Exp Biol Med Research Articles

Focus, reentry, or “focal” reentry?

the question whether tachyarrhythmias are caused by enhanced impulse formation (“focus”) or by reentrant excitation (“reentry”) has been the subject of debate for more than a century. In 1887 McWilliam ([12][1]) was the first to suggest that disturbances in impulse propagation could be

How does pulmonary surfactant reduce surface tension to very low values?

#### Background: Although initially proposed by von Neergaard in 1929 ([14][1]), direct evidence for surface-active material at the air-fluid interface of the lung was first reported by Richard Pattle and John Clements in the 1950s ([5][2], [10][3]). Pattle deduced that microbubbles formed from

Insulin-degrading enzyme hydrolyzes ATP.

We reported in a previous work that insulin degradation by insulin-degrading enzyme (IDE) was inhibited by ATP (Exp Biol Med 226:334-341, 2001). Then we studied ATP hydrolysis as a possible mechanism for reversion of this inhibition. ATP hydrolysis was determined by (32)P release after hydrolysis of gamma[(32)P]ATP. ATP hydrolysis was studied by Sephadex G200 chromatography, immunoprecipitation, and nondissociating gel electrophoresis. Purified recombinant rat IDE and extractive homogenous IDE showed similar ATP hydrolysis. All results showed concordance between insulin degradation and ATP hydrolysis, suggesting that IDE has both functions. In order to define the type of hydrolysis, we studied inhibitors of IDE, phosphohydrolases, and ATPases. Each substance studied had no effect on ATP hydrolysis, except 1 mM orthovanadate, a known inhibitor of ATPases, phosphatases, and insulin degradation. ATP hydrolysis followed a Michaelis-Menten kinetic with Vmax: 570.45 +/- 113.08 pmol Pi/hr and apparent Michaelis constant (Km): 63.13 +/- 3.48 microM. ATP binding studies strongly suggested an ATP binding site and enzyme kinetics established only one active hydrolytic ATP binding site per IDE molecule. ATP-induced enzyme aggregation changes as observed by electrophoresis mobility in nondissociating conditions and conformational changes on insulin binding as shown by IDE-insulin cross-linking. We conclude that IDEs have ATPase activity and that insulin-binding and degradation are dependent on ATP concentration; however, insulin does not modify the ATPase activity of IDE.

Mechanisms of vasoconstriction with direct skin cooling in humans

direct skin cooling causes a vasoconstriction partly dependent on α2-adrenergic function. In in vitro studies, this is a manifestation of reactive oxygen species stimulating Rho and Rho kinase to cause translocation of α2C-receptors from the Golgi to the plasma membrane. In this issue of American

Modulation of circulating substance P in Mg‐deficient (MgD) rats by NMDA receptor and neutral endopeptidase inhibitors

Changes in dietary Mg intake influences rat plasma levels of the proinflammatory neuropeptide, substance P (SP), and was associated with altered cardiac tolerance to postischemic stress (Exp Biol Med 228, 2003). In this study, the impact of NMDA receptor inhibition (MK-801, 0.5 mg/kg/day, s.c. pellet) and neutral endopeptidase inhibition (phosphoramidon [PR], 5 mg/kg/day, s.c. pellet) on plasma SP levels were assessed (ELISA) in MgD (Mg = 9% RDA) rats, along with changes in in vivo oxidative stress (RBC GSH loss). During wk 1, MgD alone induced a substantial elevation in SP (0.59 ng/ml/wk) vs Mg-normal rats. PR-treatment of MgD rats during wk 1 led to a further 1.9-fold increase in SP (1.13 ng/ml/wk) and caused a 2-fold greater loss in GSH vs MgD alone. By contrast, continuous MK-801 treatment during 3 wks of MgD caused an overall 49 % reduction in SP vs untreated, but MK-801’s impact was greatest during wk 1 (89 % lower) vs wk 2 (51 %) and wk 3 (30 %). MK-801 treatment of 2 and 3 wk MgD rats led to 38 and 56 % higher GSH levels vs MgD alone, indicating attenuation of oxidative stress. Thus, in addition to a dietary approach (Mg intake), modulating plasma SP levels during MgD by targeted inhibition of neuronal SP release (MK-801) or SP degradation (PR) can also influence the severity of in vivo oxidative stress, and may ultimately impact subsequent cardiac susceptibility to secondary (postischemic) stress. NIH-HL-66226 & HL-65178.

A Functional Polymorphism of Toll-Like Receptor 4 Gene Increases Risk of Gastric Carcinoma and Its Precursors

TLR4 is a cell-surface signaling receptor involved in the recognition and host response to Helicobacter pylori. The TLR4+896A>G polymorphism linked with impaired reactivity to bacterial lipopolysaccharide may play a role in gastric carcinogenesis. We assessed associations with premalignant gastric changes in 149 relatives of gastric cancer patients, including 45 with hypochlorhydria and gastric atrophy. We also genotyped 2 independent Caucasian population-based case-control studies of upper gastrointestinal tract cancer, initially in 312 noncardia gastric carcinoma cases and 419 controls and then in 184 noncardia gastric carcinomas, 123 cardia carcinomas, 159 esophageal cancers, and 211 frequency-matched controls. Odds ratios were computed from logistic models and adjusted for potential confounding factors. TLR4+896G carriers had an 11-fold (95% confidence interval [CI], 2.5-48) increased odds ratio (OR) for hypochlorhydria; the polymorphism was unassociated with gastric acid output in the absence of H pylori infection. Carriers also had significantly more severe gastric atrophy and inflammation. Seventeen percent of gastric carcinoma patients in the initial study and 15% of the noncardia gastric carcinoma patients in the replication study had 1 or 2 TLR4 variant alleles vs 8% of both control populations (combined OR = 2.3; 95% CI = 1.6-3.4). In contrast, prevalence of TLR4+896G was not significantly increased in esophageal squamous cell (2%, OR = 0.2) or adenocarcinoma (9%, OR = 1.4) or gastric cardia carcinoma (11%, OR = 1.4). Our data suggest that the TLR4+896A>G polymorphism is a risk factor for noncardia gastric carcinoma and its precursors. The findings underscore the role of the host innate immune response in outcome of H pylori infection.

Heat-induced antigen retrieval: Mechanisms and application to histochemistry

Since the introduction of the fluorescence-labeled antibody method by Coons et al. [Immunological properties of antibody containing a fluorescent group. Proc Soc Exp Biol Med 47, 200–2002], many immunohistochemical methods have been refined to obtain high sensitivity with low background staining at both light and electron microscopic levels. Heat-induced antigen retrieval (HIAR) reported by Shi et al. in the early 1990s has greatly contributed to immunohistochemical analysis for formalin-fixed and paraffin-embedded (FFPE) materials, particularly in the field of pathology. Although antigen retrieval techniques including enzyme digestion, treatment with protein denaturants and heating have been considered tricky and mysterious techniques, the mechanisms of HIAR have been rapidly elucidated. Heating cleaves crosslinks (methylene bridges) and add methylol groups in formaldehyde-fixed proteins and nucleic acids and extends polypeptides to unmask epitopes hidden in the inner portion of antigens or covered by adjacent macromolecules. In buffers having an appropriate pH and ion concentration, epitopes are exposed without entangling the extended polypeptides during cooling process, since polypeptides may strike a balance between hydrophobic attraction force and electrostatic repulsion force. Recent studies have demonstrated that HIAR is applicable for immunohistochemistry with various kinds of specimens, i.e., FFPE materials, frozen sections, plastic-embedded specimens, and physically fixed tissues at both the light- and electron-microscopic levels, and have suggested that the mechanism of HIAR is common to aldehyde-fixed and aldehyde-unfixed materials. Furthermore, heating has been shown to be effective for flow cytometry, nucleic acid histochemistry (fluorescein in situ hybridization (FISH), in situ hybridization (ISH), and terminal deoxynucleotidyl transferase-mediated nick labeling (TUNEL)), and extraction and analysis of macromolecules in both FFPE archive materials and specimens processed by other procedures. In this article, we review mechanism of HIAR and application of heating in both immunohistochemistry and other histochemical reactions.

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

Mutational analysis of BMP15 and GDF9 as candidate genes for premature ovarian failure

Mutational screening of the bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) genes in a population with premature ovarian failure (POF) identified no new mutations. However, three single nucleotide polymorphisms in the BMP15 gene, two in the 5' untranslated region (31T>G and 71C>G) and another in exon 1 (387G>A), were found to be common in both POF and control groups.

Ethanol and cardiac function

regular heavy ethanol consumption has been associated with a type of nonischemic dilated cardiomyopathy termed “alcoholic cardiomyopathy” (ACM), which is clinically expressed as an impairment of left ventricular function (nonsymptomatic stage) and occasionally as overt heart failure (symptomatic

Cox-2 Is Regulated by Toll-Like Receptor-4 (TLR4) Signaling: Role in Proliferation and Apoptosis in the Intestine

We recently showed that mice deficient in Toll-like receptor 4 (TLR4) or its adapter molecule MyD88 have increased signs of colitis compared with wild-type (WT) mice after dextran sodium sulfate (DSS)-induced injury. We wished to test the hypothesis that cyclooxygenase 2 (Cox-2)-derived prostaglandin E2 (PGE2) is important in TLR4-related mucosal repair. Cox-2 expression was analyzed by real-time polymerase chain reaction, immunohistochemistry, Western blotting, and luciferase reporter constructs. Small interfering RNA was used to inhibit expression of MyD88. TLR4-/- or WT mice were given 2.5% DSS for 7 days. Proliferation and apoptosis were assessed using bromodeoxyuridine staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays, respectively. PGE2 was given orally to DSS-treated mice. Intestinal epithelial cell lines up-regulated Cox-2 expression in a TLR4- and MyD88-dependent fashion. Lipopolysaccharide-mediated stimulation of PGE2 production was blocked by a selective Cox-2 inhibitor or small interfering RNA against MyD88. After DSS injury, Cox-2 expression increased only in WT mice. TLR4-/- mice have significantly reduced proliferation and increased apoptosis after DSS injury compared with WT mice. PGE2 supplementation of TLR4-/- mice resulted in improvement in clinical signs of colitis and restoration of proliferation and apoptosis to WT values. The mechanism for improved epithelial repair may be through PGE2-dependent activation of the epidermal growth factor receptor. We describe an important link between TLR4 signaling and Cox-2 expression in the gut. TLR4 and MyD88 signaling are required for optimal proliferation and protection against apoptosis in the injured intestine. Although TLR4 signaling is beneficial in the short term, chronic signaling through TLR4 may lower the threshold for colitis-associated cancer.

Soy Protein, Isoflavones, and Cardiovascular Health

This editorial summarizes the recent American Heart Association (AHA) Science Advisory on soy protein and isoflavones (phytoestrogens) published in the February 21, 2006, issue of Circulation .1 Soy protein and isoflavones have gained considerable attention for their potential role in improving risk factors for cardiovascular disease. This scientific advisory report assesses the more recent work published on soy protein and its component isoflavones. In 22 randomized trials, isolated soy protein with isoflavones compared with milk or other proteins decreased LDL cholesterol concentrations in most studies; the average effect was approximately 3%. This reduction is very small compared with the large amount of soy protein tested in these studies, averaging 50 g, approximately half the usual total daily protein intake. No significant effects were evident on HDL cholesterol, triglycerides, lipoprotein(a), or blood pressure. Among 19 studies of soy isoflavones, the average effect on LDL cholesterol and other lipid risk factors was nil. Soy protein or isoflavones have not been shown to lessen vasomotor symptoms of menopause, and results are mixed regarding slowing of postmenopausal bone loss. The efficacy and safety of soy isoflavones for preventing or treating cancer of the breast, endometrium, and prostate are not established; evidence from clinical trials is meager and cautionary as regards a possible adverse effect. For this reason, use of isoflavone supplements in food or pills is not recommended. Thus, earlier research indicating that soy protein compared with other proteins has clinically important favorable effects has not enjoyed confirmation. In contrast, many soy products should be beneficial to cardiovascular and overall health because of their high content of polyunsaturated fats, fiber, vitamins, and minerals and low content of saturated fat. In October 1999, the US Food and Drug Administration (FDA) approved labeling for foods containing soy protein as protective against coronary heart disease.2 …

Developmental stage–specific shift in responsiveness to chemokines during human B-cell development

To better understand the role of chemokines during human B-cell development in bone marrow. Differentiation stage-specific B cells (pro-B, pre-B, immature, and mature) were analyzed for chemokine receptor expression and for migration to corresponding ligands. We also hypothesized that inflammatory conditions may cause the upregulation of certain chemokine receptors on early B cells, rendering them sensitive to extramedullary chemotactic cues. To test this hypothesis, we used human pre-B 697 cells to investigate whether various inflammatory agents could modify chemokine receptor expression and function. Chemotaxis to CXCL12 was observed for all B cell subsets. However, chemotactic responses to CCL19, CCL21, CXCL13, and CCL20 were limited to late-stage, IgM+ bone marrow B cells (immature B and mature B). Chemotactic responses to corresponding ligands correlated with the pattern of chemokine receptor expression. The expression of CCR7, however, was low on early (pro-B and pre-B) B cells and did not induce chemotaxis. Interestingly, both CCL19 and CCL21 could trigger ERK1/2 phosphorylation in early B cells. Exposure of pre-B 697 cells to TNF-alpha upregulated CCR7 and CXCR5 expression, whereas it had no effect on CCR6 surface expression. Correspondingly, TNF-alpha-stimulated pre-B cells chemotaxed towards CCL19 and CXCL13, in contrast to non-TNF-alpha-stimulated controls. We postulate that CXCR5, CCR7, and CCR6 participate in bone marrow trafficking and/or bone marrow egress of late-stage B cells under steady-state conditions, whereas inflammation-induced expression of CCR7 and CXCR5 may facilitate early B-cell emigration out of the bone marrow and their positioning in secondary lymphoid organs.

The “wisdom” of lung surfactant: balancing host defense and surface tension-reducing functions

the mixture of lipids and proteins that comprises pulmonary surfactant is both complex and multifunctional. Although surfactant was originally identified as a lipoprotein complex that reduces surface tension at the air-liquid interface of the lung ([4][1], [20][2]), both in vitro and in vivo studies

Role of IL-12B Promoter Polymorphism in Adamantiades–Behcet's Disease Susceptibility: An Involvement of Th1 Immunoreactivity against Streptococcus Sanguinis Antigen

Adamantiades-Behcet's disease (ABD) is a chronic inflammatory multisystem disorder. Although the precise etiology is unclear, high prevalence of human leukocyte antigen (HLA)-B51 predisposition and predominantly involved T-helper type 1 cells (Th1)-type proinflammatory cytokines and extrinsic Streptococcal infection suggest a substantial association with an immunogenetic basis and strengthens the hypothesis that IL-12, a potent inducer of Th-1 immune reaction, is a putative candidate in its pathogenesis. These clinicopathological findings led us to examine interleukin 12 p40 (IL-12B) promoter polymorphism, for which the 4-base pair (bp) heterozygous insertion has been shown to affect the gene transcription and subsequent protein production. We analyzed IL-12B promoter genotypes in 194 Japanese subjects (92 with ABD and 102 normal controls) by PCR-based restriction enzyme digestion. The frequency of the insertion heterozygosity was significantly higher in patients than in controls (49/92, 53.3% vs 39/102, 38.2%, respectively). Comparing these with HLA haplotype data, this trend was more significant in HLA-B51-negative patients (29/42, 69.0% vs 20/50, 40.0%; P = 0.005). As assessed by semiquantitative reverse transcription-PCR and ELISA, stimulation with Streptococcal antigens specifically increased expression of IL-12 p40 mRNA and protein, in conjunction with IL-12 p70 induction, in peripheral blood mononuclear cells from heterozygous patients. Our results provide evidence for anti-bacterial host response toward Th1-immunity mediated by IL-12 in patients with ABD, and the possible insight into the genetic susceptibility that is independent of HLA background.

Expression and localization of human endothelin-converting enzyme-1 isoforms in symptomatic atherosclerotic disease and saphenous vein.

Endothelin-converting enzyme (ECE-1) is a critical enzyme in the production of the potent vasoconstrictor peptide endothelin (ET-1). It has previously been shown that the levels of both ET-1 and ECE-1 are raised in atherosclerosis, but the possible relevance of the isoforms of ECE-1 in these changes has not yet been investigated. The aim of this study was to examine the expression of the ECE-1a and ECE-1c isoforms in human atherosclerotic pathologies. Immunohistochemical analysis was carried out on sections from atherosclerotic and non-atherosclerotic vascular tissue using a combination of ECE-1 isoform-specific antibodies, anti-alpha-actin antibodies to identify smooth muscle cells (SMC) and anti-CD68 antibodies to identify macrophages. ECE-1 isoform expression was also examined in cultured SMC and in macrophages isolated from human blood. Results indicated differences in isoform expression in atherosclerotic lesions, with distinct patterns of staining for ECE-1a and ECE-1c. ECE-1c immunoreactivity was seen in macrophages, and also correlated with actin staining. ECE-1a was also localized to macrophages and SMC. Results of this study suggest that these local changes influence the expression patterns of the ECE-1 isoforms within individual cell types. Correlation of these isoform expression patterns with the stage of atherosclerosis could provide novel indicators of disease progression.

Patellofemoral Pain Syndrome in Tibetan Buddhist Monks

Patellofemoral pain syndrome is a common diagnosis in athletes and especially runners. This article discusses 3 cases of patellofemoral pain caused by pronounced inactivity and prolonged knee hyperflexion at altitude in a unique population of Tibetan Buddhist monks. In this case, the monks responded well to a program of activity modification and exercises.

Guest Editorial: Assessing Risks and Benefits of Genistein and Soy

Guest Editorial: Assessing Risks and Benefits of Genistein and Soy

Vascular Cell Locomotion

See related article, pages 1479–1489 Since the discovery of osteopontin (OPN) in the vasculature, vascular biologists have been intrigued by its potential role in vascular arteriosclerosis and vessel calcification during the aging process.1 Osteopontin is a phosphorylated acidic glycoprotein adhesion molecule, originally cloned from bone; and as in bone, its cardiovascular functions include cellular migration, adhesion, and spreading.2 The most salient features of this intriguing calcium-regulating molecule are its ability to interact with αvβ3 and other integrins on the surface of cells. Because this ligand–receptor complex is known to be involved in cell migration, it is not surprising that an antibody to the β3 subunit of this receptor effectively reduced restenosis in patients.3 Interestingly, angiotensin II and other growth factors, including platelet-derived growth factor and transforming growth factor-β, regulate OPN gene expression in vascular smooth muscle cell (VSMC) cultures and fibroblasts.1,4 Cardiac fibroblasts have been extensively reported to produce OPN in response to these growth factors, resulting in fibroblast proliferation. On the one hand, OPN is thought to cause vascular dystrophy by altering extracellular matrix formation; on the other hand, cellular motogenesis is a hallmark of this emerging vascular cytokine. Its cellular expression includes a variety of inflammatory cells, such as T cells and macrophages, as well as mesenchymal cells of the vasculature including adventitial myofibroblasts. Moreover, OPN has both pro- and antiinflammatory properties. As a proinflammatory agent it can attract and modulate the function of the above mentioned cell types. Of seemingly major importance, OPN interacts with intracellular signaling agents to promote cell migration and matrix metalloproteinases, both of which are required for cell tunneling through dense and elastin-rich vascular tissue. In fibroblasts these agents appear to include cell surface CD44 and ERM proteins located just below the plasma membrane …

CD163 Identifies Perivascular Macrophages in Normal and Viral Encephalitic Brains and Potential Precursors to Perivascular Macrophages in Blood

Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.