Serum and Plasma Levels of Vascular Endothelial Growth Factors in Relation to Quality of Glucose Control, Biomarkers of Inflammation, and Diabetic Nephropathy.

Levels of vascular endothelial growth factors (VEGF) are regulated in a complex network of adipokines, glucose control, and low grade inflammation together with activated platelets, leucocytes, and endothelial dysfunction. Increased levels of VEGF are associated with enhanced angiogenesis and impaired repair mechanisms of vascular lesions in endorgans. Little is known about the interaction of systemic VEGF levels with quality of diabetes control, biomarkers of inflammation, and diabetic nephropathy. Moreover, it is unclear, whether serum and plasma VEGF levels are similarly suited to reflect risk associated with VEGF.In this case control study, we analyzed these parameters in serum and plasma of age and sex matched controls without diabetes (n=99) and type 2 diabetes (n=302). Serum VEGF-A was significantly increased in patients with T2DM while plasma levels were in the same range as for controls. Individual levels varied in a wide range. Serum levels were 4.9 times higher in controls and 7.3 times higher in T2DM as compared to plasma levels. T2DM was associated with significantly higher levels of hsCRP, ALAT, and albumin/creatinine ratio. When calculated for tertiles of HbA1c, we observed a highly significant increase from tertile one to the upper tertile for serum VEGF-A but not for plasma VEGF-A. Correlation analysis revealed a significant relationship between VEGF-A, HbA1c, inflammation, and diabetic nephropathy. Our results indicate that increased VEGF-A levels in T2DM significantly depend on quality of HbA1c control. Serum levels of VEGF-A, with a strong contribution of platelet derived VEGF, better reflect the glycemic burden than plasma levels of VEGF-A. Mechanistic studies are needed to explore links to inflammation and diabetic nephropathy.

Effects of pioglitazone versus simvastatin on biomarkers of inflammation in patients on high cardiovascular risk.

High levels of fetuin-A has been linked to cardiovascular disease, possibly via modulating low-grade systemic inflammation. We performed a subanalysis from the PIOSTAT study to investigate a possible link between fetuin-A and the inflammatory biomarker hs-CRP. 66 nondiabetic individuals at cardiovascular risk were randomized to either pioglitazone, simvastatin, or the combination of both, and followed for 12 weeks. At study endpoint, correlations between serum fetuin-A, hs-CRP, blood lipids, PAI-1, MMP-9, HOMA-IR, and liver transaminases were investigated by Spearman rank correlation. Changes in fetuin-A concentration did not correlate to changes in hs-CRP (r=0.19, p=0.16). A positive correlation was found for change of HOMA-IR value (r=0.33, p=0.01) and for the AST/ALT ratio (p<0.05). Our data suggest that the previously observed correlation between elevated circulating fetuin-A and hs-CRP in epidemiological studies may not reflect a causal relationship in nondiabetic patients on high cardiovascular risk.

Infiltration of the brain by pathogens causes Alzheimer's disease.

Despite very numerous studies on Alzheimer's disease (AD), especially on amyloid plaques and neurofibrillary tangles, little information has been obtained thus on the causes of the disease. Evidence is described here that implicates firstly herpes simplex virus type 1 (HSV1) as a strong risk factor when it is present in brain of carriers of the type 4 allele of the gene for apolipoprotein E (APOE-4). Indirect support comes from studies indicating the role of APOE in several diverse diseases of known pathogen cause. A second putative risk factor is the bacterium, Chlamydia pneumoniae. This pathogen has been identified and localized in AD brain. Current studies aimed at "proof of principle" address the entry of the organism into the CNS, the neuroinflammatory response to the organism, and the role that the organism plays in triggering AD pathology. An infection-based animal model demonstrates that following intranasal inoculation of BALB/c mice with C. pneumoniae, amyloid plaques/deposits consistent with those observed in the AD brain develop, thus implicating this infection in the etiology of AD.

Chlamydia pneumoniae infection promotes the transmigration of monocytes through human brain endothelial cells.

We have investigated the effects of Chlamydia pneumoniae on human brain endothelial cells (HBMECs) and human monocytes as a mechanism for breaching the blood-brain barrier (BBB) in Alzheimer's disease (AD). HBMECs and peripheral blood monocytes may be key components in controlling the entry of C. pneumoniae into the human brain. Our results indicate that C. pneumoniae infects blood vessels and monocytes in AD brain tissues compared with normal brain tissue. C. pneumoniae infection stimulates transendothelial entry of monocytes through HBMECs. This entry is facilitated by the up-regulation of VCAM-1 and ICAM-1 on HBMECs and a corresponding increase of LFA-1, VLA-4, and MAC-1 on monocytes. C. pneumoniae infection in HBMECs and THP-1 monocytes up-regulates monocyte transmigration threefold in an in vitro brain endothelial monolayer. In this way, C. pneumoniae infection in these cell types may contribute to increased monocyte migration and promote inflammation within the CNS resulting from infection at the level of the vasculature. Thus, infection at the level of the vasculature may be a key initiating factor in the pathogenesis of neurodegenerative diseases such as sporadic AD.

Role of infection in Alzheimer's disease.

Alzheimer's disease (AD) is a chronic condition in which inflammation has been shown to contribute to neurodegeneration. Current thinking suggests that deposition of beta-amyloid in the brain promotes inflammation resulting in neuronal damage/death. Alternatively, our data suggest that chronic inflammation observed in late-onset sporadic AD may be stimulated by infection with the obligate, intracellular bacterium, Chlamydia pneumoniae. Our results indicate that C. pneumoniae is found in high frequency in glial cells in areas of neuropathology within the brains of patients with AD. Based on our evidence, nervous system infection with C. pneumoniae should be considered a risk factor for sporadic AD.

Transglutaminase-catalyzed formation of Alzheimer-like insoluble complexes from recombinant tau.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which abnormal filamentous inclusions accumulate in dystrophic and dying nerve cells. These inclusions have been described as neurofibrillary tangles (NFTs) of which paired helical filaments (PHFs) are the primary constituents (1-3). The PHFs primarily are composed of the microtubule-associated protein tau, which has undergone posttranslational modification such as phosphorylation (4,5), glycation (6-9), and crosslinking by transglutaminase (TGase) (10-16). Crosslinking of proteins catalyzed by TGase results in the deposition of these proteins into insoluble matrices that are resistant to proteolytic digestion and chaotropic denaturation (for review see ref. 17). In this regard, TGase has been demonstrated to be associated with NFTs from the Alzheimer brain (13,14) and to exhibit elevated activity in the AD brain as compared with normal aged-matched control subjects (16). Here we discuss important aspects of TGase and in vitro experimental approaches that address its ability to catalyze the tau protein into insoluble complexes exhibiting biophysical and immuno-logical properties similar to those of the Alzheimer PHFs and NFTs.

Sézary T-cell activating factor is a Chlamydia pneumoniae-associated protein.

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it's role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated with Chlamydia pneumoniae bacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein of C. pneumoniae or against the lipopolysaccharide of Chlamydiae spp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence of C. pneumoniae DNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of the C. pneumoniae antigens and SAF appears to be associated with active disease in that C. pneumoniae antigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is a Chlamydia-associated protein and that further investigation is warranted to determine whether SAF and C. pneumoniae play a role in the pathogenesis of CTCL.

Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.

We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.

The association of tissue transglutaminase with human recombinant tau results in the formation of insoluble filamentous structures.

To determine possible mechanisms by which NFTs are formed in Alzheimer's disease (AD), we investigated the ability of tissue transglutaminase (TGase) to convert human recombinant tau proteins into insoluble filamentous structures. TGase derived from guinea pig liver was activated by calcium to catalyze the in vitro cross-linking of the largest soluble recombinant tau isoform (htau40) into insoluble complexes as determined by electrophoresis following incubation in 4 M urea and SDS. The TGase-catalyzed formation of these insoluble complexes occurred within 15 min to 24 h and the decreased migration of the insoluble material correlated with increased calcium concentrations ranging from 2 mM to 50 mM when analyzed electrophoretically. TGase-treated human recombinant tau formed filamentous structures in vitro that were immunoreactive with antibodies to tau and TGase. These structures retained the insoluble characteristics typical of AD PHF/NFTs. Immunolabeling with the TGase antibody revealed that TGase is associated with the filaments formed from human recombinant tau in vitro as well as with PHFs isolated from NFTs from AD brains. These novel findings support an in vitro model for investigating the biophysical changes that occur in converting soluble tau proteins into an insoluble matrix consistent with the insoluble PHFs/NFTs which may contribute to neuronal degeneration and cell death in the AD brain.

Localization of transglutaminase in hippocampal neurons: implications for Alzheimer's disease.

The purpose of this investigation was to identify and localize tissue transglutaminase (TGase) within neurons from the hippocampi of normal aged individuals and of those with confirmed Alzheimer's disease (AD). This enzyme may be a factor in the molecular mechanisms of neurodegeneration and formation of insoluble macromolecular complexes found in the neurons of normal aged and AD brain tissue. An antibody made to the extracellular TGase, coagulation factor XIIIa, was found to be specific for purified intracellular guinea pig liver tissue TGase. The specificity for liver tissue TGase has enabled us to identify tissue TGase(s) within rat hippocampal neurons and within neurons from normal aged and AD hippocampal tissues. Degenerating neurons from the AD hippocampus, compared to neurons from the normal aged hippocampus, exhibited increased immunoreactivity for TGase and demonstrated co-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associated with the neurofibrillary degeneration observed in AD, thereby implicating TGase as a potential factor in the pathogenesis of Alzheimer's disease.

Analysis of paired helical filaments (PHFs) found in Alzheimer's disease using freeze-drying/rotary shadowing.

Paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brains were analyzed using freeze-drying/rotary shadowing and immunoelectron microscopy. These filaments are the major contributors to the formation of neurofibrillary tangles (NFTs) found in AD, and are composed primarily of the microtubule-associated protein tau. We have focused on the identification of PHF-tau protein within isolated PHFs using anti-tau antibodies (tau 14, 46, 60). These PHFs consisted of both helically twisted and "straight" paired filaments. With freeze-drying/rotary shadowing, we are able to demonstrate in 2-D and 3-D subtle twists within the straight filaments as well as immunolabeling of the individual filamentous strands composing the PHFs. Additionally, projections emanating from numerous filaments and bridges between PHFs often were immunolabeled with anti-tau antibodies. Our results suggest that tau proteins are present in discrete, nonconfluent patterns within the PHFs and are components of both strands composing the PHFs. Tau proteins are present in the bridges between individual PHFs and may contribute to interconnecting PHFs into a complex macromolecular network such as the NFTs found in AD.

Differences in the histogenesis of EDL and diaphragm in rat.

We have examined the histogenesis of the diaphragm and extensor digitorum muscle in rat embryos, with the aim of defining differences in developmental patterns that can be related to the functional requirements of these muscles during and after development. Patterns of interactions between myotubes and other cells, and frequency of gap junctions are quite different in the two muscles. In diaphragm, primary myotubes (at day 16 in utero) are closely associated with each other, forming parallel sheets or palisades and communicating by gap junctions. Secondary myotubes have formed by day 18, but are immature, and the frequency of gap junctions is lower. The arrangement in palisades is maintained even after fibers are separated from each other by their individual basal lamina. In EDL primary fibers at day 16 have fewer gap junctions, and the peak in communication occurs after the appearance of secondary myotubes (day 18 and 21). Secondary myotubes are more mature than in diaphragm at day 18.

Quantitation of Ca ATPase, feet and mitochondria in superfast muscle fibres from the toadfish, Opsanus tau.

The muscle band surrounding the swimbladder of the toadfish (Opsanus tau) is one of the fastest known muscles in vertebrates. Rapid, non-fused twitches are responsible for the characteristic sound produced by the organ by both male and female toadfish. We have quantitated the membrane systems (transverse (T) tubules, sarcoplasmic reticulum (SR) and mitochondria), and some of their proteins (Ca2+ ATPase, or calcium pump, and foot protein or Ca2+ release channel) in these muscle fibres. As expected from the well-known morphology, the content of Ca2+ release and Ca2+ uptake proteins are considerably higher than in slower twitch fibres (e.g. fast-twitch and slow-twitch fibres in hind legs of mammals). Unexpectedly, the increment in ATPase is much larger than the increment in foot protein. The ATPase to foot ratio in muscle fibres from the swimbladder of males and females is higher by a factor of five to seven than in guinea pig fast-twitch fibres. We conclude that calcium uptake is a limiting factor in the ability to sustain the trains of high frequency, non-fused synchronous contractions of which these fibres are capable. Sexual dimorphism is demonstrated in the content of mitochondria (higher in males) and in the density of junctional feet (higher in females). The former is probably related to the more continuous activity during the males' mating call but the latter is to be demonstrated.

[In vivo effects of high density lipoprotein rich serum fractions (HDLF) in the human. 1. Modification of circulating thrombocyte aggregates, cardiovascular parameters and lipid metabolism by HDLF]. / In-vivo-Wirkungen High-Density-Lipoprotein-reicher Serumfraktionen FFDLF) beim Menschen. 1. Mitteilung: Beeinflussung zirkulierender Thrombozytenaggregate, kardiovaskulärer Parameter und des Lipidmetabolismus durch HDLF.

By means of the modified Cohn-method 6 Cohn-IV-1-fractions were prepared in the small pool technique as sterile, pyrogen-free and HDL-rich serum fractions (HDLF), the HDL-concentrations of which were 15-20 g/l. In in-vitro investigations they inhibited the spontaneous aggregation of erythrocytes as well as the ADP-induced aggregation of thrombocytes and activated the molar and fractional initial cholesterol estirification rates as well as the prostacyclin synthesis. From HDLF intravenously applied in people as immediate effects result significant decreases of the cardiac pre- and afterload, an increase of the cardiac ejection fraction as well as a complete dissolution of circulating aggregates of thrombocytes. In 35 patients with hyperlipidaemias after infusion of 100 ml HDLF up to the 5th day increasing and for 14 days remaining increases of concentration of biliary bile acids, total phospholipids and of cholesterol were observed in lithogenic indices remaining in the normal region. The plasmatic lipo- and apolipoproteins reacted with increases, decreases or constancy of concentration which might be based on the different ethryopathogeneses of the hyperlipoproteinaemias.

Quantitation of 'junctional feet' content in two types of muscle fiber from hind limb muscles of the rat.

1. Transverse tubules in fibers from rat soleus and extensor digitorum longus (EDL) muscles of the rat were infiltrated with silver dichromate (black reaction of Golgi). This provides a faithful, high-contrast outline of the tubules, which allows distinction between segments involved in junction formation with the sarcoplasmic reticulum and segments that are free. 2. Electron micrographs of semithin transverse sections were used to quantitate T tubule parameters and to measure cross-sectional area and perimeter of individual fibers. Thin sections and data from the literature were used to obtain the contribution of caveolae to external surface area and the frequency of junctional feet along the junctional T tubule membrane. 3. From the above data we calculate the ratio of number of feet to total external surface area for a given fiber segment. The ratio is compared with data in the literature on the total amount of 'charge movement' (in nC/uF of total external surface area). 4. The average feet/surface area ratio is twice as large in EDL than in soleus fibers, while the charge movement is up to five-fold larger. Probably some of the total charge movement is not directly associated with events related to the turning on of the SR permeability to calcium.

Golgi stain identifies three types of fibres in fish muscle.

Using Golgi infiltration we have studied the structure and disposition of transverse tubules in muscle fibres from the sand dab fin musculature. Three types of fibres differ significantly from each other in the extent and disposition of junctions between transverse tubules and the sarcoplasmic reticulum. These correlate with the three groups of fibres having different relaxation times shown in the accompanying paper (Gilly & Aladjem, 1987). Fibres with very slow relaxation (tonic fibres) correspond to those which have an unusual disposition of T tubules and very rare T-SR junctions. In the fast twitch fibres the peripheral T tubules segments converge into tangentially arranged tubules before joining the plasmalemma.

[Oxygen supply and oxygen uptake by skeletal muscle in acute arterial circulatory disorders--an experimental study in the dog]. / Sauerstoffangebot und Sauerstoffaufnahme der Skelettmuskulatur bei akuter arterieller Durchblutungsstörung--eine experimentelle Studie am Hund.

The oxygen supply and the oxygen uptake of the ischemic skeletal muscle were defined in rest and by loading in experimental studies in dog. About 72% of the supplied arterial oxygen was taken up from the ischemic skeletal muscle in rest in opposite to 84.7% by loading. Therewith the authors found an essential higher value as supposed in literature so far. The absolute value of the oxygen uptake of the normally circulated skeletal muscle with 0.14 ml O2/min/100 g is small in contrast to the highly differentiated parenchymatous organs. An adaptation to an anaerobic metabolism is assumed by the authors in case of falling below the critical boundary value of 0.1 ml O2/min/100 g in skeletal muscle.

[pH measurement in tissue--a reliable criterion for the registration of acute arterial circulatory disorder in animal experiments]. / Die Gewebs-pH-Messung--ein verlässliches Kriterium zur Erfassung einer akuten arteriellen Durchblutungsstörung im Tierexperiment.

An arterial ischemia was carried out step by step in the right pelvic extremity of the dog. The ph level was measured continuously in the lateral vastus of the femoris muscle on both sides by means of a glass-one-rod measuring equipment. The pH value decreased to 6.43 ninety minutes after a total interruption of the arterial circulation. The initial value amounted to 7.20. The tissue-pH-values increase quickly during the following phase of recirculation.