Toxic Immunoglobulin Light Chain Autoantibodies are Associated with a Cluster of Severe Complications in Older Adult Type 2 Diabetes.

AIMS: To assess neuronal depolarization evoked by autoantibodies in diabetic depression compared to depolarization evoked by autoantibodies in control patients. To determine whether a subset of severe (late-onset) diabetic complications may be mediated in part by toxic immunoglobulin light chains that may increase in diabetic nephropathy. METHODS: Protein-A eluates from plasma of 21 diabetic depression patients and 37 age-matched controls were tested for depolarization in hippocampal or immature neurons. Subsets of depolarizing or non-depolarizing autoantibodies were tested for neurite outgrowth inhibition in N2A neuroblastoma cells or the ability to modulate Ca2+ release in HL-1 atrial cardiomyocytes or in endothelial cells. The stability of depolarizing autoantibodies was investigated by heat treatment (56°C × 30 minutes) or following prolonged exposure to the pro-protein convertase, furin. Gel filtration of active depolarizing autoantibodies was performed to determine the apparent molecular mass of peak neurotoxicity associated with the autoantibodies. RESULTS: Diabetic depression (n = 21) autoantibodies caused significantly greater mean depolarization in neuroblastoma cells (P < 0.01) compared to autoantibodies in diabetic (n = 15) or non-diabetic (n = 11) patients without depression. Depolarizing autoantibodies caused significantly more (P=0.011) inhibition of neurite outgrowth in neuroblastoma cells than non-depolarizing autoantibodies (n = 10) and they evoked sustained, global intracellular Ca2+ release in atrial cardiomyocytes or in endothelial cells. A subset of older diabetic patients suffering with a cluster of nephropathy, non-ischemic cardiomyopathy and/or depression demonstrated the presence of stable light chain dimers having apparent MW of 46 kD and associated with peak neurotoxicity in neuroblastoma cells. CONCLUSION: These data suggest that autoantibodies in older adult diabetic depression cause long-lasting depolarization in hippocampal neurons including adult dentate gyrus neural progenitor cells. The autoantibodies may impair adult dentate gyrus neurogenesis associated with treatment-refractory depression via several mechanisms including suppression of neurite outgrowth, and alteration of membrane excitability. Stable, toxic light chain autoantibody components may contribute to a cluster of severe (late-onset) complications characterized by dysfunction in highly vascularized tissues.

Rab3A is required for brain-derived neurotrophic factor-induced synaptic plasticity: transcriptional analysis at the population and single-cell levels.

Brain-derived neurotrophic factor (BDNF) modulates synaptic strength in hippocampal neurons, in addition to promoting survival and differentiation. To identify genes involved in trophic regulation of synaptic plasticity, we have used a multidisciplinary approach of differential display and family-specific slot blots in combination with whole-cell patch-clamp recordings of dissociated hippocampal neurons. Three hour exposure to BDNF elicited a 2.6-fold increase in synaptic charge and a concomitant induction of 11 genes as revealed by differential display, including the small GTP-binding vesicular trafficking protein Rab3A and the enzyme guanylate cyclase (GC). Slot blot analysis on a population of neurons confirmed an average of 3.1-fold induction of these clones. In contrast, individual pyramidal-like neurons that were first characterized electrophysiologically in the presence of BDNF and subjected to transcriptional analysis displayed more robust increases (4.8-fold), emphasizing the neuronal heterogeneity. Transcriptional changes of Rab3A and GC were accompanied by translational regulation, shown by Western blot analysis. Furthermore, a number of GC-associated and Rab3A effector molecules were induced by BDNF at either the gene or protein levels. The functional role of Rab3A in BDNF-induced synaptic plasticity was assessed using cells derived from Rab3A knock-out mice. These neurons failed to show an increase in synaptic charge in response to BDNF at 10 min; however a late response to BDNF was detected at 20 min. This late response was similar in time course to that induced by postsynaptic activation of glutamate receptors. Our results demonstrate a requirement for Rab3A and may reveal a temporal distinction between presynaptic and postsynaptic mechanisms of BDNF-induced synaptic plasticity associated with learning and memory.

Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling.

Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling.

Flow cytometric determination of cell proliferation in hypertensive blood vessels.

BACKGROUND: Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. METHODS: Pulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. RESULTS: An approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling. CONCLUSIONS: Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.

Remodeling of rat neonatal pulmonary artery: role of matrix metalloproteinases.

We studied whether rapid thinning of large pulmonary arteries of neonatal rats is associated with breakdown of collagen. Pulmonary artery extracts from fetal to 21 days of age were assayed for collagen content and matrix metalloproteinases. Within 3 days postpartum, no changes in collagen content, collagenolytic activity, or levels of stromelysin-l or gelatinase A were observed. After day 3, collagen content and total proteolytic activity increased with little change in matrix metalloproteinase expression. Thus, collagen was not degraded, and the late increases in collagen and total proteolytic activity were probably growth related. Unlike adult rats in which collagen is broken down after reversal of hypoxic pulmonary artery remodeling, collagen is not broken down in neonatal pulmonary arteries during adaptation to extrauterine life.

Isolation of differentially expressed genes in hypertensive pulmonary artery of rats.

Pulmonary artery remodeling is a complex biological process, and a key molecular mechanism regulating this process is selective up- and downregulation of genes. We used reverse transcriptase-polymerase chain reaction (RT-PCR) differential display in a rat model of hypoxic pulmonary hypertension to identify selectively expressed genes relevant to pulmonary artery remodeling. We characterized the pattern of gene expression in hypertensive and normal arteries. Eight differentially expressed cDNAs were selected, isolated, and characterized. Homology searches identified 4 previously identified genes and 4 novel genes that were not further characterized. The known genes were beta-glucoronidase, hemeoxygenase-2 (HO-2), glycerol-3-phosphate dehydrogenase, and cytoplasmic gamma-actin. Each of the 4 known genes was relevant to processes involved in pulmonary artery remodeling. We conclude that mRNA differential display was informative in identifying genes coding for products directly involved in pulmonary artery remodeling.

Expression of matrix-degrading enzymes in pulmonary vascular remodeling in the rat.

Exposure of rats to hypoxia causes pulmonary arterial remodeling, which is partly reversible after return to air. We hypothesized that degradation of excess collagen in remodeled pulmonary arteries in the posthypoxic period is mediated by endogenous matrix metalloproteinases (MMPs). Total proteolytic, collagenolytic, and gelatinolytic activities, levels of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1), and immunolocalization of stromelysin-1 in main pulmonary arteries were determined after exposure of rats to 10% O2 for 10 days followed by normoxia. We observed transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and expression of approximately 72-, 68-, and 60-kDa gelatinases by zymography within 3 days of cessation of hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1 level increased. Immunoreactive stromelysin-1 was localized predominantly in the luminal region of normal and hypertensive pulmonary arteries. These results are consistent with the notion that endogenous MMPs may mediate the breakdown of excess collagen in remodeled pulmonary arteries during the early posthypoxic period.

Gene expression in activated brain microglia: identification of a proteinase inhibitor that increases microglial cell number.

Microglia, the intrinsic immune cells of the central nervous system, are activated in a variety of inflammatory brain diseases in which they play a pathogenetic role. However, mechanisms underlying activation are largely unknown. To begin elucidating molecular mechanisms associated with activation, we characterized the pattern of gene expression in virtually pure dissociated microglial cultures, using RT-PCR differential display. Microglia were activated with bacterial lipopolysaccharide (LPS), a traditional stimulant, and the profile of gene expression was compared to that in basal, control cultures. Activation resulted in altered expression of six genes. The cDNAs were isolated, sequenced and characterized. Homology searches identified three novel genes, and two that exhibited very high sequence similarity to the gene encoding squamous cell carcinoma antigen (SCCA). SCCA (1 and 2) are tandemly arranged genes that encode two serine proteinase inhibitors (serpins). SCCA has been detected exclusively in cancer cells, and is a plasma marker for squamous cell carcinoma. Immunoblot analysis indicated that gene expression was accompanied by a 5-fold increase in the synthesis of SCCA protein in LPS-activated microglia. To assess potential biological actions of the SCCA serpins, SCCA1 protein was added to cultures. SCCA1 altered microglial morphology, and elicited a dramatic, 5-fold increase in cell number within 72 h. The effects appeared to be cell-specific, since the protein had no effect on other cell types: cortical astrocytes and neurons from cortex or basal forebrain were unaffected. We tentatively conclude that SCCA1 may play a cell-specific role in increasing cell number, a critical early step in microglial activation and brain inflammation. More generally, differential display of genes in the microglial model system may help define patterns of expression associated with CNS disease, thereby identifying pathogenetic mechanisms and new therapeutic targets.

Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat.

Pulmonary vascular remodeling, produced by cell hypertrophy and extracellular matrix protein synthesis in response to hemodynamic stress, regresses after reduction of blood pressure, possibly by proteolysis of structural proteins. To test this postulate, we assessed the breakdown of extracellular matrix proteins and expression of collagenase and elastase in pulmonary arteries of rats exposed to hypoxia (10% O2 for 10 d) followed by normoxia. During hypoxia, contents of collagen and elastin increased in pulmonary arteries and latent rat interstitial collagenase was expressed without increased collagenolytic activity or mRNA levels. At 3 days after normoxia, collagen and elastin contents decreased coincident with the new appearance of activated collagenase and transient increases in collagenolytic and elastolytic activities. The amount of immunoreactive collagenase, localized predominately in connective tissue-type mast cells, was increased in the adventitia and media of hypertensive vessels. We conclude that mast cells containing latent collagenase are recruited into the outer walls of pulmonary arteries during remodeling. It is possible that mast cell-derived collagenase contributes to collagen breakdown in pulmonary arteries during early recovery from hypoxia and plays a role in restoration of vascular architecture.

Effect of diet on lung structure, connective tissue metabolism and gene expression.

Nutritional impairment can adversely affect the respiratory system. The structure of the lung is altered by severe calorie-protein restriction in rodents producing an emphysema-like lesion after several weeks of severe caloric restriction. Biochemical and morphological evidence suggests destruction of collagen and elastin in nutritional emphysema. Impaired lung growth may explain the biochemical changes in growing animals. Although the molecular basis of nutritional emphysema is not known, altered gene expression by nutrients affects several metabolic pathways, and examples of the effects of nutrients on gene expression are given. Nutritional emphysema in animals may be relevant to humans because malnutrition may accelerate the progression of the disease in patients with advanced emphysema.

Aberrant splicing of the type III procollagen mRNA leads to intracellular degradation of the protein in a patient with Ehlers-Danlos type IV.

Ehlers-Danlos syndrome type IV (EDS IV) is an autosomal dominant disorder characterized by fragile skin, blood vessels, and internal organs and associated with decreased production, secretion, or thermal stability of type III procollagen. Mutations in the gene for type III procollagen have been identified in patients exhibiting decreased secretion or thermal stability of the protein, but no defect has been elucidated to explain the decreased production of type III procollagen in some patients with EDS IV. We report on a patient with a moderate case of EDS IV who produced decreased amounts of type III procollagen despite normal levels of translatable type III procollagen mRNA. S1 nuclease analysis of the type III procollagen mRNA indicated a defect in the region encoding exon 27. Sequence analysis of cDNA clones and genomic fragments generated by polymerase chain reaction amplification revealed that sequences encoded by exon 27 were absent from 3 out of 5 cDNA clones and that a G at the +5 position of the splice donor site in intron 27 was changed to an A in one allele of the patient's type III procollagen gene. Using a cDNA-genomic DNA hybrid probe in S1 nuclease analysis, fragments consistent with mRNA species containing and lacking exon 27 were detected in a 1:1 ratio. Pulse label and chase experiments in the presence or absence of brefeldin A indicated that most of the type III procollagen molecules synthesized by the patient's fibroblasts were not secreted into the medium but were degraded in the endoplasmic reticulum-Golgi compartment by a nonlysosomal mechanism.

Circulating fibroblast growth factor-like autoantibodies in two patients with multiple endocrine neoplasia type 1 and prolactinoma.

Basic fibroblast growth factor (bFGF) is a potent endothelial cell mitogen found in a variety of normal and tumor tissues. bFGF lacks a classical amino-terminal signal sequence and is not readily detectable in plasma from normal subjects. In earlier studies we showed increased bFGF-like mitogenic activity for parathyroid-derived endothelial cells and (increased) bFGF immunoreactivity (0.24-1.28 ng/mL) in plasma of subjects with multiple endocrine neoplasia type 1 (MEN-1). In the present study we examined the proliferative activity of MEN-1 and normal plasmas (applied to protein-A columns) in calf pulmonary artery endothelial cells. Protein-A-eluted activity in plasma from MEN-1 prolactinoma plasma exceeded activity from normal and MEN-1 nonprolactinoma plasma in three of eight MEN-1 subjects with untreated or recurrent prolactinoma. Protein-A-eluted active fractions from MEN-1 prolactinoma plasma had several properties of an immunoglobulin G, including affinity for antihuman immunoglobulin G (IgG) agarose, sensitivity to thiols, and (prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions) apparent mol wt corresponding to those of the heavy and light chains of IgG. The IgG fraction of MEN-1 prolactinoma plasma had far more activity in endothelial cells than did optimal concentrations of known growth factors or conditioned medium from prolactinoma cells. Endothelial cell bioactivity in protein-A-eluted fractions from MEN-1 prolactinoma plasma was neutralized 70% by rabbit antibodies to intact bFGF. These results imply novel growth stimulatory bFGF-like autoantibodies in a subset of MEN-1 patients with prolactinoma.

Effect of glucocorticoids on collagen accumulation in pulmonary vascular remodeling in the rat.

Administration of corticosteroids may attenuate the development of pulmonary hypertension by inhibiting the cell proliferation and protein synthesis that occur in early pulmonary vascular remodeling. However, in vitro studies show that corticosteroids stimulate collagen synthesis in vascular smooth muscle cells, and corticosteroid administration may be deleterious in stimulating collagen deposition. To test whether corticosteroid treatment promotes vascular collagen production in vivo, we administered triamcinolone diacetate to rats exposed to 10% O2 for 3 days and measured pro alpha 1(I) collagen mRNA and the hydroxyproline/protein ratio in the main pulmonary artery. Triamcinolone treatment (12 mg/kg intraperitoneally, once daily for 3 days) reduced mean right ventricular pressure (11 +/- 1 versus 14 +/- 1 mm Hg) and protein content of pulmonary arteries (1.8 +/- 0.1 versus 2.7 +/- 0.1 mg/vessel) (both p < 0.05). However, corticosteroid treatment produced a dose-related increase in pro alpha 1(I) mRNA levels and increased the ratio of hydroxyproline/protein (47 +/- 2 versus 38 +/- 3 micrograms/mg; p < 0.05). Thus, corticosteroid administration ameliorated the increase in pulmonary hypertension in early hypoxia, but increased the proportion of collagen in the vessel wall. Corticosteroid treatment in pulmonary vascular remodeling may be deleterious in increasing the concentration of collagen in the vessel wall.

Articular chondrocytes secrete IL-1, express membrane IL-1, and have IL-1 inhibitory activity.

Previous work from our laboratory has shown that rabbit articular chondrocytes, like macrophages, produce reactive oxygen intermediates, express Ia antigen, and can mediate immunologic functions such as antigen presentation and induction of mixed and autologous lymphocyte reactions. We were interested in seeing if these cells could secrete interleukin-1 (IL-1) or express membrane form of IL-1 (mIL-1). Using the standard C3H/HeJ thymocyte assay, neither secreted IL-1 nor mIL-1 activity was detected in untreated or LPS-treated chondrocytes. However, the D10.G4.1 proliferation assay showed that chondrocytes, stimulated with LPS, secrete IL-1 and express the mIL-1 in a dose- and time-dependent manner. The IL-1 activity in LPS-stimulated chondrocyte supernatant and on fixed cells could be inhibited by anti-IL-1 antibodies. Sephadex G-75 chromatography of pooled, concentrated LPS culture supernatant resolved into two peaks of IL-1 activity at 13-17 and at 45-70 kDa, respectively. The bioactivity of chromatographic fractions were similar using both the thymocyte and D10.G4.1 bioassays. Western blot analysis of chondrocyte supernatant detects 17-kDa IL-1 beta; no processed 17-kDa IL-1 alpha was seen but IL-1 alpha-specific reactivity was observed at 64 kDa. Immunoblot analysis of chondrocyte lysates shows that cell-associated IL-1 is IL-1 alpha and is 37 kDa in size. PCR analysis shows the presence of mRNA for IL-1 beta and IL-1 alpha in LPS-treated cells; IL-1 beta mRNA was detected in untreated chondrocytes. The inability to detect IL-1 by the thymocyte assay is due to the presence of a chondrocyte inhibitor of IL-1 that can be demonstrated in cell sonicates, supernatants, and on paraformaldehyde-fixed chondrocytes. Chromatography of LPS-stimulated supernatant showed a peak of IL-1 inhibitory activity at 21-45 kDa. Chondrocytes which secrete IL-1 and express mIL-1 could play a critical role in maintaining chronic inflammation in rheumatoid arthritis. Therefore, the ability of chondrocytes to produce both IL-1 and an inhibitor to IL-1 is important in interpreting the mechanism of cartilage matrix maintenance and degradation.

Prevalence of macrolides-lincosamides-streptogramin B resistance and erm gene classes among clinical strains of staphylococci and streptococci.

A total of 332 staphylococcal and 263 streptococcal isolates from three hospital microbiology laboratories were tested with erythromycin, clindamycin, and vernamycin B alpha to determine the prevalence of macrolides-lincosamides-streptogramin B resistance. Constitutive resistance was detected in 28 Staphylococcus aureus isolates (15.5%), 53 coagulase-negative staphylococci (35.1%), and 20 streptococci (7.6%). Inducible resistance was observed in 13 S. aureus isolates (7.2%), 25 coagulase-negative staphylococci (16.6%), and 2 streptococci (0.8%). Eleven coagulase-negative staphylococci (7.3%) exhibited a novel phenotype, namely inducible resistance to erythromycin and vernamycin B alpha but not clindamycin. Among the staphylococci, two variants of the inducible phenotype detected with the agar diffusion assay correlated with the presence of classical ermA or ermC genes, respectively, by dot-blot analysis. The prevalence of the staphylococcal phenotypes were different in the hospitals surveyed, and there was an apparent inverse correlation between the resistance observed and the use of erythromycin in each hospital.

Molecular epidemiology of macrolides-lincosamides-streptogramin B resistance in Staphylococcus aureus and coagulase-negative staphylococci.

Macrolides-lincosamides-streptogramin B (MLS) resistance is commonly found in Staphylococcus aureus and coagulase-negative staphylococci (22 and 45%, respectively, among isolates from three New Jersey hospitals). We have examined representative subsets of 107 MLS-resistant isolates for the molecular nature of the resistance determinant, the erm gene, by dot blot and Southern hybridization analysis. All of 35 S. aureus isolates examined and 39 of 42 coagulase-negative isolates examined were found to harbor the ermA or ermC evolutionary variant. Genes of the ermC class occurred exclusively on a small plasmid similar to or indistinguishable from one (pNE131) previously described in S. epidermidis. Genes of the ermA class occurred exclusively in the chromosome, and restriction patterns indicated that they were part of a transposon, Tn554, characteristic of the classical S. aureus ermA strain. Unlike S. aureus ermA strains examined previously, which harbor Tn554 at a single specific (primary) site, four of our S. aureus isolates had second inserts at different chromosomal sites. The majority of our coagulase-negative isolates had two or more inserts, neither of which occurred at the classical primary site and many of which differed from one another in location (as inferred from restriction patterns). Coagulase-negative staphylococci constitute a large reservoir of the ermA and ermC class of determinants, with clear potential for interspecies spread.