Ultrastructural localization of extracellular matrix proteins of the lymph node cortex: evidence supporting the reticular network as a pathway for lymphocyte migration.

BACKGROUND: The lymph node (LN) is a crossroads of blood and lymphatic vessels allowing circulating lymphocytes to efficiently recognize foreign molecules displayed on antigen presenting cells. Increasing evidence indicates that after crossing high endothelial venules, lymphocytes migrate within the node along the reticular network (RN), a scaffold of fibers enwrapped by fibroblastic reticular cells (FRC). Light microscopy has shown that the RN contains specific extracellular matrix (ECM) proteins, which are putative molecular "footholds" for migration, and are known ligands for lymphocyte integrin adhesion receptors. RESULTS: To investigate whether ECM proteins of the RN are present on the outer surface of the FRC and are thus accessible to migrating lymphocytes, ultrastructural immunohistochemical staining of cynomolgus monkey LN was performed using antibodies to human ECM proteins that were successfully employed at the light microscopic level. The fibrillar collagens I and III were observed primarily within the reticular network fibers themselves. In contrast, the matrix proteins laminin, fibronectin, collagen IV, and tenascin were observed within the reticular fibers and also on the outer membrane surface of the FRC. CONCLUSIONS: These findings suggest a molecular basis for how the RN functions as a pathway for lymphocyte migration within the lymph node.

The T- and L-type calcium channel blocker (CCB) mibefradil attenuates leg edema induced by the L-type CCB nifedipine in the spontaneously hypertensive rat: a novel differentiating assay.

Among the L-type calcium channel blockers (CCBs), particularly dihydropyridines like nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester], a common adverse effect is vasodilatory edema. Newer CCBs, such as the T- and L-type CCB, mibefradil [(1S,2S)-2-[2[[3-(2-benzimidazolylpropyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphthyl methoxyacetate dihydrochloride hydrate], demonstrate antihypertensive efficacy similar to that of their predecessors but seem to have a reduced propensity to cause edema. Using a magnetic resonance imaging (MRI) T(2) mapping technique, we investigated the ability of mibefradil to reduce extracellular water accumulation caused by the L-type CCB, nifedipine, in the hindleg skeletal muscle of the spontaneously hypertensive rat. Mibefradil (10 mg/kg i.v.) and nifedipine (1 mg/kg i.v.) lowered mean arterial blood pressure by 97 +/- 5 and 77 +/- 4 mm Hg, respectively. MRI edema index (expressed as percentage increase of integral T(2) over predrug control) was significantly higher with nifedipine (2606 +/- 86%; p < 0.05) than with mibefradil (981 +/- 171%) measured 30 to 60 min after the start of drug infusion. The hindleg edema caused by nifedipine was dose dependently decreased by coadministration of mibefradil (0, 0.3, or 3 mg/kg). The hindleg edema formation was not due to albumin leakage into the interstitial space based on immunostaining. However, a 4.2-fold increase in the arterial L-/T-type CC mRNA expression ratio was observed compared with the venous L/T ratio as shown by quantitative reverse transcription polymerase chain reaction. These results demonstrate the novel utility of MRI to measure extravascular water after acute exposure to CCBs and indicate that T-type CCB activity may reduce L-type CCB-induced vasodilatory edema in the skeletal muscle vasculature, possibly by a differential effect on arteriole and venule dilatation.

Acute coronary artery injury in dogs following administration of CI-1034, an endothelin A receptor antagonist.

The objective of this study was to characterize acute coronary artery injury evoked by the endothelin A receptor (ETAR) antagonist, CI-1034. Male dogs (n = 5) were intravenously administered CI-1034 at 120 mg/kg for 4 d. Control animals (n = 3) received vehicle. Macroscopically, drug-related hemorrhage was observed in the right coronary groove and atrium. Histologically, drugrelated coronary changes were characterized as medial hemorrhage and necrosis, with mixed inflammatory-cell infiltrates in the adventitia and media. Immunohistochemistry staining indicated increased expression of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and S100A8/A9 (within in monocytes and neutrophils) proteins in coronary arteries of CI-1034-treated animals. However, there were similar expression levels of endothelial nitric oxide synthase (eNOS) among control and CI-1034-treated animals. Significant drug-related nitric oxide (NO) accumulation occurred on days 1 through 4 in serum. Increased interleukin (IL)-6 and fibrinogen in plasma and serum amyloid A (SAA) occurred on days 2 through 5 in CI-1034-treated animals. Increased levels of NO accumulation in serum; increased IL-6 and fibrinogen levels in plasma; increased SAA levels; and increased expressions of iNOS, cleaved caspase-3, and S100A8/A9 complex appear to be characteristic of CI-1034-induced acute vascular injury in dogs.

Axonopathy, tau abnormalities, and dyskinesia, but no neurofibrillary tangles in p25-transgenic mice.

Neurofibrillary tangles, one of the pathologic hallmarks of Alzheimer's disease (AD), are composed of abnormally polymerized tau protein. The hyperphosphorylation of tau alters its normal cellular function and is thought to promote the formation of neurofibrillary tangles. Growing evidence suggests that cyclin-dependent kinase 5 (cdk5) plays a role in tau phosphorylation, but the function of the enzyme in tangle formation remains uncertain. In AD, cdk5 is constitutively activated by p25, a highly stable, 25kD protein thought to be increased in the AD brain. To test the hypothesis that p25/cdk5 interactions promote neurofibrillary pathology, we created transgenic mouse lines that overexpress the human p25 protein specifically in neurons. Mice with high transgenic p25 expression have augmented cdk5 activity and develop severe hindlimb semiparalysis and mild forelimb dyskinesia beginning at approximately 3 months of age. Immunohistochemical and ultrastructural analyses showed widespread axonal degeneration with focal accumulation of tau in various regions of the brain and, to a lesser extent, the spinal cord. However, there was no evidence of neurofibrillary tangles in neuronal somata or axons, nor were paired helical filaments evident ultrastructurally. These studies confirm that p25 overexpression can lead to tau abnormalities and axonal degeneration in vivo but do not support the hypothesis that p25-related induction of cdk5 is a primary event in the genesis of neurofibrillary tangles.

An immunohistochemical approach to differentiate hepatic lipidosis from hepatic phospholipidosis in rats.

Hepatocellular vacuolation can be a diagnostic challenge since cytoplasmic accumulations of various substances (lipid, water, phospholipids, glycogen, and plasma) can have a similar morphology. Cytoplasmic accumulation of phospholipids following administration of cationic amphiphilic drugs (CAD) can be particularly difficult to differentiate from nonphosphorylated lipid accumulations at the light microscopic level. Histochemical methods (Sudan Black, Oil Red-O, Nile Blue, etc.) can be used to identify both nonphosphorylated and/or phosphorylated lipid accumulations, but these techniques require non-paraffin-embedded tissue and are only moderately sensitive. Thus, electron microscopy is often utilized to achieve a definitive diagnosis based upon the characteristic morphologic features of phospholipid accumulations; however, this is a low throughput and labor intense procedure. In this report, we describe the use of immunohistochemical staining for LAMP-2 (a lysosome-associated protein) and adipophilin (a protein that forms the membrane around non-lysosomal lipid droplets) to differentiate phospholipidosis and lipidosis, respectively in the livers of rats. This staining procedure can be performed on formalin-fixed paraffin embedded tissues, is more sensitive than histochemistry, and easier to perform than ultrastructural evaluation.

Comparative methods for multiplex analysis of cytokine protein expression in plasma of lipopolysaccharide-treated mice.

Changes in circulating cytokines might serve as predictors of compound-evoked inflammatory responses. CD-1 mice were treated with lipopolysaccharide (LPS; 0.2 ml of 0.25 mg/ml, intraperitoneal) for subsequent expression measurement of plasma cytokine protein expression at 24-h post-treatment using multiple antibody Western blot, and at both 2-h and 24-h post-treatment using antibody array and suspension bead array. Antibody array provided a semi-qualitative assessment and suggested significantly increased expression of GCSF at 2-h post-treatment and GCSF, IL-6, IL-12, MCP-1, MCP-5, RANTES and sTNFR1 at 24-h post-treatment. Densitometric analysis of multiple antibody Western blots provided a semi-quantitative assessment and indicated significantly increased expression of IL-6, IL-12, IL-17, GCSF, eotaxin, and MCP-2 at 24-h post-treatment. The suspension bead array yielded statistically significant cytokine protein expression increases for IL-6, IL-10, IFNgamma and TNFalpha at both 2-h and 24-h post-treatments, while significant expression at 24-h post-treatment only was noted for IL-1beta, IL-5, IL-12 and GM-CSF. Suspension bead array provided the greatest range of detection, revealing subtle increased expression of GM-CSF, IL-1beta, IL-5, IL-10, TNFalpha and IFNgamma at 24-h post-treatment, not detected by antibody array or multiple antibody Western blot. Suspension bead array proved to be the best method for detection of LPS-evoked changes in plasma cytokine levels.