The expression of the endothelial cell antigen CD34 in demyelinating disease

In this study, the antigenic expression of CD34, a 110 kDa glycoprotein which is expressed on human haemopoletic cells and vascular endothelium, has been assessed in variety of neuropathological conditions, including infectious and demyelinating disease. Using immunop0eroxidase staining on paraffin sections, the immunohistochemical results show that CD34 antigen is expressed widely on human CNS endothelium in grey and white matter, in the eye including retina, and in the anterior and posterior lobes of the pituitary. In demyelinating disease CD34 antigen expression was not detected in old lesions. CD34 endothelial positivity was observed in areas of gliosis, vasogenic oedema, vascular disease and in Alzheimer's and Parkinson's disease pathology. A general pattern emerger, with CD34 antigen reactivity predominantly but positive in adjacent non-inflamed tissue, irrespective of myelin pathology. We conclude that perivascular inflammation is a key factor in the absence of immunoreactivity of CD34 in the CNS in demyelinating disease.     

Altered angioarchitecture in selected areas of brains with Alzheimer's disease

Summary It was the aim of this study to determine, qualitatively and quantitatively, alterations in the blood vessels of brains removed postmortem from patients with Alzheimer's disease (AD), and to compare these findings with the appearance of cerebral blood vessels in a group of individuals without brain disorders. Celloidin sections of brain tissue from four cerebral areas, pre-frontal (Brodmann's area 9), basal forebrain, sensorimotor, and hippocampus, were subjected to an alkaline phosphatase reaction to facilitate the evaluation of the vascular distribution. The vascular density in five sections was determined by counting the number of vascular intersections with a microscopic test grid of 100 squares; ten fields per section were examined in this manner. Analysis of 16 AD and 6 control brains, showed that there was a striking and statistically significant reduction in the vascular net density specifically in the basal forebrain region and the hippocampus of AD brains. In addition, vessels in the AD brains exhibited extensive topographical changes, such as kinking and looping. These results indicate that modifications in vascular density are present in AD brains with a marked regional specificity.Supported in part by the St. Louis Alzheimer's Disease and Related Disorders Association and the Department of Community Medicine. St. Louis University Medical Center     

Interleukin-2 receptor in peripheral blood lymphocytes of Alzheimer's disease patients.

In this study, we investigated the differences in the incidence rate of human leukocyte antigen-DR (HLA-DR) and interleukin 2 receptor (IL-2R) in the peripheral blood lymphocytes of 13 women who were diagnosed clinically as having Alzheimer's disease (AD group), and 13 healthy women with no mental disorders (control group). As a result, the AD group showed a markedly higher rate of lymphocyte subsets of CD4+HLA-DR+, CD4+IL-2R+, CD8+HLA-DR+, CD8-HLA-DR+ and CD8+IL-2R+. This finding indicates a possible immune reaction occurring in the peripheral blood of AD patients. In addition, these higher rates correlate well with the changes in the immune system reported in the postmortem brains of AD patients. Our findings indicate that immunological interactions exist between the central nervous system and the peripheral blood lymphocytes of AD patients, and AD might be induced by an immune reaction occurring in the brain.     

Decreased release of the angiogenic peptide vascular endothelial growth factor in Alzheimer's disease: recovering effect with insulin and DHEA sulfate

Changes of vascular endothelial growth factor (VEGF) secretion have recently been demonstrated in patients with Alzheimer's disease (AD). Since VEGF has been involved in brain angiogenesis, neuroprotection and cerebromicrovascular exchange of substrates and nutrients, the study of VEGF could have important relapses into the pathogenesis and treatment of AD. Within this context, 35 healthy subjects (16 of young and 19 of old age), 18 patients with dementia of the vascular type (VAD) and 22 with dementia of the Alzheimer's type (AD) were included in the study. VEGF levels were determined in the supernates of circulating natural killer (NK) immune cells isolated by immunomagnetic separation (pure CD16 + CD56 + NK cells at a final density of 7.75 x 10(6) cells/ml). VEGF was measured in spontaneous conditions (without modulation) and after exposure of NK cells with IL-2, lipopolysaccharide (LPS), dehydroepiandrosterone sulfate (DHEAS), LPS + insulin, amyloid-beta (Abeta) fragment 1-42, the inactive sequence Abeta(40-1) and Abeta(1-42) + insulin. A significant decrease in VEGF released by NK cells was demonstrated in AD subjects compared to the other groups. No differences of VEGF levels were found between healthy subjects of old age and the VAD group. The incubation with LPS and DHEAS significantly increased, in a dose-dependent manner, VEGF levels in AD as well as in healthy subjects of young and old age and in VAD patients. The incubation of NK cells with Abeta(1-42) completely suppressed VEGF generation in AD subjects, also reducing VEGF release in the other groups. The co-incubation of NK with LPS + insulin, at different molar concentrations, significantly restored (4- and 6-fold increase from LPS alone) VEGF in AD, also enhancing VEGF secretion in healthy subjects and the VAD group, while the co-incubation of NK with Abeta(1-42) + insulin promptly abolished the negative effects of Abeta(1-42) on VEGF release. These data might suggest that the decreased VEGF secretion by peripheral immune cells of AD subjects could have a negative role for brain angiogenesis, neuroprotection and for brain microvascular permeability to nutrients, increasing brain frailty towards hypoxic injuries. On the contrary, insulin and DHEAS could have beneficial effects in AD, as well as in VAD and in physiological aging, by increasing, in a dose-dependent fashion, VEGF availability by peripheral and resident immune and endothelial cells, so contributing to increase its circulating pool.     

Evidence that immunoglobulin-positive neurons in Alzheimer's disease are dying via the classical antibody-dependent complement pathway

A recent study provided evidence that immunoglobulins (Igs) are not only present in Alzheimer's disease (AD) brains, but are also immunohistochemically detected in and/or on a particular population of pyramidal neurons that appeared morphologically degenerative in contrast to neighboring normal-appearing Ig-negative neurons. Because little has been reported about these Ig-positive neurons, the objectives of this study were to characterize the inflammatory profile of these neurons in the AD brain by determining if they possess complement components and are associated with reactive microglia. The data showed that the Ig-positive neurons had complement C1q and C5b-9 proteins and appeared degenerative. Furthermore, D-related human leukocyte antigen (HLA-DR)-positive fibers of reactive microglia were spatially closer (p < 0.001) and often in contact with the Ig-positive neurons than the Ig-negative neurons. Collectively, these data suggest that the Ig-positive neurons detected in AD brains are dying from the processes of the antibody-induced classical complement pathway.     

Clusterin (Apolipoprotein J) Protein Levels Are Increased in Hippocampus and in Frontal Cortex in Alzheimer's Disease

We studied the multifunctional protein clusterin (apolipoprotein J, SGP-2, SP-40,40) in brain tissue using quantitative Western blotting and immunohistochemistry. The material included postmortem brains from 19 patients with Alzheimer's disease (AD), 6 with vascular dementia (VAD), and 7 age-matched control subjects. Intense clusterin staining was found in the soma of both neuronal and astroglial cells. In addition, positive staining was found in a portion of senile plaques (SP) in AD brains. Quantitative analysis showed that clusterin levels were significantly increased in AD, both in frontal cortex (150% of the control value,P = 0.002) and in the hippocampus (179% of the control value,P < 0.001), while normal clusterin levels were found in cerebellum (104% of the control value). No significant changes were found in VAD. Within the AD group, there was a significant negative correlation between clusterin levels in hippocampus and severity of dementia (r = −0.40), while no such correlation was found in frontal cortex (r = 0.12). No significant correlations were found between clusterin levels and the number of SP or neurofibrillary tangles. No significant differences in clusterin levels were found in any brain region between AD patients possessing different numbers of the ApoE4 allele. The increased clusterin levels in AD brain, together with the absence of a correlation between SP counts and clusterin levels, and the finding that clusterin is only found in a smaller portion of SP do not suggest a link between clusterin and β-amyloid dependence. Instead we hypothesize that the increase is part of a regional response in AD brain.     

Clinically diagnosed Alzheimer's disease: autopsy results in 150 cases

One hundred fifty autopsy brains from patients with clinically diagnosed Alzheimer's disease (AD) were examined pathologically. The brains were received consecutively over a 3-year period from numerous sources as part of a research program in which one brain half was frozen for biochemical studies and the other half was fixed in formalin. One hundred thirty-one (87%) of the 150 cases fulfilled histological criteria for AD, with or without additional findings, such as Parkinson's disease or stroke. At least a minimal degree of amyloid angiopathy was found in every brain showing histopathological abnormalities of AD. Twenty-three (18%) of the 131 AD brains had Lewy bodies in neurons of the substantia nigra. Thirteen of the 19 non-AD cases were diagnosed as other neurodegenerative disorders. In only 2 cases was no histological correlate for the patient's dementia found. We conclude that (1) the many physicians who diagnosed these cases did so highly accurately; (2) degenerative changes in the substantia nigra were more common in patients with AD than has been reported for the general aged population; (3) amyloid angiopathy was a constant accompaniment of AD, although its severity varied widely; (4) vascular dementia was rarely clinically misdiagnosed as AD; (5) neuropathological findings were insufficient to account for the clinical syndrome of dementia in less than 2% of cases; (6) the histological criteria established by the National Institutes of Health/American Association of Retired Persons Workshop on the Diagnosis of Alzheimer's Disease worked well in assessing this large series.     

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.     

Soluble Abeta oligomers ultrastructurally localize to cell processes and might be related to synaptic dysfunction in Alzheimer's disease brain

Soluble Abeta oligomers have recently been considered to be responsible for cognitive dysfunction prior to senile plaque (SP) formation in Alzheimer's disease (AD) brain. To investigate the ultrastructural localization of soluble Abeta oligomers, we conducted the post-embedding immunoelectron microscopic (IEM) study using an antibody against a molecular mimic of oligomeric Abeta. We examined autopsied brains from AD patients and nondemented subjects. Oligomer-specific immunoreactions detected by IEM tended to be found with higher density (1) in AD than in nondemented brains and (2) at the axon and axon terminal in AD than in nondemented brains. These findings imply that soluble Abeta oligomers might be related to synaptic dysfunction in AD brain.     

Age-related ubiquitin deposits in dystrophic neurites: an immunoelectron microscopic study

Widespread neuritic dystrophy is a hallmark of Alzheimer's disease (AD) and, in a less severe form, of brain ageing in various mammalian species. By immunohistochemistry, diffuse dot-like staining for ubiquitin (Ubq), a polypeptide involved in the degradation of abnormal and short-lived proteins, has been associated with human brain ageing. The nature of the Ubq deposits was investigated by immunogold electron microscopy on autopsy samples from aged human and dog brains. Most of the dot-like staining was localized to the white matter and corresponded to myelinated dystrophic neurites filled by Ubq-labelled lysosomal dense bodies. They did not contain paired helical filaments or multilamellar bodies. A minority of Ubq deposits was represented by amorphous densities in focal enlargements of the myelin sheaths. Our findings show that the spectrum of Ubq changes in ageing brain is wider than formerly recognized, and support the hypothesis that a defective regulation of the lysosomal system might be involved in the pathogenesis of structural abnormalities both in the ageing brain and in Alzheimer's disease.     

Expression of matrix metalloproteinase-9 and urinary-type plasminogen activator in Alzheimer's disease brain

OBJECTIVE: Urinary-type plasminogen activator (uPA) binding to uPA receptor (uPAR) promotes the activation of matrix metalloproteinase-9 (MMP-9), which degrades amyloid beta protein (Abeta) in vitro. We investigated the expression of MMP-9, uPA, and uPAR in post-mortem brains from patients with Alzheimer's disease (AD) and those with vascular dementia (VD). MATERIAL AND METHODS: We used immunohistochemistry to examine the sections of the parietal lobe and hippocampus from 4 AD and 3 VD patients. The anti-MMP-9 antibody, anti-uPA antibody, and anti-uPAR antibody were used to perform immunohistological analysis. RESULTS: In the brain tissues from the AD patients, we found expression of MMP-9 in the cytoplasm of neurons, neurofibrillary tangles, senile plaques, vascular walls and uPAR expression in the cytoplasm of neurons and vascular walls. uPA was detected only in the vascular walls. On the other hand, we could not find expression of MMP-9, uPAR and uPA in the brain tissues of the VD patients, except for the vascular walls. CONCLUSION: The neurons in the AD brains expressed MMP-9 and uPAR. MMP-9 may be produced for the degradation of Abeta, but uPA, which activates MMP-9, was not immunolocalized to the neurons in the AD brains.     

Vascular dementia in leukoaraiosis may be a consequence of capillary loss not only in the lesions, but in normal-appearing white matter and cortex as well

We investigated capillary density in 12 subjects with leukoaraiosis (LA), in 9 age-matched normal subjects, in 7 cases of Alzheimer's disease (AD), and 4 after whole-brain irradiation for brain tumors. In the LA study (which as been published), autopsy brains were evaluated by MRI. The presence of LA was indicated by confluent or patchy areas of hyperintensity in the deep white matter. We employed a stereology method using computerized image processing and analysis to determine microvascular density. Afferent vessels (arterioles and capillaries, but not veins or venules) were stained for alkaline phosphatase in 100 microm thick celloidin sections. Microvascular density in LA lesions in the deep white matter (2.56%) was significantly lower than in the corresponding deep white matter of normal subjects (3.20%, p=0.0180). LA subjects demonstrated decreased vascular density at early ages (55-65 years) when compared to normal subjects. Our findings indicate that LA affects the brain globally, with capillary loss, although the parenchymal damage is found primarily in the deep white matter. In ongoing studies of the deep white matter in AD brains, we found a pattern of decreased vascular density compared to normal, as well as an age-related decline. In the four irradiated brains, we found very low vessel densities, similar to those found in LA, without an additional age-related decline.     

Quantification of Alzheimer pathology in ageing and dementia: age-related accumulation of amyloid-beta(42) peptide in vascular dementia

Clinicopathological observations suggest there is considerable overlap between vascular dementia (VaD) and Alzheimer's disease (AD). We used immunochemical methods to compare quantities of amyloid-beta (Abeta) peptides in post mortem brain samples from VaD, AD subjects and nondemented ageing controls. Total Abeta peptides extracted from temporal and frontal cortices were quantified using a previously characterized sensitive homogenous time-resolved fluorescence (HTRF) assay. The HTRF assays and immunocapture mass spectrometric analyses revealed that the Abeta(42) species were by far the predominant form of extractable peptide compared with Abeta(40) peptide in VaD brains. The strong signal intensity for the peak representing Abeta(4-42) peptide confirmed that these N-terminally truncated species are relatively abundant. Absolute quantification by HTRF assay showed that the mean amount of total Abeta(42) recovered from VaD samples was approximately 50% of that in AD, and twice that in the age-matched controls. Linear correlation analysis further revealed an increased accumulation with age of both Abeta peptides in brains of VaD subjects and controls. Interestingly, VaD patients surviving beyond 80 years of age exhibited comparable Abeta(42) concentrations with those in AD in the temporal cortex. Our findings suggest that brain Abeta accumulates increasingly with age in VaD subjects more so than in elderly without cerebrovascular disease and support the notion that they acquire Alzheimer-like pathology in older age.     

Localization and expression of cdc2 and cdk4 in Alzheimer brain tissue.

Two regulators of the eukaryotic cell cycle, cell division cycle 2 (cdc2) and cyclin-dependent kinase 4 (cdk4), have been reported to be related to Alzheimer's disease (AD) pathology, and especially to hyperphosphorylated tau protein. Using well-characterized polyclonal antibodies which recognize the C termini of cdc2 kinase and cdk4, we examined by immunohistochemistry brain tissues from patients with non-neurological conditions, AD and cerebral infarction. Semiquantitative mRNA analysis by RT-PCR was also done using non-neurological and AD brains. In AD, as previously reported, the antibody to cdc2 showed positive staining of a few intracytoplasmic neurofibrillary tangles (NFTs). In addition, this antibody gave positive immunolabelling in astrocytes and capillaries in all brains studied. In both AD and cerebral infarct cases, the staining of astrocytes was more intense than in non-neurological brain tissue. In all cases, the antibodies to cdk4 showed positive immunolabelling in the nuclei of all cell types except neurons. In AD tissue, the antibody showed additional staining of neuronal nuclei and cytoplasm. In contrast to a previous report, we did not find positive labelling of NFTs with the anti-cdk4 antibody. In infarct areas, particularly strong nuclear staining in glial cells was seen. The relative levels of cdk4 mRNA in AD brains were higher than those in controls. These data suggest that cdc2 kinase appears in glial cells capable of cell division and may play a role in the regulation of amyloid precursor protein processing and NFT formation in neurons. As suggested in a report on rat brain, neuronal expression of cdk4 may reflect some pathological process in damaged cells in AD.     

Topographic heterogeneity of amyloid B-protein epitopes in brains with various forms of neuronal ceroid lipofuscinoses suggesting defective processing of amyloid precursor protein

Summary To verify our hypothesis of defective protease inhibitor domains that are encoded by abnormal processing of amyloid precursor protein (APP) in brains of patients with neuronal ceroid lipofuscinoses (NCL), immunohistochemical and cytochemical studies were performed with monoclonal antibodies (mAbs) directed against various domains of APP. For the studies, 22 autopsy brains were used: 12 with different forms of NCL, and 10 control brains. The staining procedure for the avidin-biotin complex (ABC) technique and the postembedding gold-labelled procedure for electron microscopy (EM) were employed. Of all mAbs used for the study, only mAbs generated against amyloid B-protein bound to neural tissue were affected with NCL. The strongest immunostaining of neurons and of some reactive glial cells was found in brains with the juvenile form of NCL. Only in the infantile form of the disease were some neurons overloaded with storage material weakly immunoreactive. In brains of patients with the adult form of NCL, immunoreactivity was found in affected neurons and in extracellularly deposited material of senile plaques. The results of EM study showed that the immunoreactivity was restricted to lysosomal cytosomes in neural tissue with any form of NCL selectively localized on the curvilinear and fingerprint proteinaceous component of ceroid lipofuscin. Studies performed on control aging brains and Alzheimer's disease (AD) brains confirmed previous observations of immunoreactivity being found diffusely in the protein component of some neurons containing lipopigment. The defective processing of APP in brains with NCL and AD and in ageing brains is discussed. Our present results support the notion of heterogeneity of ceroid lipofuscin storage material in various forms of NCL and underline the hypothesis that abnormalities found in the protease inhibitors or APP in the proteinaceous composition of storage lipopigment could be a key to the unknown etiology of NCL.Supported by NIH grant NS23717     

The use of real-time PCR analysis in a gene expression study of Alzheimer's disease post-mortem brains

The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.     

Fas antigen expression in brains of patients with Alzheimer-type dementia

Fas antigen (CD95) is a cell surface protein that mediates apoptosis. We have investigated the immunohistochemical localization of Fas antigen in postmortem brain tissue from control subjects, patients with Alzheimer-type dementia (ATD), and from a few patients with diffuse Lewy body disease, progressive supranuclear palsy and adrenoleukodystrophy. In all brains, including controls, vascular endothelial cells and residual blood plasma were weakly stained. In ATD brains, senile plaques and a small number of star-like cells were also stained for Fas. Fas-positive cells were identified as astrocytes on a morphological basis. Fas-positive astrocytes were also present in brains of patients with neurological diseases other than ATD. In double immunostaining for Fas and glial fibrillary acidic protein (GFAP). a small number of cells were positive for both antigens. The majority of Fas-positive astrocytes were, however, negative for GFAP. This implies the downregulation of GFAP production in these cells. Doubly labeled astrocytes were also fouond around senile plaques, suggesting that the Fas immunoreactivity in senile plaques was derived from astrocytic membranes. The results of this study indicate that Fas antigen is expressed by a subset of reactive astrocytes in degenerative neurological diseases. Such astrocytes may undergo the Fas-mediated apoptotic process.     

Synaptic loss in Alzheimer's disease and other dementias

The extent and location of neuronal losses necessary or sufficient to produce dementia in patients with Alzheimer's Disease (AD) is unknown. To approach this question, we studied synaptic terminals in postmortem brain tissue utilizing immunohistochemical techniques. We used antibodies against two proteins found in synaptic terminals--synapsin I and synaptophysin--as synaptic markers in the hippocampal complexes of eight patients with autopsy-proven AD and eight nondemented control subjects. Quantitative microscopy measured the regional density of synaptic staining. All AD patients showed a striking decrease in synaptic staining in the outer half of the molecular layer of the dentate gyrus compared with control brains, where the density of synaptic terminals was uniform throughout. In an additional patient with progressive degenerative dementia but without plaques or tangles on neuropathologic examination, similar depletion of synaptic staining was seen in the dentate gyrus. Quantitative densitometric analyses confirmed the focal decrease in synaptic staining in the outer half of the molecular layer in demented patients. We also found a slight increase in synaptic staining in the inner half of this layer.     

Histochemically-reactive zinc in amyloid plaques, angiopathy, and degenerating neurons of Alzheimer's diseased brains

Excess brain zinc has been implicated in Alzheimer's neuropathology. Here we evaluated that hypothesis by searching the brains of Alzheimer's patients for abnormal zinc deposits. Using histochemical methods, we found vivid Zn2+ staining in the amyloid deposits of dense-core (senile) plaques, in the amyloid angiopathy surrounding diseased blood vessels, and in the somata and dendrites of neurons showing the characteristic neurofibrillary tangles (NFT) of Alzheimer's. In contrast, brains from age-matched, non-demented subjects showed only occasional staining for Zn2+ in scattered neurons and possible plaques. A role of abnormal zinc metabolism in Alzheimer's neuropathology is suggested.     

Pick's disease immunohistochemistry: new alterations and Alzheimer's disease comparisons

Pick's disease (PD) brains were examined immunohistochemically for the expression of antigens known to be associated with Alzheimer's disease (AD) lesions. Most antibodies which label intracellular neurofibrillary tangles (NFTs) in AD were found to stain Pick bodies (PBs). Among them was the monoclonal antibody A2B5, which is known to recognize neuronal surface gangliosides. This result indicates that membrane proteins are probably incorporated into PBs as into NFTs. However, PBs, in contrast to NFTs, showed a paucity of staining for heparan sulfate glycosaminoglycan and basic fibroblast growth factor (bFGF). Staining for midline, seen in senile plaques in AD, was not seen in PD. The relative lack of staining for these two neurotrophic factors in PD brain may reflect underlying mechanisms which are distinct from those in AD. We also describe two glial abnormalities in PD: glial fibrillary tangles and clusters of granules positive for the complement protein C4d in the hippocampal dentate fascia. These are presumably related to complement-activated oligodendroglia, and both pathological structures are more abundant in advanced cases, suggesting that they may be hallmarks of the disease progression.