Peripheral distribution of dermatan sulfate proteoglycans (decorin) in amyloid-containing plaques and their presence in neurofibrillary tangles of Alzheimer's disease.

We used a polyclonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the small dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristic lesions in Alzheimer's brain. All antibodies demonstrated positive decorin immunostaining in both the amyloid deposits of neuritic plaques (NPs) and the filamentous structures within neurofibrillary tangles (NFTs). Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout NPs containing amyloid fibrils, decorin was primarily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorin was also immunolocalized to the paired helical and straight filaments within NFTs and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque.

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis.

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Early accumulation of heparan sulfate in neurons and in the beta-amyloid protein-containing lesions of Alzheimer's disease and Down's syndrome.

A monoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha 1----4 glucuronic acid-containing determinant in heparan sulfate (HS) chains of a basement membrane-derived heparan sulfate proteoglycan identified and immunolocalized HS specifically to the amyloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle-bearing neurons in the brains of Alzheimer's and Down's syndrome (DS) patients. Ultrastructural immunohistochemistry demonstrated that HS within neurons of Alzheimer's disease (AD) brain was localized to lipofuscin granules, an aging pigment previously shown also to contain beta-amyloid protein (BAP). Heparan sulfate also was localized to neurite-containing, nonfibrillar 'primitive' plaques that also demonstrated positive BAP immunoreactivity in both AD and DS brains. Antibodies to laminin, fibronectin, and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurons of the hippocampus and amygdala as early as 1 day after birth. Young age-matched controls did not demonstrate similar positive HS immunoreactivity in neurons, whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest deposition of BAP was first observed as 'amorphous' or 'diffuse' cortical deposits in DS brain in patients aged 18 and 24 years before the accumulation of fibrillar amyloid (observed in DS patients who are 35 years and older). These cortical deposits also contained positive HS immunoreactivity, implying that HS accumulation in conjunction with the BAP is an early event that ultimately may contribute to the early age-related accumulation (ie, as early as 35 years of age in DS) of NPs, NFTs, and/or CA. Furthermore the colocalization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may be important in lesion development in these two diseases.

Congo red staining on 1 micron de-plasticized sections for detection of lesions in Alzheimer's disease and related disorders.

Neuritic plaques (NPs), neurofibrillary tangles (NFTs) and congophilic angiopathy (CA), the three characteristic lesions in Alzheimer's disease, are easily detected in paraffin sections using light microscopy and specific staining methods including Congo red and Thioflavin S. Identification of these lesions in plastic thick sections (1 micron) is more tedious and relies essentially on morphological criteria. This causes investigators to subsequently analyze large numbers of thin sections under the electron microscope. Since many researchers use electron microscopy for various aspects of Alzheimer's disease and related research, it would be advantageous to have a rapid method enabling the investigator to quickly and reliably identify in thick sections the characteristic NPs, NFTs and/or CA, which can then be used for further analysis at the ultrastructural level. In this context, the present study describes a dependable technique for identifying NPs, NFTs and/or CA in Alzheimer's disease and related disorders and involves Congo red staining on one micron sections after plastic removal.

The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease.

Two immunocytochemical probes were used to specifically identify and localize heparan sulphate proteoglycans (HSPGs) in 17 cases of Alzheimer's disease (AD). A monoclonal (HK-102) and an affinity-purified polyclonal antibody, each recognizing specific domains on the protein core of a basement membrane-derived HSPG, localized HSPGs to the amyloid fibrils present in neuritic plaques (NPs) and congophilic angiopathy (CA) in the brains of Alzheimer's patients, with weak to no immunostaining in neurofibrillary tangles from the same tissues. HSPGs were also demonstrated in "primitive plaques," suggesting that their accumulation takes place during early stages of plaque development. Immunolocalization of HSPGs to subsets of astrocytes and neuronal cells, particularly those in close proximity to NPs and CA, suggested possible involvement of these two cell types in deposition of HS-PGs into the amyloidotic lesions. The current study not only identifies a new component (HSPGs) present in the amyloid deposits of NPs and CA but also suggests that astrocytes, neurons, or both may be involved in its deposition at these sites.

Colloidal gold immunostaining on deplasticized ultra-thin sections.

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.

Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody.

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.

Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold.

Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.

Differences between mentally retarded and nonretarded persons' efficiency of auditory sentence processing.

Mentally retarded adolescents and MA-matched nonretarded children participated in three experiments designed to examine differences in language-processing efficiency. A compressed speech technique was used in Experiments 1 and 2. Experiment 3 relied on a sentence-picture verification procedure. Our results suggest that retarded and nonretarded individuals differ in the speed with which they are able to execute the semantic-analytic processes but not necessarily the phonological encoding processes that are involved in auditory language comprehension. In addition, the data suggest a possible group difference in the quality of the semantic representation encoded during sentence processing.

Peptide-carrier interaction: induction of liposome fusion and aggregation by insulin.

As the roles of peptidic agents in therapy expand, the need for gaining the knowledge for formulating peptides and/or polypeptides becomes increasingly urgent. In an attempt to study various approaches to formulating peptidic agents for therapeutic applications, we investigated the interactions between drug carriers and peptides, using liposomes and insulin as a model. The fusion and aggregation properties of dipalmitoylphosphatidylcholine (DPPC) small, unilamellar liposomes, on the binding of insulin was studied by the techniques of resonance energy transfer of fluorescent labeled lipids, electron microscopy, and right-angle scattering. Within 1 h of adding insulin to DPPC liposomes at 25 degrees C, the average size of the liposomes increased from 239 to 361 A in diameter. There was no further increase in the size of the liposomes after the fused liposomes reached this size. However, the aggregation of the fused liposomes continued to increase for several hours after the insulin-induced fusion stopped. Our results suggest that insulin induces the aggregation of newly fused liposomes, when the temperature is below the gel----liquid crystalline phase-transition temperature (Tc) of the liposomes. The aggregation of fused liposomes is markedly affected by not only the zinc content of insulin but also the pH and ionic strength of the solution. The results of the present study demonstrate that an amphyphilic molecule, such as insulin, could induce the fusion of liposomes via hydrophobic interaction and facilitate liposome aggregation via hydrophilic interaction. Thus, when entrapping insulin by small, unilamellar liposomes, care should be taken to avoid fusion and aggregation.

Semantic processing can facilitate free recall in mildly retarded adolescents.

The efficacy of semantic processing in free recall was investigated in two experiments with EMR adolescents. In Experiment 1, they were taught to use one of two semantic strategies for memorizing a 15-word list. Compared with controls, neither strategy helped recall either in original learning or transfer. In Experiment 2, one of the semantic strategies, a story mnemonic, was investigated further. Rather than being taught to construct their own stories as in Experiment 1, subjects in Experiment 2 were provided with experimenter-composed stories. They showed better immediate recall and retention after 2 months than did no-strategy controls. However, about 1 year after original learning, the retention of experimental and control subjects no longer differed. Discussion focused on the story mnemonic's potential utility and the criteria for judging such potential, e.g., amount of facilitation, ease of training and performance of the strategy, and the degree of its generalizability.

Continuous ambulatory peritoneal dialysis in children: comparison with hemodialysis.

The clinical and biochemical effects of continuous ambulatory peritoneal dialysis in 20 children and of hemodialysis in 16 children were compared over a 2 1/2-year period. Statistically significant differences between the treatment groups included higher hematocrit, higher serum carbon dioxide and cholesterol levels, large intake of calories and protein, and lower systolic blood pressure and rates of transfusion in the patients receiving continuous ambulatory peritoneal dialysis. These patients had more complications than the patients receiving hemodialysis, but hospitalization rates in the two groups were similar. The cost of continuous ambulatory peritoneal dialysis was +19,600 per patient-year; the cost of hemodialysis was +54,300 per patient-year; the cost of hemodialysis was +54,300 per patient-year. There were four treatment failures with continuous ambulatory peritoneal dialysis and one with hemodialysis. Patients treated with both forms of dialysis preferred continuous ambulatory peritoneal dialysis. We conclude that continuous ambulatory peritoneal dialysis is an important alternative to hemodialysis in children.

Electron microscopic immunoperoxidase staining of insulin using 4-chloro-1-naphthol after osmium fixation.

Ultrastructural localization of insulin in B cells of guinea pig pancreas was accomplished after osmium fixation with an immunoperoxidase procedure that utilized 4-chloro-1-naphthol (CN) in the substrate solution. The principal features of this protocol were: a) osmium tetroxide postfixation; b) omission of hydrogen peroxide "etching"; c) use of CN instead of diaminobenzidine in the substrate solution; d) elimination of osmium tetroxide after the substrate reaction; e) uranyl acetate and lead citrate counterstaining. This procedure produces intense specific staining with low background using highly dilute antiserum, and appears to be useful for postembedding immunoperoxidase staining of a variety of peptide antigens in osmium-fixed tissue.

Extended recency effect extended: blocking, presentation mode, and retention interval.

The effect of blocking of stimulus items on the free recall of EMR adolescents was examined. In Experiment 1 a multitrial free-recall list of 15 pictures was presented either simultaneously in groups of 3, or sequentially, one at a time. Consistent ordering was used in both conditions, so that on each trial, each item in each set of 3 pictures was presented contiguously with the other 2 items from that set. In addition, recall came immediately or after a filled or unfilled delay of 24.5 seconds. Results showed that simultaneous presentation led to higher recall, subjective organization, and clustering than did sequential presentation, but analysis of serial-position curves showed a much reduced extended recency effect in comparison with previous studies. Experiment 2 was designed to determine whether the cause of the reduced extended recency was the use of pictures rather than words as stimuli. Stimuli were presented either as pictures, as pictures with auditory labels, or as words with auditory labels, with both simultaneous and consistent ordering for all conditions. Results indicated a strong extended recency effect for all groups, eliminating presentation mode as a causal factor in the data of Experiment 1. We concluded that blocking leads to increased organization and recall over a variety of presentation modes, rates, and block sizes.

Cognitive processes and academic achievement in EMR adolescents.

Twelve cognitive process variables were investigated as predictors of reading and arithmetic achievement and studied for internal structure, with 115 EMR adolescent subjects. Stepwise regression and factor analyses were employed to study prediction and structure, respectively. Memory variables were the most important predictors of reading. They were also involved in arithmetic but to a lesser extent. The data suggested that the ability to generate and utilize strategies facilitating the recall of unstructured material as well as the capacity to be sensitive to strategy-relevant structure embedded in stimuli were important prerequisites for the acquisition of reading skills. An oddity task, measuring the ability to maintain the same relational focus for successive applications to new stimulus material, was found to be most pertinent for predicting arithmetic computational skills.