicate that the LFA-1 binding sites for ICAM-1 and ICAM-2 and -3, although in close vicinity, are distinct. Furthermore, effects of M18/2 on metastasis cannot be ascribed to blocking of any known beta2-integrin activity.

Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity.

T-cell hybridomas are highly metastatic, and their in vitro invasiveness correlates with metastatic capacity. Invasion is blocked by pertussis toxin (PT), which adenosine diphosphate (ADP)-ribosylates G1-proteins, and we have provided evidence that the PT-sensitive signal stimulates leukocyte function-associated antigen-1 (LFA-1)-mediated adhesion required for invasion. PT pretreatment of TAM2D2 T-cell hybridoma cells reduced metastasis, but only to a limited extent. In the present study, we have transfected the cDNA of the PT ADP-ribosyltransferase S1 subunit into TAM2D2 cells to abrogate G1-protein function permanently. We report here a substantial reduction in the metastatic capacity of two transfectants, S05 and S09, in which 88% and 95% of the G1-proteins was ADP-ribosylated. Two-thirds of the mice injected with S09 cells were tumor-free. Metastasis to the liver was almost completely prevented and less metastases were formed in the spleen and kidneys. Metastasis formation by S05 cells in liver and spleen was much reduced, but in lymph nodes and peritoneal tissues, metastases occurred with a frequency similar to that of controls. We conclude that G1-proteins play an important role in T-cell hybridoma metastasis. We propose that the reduction in metastasis is due to diminished entry of tumor cells from the blood into tissues.

Chromosomal changes in renal oncocytomas. Evidence that t(5;11)(q35;q13) may characterize a second subgroup of oncocytomas.

Many of the reported oncocytomas have different chromosome abnormalities, indicating that they comprise a cytogenetically heterogenous group of tumors consisting of potentially cytogenetic subgroups. We have performed cytogenetic studies on nine renal oncocytomas. Clonal abnormalities were present in eight tumors. The findings most observed were the loss of the Y chromosome, and abnormalities of chromosomes 1 and 22. We also observed telomeric associations (tas) in two tumors and structural aberrations of chromosomes 9p and 19q, as well as monosomy 10. In two cases we found a similar reciprocal t(5;11)(q35;q13) in two cases. Review of the literature disclosed one other oncocytoma with a t(5;11) (q35;q13). This suggests that t(5;11)(q35;q13) defines a (second) subset of oncocytomas apart from the subgroup specifically associated with the loss of chromosomes 1 and Y.

Adhesion mechanisms in liver metastasis formation.

Our results strongly indicate that integrin mediated adhesion between metastasizing tumour cells and hepatocytes has a decisive role in the formation of liver metastases. However, it is also clear that tumour cells may use different adhesion pathways and furthermore that the adhesion may be modulated by several factors. The role of adhesion has been demonstrated most clearly for LFA1 on T cell hybridomas, which interacts with ICAM1 present in the liver. Alternative pathways must exist, however, given the high invasive capacity of ESb cells, which is apparently LFA1 independent. A possible alternative is adhesion to fibronectin, which is present in abundance on the hepatocyte surface, both in vivo and in vitro. As there is no basement membrane under the endothelium of liver microvessels, so that tumour cells cannot adhere to laminin and collagen type IV as in other organs, adhesion to this fibronectin may be particularly important for metastasis to the liver. Many tumour types can use this pathway, and many different fibronectin receptors may be involved, including VLA-4, VLA-5 and several alpha V integrins. For carcinomas there is another possibility: Fibronectin receptor deficient cells may still adhere using the integrin alpha 6 beta 4, which binds to an unknown ligand present on the hepatocyte surface. Modulation of adhesion can occur in several ways. One example is steric hindrance by mucins that may strongly affect and even abrogate adhesion, despite high levels of appropriate integrins. Another is the activation required for integrins on lymphoma cells, the best known example of which is the activation of LFA1 on T cell hybridomas. It will be evident, therefore, that the role of adhesion in the formation of liver metastases can only be fully understood if the complete set of adhesion molecules on the tumour cells is known as well as their functional status and the possible effects of both cellular and extracellular modulating factors.

LFA-1 integrin redistribution during T-cell hybridoma invasion of hepatocyte cultures and manganese-induced adhesion to ICAM-1.

We have reported previously that the integrin LFA-1 is essential for metastasis of T-cell hybridomas to the liver. We show here that hepatocytes isolated from normal non-inflamed rat liver express intercellular adhesion molecule-1 (ICAM-1) at the dorsal surface and more prominently at the lateral and substratum-adherent surfaces. Anti-rat ICAM-1 mAb inhibited adhesion of TAM8C4 T-cell hybridoma cells to hepatocytes. Invasion between hepatocytes was not affected, but this is probably due to lack of penetration of the mAb between the hepatocytes. In all hepatocyte-adherent TAM8C4 cells, LFA-1 was concentrated at the adhesion site. Redistribution of ICAM-1 to the interacting hepatocyte membrane was also seen, but only for part of the adherent TAM8C4 cells. LFA-1 was highly concentrated on pseudopods of invading TAM8C4 cells inserted between hepatocytes, and on the upper surface of invaded TAM8C4 cells located under the hepatocytes. ICAM-1 was concentrated in the hepatocyte membrane overlying TAM8C4 cells located underneath the monolayer. These results suggests that ICAM-1 is of major importance for liver invasion by these lymphoma cells. For optimal adhesion to ICAM-1, LFA-1 on T-cell hybridomas requires activation, which apparently occurs upon contact with cell layers that are invaded (G. La Rivière et al., J. Cell Sci. 107, 551-559, 1994). LFA-1 can be activated artificially by Mn2+. To study LFA-1 redistribution upon ICAM-1 interaction with higher resolution, we performed immuno-EM on cells before and after Mn(2+)-induced adhesion and spreading on immobilized ICAM-1. By immune fluorescence, LFA-1 was observed to redistribute to the ICAM-1-adherent surface, and to be concentrated in lamellipodia of spreading TAM8C4 cells. By immuno-EM, LFA-1 was localized in microclusters of approximately 10 gold particles. This was seen in cells fixed in suspension, and the size of these clusters did not change upon adhesion to ICAM-1. LFA-1 was present at high density in thin filopodia, but again in microclusters of similar size. Comparable results were obtained with a cytotoxic T-cell clone. We conclude that Mn(2+)-induced activation of LFA-1 is not associated with the formation or enlargement of LFA-1 clusters.

Pertussis toxin inhibition of T-cell hybridoma invasion is reversed by manganese-induced activation of LFA-1.

Pertussis toxin (PT) inhibits invasiveness of T-cell hybridomas in vitro and metastasis formation in vivo. We present evidence for the hypothesis that PT interferes with functional activation of LFA-1. Invasion by TAM2D2 T-cell hybridoma cells of fibroblast monolayers was completely blocked by PT pretreatment, but the cells regained invasiveness in the presence of Mn2+, which activates LFA-1. This invasion was blocked by anti-LFA-1 mAb, and Mn2+ did not stimulate invasiveness of LFA-1-deficient TAM2D2 mutants. TAM2D2 cells did not adhere to surfaces coated with the LFA-1 counterstructure ICAM-1, but Mn2+ induced adhesion. Hence, LFA-1 on TAM2D2 cells requires activation before it can participate in the invasion process. The hypothesis is further supported by the slightly different results obtained with the TAM8C4 T-cell hybridoma. PT inhibited invasion strongly but not completely. This reduced invasion was increased by Mn2+. TAM8C4 cells did adhere to ICAM-1, but Mn2+ enhanced adhesion. Thus, part of LFA-1 on TAM8C4 cells is constitutively active, allowing for some PT-insensitive invasion, but further activation is required for optimal adhesion and invasion. PT blocks G-protein-mediated signals, suggesting that an extracellular factor is involved. This is not a serum component or an autocrine motility factor, since the PT effect was serum-independent, and PT did not inhibit motility. Therefore, it is probably produced by the fibroblasts, and either secreted or associated with the cell surface. These results are in line with the hypothesis that a fibroblast constituent activates LFA-1 via a PT-sensitive G-protein and thus stimulates invasion of T-cell hybridomas into the fibroblast monolayer.

The role of integrins and integrin activation in liver metastasis.

An overview is presented of our studies on the interaction between blood-borne tumor cells and the tissues where metastases are formed, in particular the liver. Using blocking antibodies and tumor cell mutants, we have identified the adhesion molecules involved, which so far are all integrins. Strikingly, tumor cell lines that are quite similar, and invade in a comparable fashion, use distinct integrins. Lymphomas that invade the liver massively and diffusely use LFA-1 or fibronectin receptors to adhere to hepatocytes. We have obtained evidence that LFA-1 is activated during the interaction by factors that act through G-protein-coupled receptors, and preliminary results suggest that the same may be true for the fibronectin receptors. Whereas TA3/Ha murine mammary carcinoma cells adhere to hepatocytes via alpha 6 beta 4, TA3/St variant cells of the same tumor bind via the fibronectin receptor alpha 5 beta 1. Adhesion of the TA3/Ha cells appears to be impaired by the mucin epiglycanin that is abundantly present on the surface of these cells.

In vitro invasiveness of CTL clones and in vivo dissemination of CTL hybridomas.

Activated spleen T cells are invasive in hepatocyte and fibroblast cultures, and this property is dominantly expressed in T cell hybridomas. The invasive potential of the hybrids correlates with their capacity to disseminate in vivo. We have used this model to study the invasive and migratory properties of cytotoxic T lymphocytes (CTLs). Two murine CTL clones were highly invasive, independent of their state of activation. CTL hybridomas, derived from one of the clones, were similarly invasive. In vivo, CTL hybridoma cells disseminated to extravascular sites in the liver, kidneys, lungs, ovaria, tubae, uterus, and lymphoid, mesenchymal, and fat tissues. Within 7 to 14 days, 10(6) cells were lethal in 100% of mice. The adhesion molecules CD2, CD8, CD54, L-selectin, and CD49d (VLA-4 and LPAM-1 alpha-chain) were not expressed by all CTL hybridomas and therefore not indispensable for invasion in vitro and dissemination in vivo. In contrast, LFA-1 (CD11a/CD18), CD44, and VLA-6 (CD49f/CD29) were expressed on all hybrids. LFA-1 antibodies inhibited CTL hybridoma invasion in vitro, but antibodies inhibiting CD44-hyaluronate and VLA-6-laminin interaction had no effect. These results suggest that migration of cytotoxic T cells into noninflamed tissues is independent of their activation state and does not require L-selectin, LPAM-1, CD2, and VLA-4.

A clinicopathological study of 30 melanomas of the vulva.

Thirty cases of malignant melanomas of the vulva were studied for prognostic factors. Ulceration, tumor thickness, and positive inguinal lymph nodes were the most important prognostic factors. Morphometry did not demonstrate any prognostic meaning. Traditionally a radical vulvectomy and bilateral inguinal lymph node dissection were the therapy of choice, but this treatment modality did not show a better survival than less radical treatment. A low-risk and a high-risk group of patients have been identified for recurrence. The low-risk patient has a nonulcerative tumor, less than 3 mm thick, without clinical evidence of inguinal lymph node metastases, and should be treated by local excision with a 2- to 3-cm margin. The high-risk patient has a tumor which is ulcerative and/or more than 3 mm thick and should also be treated by local excision without elective inguinal node dissection. If clinical suspicion of inguinal lymph node metastases exists, an inguinal node dissection is advocated for better local control of the disease.

Putative invasion-specific proteins in mouse T-cell hybridomas that differ in invasive and metastatic potential.

Fusion of invasive, activated T-lymphocytes with non-invasive BW5147 T-lymphoma cells mainly yields highly invasive (HI), highly metastatic T-cell hybridomas. In addition, several non-invasive (NI), non-metastatic hybrids have been obtained, probably due to loss of involved gene(s) by chromosome segregation. Here we have compared a panel of HI and NI hybrids in a search for proteins specifically expressed by either cell type. MAbs were raised against HI hybrids, but out of more than 1,000 none bound exclusively to HI cells. Furthermore, polyclonal rat, rabbit and chicken antisera did not immunoprecipitate specific proteins from total lysates, and the expression of 18 (T-cell) surface markers did not correlate with invasiveness. These results indicated that the number of differences between HI and NI hybridomas was surprisingly small. This notion was confirmed by 2-dimensional gel electrophoresis. Among 1,000 detectable spots, we found only 2 clear-cut differences between HI and NI T-cell hybridomas, whereas multiple differences were found between individual hybrids. One protein (p130) was expressed at much higher levels by HI than by NI hybrids in this panel, whereas the other (p15) was only seen in NI hybrids. These proteins are primary candidates for a role in invasion.

The heart in Becker muscular dystrophy, facioscapulohumeral dystrophy, and Bethlem myopathy.

We report a study, assessing involvement of the heart in 33 familial cases of Becker muscular dystrophy (BMD), 31 familiar cases of facioscapulohumeral (FSH) dystrophy, and 27 familial cases of Bethlem myopathy. In the patients with BMD, correlations of myocardial involvement with age and extent of musculoskeletal involvement were made. We performed physical examination, chest x-ray, electrocardiographic (EKG), and echocardiographic examination on all patients, and continuous EKG Holter-monitoring in the patients with FSH dystrophy. Thirteen patients with BMD (45%) showed EKG changes similar to those found in Duchenne muscular dystrophy. Only 1 of the 13 individuals with cardiac involvement was wheelchair-bound. We found no evidence of cardiac changes in the patients with FSH dystrophy. In Bethlem myopathy, only 1 patient had a form of hypertrophic cardiomyopathy (asymmetrical septal hypertrophy).

Tumour necrosis factor-alpha stimulates invasiveness of T-cell hybridomas and cytotoxic T-cell clones by a pertussis toxin-insensitive mechanism.

Tumour necrosis factor-alpha (TNF-alpha) stimulated invasion by mouse T-cell hybridomas and cytotoxic T-lymphocyte clones into rat embryo fibroblast monolayers. The effect on these highly invasive cells was limited: invasion was stimulated maximally to 130% of controls. However, when cells were pretreated with pertussis toxin (PT), which inhibits invasion to +/- 20% of controls, a clearcut effect was observed: 400 U TNF-alpha per ml stimulated invasion usually two- to threefold, and sometimes even up to 10-fold. Therefore, experiments were done with PT-pretreated cells. Stimulation was dose dependent and maximal at 200-400 U TNF-alpha per ml. An anti-TNF-alpha monoclonal antibody completely abolished TNF-alpha-induced invasion. The effect was maximal 30 min after addition of cells and TNF-alpha to the monolayer and then declined. TNF-alpha preincubation of T-cell hybridoma cells, but not of fibroblasts, had a similar stimulatory effect, which was also maximal after 30 min. This shows that TNF-alpha acts directly on the T-cell hybridoma cells. Invasive T-cell hybridomas colonize many tissues from the blood similarly as normal T cells. Our data thus suggest that TNF-alpha can stimulate migration of normal T lymphocytes into inflamed tissues and can promote metastasis of malignant T lymphomas. The signals involved are transmitted via a pertussis toxin-insensitive pathway.