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A model system for testing the efficacy of chemotherapy protocols for metastatic melanoma was established using cell cultures from two brain and three lymph node metastases of melanoma from five different patients. Continuously growing cultures which were positive for tyrosinase activity were analysed regarding their proliferation rate by continuous bromodeoxyuridine (BrdU) labelling and subsequent Hoechst-33258/ethidium bromide flow cytometry. Melanoma cell cultures exhibit a strong sensitivity to BrdU: at 5% oxygen, 50% growth inhibition is attained with 360 +/- 130 microM BrdU (range: 130-520; n = 11) vs 650 +/- 50 microM BrdU (n = 3) for diploid human fibroblasts and 570 +/- 20 microM BrdU (n = 6) for human lymphoid cell lines. Moreover, BrdU sensitivity of melanoma cells is clearly oxygen dependent: 50% growth inhibition at 200 +/- 55 microM (range: 65-400 microM) for 20% oxygen vs 360 +/- 130 microM BrdU for 5% oxygen. The cell cycle kinetic mechanism of BrdU-induced growth inhibition is accumulation of cells in the first cycle G2 phase. On the basis of these results we suggest testing BrdU in chemotherapy protocols for the treatment of metastatic melanoma.

Transcranial color-coded real-time sonography (TCCS) was performed in 57 patients with primary intracranial brain tumors (n = 49) or arteriovenous malformations (n = 8) to evaluate its diagnostic potential. In 46 patients (81%), lesions could be identified employing this technique. In 7 patients, transcranial ultrasound examination was not feasible because of bone thickness; in the remaining 4 patients, the tumor was indistinguishable from adjacent brain tissue despite sufficient insonation, suggesting that these neoplasms are isoechogenic. The sonographic features of brain tumors were very similar: a hyperechogenic matrix of the lesion was interspersed by hypoechogenic pixels. Larger hypoechogenic areas (0.5-1 cm) gave evidence of tumor necrosis. Differences between the findings of TCCS and computed tomography concerning tumor size were found in 7 patients, in whom TCCS revealed an area of smaller extension within the corresponding hypodense area on the computed tomographic scan. Perifocal brain edema could not be detected by ultrasound examination. In 13 patients, a thin, hypoechogenic peritumoral halo was disclosed that did not correlate with perifocal brain edema identified by computed tomography and that may have been due to compression of adjacent parenchyma. In patients with arteriovenous malformations, TCCS permitted the identification of the main feeders, the nidus, and the draining venous system by color-coded depiction of intravascular blood flow. In conclusion, TCCS is an additional method for initial diagnosis and highly suitable for follow-up in tumor patients and provides valuable information about tissue characteristics and blood flow.

Single bolus doses of glycosylated human interleukin-2 (n IL-2) in the range of 2.8 x 10(3) to 2.0 x 10(6) IU/kg were administered to anesthesized cats via the cephalic vein (n = 10) or using suboccipital puncture (n = 8). CSF (cerebrospinal fluid) and blood samples were collected by repeated puncture. The n IL-2 concentration in four cats was determined on the basis of its biologic activity using 3H-thymidine incorporation into human ConA-blasts and by radioimmunoassay. In additional experiments radioactivity was determined in cerebrospinal fluid and serum after intravenous and intrathecal (i.th.) application of 5.8 x 10(3) - 3.2 x 10(3) IU/kg of 14C-acetyl-n IL-2 in regular time intervals. CSF and serum concentration time-profiles show a biexponential decline in the plasma elimination phase with half-lives of 4 min (alpha-phase) and 90 min (beta-phase) after intravenous and 20-120 min (alpha-phase) and 2-16 hours (beta-phase) after intrathecal application. There is a trend towards longer terminal elimination half-lives with increasing doses. Interleukin-2 is able to penetrate the blood brain barrier from the circulation into the cerebrospinal fluid and vice versa. Due to a slow rate of penetration and rapid elimination from blood only traces of n IL-2 (2-8 IU/ml) are detected in CSF after i.v. injection of 2 x 10(6) IU/kg, whereas concentrations between 400 and 1600 IU/ml are maintained in CSF for several hours following i.th. administration of 2-10 x 10(5) IU/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined three groups of adult patients to evaluate the clinical application of transcranial colour-coded real-time sonography (TCCS): 28 patients suffering from intracerebral haemorrhage, 36 patients with subarachnoid haemorrhage and 38 patients with primary brain tumours. All sonographic examinations were performed through the intact skull. Cerebral haemorrhages were visualised via TCCS as hyperechogenic areas, which were closely correlated in size and location to CT findings. Apart from the imaging of subarachnoid haemorrhages and cerebral vasospasms, TCCS permitted the identification of the angiographically confirmed aneurysms in 76%. Brain tumours were delineated in the vast majority of the patients examined. In this type of pathology TCCS provided additional information on tumour extension and macroscopic appearance.

Presented are real-time ultrasound findings in partially and completely denervated muscles of 30 patients with focal neuropathy and various other disorders of the second motor neuron. Sonographic scans of affected muscles are analyzed in conjunction with unaffected muscles of the same individual, under identical examination conditions. Initial pathological ultrasound changes could be detected as soon as 2 weeks after an acute neurogenic lesion. In denervation, the echodensity of the muscle was high and the normal intramuscular pattern was decomposed. Findings were more intense in severe and longstanding denervation. Ultrasound-indicated pathology correlated highly (chi-square: P less than 0.001) with pathological spontaneous activity detected by electromyography. Focal and systemic neuropathies showed no differences in ultrasound pathology. Six cases with central motor palsy had normal sonograms. Poor spatial resolution of real-time ultrasonography--as compared with CT and MRI--is compensated by its bedside availability, frequent repeatability without patient risk and discomfort, and its in vivo correlation of muscle morphology with muscle function.

Thirty-six patients with acute spontaneous subarachnoid hemorrhage (26 caused by rupture of an aneurysm) were examined by transcranial color-coded real-time sonography by using a 2.25-MHz ultrasound transducer. In 20 of these 26 patients (76%), the aneurysm could be identified by a characteristic abnormal blood flow pattern within the aneurysm in coronal and axial scanning planes by transcranial color-coded real-time sonography. Blood within the basal cisterns, on top of the tentorium, and within the ventricles and parenchyma was sonographically detected by increased echodensity in 75%. In addition, cerebrospinal fluid circulation disturbances and cerebral vasospasm were detected in two-dimensional B-mode images in 85% and 100%, respectively. In Doppler mode, intravascular blood flow velocity could be quantified. We conclude that transcranial color-coded real-time sonography, a new, noninvasive method for diagnosis and follow-up of patients with subarachnoid hemorrhage, allows detection of the primary vascular lesion and monitoring of complications.

Transcranial colour-coded real time sonography (TCCS) is a new, non-invasive diagnostic bedside procedure allowing two-dimensional imaging of cerebral parenchymatous structures and large intracranial arteries through the intact skull. So far we examined a population of 183 patients und 53 normal subjects, all above 18 years of age. 24 (10%) subjects were not suitable for examination. Two-dimensional imaging of telencephalic, diencephalic, mesencephalic and pontine structures is feasible. Intravascular blood flow phenomena may be analysed quantitatively by an integrated pulsed wave Doppler system. The diagnosis of arterial stenosis and occlusion could be established in 14 patients each by analysing the colour-coded B-mode image and Doppler frequency spectrum. TCCS may complement diagnostic tools in neurology and may improve standardisation of transcranial Doppler measurements.

Combination chemotherapy is widely employed in clinical oncology; however, there is no generally accepted model to evaluate individual tumour susceptibility to a given drug combination protocol. We therefore investigated the drug interaction of ifosfamide (4-hydroxyperoxy-ifosfamide) and ACNU in a recently developed in vitro model of paired sequential combination chemotherapy in primary and metastatic malignant brain tumours. A long-term standard [6,3-3H]-thymidine-incorporation assay, employing a liquid scintillation counting protocol, was selected to assess the drug sensitivity of human tumours. In vitro drug exposures were derived from correlating in vivo-(systemic and CNS) and in vitro-pharmacokinetic drug parameters. In combination experiments tumour cells were treated sequentially by two drugs in both sequences: drug exposures were calculated for 2 h with a 1-h drug-free interval in between. "Cut-off" concentrations (maximum in vitro exposure doses) were calculated as 1.74 microM (for primary CNS tumours: 0.58 microM) for ifosfamide and 5.4 microM (for primary CNS tumours: 1.33 microM) for ACNU. Dose/response relations were derived from isotope incorporation rates after cells had grown for approximately five population doubling times. Combination isoboles were plotted after drug doses had been transformed into "equieffective doses", enabling comparison of drug combination effects. In all three glioblastomas (with CNS exposure dose) an additive or supra-additive effect could be observed in either sequence (in one tumour a biphasic additive isobole was found for both sequences). Out of three bronchial carcinomas (small-cell type, brain metastases) in two non-identical sequences a supra-additive effect was observed in two tumours, with antagonistic effects in the third tumour. In all three malignant melanomas and in one renal carcinoma antagonistic effects were observed, whereas in a second renal carcinoma supra-additive effects were demonstrated for both sequences. We conclude that drug combination chemotherapy effects at the cellular level may be extremely heterogeneous.

We investigated the diagnostic potential of transcranial color-coded real-time sonography in 52 individuals using a phased-array ultrasound system with color-coded blood flow representation. Ultrasound scans in the axial and coronal planes were feasible through temporal acoustic bone windows in 49 subjects, enabling depiction of the main parenchymal and vascular structures as well as the ventricular system. Color-coded representation of blood flow in the cerebral vessels allowed unequivocal identification of the circle of Willis within the anatomic black-and-white B-mode image of the parenchymal structures. In Doppler mode, vascular blood flow phenomena may be analyzed semiquantitatively using the Doppler frequency spectrum. This noninvasive, serially applicable, mobile beside method may complement conventional neuroradiologic imaging methods, allowing on-line studies of functional processes within the adult brain.

Autocrine-secreted melanoma tumor growth-inhibiting activity (MIA, approximately Mr 8000) was isolated from supernatants of a malignant melanoma cell line HTZ-19 dM, established from a central nervous system-melanoma metastasis. Cell cycle kinetic analysis performed with bromodeoxyuridine/Hoechst flow cytometry revealed a MIA-sensitive period at the G0/G1 to S traverse; MIA mediated prolongation of the S-phase and increased arrest of cells in the G2 compartment. Growth inhibition by MIA is cell-density dependent; maximal effect is seen at low densities, and the effect may be partially antagonized by whole serum. MIA may cause growth stimulation at high cell densities and low MIA concentrations. The effect of MIA on different histological neuroectodermal cell types was compared by the same methodology: proliferation of a second malignant melanoma was inhibited, and no effect was observed with an ependymoma; 2 glioblastomas were slightly stimulated. Effects on human fibroblast-like cell strains were inconsistent. The mechanism of MIA is discussed in relation to other endogenous autocrine growth inhibitors.

In standard concentric needle electromyography the typical triphasic potential of a motor unit (MUP) consists of the spike component and the positive initial and terminal parts. Whereas the main spike is generated by a few muscle fibers located close to the tip of an electrode, the initial and terminal parts represent the sum of activities of a large proportion of fibers within the motor unit. The size of the initial and terminal part is assessed by measuring the duration of the MUP. In addition to the duration we calculated the mean absolute voltage outside the spike component: The signal was digitized and several individual MUPs of the same motor unit were averaged. The point of maximal negative rise was identified. Then the mean absolute voltage between -5 ms and +5 ms around this point, excluding the interval from -1 ms to +1 ms, was calculated. This parameter is exactly defined and unambiguous. The biceps brachii muscles of 30 healthy volunteers were examined in order to establish normal values. In 24 patients suffering from various myopathies 18 had significant (p less than 0.01) reduced initial and terminal parts, whereas only 13 patients showed decreased potential duration (according to Buchthal 1957).

Transcranial color-coded real-time sonography (TCCS) is a new diagnostic method allowing the non-invasive imaging of parenchymal and vascular structures of the adult brain through the intact skull. A description of the sonographic findings in two patients presenting with a radiologically ascertained arterio-venous malformation is provided. The AVM's can be depicted in the two-dimensional B-mode image as echodense areas interspersed with zones of lower echodensity. Their extension correlated with corresponding MRI images. The color-coded illustration of intravascular flow phenomena allowed the distinct identification of the major afferent feeding vessels, the venous drainage and of the vascular convolution of both AVM's. Information on hemodynamics, as e.g. the blood supply of the angioma by the contralateral internal carotid artery, are obtained by color-coded identification of intravascular flow direction including analysis of the Doppler frequency spectrum. All findings obtained by TCCS complied with those established by angiography. We conclude, that TCCS is a suitable method for early diagnosis and long term follow up of cerebral AVM's.

In 3 patients with low-grade astrocytomas clinical pharmacology of interferon-beta (10(7) U/mg protein) was investigated. Interferon-beta with escalating dosage (2.3, 6.9, 23, 69 X 10(6) U/patient) was given to each patient in 4 infusions at weekly time intervals. In these patients dose-dependent plasma-levels of interferon-beta of up to 5800 IU/ml were achieved. Plasma concentrations showed a biphasic decline (T1 1/2:0.095-0.49 hrs and T2 1/2: 5-14.5 hrs). Side effects were: mild fatigue, myalgia, tachycardia, hypertension, and fever; the latter was well controlled by pretreatment application of paracetamol. Hematological changes included lymphopenia (2-6 hrs after infusion) and granulocytosis (3-6 hrs after infusion). Natural Killer cell activity was also monitored: 6 hours after infusion a drop of activity - not clearly dose dependent - was observed to a minimum of 1% pretreatment activity; 24 hrs after infusion activity increased up to a maximum of 400%. In this phase I study high biological activity of interferon-beta could be detected in plasma of astrocytoma patients - clinical tolerance was good and only mild toxicity was observed.

BCNU (carmustine), 5-Fluorouracil (5-FU) and Vidarabin-monophosphate (ARA-A5'P) were compared in their activities against 30 cell lines of primary (n = 21) and metastatic (n = 9) brain tumors, which were characterized in tissue culture by cytochemical, immunological and cytogenetic criteria. In vivo achievable concentration-time products were correlated with in vitro pharmacokinetic data. A micro assay was employed to screen for drug toxicity in individual tumor cell lines; cells were exposed to the drugs at exposure doses relevant to in vivo pharmacokinetics. After 5-8 population doubling times of untreated controls, RNA-synthesis, as a parameter of cell metabolism and proliferation, was determined by incorporation of (5, 6-3H)-uridine into cellular RNA (liquid scintillation counting protocol). A tumor stem cell assay was performed under similar conditions. The cytotoxic effect of each drug on individual cell lines was expressed in terms of a sensitivity index SI (SI = 1 indicating complete resistance) to compare effects of different drugs on the individual tumor cell lines. Mean sensitivity indices for ARA-A5'P, BCNU and 5-FU in brain tumor cell lines (in brackets: primary CNS-tumors) were 0.64 (0.59), 0.89 (0.82) and 0.35 (0.33) respectively. 5-FU was significantly more active than BCNU and ARA-A5'P (P less than 0.001), whereas BCNU was significantly less active than ARA-A5'P (P less than 0.001). ARA-A5'P had a suppressive effect on formation of brain tumor stem cell colonies. There was no cross-resistance of ARA-A5'P to either BCNU or 5-FU. We conclude that ARA-A5'P and 5-FU are potent agents in experimental therapy of human brain tumors, compared with BCNU.

In 3 patients with Lyme radiculomyelitis, cellular immune reactions of cerebrospinal fluid (CSF) lymphocytes were analyzed. Phenotypic analysis of CSF cells demonstrated that the majority were T cells (CD3+) of the helper/inducer subset (CD4+). These T cells were directly expanded from the CSF by limiting dilution. A total of 505 T-cell lines were tested for Borrelia burgdorferi (Bb)-specific proliferation and also partly tested for reactivity to a panel of central and peripheral nervous system antigens. Proliferative assays revealed 33 of them to be Bb specific, 16 to be specific for myelin basic protein, 16 to be specific for peripheral myelin, 1 to be specific for cardiolipin, and 2 to be specific for galactocerebrosides. The antigen-specific proliferation was restricted by autologous human leukocyte antigen (HLA) class II molecules. The majority of CSF-derived T-cell lines stained positively for CD3, CD4, and HLA class II antigens and negatively for CD8 (cytotoxic/suppressor subset). One T-cell line provided help for the production of specific IgG by autologous B cells and secreted gamma-interferon upon stimulation with Bb antigen in the presence of autologous antigen-presenting cells. These data show that in patients with severe neurological manifestations of late Lyme disease, not only Bb-specific T-cell lines but also T cells reactive to central or peripheral nervous system autoantigens can be found.

Analysis of the local cell-mediated immunity within the central nervous system is limited by the small amount of material available. In the present report we describe that T lymphocyte clones can be expanded directly from the cerebrospinal fluid (CSF) using T cell growth factor and irradiated feeder cells. Without in vitro restimulation such clones can reproducibly be generated from fresh or cryopreserved CSF lymphocytes. From a patient with tuberculous meningitis, T lymphocyte clones were obtained that showed tuberculin (PPD) specific proliferative responses restricted by a single HLA-DR antigen. One of these clones was restricted by an antigen different from the serologically defined HLA-DR since this clone recognized PPD only on autologous but not on HLA-DR matched monocytes. These experiments show that T lymphocytes involved in the in situ immune response can be cloned directly from the site of inflammation.

Tumor cell proliferation and morphological changes in tumor cells under the influence of different cytostatic agents were measured in an in vitro assay to determine chemosensitivity of human malignant brain tumors. Aliquots of 2 to 5 x 10(4) cells of a tumor cell suspension (prepared from biopsy specimen by mechanical and enzymatic disintegration) were incubated for 72 hr in the presence of different cytostatic agents. After another nine days of incubation in fresh medium, cell proliferation was calculated by incorporation of 14C-leucine and 3H-uridine and measurement in a liquid scintillation counter. Morphological changes in tumor cells were evaluated in Labtek tissue culture slides cultured under identical conditions. All drugs were tested in pharmacologically achievable concentrations. In vitro BCNU-sensitivity of 7/17 patients correlated with a postoperative recurrence free interval of 15.1 months, in vitro BCNU-resistance of 10/17 patients correlated with a postoperative recurrence free interval of 6.38 months (p less than 0.001). Tumors of lower grades of malignancy (astrocytoma III, malignant ependymoma, medulloblastoma) in our series have a statistically significant higher median BCNU-sensitivity (63.5%, p less than 0.05) and are sensitive to more drugs (3.6 drugs, median out of six selected drugs, p less than 0.01) than tumors of higher grades of malignancy (astrocytoma IV, glioblastoma multiforme; 29.54% BCNU-sensitivity, 1.4 effective drugs). In our series differences of BCNU-tumor sensitivity and number of effective drugs were not statistically significant if related to age and sex of tumor bearing patients. We think this assay provides the opportunity to test a large number of drugs in a very short period of time. Results may indicate alternative drug regimen for those patients resistant to conventional chemotherapy.

This paper reports on 10 patients (4 male, 6 female) with primary non-Hodgkin's lymphomas of the brain (CNS-NHL--mean age 46.8 years, mean postdiagnostic survival 10 months). Pathological CSF (cerebrospinal fluid) was found in all 8 patients examined (positive cytology in 7/8 cases). Solitary tumors, diffuse periventricular infiltration or diffuse cerebral infiltration were demonstrated in cerebral computer-assisted tomography (CAT). Angiographical findings were unspecific. The histologic subtypes were lymphoplasmacytoid immunocytoma (4), unclassified low grade (1), centroblastic (1), B-immunoblastic (1), T-immunoblastic (1), lymphoblastic convoluted T-cell type (1), unclassified high grade (1) NHL. Patients who had received radiotherapy (+/- surgery) in this group had a mean survival of 15.66 months (sigma = 7.63). In addition, an overview of 83 well-documented, cases of the literature tries to characterize main histological and topographical distributions, histology-, patient's age-, and therapy-related survival. Patients with primary CNS-NHL have a 5-year survival expectancy of 30% compared with 2.3% in secondary CNS-manifestations of systemic non-Hodgkin's lymphomas. In this report, the beneficial effect of radiotherapy (mean survival 30.3 months) compared to surgery or symptomatic treatment (3.6 or 3.3 months) could be confirmed. It is concluded that primary CNS-NHL frequently present with atypical neuropsychiatric syndromes; diagnosis should be established preferentially with CAT and CSF-examinations or stereotactic biopsies, whereas open surgery should be avoided. An approach to exact classification should be attempted, as survival is clearly related to histological subtypes.

Autocrine-secreted tumor cell growth-inhibiting activities were isolated from supernatants of a malignant melanoma cell line, HTZ 19-dM, established from a central nervous system melanoma metastasis. HTZ 19-dM was characterized by cyto- and immunocytochemistry and karyotyping; cells were propagated in defined serum-free tissue culture medium for up to 8 months. Supernatants were ultrafiltrated, dialyzed, lyophilized, and purified by Bio-Gel P-10 gel permeation chromatography, leading to three active fraction pools, MIAI [melanoma-inhibiting activity (MIA), 2 kDa), MIAII (Mr 11,500-17,000) and MIAIII (proteins at the cutoff of Bio-Gel P-10) inhibiting growth of 19-dM cells with 50% inhibitory concentrations of 0.79 microgram/ml (MIAI), 0.13 microgram/ml (MIAII), and 16.7 micrograms/ml (MIAII). MIAII could be further purified by reverse phase high pressure liquid chromatography; the main activity displayed a 50% inhibitory concentration of 0.33 microgram/ml. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis one major band (molecular weight about 14,000) and two minor bands (up to Mr 17,000) were identified. Macromolecular synthesis was inhibited in 19-dM cells up to greater than 99.5%; tumor stem cell colony formation was reduced by 99.89%; the inhibitory effect of MIAII was irreversible, nonsaturable, and partially antagonized by a serum factor (depending on purification stage). MIAII was heat stable (3 min at 100 degrees C) and trypsin labile. The effect of MIAII on allogeneic neuroectodermal tumors was also investigated; proliferation of two of three malignant melanomas and two of four glioblastomas was inhibited up to 85.2%; proliferation of a neuroblastoma cell line could be inhibited to 33.8%, whereas normal fibroblasts and low grade gliomas were not influenced in their proliferation.

BCNU (carmustine), VM26 (teniposide) and ARA-A5'P (vidarabin-monophosphate) were compared in their activity against 30 cell lines of primary (N = 21) and metastatic (N = 9) human brain tumours, which were characterized in tissue culture by cytochemical, immunological and cytogenetic criteria. In vivo achievable concentration-time products c X t were correlated with in vitro pharmacokinetic data in order to evaluate in vitro drug sensitivity at relevant exposure doses. A microcytotoxicity assay was employed to screen for drug toxicity in individual tumour cell lines. Following drug exposure and 5 to 8 population doubling times of untreated controls, RNA-synthesis - as a parameter of cell metabolism and proliferation - was determined by incorporation of [5,6-3H]-uridine into cellular RNA (liquid scintillation counting protocol). The cytotoxic effect of each drug on individual cell lines was expressed in terms of a sensitivity index (SI); by these means effects of different drugs on individual tumour cell lines could be compared. Mean sensitivity indices of ARA-A5'P, BCNU and VM26 for primary brain tumour cell lines were 0.59, 0.82 and 0.54. ARA-A5'P and VM26 had almost similar activities against brain tumour cell lines, whereas BCNU was significantly (P less than 0.001) less active. High grade gliomas were less sensitive to all three agents than low grade and infratentorial gliomas. ARA-A5'P was also able to effectively reduce colony formation in brain tumour cell lines. A cross-resistance of ARA-A5'P to either BCNU or VM26 could not be observed. Clearly, ARA-A5'P is an effective drug in treatment of brain tumour cells in vitro.