Antigen-presenting properties of human vascular endothelial cells.

Human endothelial cells were obtained by collagenase digestion of the umbilical vein wall, explanted into tissue culture, and grown as monolayers of cells. Endothelial cells extracted from these monolayers were specifically lysed by anti-HLA-DR alloantisera. Moreover, the stimulating capacity of these endothelial cells toward allogeneic peripheral blood mononuclear lymphocytes was specifically and significantly inhibited by the presence of relevant anti-HLA-DR antisera. Endothelial cells that expressed HLA-DR determinants were also capable of substituting for macrophages in the lymphoproliferative response of purified T cells to soluble protein antigens. Moreover, in concordance with the results previously reported in which macrophages were employed, the endothelial cell donor should share HLA-DR determinants with the T cell donor for an optimal response to occur.

Clonal distribution of HLA-restricted antigen-reactive T cells in man.

The [3H]thymidine suicide technique was used to test the hypothesis that the response of immune human T cells from HLA-D/DR heterozygous donors to the soluble protein antigen purified protein derivative (PPD) is clonally expressed and consists of the concurrent proliferation of at least two separable subpopulations of lymphocytes. The results showed that each of the two subpopulations react to one or the other of the HLA-D/DR antigens presented together with PPD by allogeneic monocytes. In addition, using in vitro priming techniques of in vivo sensitized lymphocytes from heterozygous donors, it was possible to generate specific memory cells capable of recognizing the priming soluble protein antigen together with the HLA-D/DR determinant present in the initial sensitizing culture.

Survival, prognostic factors, and therapeutic efficacy in low-grade glioma: a retrospective study in 379 patients.

PURPOSE: We report survival, prognostic factors, and treatment efficacy in low-grade glioma. PATIENTS AND METHODS: A total of 379 patients with histologic intracranial low-grade glioma received post-operative radiotherapy (n = 361) and intraarterial carmustine (BCNU) chemotherapy (n = 153). Overall survival and prognostic factors were evaluated with the SPSS statistical program (SPSS Inc, Chicago, IL). RESULTS: Median survival (all patients) was 100 months (95% confidence interval [CI], B7 to 113); in age group 0 to 19 years (n = 41), 226 months; in age group 20 to 49 years (n = 263), 106 months; in age group 50 to 59 years (n = 49), 76 months; and for older patients (n = 26), 39 months. Projected survival at 10 and 15 years was 42% and 29%, respectively. Patient age, World Health Organization (WHO) performance status, tumor computed tomography (CT) contrast enhancement, mental changes, or initial corticosteroid dependency were significant independent prognostic factors (p < .05), while histologic subgroup, focal deficits, presence of seizures, prediagnostic symptom duration, tumor category, and tumor stage were not. Patients aged 20 to 49 years with no independent negative prognostic factors (n = 132) had a median survival time of 139 months versus 41 months in patients with two or more factors (n = 33). Patients who presented with symptoms of expansion (n = 97) survived longer when resected (P < .03); otherwise no survival benefit was associated with initial tumor resection compared with biopsy. Intraarterial chemotherapy and radiation doses more than 55 Gy were not associated with prolonged survival. Among 66 reoperated patients, 45% progressed to high-grade histology within 25 months. CONCLUSION: Prognosis in low-grade glioma following postoperative radiotherapy seems largely determined by the inherent biology of the glioma and patient age at diagnosis.

Prevalence and prognostic significance of epilepsy in patients with gliomas.

The aim of this study was to evaluate the prevalence and prognostic significance of epilepsy in 1028 patients diagnosed in the computer tomography (CT) era with histological low- or high-grade intracranial gliomas. Survival analysis included Kaplan-Meier plots, log-rank tests, logistic regression and Cox's analysis as implemented in the SPSS statistical package. Epilepsy was a positive univariate (P < 0.0001) and multivariate, (P < 0.03) prognostic factor for survival in the total patient group (n = 1028, relative risk of death 0.83, 95% confidence interval (CI) 0.70-0.98) as well as in the high-grade patient group (n = 649, relative risk of death 0.80, 95% CI 0.66-0.96), but not in the group of low-grade glioma patients (P > 0.2). The prevalence of epilepsy in glioblastoma patients was 251/512 (49%), 95/137 (69%) in anaplastic gliomas, and 322/379 (85%) in patients with low-grade gliomas, with 97 of the 102 T1 low-grade subgroup (95%) having epilepsy, indicating that the presence of epilepsy may select patients for early radiological diagnosis. The frequency of epilepsy at presentation decreased with age in high-grade glioma patients, and increased with age in low-grade glioma patients to a plateau in the fourth decade of life (P < 0.01). The prevalence of epilepsy in patients with histological intracranial gliomas varied with patient age and tumour histology, with low-grade patients having the highest prevalence. Epilepsy was a significant positive prognostic factor except in patients with low-grade gliomas, and may select low-grade patients for early diagnosis.

Radioimmunoassay in microtiter plates. A convenient method for determining bound radiolabelled ligand.

A radioimmunoassay was performed in the wells of casein-coated microplates employing 125I-labelled sheep anti-human second antibody. The antigen-antibody complexes were thereafter dislodged from the well walls using the detergent sodium dodecyl sulfate (SDS) and the entire contents of the wells were simultaneously absorbed into 48 cellulose acetate-absorbing cartridges. All 48 cartridges were transferred to counting vials and the radioactivity determined by standard gamma counting techniques. The particular advantage of the method described here is the ease with which the supernatants can be collected and transferred to counting vials with minimal handling of radioactive samples.

Cell mediated lympholysis; a modified technique using 111indium-oxine-labelled targets.

The isotope 51Cr generally used in the cell mediated lympholysis (CML) assay suffers from the disadvantage of low specific activity, poor incorporation and high spontaneous release, limiting the CML assay to 4-6 h. We have labelled PHA derived human lymphoblasts with the isotope 111indium (using 111indium-oxine) and evaluated these cells as targets in CML. The level of 111In-oxine incorporation decreased rapidly in the presence of serum; in the absence of serum approximately 85% of the available isotope in the supernatant was incorporated into the blasts. Under the labelling conditions used, spontaneous release was 1.6-2%/h on average allowing an effector phase of 18 h. About 5-8% of the released isotope was reutilized by the effector cells during an 18 h incubation period. Extending the CML assay from 6 to 18 h greatly increased the cytotoxicity. At an effector to target ratio of 25:1, the average per cent specific release increased from 15 to 50%. The use of 111In-oxine labelled targets in the CML therefore increases the sensitivity of the test and allows fewer effector and target cells to be used as compared with 51Cr techniques.

Cell mediated lympholysis: CML. a microplate technique requiring few target cells and employing a new method of supernatant collection.

A micro method for the 51Cr release assay is described. Allogeneically induced cytotoxic lymphocytes are generated in micro mixed lymphocyte cultures in the wells of micro plates. Their cytotoxic capacity is assayed by adding 51Cr labelled PHA derived lymphoblasts directly into the micro cultures with no pooling or transfer of the cytotoxic effector cells being required. The 51Cr isotope released into the cell supernatants is collected by inserting a cellulose acetate absorption cartridge into each well. A glass fiber filter attached to the cartridge effectively separates the supernatant from the cellular elements. This system allows the simultaneous collection of the supernatant from 96 wells, and can be used with either adherent or non-adherent target cells.

In vitro immunosuppressive effects of cytotoxic agents conjugated to antihuman lymphocyte globulin.

Antihuman lymphocyte globulin (ALG) was either coupled to the lymphocytoxic drug chlorambucil or covalently bound to the cytotoxic alkalating agent melphalan via a polyglutamic acid carrier. Both types of complexes strongly inhibited the proliferative response in human mixed lymphocyte cultures and the ability of mixed lymphocyte culture-activated T effector cells to lyse 51Cr-labelled lymphoblast target cells, and were more potent than ALG or drug alone. These experiments indicate that it is possible to bind cytotoxic agents to ALG without destroying either the properties of the drug or the ability of the antibody to bind to lymphoid cells.

Specific destruction of human endothelial cell monolayers by anti-DRw antisera.

Human endothelial cells were separated by collagenase digestion from the umbilical vein of newborns. The cytotoxic effect of antisera against HLA-DRw antigens was tested on monolayers of endothelial cells, using a 51Cr retention microcytotoxicity assay in the presence of rabbit complement. Of the endothelial monolayers tested, significant concordance between the typing results obtained using cord blood-derived B lymphocytes and endothelial cell monolayers was observed. A rabbit anti-human polyspecific B cell antiserum was also capable of completely lysing the endothelial monolayers tested. In addition, 51Cr-labelled dissociated endothelial cells were lysed by nonimmune peripheral blood mononuclear cells in the presence of DRw antisera having a specificity for the target endothelium.

Cell-mediated cytotoxicity toward the donor in patients with well-functioning kidney grafts. Possible mechanism of specifically reduced cytotoxic response.

Patients with well-functioning kidney allografts from one-HLA-haplotype--mismatched related donors have strongly reduced donor-specific cell-mediated cytotoxicity 2-10 years after having received their grafts. This could also be demonstrated in secondary in vitro cell-mediated lympholysis (CML). Even though secondary donor-specific CML did not exceed 10% at an effector-to-target cell ratio of 50:1, specific lysis of donor target cells increased significantly with increasing effector cell concentrations. The weak cytotoxicity toward the donor could be enhanced neither through pool-cell stimulation nor by adding exogenous Interleukin 2 (IL-2) during the induction phase. Furthermore, growth factors were produced in the cultures during the induction of cytotoxic cells in concentrations comparable to those of the sibling control. Thus no evidence could be obtained that lack of IL-2 was causing the decreased cytotoxicity in these patients. Our studies indicate that in vivo depletion of cytotoxic cells with high lytic efficiency is probably the reason for the strongly reduced donor-specific cytotoxicity.

Effects of methylprednisolone on the in vitro generation of human secondary cytotoxic lymphocytes.

The effects of methylprednisolone on the induction of secondary proliferative and cytotoxic lymphocytes in human mixed lymphocyte cultures (MLCs) have been studied. Concentrations of methylprednisolone (MP) as low as 0.01 microgram/ml proved highly effective in inhibiting the generation of cytotoxic memory cells if the steroid was present during the first 5 days of the priming mixed lymphocyte culture. The generation of cytotoxic lymphocytes was also inhibited if the steroid was added along with the restimulating cells on day 10, although the degree of inhibition was not as great as that seen when steroids were added to the cultures at their initiation. Cultures containing 1 microgram of MP per ml during the priming phase required 10 times as many effector cells to achieve a comparable level of cytotoxicity compared to control cultures without steroids. Our results indicate that an important aspect of the immunosuppressive role of steroids is the prevention of the generation of specific memory cells following exposure to alloantigens.

Donor-specific decreased cell-mediated cytotoxicity in recipients of well functioning, one HLA haplotype-mismatched kidney allografts.

Patients with well functioning kidney grafts from one HLA haplotype-mismatched related donors were studied 2 to 10 years after transplantation. No direct cell-mediated lympholysis (CML) toward donor cells was found. After in vitro sensitization in mixed lymphocyte culture, the recipient effector cells were not able to significantly kill donor target cells at an effector to target cell ratio of 50:1, while the HLA-A,B,D/DR-identical sibling of the recipient could generate strong cytotoxicity toward the donor at similar effector to target cell ratios. Nevertheless, the patient developed strong cytotoxicity toward third-party targets. Our findings indicate in vivo depletion of cytotoxic cells with specificity for donor antigens. No correlation between the mixed lymphocyte culture (MLC) response and the ability to generate cytotoxic cells could be found in recipient-donor combinations. In looking for in vivo generated suppressor cells toward the donor, a moderate suppression was found in three of six patients studied.

The human mixed lymphocyte-endothelium culture interaction.

Cells separated from the wall of the umbilical cord vein by collagenase digestion could be identified as endothelial by their characteristic ultrastructure, their growth pattern in culture, and their microscopical morphology. These cells, both freshly explanted and after long-term culturing, were capable of stimulating allogeneic lymphocytes in vitro. Control experiments indicated that this stimulation was not attributable to contamination of the endothelial cell suspensions by foetal fibroblasts or passenger lymphocytes. The dose response characteristics and kinetics of the lymphoproliferative response using endothelial stimulating cells was similar to mixed lymphocyte cultures. Sera which were capable of inhibiting the mixed lymphocyte culture response were relatively ineffective in inhibiting the stimulation caused by endothelial cells.

Presentation of viral antigens by human vascular endothelial cells in vitro.

Endothelial cells (EC) separated from the umbilical vein were shown to be free of contaminating monocytes. EC could replace peripheral blood-derived macrophages as antigen-presenting cells for in vivo sensitized T cells towards a variety of viral antigens. The T-cell--CE-antigen response was also specificity inhibited by anti-HLA-DR antisera. T cells primed by antigen together with autologous macrophages could be restimulated by antigen pulsed HLA-D/DR identical EC in an antigen specific secondary response, indicating a similar mechanism for antigen presentation by EC or macrophages.

Combined intra-arterial and systemic chemotherapy for recurrent malignant brain tumors.

Seventy-nine patients harboring recurrent brain tumors received four cycles of infraophthalmic carotid injections of 160 mg of carmustine. Two milligrams of intravenous vincristine and 50 mg of oral procarbazine was also administered three times daily for 1 week in conjunction with each BCNU treatment. The response rate was 60% with a median survival for patients with astrocytomas, anaplastic astrocytomas, and glioblastomas of 32, 20, and 6.5 months, respectively. The median survival of the responding patients was 20 months, and the survival at 30 months was 45%. The survival in patients not responding to treatment was 5 months, reflecting the natural history of the tumor. There have been no deaths related to the treatment procedure. No incidents of severe or permanent eye complications or leukoencephalopathy were observed. Based on multivariate survival analysis, only patients with a good performance status who are not steroid dependent are candidates for this treatment.

Photodynamic therapy of human glioma spheroids using 5-aminolevulinic acid.

The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (< or = 50 J cm-2), lower fluence rates are more effective. At a fluence of 50 J cm-2, near-total spheroid kill is observed at fluence rates of as low as 10 mW cm-2. The fluence rate effect is not as pronounced at higher fluences (> 50 J cm-2), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.

Localization of brain tumors with a simple scalp-mounted fiducial device. Technical note.

A method is described for marking the site of a tumor on the scalp based on information from computerized tomography (CT) scans. The technique employs a syrinx-shaped array of radiopaque catheters of varying length taped to the patient's scalp for visualization on the CT scan. Fiducial markings on the CT images allow the transfer of the tumor's location directly onto the scalp. The device can be placed anywhere on the scalp, including in a parasagittal position.

C-reactive protein levels in the differential diagnosis of brain abscesses.

C-reactive protein (CRP) is a protein found in plasma at elevated concentrations during acute or chronic infections. As an aid in the differential diagnosis between brain tumor and abscess, the CRP levels were measured in 20 patients with intracranial mass lesions and the appearance of ring-like contrast enhancement on computerized tomography (CT) scans. In nine of these patients, the final diagnosis was abscess, based on either biopsy of the mass (eight patients) or the clinical course (one patient). In seven of the nine patients, there was a significant increase in CRP levels in two consecutive measurements. In particular, patients with cerebritis who were examined early in the course of the disease and who showed nonspecific CT scans exhibited extremely high levels of CRP. Two patients had no measurable CRP activity although they both had brain abscesses. In 12 patients harboring either gliomas or metastatic intracerebral tumors, CRP levels were significantly lower than those found in patients with brain abscesses but were nevertheless higher compared to those of a group of patients with benign tumors. It is concluded, therefore, that the measurement of CRP can have some value in the differential diagnosis between brain abscess and brain tumor. The measurement technique is inexpensive and is available in the clinical laboratories of most hospitals with a neurosurgical department.

Comparison of in vitro glioma cell cytotoxicity of LAK cells from glioma patients and healthy subjects.

Peripheral blood mononuclear cells from 11 glioma patients and 11 healthy control subjects were cultured in medium containing recombinant interleukin-2 for a period of 5 days. The cytotoxicity of these lymphokine-activated killer (LAK) cells was tested on chromium-51-labeled freshly prepared allogeneic glioblastoma cells, and on the cell lines K562 (natural killer cell (NK)-sensitive) and Daudi (NK-resistant). Peripheral blood mononuclear cells from all subjects showed high levels of cytotoxicity against these targets. There was no significant difference between the patients and the control group when LAK cytotoxicity was compared. Thus, although glioma patients are known to have depressed immunological reactivity, the cytotoxic capacity of LAK cells derived from glioma patients is similar to that of LAK cells from healthy control subjects. However, the glioma patients had significantly reduced numbers of mononuclear cells in their peripheral blood, possibly due to steroid treatment. Therefore, the volume of blood required to generate the same number of LAK cells was approximately three times larger from the glioma patients than from control subjects.

Immunomagnetic separation of infiltrating T lymphocytes from brain tumors.

Tumor-infiltrating lymphocytes (TIL's) were isolated from human glioma biopsy specimens by immunomagnetic separation using T cell-specific monoclonal antibodies coupled to paramagnetic beads, and were expanded in culture with feeder cells and interleukin-2 (IL-2). The infiltrating cells from five of seven patients proliferated in culture. When tested after 2 to 3 weeks of culture, virtually all of the cells stained with antibodies against the CD2 and CD3 antigens. Most cells also expressed human leukocyte antigen class II molecules, while varying percentages of cells stained with antibodies against the IL-2 receptor and the CD4 and CD8 antigens. The cytotoxicity of the cultured TIL's against autologous and allogeneic glioma cells and the K562 and Daudi cell lines was measured and compared with that of lymphokine-activated killer (LAK) cells from the same patients. None of the TIL's showed significant cytotoxicity against these targets, whereas LAK cells lysed all of the targets.