The binding of a monoclonal antibody reactive with pp60v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synaptic region

A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.     

Relationship between clinical and radiological diagnostic criteria for Alzheimer's disease and the extent of neuropathology as reflected by 'stages': a prospective study

The distribution of pathology related to Alzheimer's disease (AD) is not uniform throughout the brain. Sites which have a predilection for the development of Alzheimer-type pathology are the limbic regions and neocortical association areas. The changes in these areas of the brain develop gradually, following a well-determined sequence that allows a pathological staging of the disease process. According to the staging hypothesis, the first pathological alterations develop in the transentorhinal and entorhinal regions. The neurofibrillary pathology then spreads into the hippocampus, but not until the final stages does it affect the neocortex. In this study we analyse the relationship between the pathological stages of AD, according ot the staging hypothesis, and the clinical diagnosis in a prospectively assessed patient group. Prediction of any given pathological stage from the clinical diagnosis was found to be poor. This may be partly due to the fact that additional pathologies can alter the clinical picture and severity of dementia in patients who are only in the initial stages of AD. Nevertheless, the NINCDS-ADRDA clinical criteria had a high sensitivity for detection of AD-related pathology: the 'probable AD' category included 22/38 (57.9%) of those in the late isocortical stage, while the 'possible AD' category included 19/23 (82.6%) of those in the limbic stage. Using proposed neuro-imaging protocols for improved identification of patients with AD-related pathology, we largely identified subjects in whom the extent of pathology had spread to the neocortex.     

Monocyte-derived HLA-G acts as a strong inhibitor of autologous CD4 T cell activation and is upregulated by interferon-beta in vitro and in vivo: rationale for the therapy of multiple sclerosis

Peripheral antigen presenting cells (APCs) contribute to the maintenance of immune tolerance and are considered to play a critical role in promoting the (re)activation of autoreactive T cells in multiple sclerosis (MS). Interferon-beta (IFN-beta) is the principle immune-modulatory agent used in the treatment of MS, but its mechanism of action remains elusive. HLA-G is a non-classical MHC molecule (MHC class Ib) attributed chiefly immune-regulatory functions. We here investigated the role of monocyte-derived HLA-G in the immune-regulatory processes of MS and its implications for current immune-modulatory therapies. Monocytes constitutively express cell surface HLA-G1 and soluble HLA-G5. Comparison of monocytic HLA-G expression between patients with relapsing-remitting MS (n=17) and healthy donors (n=20) revealed significantly lower levels of HLA-G1 protein in MS patients. However, both groups showed a significant upregulation of HLA-G in response to IFN-beta in vitro. Serial measurements of HLA-G mRNA levels in MS patients before and during IFN-beta therapy corroborated the relevance of these results in vivo: 1 month after initiation of IFN-beta1b therapy (n=9), HLA-G1 and HLA-G5 were significantly increased compared to baseline levels and remained elevated during treatment for 6 months (n=3). Importantly, functional experiments demonstrated that monocyte-derived HLA-G inhibits both Th1 (IFN-gamma, IL-2) and Th2 (IL-10) cytokine production by antigen-stimulated autologous CD4 T cells. Soluble HLA-G added to antigen-specific T cell lines (TCLs) has similar effects on the release of cytokines and reduces T cell proliferation. Although both IFN-beta and IFN-gamma strongly enhance HLA-G1 and HLA-G5 expression by monocytes in vitro, IFN-beta leads to a stronger relative upregulation of HLA-G compared to classical MHC class I molecules than stimulation with IFN-gamma. Taken together, monocyte-derived HLA-G mediates the inhibition of autologous CD4 T cell activation and might be involved in immune-regulatory pathways in the pathogenesis of MS. We conclude that some desirable immune-modulatory effects of INF-beta might be accomplished via the upregulation of the immune-tolerogenic molecule HLA-G.