Inhibition of neurogenic inflammation by the Amazonian herbal medicine sangre de grado.

UNLABELLED: This study was designed to determine if the Amazonian medicinal sangre de grado, confers benefit by suppressing the activation of sensory afferent nerves. METHODS: (i) vasorelaxation of rat mesenteric arteries in response to calcitonin gene-related peptide; (ii) rat paw edema in response to protease- activating peptide receptor 2-activating peptide; (iii) rat paw hyperalgesia in response to low-dose protease-activating peptide receptor 2-activating peptide or prostaglandin E2; (iv) gastric hyperemia in response luminal capsaicin; (v) a clinical trial of a sangre de grado balm in pest control workers. The parent botanical was fractionated for evaluation of potential active components. In preconstricted rat mesenteric arteries, highly diluted sangre de grado (1:10,000) caused a shift to the right of the calcitonin gene-related peptide dose-response curve (p < 0.01). Paw edema in response to protease-activating peptide receptor 2-activating peptide (500 microg) was reduced by as single topical administration sangre de grado balm (1% concentration, p < 0.01) for at least 6 h. Hyperalgesia induced by either low-dose protease-activating peptide receptor 2-activating peptide (50 microg) or prostaglandin E2 was prevented by sangre de grado balm. A fraction possessing analgesic and capsaicin antagonistic properties was isolated and high-performance liquid chromatography and gas chromatography-mass spectrometry analysis indicated that it was a proanthocyandin oligomer. In pest control workers, sangre de grado balm (Zangrado) was preferred over placebo, for the relief of itching, pain, discomfort, edema, and redness in response to wasps, fire ants, mosquitoes, bees, cuts, abrasions, and plant reactions. Subjects reported relief within minutes. We conclude that sangre de grado is a potent inhibitor of sensory afferent nerve mechanisms and supports its ethnomedical use for disorders characterized by neurogenic inflammation.

Treatment of gastric ulcers and diarrhea with the Amazonian herbal medicine sangre de grado.

Sangre de grado is an Amazonian herbal medicine used to facilitate the healing of gastric ulcers and to treat gastritis, diarrhea, skin lesions, and insect stings. This study was designed to evaluate the gastrointestinal applications. Gastric ulcers were induced in rats by brief serosal exposure of the fundus to acetic acid (80%). Sangre de grado was administered in drinking water at 1:1,000 and 1:10,000 dilutions from the postoperative period to day 7. Guinea pig ileum secretory responses to capsaicin, electrical field stimulation, and the neurokinin-1 (NK-1) agonist [Sar(9),Met(O(2))(11)]substance P were examined in Ussing chambers. Sangre de grado facilitated the healing of experimental gastric ulcer, reducing myeloperoxidase activity, ulcer size, and bacterial content of the ulcer. The expression of proinflammatory genes tumor necrosis factor-alpha, inducible nitric oxide synthase (iNOS), interleukin (IL)-1beta, IL-6, and cyclooxygenase-2 was upregulated by ulcer induction but reduced by sangre de grado treatment, particularly iNOS and IL-6. In Ussing chambers, sangre de grado impaired the secretory response to capsaicin but not to electrical field stimulation or the NK-1 agonist. We conclude that sangre de grado is a potent, cost-effective treatment for gastrointestinal ulcers and distress via antimicrobial, anti-inflammatory, and sensory afferent-dependent actions.

Rat somatosensory cerebropontocerebellar pathways: spatial relationships of the somatotopic map of the primary somatosensory cortex are preserved in a three-dimensional clustered pontine map.

In the primary somatosensory cortex (SI), the body surface is mapped in a relatively continuous fashion, with adjacent body regions represented in adjacent cortical domains. In contrast, somatosensory maps found in regions of the cerebellar hemispheres, which are influenced by the SI through a monosynaptic link in the pontine nuclei, are discontinuous ("fractured") in organization. To elucidate this map transformation, the authors studied the organization of the first link in the SI-cerebellar pathway, the SI-pontine projection. After injecting anterograde axonal tracers into electrophysiologically defined parts of the SI, three-dimensional reconstruction and computer-graphic visualization techniques were used to analyze the spatial distribution of labeled fibers. Several target regions in the pontine nuclei were identified for each major body representation. The labeled axons formed sharply delineated clusters that were distributed in an inside-out, shell-like fashion. Upper lip and other perioral representations were located in a central core, whereas extremity and trunk representations were found more externally. The multiple clusters suggest that the pontine nuclei contain several representations of the SI map. Within each representation, the spatial relationships of the SI map are largely preserved. This corticopontine projection pattern is compatible with recently proposed principles for the establishment of subcortical topographic patterns during development. The largely preserved spatial relationships in the pontine somatotopic map also suggest that the transformation from an organized topography in SI to a fractured map in the cerebellum takes place primarily in the mossy fiber pontocerebellar projection.

Spatial correspondence between tactile projection patterns and the distribution of the antigenic Purkinje cell markers anti-zebrin I and anti-zebrin II in the cerebellar folium crus IIA of the rat.

We have compared the band-like distribution of the Purkinje cell-specific polypeptides zebrin I and zebrin II with the spatial organization of tactile projections to crus IIa in the cerebellar hemisphere of the rat. Maps of tactile responses in the granular layer of the cerebellar hemispheres are fractured into discontinuous regions, termed "patches". High-density micromapping was used to identify specific patches and their boundaries within this fractured somatotopic map. In one series of experiments, medial and lateral boundaries of the large central ipsilateral upper lip-related patch were identified and labeled with either Fast Blue or India Ink. Following immunocytochemical processing, the band-like distribution of immunostained Purkinje cells (zebrin-positive bands) and the identified patch boundaries were digitized and reconstructed in three dimensions. Comparisons between these two features demonstrate a spatial correspondence between zebrin transitions and the boundaries of the electrophysiologically defined upper lip-related patch. In another series of experiments, we outlined the boundaries or centers of several smaller patches consistently located in the medial portion of the folium. Again, we found a correspondence between the distribution of granule cell layer tactile patches and the zebrin staining pattern. The correspondence between tactile projection patterns and molecular features demonstrated in the present study implies that there is a distinct and largely fixed spatial pattern of organization in the cerebellar hemispheres. We discuss possible causal connections and developmental determinates, as well as the physiological significance of the correspondence between the two features.

Localization of inducible nitric oxide synthase mRNA in inflamed gastrointestinal mucosa by in situ reverse transcriptase-polymerase chain reaction.

Immunohistochemistry has been critical in determining the tissue localization of inducible nitric oxide synthase (iNOS). However, this technique suffers from nonspecific staining which may lead to false-positive results and the failure of antisera to recognize iNOS from different species. We developed a technique to determine the localization of iNOS mRNA, as opposed to protein, in tissue sections using an in situ RT-PCR (IS RT-PCR) technique. Sections of inflamed gastrointestinal mucosa were used because they were known to be positive for iNOS. The IS RT-PCR technique localized iNOS mRNA to the same sites as immunoreactive iNOS in human gastritis associated with Helicobacter pylori infection, Crohn's disease, and experimental inflammatory bowel disease induced by the hapten TNBS, in rat colon and in guinea pig ileum. The detection of mRNA had an excellent signal-to-noise ratio. Preservation of tissue morphology was poorer than that with immunohistochemistry due the cycles of heating required in the PCR process. This method could be very useful in detecting iNOS gene expression in situations of excessive nonspecific staining with immunohistochemistry or of failure of antibodies to react because of species differences. This technique is also readily applicable to detect RNA and DNA markers of other disease processes.

Interleukin-10 attenuates experimental fetal growth restriction and demise.

Premature labor, fetal demise, and fetal growth restriction are accompanied by indices of inflammation or infection of the uteroplacental unit. To understand whether these events are causally related, we established an animal model of fetal demise and growth restriction and evaluated the potential utility of the anti-inflammatory cytokine interleukin-10 (IL-10). We administered low-dose endotoxin (lipopolysaccharide, or LPS, 100 microg/kg, i.p.) to third trimester rats (gestational days 14-20). Control rats received normal saline. A third group received IL-10 (100 microg/kg; s.c.) concomitantly with LPS for 7 prenatal days. Cytokine gene expression (IL-10 and TNF-alpha) was evaluated by RT-PCR and tissue levels (TNF-alpha) were determined by ELISA. Apoptosis was evaluated by TdT-mediated dUTP nick end labeling immunohistochemistry, and nitric oxide (NO) levels were quantified by microelectrode electrochemical detection in explants in culture media. LPS exposure resulted in 43% fetal demise and reduced the size of the surviving fetuses. Placental weight was not altered by LPS. IL-10 attenuated the LPS-induced fetal death rate (to 22%) and growth restriction (P<0.05). In normal rats, IL-10 did not affect fetus size or the incidence of resorptions, although placental size was marginally smaller. Increased uterine TNF-alpha content and NO release and apoptosis of uterine epithelia and muscularis were hallmarks of the LPS model. All were normalized by IL-10. IL-10 may represent a new therapeutic option for the treatment of a variety of perinatal complications. Benefit may result from the suppression of TNF-alpha- and NO-mediated cell death.

Antiinflammatory actions of cat's claw: the role of NF-kappaB.

BACKGROUND: Uncaria tomentosa is a vine commonly known as cat's claw or 'uña de gato' (UG) and is used in traditional Peruvian medicine for the treatment of a wide range of health problems, particularly digestive complaints and arthritis. PURPOSE: The aim of this study was to determine the proposed anti-inflammatory properties of cat's claw. Specifically: (i) does a bark extract of cat's claw protect against oxidant-induced stress in vitro, and (ii) to determine if UG modifies transcriptionally regulated events. METHODS: Cell death was determined in two cell lines, RAW 264.7 and HT29 in response to peroxynitrite (PN, 300 microM). Gene expression of inducible nitric oxide synthase (iNOS) in HT29 cells, direct effects on nitric oxide and peroxynitrite levels, and activation of NF-kappaB in RAW 264.7 cells as influenced by UG were assessed. Chronic intestinal inflammation was induced in rats with indomethacin (7.5 mg/kg), with UG administered orally in the drinking water (5 mg/mL). RESULTS: The administration of UG (100 microg/mL) attenuated (P < 0.05) peroxynitrite-induced apoptosis in HT29 (epithelial) and RAW 264.7 cells (macrophage). Cat's claw inhibited lipopolysaccharide-induced iNOS gene expression, nitrite formation, cell death and inhibited the activation of NF-kappaB. Cat's claw markedly attenuated indomethacin-enteritis as evident by reduced myeloperoxidase activity, morphometric damage and liver metallothionein expression. CONCLUSIONS: Cat's claw protects cells against oxidative stress and negated the activation of NF-kappaB. These studies provide a mechanistic evidence for the widely held belief that cat's claw is an effective anti-inflammatory agent.

Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis.

Therapeutic efficacy of interleukin-10 administration in colonic inflammation was assessed in rats. Following intracolonic instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS), subcutaneous administration of 1-1000 micrograms/kg per day interleukin-10, or a placebo (0.9% NaCl) was commenced and continued for 5 days. Interleukin-10 administered at 1, 10 and 100 micrograms/kg per day significantly reduced myeloperoxidase activity by 34, 57, and 28%, respectively, compared to the placebo-treated group, which was paralleled by an attenuation of colonic tumor necrosis factor alpha (TNF-alpha) content. In contrast, the severity of mucosal necrosis was not affected by interleukin-10 administration at the dose range used. In addition, the 10-fold elevation in nitric oxide release, 5-fold rise in colonic nitrite production and enhanced expression of inducible nitric oxide synthase, associated with TNBS colitis, was not suppressed by interleukin-10. Interleukin-10 gene expression was elevated during the first 14 days of TNBS colitis. We conclude that 5 days administration of interleukin-10 in TNBS colitis displays mild anti-inflammatory properties which were not mediated via a nitric oxide-dependent pathway, but may involve TNF-alpha.

Inducible nitric oxide synthase and the regulation of central vessel caliber in the fetal rat.

BACKGROUND: The purpose of this study was to evaluate the possibility that inducible nitric oxide synthase (iNOS) regulates the fetal circulation. METHODS AND RESULTS: Positive evidence for iNOS gene expression was noted in heart central vessels and placenta of untreated rat fetuses. Rats in the last week of pregnancy were treated for 5 days with L-NG-(1-Iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS, at 1, 10, and 100 micrograms/mL in the drinking water. To raise NO levels, lipopolysaccharide (LPS) 30 micrograms/kg was given by intraperitoneal injection, and sodium nitroprusside (SNP) was placed in mini-osmotic pumps to deliver 10 micrograms/kg per minute. Control animals were undisturbed. On day 21 of gestation, dams were anesthetized and fetuses were delivered by cesarean section and rapidly frozen in isopentane chilled in liquid nitrogen. Frozen sections (10 microns) were used to reconstruct a computer-generated three-dimensional image of the great vessels and ductus arteriosus. Significant constriction of the great vessels and ductus arteriosus was observed with L-NIL, whereas both LPS and SNP dilated these vessels. The vasorelaxant effect of LPS was blocked by L-NIL. NO release from placental explants was 633 +/- 41 nmol/L under basal conditions, increasing to 4.0 +/- 0.4 mumol/L with LPS administration, although placental iNOS message and protein levels were unchanged. CONCLUSIONS: We suggest that nitric oxide, generated by iNOS, plays a significant role in control of major vessel and ductus arteriosus caliber in the rat fetus. In regard to the nitrergic regulation of the circulation, the fetus is clearly different from the adult.

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase.

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.

Fetal growth retardation in rats may result from apoptosis: role of peroxynitrite.

Administration of the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) results in fetal growth retardation. This study was designed to further examine the influence of NO on fetal growth, specifically, the potential role of inducible NOS and to evaluate the possibility that apoptosis contributed to uteroplacental dysfunction. L-NAME administration caused a paradoxical increase in NO synthesis determined by direct detection of NO by electrochemistry, nitrite accumulation, and cGMP levels, indicating that a lack of NO was not the cause of the fetal growth retardation. Additionally, supplemental L-arginine or NO donors failed to reverse the effects of L-NAME on fetal and placental size. Administration of low dose endotoxin (30 micrograms/kg IP daily for 6 d) also caused significant reductions in fetal and placental size and increased NO synthesis comparable to that seen with L-NAME. Inducible NOS was constitutively expressed in the pregnant uterus (smooth muscle and epithelia) and placenta (sinusoids and macrophages) but was absent in the nonpregnant state as determined by RT-PCR and immunohistochemistry. Neither L-NAME nor endotoxin modified the expression of iNOS. In situ evidence for apoptosis (DNA fragmentation) was minimal to absent in control pregnant rats, but markedly evident in the placenta (decidua) and uterus of rats treated with L-NAME or endotoxin. Immunohistochemical evidence for nitrotyrosine, a marker for peroxynitrite formation, was absent in control rats but colocalized with apoptosis in the L-NAME and LPS groups. We conclude that L-NAME-induced fetal growth retardation is not due to a lack of NO, but as for endotoxin, results from a net reduction in cellular proliferation due to the induction of apoptosis, possibly in response to peroxynitrite formation.

Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis.

BACKGROUND & AIMS: Inflammatory bowel disease is characterized by increased synthesis of nitric oxide. The aim of this study was to determine if inducible NO synthase (iNOS) was responsible for tissue injury, potentially via peroxynitrite formation, in the guinea pig model of gut inflammation. METHODS: Inflammation was induced in guinea pig ileum by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. iNOS gene expression was assessed by reverse-transcriptase polymerase chain reaction and Western blotting, immunohistochemistry was determined by its localization, and activity was inhibited with the specific inhibitor aminoguanidine administered via the drinking water for 7 days. Nitration of tyrosines was assessed by immunohistochemistry. RESULTS: In control animals, iNOS gene expression was minimal to absent, whereas, in hapten, inflammation-marked iNOS gene expression was evident from day 1 to 7. Nitrotyrosine and iNOS immunohistochemistry were colocalized, and positive staining was most intense in epithelia and neurons. Inhibition of NO formation prevented nitrotyrosine formation. Aminoguanidine inhibited the inflammatory response and restored morphology. CONCLUSIONS: The colocalization of tyrosine nitration with iNOS immunoreactivity suggests that iNOS may be responsible for tissue injury and the formation of NO-dependent nitrating species, potentially peroxynitrite. Inhibition of iNOS may afford a new therapeutic approach to the treatment of inflammatory bowel disease.

Potential role of nitric oxide in a model of chronic colitis in rhesus macaques.

BACKGROUND/AIMS: Excess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis. METHODS: NO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction. RESULTS: Plasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity. CONCLUSIONS: It is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model.

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant.

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.

Entamoeba polecki infection in Southeast Asian refugees: multiple cases of a rarely reported parasite.

Recently, Entamoeba polecki was identified for the first time in our parasitology laboratory in stool specimens from eight Southeast Asian refugees. This ameba has been reported infrequently in the Western world; most reported cases have been from the New Guinea region. In most previously described patients and in our patients, no definite gastrointestinal symptoms could be directly attributed to E. polecki infection. Morphologically, E. polecki may mimic the pathogen E. histolytica and also nonpathogens such as E. coli. These species are most readily distinguished by studying encysted forms. In contrast to E. histolytica and E. coli, E. polecki characteristically has uninucleate cysts. Both pigs and monkeys naturally harbor E. polecki, but four of the patients in this series had no apparent contact with these animals. Other modes of infection may be human-to-human transmission or acquisition from other domestic animals. Six of our eight patients were treated successfully with one course of metronidazole in a regimen similar to that used for E. histolytica infection. In the two other patients, repeated courses of therapy eradicated the infection. Because of the recent increase in number of Southeast Asian immigrants to the United States, E. polecki may be identified more frequently than in the past. Physicians and laboratory personnel should be familiar with this organism, because it may be confused with E. histolytica or may act as a pathogen.

The impact of the IRB on medical centers.

The role of the IRB in a Medical Center is presented with respect to investigations of medical device safety and effectiveness involving human subjects. The prime points presented and discussed are: the reasons (governmental, social, economic, legal-liability, scientific and moral) for the existence of an IRB; the analytical and descriptive documentation which should always precede experimentation; the concepts governing an application to a "typical" IRB; a practical, detailed outline of some special facts and circumstances typically most important to an IRB; and, the question of confidentiality of trade secrets.

Infectious diseases of Indochinese refugees.

The recent Mayo Clinic experience in the diagnosis and treatment of infectious diseases in Indochinese refugees is discussed. One hundred patients from whom stool and blood specimens were submitted for parasitic examination showed a high percentage of parasitic infection, often with multiple agents. An outline for the initial medical examination and treatment of the parasitic agents found is presented.

Visceral leishmaniasis (kala-azar) as a cause of fever of unknown origin.

A patient with a rare cause of fever of unknown origin, visceral leishmaniasis (kala-azar), is reported. The diagnosis was made by exploratory laparotomy and splenectomy after diagnostic studies had failed to reveal the cause of the fever. The patient was cured with a 6-day course of therapy with Pentostam (sodium antimony gluconate). Visceral leishmaniasis should be considered in the differential diagnosis of patients with obscure fever who have traveled in endemic areas.