Key importance of the Helicobacter pylori adherence factor blood group antigen binding adhesin during chronic gastric inflammation.

Helicobacter pylori has been assigned as a class I carcinogen because of its relation to gastric adenocarcinoma. Chronic H. pylori infection may lead to severe gastritis, glandular atrophy (AT), and intestinal metaplasia (IM). Strains secreting the vacuolating toxin VacA and producing the cytotoxin-associated antigen CagA (type 1 strains), as well as the blood group antigen binding adhesin (BabA) targeting Lewis(b) antigens, have been associated previously with distal gastric adenocarcinoma (M. Gerhard et al., Proc. Natl. Acad. Sci. USA, 96: 12778-12783, 1999) and may therefore also be related to lesions preceding gastric cancer. Antral and corpus biopsies were collected from 451 patients; 151 were H. pylori positive, as determined by PCR. Gastric biopsies were histologically evaluated for activity of gastritis (G0-G3, granulocyte infiltration), chronicity of gastritis (L1-L3, lymphocyte infiltration), and the presence of IM and/or AT according to the Sydney classification. Simultaneously, the presence of bacterial genes encoding virulence and adherence factors (racAs1/s2, cagA, and babA2) was determined by PCR. The presence of cagA+ and vacAs1 (alone or combined) both correlated with activity and chronicity of gastritis (P < 0.05); however, the overall prevalence of these genes was 60 or 72%, respectively, and was thus relatively frequent. The babA2 gene, encoding the adhesin BabA, was detected in 38% of infected patients and was correlated with the activity of gastritis in antrum and corpus (P < 0.005). cagA+/vacAs1+ strains (suggesting the presence of type 1 strains) that were also babA2 positive were detected more frequently in patients with severe histological alterations (such as G3, IM, or AT) compared with subjects without these changes (P < 0.01). cagA+/vacAs1+ strains that were babA2 negative, however, lacked a significant correlation with severe histological changes, activity, or chronicity of gastritis in antrum and corpus. Adherence of H. pylori via BabA appears to be of importance for efficient delivery of VacA and CagA and may play a special role in the pathogenesis of severe histological changes.

Growth inhibition of experimental glioma grafts by monoclonal antibody treatment.

The effects of 14AC1 monoclonal antibody (McAb) on 79FR-G-41 rat glioma cells in vitro, on the formation of metastases in lung by antibody coated glioma cells, and on the growth of glioma grafts in BALB/c-nu/nu mice were investigated. The 14AC1 antibodies - isotyped as IgG2a - were obtained from a hybridoma clone established after fusion of X63-Ag8.653 myeloma cells and spleen cells of BALB/c mice hyperimmunized with 79FR-G-41 glioma cells. Antibody treatment of glioma cells in vitro caused evident cell surface alterations and pronounced growth depression of most cells. However, a few tumor cells remained unchanged in morphology and continued to proliferate. Moreover, 14AC1 antibodies drastically reduced lung metastasis by pretreated and i.v. delivered glioma cells. Additionally, 14AC1 antibodies suppressed the growth of transplanted rat gliomas in nude mice as evidenced by a longer latency period and a smaller volume of glioma grafts in treated than in control tumor bearers. Nevertheless, glioma grafts showed accelerated growth after termination of antibody treatment. Further experimental investigation is required in order to identify the precise mechanisms of the effects of McAbs on tumor cells in vitro and in vivo.

Monoclonal antibodies against human astrocytomas and their reactivity pattern.

The establishment of hybridomas after fusion of X63-Ag8.653 mouse myeloma cells and splenocytes from BALB/c mice hyperimmunized against human astrocytomas is presented. The animals were primed with 5 X 10(6) chemically modified uncultured or cultured glioma cells. Six weeks after the last immunization step an intrasplenal booster injection was administrated and 3 days later the spleen cells were prepared for fusion experiments. According to the specificity analysis of the generated antibodies 7 hybridoma products (MUC 7-22, MUC 8-22, MUC 10-22, MUC 11-22, MUC 14-22, MUC 15-22 and MUC 2-63) react with gliomas, neuroblastomas and melanomas as well as with embryonic and fetal cells but do not recognize non-neurogenic tumors. The selected monoclonal antibodies (McAbs) of IgG1 and IgG2a isotypes are not extensively characterized but these antibodies have been demonstrated to be reactive with a panel of glioma cell lines with varying patterns of antigen distribution. Using the McAbs described above and a series of cryosections of glioma biopsies and paraffin sections of the same material as well as glioma cultures established from these, variable antigenic profiles among glioma cell populations could be demonstrated. From these results it is evident that there is not only a distinct degree of antigenic heterogeneity among and within brain tumors, but also that the pattern of antigenic expression can change continuously. Some of the glioma associated antigens recognized by the selected antibodies persist after fixation with methanol/acetone and Karnovsky's fixative and probably are oncoembryonic/oncofetal antigen(s). The data suggest that the use of McAbs recognizing tumor associated oncofetal antigens in immunohistochemistry facilitates objective typing of intracranial malignancies and precise analysis of fine needle brain/tumor biopsies in a sensitive and reproducible manner.

Expression of plakoglobin in renal cell carcinoma.

Little is known about the role of molecules involved in cell-cell interactions during the progression of renal cell carcinoma (RCC). We investigated the expression of plakoglobin (a component of the cadherin-catenin adhesion system) in 94 samples of normal kidney tissue from patients with RCC, in 109 primary renal cell carcinomas and in 16 metastases by immunohistochemistry. Expression of plakoglobin was significantly diminished in tumor tissue, particularly in metastatic lesions, as compared to normal kidney tissue (p < 0.001). Follow-up data were available from 87 patients. Patients with a diffuse plakoglobin expression (91-100% positive cells) in primary tumor tissue had a significant better survival rate than patients with a disturbed plakoglobin expression (p < 0.05) as determined by the log rank test. These results indicate that loss of plakoglobin may play an important role in malignant transformation of renal cells. Plakoglobin expression status could give additional information about the individual prognosis.

Antigen variation in a human glioblastoma: from the primary tumor to the second recurrence, permanent cell line and xenotransplantation tumors.

Antigen expression in a human glioblastoma was investigated by immunochemical methods in the primary tumor, the first and second recurrence, a permanent cell line derived from the first recurrence and in its xenotransplantation tumors. In the primary tumor, GFAP, vimentin, S100, Leu-7 and glioma-associated antigens (GAA) as defined by the monoclonal antibodies (mAbs) MUC 2-39, MUC 8-22 and MUC 2-63 were markedly expressed. In the recurrences, gradual loss of GFAP and Leu-7 could be observed, whereas S100, vimentin and GAA gave similar results to those in the primary tumor. In contrast, fibronectin and collagen IV, which were restricted to the vessel walls in the primary tumor, were represented in sarcomatous areas of the recurrences. In some of these areas, co-expression of glial cell markers was observed. In short-term cell cultures, expression of glia- and glioma-associated antigens as well as fibronectin and collagen IV was comparable to that of the recurrent tumor tissue. In long-term passages, immunoreactivity of GFAP, Leu-7 and S100 decreased, whereas GAA, vimentin and fibronectin increased. Collagen IV positive cells were not visible beyond passage 15. Transplantation tumors were only partly positive for glial cell markers, but revealed strong immunoreactivity for GAA, fibronectin and collagen IV. With these observations we confirm that the phenotypic variability of glioma cells makes it difficult to identify the origin of cells in human glioblastomas from their antigenicity.

Radioimmunodetection of human glioma xenografts by radiolabelled monoclonal antibodies.

Radiolabelled monoclonal antibodies (131I-MUC 8-22, 131I-MUC 2-63) were used for external scintigraphy of human glioma xenografts. To induce transplantation tumors. 5 x 10(6) cells (85HG-66) of an in vitro established human malignant astrocytoma (N66/85) were inoculated s.c. in BALB/c-nu/nu mice. The labelling of the immunoglobulins with 131iodine was carried out according to the iodogen method, the nude mice, bearing xenograft, received 30 m. 131I-labelled intact monoclonal immunoglobulins (200mCi: 7,4MBq) and the imaging was performed on days 4, 8 and 12 after the application. After 4 days, a clear tumor accumulation of iodinated MUC 2-63 antibodies recognizing surface determinants was visible. This enrichment of monoclonal antibodies (MAbs) led to a characteristic tumor presentation on day 8. Obviously, the MUC 2-63 antibodies remain in the tumor tissue for a long time, so that even on day 12 satisfactory tumor imaging is possible. On the other hand, neither with normal mouse IgG nor with MUC 8-22 antibodies - which react with intracellular structures - could a tumor localization be achieved. The result of the studies on the distribution of 131I-MUC 2-63 on day 19 was that the activity in the tumor tissue was about 4.4 times higher than in the blood and even more times higher than in solid organs.

Radioimmunodetection of gliomas by administration of radiolabelled monoclonal antibodies. Experimental data.

Radiolabelled monoclonal antibodies (McAbs) raised against membrane components of an experimental rat glioma (79FR-G-41) were administered parenterally to immunodeficient mice bearing glioma grafts for tumor radioimmunodetection by external imaging. Purified McAbs (14AC1) of IgG2a isotype were labelled with Na131I (2mCi/50ml) using the Chloramin-T method. As control, for non-specific uptake of proteins in the tumor, normal mouse IgG were also iodinated. For radioimaging, nude mice bearing gliomas in the thigh muscle were injected intravenously with 15 micrograms of the 131I-McAb with an activity of approximately 150 mu Ci. Control tumor-bearing animals received the same amount of mouse 131I-IgG. Scans obtained immediately after injecting the intact 131I-14AC1 antibody and at 24, 48, 72, and 96 hours demonstrated accumulation in the tumor. The tumor was clearly visible 48 hours following injection of 131I-labelled antibody. At 96 hours after injection, the McAb showed a clearly higher uptake into the tumor as the control IgG. The biodistribution of the injected antibody was studied at 96 hours after injection following the last gamma-imaging. At this time the blood activity was still high, but the maximum activity was found in the tumor for the specific McAb. Using the 131I-14AC1 to image glioma transplants, it could be shown that grafts are permeable for the McAb. The time-course experiments administering 131I-14AC1 antibody and normal mouse 131I-IgG, demonstrated that the localization of 131I-I4AC1 antibody in glioma grafts is the result of specific antigen binding. The scintigrams using intact antibody without background subtraction provided adequate tumor visualization, but the activity in the blood was high even 96 hours after injection. More rapid clearance of blood - pool radioactivity would possibly be achieved with F(ab')2 fragments. These in vivo glioma imaging studies, together with related in vitro binding tests, indicate the potential value of monoclonal antiglioma antibodies not only for clinical tumor radioimmunodetection, but also for the evaluation of immunotherapeutic approaches to the glioma disease of man.

Relationship between Ki-67 positive cells, growth rate and histological type of human intracranial tumors.

The proliferation rate of 40 intracranial neoplasms (30 gliomas, 1 hemangioblastoma, 3 meningiomas, 1 neurinoma and 5 brain metastases) was investigated using the monoclonal antibody Ki-67. In eleven of the gliomas recurrences could be observed, and two of them recurred for second time. In total the Ki-67 labelling indices of 53 specimens were investigated. The Ki-67 nuclear antigen was demonstrated in frozen sections by application of the appropriate monoclonal antibodies according to a modified alkaline phosphatase-antialkaline phosphatase (APAAP) technique. The proliferation rate was evaluated by cell count calculation of the staining index. Ki-67-labelled glioma cells varied from 0.2 percent in one meningioma (WHO-grade I) to 9.1 percent in one glioblastoma. In ten glioma recurrences, higher Ki-67 staining indices could be observed than in their primaries, even when the histological grading did not change substantially. In a cerebellar hemangioblastoma, a trigeminal neurinoma and two endotheliomatous meningiomas the fraction of stained nuclei was less than one percent; however, one recurrent transitional meningioma without any histological sign of malignancy showed a staining index of 2.4 percent. The staining indices of five brain metastases of different malignancies ranged from 1.5 percent in a malignant melanoma to 6.1 percent in bronchial carcinoma. In the majority of the cases examined, the percentage of Ki-67 labelled cells was in accordance with the histologic grade of the neoplasm. In general, there was a direct relationship between the number of stained nuclei and the frequency of mitoses (mitotic index) evaluated in hematoxylin-eosin stained frozen sections. Interestingly, the frequency of mitosis and stained nuclei were higher in tumor recurrences than in the primaries. The results of this study imply that immunohistological labelling of the proliferating cell fraction should become an important additional criterion to predict the biological behaviour of human nervous system neoplasms.

Radioimaging of experimental glioma grafts using F (ab')2-fragments of monoclonal antibodies.

Iodinated F (ab')2-fragments of monoclonal IgG2a-antibodies (McAbs) 14 AC1 raised against membrane components of an experimental rat glioma (79FR-G-41) were used for the radioimmunodetection of glioma xenografts in athymic mice. For gamma imaging, nude mice with intramuscular glioma grafts received 10-20 micrograms of 131I-labelled whole immunoglobulins and F (ab')2-fragments of the 14AC1 McAb, respectively, with an activity of 50-150 mu Ci (1.85-5.55 MBq). Scans obtained at 24, 48, 72 and 96 hours after injection demonstrated tumor accumulation of F (ab')2-fragments at 24 hours, and adequate tumor visualization without background subtraction at 48 hours. The rapid blood clearance of F (ab')2-fragments resulted in a whole body half life of 6 hours. Scintigrams using intact antibody provided comparable tumor localization at 96 hours, but also high bloodpool radioactivity and a half life period of more than 4 days. 131I-labelled normal mouse-IgG as control for non-specific uptake of immunoglobulins in the tumor revealed no tumor visualization at any time. Rapid tumor permeation, short blood clearance and a markedly reduced background radioactivity let F (ab')2-fragments appear superior to whole immunoglobulins in immunoscintigraphy, thus indicating their potential value for the improvement of radioimmunodiagnosis of glioma disease.

Antigenic heterogeneity of human brain tumors defined by monoclonal antibodies.

The antigenic profiles of human gliomas and in vitro established cell lines were investigated using the monoclonal antibodies (MABs) MUC 8-22 and MUC 2-63. The reactivity with tissue samples and cytospin preparations obtained from 45 brain tumors was estimated by the indirect immunoperoxidase technique. In addition, computer-assisted cytofluorometry was used to quantify the intensity and distribution of antibody-binding. Various degrees of antibody-binding among and within gliomas and glioma-derived cell lines were observed. The data show that a variable percentage of cells are not labeled with the employed MABs. The spectrum of reactivity of the selected antibodies was independent of the histological grading of gliomas. However, there were significant differences in various stages of subcultivation of glioma lines. In most cases, the heterogeneity of antigen expression decreased during successive in vitro propagation of glioma cells. The extent of variation in staining intensity values differed within cell populations and reflected the antigenic heterogeneity of human brain tumors. The findings presented here suggest that the use of MABs which recognize glioma-associated antigens facilitates the objective analysis of brain tumors and is of potential value for immunohistochemical application in surgical neuropathology.

[Histological changes following endovesical neodymium-YAG-laser irradiation (author's transl)]. / Histologische Grundlagen der endovesicalen Neodym-YAG-Laserbestrahlung.

Experimental animal research was used to compare the coagulatory qualities of the argon laser and the Neodymium-YAG laser to results obtained through electrocoagulation. The argon laser effects a necrosis near the urinary bladder wall surface, and when the energy density is increased, it may ablate the tissue to the point of perforation. In contrast, the Neodymium-YAG laser effects a bandshaped necrosis that penetrates all layers of the urinary bladder wall without primarily resulting in an ablation of tissue. Electrocoagulation results in irregularly demarcated necroses of varying depths, which can be explained by the uncontrolled flow of the high-frequency currents according to the paths of least resistance. The Neodymium-YAG laser is the one most suited for transmural coagulation of defined areas within the urinary bladder wall due to the even spread of its thermal energy that is independent of the affected tissue structure, as well as to its deep coagulatory qualities which in turn are due to the laser's minimal tissue absorption. The Neodymium-YAG laser allows the setting of reproduceable, sharply demarcated necroses into the bladder wall with an ensuing complete destruction of all wall layers. The wall framework within the necrotic area, however, remains intact, so that the danger of perforation is kept at a minimum.

[Dosimetry of the neodymium-YAG laser endovesical irradiation of bladder tumors. Animal experiments (author's transl)]. / Dosierung der Neodym-YAG-Laserstrahlung zur endovesikalen Anwendung bei Blasentumoren. Tierexperimentelle Untersuchungen.

Therapy of urinary bladder tumors with the aid of the Neodymium-YAG-laser is improved by two following demands. A homogeneous, deep necrosis must be obtained for the tumor cells to be devitalised completely, and neighbouring organs must be protected adequately. Both depth and shape of the necrosis were determined on liver tissue at different conditions, e.g. laser application with air, water and various time/power ratios. Measurements of temperature profiles at the bladder back wall reveal that the parameters necessary for sufficient transmural destruction of bladder tumors do not affect neighbouring organs. In clinical application it is not necessary to control temperatures occurring at the bladder wall during irradiation by means of temperature probes. It is sufficient to adjust the dosis of laser on the basis of superficial, visual discoloration. Carbonisation should be avoided by all means. Comparable effects were obtained by clinical application of data obtained during animal experimentation.

[Interstitial laser coagulation of benign prostatic hyperplasia]. / Die interstitielle Laserkoagulation der benignen Prostatahyperplasie.

We report on the new method of interstitial laser coagulation for the treatment of benign prostatic hyperplasia (BPH). The procedure is based on the interstitial application of Nd:YAG laser irradiation, delivered through a new light guide system. Such light applicators coagulate constant tissue volumes in a homogeneous manner, as proven by in vitro studies in different tissues, including surgically removed prostate adenoma. The extent of the coagulation is determined by laser power and irradiation time. At 5 W, for example, and during a 10-min period, this zone reached a diameter of up to 20 mm. Temperatures generated in the process were over 100 degrees C, as measured by time/space resolution. These results were confirmed by in vivo studies in canine prostates. In the course of 7 weeks, the coagulated areas formed scars with degeneration and fibrosis, accompanied by marked shrinking. Neighbouring organs were not affected. The method was successfully transferred to clinical practice. The application of the light guides to the lateral lobes was performed percutaneously from the perineum under transrectal ultrasound guidance. The median lobe was punctured transurethrally under direct vision. Twenty-seven patients with an average age of 67.7 years were treated between July 1991 and March 1992. At the time of evaluation 15 patients had a follow-up of more than 2 months. They experienced a mean increase of peak flow rate from 6.6 to 15.2 ml/s and a mean decrease of residual volume from 206 to 38 ml. This was accompanied by a marked lessening of symptoms. The average prostate weight decreased from 63 to 44 g. Sexually active patients did not experience retrograde ejaculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological fundamentals in the treatment of tumors with the neodymium-YAG laser.

On the basis of experimental animal research regarding type, distribution, and depth of necrosis in urinary bladder under neodymium-YAG (Nd:YAG) laser irradiation, identical results have been confirmed in human tissue. Bladder tumors indicated for cystectomy have first been treated with the Nd:YAG laser and histologically examined. Destruction of tumor tissue is demonstrated. Particular attention was paid to the temporary closing of the lymphatic drainage system due to the deep coagulation capability of the Nd:YAG laser irradiation. Nd:YAG laser irradiation is especially suitable for tumor coagulation, since simultaneous interception of lymphatic drainage will inhibit the spreading of tumor cells.

Use of the neodymium-YAG laser in the treatment of ureteral tumors and urethral condylomata acuminata. Clinical experience.

The further development of endoscopic instruments has made neodymium-YAG laser irradiation of ureteral tumors possible. To date a total of 13 ureteral tumors in 10 patients have been photocoagulated. Over a mean follow-up period of 23 months, only 1 heterotopic recurrence was found after 14 months. In view of the results achieved so far, nephroureterectomy as the first therapeutic step appears to have been made obsolete. Since 1979 we have treated 48 patients with the neodymium-YAG laser. Seven patients with excessive condyloma involvement were followed postoperatively. Our experience indicates that, in view of its low complication and recurrence rates, endoscopic laser coagulation of urethral condylomata is the method of choice. A short-term ureteroscopic follow-up examination, however, is required to detect any condylomata that might have been overlooked.

[Experimental induction of maligant tumors in the urinary bladder of the rabbit]. / Experimentelle Erzeugung von malignen Tumoren an der Kaninchen-Harnblase.

Suspended material of Brown-Pearce carcinoma is qualified for producing bladder tumors in rabbits after injection into the wall of the exposed, but not opened bladder. The advantages are the rapid growing and the possibility of controlling by endoscopy and radiography. At present we use this tumor-pattern for examination of the effect of different laser beams in experimental bladder tumors.

Endoscopic Neodymium-YAG laser application for destroying bladder tumors.

302 bladder tumors to a size of a thumb tip were treated with a Nd: YAG laser. 196 large bladder tumors were removed by transurethral resection combined with laser irradiation 1 week later. In all cases tumors were classified as pTA to pT3a, No, Mo tumors. 8 weeks after treatment we found local recurrences in 9% using the rigid laser endoscopes. On the contrary, the local recurrence was 2.1% using laser endoscope with modified Albarran insert. In proven cases the Nd:YAG laser application is an effective and careful procedure to destroy bladder tumors.

[Necrotising vasculitis in lympho-plasmocytoid immunocytoma (author's transl)]. / Nekrotisierende Vaskulitis bei lympho-plasmozytoidem Immunozytom.

A 42-year-old patient died of fulminant necrotising vasculitis with acute renal failure. During life neither paraproteins nor immune complexes or cryo-globulins could be demonstrated in the blood. At necropsy an intraabdominal lympho-plasmocytoid immunocytoma was found as underlying disease.

Experimental investigation of neodymium-YAG laser induced shock waves for lithotripsy.

Experiments on the endoscopic destruction of kidney stones by neodymium-YAG laser, either thermically or mechanically, are being carried out with various results. The thermal destruction is clinically not feasible, and the risk of damaging neighboring tissue is inevitable. The direct generation of shock waves with pulsed Q-switch systems seems to represent the only feasible endoscopic method subject to further development of suitable devices for clinical tests.