Analysis of tumor-infiltrating lymphocyte in primary malignant lymphoma of the central nervous system using double immunofluorescence staining method]

Three cases of malignant lymphoma were examined immunohistochemically using anti-Leu monoclonal antibodies (MAB) against B lymphocytes, T lymphocytes subsets, NK cells, IL-2 receptor. In all three cases tumor cells were stained with anti-B cell's MABs, that is they were diagnosed as B cell type of malignant lymphoma. Moderate tumor-infiltrating lymphocytes in the parenchyma or around blood vessels were observed, and majority of these cells were T lymphocytes, demonstrating Leu 2a or Leu 3a + 3b positive phenotypes. IL-2 receptor positive cells also found in the parenchyma of these tumors. T lymphocyte subsets, such as Leu 2a positive cells and Leu 3a + 3b positive cells, were analyzed by means of double immunofluorescence staining method using the combination of Leu 2a and Leu15, or Leu 3a + 3b and Leu 8 MABs. This method demonstrated that most of Leu 2a positive cells were negative for Leu 15 and that the majority of Leu 3a + 3b positive cells were negative for Leu 8. These results suggested that tumor-infiltrating lymphocytes in the malignant lymphoma were mainly activated cytotoxic or helper T cells.     

Proliferation kinetics of human brain tumors by in situ double labeling with bromodeoxyuridine and iododeoxyuridine]

Nineteen patients with human brain tumors (9 gliomas, 6 metastatic brain tumors, 3 meningiomas, 1 neurinoma) received intravenous infusions of iododeoxyuridine (IUdR) and bromodeoxyuridine (BUdR) at different time sequences, to estimate the duration of S-phase (Ts) and the potential doubling time (Tp) of individual tumors. Excised tumor specimens were reacted with Br-3, a monoclonal antibody that identifies only BUdR, and with IU-4, a monoclonal antibody that recognizes both IUdR and BUdR, and then were stained immunohistochemically. The BUdR LIs varied from 0.9% to 26.0%, reflecting the malignancy of each tumor. Despite the difference in LIs, however, the Ts measured was fairly uniform. The Ts was 8.9 +/- 1.8 hrs (mean +/- SD) in malignant gliomas, 9.2 +/- 2.5 hrs (mean +/- SD) in metastatic brain tumors and 9.2 +/- 0. 3 hrs (mean +/- SD) in meningiomas, respectively. In contrast, the Tp varied from 1.3 to 12.4 days in malignant gliomas and from 1.2 to 4.4 days in metastatic brain tumors. Double logarithmic regression analysis showed a close correlation between the BUdR LI and Tp in human brain tumors with LI > 1%. Double labeling studies with BUdR and IUdR allow some of the proliferation characteristics to be determined from a single biopsy specimen and provide more useful information of each tumors than can be obtained by single-labeling studies with BUdR.     

Immunohistochemical staining of human brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen

Using immunoperoxidase histochemistry, human brain sections obtained at biopsy were labeled with monoclonal antibodies which identify human lymphocytes subsets, monocytes, and the Ia antigen. Staining of a population of cells in white matter was present with the anti-Ia and the anti-M1 (monocyte-associated) antibodies but not with any of the 8 monoclonal antibodies which react with human T-cell subsets (anti-T1, 3, 4, 5, 6, 8, 10 and 12). The Ia antigen was present on 1–2% of cells in white matter, and approximately 5% of cells in white matter were M1-positive. Ia-positive cells demonstrated a pattern of diffuse surface membrane staining whereas the M1 antigen appeared to cluster at proximal cell processes. Definitive identification of these cells as microglial cells, astrocytes or oligodendrocytes was not possible.These findings demonstrate that: (1) cells which beam the Ia and M1 determinants can be found in histologically normal human white matter, and (2) human oligodendrocytes do not react with monoclonal antibodies (anti-T5 and anti-T8) that identify human suppressor/cytotoxic cells.     

Calmodulin content in human brain tumors

Using a radioimmunoassay method, the particulate and soluble calmodulin levels were determined in biopsied specimens from normal human brain and from various human brain tumors. Both in normal and pathological tissues the major portion of calmodulin was revealed in the cytosol. The chromatographic elution profiles of calmodulin obtained from soluble and particulate fractions of the same specimen were identical, thus suggesting an identity of the supernatant and particulate form of calmodulin. In all the examined oncotypes, the calmodulin content was lower than in normal extracts and this biochemical feature could not have been correlated with the degree of malignancy of the neoplasia. Furthermore, the translocation of calmodulin from the particles to the cytoplasm, reported in other rapidly growing tumors, lacks in human cerebral ones. Our findings indicate that in human brain oncotypes the calmodulin distribution is quite different from that found in tumors taken from other tissues, where its level is increased and a positive correlation between calmodulin concentration and growth rate of neoplastic tissue has been revealed.     

P－糖蛋白在颅脑肿瘤中表达的研究

采用Ｐ－糖蛋白单抗和免疫荧光染色法检测４５例颅脑肿瘤手术标本，Ｐ－糖蛋白阳性表达率如下：原发性胶质瘤１３／２８，脑转移癌１／２，颅骨恶性肿瘤２／２，脑膜瘤、听神经瘤和垂体腺瘤等良性肿瘤Ｏ／１３。在胶质瘤中Ｐ－糖蛋白的表达强度与肿瘤的恶性程度呈正相关。在同一肿瘤中，Ｐ－糖蛋白的分布很不均一，呈明显的异质性。     

Magnetic resonance imaging of human brain macrophage infiltration

Macrophage tracking by magnetic resonance imaging (MRI) with iron oxide nanoparticles has been developed during the last decade for numerous diseases of the CNS. Experimental studies on animal models were confirmed by first clinical applications of MRI technology of brain macrophages for multiple sclerosis, ischemic stroke lesions, and tumors. As activated macrophages act in concert with other immune competent cells, this innovative MRI approach provides new functional data on the immune reaction in these CNS diseases. The MRI detection of brain macrophages defines precise spatial and temporal patterns of macrophage involvement that helps to characterize individual neurological disorders. This approach is being explored as an in vivo marker for the clinical diagnosis of cerebral lesion activity, in experimental models for the prognosis of disease development, and to determine the efficacy of immunomodulatory treatments under clinical evaluation. Comparative brain imaging follow-up studies of blood-brain barrier leakage by MRI with gadolinium-chelates, microglia activation by positron emission tomography with radiotracer ligand PK11195 and MRI detection of macrophage infiltration provide more precise information about the pathophysiological cascade of inflammatory events in cerebral diseases. Such multimodal characterization of the inflammatory events should help in the monitoring of patients, in defining precise time intervals for therapeutic interventions, and in developing and evaluating new therapeutic strategies.     

Ultrastructure of capillary walls in human brain tumors

Summary Changes in capillary walls between human glial, non-glial and metastatic brain tumors were studied with conventional ultrathin section and freeze-fracture replica techniques. The following results were obtained. (1) In glial tumors, ultrathin section studies showed cell junctions of the capillaries were either short or elongate. Moreover, endothelial hyperplasia, surface infolding of endothelial cells, irregularity of the basal lamina and a large extravascular space were observed. Freeze-fracture replicas of capillary endothelium showed tight junctions as two to seven strands. In addition, pinocytotic vesicles had increased markedly and were an average of 25 per m². Both ultrathin and freeze fracture studies showed that, in contrast to malignant gliomas, there were only slight changes in benign astrocytomas. (2) In non-glial tumors, ultrathin sections showed surface infoldings, increased vesicles, many fenestrations of endothelial cells, irregularity of basal lamina and enlarged perivascular space. Freeze-fracture replicas of vascular endothelium, showed that the average number of pinocytotic vesicles and fenestrations were 25 and 22 per m², respectively. Moreover, the tight junction was composed of one or two strands which appeared to be a discontinuous array of particles. (3) In metastatic brain tumors, ultrathin studies showed capillary endothelia were proliferated, had marked infolding, and showed an increased number of pinocytotic vesicles and many fenestrations. Moreover, short and elongate intercellular junctions were presented but no open junction was detected. Finally the basal lamina lost its three-layered appearance and was irregular in width. Freeze-fracture replicas showed pinocytotic vesicles had increased and were 24 per m² on average in four cases, but fenestrations and tight junctions could not be detected. The most fundamental feature of vessels in these three different kinds of tumors was whether they were fenestrated or not. Glial tumors were non-fenestrated, whereas non-glial and metastatic tumors were fenestrated.     

Micromapping of the human brain: Three-dimensional imaging of immunofluorescence and dendritic morphology using dual-channel confocal laser scanning microscopy

A strategy for investigating neuronal networks at the microscopic level is described. The Lucifer Yellow microinjection technique was combined with immunofluorescence on human brain material, which was then studied in a confocal laser scanning microscope with dual-channel scanning and equipped with an argon/krypton laser. The three-dimensional architecture of the Lucifer Yellow-injected neurons was investigated after transfer of the scanned frames in file format to a Silicon Graphics IRIS computer, using VoxelView software from Vital Images Inc. Microinjection of Lucifer Yellow revealed the dendritic morphology of various types of cells in different brain areas. Indirect immunofluorescence, with Texas Red as the secondary label, was used to determine the distribution of various categories of macromolecules (enzymes, receptor protein, and synaptic vesicle proteins) in the brain slices. We used single- as well as dual-channel confocal laser scanning microscopy for imaging these double-stained fluorescent specimens. Using these techniques in combination, we have created and saved three-dimensional confocal images of detailed morphology (axons and dendrites with spines and varicosities) of individual cells, together with the localization of immunofluorescence. These three-dimensional confocal images will be collected in a database for probable future use in human brain mapping. © 1994 Wiley-Liss, Inc.     

Immunohistochemical studies on human brain tumors using anti-Leu 7 monoclonal antibody in paraffin-embedded specimens

Summary Using the four-step peroxidase-antiper-oxidase (PAP) method, the presence of the antigen recognized with anti-Leu 7 monoclonal antibody was investigated in paraffin-embedded human brain tissue and tumors. The antigen was demonstrated in the myelin sheaths, oligodendrocytes, and some choroid plexus cells in normal brain and in oligodendrogliomas, some astrocytomas and choroid plexus papillomas. The technique can be used to identify hormal and neoplastic oligodendrocytes.     

Metabolic underpinnings of activated and deactivated cortical areas in human brain

Neuroimaging with functional MRI (fMRI) identifies activated and deactivated brain regions in task-based paradigms. These patterns of (de)activation are altered in diseases, motivating research to understand their underlying biochemical/biophysical mechanisms. Essentially, it remains unknown how aerobic metabolism of glucose to lactate (aerobic glycolysis) and excitatory-inhibitory balance of glutamatergic and GABAergic neuronal activities vary in these areas. In healthy volunteers, we investigated metabolic distinctions of activating visual cortex (VC, a task-positive area) using a visual task and deactivating posterior cingulate cortex (PCC, a task-negative area) using a cognitive task. We used fMRI-guided J-edited functional MRS (fMRS) to measure lactate, glutamate plus glutamine (Glx) and γ-aminobutyric acid (GABA), as indicators of aerobic glycolysis and excitatory-inhibitory balance, respectively. Both lactate and Glx increased upon activating VC, but did not change upon deactivating PCC. Basal GABA was negatively correlated with BOLD responses in both brain areas, but during functional tasks GABA decreased in VC upon activation and GABA increased in PCC upon deactivation, suggesting BOLD responses in relation to baseline are impacted oppositely by task-induced inhibition. In summary, opposite relations between BOLD response and GABAergic inhibition, and increases in aerobic glycolysis and glutamatergic activity distinguish the BOLD response in (de)activated areas.     

Immunohistochemical demonstration of immunoglobulins and albumin in human brain tumors

Routinely processed biopsy material, including 56 gliomas of varying malignancy, 10 meningiomas, 10 brain metastases and 12 brain abscesses, was examined for the presence and distribution of IgG, IgA, IgM, IgD and albumin using the unlabeled antibody peroxidase-antiperoxidase technique. In all specimens the deposition of stained immunoglobulins (Ig) was strictly associated with that of albumin even on cell surfaces. Thus there was no evidence for specific membrane binding or cytotoxicity. The interstitial proteins demonstrated are most likely derived from the plasma by blood-brain barrier breakdown which occurs in nearly all tumors and abscesses. Obvious intracellular staining for Ig and albumin was seen in glioma cells and astrocytes only. This is suggested to be due to active protein uptake as a specific feature of astrocyte differentiation which decreases with malignancy and is lost in glioblastomas. Evidence for local Ig production was found in 8 out of 10 metastases with striking IgG- and IgA-positive plasma cells within lymphocytic infiltrations and in one meningioma showing conspicuous plasma cells components. No glioma contained Ig-bearing plasma cells, though round cell infiltrations were present in 64% of the unselected cases. The significance of these findings regarding the immunological situation in brain tumors is briefly discussed.     

Acid fast staining of oxidized neuromelanin and lipofuscin in the human brain.

Acid fast staining of the bleached residuum of substantia nigra neuromelanin and of oxidized inferior olive lipofuscin was demonstrated in paraffin and frozen sections stained with the acetic acid, carbol fuchsin method of Barbeito-Lopez and the aldehyde fuchsin method of Gomori. Acid fast staining occurred when sections were exposed to a prestain oxidation with potassium permanganate in conjunction with a poststain differentiation with dilute hydrochloric acid. The acid fast staining with acetic acid, carbol fuchsin was differentiable and in contrast to the acid fast staining with aldehyde fuchsin which was nondifferentiable. A possible histochemical basis for differentiable and nondifferentiable acid fast staining was discussed. The identify of the bleached residuum of neuromelanin as lipofuscin was also discussed.     

Lipid composition of human malignant brain tumors

Malignant transformation is characterized by the uncontrolled proliferation of cells. And changes in the composition of glycolipids, cell surface component which may be involved in regulation of cell growth, were often observed in the malignant transformation. In this study, cholesterol, lipid-bound phosphorus, cerebroside, sulfatide and ganglioside were quantitated in the tissue of 20 human malignant brain tumors (malignant glioma, 8; low grade glioma, 4; metastatic tumor, 7; malignant meningioma, 1). As compared with normal brain, all tumor tissue contained lower cholesterol, sialic acid, cerebroside and sulfatide. Metastatic brain tumor or glioma showed characteristic patterns in the content of ganglioside, cerebroside and sulfatide respectively. The ganglioside patterns of metastatic tumor or glioma contained a greater proportion of structurally simpler gangliosides than normal brain. And in metastatic tumor, GM3 was a major ganglioside. On the contrary, glioma had increased proportion of GM3 and GD3 gangliosides. High grade glioma such as Grade 3-4 contained higher proportion of GM3 and GD3, whereas low grade glioma (Grade 1-2) contained less proportion of GM3 and GD3.     

Freeze-fracture study of human brain tumors.

This freeze-fracture study was performed in 3 astrocytomas, 6 glioblastomas, 2 ependymomas, 3 medulloblastomas, 1 cerebellar sarcoma, 3 germinomas, and 1 medulloepithelioma. The number of nuclear pores/mum2 nuclear membrane was not correlated with biological malignancy. Fracture faces A and B were discernible in nuclear, Golgi and rough endoplasmic reticulum (ER), mitochondrial surface, and plasma membranes. Fenestrae were evident in Golgi and ER membranes. The transitional zone of cristae from the inner surface membrane appeared as a circular hole and broken-off neck on faces A and B of the inner surface membrane, respectively. The decrease in number of membrane particles in the plasma membrane seemed to correlate with the frequency of metastases, and, in addition, the membrane particles appeared to cluster in glioblastoma, medulloblastoma, and medulloepithelioma. The gap junctions were abundant in astrocytomas, moderate in number in ependymomas and germinomas, and rare in glioblastomas, cerebellar sarcoma, and medulloepithelioma. Tight junctions were often found in germinomas and medulloepithelioma, and rarely in ependymomas.