Maciej Poltorak

Immunological reactions induced by intracerebral transplantation: Evidence that host microglia but not astroglia are the antigen-presenting cells

The immunological reactions to embryonic cerebellar xenografts (n = 16) and allografts (n = 8) in host rat brain were studied after 2, 4, and 6 weeks of survival and compared to a control group consisting of 10 rats with isografts. Indirect immunofluorescence was performed on fresh frozen brain sections using antibodies against antigen presenting cells (Ia/Ox-6+ cells) and T helper (W3/25+) cells. Massive infiltrations of both cell types were found within xenografts. Ia antigen was present in the walls of small vessels near the transplant as well as in the ventricles on supra- and subependymal cells. In host tissue surrounding the grafts, Ox-6+ immunoreactivity was also observed in a population of cells ranging from an irregular rod-like shape with short branching processes to more rounded cell bodies with retracted processes. The appearance of these cells was characteristic of microglia. These cells were GFAP-negative. These cellular reactions were associated with rejection of the grafts. In contrast, the allografts survived, but nevertheless cells expressing Ox-6+ and to a lesser extent W3/25+ immunoreactivity were found along the injection needle tract and in damaged host tissue surrounding the grafts. No Ox-6+ perivascular infiltrations were seen. Some staining was also found within the allografts, mainly associated with damaged tissue. Ox-6+ ramified cells were also observed. Both Ox-6+ and W3/25+ immunoreactivity decreased with the time of survival. Host and donor GFAP-positive astrocytes did not express Ox-6+ molecules, and therefore probably were not involved in presenting antigen to effector cells. The control isografts also survived very well, but nevertheless Ox-6+ and less widespread W3/25+ cells were present in surrounding injured host tissue. Ox-6+ perivascular infiltration was not found in the host brain of animals with isografts. Ox-6+ and W3/25+ immunoreactivities were present primarily in graft areas that appeared damaged, often closely associated with injured host tissue. These results indicate that the process of implantation of grafts and associated brain injury induces enhanced Ia/Ox-6+ immunoreactivity, primarily on microglia in brain parenchyma surrounding grafts, and suggest that host microglia may substantially contribute to the initiation of immune reactions against intracerebral grafts. Despite this predisposition to an immunological response, only in the case of xenografts did these reactions, with the addition of Ox-6+ perivascular cuffing and cell infiltrations within the grafts, lead ultimately to graft rejection.

Disturbances in cell recognition molecules (N-CAM and L1 antigen) in the CSF of patients with schizophrenia

Although the pathogenesis of schizophrenia is unknown, there are data which indicate that the disease may be due to neurodevelopmental disturbances. Cell recognition molecules such as N-CAM and L1 antigen are involved in cell-cell interactions during development and in plasticity of the nervous system and could therefore be altered in relation to ongoing or established pathological processes. Using the Western blot technique, we found significant increases in N-CAM immunoreactive proteins and decreases in L1 antigen in the CSF of schizophrenic patients as compared to normal controls. The decrease in L1 antigen was observed in the 140-kDa band, and N-CAM was increased only in the 120-kDa band. The 120-kDa band of N-CAM and the 140-kDa band of L1 antigen were prominent components of CSF, but in serum these bands were minor or not detectable. Neuroleptic treatment did not significantly change either N-CAM or L1 antigen concentrations in CSF. It is possible that these CSF proteins are derived from CNS cells as secreted soluble N-CAM isoforms and L1 peptides. Our results suggest the possibility of a specific pattern of abnormal cellular function in the CNS in schizophrenia.

Increased Neural Cell Adhesion Molecule in the CSF of Patients with Mood Disorder

Abstract: Neural cell adhesion molecule (N‐CAM) is involved in cell‐cell interactions during synaptogenesis, morphogenesis, and plasticity of the nervous system. Disturbances in synaptic restructuring and neural plasticity may be related to the pathogenesis of several neuropsychiatric diseases, including mood disorders and schizophrenia. Disturbances in brain cellular function may alter concentrations of N‐CAM in the CSF. Soluble human N‐CAM proteins are detectable in the CSF but are minor constituents of serum. We have recently found an increase in N‐CAM content in the CSF of patients with schizophrenia. Although the pathogenesis of both schizophrenia and mood disorders is unknown, ventriculomegaly, decreased temporal lobe volume, and subcortical structural abnormalities have been reported for both disorders. We have therefore measured N‐CAM concentrations in the CSF of patients with mood disorder. There were significant increases in amounts of N‐CAM immunoreactive proteins, primarily the 120‐kDa band, in the CSF of psychiatric inpatients with bipolar mood disorder type I and recurrent unipolar major depression. There were no differences in bipolar mood disorder type II patients as compared with normals. There were no significant effects of medication treatment on N‐CAM concentrations. It is possible that the 120‐kDa N‐CAM band present in the CSF is derived from CNS cells as a secreted soluble N‐CAM isoform. Our results suggest the possibility of latent state‐related disturbances in N‐CAM cellular function, i.e., residue from a previous episode, or abnormal N‐CAM turnover in the CNS of patients with mood disorder.

Immortalized GABAergic Cell Lines Derived from Rat Striatum Using a Temperature-Sensitive Allele of the SV40 Large T Antigen

Embryonic striatal cells were immortalized using the A58 temperature-sensitive allele of the SV40 large T antigen. Two cell lines, M213-2O and M26-1F, with gamma-aminobutyric acid (GABA)ergic properties were selected from 85 clones thus developed. M213-2O is a multipolar, polygonal cell line which expresses SV40 large T antigen and glutamate decarboxylase (GAD) at the permissive temperature (33 degrees C) and GAD and MAP-2 immunoreactivity at the nonpermissive temperature (39.3 degrees C). M26-1F has a fibroblast-like morphology and expresses SV40 large T antigen and GAD both at the permissive and nonpermissive temperatures and MAP-2 immunoreactivity at the nonpermissive temperature. Both lines contain GABA as measured by reversed-phase HPLC and M213-2O expresses nipecotic-sensitive [14C]GABA uptake.

Certain host-donor rat strain combinations do not reject brain allografts after systemic sensitization

To investigate the factors related to allogenic brain graft rejection, rat cortex from either LEW-RT1l or BN-RT1n rats was transplanted into brains of several isogenic and allogenic inbred strains: F344-RT1l, LEW-RT1l, BN-RT1n, AO-RT1u, PVG-RT1c, PVG-RT1u, and PVG-RT1l. Each donor and host combination was subsequently subdivided into two subgroups. One group was systematically sensitized twice with donor skin tissue and another group served as a sham-sensitization control. Four weeks after the second sensitization, host brains were examined histologically, and the percentage volume of each graft that showed increased cellularity was estimated. Expression of major histocompatibility complex (MHC) and T cell antigens was also studied immunocytochemically. Almost all grafts in sham-sensitized animals from all groups were accepted with minimal or no reaction. However, two strains (AO-RT1u and F344-RT1l) showed considerable cell infiltration and expression of MHC antigens even without sensitization. After sensitization, almost all allogenic strain combinations showed greater signs of rejection-related responses. The severity of the tissue reaction, however, varied considerably between groups. All grafts from BN-RT1n donors were rejected severely in all host strains. For LEW-RT1l donors, grafts survived well in some host strains (BN-RT1n, AO-RT1u, PVG-RT1c, and PVG-RT1u), even with MHC + non-MHC disparity. Curiously, F344-RT1l hosts rejected LEW-RT1l grafts even though the two strains have the same MHC loci, but different minor histocompatibility (mH) loci. For both donor strains, experimental autoimmune encephalomyelitis-susceptible hosts tended to show more vigorous rejection responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Monozygotic twins discordant for schizophrenia are discordant for N-CAM and L1 in CSF

While schizophrenia has a genetic component, its pathogenesis is unknown. Abnormal concentrations of two cell recognition molecules (CRMs), neural-cell adhesion molecule (N-CAM) and L1 antigen have been described in the cerebrospinal fluid (CSF) of patients with schizophrenia. Studies of monozygotic twins discordant for schizophrenia may help separate genetic and environmental contributions to the disease. In the present study of monozygotic twins discordant for schizophrenia, the affected twins had increased N-CAM and decreased L1 antigen in their CSF. Non-affected twins were not different from normals. Although processes related to genetic instability cannot be entirely ruled out, these results suggest that these abnormalities are not a part of the genetic predisposition to become schizophrenic. Thus the changes in N-CAM and L1 antigen may reflect either the events which precipitated the onset of schizophrenia, or events which are associated with the experience of having the disease.

Effects of adrenal medulla grafts on plasma catecholamines and rotational behavior

The mechanisms by which adrenal medulla grafts influence the function of host brains in animal models of Parkinsons disease are unclear. To explore this issue, fragments of adrenal medulla or sciatic nerve were transplanted into the lateral ventricle of bilaterally adrenalectomized (ADX) or sham-ADX rats with unilateral 6-hydroxydopamine lesions of the substantia nigra. Additional control group received sham-transplantation surgery. Behavioral effects of these procedures were tested following administration of apomorphine, amphetamine, or nicotine. Plasma catecholamines were measured before and after transplantation surgery. In both ADX and sham-ADX rats, adrenal medulla grafts produced greater decreases in apomorphine-induced rotational behavior than did sciatic nerve grafts or sham-transplanted groups. Decreases in rotation were smaller in ADX than in sham-ADX animals, regardless of graft treatment. Plasma catecholamines increased after transplantation surgery in each of the sham-ADX groups, regardless of graft type. Increases in plasma dopamine concentrations were associated with decreases in rotational behavior. Five months after transplantation, grafted chromaffin cells demonstrated catecholamine fluorescence, tyrosine hydroxylase (TH) and chromogranin A immunoreactivities, and expression of TH mRNA. It is concluded that adrenal medulla grafts produce decreases in apomorphine-induced rotation through a combination of two independent effects. One is a specific effect of adrenal medulla grafts. The second is a nonspecific effect that requires an intact adrenal gland and may be related to increases in plasma catecholamine concentrations.

Chapter 30 Intraventricular brain allografts and xenografts: studies of survival and rejection with and without systemic sensitization

Publisher Summary An understanding of the immunological properties of the brain ventricular system is important for understanding the possible forms of brain grafting procedures, which can be applicable clinically. This chapter describes a series of experiments, and examines: (1) the survival of allografts and xenografts in the lateral ventricle, (2) the vulnerability of established brain allografts to rejection following systemic immunization, and (3) the histochemical features of the immune response to intraventricular allografts and xenografts. When brain allografts are left in place for more than one year, there is little or no evidence of graft destruction. However, sensitization by subcutaneous implantation of skin or adult brain tissue can provoke a partial immune response, including the appearance of cells with immunoreactivity to immune response gene-associated antigen (Ia) and helper T antigen. The immune response to xenografts includes accumulation of both Ia and helper T immunoreactive cells. The immunological privilege for allografts can be abrogated by systemic immunological sensitization. The data presented in the chapter suggests that intraventricular brain tissue grafts are afforded immunological privilege at least in part because of the unusual properties of the brain ventricular system.

Cell adhesion molecules (CAMs) in adrenal medulla in situ and in vitro: Enhancement of chromaffin cell L1Ng-CAM expression by NGF

We have studied the expression of the cell adhesion molecules (CAMs) L1/Ng-CAM, N-CAM, J1/tenascin, and myelin-associated glycoprotein and their common carbohydrate L2/HNK-1 epitope in normal rat adrenal gland sections as well as in adrenal medulla cell culture with and without NGF stimulation. In situ L1/Ng-CAM was observed on the surface of some but not all chromaffin cell clusters, including their closely associated extracellular matrix (ECM). N-CAM immunoreactivity was present on all chromaffin cells and ECM. The ECM of whole medullas also expressed J1/tenascin molecules. In long-term cultures, nerve growth factor (NGF) stimulation enhanced L1/Ng-CAM, N-CAM, and Thy 1.1 immunolabeling on chromaffin cells and their processes. Process outgrowth was greater from chromaffin cell clusters containing S-100 positive Schwann cells as compared to dispersed single chromaffin cells. When long bundles of chromaffin cell fibers were present, S-100, L1/Ng-CAM, and N-CAM positive Schwann cells were always found and were grouped in distinct clusters in the intervals between the chromaffin cells. In some areas, however, after NGF stimulation some chromaffin cell process development occurred despite an apparent lack of close contact with Schwann cells. NGF-activated chromaffin cells also demonstrated neurofilament- and vimentin-like-immunoreactive filaments within cell bodies and their processes. Chromaffin cells were usually found on a layer of N-CAM and fibronectin positive fibroblasts, and often were associated with laminin-immunoreactive material. These data suggest a possible role of N-CAM and L1/Ng-CAM as well as ECM laminin in process outgrowth from chromaffin cells.

Human cortical neuronal cell line (HCN-1): Further in vitro characterization and suitability for brain transplantation

The human neuronal cell-1 (HCN-1) line has recently been established. Under favorable conditions, these cells differentiate into mature neuronal phenotypes. Here we report on further characterization of these cells. Cultured HCN-1 cells express fibronectin immunoreactivity and grow well on fibronectin substrate but do not respond to human bFGF. In the undifferentiated state, some HCN-1 cells show MHC class I antigen expression. After differentiation, HCN-1 cells and their processes are MHC class I negative. On the other hand, interferon-γ stimulation enhances MHC class I expression but does not induce MHC class II immunoreactivity. Our in vitro data indicate that HCN-1 cells express mixed characteristics, including both neuronal and mesenchymal markers, and are consistent with the suggestion that the HCN-1 cell line resembles an immature neuroepithelial cell precursor with a complex origin. One possible application of the use of the HCN-1 cells includes intracerebral transplantation. We also examined the survival of dissociated HCN-1 cells implanted into rat brain parenchyma. The host animals were not immunosuppressed. Despite expression of MHC class I antigens, small clusters of HCN-1 cells survived in the rat brain. These xenografts did not induce distinct immunological responses within the host brain tissue. Surviving HCN-1 cells demonstrated similar features to those observed in culture. Our preliminary results suggest that the HCN-1 cell line would be suitable for intracerebral transplantation in primates or humans. However, it may be that short-term host immunosuppression or addition of HCN-1 cell differentiation factors would be beneficial for enhanced cell survival.