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Human gut microbial dynamics are highly individualized, making it challenging to link microbiota to health and to design universal microbiome therapies. This individuality is typically attributed to variation in host genetics, diets, environments and medications but it could also emerge from fundamental ecological forces that shape microbiota more generally. Here, we leverage extensive gut microbial time series from wild baboons-hosts who experience little interindividual dietary and environmental heterogeneity-to test whether gut microbial dynamics are synchronized across hosts or largely idiosyncratic. Despite their shared lifestyles, baboon microbiota were only weakly synchronized. The strongest synchrony occurred among baboons living in the same social group, probably because group members range over the same habitat and simultaneously encounter the same sources of food and water. However, this synchrony was modest compared to each host's personalized dynamics. In support, host-specific factors, especially host identity, explained, on average, more than three times the deviance in longitudinal dynamics compared to factors shared with social group members and ten times the deviance of factors shared across the host population. These results contribute to mounting evidence that highly idiosyncratic gut microbiomes are not an artefact of modern human environments and that synchronizing forces in the gut microbiome (for example, shared environments, diets and microbial dispersal) are not strong enough to overwhelm key drivers of microbiome personalization, such as host genetics, priority effects, horizontal gene transfer and functional redundancy.

The process of glutamate release, activity, and reuptake involves the astrocyte, the presynaptic and postsynaptic neuron. Glutamate is released into the synapse and may occupy and activate receptors on both neurons and astrocytes. Glutamate is rapidly removed from the synapse by a family of plasma membrane excitatory amino acid transporters (EAATs), also localized to neurons and astrocytes. The purpose of the present study was to examine EAAT labeling in postmortem human cortex at the light and electron microscopic levels. Postmortem prefrontal cortex was processed for EAAT1 and EAAT2 immunohistochemistry. At the light microscopic level, EAAT1 and EAAT2 labeling was found in both grey and white matter. Most cellular labeling was in small cells which were morphologically similar to glia. In addition, EAAT1 labeled neurons were scattered throughout, some of which were pyramidal in shape. At the electron microscopic level, EAAT1 and EAAT2 labeling was found in astrocytic soma and processes surrounding capillaries. EAAT labeling was also found in small astocytic processes adjacent to axon terminals forming asymmetric (glutamatergic) synapses. While EAAT2 labeling was most prevalent in astrocytic processes, EAAT1 labeling was also present in neuronal processes including the soma, axons, and dendritic spines. Expression of EAAT1 protein on neurons may be due to the hypoxia associated with the postmortem interval, and requires further confirmation. The localization of EAATs on the astrocytic plasma membrane and adjacent to excitatory synapses is consistent with the function of facilitating glutamate reuptake and limiting glutamate spillover. Establishment that EAAT1 and EAAT2 can be measured at the EM level in human postmortem tissues will permit testing of hypotheses related to these molecules in diseases lacking analogous animal models.

Typically anadromous, the twaite shad (Alosa fallax) can become landlocked and adapt to a fully freshwater life. The only landlocked shad population in Northwestern Europe is found in a lake in Ireland, Lough Leane. The Killarney shad, Alosa killarnensis (or Alosa fallax killarnensis, as it is mostly referred to) displays a level of morphological divergence that indicates a long-term isolation in the lake. Microsatellites and mtDNA control region sequences were used within a coalescent framework (BEAST and Approximate Bayesian Computation (ABC)) to investigate its colonization history and to clarify its taxonomic status. Results indicate that the lake was likely colonized in two independent events, the first coinciding with the retreat of the ice sheet from the area after the Last Glacial Maximum and the second after the Younger Dryas. Microsatellite data has shown that these two landlocked lineages have completely admixed within the lake, and have started diverging significantly from their closest ancestor, the twaite shad. We argue that our molecular results (together with the life-history and physiological divergence between Killarney and twaite shad) are conspicuous enough to view the landlocked population as a new species, and one whose future existence would certainly not be insured by its sister taxon.

Enteroaggregative Escherichia coli (EAEC) is a major pathogen worldwide, associated with diarrheal disease in both children and adults, suggesting the need for new preventive and therapeutic treatments. We investigated the role of the micronutrient zinc in the pathogenesis of an E. coli strain associated with human disease. A variety of bacterial characteristics-growth in vitro, biofilm formation, adherence to IEC-6 epithelial cells, gene expression of putative EAEC virulence factors as well as EAEC-induced cytokine expression by HCT-8 cells-were quantified. At concentrations (≤ 0.05 mM) that did not alter EAEC growth (strain 042) but that are physiologic in serum, zinc markedly decreased the organism's ability to form biofilm (P0.001), adhere to IEC-6 epithelial cells (P0.01), and express putative EAEC virulence factors (aggR, aap, aatA, virK) (P0.03). After exposure of the organism to zinc, the effect on virulence factor generation was prolonged (3 h). Further, EAEC-induced IL-8 mRNA and protein secretion by HCT-8 epithelial cells were significantly reduced by 0.05 mM zinc (P0.03). Using an in vivo murine model of diet-induced zinc-deficiency, oral zinc supplementation (0.4 µg/mouse daily) administered after EAEC challenge (10 (10) CFU/mouse) significantly abrogated growth shortfalls (by90%; P0.01); furthermore, stool shedding was reduced (days 9-11) but tissue burden of organisms in the intestine was unchanged. These findings suggest several potential mechanisms whereby physiological levels of zinc alter pathogenetic events in the bacterium (reducing biofilm formation, adherence to epithelium, virulence factor expression) as well as the bacterium's effect on the epithelium (cytokine response to exposure to EAEC) to alter EAEC pathogenesis in vitro and in vivo. These effects may help explain and extend the benefits of zinc in childhood diarrhea and malnutrition.

Glycogen synthase kinase-3β (GSK3β) activity has been previously linked to Alzheimer's disease (AD) by its phosphorylation of tau and activation by amyloid. GSK3β intracellular distribution is important in regulating its activity by restricting access to compartment-specific substrates. This study investigated regional and intracellular distribution of GSK3β in a mouse model of AD, a bigenic mouse with combined amyloid and tau pathology (BiAT), and controls (FVB). At two different ages, the entire rostrocaudal extent of each brain was examined. Young (6-months-old) FVB and BiAT mice did not differ in GSK3β expression and localization. In old (13-month-old) BiAT mice, neurons showed increased GSK3β expression only in AD-relevant brain regions as compared with modest staining in region- and age-matched controls. Two regions with the most robust changes between FVB and BiAT mice, the amygdala and piriform cortex, were quantified at the light microscopic level. In both regions, the density of darkly labeled neurons was significantly greater in the old BiAT mice vs. the old FVB mice. Electron microscopy of the piriform cortex showed neuronal GSK3β labeling in the rough endoplasmic reticulum, on ribosomes, and on microtubules in dendrites in both strains of mice. In old BiAT mice, GSK3β labeling was qualitatively more robust compared to age-matched controls, and GSK3β also appeared in neurofibrillary tangles. In conclusion, GSK3β expression was increased in specific intracellular locations and was found in tangles in old BiAT mice, suggesting that GSK3β overexpression in specific brain areas may be intrinsic to AD pathology.

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children., Author Summary Cryptosporidium attributable morbidities in malnourished children are increasingly recognized. Exactly how malnutrition interferes with host mucosal immunity to diarrheal pathogens and mucosal vaccine responses remains unclear. Dissecting these interactions in an experimental model of cryptosporidiosis can uncover new insights into novel therapeutic approaches against a pathogen for which effective therapies and vaccines are currently unavailable. We demonstrate that although malnutrition diminishes baseline (primary) Th1-type mucosal immunity these deficits can be partially overcome via non-specific mucosal strategies (S. Typhi and CpG) and completely restored after a sub-clinical (low-dose) exposure to viable C. parvum. These results add insight into preventive strategies to help alleviate Cryptosporidium-specific diarrhea in children in low-resource settings and abrogate prolonged post-infection sequelae.

Bryde's whales (Balaenoptera edeni) are commonly found in tropical and subtropical regions of the Pacific Ocean, but few studies have explored the presence of Bryde's whales at the boundary of their distribution range. Such studies are increasingly relevant as climate impact models predict the range expansion of warm water species towards the poles in response to ocean warming. Like other baleen whales, Bryde's whales produce distinct low frequency (

Summary In Ireland, the Water Framework Directive (WFD) Freshwater Morphology Programme of Measures and Standards (POMS) identified barriers to fish migration as one of the principal issues placing channels ‘at risk’ in terms of failing to achieve hydromorphology status. To quantify the degree of risk posed, and to develop methods to appraise and quantify risk associated with barriers, the POMS undertook a pilot study on the River Nore catchment. This provided an opportunity to compare the outcomes of an intensive field-based qualitative assessment of barriers to fish migration with a rules-based appraisal of risk, based on layers of geo-referenced data, in a Geographical Information System (GIS) analysis. The underlying aim of this risk-based assessment was the restoration of the fisheries resource, in the context of WFD, grounded on knowledge-based assessment. The comparison focused on the Atlantic salmon (Salmo salar L.). A rapid-assessment field survey compiled a geo-referenced inventory of 508 structures (497 artificial and 11 natural) and classified each with regard to presumed risk of preventing the upstream migration of adult Atlantic salmon. This inventory was then subjected to a rigorous GIS-based analysis, using field-generated datasets on Atlantic salmon ecology in the Nore catchment. The rules-based analysis provided a very substantial re-classification of artificial barriers with a major reduction in the numbers classified as impassable or high-risk. It confirmed that no ‘high risk’ barriers to adult salmon occurred on the R. Nore main stem but identified clusters of high risk barriers in the Dinin subcatchment and a series of moderate risk obstructions in the King’s River system. The GIS-based approach enabled an examination of the costs and benefits of stepwise removal of barriers, in terms of additional wetted habitat and salmon spawning area made accessible upstream of each barrier. A multi-species approach was then explored, examining the risk to upstream passage for eel (Anguilla Anguilla L.), sea lamprey (Petromyzon marinus L.) and Twaite shad (Alosa fallax Lacepede). Substantial clustering of sea lamprey spawning effort was reported downstream of the lowermost weirs on the R. Nore. A combination of barriers identified from a salmon perspective and via the multi-species approach provided a listing of priority barriers for attention. The strategy of step-wise removal or modification of artificial barriers in an upstream direction would provide optimal conservation value for the relevant species. The study identified the primacy of the field survey in geo-referencing site locations. However, it also identified the shortcomings of the qualitative, ‘expert-judgement’ approach to risk category allocation of the barriers and, hence, the need for more rigorous criteria in implementing a field-based rapid assessment.

The role of the liver in fatal intoxication with the binary toxin ricin is undefined. Means to reverse pathological events in the liver and to gain host survival after exposure to this toxin have not been described. We report a robust 7/4+ neutrophil influx into the liver of C57BL/6 mice after parenteral ricin challenge. This influx occurs in peri-portal and centro-lobular hepatic areas within 2 hours, followed by the abrupt disappearance of hepatic macrophages/Kupffer cells at 8–12 hours. Microarray, ribonuclease protection assays, Northern blotting, and enzyme-linked immunosorbent assays showed chemokine mRNA and protein for neutrophils (CXCL1/KC, CXCL2/MIP-2) and macrophages (CCL2/MCP-1) increased in the liver by 2 hours and persisted for more than 40 hours after ricin injection. A specific sequence of intra-hepatic pathophysiological events followed ricin injection, including red blood cell pooling (8–12 h), loss of hepatocyte glycogen (8–48 h) associated with progressive hypoglycemia, and fibrin deposition (24–48 h). Monoclonal antibody to ricin A chain administered intravenously blunted hypoglycemia and abrogated death. This outcome was observed when anti-ricin antibody was present prior to toxin exposure or when administered up to 10 hours post-toxin exposure. Targeting antibody to specific amino acid sequences on the ricin A chain (HAEL and QXXWXXA) was critical to the therapeutic effect. Re-emergence of liver macrophages/Kupffer cells and replenishment of glycogen in previously depleted hepatocytes preceded full recovery of the host. These data define the pathobiology of liver injury in ricin intoxication, as well as a new means and specific targets for post-exposure therapeutic intervention.

Notes MARINE MAMMAL SCIENCE, **(*): ***–*** (*** 2014) © 2014 The Authors. Marine Mammal Science published by Wiley Periodicals, Inc. on behalf of Society for Marine Mammalogy This is an open access article under the terms of the Creative Commons Attribution- NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1111/mms.12189 North Pacific right whales (Eubalaena japonica) recorded in the northeastern Pacific Ocean in 2013 A NA S IROVI C , 1 S ARAH C. J OHNSON , L AUREN K. R OCHE , L EAH M. V ARGA , S EAN M. W IGGINS and J OHN A. H ILDEBRAND , Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive MC 0205, La Jolla, California 92093-0205, U.S.A. The North Pacific right whale (Eubalaena japonica) is among the most critically endangered marine mammals in the world (Brownell et al. 2001). Its population was drastically reduced by 19th and 20th century whaling and its recovery was addition- ally slowed by illegal Soviet whaling that occurred during the 1960s (Clapham et al. 2004, Ivashchenko and Clapham 2012). With a severe decrease in the population also came a restriction in range, from extensive distribution across the North Pacific, to a constricted presence of two different populations, one in the Sea of Okhotsk and along the Western Pacific, and the other in the eastern Bering Sea (Scarff 1986, Clap- ham et al. 2004, Josephson et al. 2008). The abundance estimates for the eastern pop- ulation are around 30 animals (Wade et al. 2006, 2011b; Marques et al. 2011), while the western population is considered to be larger with current estimates of probably no more than 300 animals (Brownell et al. 2001). Most baleen whales undertake seasonal migrations, spending summers feeding in high latitude, productive areas and winters in warmer, lower latitude breeding grounds (Kellogg 1929). North Pacific right whales spend their summers in the Ber- ing Sea and, to a lesser extent, the Gulf of Alaska, the Aleutian archipelago, and off Kodiak (Scarff 1986, Brownell et al. 2001, Clapham et al. 2004, Mellinger et al. 2004, Shelden et al. 2005, Wade et al. 2011a). Most current research effort has been focused within the area in the Southeastern Bering Sea where North Pacific right whales have been sighted most frequently (Wade et al. 2006, Munger et al. 2008). Over the years, however, a small number of sightings of single right whales have been Corresponding author (e-mail: asirovic@ucsd.edu).

We investigated the effect of multiple variables on the amplification success rate of microsatellite DNA extracted from faeces of wild Eurasian otters. The success rate was affected by (i) type of sample, with higher success rates in anal jelly samples than faeces, and (ii) temperature, with a negative effect of increased temperature at time of collection. To increase the effectiveness of microsatellite genotyping of otter faeces, we recommend collecting samples in cold months and early in the morning, preferably in a frozen state, and the collection of anal jelly samples, or the jelly part from faeces, whenever possible.

Trefoil factor-1 (Tff1) expression is remarkably down-regulated in nearly all human gastric cancers. Therefore, we used the Tff1 knockout mouse model to detect molecular changes in preneoplastic gastric dysplasia. Oligonucleotide microarray gene expression analysis of gastric dysplasia of Tff1 −/− mice was compared to that of normal gastric mucosa of wild-type mice. The genes most overexpressed in Tff1−/− mice included claudin-7 (CLDN7), early growth response-1 (EGR1), and epithelial membrane protein-1 (EMP1). Quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry showed that Cldn7 was overexpressed in all 10 Tff1−/− gastric dysplasia samples. Comparison with our serial analysis of gene expression database of human gastric cancer revealed similar deregulation in human gastric cancers. Quantitative real-time reverse transcriptase-polymerase chain reaction of human gastric adenocarcinoma samples indicated that, of these three genes, CLDN7 was the most frequently up-regulated gene. Using immunohistochemistry, both mouse and human gastric glands overexpressed Cldn7 in dysplastic but not surrounding normal glands. Cldn7 expression was observed in 30% of metaplasia, 80% of dysplasia, and 70% of gastric adenocarcinomas. Interestingly, 82% of human intestinal-type gastric adenocarcinomas expressed Cldn7 whereas diffuse-type gastric adenocarcinomas did not (P < 0.001). These results suggest that Cldn7 expression is an early event in gastric tumorigenesis that is maintained throughout tumor progression.

Reelin is a glycoprotein that plays a critical role in brain development, including proper cortical lamination. In adult animals, reelin continues to be expressed in different neuronal populations in many brain regions. We performed labeling for reelin immunoreactivity (-i) in post-mortem cerebral cortex from five adults and two fetuses with three different antibodies. The tissue was then processed for light and electron microscopy. In cell bodies, reelin-i was found in pyramidal and nonpyramidal neurons on the outer nuclear membrane, rough endoplasmic reticulum (rER), and ribosomes. In dendrites, labeling was found in the rER and ribosomes and was diffusely distributed in spines. In the neuropil, diffuse labeling was seen in small axon terminals and unmyelinated axons, and the postsynaptic density (PSD) frequently had discrete labeling. Reelin-i was also found in glial somata and in small astrocytic processes. With rare exceptions, reelin-i in the adult was conspicuously absent from both the extracellular matrix (ECM) and the subcellular organelles, where secreted proteins are modified and taken back into the cell. Labeling in fetal cortex was similar to that in the adult except for prominent labeling in the ECM. The presence of reelin in adult spines, PSD, and terminals suggests that in the adult human reelin has a role in synaptic remodeling, which is consistent with the evidence for its role in long-term potentiation in the adult brain. J. Comp. Neurol. 482:294–308, 2005. © 2004 Wiley-Liss, Inc.

BACKGROUND Gastric carcinoma is one of the leading causes of cancer-related death worldwide, but the mechanisms underlying its development and progression still remain largely uncharacterized. As loss of trefoil factor 1 (TFF1) expression leads to neoplastic growth in the antropyloric mucosa of mice, the authors sought to comprehensively study the human TFF1 gene in primary gastric carcinomas. METHODS The authors studied the human TFF1 gene in primary gastric carcinomas and normal gastric mucosa at the DNA, RNA, and protein levels through DNA sequencing, quantitative real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry. RESULTS Strikingly, TFF1 mRNA expression was significantly decreased in all 37 gastric carcinomas studied compared with normal gastric mucosa. Furthermore, six tumor/normal pairs with available histologic samples demonstrated a marked decrease in protein expression in tumor samples. Screening of the entire TFF1 coding region in a panel of 42 human gastric tumors did not reveal any somatic mutations, although a few rare germline sequence variants were identified. CONCLUSIONS These findings demonstrated a significant decrease in the TFF1 transcript in the majority of human gastric carcinomas along with a corresponding reduction in protein expression, both of which occurred in the absence of gene mutation. Dysregulation of TFF1 expression at the transcript level was a critical event in the development of most gastric carcinomas. Cancer 2003. © 2003 American Cancer Society.

In this work, the use of MRI to stage gastric neoplasia in a mouse model of spontaneous gastric cancer is demonstrated. The methodology involves 1) the use of deuterated water (2H2O) to distend the stomach, and to provide negative contrast between the stomach and the lumen without inducing susceptibility-based field shifts; 2) GlucaGen® to minimize motion artifacts that arise from peristalsis; and 3) Gd-DTPA to enhance contrast between the dysplasia/tumor and the normal wall. Initial images are presented from a Tff1 −/− homozygous knock-out model of gastric cancer and a heterozygous C57BL6/J control mouse. There are clear differences between the two types of animals in the MR appearance of the stomach. The distended stomach of the control mouse has a smooth perimeter and a thin wall, and an absence of nodules. The stomach of the Tff1 −/− mouse demonstrates a thickened wall; a jagged, irregular surface; and protruding nodules that are bright after injection of Gd-DTPA. Magn Reson Med 49:1033–1036, 2003. © 2003 Wiley-Liss, Inc.

The biology of prostanoids in the normal human colon is only beginning to be understood. We used in situ and in vitro techniques to define the lineage, number, per cell enzyme content, and epithelial functional effect of prostaglandin-generating cells, identified by the presence of cyclooxygenase 1 (COX 1). Immunohistochemical results were quantitated densitometrically, and cell surface staining in situ was verified by flow cytometry of isolated cells and by Western blotting. Three populations of COX 1(+) mucosal cells were identified, based on their morphology and local distribution in human mucosa; these were in the intra-epithelial, crypt apical, and lamina propria regions, with each containing a similar amount of COX 1 protein on a per cell basis. The most numerous were COX 1(+) mononuclear cells in the lamina propria, identified as CD3(+) T lymphocytes, both in situ and ex vivo. In toto, 21% of lamina propria mononuclear cells were COX 1(+), and over 50% of these cells were CD3(+) T cells. Findings were similar in the colon with mild-moderate inflammation due to ulcerative colitis. Using established surface markers, intra-epithelial and crypt apical COX 1(+) cells were non-lymphoid CD45(+) leukocytes; neither IgA (B-lymphocytes) nor alpha-smooth muscle actin (myelofibroblasts) was co-expressed on these COX 1(+) cells. Examining the effect of a major product of COX 1 in an in vitro system of human colonic epithelial monolayers, prostaglandin E(2) (PGE(2)) in low concentration (10(-6) M) enhanced epithelial barrier function and partially protected epithelia from the barrier-disruptive consequences of a pro-inflammatory cytokine, IFN-gamma. We conclude that the human colon contains three tiers of cell types for local synthesis of prostanoids, distinguishable by their location, morphology, and cell lineage. Further, maintenance of the barrier function of colonic epithelium may be added to other cell functions in mucosa regulated, in part, by prostanoids.

Apolipoliprotein E (apoE), a critical targeting protein in lipid homeostasis, has been found to have immunoinflammatory effects on murine models of infection and malnutrition. The effects of apoE in undernourished and Cryptosporidium parvum-infected mice have not been investigated. In order to study the role of apoE in a model of C. parvum infection, we used the following C57BL6J mouse genetic strains: APOE-deficient, wild-type controls, and APOE targeted replacement (TR) mice expressing human APOE genes (E3/3; E4/4). Experimental mice were orally infected with 10(7)-unexcysted-C. parvum oocysts between post-natal days 34-35 followed by malnutrition induced with a low-protein diet. Mice were euthanized seven days after C. parvum-challenge to investigate ileal morphology, cytokines, and cationic arginine transporter (CAT-1), arginase 1, Toll-like receptor 9 (TLR9), and inducible nitric oxide synthase (iNOS) expression. In addition, we analyzed stool oocyst shedding by qRT-PCR and serum lipids. APOE4/4-TR mice had better weight gains after infection plus malnutrition compared with APOE3/3-TR and wild-type mice. APOE4/4-TR and APOE knockout mice had lower oocyst shedding, however the latter exhibited with villus blunting and higher ileal pro-inflammatory cytokines and iNOS transcripts. APOE4/4-TR mice had increased ileal CAT-1, arginase-1, and TLR9 transcripts relative to APOE knockout. Although with anti-parasitic effects, APOE deficiency exacerbates intestinal inflammatory responses and mucosal damage in undernourished and C. parvum-infected mice. In addition, the human APOE4 gene was found to be protective against the compounded insult of Cryptosporidium infection plus malnutrition, thus extending our previous findings of the protection against diarrhea in APOE4 children. Altogether our findings suggest that apoE plays a key role in the intestinal restitution and immunoinflammatory responses with Cryptosporidium infection and malnutrition.

The tissue concentration of PGE(2)is heightened during mucosal inflammation. Nevertheless, the cellular targets of this prostanoid and its effects on epithelial cell physiology are incompletely understood. We used a panel of specific immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2)on cells of histologically normal and inflamed human colonic mucosa, and then examined the physiological consequences for the epithelial component of intestine, with special attention to its barrier function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells, and differed markedly between normal and inflamed tissue. In non-inflamed mucosa, EP(2)and EP(3)were expressed on epithelia at the apex of crypts; while EP(4)was expressed on surface and lateral crypt epithelia. Dual immunostaining and in situ hybridization with digoxygenin-labelled RNA probes largely confirmed the epithelial localization of EP(4). On the other hand, during inflammation, lateral crypt (non-surface) epithelial cells newly and significantly expressed prostanoid receptors EP(2)and EP(3)(p

Exposure to oocysts of the protozoanCryptosporidium parvumcauses intestinal epithelial cell dysfunction in vivo and in vitro, but effective means by which mucosal injury might be prevented remain unclear. We examined the ability of transforming growth factor β1 (TGF-β1)—a cytokine synthesized and released by cells in the intestine—to preserve the barrier function of human colonic epithelia when challenged withC. parvumoocysts and then studied the mechanisms involved. Epithelial barrier function was monitored electrophysiologically, receptors for TGF-β1 were localized by confocal microscopy, and TGF-β1-induced protein kinase C activation was detected intracellularly by translocation of its α isozyme. TGF-β1 alone enhanced intestinal epithelial barrier function, while exposure toC. parvumoocysts (≥105/monolayer) markedly reduced barrier function to ≤40% of that of the control. When epithelial monolayers were pretreated with TGF-β1 at 5.0 ng/ml, the barrier-disrupting effect ofC. parvumoocysts was almost completely abrogated for 96 h. Further investigation showed that (i) the RI and RII receptors for TGF-β1 were present on 55 and 65% of human epithelial cell line cells, respectively, over a 1-log-unit range of receptor protein expression, as shown by flow cytometry and confirmed by confocal microscopy; (ii) only basolateral and not apical TGF-β1 exposure of the polarized epithelial monolayer resulted in a protective effect; and (iii) TGF-β1 had no direct effect on the organism in reducing its tissue-disruptive effects. In exploring mechanisms to account for the barrier-preserving effects of TGF-β1 on epithelium, we found that the protein kinase C pathway was activated, as shown by translocation of its 80-kDa α isozyme within 30 s of epithelial exposure to TGF-β1; the permeability of epithelial monolayers to passage of macromolecules was reduced by 42% with TGF-β1, even in the face of active protozoal infection; and epithelial cell necrosis monitored by lactate dehydrogenase release was decreased by 50% 70 h after oocyst exposure. Changes in epithelial function, initiated through an established set of surface receptors, likely accounts for the remarkable barrier-sparing effect of nanogram-per-milliliter concentrations of TGF-β1 when human colonic epithelium is exposed to an important human pathogen,C. parvum.

Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium—its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-β1 (TGF-β1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-β1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-γ), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-β1. Receptors (RI and RII) for TGF-β1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products. J. Cell. Physiol. 181:55–66, 1999. © 1999 Wiley-Liss, Inc.

We examined the concentration- and time-dependent effects of two related protein kinase inhibitors, KT5926 and K-252a, on neurite formation and nerve growth cone migration of chick embryo sensory neurons. The effects of these drugs on neurite formation over an 18-h period were dissimilar. KT5926 stimulated neurite formation at concentrations between 100 and 500 nM and inhibited neurite formation at 5 μM. K-252a had no stimulatory effects on neurite formation, and it inhibited neurite formation at concentrations above 50 nM. This difference may occur because K-252a inhibits activation of the nerve growth factor receptor trk A, while KT5926 does not inhibit trk A. Both drugs, however, had similar immediate effects on growth cone migration. Growth cone migration and lamellipodial spreading were rapidly stimulated by 500 nM concentrations of KT5926 and K-252a. At 2 μM levels of either drug, growth cone spreading was still stimulated, but growth cone migration was inhibited by both drugs. These results show that changes in protein phosphorylation/dephosphorylation can rapidly regulate the cellular machinery that is responsible for driving growth cone migration and neurite elongation. The different effects of 2 μM concentrations of either KT5926 or K-252a on growth cone spreading versus migration suggests that the actin-dependent protrusive motility of the growth cone leading margin is regulated differently by changes in protein phosphorylation and dephosphorylation than the cytoskeletal mechanism that drives neurite elongation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 161–171, 1997

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a common cause of diarrhoea in healthy, malnourished and immune-deficient adults and children. There is no reproducible non-neonatal animal model for longitudinal studies of disease mechanism or therapy. Using two strains of human-derived EAEC to challenge weaned C57BL/6 mice, we explored an in vivo model of EAEC infection in mice, in which disease was monitored quantitatively as the growth rate, stool shedding and tissue burden of organisms; nutritional status was varied, and a new class of therapeutics was assessed. A single oral challenge of EAEC strain 042 resulted in significant growth shortfalls (5–8 % of body weight in 12 days), persistent shedding of micro-organisms in stools [>103.2 c.f.u. (10 mg stool)−1 for at least 14 days] and intestinal tissue burden [~103 c.f.u. (10 mg tissue)−1 detectable up to 14 days post-challenge]. Moderate malnourishment of mice using a ‘regional basic diet’ containing 7 % protein and reduced fat and micronutrients heightened all parameters of infection. Nitazoxanide in subMIC doses, administered for 3 days at the time of EAEC challenge, lessened growth shortfalls (by >10 % of body weight), stool shedding [by 2–3 logs (10 mg stool)−1] and tissue burden of organisms (by >75 % in the jejunum and colon). Thus, weaned C57BL/6 mice challenged with EAEC is a convenient, readily inducible model of EAEC infection with three highly quantifiable outcomes in which disease severity is dependent on the nutritional status of the host, and which is modifiable in the presence of inhibitors of pyruvate ferredoxin oxidoreductase such as nitazoxanide.

SUMMARY The adhesive interactions of nerve growth cones stabilize elongating nerve fibers and mediate transmembrane signaling to regulate growth cone behaviors. We used interference reflection microscopy and immunocytochemistry to examine the dynamics and composition of substratum contacts that growth cones of chick sensory neurons make with extracellular adhesive glycoproteins, fibronectin and laminin. Interference reflection microscopy indicated that sensory neuronal growth cones on fibronectin-treated substrata, but not on laminin, make contacts that have the appearance and immobility of fibroblastic focal contacts. Interference reflection microscopy and subsequent immunocytochemical staining showed that pl integrin and phosphotyrosine residues were concentrated at growth cone sites that resemble focal contacts. Two other components of focal contacts, paxillin and zyxin, were also co-localized with concentrated phosphotyrosine residues at sites that resemble focal contacts. Such staining patterns were not observed on laminintreated substrata. Growth cone migration on fibronectintreated substrata was inhibited by herbimycin A, a tyrosine kinase inhibitor. We conclude that sensory neuronal growth cones distinguish fibronectin from laminin by making contacts with distinct organization and regulation of cytoskeletal components at the adhesive sites. This finding suggests that growth cone interactions with different adhesive molecules lead to distinctive transmembrane organization and signaling to regulate nerve fiber elongation. 0 1996 John Wiley & Sons, Inc.

Background. Although several animal models of cryptosporidiosis have been reported, most involve genetically or pharmacologically immune-suppressed hosts. Methods. We report challenge with excysted (in vitro and in vivo) and unexcysted (in vivo) Cryptosporidium parvum oocysts in human colonic adenocarcinoma (HCT-8) cells and weaned nourished and malnourished C57BL/6 mice, following outcomes of growth rate, stool shedding, and tissue burden. We tested treatment with an oligodeoxynucleotide containing unmethylated CpG motif (CpG-ODN) and alanyl-glutamine in vivo and in vitro. Results. C. parvum–challenged mice showed prolonged weight loss (.10% over 4 days), robust stool shedding (.3 logs/d over 7 days), and epithelial infection in the ileum, cecum, and colon. Of 2 potential therapeutic compounds evaluated in the model, CpG-ODN reduced body weight loss (to ,6% on days 3–7 after challenge), reduced shedding of organisms (by 25% on days 1 and 3 after challenge), and decreased the burden of parasites in the ileum. Alanyl-glutamine showed similar benefits. In vitro findings suggested that effects on the epithelial component of the mucosa probably likely responsible for beneficial effects seen in vivo. Conclusions. Weaned mice provide a convenient and reproducible model of cryptosporidial disease, including its vicious cycle with body weight loss and heavier infection with malnutrition, and this model may be useful in exploring innovative therapeutic solutions for this challenging infectious disease.

Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause nosocomial diarrhea, colitis or even death. Peroxisome proliferator-activated receptor (PPAR) γ has been implicated in the prevention of inflammation in autoimmune and infectious diseases; however, its role in the immunoregulatory mechanisms modulating host responses to C. difficile and its toxins remains largely unknown. To characterize the role of PPARγ in C. difficile-associated disease (CDAD), immunity and gut pathology, we used a mouse model of C. difficile infection in wild-type and T cell-specific PPARγ null mice. The loss of PPARγ in T cells increased disease activity and colonic inflammatory lesions following C. difficile infection. Colonic expression of IL-17 was upregulated and IL-10 downregulated in colons of T cell-specific PPARγ null mice. Also, both the loss of PPARγ in T cells and C. difficile infection favored Th17 responses in spleen and colonic lamina propria of mice with CDAD. MicroRNA (miRNA)-sequencing analysis and RT-PCR validation indicated that miR-146b was significantly overexpressed and nuclear receptor co-activator 4 (NCOA4) suppressed in colons of C. difficile-infected mice. We next developed a computational model that predicts the upregulation of miR-146b, downregulation of the PPARγ co-activator NCOA4, and PPARγ, leading to upregulation of IL-17. Oral treatment of C. difficile-infected mice with the PPARγ agonist pioglitazone ameliorated colitis and suppressed pro-inflammatory gene expression. In conclusion, our data indicates that miRNA-146b and PPARγ activation may be implicated in the regulation of Th17 responses and colitis in C. difficile-infected mice.

The article of record as published may be found at https://doi.org/10.1371/journal.pone.0032681 Anthropogenic noise may significantly impact exposed marine mammals. This work studied the vocalization response of endangered blue whales to anthropogenic noise sources in the mid-frequency range using passive acoustic monitoring in the Southern California Bight. Blue whales were less likely to produce calls when mid-frequency active sonar was present. This reduction was more pronounced when the sonar source was closer to the animal, at higher sound levels. The animals were equally likely to stop calling at any time of day, showing no diel pattern in their sensitivity to sonar. Conversely, the likelihood of whales emitting calls increased when ship sounds were nearby. Whales did not show a differential response to ship noise as a function of the time of the day either. These results demonstrate that anthropogenic noise, even at frequencies well above the blue whales' sound production range, has a strong probability of eliciting changes in vocal behavior. The long-term implications of disruption in call production to blue whale foraging and other behaviors are currently not well understood. Naval Postgraduate School Chief of Naval Operations N45 Pacific Fleet

A monolayer of mature polarized colonic epithelial cells (T84) able to generate and maintain a barrier to macromolecular flow was used to study pathophysiologic events that occur on microvillus cell exposure to Cryptosporidium parvum. By 24-48 h, several life cycle forms were seen in parasitophorous vacuoles near the apical cell surface, along with a time- and oocyst dose-dependent reduction in epithelial barrier function. As few as 10(5) organisms constituted a successful infecting dose, and heat inactivation of organisms markedly reduced the monolayer barrier alteration. Horseradish peroxidase flux studies demonstrated a substantial increase in macromolecular permeability of the monolayer, and lactate dehydrogenase determinations indicated modest injury of the T84 epithelial cells on exposure to oocysts. Thus, disruption of the epithelial cell barrier, not just opening of transcellular channels for ion flow as reported previously, is responsible for the effects of C. parvum oocysts on intestinal epithelium.

Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3-12 days, then were infected with 5 × 10(7) excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height-crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

Meristic identification, mitochondrial DNA and a suite of microsatellite markers were employed to estimate the incidence of hybridization in wild populations of anadromous Allis shad Alosa alosa and twaite shad Alosa fallax in southern Irish riverine and estuarine waters. It was shown that 16% of the fishes examined were misclassified using meristic count of gill rakers. Next, a significant proportion of fishes that were robustly assigned to a species using nuclear markers were shown to possess the mtDNA of the other. The genomes of A. alosa and A. fallax in Ireland are extensively introgressed, which suggests a complex history of hybridization between these species, which can only partially be explained by recent man-made habitat changes.

Diarrhea is a major public health problem that affects the development of children. Anthropometric data were collected from 274 children with (N = 170) and without (N = 104) diarrhea. Stool specimens were analyzed by conventional culture, polymerase chain reaction for enteroaggregative Escherichia coli (EAEC), Shigella, Cryptosporidium, Entamoeba, and Giardia species, and by enzyme-linked immunosorbent assay for fecal lactoferrin levels. About 50% of the study population was mildly to severely malnourished. Fecal lactoferrin levels were higher in children with diarrhea (P = 0.019). Children who had EAEC infection, with or without diarrhea, had high mean lactoferrin levels regardless of nutritional status. The EAEC and Cryptosporidium were associated with diarrhea (P = 0.048 and 0.011, respectively), and malnourished children who had diarrhea were often co-infected with both Cryptosporidium and EAEC. In conclusion, the use of DNA-biomarkers revealed that EAEC and Cryptosporidium were common intestinal pathogens in Accra, and that elevated lactoferrin was associated with diarrhea in this group of children.

Enteroaggregative Escherichia coli is emerging as an increasingly recognized cause of persistent, mildly inflammatory diarrhea in the United States, especially among patients with AIDS, as well as among children, for whom it is accompanied by growth shortfalls.We describe a novel model of disease induced in neonatal and weaned C57BL/6 mice by pathogenic strains of enteroaggregative E. coli 042 and JM221.Enteroaggregative E. coli caused growth impairment (up to 47%), persistent stool shedding (for3 weeks), and a substantial tissue burden of organisms (150,000 organisms per milligram of tissue), as well as histopathological changes in the colonic epithelium (days 4 and 6 of infection) using the model. Undernutrition in neonatal mice, as well as in weaned mice, intensified infection by 1-4 logs, as assessed by quantitative polymerase chain reaction for fecal shedding of organisms. Growth impairment was dependent on both microorganism burden and challenge dose.Both neonatal and weaned mice provide models for a vicious cycle of enteroaggregative E. coli infection that causes growth shortfalls and undernutrition, thus worsening infection. Hence, these neonatal and weaned mice provide the opportunity to dissect mechanisms of this cycle in childhood malnutrition, as well as to define the role played by innate and acquired host defenses in this important infection.

Recent evidence suggests that growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after 2 h. When protein synthesis inhibitors were added 15 min before adding guidance cues, we found no changes in the typical responses of retinal, sensory, and sympathetic growth cones. In the presence of cycloheximide or anisomycin, ephrin-A2, slit-3, and semaphorin3A still induced growth cone collapse and loss of actin filaments, nerve growth factor (NGF) and neurotrophin-3 still induced growth cone protrusion and increased filamentous actin, and sensory growth cones turned toward an NGF source. In compartmented chambers that separated perikarya from axons, axons grew for 24–48 h in the presence of cycloheximide and responded to negative and positive cues. Our results indicate that protein synthesis is not strictly required in the mechanisms for growth cone responses to many guidance cues. Differences between our results and other studies may exist because of different cellular metabolic levels inin vitroconditions and a difference in when axonal functions become dependent on local protein synthesis.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.

In recent years, the relationship between human papillomaviruses (HPV) and genital neoplasia has been explored intensively, and a molecular basis for the role of HPV in the genesis of these diseases has been convincingly demonstrated. These findings have provided justification for efforts to apply this molecular information to the early detection and possible prevention of HPV-related neoplasia. The technology of detecting viral nucleic acids in genital fluids brought with it initial hopes that it would serve to identify women at risk for having or developing precancers or cancers of the cervix. Subsequent studies, however, have demonstrated limitations of the technology for predicting future disease. Recently, molecular immunology has complemented these prior efforts, with the intent to identify serological indices of exposure to HPV and perhaps delineate individuals at risk. The molecular basis for this approach, its limitations, and future prospects for immunodiagnosis are the subject of this review.

Although the E4 open reading frame (ORF) of human papillomaviruses (HPV) encodes an abundant protein in cutaneous warts, the location and extent of HPV E4 expression in genital precancers, specifically those associated with HPV-16, has not been described. Expression plasmids (pATH) containing segments of the HPV-16 E4 (3401–3620) and L1 (6151–6792) open reading frames (ORFs) were induced and expressed in bacteria and the resulting fusion proteins were used to elicit antisera in rabbits. Antisera reacting to the E4 and L1 components of the fusion proteins were used to screen biopsies from 150 cervical precancers (cervical intraepithelial neoplasia) and condylomata. Six biopsies exhibited specific immunostaining with the anti-E4 sera. Staining was cytoplasmic, and occurred virtually always in foci containing immunostaining for L1 proteins. Moreover, analysis of these 6 cases and 22 others for HPV-16 RNA by RNA-RNA in situ hybridization demonstrated a similar correlation between E4 immunostaining and the presence of abundant transcripts specific to HPV-16. These data are consistent with the hypothesis that expression of the HPV-16 E4 ORF is dependent upon viral replication and epithelial differentiation, similar to L1 expression, and that the E4 epitopes identified by the rabbit antisera may be unique to HPV-16 relative to other common cervical papillomaviruses.

Neutrophilia is a characteristic of hemolytic uremic syndrome caused by Shiga toxin (Stx2)-producing Escherichia coli. However, the role of neutrophils in the toxin-induced renal injury occurring in enterohemorrhagic E. coli infection remains undefined. We report the trafficking of neutrophils to the kidney of C57BL/6 mice throughout a 72-hour time course after challenge with purified E. coli Stx2 and lipopolysaccharide (LPS). Increased neutrophils were observed in the renal cortex, particularly within the glomeruli where a more than fourfold increase in neutrophils was noted within 2 hours after challenge. Using microarray analysis, an increased number of transcripts for chemoattractants CXCL1/KC (69-fold at 2 hours) and CXCL2/MIP-2 (29-fold at 2 hours) were detected. Ribonuclease protection assays, Northern blotting, enzyme-linked immunosorbent assay, and immunohistochemistry confirmed microarray results, showing that both chemokines were expressed only on the immediate periglomerular epithelium and that these events coincided with neutrophil invasion of glomeruli. Co-administration of Stx2 with LPS enhanced and prolonged the KC and MIP-2 host response (RNA and protein) induced by LPS alone. Immunoneutralization in vivo of CXCL1/KC and CXCL2/MIP-2 abrogated neutrophil migration into glomeruli by 85%. These data define the molecular basis for neutrophil migration into the kidney after exposure to virulence factors of Shiga toxin-producing E. coli O157:H7.

Over the latter part of the 20th century, Eurasian otter Lutra lutra populations suffered dramatic declines, resulting in extinction or fragmentation of populations in many western and central European countries. Part of the Czech otter population became totally isolated while the Slovak population remained partly connected to the relatively continuous central and eastern European otter distribution range. This paper examines the genetic structure and past demographic history of otters in the Czech and Slovak Republics, using microsatellite nuclear markers amplified from DNA extracted from tissue and faecal samples. A relatively high level of genetic differentiation was found between the Czech and Slovak populations (FST=0.154, P=0.0002), supported by a perfect assignment in Bayesian cluster analysis. Both the Czech and Slovak populations showed significant heterozygosity excess (assuming an infinite allele model), indicating recent population bottlenecks. A very recent population decline was also suggested by coalescent analysis, inferring a drop to c. 25% of past effective population size in both populations. The timing of the decline was in accordance with published data from otter surveys, suggesting that the strongest decline probably occurred between the 1970s and the mid-1990s. The results of this study confirm that otter populations remain vulnerable to any violent demographic change and, despite the claims of fish-farmers and anglers for legal culls, it is highly desirable that they remain a strongly protected species in both countries. The spreading and re-connection of otter populations observed recently is essential for the future health of the populations, and should be supported through habitat conservation.

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.

Although the tissue concentration of PGE(2) is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2) to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells; EP(4) alone was expressed on lamina propria mononuclear cells. Dual immunostaining in situ identified the CD3(+) T lymphocyte as a major EP(4) receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP(4)(+), and almost 30% of these were CD3(+). In situ hybridization with digoxygenin-labeled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a significant enhancement in EP(4) immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8 +/- 1.8 to 8.6 +/- 1.8 (p < 0.04). The effects of PGE(2) included a 35% reduction in T lymphocyte IL-2 secretion. COX 1(+) lamina propria cells nearly doubled in number during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. The number of newly appeared COX 2(+) lymphocytes remained

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-γ mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors. (Otolaryngol Head Neck Surg 1999;120:869-75.)

Baleen whales are an important contributor to the Southern California Bight soundscape. Calls from some species only contribute seasonally, while others are part of the soundscape year-round, albeit at varying levels. Passive acoustic monitoring has been conducted at the U.S. Navy’s Southern California Offshore Range (SCORE) since 2009. Data were collected at two sites concurrently using High-frequency Acoustic Recording Packages (HARPs). Analysis in the low frequency band (10 Hz—1 kHz) has yielded results on the seasonal and interannual variation in the presence of calling blue (Balaenoptera musculus), fin (B. physalus), Bryde’s (B. edeni), and humpback whales (Megaptera novaeangliae). Calls of most species (blue, Bryde’s, and humpback whales) were only present during part of the year, indicating seasonal migration common for these species. On the contrary, fin whale calls were prevalent year-round, although the abundance of their 20 Hz calls tended to decrease in the summer. The link between the relativ...

Background Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of persistent diarrhea and enteric disease worldwide. Mucosal immunity towards EAEC infections is incompletely understood due in part to the lack of appropriate animal models. This study presents a new mouse model and investigates the role of peroxisome proliferator-activated receptor gamma (PPARγ) in the modulation of host responses to EAEC in nourished and malnourished mice. Methods/Principal Findings Wild-type and T cell-specific PPARγ null C57BL/6 mice were fed protein-deficient diets at weaning and challenged with 5×109cfu EAEC strain JM221 to measure colonic gene expression and immune responses to EAEC. Antigen-specific responses to E. coli antigens were measured in nourished and malnourished mice following infection and demonstrated the immunosuppressive effects of malnutrition at the cellular level. At the molecular level, both pharmacological blockade and deletion of PPARγ in T cells resulted in upregulation of TGF-β, IL-6, IL-17 and anti-microbial peptides, enhanced Th17 responses, fewer colonic lesions, faster clearance of EAEC, and improved recovery. The beneficial effects of PPARγ blockade on weight loss and EAEC clearance were abrogated by neutralizing IL-17 in vivo. Conclusions Our studies provide in vivo evidence supporting the beneficial role of mucosal innate and effector T cell responses on EAEC burden and suggest pharmacological blockade of PPARγ as a novel therapeutic intervention for EAEC infection.

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E2 in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E2 is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E2 modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E2's regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E2 concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E2, when present in as low a concentration as 10(-10) M. PGE2 also inhibited (by > or = 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E2 dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [3H] thymidine uptake studies suggested this was not due to a prostaglandin E2-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E2, e.g., high concentrations (10(-6) M) of prostaglandin E2 inhibited IL-3 release by only 41%. We conclude that prostaglandin E2 in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there.

A cluster-sampling, cross-sectional study was conducted for assessing the prevalence of Cryptosporidium infection in children less than 16 years of age from three villages, Dondian, Linshan, and Fuziyin, in rural Anhui in eastern China. Among 320 apparently healthy children less than 10 years of age from Dondian who had stool specimens collected, cryptosporidial oocysts were found in stools of three children from Dondian, and no positive specimens were found in 239 children studied from Linshan. In addition, a total of 610 serum samples from children in these three villages were tested for specific IgG antibody to Cryptosporidium with an enzyme-linked immunosorbent assay (ELISA) and the prevalence rates were 42.3%, 51.7%, and 57.5%, respectively, in Dondian, Linshan, and Fuziyin. Seroprevalence increased progressively with age. No detectable antibody was found in infants between two and six months of age, and seropositivity steadily increased after one year of age. Among 36 sera from adults 15-60 years of age without diarrheal illness in Huanglu villages of rural Chaohu, 50% (18 of 36) were positive. As expected, a good correlation was found in the specific IgG antibody between the paired serum specimens from 30 matched mother-neonates who showed transplacental transfer of IgG. However, little or no IgM antibody was seen in the neonates even though several mothers had a positive anticryptosporidial IgM enzyme-linked immunoassay result. Forty randomly selected serum samples from children less than four years of age in a similarly impoverished semiurban community in Fortaleza, Brazil, where the majority of households also have pit toilets and shared community water supplies and 172 serum samples from patients one month to 29 years of age admitted to the University of Virginia Hospital without diarrhea were also examined. In Fortaleza, almost all children acquired antibody by their second year of life, demonstrating the high prevalence of this infection. In rural Anhui, only about half the children were infected by 5-7 years of age. The overall prevalence rate (16.9%) of seropositivity among children and young adults in Virginia was much lower than in China and Brazil. These results indicate that cryptosporidial infection is ubiquitous, and is highly endemic in these impoverished communities. The difference between China and Brazil may reflect earlier weaning, hygiene practices, poorer water or sanitation, multiple siblings in family and geographic environment in Brazil.

Mucosal and submucosal mast cell hyperplasia is a feature of the chronic inflammatory bowel diseases--ulcerative colitis and Crohn's disease. The mast cells are often seen to be degranulated in areas of active disease, suggesting that the inflammatory mediators released from these cells contribute to the pathophysiology of these disorders. We examined the hypothesis that epithelial cell-derived proteins, intestinal epithelial cell-associated components (ECAC), interact with the mast cells of patients with chronic inflammatory bowel disease to trigger the local release of mast cell mediators. Aliquots of human intestinal mucosal mast cell suspensions obtained from surgically resected specimens of colon or small intestine (ulcerative colitis, 12; Crohn's disease, 3; histologically normal controls, 8) were incubated with 1-100 micrograms/ml of colon or small bowel-derived murine ECAC or control kidney protein, or 1 microgram/ml goat anti-human IgE positive control for 30 min at 37 degrees C. Supernatants were analyzed in duplicate for histamine content by fluorometric assay. The median percent total histamine released by chronic inflammatory bowel disease mast cell suspensions to colonic epithelium-derived protein (ECAC-C) was 4% histamine (range 0-20%), such that the distribution of histamine release values in inflammatory bowel disease specimens was significantly different from the distribution of values in mast cells taken from normal mucosa (median 0%, P < 0.05). The median histamine release by all chronic inflammatory bowel disease specimens was also increased in response to the ECAC preparations derived from small bowel epithelium in that a third of the inflammatory bowel disease specimens showed greater than 10% histamine release to ECAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA · DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%;P = 0.04) and pATH-L2 (48% vs 18%;P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.

Near-total pancreatectomy in a neonate presenting with persistent hypoglycemia offered an unusual opportunity to grow preparations enriched in beta-cells. Morphology, chromosomal analysis, and immunohistochemistry were used to characterize a subculture (Nesi B) that remained stable through passage 11. Insulin secretion of Nesi B was constitutive at 38-74 nU/microliters of medium/24 h, increasing modestly in the presence of isobutylmethylxanthine, an inhibitor of cyclic AMP phosphodiesterase. Endocrine cells, exclusive of those in islet regions, were widely distributed throughout the original tissue section, and immunostaining of the Nesi B subculture demonstrated well-differentiated heavily granulated insulin-positive cells, each with a normal modal number of chromosomes (46). Nesi B cell-associated macromolecules were isolated in 3 x 10(-3) ethylenedinitrilotetraacetate/phosphate-buffered saline and were found to be reactive with a heterologous immune serum elicited to a cloned rat pancreatic beta-cell line (RIN-5F). Western (immuno) blotting showed this immunoreactivity to reside primarily in a 95-kDa fraction of Nesi B-derived components. These results indicate that human nesidioblasts can be cultured for a sufficient number of passages to allow isolation and immunochemical characterization of pancreatic beta-cell macromolecules shared between rat and human and that may serve as organ-specific antigens for inflammatory disorders of the pancreas.

In order to develop an experimental model of symptomatic cryptosporidiosis in an immunosuppressed mammal, we investigated the pathophysiology of infection with Cryptosporidium and the humoral and cellular host responses in rnu/rnu (athymic) rats and their heterozygous (rnu/+) littermates by challenging suckling rats with greater than or equal to 2.5 x 10(6) Cryptosporidium oocytes oro-gastrically. Normal and immunodeficient animals were followed for onset and duration of infection (fecal oocysts), physiologic consequences (diarrhea, impaired weight gain, brush-border enzyme activities), and immunologic response (both B- and T-lymphocyte-mediated). Homozygosity for the rnu gene was associated with protracted cryptosporidial infections; shedding for up to 52 days occurred, and delay in weight gain was noted in rnu/rnu-infected compared with rnu/rnu-uninfected rats (p less than 0.05). In contrast, cryptosporidial challenge of rnu/+ rats resulted in self-resolving infections, occasionally with transient diarrhea lasting four days or less occurring 10-15 days after oro-gastric challenge. The latter animals mounted a cell-mediated immune response to Cryptosporidium: three months after challenge, five of five rnu/+ rats demonstrated positive skin test responses to a subcutaneous 3.5 micrograms dose of cryptosporidial antigen. Further, sera from 6 rnu/+ rats taken two to three months after oro-gastric oocyst challenge exhibited specific anticryptosporidial immunoglobulin binding (A405 = 0.96), compared to that of seven uninfected rnu/+ controls (A405 = 0.09, P less than 0.02). Macromolecules of 150, 105, and 88 kD in the Cryptosporidium antigen preparation were bound by serum immunoglobulin from previously infected, recovered rnu/+ rats. Two brush-border enzymes (lactase and alkaline phosphatase) were markedly reduced in the ileum 8-10 days after oro-gastric challenge in rats with diarrhea and oocyst shedding. We find the rnu/rnu (athymic, nude) rat provides a useful model for study of prolonged cryptosporidial infection with impaired weight loss, brush-border enzyme alteration and intermittent diarrhea. These studies further suggest that a T-lymphocyte population is involved in recovery from Cryptosporidium infection and that this recovery is associated with both cellular and humoral immune responses to specific cryptosporidial antigenic macromolecules. This model should open further avenues for the study of the pathogenesis and protective immunity in cryptosporidial infection.

Studies of cervical secretions as well as cells composing the endocervix have provided evidence for a functional and potentially important immunological system in the mucosa of that organ. The availability of the tools of cell biology as well as three agents that may be used as probes to infect cervical mucosa experimentally has made possible a detailed approach to define the structural and functional characteristics of local cervical immunity. A long-term goal of these studies is to determine how the cervical immune response may be regulated to reduce local viral replication and virus-associated diseases. With Langerhans cells for antigen presentation, cervical immune responses generally remain detectable for more than 30 days, are predominantly of the IgA isotype, can be influenced by estrogen or progesterone, and are best elicited by local rather than systemic exposure to antigen. Cervical immune responses to the human papillomaviruses (HPV) are of particular importance in this regard because this virus is associated with cervical neoplasia. While responses in serum to HPV-16 proteins L1, E4, and E7 has been found in up to 78% of persons with HPV-associated cervical neoplasms, data showing that a local response of comparable frequency consistently occurs have yet to be confirmed. The current status of local HPV-16-specific immunoglobulin as a potentially useful indicator of HPV-16-related infection or pre-cancer is controversial, and is confounded by several potentially important factors, including patient age, estrogen/progesterone level, smoking status, and sample admixture with serum immunoglobulin.