The Effect of Cortex/Medulla Proportions on Molecular Diagnoses in Kidney Transplant Biopsies: Rejection and Injury Can Be Assessed in Medulla.

Histologic assessment of kidney transplant biopsies relies on cortex rather than medulla, but for microarray studies, the proportion cortex in a biopsy is typically unknown and could affect the molecular readings. The present study aimed to develop a molecular estimate of proportion cortex in biopsies and examine its effect on molecular diagnoses. Microarrays from 26 kidney transplant biopsies divided into cortex and medulla components and processed separately showed that many of the most significant differences were in glomerular genes (e.g. NPHS2, NPHS1, CLIC5, PTPRO, PLA2R1, PLCE1, PODXL, and REN). Using NPHS2 (podocin) to estimate proportion cortex, we examined whether proportion cortex influenced molecular assessment in the molecular microscope diagnostic system. In 1190 unselected kidney transplant indication biopsies (Clinicaltrials.govNCT01299168), only 11% had <50% cortex. Molecular scores for antibody-mediated rejection, T cell-mediated rejection, and injury were independent of proportion cortex. Rejection was diagnosed in many biopsies that were mostly or all medulla. Agreement in molecular diagnoses in paired cortex/medulla samples (23/26) was similar to biological replicates (32/37). We conclude that NPHS2 expression can estimate proportion cortex; that proportion cortex has little influence on molecular diagnosis of rejection; and that, although histology cannot assess medulla, rejection does occur in medulla as well as cortex.

The autoactivation of rabbit Hageman factor.

Proteolytic cleavage and activation of isolated, single chain, zymogen Hageman factor was observed in the presence of kaolin alone. The rate of cleavage of kaolin-bound Hageman factor was enhanced 50-fold by the presence of prekallikrein and high molecular weight kininogen. The two-chain 82,000 dalton form of activated Hageman factor (alpha-HF(a)) also cleaved kaolin- bound single-chain Hageman factor in a dose-dependent manner, yielding fragments of 28,000 and, 50,000 dahons under reducing conditions. Cleavage of kaolin-bound single-chain Hageman factor was not inhibited by preincubation with diisopropylfluorophosphate (12 mM) for 10 min, but long-term incubation of Hageman factor with diisopropylfluorophosphate (up to 48 h) resulted in inhibition of cleavage of kaolin-bound Hageman factor to an extent proportional to the inhibition of procoagulant Hageman factor activity. Hageman factor cleavage was maximal when the kaolin concentration was {approximately} 10-fold greater than the Hageman factor concentration (wt:wt), and was partially inhibited by high molecular weight kininogen. Kaolin-bound Hageman factor cleaved clotting factor XI in an amount which correlated with the extent of cleavage of the Hageman factor. These findings are compatible with the concept that single-chain Hageman factor and alpha- HF(a), are both capable of cleaving and activating kaolin-bound Hageman factor and that a close molecular association of kaolin-bound Hageman factor molecules is required for this reaction.

Chemotactic activity generated from the fifth component of complement by plasma kallikrein of the rabbit.

Rabbit plasma kallikrein incubated with rabbit C5 resulted in the generation of chemotactic and secretagogue activity for rabbit neutrophils. This effect on C5 appeared to be due to kallikrein itself and not to a contaminating enzyme, because it could be inhibited by anti-kallikrein IgG or by soybean trypsin inhibitor to the same extent the kinin generation by the same kallikrein preparation was inhibited by these agents. The chemotactic response was consistent with the generation of a C5a-like peptide from C5 because the effect could be partially inhibited by carboxypeptidase N and was related to the generation of a small (approximately 14,000 mol wt) fragment of C5. No direct chemotactic response was detectable for kallikrein, activated Hageman factor, high-molecular weight kininogen, or intact C5. Incubation of Kallikrein, high-molecular weight kininogen, and Hageman factor together, so that activation of all three proteins occurred, did not results in the generation of detectable chemotactic activity.

Pattern of myelin breakdown during sciatic nerve Wallerian degeneration: reversal of the order of assembly.

Myelin sheaths of rapidly growing rats were sequentially labeled with the 3H and 14C isotopes of leucine as precursors of protein synthesis. The two injections were separated by time intervals ranging from 2 to 12 d. Wallerian degeneration was initiated by sciatic nerve neurotomy at 2 or 10 d after the second injection of radioactivity. After 5 d of degeneration, myelin was purified and the ratio of isotopes was determined in the delipidated protein. Regardless of the order in which the two isotopes were administered, the relative recovery of radioactivity resultant from the second injection was greatly reduced in degenerating nerves compared with sham-operated controls. Radioactivity incorporated from the first injection was also reduced, but to a lesser extent. Consequently, the isotope ratio corresponding to the first/second injection was greater in degenerating nerves than in controls, and the ratio increased in proportion to the time interval separating the two injections. The magnitude of the effect of degeneration was only slightly greater when degeneration was initiated 2 d after the second injection than when initiated 10 d after the last injection. Consequently, myelin disintegration rather than diminished incorporation of radioactivity accounts for the losses of radioactivity. Furthermore, the pattern of myelin degeneration preferentially involves the last myelin to be formed.

Analysis of renal fibrosis in a rabbit model of crescentic nephritis.

The pathogenesis of renal fibrosis in crescentic nephritis is incompletely understood. To improve our understanding of this process, crescentic nephritis was induced in New Zealand White rabbits by administration of guinea pig antiglomerular basement membrane IgG after sensitization with guinea pig IgG, and their kidneys were analyzed for the development of fibrosis. Collagen synthesis in renal cortical tissue was significantly elevated by day 3, peaked at days 7-15, and returned towards baseline by day 21. Collagen content of both glomeruli and cortex were increased starting on days 14-16, and remained constant in cortex thereafter. Light microscopic analysis was much less sensitive, revealing fibrosis only after day 21. Immunofluorescence revealed that type IV collagen was distributed primarily in the glomerulus, while types I and III were increased in the glomerulus and interstitium. Thus, in this model of crescentic nephritis, fibrosis, as assessed biochemically, developed early at time points when morphologic analysis failed to detect such a development. Hence early therapeutic intervention, before morphologic evidence of fibrosis is evident, may be more successful in arresting the progression of this disease before it reaches irreversible terminal stages.

Activation of rabbit Hageman factor by homogenates of cultured rabbit endothelial cells.

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Tumor necrosis factor-a (TNF-a) production and localization of macrophages and T lymphocytes in the rabbit corpus luteum.

Utilizing immunocytochemistry numerous macrophages were localized in regressing corpora lutea. In contrast, few macrophages were observed in young corpora lutea. Regressing corpora lutea readily produced TNF-a in vitro in response to lipopolysaccharide, whereas young corpora lutea produced significantly less TNF-a. T lymphocytes were identified in young corpora lutea preceding the appearance of macrophages. These observations suggest that cells of the immune system and cytokines could be important participants in physiological regression of the corpus luteum.

Prolonged daily inhalation of halothane modifies the dose-response pattern to acute administration of halothane. An electrophysiological study.

Sensory-evoked field potentials were obtained from freely moving rats implanted sterotaxically with permanent electrodes in the parafasciculus thalami (PF), mesencephalic central gray (CG), ventromedial hypothalamus (VMH) and somatosensory cortex (SCX). Animals were exposed to chronic, subanesthetic inhalation of halothane (0.5%, 3 hr/day, 5 days/week) for 56 days. The averaged acoustic evoked responses (AAER) were recorded on day 0, as well as at 28 and 56 days after a 48-hr halothane-free period ("control") and after acute doses of halothane (0.25, 0.5 and 1.5%). In general, the averaged sensory-evoked responses from each structure were affected at day 0 of the experiment in dose-response manner, and suppression of the responses was the main effect of halothane. Chronic exposure to subanesthetic inhalation of halothane produced marked alteration of the "control" recording from 3 CNS structures; mainly from the mesencephalic central gray, the parafasciculus thalami and the somatosensory cortex and the direction (increase or decrease) of the averaged acoustic evoked responses in all the four CNS sites studied. The total responsiveness was modified as well, i.e. the recordings obtained from the mesencephalic central gray and somatosensory cortex exhibited hypersensitivity while the recordings obtained from the parafasciculus thalami and ventromedial hypothalamus exhibited tolerance. It is concluded that prolonged and intermittent inhalation of halothane can alter the electrophysiological properties of the four structures investigated.

Effects of microiontophoretic application of cocaine, alone and with receptor antagonists, upon the neurons of the medial prefrontal cortex, nucleus accumbens and caudate nucleus of rats.

The spontaneous extracellular electrical activity of 102 neurons, within the caudate nucleus (CN), medial prefrontal cortex (MPC), nucleus accumbens (NAc) and a control site, the lateral thalamic nucleus (LT), was studied. Cocaine depressed spontaneous activity in the majority of the cells studied from all regions except the lateral thalamus. Desipramine, which has been used clinically for the treatment of withdrawal of cocaine, also depressed neuronal activity in the caudate nucleus. In addition, of the three receptor antagonists tested, sulpiride, methysergide and naloxone, only the dopamine antagonist (sulpiride) affected cocaine-induced neuronal responses. This study further emphasizes the emerging importance of midbrain dopaminergic systems in the pharmacological effects of this important drug of abuse.

Up-regulation of transcription of smooth muscle myosin alkali light chain by ethanol in human breast cancer cells.

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and that chronic ethanol exposure enhances metastasis of breast cancer cells, and with an in vitro model (T47D human breast cancer cells), we have previously demonstrated that ethanol exposure stimulated the migration of breast cancer cells. In the present study, differential display reverse transcription polymerase chain reaction was used to identify ethanol-responsive genes in T47D cells. Three differentially displayed, ethanol-responsive gene fragments were identified, and their expression was confirmed by Northern blot hybridization. Sequence analysis revealed that one cDNA fragment represented the myosin alkali light chain (MLC 1sm) of human smooth muscle. The expression of MLC 1sm was found to be significantly higher in breast cancer cells than in normal mammary epithelial cells. With T47D cells, ethanol induced an additional duration- and concentration-dependent up-regulation of MLC 1sm. At 400 mg/dl, an ethanol-mediated increase was evident at 6 h (55% increase), peaked at 24 h (2.7-fold increase) following exposure, and diminished thereafter. At pharmacologically relevant concentrations (e.g., 100 mg/dl), ethanol produced a significant increase of MLC 1sm expression, and progressively higher ethanol concentrations resulted in more up-regulation. The half-life of MLC 1sm mRNA was not altered, however, the transcription rate of MLC 1sm was significantly increased by ethanol. MLC is a structural component of the cytoskeleton of eukaryotic cells, and it plays critical roles in the regulation of cell shaping, movement, and growth. Thus, ethanol-mediated up-regulation of MLC may be an underlying molecular mechanism for its tumor promoting effect.

Myelin isolation: comparison of sedimentation and flotation techniques.

Brains from young (20 day old) and adult rats were used to compare myelin yields obtained by sedimentation and flotation techniques. The flotation method consistently gave approx 70% higher yields of myelin than the sedimentation method. Both myelin preparations have virtually identical protein composition as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoretic analysis revealed substantial concentrations of myelin proteins in the non-myelin particulate fraction obtained by the sedimentation but not by the flotation method. The study indicates that the paradigm of the sedimentation method results in a significant loss of myelin during isolation, and that this loss can be avoided or minimized by employing the flotation method.

How much undernourishment is required to retard brain myelin development.

This study employs a large population of developing rats designed to range continuously from above a normal, average weight to severely undernourished. The purpose of the study is to determine if brain myelin development is vulnerable to mild growth retardation from chronic hunger, or if brain myelin development is altered only after a certain tolerable amount of growth retardation is exceeded. The brains were examined at a landmark age, weaning, since myelination is one of the most vulnerable features of brain development and its rate of synthesis is highest at this age. Brain size was logarithmically related to body weight, and brain growth retardation increased as the severity of food deprivation increased. There was an additional reduction in the concentration of brain myelin. In contrast to brain weight, the reduction in myelin concentration was linearly related to body size over the full range from well nourished to undernourished. From a population perspective, these data indicate growth retardation from undernourishment in any amount slows brain growth and additionally lowers the concentration of brain myelin; however, individuals, in both well nourished and undernourished groups, vary widely. Implications and cautions of extrapolation to human populations are discussed.

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments.

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Myelin development and nutritional insufficiency.

Postnatal undernourishment does not greatly retard the generation of rat brain cells, although there is a slight reduction in total cell numbers and brain size. Possibly the maturation of cells is more severely affected. The ratio of myelinated to non-myelinated fibers is greatly reduced in the corpus callosum and pyramidal tract, and presumably in other areas as well. There is only a slight reduction in the numbers of myelin lamellae for axons of a given size. The recovery of brain myelin and the incorporation of radioactive precursors into purified myelin proteins and lipids are all greatly reduced, leading to a comparatively severe reduction in the brain myelin concentration. The myelin composition is only slightly altered, possibly as a result of delay in its normal chemical maturation. The actual vulnerable period that produces a lasting myelin deficit is the early period that includes oligodendroglia cell proliferation, whereas undernutrition restricted to a later period that includes the actual peak of myelin does not cause a lasting reduction in the brain myelin concentration. The belief that stunting the postnatal proliferation of oligodendroglia largely accounts for the myelin effect has not been substantiated by direct analysis of cell numbers. Consequently, the observed hypomyelination likely results from a failure of oligodendroglia to mature and to initiate myelin formation. The myelin deficit appears largely uniform throughout the brain. Indirect evidence in human studies indicate that comparable effects appear in undernourished infants.

Relative synthesis of myelin in different brain regions of postnatally undernourished rats.

We used a double isotope procedure and starved and normal littermate rats to compare relative protein synthesis in the cerebellar nuclear, myelin, synaptosomal, mitochondrial, and microsomal subfractions of postnatally starved animals. The remaining brain tissue was dissected into 6 additional regions (cerebral cortex, medulla oblongata, midbrain, hippocampus, striatum, and hypothalamus) and these were frozen for similar subcellular fractionation and analysis at a later date. The microsomal fraction derived from frozen tissues was discarded. The results show that early postnatal starvation specifically depresses myelin synthesis to about the same extent in all major brain regions at 18 and 21 days of age.

A possible effect of the methylxanthines caffeine, theophylline and aminophylline on postnatal myelination of the rat brain.

A double isotope methodology was used to assess the effect of methylxanthine administration on membrane protein synthesis in developing rat brain. Rat pups were given either aminophylline, theophylline, or caffeine in a dosage of 40 mg/kg or 80 mg/kg daily from the second postnatal day through 20 days of age. Results show depressed myelin protein synthesis at 21.24 days by theophylline (80 mg/kg) and caffeine (40 and 80 mg/kg). Synthesis was essentially normal at 27-28 days of age, indicating a possible delay in development followed by a 'catch-up' phenomenon.

A morphometric analysis of pyramidal tract structures during postnatal undernourishment and recovery.

Rats were postnatally undernourished during the suckling period (up to 20 days) and the brainstems of the perfused rats were dissected and prepared for electronmicroscopy at 21, 35 and 63 days of age. The effects on myelin were relatively mild and consisted primarily of a slight reduction in the relative numbers of myelinated fibers, most likely caused by a lag in the rate of loss of non-myelinated fibers, and fewer lamellae in myelinated axons of less than 2.5 micron circumference. Organelles were examined in the interfasicular oligodendroglia and in paragigantocellular reticular neurons immediately dorsal to the pyramidal tract. The numbers of mitochondrial particles in neuronal perikarya were significantly increased by postnatal undernourishment, although the numbers of other organelles appeared normal. Increased numbers of mitochondria persisted in nutritionally rehabilitated rats. Mitochondrial particles in oligodendroglia were not altered.

Abnormal upregulation of myelin genes underlies the critical period of myelination in undernourished developing rat brain.

Since myelin gene expression is suppressed during active myelination of the undernourished brain, this study was designed to determine the effects of undernourishment on the upregulation of myelin genes and the relationship between upregulation and the 'critical period' associated with permanent hypomyelination of the brain. Long-Evans rat dams were given either ad libitum or restricted access to rat chow to produce two populations of developing offsprings. The food deprivation schedule was designed to produce a degree of growth retardation comparable to our earlier studies of hypomyelination in undernourished brain. The expression of myelin genes, at various developmental ages, was determined in the forebrains from undernourished and normal, well fed controls by Northern analysis. In well nourished forebrain, proteolipid protein (PLP), myelin associated glycoprotein (MAG), and basic protein (BP) messages began to increase polynomially after day 8 post partum, leading to a rapid accumulation of message during the following several days. In undernourished forebrain, PLP, MAG, and BP messages did not show any increase until day 10, and then increased at a diminished rate as compared to well nourished forebrain. Additionally, the two PLP messages (1.6 kb and 3.2 kb) showed different vulnerabilities to protein-calorie undernourishment, which explains the abnormal ratio of the 3.2 and 1.6 kb forms we previously found in undernourished brain. This study shows a pattern of temporal specificity when the myelin PLP, MAG, and BP genes are synchronously upregulated in the normal forebrain to a high rate of transcription between day 7 to 9, which is several days before the onset of rapid myelination of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance of myelin proteins in rat sciatic nerve during development.

Rats between 5 and 45 days of age were sacrificed and their sciatic nerves dissected. Myelin was prepared from these sciatic nerves by a procedure involving purification on discontinuous sucrose gradients. The proteins of whole sciatic nerves at different ages and the proteins derived from the myelin isolated from these sciatic nerves were examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulfate. Over half of the proteins of sciatic nerve myelin migrated in a single band on the gel (P0). There were only minor changes in the protein distribution of sciatic nerve mylein during development. In contrast, the polyacrylamide gel patterns of whole sciatic nerve homogenate changed markedly during development between 5 and 15 days of age. The amount of P0 protein as a proportion of the total sciatic nerve protein increased from 3% at 5 days of age to 13% at 15 days of age after which it remained constant. Several other proteins which were also characteristic of the isolated myelin increased in relative importance during this time period. Parallel experiments dealing with a metabolic parameter of myelinogenesis, incorporation of intraperitoneally injected [35S]sulfate into sulfatide, were conducted. The maximum synthesis of sulfatide occurred between 6 and 16 days of age, coincident with the marked accumulation of myelin proteins in sciatic nerve.