Effects of systemic recombinant interleukin-2 on natural killer and lymphokine activated killer activity of human tumor infiltrating lymphocytes.

The purpose of these studies was to compare local and systemic human lymphokine activated killer (LAK) and natural killer (NK) cytotoxicity and to determine its modulation by the systemic administration of recombinant interleukin-2 (rIL-2). After preoperative systemic rIL-2, we extracted tumor infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) from patients with pulmonary tumors and compared pre- and posttreatment spontaneous NK activity and their response to in vitro rIL-2. Spontaneous TIL NK activity was increased in patients receiving 15,000 units/kg rIL-2 preoperatively [6.6 lytic units (LU)] compared to those receiving 1,000-10,000 units/kg (0.8 LU) or no rIL-2 (1.4 LU). After 3 days incubation with 1,000 units/ml rIL-2, TIL NK cytotoxic activity was increased in patients receiving 15,000 units/kg rIL-2 (65.4 LU) compared to those receiving 1,000-10,000 units/kg (6.0 LU) or no treatment (24.9 LU). Spontaneous TIL LAK activity was low overall (1.1 LU) with the exception of two patients receiving 15,000 units/kg who had 3.1 and 3.7 LU spontaneously. TIL LAK precursor activity was only slightly increased in patients receiving 1,000-10,000 units/kg rIL-2, whereas those receiving 15,000 units/kg rIL-2 had an average of 22.8 LU. Systemic rIL-2 also increased spontaneous PBL NK activity. Reincubation of PBL obtained at time of surgery or 3 days after discontinuing systemic rIL-2 resulted in significant increases in cytotoxic response to in vitro rIL-2 compared to pre-IL-2 in vitro responses. Systemic rIL-2 had no effect on spontaneous PBL LAK activity. Thus, the immunosuppressive tumor environment can be partially reversed with 15,000 units/kg systemic rIL-2. Higher doses of systemic rIL-2 also increased spontaneous PBL NK activity at time of surgery and 3 days after discontinuing rIL-2. Both TIL and PBL inducible cytotoxicity were boosted in vitro following higher doses of systemic rIL-2.

Lymphoid cell subpopulations infiltrating into autologous rat tumors undergoing rejection.

Lymphoid cell subpopulations infiltrating into autografts of methylcholanthrene-induced sarcomas in rats immunized with autologous tumor cells were identified in terms of immunohistochemical and cytofluorographic techniques using various monoclonal antibodies raised against different classes of rat lymphohemopoietic cells. These antibodies included in this study directed to rat T-cell antigens corresponding to mouse Lyt-1 (RLyt-1) and Lyt-2,3 antigens (RLyt-2) and to W3/25 antigen expressed on a particular subset of rat T-cells with helper function, as well as to rat granulocyte-macrophage-specific antigen (RGM-1). Histological studies demonstrated that the autografts of highly antigenic tumors introduced to the primary hosts were completely rejected following massive immigration of lymphoid cells into the tumor sites, which was not observed in progressively growing, minimally antigenic tumors. These lymphoid cells found within regressing highly antigenic tumor autografts were identified mostly to be T-cells bearing RLyt-1 (approximately 70%), and more than two-thirds of these T-cells expressed RLyt-2 antigen. In contrast to T-cells, macrophages and B-cells, each of which could be recognized by the presence of either RGM-1 antigen or immunoglobulin on their cell surfaces, appeared to have a minimal role in the rejection of autochthonous tumors, as reflected by their less frequent appearance within the tumor tissues during the rejection process.

Human blood group B-active ganglio-glycosphingolipid in rat glioma.

A human blood group B-active glycosphingolipid, belonging to the ganglio-series, was isolated from rat glioma cell line RG2 subcutaneous isografts. The oligosaccharide structure of the glycosphingolipid was completely characterized as Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc beta 1- 1'ceramide by NMR spectrometry, negative fast atom bombardment-mass spectrometry, sequential degradation by glycosidases and methylation analysis. Human blood group B antigenicity and the activity of this glycosphingolipid were confirmed by immunostaining on thin-layer chromatography and the inhibition of hemagglutination, respectively. Although the lipid has been detected in rat granuloma, bone marrow cells, spleen, thymus, ascites hepatoma cells and gastric mucosa, this is the first report of the occurrence of the B-active lipid in glioma.

Novel modification of ceramide: rat glioma ganglioside GM3 having 3-O-acetylated sphingenine.

A novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardment-mass spectrometries. In addition, the O-acetyl GM3 showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3.

Interleukin 4 enhances ganglioside GD3 expression on the human fibroblast cell line WI-38.

Human fibroblast cell line WI-38 cultured in vitro was treated with a human recombinant IL-4 at concentrations of 1 to 100 U/ml to examine the alteration of glycosphingolipid (GSL) expression of the cells. Neutral GSL of non-treated WI-38 cells consisted of CMH (GlcCer), CDH, CTH, and Gb4Cer; CMH and CTH were the major components. The acidic GSL were composed of GM3 as the predominant component and other minor gangliosides including GD3. The neutral GSLs did not change in profile during the treatment with IL-4, while the acidic GSLs showed a prominent change, an increase of GD3 content. The increase of GD3 was detectable with IL-4 concentrations over 1 U/ml, and reached a plateau at 10 U/ml, where the amount of GD3 was almost equal to that of GM3. The GD3 increase occurred at 24 h after the IL-4 treatment, and lasted for at least 96 h, as long as IL-4 remained present in the culture media. The GD3 synthase (sialyltransferase) level was found to be increased in an IL-4 dose-dependent manner. IL-4 did not influence the growth or morphological appearance of WI-38 cells. The results demonstrate a novel biological effect of IL-4, modulating GSL in non-hematopoietic cells.

Natural killer activity of lymphocytes infiltrating human lung cancers following preoperative systemic recombinant interleukin 2.

Tumor-infiltrating lymphocytes (TILs) show depressed natural killer (NK) activity compared with peripheral blood lymphocytes (PBLs). To determine if TIL NK function can be reactivated in vivo, 11 patients with tumors metastatic to the lung were treated with systemic recombinant interleukin 2 (rIL-2). Spontaneous TIL NK activity and NK activity after three days' incubation with 100 U/mL of rIL-2 were increased in patients receiving 15,000 U/kg of rIL-2 preoperatively compared with those receiving between 1000 and 10,000 U/kg. Histologically, higher doses of rIL-2 increased the number of intratumoral lymphocytes, the level of peritumoral lymphocytic transferrin, and the expression of HLA-DR. Spontaneous PBL NK activity in patients receiving between 10,000 and 15,000 U/kg of rIL-2 was also increased and was further increased by in vitro culture with rIL-2. Thus, PBL NK activity and TIL NK function in vivo can be augmented with 15,000 U/kg of systemic rIL-2. Both TIL- and PBL-inducible cytotoxicities were further enhanced by in vitro culture with rIL-2.

Construction of rat-mouse T-cell hybridomas that constitutively produce rat IL-2.

We have established rat-mouse T-cell hybridomas that constitutively produce rat Interleukin-2 (IL-2). T-cell hybridomas cannot be boosted to a higher level of IL-2 production by Con A stimulation. IL-2 prepared from T-cell hybridomas and from Con A activated rat spleen cells was partially purified using Ultrogel AcA 54 chromatography and ion exchange chromatography on Mono Q or chromatofocusing on Mono P. When analyzed on Mono P, IL-2 activity derived from IA2-B10 T-cell hybridoma eluted as a single peak with pH range 6.9-7.1, whereas IL-2 derived from Con A activated spleen cells resolved into four peaks within the following pH range: 7.1-7.2, 6.5-6.6, 6.1-6.2, and 5.6-5.7. Neuraminidase-treated IL-2 derived from Con A activated spleen cells resolved into single peaks appearing in the pH range 7.1-7.2. In contrast, neuraminidase treatment did not change the elution profile of IL-2 derived from the IA2-B10 hybridoma. IL-2 activity derived from the 3D6-B1 T-cell hybridoma also eluted as a single peak with the pH range 7.1-7.2. Neuraminidase treatment did not change the elution profile of IL-2. These data demonstrate that heterogeneity of IL-2 might be due to differences in the degree of glycosylation of IL-2 and differences in the sources of T-cells from which the IL-2 has derived.

Effect of local administration of lymphokine-activated killer cells and interleukin-2 on malignant brain tumor patients.

Nine patients with malignant brain tumors were treated with intratumoral infusion of lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2). LAK cells were generated from macrophage-depleted peripheral blood lymphocytes by culturing with IL-2 for 4 days. The resulting LAK cells showed strong cytotoxic activity against tumor target cells. Three patients received sufficient LAK cells (> or = 5.76 x 10(8)) to show partial tumor response by computed tomography and clinical signs. No severe neurological side effects occurred in any patient. Intratumoral administration of LAK cells and IL-2 can be effective in patients with malignant brain tumors.

[Clinicopathological findings in symptomatic Rathke's cleft cyst: correlation between enhancement effects on MRI and histopathology of the cyst wall].

We have studied MR images and the histopathology of eight patients with symptomatic Rathke's cleft cysts. Six cases showed visual disturbance and two showed galactorrhea. In five, the cyst fluid had low signal intensity on T1-weighted images and high intensity on T2-weighted images; in 2, the cyst fluid had high intensity on both T1 and T2-weighted images; in 1, the cyst fluid had high intensity on T1-weighted images and low intensity on T2-weighted images. Enhancement of the cyst wall by Gd-DTPA was able to be distinguished in six cases: two patients showed no enhancement, two showed thin enhancement and the remaining two, thick enhancement. Fluid aspiration and total resection of the cyst wall was performed in all patients (three cases by the transcranial approach and five by the transsphenoidal approach). Normal pituitary glands were found in all cases during the operations. Histopathologically, ciliated epithelium with goblet cells was recognized in three cases. Non-ciliated epithelium was recognized in the other five. Stratified squamous component was recognized in one case and secondary inflammation, in another. Normal pituitary tissue was recognized in five cases. Immunohistochemically, ciliated and non-ciliated epithelium was successfully stained for detecting antibody against epithelial membrane antigen and/or carcinoembryonic antigen. Two cases with no enhancement of the cyst wall by Gd-DTPA showed only ciliated epithelium. Two cases with thin enhancement of the cyst wall had single layer epithelium with normal pituitary tissue. Two cases with thick enhancement of the cyst wall showed single layer epithelium with its stratified squamous component or with secondary inflammation. A close relationship was suggested between the enhancement effect on MRI and histopathology of the cyst wall.

[Clinical analysis of the fatal cases of adult malignant gliomas after aggressive treatment].

Six patients operated on for supratentorial malignant astrocytomas and seven patients operated on for glioblastoma multiforme were analyzed to evaluate the effect of aggressive surgical resection on the length of survival and causes of death. Early postoperative contrast enhanced CT scan was used to assess the extent of surgical resection. A gross total resection was considered to have been accomplished when there was no evidence of any residual enhanced mass. When 10% or less of the preoperative enhanced mass remained, the resection was classified as a subtotal resection. Subsequent follow-up CT scan showed that a gross total resection was accomplished in nine patients, and a subtotal resection was attained in four patients. The patients' ages ranged from 40 to 78 years (mean, 59 years). The median survival after the first aggressive surgical resection was 18.0 months in patients with malignant astrocytoma and 13.6 months in those with glioblastoma multiforme. The median duration between first operation and recurrence of tumor was 8.8 months in patients with malignant astrocytoma and 11.5 months in those with glioblastoma multiforme. A second aggressive surgical resection for recurrent malignant astrocytoma or glioblastoma multiforme was carried out in four patients (40%) of the evaluated ten patients. The median survival of these patients after reoperation was 8.25 months. Accordingly, aggressive surgical resection of malignant astrocytoma and glioblastoma multiforme is correlated with longer survival and is advocated in the treatment of recurrent tumors. Leptomeningeal dissemination was diagnosed in nine patients (90%) of evaluated ten patients.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hypothalamic histiocytosis X--report of a case].

Histiocytosis X is a non-neoplastic disorder of unknown etiology characterized by a mass of proliferating histocytes, plasma cells and inflammatory cells forming a granuloma within the reticuloendothelial elements of any organ system in the body. The three clinical syndromes of this condition, eosinophilic granuloma, Hand-Schüller-Christian disease and Letterer-Siwe disease were unified into a single nosological entity by Lichtenstein in 1953. The complication of the central nervous system is shown in the cerebrum, cerebellum, sella turcica, hypothalamus and so on. Hypothalamic histiocytosis X is often found in cases of disseminated histiocytosis X, but an isolated histiocytosis X of the hypothalamus is very rare. Including our case, 16 cases of hypothalamic histiocytosis X were reviewed in this paper. Age at onset was older than disseminated histiocytosis X. The sex ratio was almost equal. Diabetes insipidus was equally found as in other suprasellar masses and it was important as an initial symptom. The CT finding was shown as a homogeneously enhanced mass but no specific finding of CT was seen as suprasellar histiocytosis X. Therefore, differential diagnosis was very difficult. Treatments for this disease were variable. Chemotherapy, irradiation or combination of both were reported. The combination therapy was thought to be the most effective treatment. The relation between this disease and disseminated histiocytosis X is not known. But possibility of transformation of this disease into disseminated histiocytosis X was reported in some papers. Therefore, early diagnosis by biopsy of the hypothalamic lesion was stressed.

[A case of dural type of histiocytosis X presenting as a mass lesion in the tentorium cerebelli].

Histiocytosis X is a disease of unknown etiology, characterized by a mass of proliferating histiocytes, plasma cells and inflammatory cells foaming a granuloma within the reticuloendothelial elements of any organ in the body. In the central nervous system (CNS), hypothalamic disorder of histiocytosis X is often found, but histiocytosis X in other regions is quite rare. We report a case of a 5-year-old girl with histiocytosis X of the zygoma presenting as a mass lesion in the tentorium cerebelli. A computed tomographic (CT) scan demonstrated a tumor at the left tentorial region, extending along the dura mater of the tentorium cerebelli. Magnetic resonance imaging (MRI) revealed a low signal intensity region on both T1 and T2-weighted images. MRI with Gd-DTPA showed a homogeneous enhanced mass extending to right and inferior sites with a thickened tentorium. As the thickened dura matter continued from the left middle fossa to the mass lesion, the tumor was considered to arise from the left zygoma and extend to the tentorium cerebelli. CNS extension of histiocytosis X is manifested either as (1) the cerebral type or (2) the dural type. Many cases of cerebral type histiocytosis X including hypothalamic disorder have been reported. Only 6 cases of the dural type of histiocytosis X have been described. Although the lesions of the cerebral type of histiocytosis X show prolonged T1 and T2 values on MRI, the MRI findings of the dural type have not been reported. The present case is the first report of the appearance of the lesion on MRI.(ABSTRACT TRUNCATED AT 250 WORDS)

[Prognosis after total removal of craniopharyngiomas via the frontobasal interhemispheric approach].

Prognosis after total removal of craniopharyngiomas via the frontobasal interhemispheric approach is reviewed. Seventeen patients with craniopharyngiomas were operated on in Sapporo Medical University Hospital between January, 1985 and December, 1993. In eleven patients, lamina terminalis was incised and in the last six patients, it was left intact. Tumors were completely resected in all patients. After removal of the tumor, hypothalmic--pituitary functions, visual functions and psychometric functions were examined. Two of the 17 cases showed hypernaturemia and fourteen (82%) had permanent DI. Fourteen patients are receiving DDAVP and all are receiving endocrine replacement. Of fourteen patients who had disturbance of their visual acuity, nine (64%) improved. Five of six patients (83%) who presented visual field defect showed improvement in their deficits. Thirteen patients had a psychometric assessment at the time of follow-up examination. Full-scale intelligence quotient scores were distributed as follows: three above 120, five between 90 and 109, three between 70 and 79 and two below 69. Four (32%) had some impairment of memory. Concerning the QOL after total removal of craniopharyngiomas via the frontobasal interhemispheric approach, thirteen patients (76%) are leading normal lives, and three (18%) are leading nearly normal lives but require some help to overcome mild deficits. One (6%) has suffered a significant handicap.

A case of calcification of the cervical ligamentum flavum.

We report a case of cervical myelopathy caused by the calcification of the cervical ligamentum flavum. A 42-year-old woman with gait disturbance and an episode of dysuria was transferred to our hospital on June 30, 1994. Neurological examination revealed only a mild right hemiparesis. A plain neck X-ray and a tomogram revealed a nodular calcification in the posterior part of the spinal canal at the level of C5/6. Three dimensional computed tomography clearly demonstrated that the mass consisted of three nodular structures on the vertebral lamina. Magnetic resonance images demonstrated severe compression of the spinal cord by the mass whose intensity was low. The mass was removed en bloc together with the ligamentum flavum and C5 and C6 lamina. The mass showed no continuity to the dura mater. The calcification was confined within the ligament. The patient's neurological deficits were resolved two weeks after the surgery. X-ray diffraction study demonstrated the component of the mass was found to be pure hydroxyapatite. Clinical features of calcification of the ligamentum flavum are reviewed from 85 reported cases including ours, and the difference between this calcification and the ossification of the ligament is emphasized. Calcification of the ligamentum flaum is a distinct clinical entity.

[A case report of interstitial pneumonia caused by granulocyte colony-stimulating factor].

Several clinical trials have demonstrated that granulocyte colony-stimulating factor (G-CSF) accelerates the recovery of neutropenia in chemotherapy-induced bone marrow suppression. In this report, we describe a 46-year-old female with glioblastoma multiforme who developed interstitial pneumonia due to administration of G-CSF during the phase of immunochemoradiotherapy-induced neutropenia. Thirty-three days after starting immunochemoradiotherapy (ACNU, VCR, IFN -beta, radiation), she developed neutropenia (1,000/microliters). Administration of G-CSF at doses of 125-250 micrograms/day led to an increase of peripheral neutrophil counts. Eleven days later, the patient developed sudden severe respiratory failure and cyanosis with worsening of lung shadows. Blood gas levels on room air were PaO2 49.3mmHg, PaCO2 28.0mmHg, and pH 7.46. At this time, her neutrophil count had risen to 26,080/microliters. LDH and alpha - HBD had also increased to 1,439 IU/l and 1,117IU/l respectively. Chest radiograph and CT scan demonstrated interstitial pneumonia. After treatment with methyl prednisolone, her respiratory symptoms were gradually resolved. A number of side-effects have been reported with granulocyte-macrophage colony-stimulating factor (GM-CSF). These include fluid retention with pericardial and pleural effusion, fever, bone pain, fatigue, and rash. This report also suggests that G-CSF might be a cause of interstitial pneumonia during the phase of immunochemoradiotherapy-induced neutropenia.

[A case of ANCA negative Wegener's granulomatosis with pulmonary giant bulla showing high level of soluble ICAM-1].

This paper reports a rare case of the limited form of Wegener's granulomatosis (WG) with pulmonary giant bulla, which was pathologically identified only in the lung and showed a high level of soluble intercellular adhesion molecule-1 (ICAM-1) but not anti-neutrophil cytoplasmic antibody (ANCA). A 46 year-old male was admitted for the further examination of right anterior chest pain and dyspnea. Chest X-ray and CT examination showed giant bulla accompanied with the invasive lesions in upper lobe of right lung. Partial lobectomy was done. Granulomatous and necrotizing vasculitis characteristics of WG were confirmed by the histopathology of resected tissues. Immunological analysis on admission showed a high level of soluble ICAM-1 and ANCA negative, but that of soluble ICAM-1 was reduced after surgical operation and remained within normal range during an administration of prednisolone and cyclophosphamide. These results suggest that soluble ICAM-1 may be one of the useful parameters for a diagnosis of WG and for an estimation of its treatments, especially in the cases of ANCA negative WG.

[Diagnosis of early cancer with monoclonal antibodies].

Monoclonal antibodies which have been utilized in the field of early diagnosis for cancer were reviewed. Monoclonal antibodies to ras oncogene product and to very high-molecular-weight MUSE 11 antigen were found to be useful for diagnosing some of the early carcinomas.

The regulatory effect of adherent cells on lymphokine activated killer cells.

We analyzed the effect of adherent cells on the induction of lymphokine-activated killer (LAK) cell activity by depleting adherent cells from peripheral blood lymphocytes (PBL) or by adding adherent cells to PBL before culture with interleukin-2. We found that adherent cells clearly down-regulate LAK cell induction. These inhibitory effects are dependent on the number of adherent cells added. Inhibitory effects of adherent cells are abolished by the addition of indomethacin to the LAK culture. Soluble factors derived from adherent cells, such as interferons and interleukin-1, have a slight enhancing effect on LAK induction. In contrast, adherent cells appear to inhibit LAK induction primarily by producing prostaglandin E2(PGE2). PGE2 in turn inhibits the induction of LAK effector cells by inhibiting the expression of the transferrin receptor on LAK cells. These effects are manifested most strikingly in the early phases of LAK induction.

Establishment of rat-mouse T cell hybridomas that constitutively produce a soluble factor that is needed for the generation of cytotoxic cells: biochemical and functional characterization.

Spleen cells from Lewis rats were cultured with 4 micrograms/ml Con A. These cells were then fused with BW 5147 mouse T lymphoma cells. Two hybrid clones (6B2-B8 and 6B2-E6) obtained by fusion formed CGF effectively. It was found that hybrid cells can be boosted to produce higher levels of CGF upon stimulation with Con A. 6B2-B8 express rat T cell markers. CGF formed by 6B2-B8 had a m.w. of 23,000 and 40,000. CGF was eluted from a Mono Q anion-exchange column with an FPLC system at 0.4 to 0.6 M NaCl as a major peak and at 0.8 M NaCl as a minor peak. CGF was eluted as three peaks with pH 4.1, 4.8, and 5.2 from a Mono P chromatofocusing column. CGF from 6B2-B8 does not contain IL-1, IL-2, IL-3, or CSF.

Functional analysis of mononuclear cells infiltrating into tumors: differential cytotoxicity of mononuclear cells from tumors of immune and nonimmune rats.

A million syngeneic gliosarcoma (T-9) cells injected subcutaneously are sufficient to kill Fisher rats within 2 mo. Fisher rats became resistant to T-9 cells by surgical removal of the implanted tumor and repeated immunization with MMC-treated T-9 cells. Histopathologic studies revealed massive accumulation of mononuclear cells in tumor tissues of immune rats that were rejecting syngeneic T-9 tumors, whereas very few mononuclear cells were found in tumor tissues of nonimmune rats. Cell populations infiltrating into tumor tissues were identified by immunohistochemical techniques. Mononuclear cells found within the regressing tumors of immune rats were identified mostly to be T cells, and two-thirds of these T cells were OX-8 positive. In contrast, mononuclear cells found within the growing tumors of nonimmune rats were identified to be mixtures of macrophages and T cells, and very few OX-8 positive cells were found. Mononuclear cells were isolated from implanted T-9 tumors to determine whether mononuclear cells lysed T-9 cells specifically. Significant tumoricidal activity was seen when mononuclear cells from tumors of immune rats were used, whereas no detectable tumoricidal activity was observed with mononuclear cells from tumors of nonimmune rats. Winn assays confirmed in vitro 51Cr release assays by showing that tumors were rejected only when T-9 cells were implanted into normal Fisher rats along with mononuclear cells from tumors of immune rats.