Assessment of the purity of isolated cell populations for lineage-specific chimerism monitoring post haematopoietic stem cell transplantation.

Following haematopoietic stem cell transplantation, monitoring the proportion of donor and recipient haematopoiesis in the patient (chimerism) is an influential tool in directing further treatment choices. Short tandem repeat (STR) analysis is a method of chimerism monitoring using DNA isolated from peripheral blood, bone marrow or specific isolated cell lineages such as CD3+ T cells. For lineage-specific STR analysis on cell populations isolated from peripheral blood, a qualitative estimation of the purity of each isolated population is essential for the correct interpretation of the test data. We describe a rapid, inexpensive method for the determination of purity using a simple flow cytometry method. The method described for assessing the purity of sorted CD3+ cells can be applied to any cell population isolated using the same technology. Data obtained were comparable to results from a commercial polymerase chain reaction (PCR)-based method for the assessment of purity (Non-T Genomic Detection Kit, Accumol, Calgary, AB, Canada) (P = 0.59). Of the 303 samples tested by flow cytometry, 290 (95.7%) exceeded 90% purity, and 215 (70.95%) were over 99% pure. There were some outlying samples, showing diversity between samples and the unpredictability of purity of isolated cell populations. This flow cytometry method can be easily assimilated into routine testing protocols, allowing purity assessment in multiple-sorted cell populations for lineage-specific chimerism monitoring using a single secondary antibody and giving results comparable to a PCR-based method. As purity of isolated cell lineages is affected by time after venepuncture and storage temperature, assessment of each sample is recommended to give a reliable indication of sample quality and confidence in the interpretation of the results.

Use of physician and acute care services by persons with and without Alzheimer's disease: a population-based comparison.

OBJECTIVE: To estimate differences in use of acute care services between persons with and without Alzheimer's disease (AD). STUDY DESIGN: Population-based historical cohort study. SETTING/SUBJECTS: All Rochester, Minnesota, residents with AD onset between January 1, 1980, and December 31, 1984 (n = 301), plus one age- and sex-matched nondemented control per case, were identified with a retrospective review of community-based medical records. MEASUREMENTS: Cases and controls were followed in their medical records for number of acute care encounters in the year before January 1 of the index year (year of onset for AD case and their matched control) and in the 4 years following December 31 of the index year. Encounters included clinician visits (office or nursing home), emergency room (ER) visits, hospitalizations (inpatient and outpatient), and inpatient days. Multivariate regression analyses were adjusted for age, sex, pre-index level of illness, and follow-up time. RESULTS: In the pre-index period, cases and controls were similar with respect to level of illness, number of office visits, ER visits, and hospitalizations. In the year before AD onset, 17 cases (7%) had a clinician visit in the nursing home compared with no controls. In the 4 years after the index year, mean length of follow-up was 3.4 years for both cases and controls. The numbers of ER visits, hospitalizations, and inpatient days were similar for cases and controls. Sixty-four percent of AD cases had a clinician visit in a nursing home versus 1% of controls. Controls experienced more office visits than cases (median = 16 vs 10, P < .001). CONCLUSIONS: The onset of AD is not associated with greater use of acute care services. However, neither is the high use of nursing home care offset by fewer ER or hospital encounters.

Regulation of the neuron-specific exon of clathrin light chain B.

Clathrin light chain B (LCB) is a major component of clathrin coated vesicles, which are structures involved in intracellular transport. A neuron-specific isoform of LCB is generated by incorporation of a single exon (EN) using an alternative splicing mechanism that reflects the special demands of neurons, such as axonal transport and synaptic neurotransmission. Here, we demonstrate that this neuron-specific exon is developmentally regulated and is excluded in non-neuronal cells because its 5' and 3' splice sites deviate from the mammalian consensus sequences. A gel retardation assay indicated the presence of a developmentally regulated factor in brain that binds to the neuronal exon. In addition, EN usage is repressed by increasing the concentration of htra2-beta1, a splice factor whose isoform expression is influenced by neuronal activity. We propose that a brain-specific factor is involved in EN recognition during development and adulthood. In addition, ubiquitously expressed splicing factors such as htra2-beta1 are involved in regulating EN expression in the adult brain.

PMA-induced reduction in invasiveness is associated with hyperphosphorylation of MARCKS and talin in invasive bladder cancer cells.

Protein kinase C (PKC) plays a critical role in signal transduction for a variety of cell activation processes. Enhanced PKC activity is often found in cancer cells that show marked invasive and/or metastatic potential. Thus, a specific PKC inhibitor may serve as a tool to reduce invasive or metastatic potential of cancer cells. We show here that phorbol 12-myristate 13-acetate (PMA), a PKC activator, also reduces invasiveness of EJ invasive transitional carcinoma cells. PMA-induced reduction in invasiveness was parallel with inhibition of cell motility. PMA neither induced E-cadherin expression nor augmented cell-matrix adhesion of EJ cells. PMA caused retraction of microspikes from the rim of the cells and consequently rounding of the cellular rim, and the disappearance of microfilaments from the cytoplasm. PMA at 10(-7) M, at which concentration the motility of EJ cells was completely inhibited, down-regulated PKC activity over 5 hr after transient translocation of PKC activity to the membrane fraction. At the same time, PMA induced hyperphosphorylation of MARCKS and talin. During the process of cell movement, actin-binding proteins are in a cycle of phosphorylation and dephosphorylation. Once this cycle is interrupted, cells can no longer maintain the dynamics of cytoskeletal structure. We suggest that retention of the hyperphosphorylated state of MARCKS and talin is responsible for the mechanism(s) by which PMA produces inhibitory activity against invasiveness of EJ cells.

Risk of dementia among persons with diabetes mellitus: a population-based cohort study.

It is unclear whether persons with diabetes are at increased risk for dementia, including Alzheimer's disease. Existing studies are limited by small sample size, selection bias, and case-control designs. This population-based historical cohort study provides estimates of the risk of dementia and Alzheimer's disease associated with adult onset diabetes mellitus (AODM). The sample included all persons with AODM residing in Rochester, Minnesota, on January 1, 1970, plus all persons diagnosed in Rochester or who moved to Rochester with the diagnosis between January 1, 1970, and December 31, 1984. Individuals were followed through review of their complete medical records from AODM diagnosis until dementia onset, emigration, death, or January 1, 1985. Standardized morbidity ratios for dementia and Alzheimer's disease were calculated, using an expected incidence based on age- and sex-specific rates for the Rochester population. Poisson regression was used to estimate risks for persons with AODM relative to those without. Of the 1,455 cases of AODM followed for 9,981 person-years, 101 developed dementia, including 77 who met criteria for Alzheimer's disease. Persons with AODM exhibited significantly increased risk of all dementia (Poisson regression relative risk (RR) = 1.66, 95% confidence interval (CI) 1.34-2.05). Risk of Alzheimer's disease was also elevated (for men, R = 2.27, 95% CI 1.55-3.31; for women, RR = 1.37, 95% CI 0.94-2.01). These findings emphasize the importance of AODM prevention and prompt additional investigation of the relation between AODM and dementia.

Factor VIII:C activity in cryoprecipitate prepared at the Blood Bank, University Hospital of the West Indies, Jamaica - abstract

Cryoprecipitate is the blood product rich in factor VIII: C activity used in the treatment of haemophhlia A. The University Hospital Blood Bank makes most of this product used in Jamaica. Commercially prepared factor VIII concentrates are expensive. We examined the effect of donor variables and techniques of preparation on the potency of cryoprecipitates in order to determine the most efficient production method. Factor VIII: C activity of cryoprecipitate was measured using the activated partial thromboplastin time with a normal plasma pool being used as reference plasma. Donor age and method of freezing the plasma had no effect on potency. Blood group B had a higher yield of factor VIII: C than groups A and O. Potency was decreased by prolonged storage of blood prior to processing (p = 0.015) but was increased by increasing volumes of cryprecipitate (p<0.01). The mean potency of the factor VIII: C was 184 i.u., surprisingly higher than the usually assumed 70 i.u. used for calculating the requirements of our haemophiliacs. We recommend that plasma from fresh tested blood, frozen in - 40§C be used for preparing cryoprecipitate and that a higher value than 70 i.u. of factor VIII: C be used for each bag of cryoprecipitate (AU)

Neuromuscular junctions contain NP185: the multifunctional protein is located at the presynaptic site.

The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal plasma membrane (Su et al., 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals (Perry et al., 1991). In this report we describe double-labelling experiments with avian embryonic striated muscle fibers demonstrating the exclusive presence of the brain-specific protein at the neuromuscular junction. We used indirect rhodamine immunofluorescence labeling with a monoclonal antibody (mAb-8G8) to mark the location of NP185 in muscle combined with fluorescein-alpha-bungarotoxin to mark the postsynaptic location of the acetylcholine receptors (AChRs). We show that the distribution of both NP185 and AChRs has an overall correlation, but the location of NP185 is circumscribed to presynaptic structures adjacent but not overlapping with postsynaptic structures displaying the AchRs. To confirm the identity of NP185, the molecule was extracted from both tissues, partially purified, immunoprecipitated, and identified in Western blots with the mAb 8G8. The mAb reacted with an identical 185 kD protein band purified from both tissues. Based on its properties and specific neuronal location, the NP185 molecule may function in motor nerve terminals by screening membrane proteins, identifying areas of the synaptic plasma membrane, and to anchor these elements with structural proteins for their recycling and transport within the neuronal cellular compartments.

Systemic lupus erythematosus patients with central nervous system involvement show autoantibodies to a 50-kD neuronal membrane protein.

An antibody was detected in the sera from patients with systemic lupus erythematosus (SLE) and central nervous system (CNS) involvement that reacted with a 50-kD antigen in the plasma membrane of brain synaptic terminals. The 50-kD antigen was solubilized with Triton X-100 from preparations enriched with synaptic plasma membranes, and was partially purified by molecular sieve filtration column chromatography. The sera of 19 of 20 CNS-SLE patients showed strong to moderate immunoreactivity with the 50-kD protein in Western blots. Immunoreactivity with the 50-kD protein was also detected in the cerebrospinal fluid of CNS-SLE patients. Control sera from healthy individuals did not react with the 50-kD protein. Low to background reactivity was detected in 35% of a group of SLE patients without CNS manifestations, and in 3% of patients displaying other connective tissue diseases. A total of 100 individuals were tested in this study. Purified autoantibodies to the 50-kD protein from CNS-SLE patients were used for immunofluorescent labeling of neuroblastoma cells. The immunofluorescent staining revealed a distinct macular distribution pattern on the surface of the cell membrane. Taken together, the data suggest that the 50-kD protein may be an important target for autoantibodies, preponderantly found in CNS-SLE patients, and that the antigen may play a role in the pathogenesis of some neurological manifestations in SLE.

Lupus nephritis: prognostic factors in children.

As newer treatment modalities become available for patients with severe lupus nephritis, it becomes increasingly important to identify patients at risk for renal failure. In this study, the records of 90 children presenting with systemic lupus erythematosus over a 13-year period were reviewed. Nineteen were lost to follow-up prior to completion of the study. Of the 71 remaining children, 16 (22%) progressed to chronic renal failure. Persistent hypertension lasting greater than 4 months, anemia, abnormalities of the urinalysis, and elevated serum creatinine level were significantly associated with progression to renal failure. Sex, race, age, abnormalities of creatinine clearance, and 24-hour urine protein collection were not associated with progression to renal failure. Renal biopsies were obtained in 45 children. Biopsies were initially classified according to World Health Organization criteria. Diffuse proliferative glomerulonephritis was significantly associated with progression to renal failure. The 45 biopsies available were reviewed by one of the authors and categorized by activity and chronicity indices. Both the active lesions of fibrinoid necrosis, synechiae, tubular casts, and vasculitic lesions and the chronic lesion of glomerular sclerosis correlated with progression to renal failure. Of the 16 children who progressed to renal failure, 2 had cadaver kidney transplants and are well 5 years posttransplant; 4 had fulminant lupus and died within 1 month of commencing dialysis; 10 began chronic dialysis. Five of the 10 children on chronic dialysis died from sepsis. These data suggest that children with systemic lupus erythematosus who undergo dialysis do poorly.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal protein NP185 is developmentally regulated, initially expressed during synaptogenesis, and localized in synaptic terminals.

Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.

Neuronal protein NP185 in avian and murine cerebellum: expression during development and evidence for its presence in nerve endings.

The neuronal protein NP185 is a neural tissue-specific protein isolated from clathrin-coated vesicles in brain. Using 8G8, a monoclonal antibody (MAb) characterized in our laboratory, we studied the expression and distribution of neuronal protein NP185 in developing avian cerebellum and in mature murine cerebellum. Furthermore, we compared these parameters to that of synapse-specific neuronal protein, synaptophysin, and an axon-specific (i.e., non-synaptic) neuronal protein, neurofilament NF68. We found that NP185 expression temporally and spatially corresponds to avian cerebellar synaptogenesis. In addition, NP185 distribution parallels synaptophysin distribution throughout development, while differing from that of either unassembled or filamentous forms of NF68. The evidence also suggests that embryonic NP185 expression coincides with synaptogenesis, and that NP185 remains concentrated in the terminal boutons of mature neurons. The synapse specificity of NP185 and the recent biochemical properties reported for this protein support the postulate that this molecule may trigger synaptic events and distinguish structurally and functionally active synapses.

Neuronal specific protein NP185 is enriched in nerve endings: binding characteristics for clathrin light chains, synaptic vesicles, and synaptosomal plasma membrane.

The neuronal specific protein NP185, found associated with brain clathrin-coated vesicles, formed a complex with unphosphorylated, but not with phosphorylated, clathrin light chains. The NP185-clathrin light chain complex was associated with casein kinase II activity, which, in the presence of polylysine, phosphorylated clathrin light chain b but not the NP185. The dissociation of this complex with 50% ethylene glycol pH 11.5 suggests that NP185 binds to hydrophobic domains of clathrin light chains. When NP185 molecules were retained by monoclonal antibody-linked Sepharose beads, they bound synaptic vesicles, decoated vesicles and synaptosomal plasma membrane. Immunohistochemistry on mouse cerebellar tissue sections using 8G8, a monoclonal antibody raised against NP185, showed neuronal specific labeling closely following synaptic distribution. In immunoblots, NP185 shares similar epitopes to those detected in another assembly polypeptide, AP-180, an indication that both proteins are identical. It appears that NP185 plays a specific role in nerve ending functions through its ability to induce clathrin to polymerize into cages, its interaction with synaptic vesicles, with the plasma membrane and with clathrin coat components.

Recall of order information by deaf signers: phonetic coding in temporal order recall.

To examine the claim that phonetic coding plays a special role in temporal order recall, deaf and hearing college students were tested on their recall of temporal and spatial order information at two delay intervals. The deaf subjects were all native signers of American Sign Language. The results indicated that both the deaf and hearing subjects used phonetic coding in short-term temporal recall, and visual coding in spatial recall. There was no evidence of manual or visual coding among either the hearing or the deaf subjects in the temporal order recall task. The use of phonetic coding for temporal recall is consistent with the hypothesis that recall of temporal order information is facilitated by a phonetic code.

Novel proteins mediate an interaction between clathrin-coated vesicles and polymerizing actin filaments.

A monoclonal antibody, A-7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn-coated vesicles. The 40-kDa antigen was purified and found to display actin-binding properties. Negative-staining electron microscopy showed that one of the antigens reactive with A-7C11 appears to mediate the association of isolated clathrin-coated vesicles with assembling actin filaments in vitro. Immunofluorescence microscopy of cultured fibroblasts with A-7C11 revealed the antigens aligned with both actin filaments and as punctate structures near the plasma membrane. The data suggest that the interaction between clathrin-coated vesicles and the actin cytoskeleton is mediated by antigens identified by monoclonal antibody A-7C11.

Prevalence and concentration of IgM rheumatoid factor in polyarticular onset disease as compared to systemic or pauciarticular onset disease in active juvenile rheumatoid arthritis as measured by ELISA.

The prevalence and concentration of IgM rheumatoid factor (RF) in children with juvenile rheumatoid arthritis (JRA) and its major disease onset groups remains uncertain. In our study enzyme linked immunoabsorbent assay (ELISA) of 68 children with active JRA showed IgM RF in the area of 67% (16/24) of those with polyarticular onset disease, 26% (7/27) of those with systemic onset disease, and 6% (1/17) of those with pauciarticular onset disease. The median IgM RF concentration was 50-fold higher in polyarticular disease compared to systemic disease. The prevalence of IgM RF in polyarticular disease was greater in those with severe disease (functional classes and 3 and 4), with 90% (9/10) seropositive. By agglutination assay, the prevalence of IgM RF in JRA was significantly less than by ELISA, with 33% of the polyarticular group positive for IgM RF, and none of the systemic group positive, Relatively low concentration IgM RF similar to that seen in systemic JRA was also found in high prevalence in the area of children with non-JRA, systemic rheumatic disease (n = 8). In summary, our study shows by ELISA that high concentrations of IgM RF are found essentially only in the sera of children with polyarticular onset JRA and especially in those with severe disease.

Short-term memory coding by deaf signers: the primary language coding hypothesis reconsidered.

Shand (Cognitive Psychology, 1982, 14, 1-12) hypothesized that strong reliance on a phonetic code by hearing individuals in short-term memory situations reflects their primary language experience. As support for this proposal, Shand reported an experiment in which deaf signers' recall of lists of printed English words was poorer when the American Sign Language translations of those words were structurally similar than when they were structurally unrelated. He interpreted this result as evidence that the deaf subjects were recoding the printed words into sign, reflecting their primary language experience. This primary language interpretation is challenged in the present article first by an experiment in which a group of hearing subjects showed a similar recall pattern on Shand's lists of words, and second by a review of the literature on short-term memory studies with deaf subjects. The literature survey reveals that whether or not deaf signers recode into sign depends on a variety of task and subject factors, and that, contrary to the primary language hypothesis, deaf signers may recode into a phonetic code in short-term recall.

High prevalence of IgA rheumatoid factor in severe polyarticular-onset juvenile rheumatoid arthritis, but not in systemic-onset or pauciarticular-onset disease.

The presence of IgA rheumatoid factor (IgA-RF) has been correlated with severe joint disease in adult rheumatoid arthritis (RA), but IgA-RF has not been reported in juvenile rheumatoid arthritis (JRA). In the present study, IgA-RF was assayed by an enzyme-linked immunosorbent assay and was found in the sera of 14 of 24 children (58%) with active polyarticular JRA. The presence of IgA-RF correlated with the degree of functional disability. In contrast, IgA-RF was not found in the sera of systemic-onset disease patients, regardless of the degree of dysfunction. IgA-RF was detected in only 1 patient with pauciarticular disease, despite the fact that several patients in this group had severe disease. The presence of IgA-RF in polyarticular JRA did not correlate with serum IgA levels, but did correlate with the presence and the level of serum IgM-RF. Thus, the presence of IgA-RF appears to be specific for polyarticular JRA, and shows a correlation with severe disease in this group.

Novel regulatory role of phosphorylated clathrin light chain beta in bovine brain coated vesicles.

The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50-kDa polypeptide in CCVs with phosphorylated LC beta results in a rapid dephosphorylation of the 50-kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LC beta prevent the modulatory effect of phosphorylated LC beta on the 50-kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LC beta has a modulatory role in CCVs. The data also suggest that phosphorylated LC beta promotes activation of a coated vesicle phosphatase.

Interactive video for bilingual ASL/English instruction of deaf children.

Advances in technology make possible certain instructional approaches that heretofore were difficult to implement. One of these advances is the use of computers to present video instructional materials for student-directed learning. In the experimental program described here, we use a bilingual approach to teach aspects of English to deaf children who are fluent in ASL. The goal of this project is to explore ways that ASL and English can be used cooperatively to help deaf students learn more about English.