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A hallmark of infantile hemangioma, the most common tumor of infancy, is its dramatic growth after birth, by diffuse proliferation of immature endothelial cells, followed by spontaneous regression. The growth and involution of infantile hemangioma is quite different from other vascular anomalies, which do not regress and can occur at any time during life. Some hemangioma lesions can be extremely disfiguring and destructive to normal tissue and may even be life-threatening. Unfortunately, existing therapeutic approaches have limited success and significant adverse effects of some treatment modalities limit their use. Better understanding of the pathogenesis of hemangioma will enable the development of better therapeutic strategies. Herein, we review recent studies and new hypotheses on the pathogenesis of the tumor. Detailed mechanisms of activated vascular endothelial growth factor (VEGF) signaling in tumor cells, identification of their origin and characterization of multipotent stem cells that can give rise to infantile hemangioma are shedding new light on this intriguing vascular tumor.

During a study of the prevalence of Campylobacter jejuni in black-headed gulls (Larus ridibundus) in Sweden, three isolates, strains LMG 24379, LMG 24380T and LMG 24381, were initially identified as Campylobacter lari. Further characterization by both AFLP and whole-cell protein SDS-PAGE analyses revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by phenotypic characterization, 16S rRNA and hsp60 gene sequence analysis and DNA–DNA hybridizations. The combined data confirm that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter volucris sp. nov. is proposed. The type strain is LMG 24380T (=CCUG 57498T).

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga . Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.

A hallmark of infantile hemangioma, the most common tumor of infancy, is its dramatic growth after birth, by diffuse proliferation of immature endothelial cells, followed by spontaneous regression. The growth and involution of infantile hemangioma is quite different from other vascular anomalies, which do not regress and can occur at any time during life. Some hemangioma lesions can be extremely disfiguring and destructive to normal tissue and may even be life-threatening. Unfortunately, existing therapeutic approaches have limited success and significant adverse effects of some treatment modalities limit their use. Better understanding of the pathogenesis of hemangioma will enable the development of better therapeutic strategies. Herein, we review recent studies and new hypotheses on the pathogenesis of the tumor. Detailed mechanisms of activated vascular endothelial growth factor (VEGF) signaling in tumor cells, identification of their origin and characterization of multipotent stem cells that can give rise to infantile hemangioma are shedding new light on this intriguing vascular tumor.

The family Chlamydiaceae contains several bacterial pathogens of important human and veterinary medical concern, such as Chlamydia trachomatis and Chlamydophila psittaci. Within the order Chlamydia ...

SummaryThe primary cilium acts as a transducer of extracellular stimuli into intracellular signaling [1, 2]. Its regulation, particularly with respect to length, has been defined primarily by genetic experiments and human disease states in which molecular components that are necessary for its proper construction have been mutated or deleted [1]. However, dynamic modulation of cilium length, a phenomenon observed in ciliated protists [3, 4], has not been well-characterized in vertebrates. Here we demonstrate that decreased intracellular calcium (Ca2+) or increased cyclic AMP (cAMP), and subsequent protein kinase A activation, increases primary cilium length in mammalian epithelial and mesenchymal cells. Anterograde intraflagellar transport is sped up in lengthened cilia, potentially increasing delivery flux of cilium components. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases mechanotransductive signaling. This adaptive response is blocked when the autosomal-dominant polycystic kidney disease (ADPKD) gene products, polycystin-1 or -2, are reduced. Dynamic regulation of cilium length is thus intertwined with cilium-mediated signaling and provides a natural braking mechanism in response to external stimuli that may be compromised in PKD.

The VWA domain-containing extracellular matrix protein AMACO has not been extensively characterized and its function remains unknown. It has been proposed as a potential cancer marker and carries a rare O-glucosylation and O-fucosylation on its first EGF-like domain. AMACO is a basement membrane associated protein, however its exact localization has not been determined. Here we show by immunogold electron microscopy of mouse kidney and skin that AMACO does not occur within the basement membrane but rather subjacent to the basement membrane at its stromal surface. In skin, AMACO often colocalizes with triple-helical domains of collagen VII containing anchoring fibrils as they emerge from the basal lamina. However, the immunogold patterns for AMACO and the C-terminal end of collagen VII show discrete differences, indicating that AMACO and collagen VII do not colocalize at anchoring plaques. In contrast, the localization pattern of AMACO partially overlaps with that for collagen XVIII. In addition, mouse AMACO was shown to support beta1 integrin-mediated adhesion of a keratinocyte-like cell line, HaCaT, and a fibroblast cell line, Wi26, in an RGD-dependent manner, most likely using an RGD-motif near the C-terminus of AMACO. However, the loss of cell adhesion to the C-terminal part of the human AMACO, due to the unique absence of an RGD sequence in the human protein, suggests that cell adhesion is not AMACO's major function.

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. alpha5beta1 and alphavbeta3 integrins form stable complexes with immobilized endostatin (KD=approximately 1.8x10(-8) M, two-state model). Two arginine residues (Arg27 and Arg139) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the alphavbeta3 integrin at the interface between the beta-propeller domain of the alphav subunit and the betaA domain of the beta3 subunit. In addition, we report that alpha5beta1 and alphavbeta3 integrins bind to heparin/heparan sulfate. The ectodomain of the alpha5beta1 integrin binds to haparin with high affinity (KD=15.5 nM). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and alpha5beta1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.

Mechanical stress is known to modulate postnatal skeletal growth and development. However, the mechanisms underlying the mechanotransduction are not fully understood. Polycystin-1 (PC1) is a promising candidate among proteins that may play a role in the process as it has been shown to function as a flow sensor in renal epithelium and it is known to be important for skeletal development. To investigate whether PC1 is involved in mechanotransduction in skeletal tissues, mice with a conditional deficiency for PC1 in neural crest cells, osteoblasts or chondrocytes were subjected to midpalatal suture expansion. Dynamic bone labeling revealed that new bone formation in response to expansion was significantly reduced in Wnt1Cre;Pkd1 mice, as the suture area containing new bone was 14.0+/-3.4% in mutant mice versus 65.0+/-3.8% in control mice at 2 weeks (p

Coronaviruses (CoVs) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, “Multiprobe QPCR”, which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan®) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR (“NQPCR”) for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in 7 of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.

OBJECTIVE Key features of diabetic nephropathy include the accumulation of extracellular matrix proteins. In recent studies, increased expression of type VIII collagen in the glomeruli and tubulointerstitium of diabetic kidneys has been noted. The objectives of this study were to assess whether type VIII collagen affects the development of diabetic nephropathy and to determine type VIII collagen–dependent pathways in diabetic nephropathy in the mouse model of streptozotocin (STZ)-induced diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by STZ injections in collagen VIII–deficient or wild-type mice. Functional and histological analyses were performed 40 days after induction of diabetes. Type VIII collagen expression was assessed by Northern blots, immunohistochemistry, and real-time PCR. Proliferation of primary mesangial cells was measured by thymidine incorporation and direct cell counting. Expression of phosphorylated extracellular signal–regulated kinase (ERK1/2) and p27Kip1 was assessed by Western blots. Finally, Col8a1 was stably overexpressed in mesangial cells. RESULTS Diabetic wild-type mice showed a strong renal induction of type VIII collagen. Diabetic Col8a1−/Col8a2− animals revealed reduced mesangial expansion and cellularity and extracellular matrix expansion compared with the wild type. These were associated with less albuminuria. High-glucose medium as well as various cytokines induced Col8a1 in cultured mesangial cells. Col8a1−/Col8a2− mesangial cells revealed decreased proliferation, less phosphorylation of Erk1/2, and increased p27Kip1 expression. Overexpression of Col8a1 in mesangial cells induced proliferation. CONCLUSIONS Lack of type VIII collagen confers renoprotection in diabetic nephropathy. One possible mechanism is that type VIII collagen permits and/or fosters mesangial cell proliferation in early diabetic nephropathy.

The infection of wild birds by highly pathogenic strains of avian influenza (Al) virus was virtually unknown--apart from one instance of the disease appearing in common terns in South Africa in 1961--before the Asian strain of highly pathogenic AI virus (AIV), H5N1, began to expand across the world. Outbreaks of clinical disease in Eurasia have resulted in visible mortality among populations of free-ranging wild birds in a multitude of species. The circulation pattern of influenza viruses in natural ecosystems results from a selection pressure towards strains which are indirectly transmitted by droppings from water birds and contaminated fomites, and which exhibit low pathogenicity. Some of these viruses, of the subtypes H5 or H7, can mutate into highly pathogenic strains after being introduced into domestic poultry farms. The maintenance of highly pathogenic AIV (HPAIV) H5N1 in several parts of the world exposes wild birds to infected poultry, resulting in long-distance virus transmission. There is great concern that these wild birds may, in turn, propagate these HPAIV or introduce them into domestic birds. Rigorous disease control and biosecurity measures to protect poultry farms are the only solution presently available to mitigate such a risk.

The antiviral drug Tamiflu (Oseltamivir Phosphate, OP), has been indicated by the World Health Organization as a first-line defense in case of an avian influenza pandemic. Recent studies have demonstrated that Oseltamivir Carboxylate (OC), the active metabolite of the prodrug OP, has the potential to be released into water bodies. The present laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, addition of 5% of sediments resulted in rapid OC degradation. Estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 degrees C, more than 8% of (14)C-OC evolved as (14)CO(2) from water/sediment samples of the River Po and Venice lagoon. At the end of the 21-day incubation period, more than 65% of the (14)C-residues were recovered from the liquid phase of both Po and Venice water/sediment samples. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice lagoon treated with (14)C-OC, more than 30% of the (14)C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that presence of sediments would not reduce its bioavailability to microbial degradation.

The natural reservoir of influenza A virus is waterfowl, particularly dabbling ducks (genusAnas). Although it has long been assumed that waterfowl are asymptomatic carriers of the virus, a recent study found that low-pathogenic avian influenza (LPAI) infection in Bewick's swans (Cygnus columbianus bewickii) negatively affected stopover time, body mass and feeding behaviour. In the present study, we investigated whether LPAI infection incurred ecological or physiological costs to migratory mallards (Anas platyrhynchos) in terms of body mass loss and staging time, and whether such costs could influence the likelihood for long-distance dispersal of the avian influenza virus by individual ducks. During the autumn migrations of 2002–2007, we collected faecal samples (n=10 918) and biometric data from mallards captured and banded at Ottenby, a major staging site in a flyway connecting breeding and wintering areas of European waterfowl. Body mass was significantly lower in infected ducks than in uninfected ducks (mean difference almost 20 g over all groups), and the amount of virus shed by infected juveniles was negatively correlated with body mass. There was no general effect of infection on staging time, except for juveniles in September, in which birds that shed fewer viruses stayed shorter than birds that shed more viruses. LPAI infection did not affect speed or distance of subsequent migration. The data from recaptured individuals showed that the maximum duration of infection was on average 8.3 days (s.e. 0.5), with a mean minimum duration of virus shedding of only 3.1 days (s.e. 0.1). Shedding time decreased during the season, suggesting that mallards acquire transient immunity for LPAI infection. In conclusion, deteriorated body mass following infection was detected, but it remains to be seen whether this has more long-term fitness effects. The short virus shedding time suggests that individual mallards are less likely to spread the virus at continental or intercontinental scales.

Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. "Cherubism mice", which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-alpha. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.

Infantile hemangiomas are localized and rapidly growing regions of disorganized angiogenesis. We show that expression of vascular endothelial growth factor receptor-1 (VEGFR1) in hemangioma endothelial cells (hemECs) and hemangioma tissue is markedly reduced compared to controls. Low VEGFR1 expression in hemECs results in VEGF-dependent activation of VEGFR2 and downstream signaling pathways. In hemECs, transcription of the gene encoding VEGFR1 (FLT1) is dependent on nuclear factor of activated T cells (NFAT). Low VEGFR1 expression in hemECs is caused by reduced activity of a pathway involving beta1 integrin, the integrin-like receptor tumor endothelial marker-8 (TEM8), VEGFR2 and NFAT. In a subset of individuals with hemangioma, we found missense mutations in the genes encoding VEGFR2 (KDR) and TEM8 (ANTXR1). These mutations result in increased interactions among VEGFR2, TEM8 and beta1 integrin proteins and in inhibition of integrin activity. Normalization of the constitutive VEGFR2 signaling in hemECs with soluble VEGFR1 or antibodies that neutralize VEGF or stimulate beta1 integrin suggests that local administration of these or similar agents may be effective in hemangioma treatment.

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the “New Head”.

In vertebrates, coordinated embryonic and postnatal growth of the craniofacial bones and the skull base is essential during the expansion of the rostrum and the brain. Identification of molecules that regulate skull growth is important for understanding the nature of craniofacial defects and for development of non-invasive biologically based diagnostics and therapies. Here we report on spatially restricted growth defects at the skull base and in craniofacial sutures of mice deficient for polycystin-1 (Pkd1). Mutant animals reveal a premature closure of both presphenoid and sphenooccipital synchondroses at the cranial base. Furthermore, knockout mice lacking Pkd1 in neural crest cells are characterized by impaired postnatal growth at the osteogenic fronts in craniofacial sutures that are subjected to tensile forces. Our data suggest that polycystin-1 is required for proliferation of subpopulations of cranial osteochondroprogenitor cells of both mesodermal and neural crest origin during skull growth. However, the Erk1/2 signalling pathway is up-regulated in the Pkd1-deficient skeletal tissue, similarly to that previously reported for polycystic kidney.

Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23−/− or Klotho−/− knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23–Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1α-hydroxylase expression and phosphoinositide-3 kinase–dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.

Farmers around the world have been urged to eliminate barn swallows (Hirundo rustica) from their buildings, based on the unproven assumption that these birds amplify and spread Salmonella to livestock, humans, and stored food. However, we tested over 500 barn swallows in Sweden and found no evidence that they carry Salmonella. Our results cast doubt on the hypothesis that barn swallows are competent reservoirs of Salmonella and suggest that their role, if any, is limited to being accidental hosts of the bacterium.

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.

Faecal bacteria from 49 Gentoo penguins on the Antarctic Peninsula were identified by biochemical methods and sequencing, and tested for antibiotic susceptibility using agar dilution. Of the 42 Enterobacteriaceae isolates found, 39 belonged to the genus Edwardsiella. All isolates were susceptible to the 17 antibiotics tested. This implies that antibiotic selection pressure is a prerequisite to a high prevalence of antibiotic resistance, and in the absence of contact with human activities, antibiotic resistance in Enterobacteriaceae remains undetectable.

The transcription factor, Runx2, is known to play crucial roles in skeletal and tooth morphogenesis. Here we document that Runx2 has a regulatory role in skin and hair follicle development. The expression of Runx2 is restricted to hair follicles and is dynamic, pari passu with follicle development. Follicle maturation is delayed in the absence of Runx2 and overall skin and epidermal thickness of Runx2 null embryos is significantly reduced. The Runx2 null epidermis is hypoplastic, displaying reduced expression of Keratin 14, Keratin 1 and markers of proliferation. The expression pattern of Runx2 in the bulb epithelium of mature hair follicles is asymmetric and strikingly similar to that of Sonic hedgehog. This suggests that Runx2 may be a regulator of hedgehog signaling in skin as it is in bones and teeth. Supporting this possibility, we demonstrate that Sonic hedgehog, Patched1 and Gli1 transcripts are reduced in the skin of Runx2 null embryos. Moreover, we document Patched1 expression in epidermal basal cells and show that the skin of Sonic+/− embryos is thinner than that of wild-type littermates. These observations suggest that Runx2 and hedgehog signaling are involved in the well known, but unexplained, coupling of skin thickness to hair follicle development.

OBJECTIVE. Patients with Down syndrome have a reduced risk of developing solid tumors. This protective effect has been attributed to increased gene dosage from an additional copy of chromosome 21, and elevated expression of endostatin has been implicated. We hypothesized that vascular anomalies, including infantile hemangioma, an angiogenesis-dependent vascular tumor, and vascular malformations might be similarly inhibited in patients with Down syndrome. PATIENTS AND METHODS. The Children's Hospital Boston Vascular Anomalies Center database was searched for patients with Down syndrome between 1999 and 2007. In addition, the records of patients with Down syndrome treated at Children's Hospital Boston and the National Birth Defects Center between 1985 and 2007 were reviewed to find concurrent vascular anomalies. Two-sided exact binomial tests were used to evaluate whether patients with vascular anomalies are at reduced risk for Down syndrome or if patients with Down syndrome are at less risk for vascular anomalies compared with the general population. Ninety-five–percent confidence intervals were calculated on the basis of the risk of Down syndrome (1 in 800) and vascular anomalies (1 in 22) in the general population. RESULTS. Two of the 7354 patients evaluated in our vascular anomalies unit had Down syndrome. Both patients had a lymphatic malformation: one in the orbit and the other in the lower extremity. Six of the 633 patients with Down syndrome had a vascular anomaly (infantile hemangioma [n = 4] or lymphatic malformation [n = 2]). The risk of concurrent Down syndrome and vascular anomalies was different from the corresponding risk in the general population. CONCLUSIONS. Patients with Down syndrome have a reduced risk of vascular anomalies compared with the general population. Elevated expression of antiangiogenic proteins may protect these patients from developing vascular anomalies, as well as solid tumors.

Mycobacterium avium is the most common pathogen in children presenting as non-tuberculous mycobacterial lymphadenitis. In Sweden non-tuberculous mycobacterial infection is a notifiable disease. The goal of our study was to determine the annual incidence of M. avium infection in Swedish children, 1998-2003, and describe clinical features related to age and treatment of children with M. avium lymphadenitis. To do this, we retrospectively analysed patient records of 162 children less than 7 y of age from the entire country with culture-verified M. avium disease. The incidence of M. avium infection in Sweden was lower in 2000-2003 than in 1998-1999. Young children (

Analysing datasets from hunting statistics and human cases of tick-borne encephalitis (TBE), we found a positive correlation between the number of human TBE cases and the number of red fox (Vulpes vulpes). Time lags were also present, indicating that high numbers of red fox in 1 y translated into high numbers of human TBE cases the following y. Results for smaller predators were mixed and inconsistent. Hares and grouse showed negative correlations with human TBE cases, suggesting that they might function as dilution hosts. Combining our findings with food web dynamics, we hypothesize a diversity of possible interactions between predators and human disease - some predators suppressing a given disease, others enhancing its spread, and still others having no effect at all. Larger-sized predators that suppress red fox numbers and activity (i.e. wolf, Canis lupus; European lynx, Lynx lynx) were once abundant in our study area but have been reduced or extirpated from most parts of it by humans. We ask what would happen to red foxes and TBE rates in humans if these larger predators were restored to their former abundances.

Since prehistoric times, the Bering Strait area (Beringia) has served as all avenue of dispersal between the Old and the New Worlds. On a field expedition to this area, we collected fecal samples from dabbling ducks, geese, shorebirds, and gulls on the Chukchi Peninsula, Siberia, and Pt. Barrow, Alaska, and characterized the sub-types of avian influenza virus present in them. Four of 202 samples (2%) from Alaska were positive for influenza A virus RNA in two independent polymerase chain reaction (PCR)-based screening assays, while all shorebird samples from the Chukchi Peninsula were negative. Subtypes H3N8 and H6N1 were recorded once, while subtype H8N4 was found in two samples. Full-length sequences were obtained from the three unique isolates, and phylogenetic analysis with representative sequences for the Eurasian and North American lineages of influenza A virus showed that one HA gene Clustered with the Eurasian rather than the North American lineage. However, the closest relative to this sequence was a North American isolate from Delaware described in 2002, indicating that a H6 spillover from Asia has established itself in North America.

In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba - Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga . Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni , C. coli , C. lari , and C. hyointestinalis , were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter -selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10 4 CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.

Osteoarthritis (OA) is the most common form of arthritis (1). Although the precise etiology of the disease in most cases is unknown, it is generally accepted that OA is a multifactorial disorder involving both genetic and environmental components (2). Genetic factors are either mutations or variations in genes that result in defects or variability in cartilage matrix properties and chondrocyte metabolism. In the case of matrix defects caused by mutations in matrix genes, age-dependent cartilage degeneration may occur as a result of even normal mechanical stresses on a joint. In the case of inherited sequence variations in genes, some variations may confer increased risk for OA (3). Environmental factors include obesity, over-loading on joints, repetitive injury involving ligaments and menisci, loss of muscle strength and joint malalignment. These conditions can result in abnormal mechanical stresses on the joint, leading to cartilage degeneration. Since OA can occur as a result of one or a combination of these factors, investigating the detailed pathogenic mechanisms in all these conditions remains a formidable challenge. However, regardless of the nature of the factor(s) that initiate the disease, the pathological progression of OA follows a typical pattern (4). The earliest indication of pathological change is chondrocyte clustering as a result of increased cell proliferation and a general up-regulation of synthetic activity. Increased expression of cartilage-degrading proteinases and matrix proteins suggests an attempt at repair. Gradual loss of proteoglycans appears in the surface region of articular cartilage and this is followed by type II collagen degradation. Cracks develop along the articular surface, producing the histological image termed fibrillation. At later stages of the disease, fibrocartilage forms, probably as a consequence of unsuccessful attempts by chondrocytes to fill in the cracks. Finally, osteophytes, bony structures at the periphery of the joint surface, form.

Hedgehog signaling plays an essential role in patterning of the vertebrate skeleton. Here we demonstrate that conditional inactivation of the Kif3a subunit of the kinesin-2 intraflagellar transport motor in mesenchymal skeletal progenitor cells results in severe patterning defects in the craniofacial area, the formation of split sternum and the development of polydactyly. These deformities are reminiscent of those previously described in mice with deregulated hedgehog signaling. We show that in Kif3a-deficient mesenchymal tissues both the repressor function of Gli3 transcription factor and the activation of the Shh transcriptional targets Ptch and Gli1 are compromised. Quantitative analysis of gene expression demonstrates that the Gli1 transcript level is dramatically reduced, whereas Gli3 expression is not significantly affected by kinesin-2 depletion. However, the motor appears to be required for the efficient cleavage of the full-length Gli3 transcription factor into a repressor form.

During spring and autumn 2001, we screened 13,260 migrating birds at Ottenby Bird Observatory, Sweden, and found 3.4% were infested with ticks. Four birds, each a different passerine species, carried tickborne encephalitis virus (TBEV)–infected ticks (Ixodes ricinus). Migrating birds may play a role in the geographic dispersal of TBEV-infected ticks.

We investigate the properties of second-harmonic generation (SHG) tissue imaging for the functional biological unit fascia, skeletal muscle, and tendon. Fascia and Achilles tendon primarily consist of similar collagen type I arrays that can be imaged using SHG microscopy. For muscle, it is the myosin molecules represented within the A bands. For fascia and tendon tissue samples, we observe, in addition to a stronger signal in forward images, vastly different features for the backward versus the forward images. In vivo as well as intact ex vivo thick tissue imaging requires backward detection. The obtained image is a result of the direct backward components plus a certain fraction of the forward components that are redirected (backscattered) toward the objective as they propagate within the tissue block. As the forward and the backward images are significantly different from each other for the imaged collagen type I tissue, it is crucial to determine the fraction of the forward signal that contributes to the overall backward signal. For intact ex vivo SHG imaging of Achilles tendon, we observe a significant contribution of forward features in the resulting image. For fascia, the connective tissue immediately surrounding muscle, we only observe backward features, due to low backscattering in muscle.

Mechanical stress is an important epigenetic factor for regulating skeletal remodeling, and application of force can lead to remodeling of both bone and cartilage. Chondrocytes, osteoblasts and osteoclasts all participate and interact with each other in this remodeling process. To study cellular responses to mechanical stimuli in a system that can be genetically manipulated, we used mouse midpalatal suture expansion in vivo. Six-week-old male C57BL/6 mice were subjected to palatal suture expansion by opening loops with an initial force of 0.56 N for the periods of 1, 3, 5, 7, 14 or 28 days. Periosteal cells in expanding sutures showed increased proliferation, with Ki67-positive cells representing 1.8+/-0.1% to 4.5+/-0.4% of total suture cells in control groups and 12.0+/-2.6% to 19.9+/-1.2% in experimental/expansion groups (p

Endostatin, a fragment of the basement membrane component collagen XVIII, exhibits antiangiogenic properties in vitro and in vivo when high doses are administered. It is not known whether endogenous endostatin at physiological levels has a protective role as an inhibitor of pathological angiogenesis, such as choroidal neovascularization (CNV) in age-related macular degeneration. Using a laser injury model, we induced CNV in mice lacking collagen XVIII/endostatin and in control mice. CNV lesions in mutant mice were approximately 3-fold larger than in control mice and showed increased vascular leakage. These differences were independent of age-related changes at the choroid-retina interface. Ultrastructural analysis of the choroidal vasculature in mutant mice excluded morphological vascular abnormalities as a cause for the larger CNV lesions. When recombinant endostatin was administered to collagen XVIII/endostatin-deficient mice, CNV lesions were similar to those seen in control mice. In control mice treated with recombinant endostatin, CNV lesions were almost undetectable. These findings demonstrate that endogenous endostatin is an inhibitor of induced angiogenesis and that administration of endostatin potently inhibits CNV growth and vascular leakage. Endostatin may have a regulatory role in the pathogenesis of CNV and could be used therapeutically to inhibit growth and leakage of CNV lesions.

We examine the major genes in mice and humans involved in the pathogenesis of monosodium urate, calcium pyrophosphate dihydrate and hydroxyapatite crystal-induced arthritis.Several genetic causes of renal disease associated with hyperuricemia and gout provide insight into genes involved in renal urate handling. Mutations or polymorphisms in exons 4 and 5 and intron 4 of urate transporter 1 may be independent genetic markers of hyperuricemia and gout. Genetic analysis supports the role of ANKH mutations in calcium pyrophosphate dihydrate-induced arthritis. ANKH gain-of-function mutations were confirmed by functional studies; however, the crystals formed in ATD5 cells were basic calcium phosphate, not calcium pyrophosphate dihydrate, underlying the significance of chondrocyte differentiation state and the factors regulating normal and pathological mineralization. Animal models have implicated a general model of crystal-induced inflammation involving innate immunity through the NALP3 (Natch domain, leucine-rich repeat, and PYD-containing protein 3) inflammasome signaling through the interleukin-1 receptor and its signaling protein myeloid differentiation primary response protein 88.Genetic analysis has elucidated genes responsible for crystal formation and animal models have unveiled mechanisms in the development of crystal-induced arthritis. Future studies will hasten understanding of the pathology of crystal-induced arthritis and provide new therapies.

Ducks may maintain influenza virus from 1 year to the next., We conducted large-scale, systematic sampling of influenza type A virus in migratory waterfowl (mostly mallards [Anas platyrhynchos]) at Ottenby Bird Observatory, southeast Sweden. As with previous studies, we found a higher prevalence in fall than spring, and among juveniles compared with adults. However, in contrast to other studies, we found that prevalence in spring was sometimes high (mean 4.0%, highest 9.5%). This finding raises the possibility that ducks are capable of perpetuating influenza A virus of different subtypes and subtype combinations throughout the year and from 1 year to the next. Isolation of the H5 and H7 subtypes was common, which suggests risk for transmission to sensitive domestic animals such as poultry. We argue that wild bird screening can function as a sentinel system, and we give an example of how it could have been used to forecast a remote and deadly outbreak of influenza A in poultry.

Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus–host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds., Author Summary Significant gaps in our knowledge of the ecology of avian influenza in wild migratory birds have become apparent during recent outbreaks of H5N1 highly pathogenic avian influenza, in particular in relation to the risk of virus spread by wild birds. An eight-year surveillance study, which included more than 36,000 wild birds tested for low pathogenic avian influenza, provides new information on host species, prevalence, and temporal and geographical variation of avian influenza in wild migratory birds in Europe. Dabbling ducks harbored nearly all known influenza virus subtypes, with the exception of H13 and H16, which were found primarily in gulls. In contrast to American studies, waders did not play a role in the epidemiology of avian influenza in Europe. This study provides important information on the ecology and epidemiology of avian influenza A virus and could assist in the design of new surveillance studies for high and low pathogenic avian influenza in wild birds.

Migratory birds have been suggested to contribute to long-distance dispersal of antimicrobial resistant bacteria, but tests of this hypothesis are lacking. In this study we determined resistance profiles and genotypes of ESBL-producing bacteria in randomly selected Escherichia coli from Franklin's gulls (Leucophaeus pipixcan) at breeding sites in Canada and compared with similar data from the gulls' wintering grounds in Chile. Resistant E. coli phenotypes were common, most notably to ampicillin (30.1%) and cefadroxil (15.1%). Furthermore, 17.0% of the gulls in Canada carried ESBL producing bacteria, which is higher than reported from human datasets from the same country. However, compared to gulls sampled in Chile (30.1%) the prevalence of ESBL was much lower. The dominant ESBL variants in Canada were bla(CTX-M-14) and bla(CTX-M-15) and differed in proportions to the data from Chile. We hypothesize that the observed differences in ESBL variants are more likely linked to recent exposure to bacteria from anthropogenic sources, suggesting high local dissemination of resistant bacteria both at breeding and non-breeding times rather than a significant trans-hemispheric exchange through migrating birds.

Background : Campylobacter jejuni is a common cause of human bacterial diarrhea in most parts of the world. Most C. jejuni infections are acquired from contaminated poultry, milk, and water. Due to health care costs and human suffering, it is important to identify all possible sources of infection. Unpasteurized milk has been associated with several outbreaks of C. jejuni infection. Campylobacter has been identified on fresh fruit, and other gastrointestinal pathogens such as Salmonella, E. coli O157:H7 and Cryptosporidium have been involved in fruit juice outbreaks. C. jejuni is sensitive to the acidic environment of fruit juice, but co-cultures with the amoeba, Acanthamoeba polyphaga , have previously been shown to protect C. jejuni at low pH. Methods : To study the influence of A. polyphaga on the survival of C. jejuni in milk and juice, the bacteria were incubated in the two products at room temperature and at 4°C with the following treatments: A) C. jejuni preincubated with A. polyphaga before the addition of product, B) C. jejuni mixed with A. polyphaga after the addition of product, and C) C. jejuni in product without A. polyphaga . Bacterial survival was assessed by colony counts on blood agar plates. Results : Co-culture with A. polyphaga prolonged the C. jejuni survival both in milk and juice. The effect of co-culture was most pronounced in juice stored at room temperature. On the other hand, A. polyphaga did not have any effect on C. jejuni survival during pasteurization of milk or orange juice, indicating that this is a good method for eliminating C. jejuni in these products. Conclusion : Amoebae-associated C. jejuni in milk and juice might cause C. jejuni infections. Keywords: unpasteurized milk; fruit juice; C. jejuni infection; co-culture ; Campylobacter survival; gastrointestinal pathogens (Published: 18 September 2015) Citation: Infection Ecology and Epidemiology 2015, 5: 28675 - http://dx.doi.org/10.3402/iee.v5.28675

Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment.

Understanding the mechanisms by which bone marrow mesenchymal stem cells (BMSCs) differentiate into bone-forming osteoblasts and marrow adipocytes is crucial to develop strategies for the treatment of several bone diseases. Age-related bone loss resulting in osteopenia and osteoporosis has been associated with reduced numbers of osteoblasts and increased numbers of adipocytes, likely originating from differentiation defects in BMSCs. Although many factors involved in the complex regulation of osteoblast and adipocyte cell lineages have previously been identified, their functional interactions in the context of BMSC differentiation and maintenance of bone homeostasis during ageing are unknown. Recent discoveries have provided important new insights into the mechanisms by which the nuclear envelope protein lamin A and vascular endothelial growth factor A (VEGF) mutually control BMSC fate. Particularly interesting is the finding that VEGF in this context functions as an intracellular protein, unaffected by neutralizing antibodies, and not as a secreted growth factor. These insights may not only facilitate the identification of new targets for treating bone diseases but also lead to improved design of tissue engineering approaches aimed at stimulating bone regeneration and repair.

Courtney Aldrich USA Praveen Ballabh USA Markus Biburger Germany Trevor Birmingham Canada Frederick Colbourne Canada Peter Crouch Australia Jean-Pierre David Germany Solange L. De Castro Brazil Chetana S. Deshpande India Julia Etich Germany Yoshiaki Furukawa Japan Kolja Gelse Germany Felipe Goni-de-Cerio Spain Bettina Groetsch Germany Ulrike Harre Germany Gordon Howarth Australia Dirk Hubmacher USA Pan Jihong China Dimitrios Kanakis Greece Masashi Kanayama USA Shunsuke Kanbara Japan Andre Pascal Kengne South Africa Michio Kurosu USA Alexander Benedikt Leichtle Switzerland Roberto Marci Italy David McGhee UK Dirk Mielenz Germany Tetsuryu Mitsuyama Japan Evelyn Marielle Monninkhof Netherlands Jennifer Moylan USA Takashi Nagasawa Japan Edin Nevzati Switzerland Nikoletta Papadopoulou Germany Rui Prediger Brazil Haibo Qiu China Jaqueline Rinaldi Brazil Juan Rodriguez Argentina Celena Scheede-Bergdahl Canada Je Kyung Seong South Korea Vishnumurthy Shushrutha Hedna USA Mitsutoshi Tsukimoto Japan Stephanie Wohlgemuth USA Fumihiko Yoshino Japan Ti-Fei Yuan China

Background : The zoonotic bacterium Campylobacter jejuni has a broad host range but is especially associated with birds, both domestic and wild. Earlier studies have indicated thrushes of the genus Turdus in Europe to be frequently colonized with C. jejuni , and predominately with host-associated specific genotypes. The European Blackbird Turdus merula has a large distribution in Europe, including some oceanic islands, and was also introduced to Australia by European immigrants in the 1850s. Methods : The host specificity and temporal stability of European Blackbird C. jejuni was investigated with multilocus sequence typing in a set of isolates collected from Sweden, Australia, and The Azores. Results : Remarkably, we found that the Swedish, Australian, and Azorean isolates were genetically highly similar, despite extensive spatial and temporal isolation. This indicates adaptation, exquisite specificity, and stability in time for European Blackbirds, which is in sharp contrast with the high levels of recombination and mutation found in poultry-related C. jejuni genotypes. Conclusion : The maintenance of host-specific signals in spatially and temporally separated C. jejuni populations suggests the existence of strong purifying selection for this bacterium in European Blackbirds. Keywords: gastrointestinal pathogen ; Campylobacter jejuni; Blackbird; MLST; population structure (Published: 2 December 2015) Citation: Infection Ecology and Epidemiology 2015, 5: 28383 - http://dx.doi.org/10.3402/iee.v5.28383

This chapter describes the basic principles of research ethics as they apply to biomedical research on human subjects and experimental animals. It builds on the more general introduction to ethics and scientific conduct presented in Chapter 2. It will first introduce two influential international regulatory documents on human biomedical research ethics and discuss specific problems raised when individuals are unable to give consent to participate in research projects, when placebos are used in randomized controlled trials, and when research involves vulnerable populations. It will also discuss the role of research ethics committees and how biomedical research on human subjects is regulated in most countries and at the international level. Although the details of regulatory protocols differ from country to country, the existence of long-standing international regulatory documents has resulted in significant harmonization of national regulations and an almost universal acceptance of certain principles of research ethics at the regulatory level.The controversial problems raised by research on embryos or fetuses will not be discussed, as this issue will be relevant to very few readers of this book, and the discussion would require an in-depth analysis of the moral status of embryos and fetuses.1 The last part of the chapter will concern biomedical research on experimental animals, the justification of animal research, the obligation to minimize animal suffering, and the question of whether certain species require specific protections (see also Chapter 6). Summaries and key points are highlighted in boxes and a table.

This chapter describes how to plan, prepare for, develop, and carry out basic medical research projects, which aim to enhance the understanding of fundamental, normal, and pathological processes at the organismic, organ, cellular, or molecular levels. The various steps that lead to the identification of important research questions, clearly stated research problems, and hypotheses, as well as specific advice on choosing a research problem, will be provided. This will be followed by a description of the experimental strategies and methods currently available at the organismic and cellular levels, and their strengths and limitations. Some of the experimental strategies made possible by technical advances in genetics and molecular biology will be described for studies using mice, but information will also be provided for other commonly used experimental animals. Ethical and legal issues related to research on experimental animals will be discussed. The discussion of experimental strategies at the cellular level will reflect the importance of cell and tissue culture as both an alternative and complement to research on experimental animals. It will include a brief history of cell culture and a review of more recent technical advances. Summaries, key points of advice, and some useful data and technical examples are highlighted in boxes.

Background The need for new antibiotic drugs increases as pathogenic microorganisms continue to develop resistance against current antibiotics. We obtained samples from Antarctica as part of a search for new antimicrobial metabolites derived from filamentous fungi. This terrestrial environment near the South Pole is hostile and extreme due to a sparsely populated food web, low temperatures, and insufficient liquid water availability. We hypothesize that this environment could cause the development of fungal defense or survival mechanisms not found elsewhere. Results We isolated a strain of Penicillium nalgiovense Laxa from a soil sample obtained from an abandoned penguin’s nest. Amphotericin B was the only metabolite secreted from Penicillium nalgiovense Laxa with noticeable antimicrobial activity, with minimum inhibitory concentration of 0.125 μg/mL against Candida albicans. This is the first time that amphotericin B has been isolated from an organism other than the bacterium Streptomyces nodosus. In terms of amphotericin B production, cultures on solid medium proved to be a more reliable and favorable choice compared to liquid medium. Conclusions These results encourage further investigation of the many unexplored sampling sites characterized by extreme conditions, and confirm filamentous fungi as potential sources of metabolites with antimicrobial activity.

Candidatus Neoehrlichia mikurensis (CNM; family Anaplasmataceae) was recently recognized as a potential tick-borne human pathogen. The presence of CNM in mammals, in host-seeking Ixodes ticks and in ticks attached to mammals and birds has been reported recently. We investigated the presence of CNM in ornithophagous ticks from migrating birds. A total of 1,150 ticks (582 nymphs, 548 larvae, 18 undetermined ticks and two adult females) collected from 5,365 birds captured in south-eastern Sweden was screened for CNM by molecular methods. The birds represented 65 different species, of which 35 species were infested with one or more ticks. Based on a combination of morphological and molecular species identification, the majority of the ticks were identified as Ixodes ricinus. Samples were initially screened by real-time PCR targeting the CNM 16S rRNA gene, and confirmed by a second real-time PCR targeting the groEL gene. For positive samples, a 1260 base pair fragment of the 16S rRNA gene was sequenced. Based upon bacterial gene sequence identification, 2.1% (24/1150) of the analysed samples were CNM-positive. Twenty-two out of 24 CNM-positive ticks were molecularly identified as I. ricinus nymphs, and the remaining two were identified as I. ricinus based on morphology. The overall CNM prevalence in I. ricinus nymphs was 4.2%. None of the 548 tested larvae was positive. CNM-positive ticks were collected from 10 different bird species. The highest CNM-prevalences were recorded in nymphs collected from common redpoll (Carduelis flammea, 3/7), thrush nightingale (Luscinia luscinia, 2/29) and dunnock (Prunella modularis, 1/17). The 16S rRNA sequences obtained in this study were all identical to each other and to three previously reported European strains, two of which were obtained from humans. It is concluded that ornithophagous ticks may be infected with CNM and that birds most likely can disperse CNM-infected ticks over large geographical areas. Funding for this study came from the Medical Research Council of Southeast Sweden (grant numbers FORSS-307591 and FORSS-387231), Carl Trygger's Foundation for Scientific Research, Langmanska kulturfonden, Magnus Bergvall's Foundation for Scientific Research, Helge Ax:son Johnson's Foundation and the EU Interreg IVA project ScandTick.

Extended spectrum β-lactamases (ESBLs), a group of enzymes conferring resistance to third generation cephalosporins have rapidly increased in Enterobacteriacae and pose a major challenge to human health care. Resistant isolates are common in domestic animals and clinical settings, but prevalence and genotype distribution varies on a geographical scale. Although ESBL genes are frequently detected in bacteria isolated from wildlife samples, ESBL dissemination of resistant bacteria to the environment is largely unknown. To address this, we used three closely related gull species as a model system and collected more than 3000 faecal samples during breeding times in nine European countries. Samples were screened for ESBL-producing bacteria, which were characterized to the level of ESBL genotype groups (SHV, TEM), or specific genotypes (CTX-M). ESBL-producing bacteria were frequently detected in gulls (906 of 3158 samples, 28.7 %), with significant variation in prevalence rates between countries. Highest levels were found in Spain (74.8 %), The Netherlands (37.8 %) and England (27.1 %). Denmark and Poland represented the other extreme with no, or very few positive samples. Genotyping of CTX-M isolates identified 13 different variants, with bla CTX-M-1 and bla CTX-M-14 as the most frequently detected. In samples from England, Spain and Portugal, bla CTX-M-14 dominated, while in the rest of the sampled countries bla CTX-M-1 (except Sweden where bla CTX-M-15 was dominant) was the most frequently detected genotype, a pattern similar to what is known from studies of human materials. CTX-M type ESBLs are common in the faecal microbiota from gulls across Europe. The gull ESBL genotype distribution was in large similar to published datasets from human and food-production animals in Europe. The data suggests that the environmental dissemination of ESBL is high from anthropogenic sources, and widespread occurrence of resistant bacteria in common migratory bird species utilizing urban and agricultural areas suggests that antibiotic resistance genes may also be spread through birds.

Translational medical research in academia and research and development in drug companies will be discussed. The dependence of translational medical research on basic medical research in both settings will be emphasized. A description of the history of aspirin, one of the most cost-effective and widely used drugs in medicine, will be provided as an example of the time and effort that the pharmaceutical industry puts into the development of a successful drug and highlighting the role of market needs and return on investment in for-profit drug development. Using aspirin and Plavix, an anti-platelet drug, as examples, the chapter will also cover the importance of understanding drug metabolism and cellular and molecular drug targets. This will be followed by an argument for the need for more and better translational medical research and the strengthening of translational drug discovery at academic institutions, including the steps taken in the United States to improve the funding mechanisms for such research, and what is being done internationally to develop drugs for the treatment of patients with rare disorders. Examples are provided to illustrate how research into the causes of rare disorders can lead to drug development for both rare and common disorders. A discussion of the water channel called aquaporin illustrates the difficulties associated with developing drugs for specific cellular or molecular targets and the frustrations and rewards that are associated with translational medical research. Finally, a description of the training environment for students interested in a career in translational medical research is given, followed by a summary of current efforts to substantially enhance collaborations between academia and the pharmaceutical industry and the underlying trends that drive these efforts.

Influenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In this in vivo mallard ( Anas platyrhynchos ) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.