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Unlabelled: Enterococcus faecium has recently emerged as an important multiresistant nosocomial pathogen. Defining population structure in this species is required to provide insight into the existence, distribution, and dynamics of specific multiresistant or pathogenic lineages in particular environments, like the hospital. Here, we probe the population structure of E. faecium using Bayesian-based population genetic modeling implemented in Bayesian Analysis of Population Structure (BAPS) software. The analysis involved 1,720 isolates belonging to 519 sequence types (STs) (491 for E. faecium and 28 for Enterococcus faecalis). E. faecium isolates grouped into 13 BAPS (sub)groups, but the large majority (80%) of nosocomial isolates clustered in two subgroups (2-1 and 3-3). Phylogenetic and eBURST analysis of BAPS groups 2 and 3 confirmed the existence of three separate hospital lineages (17, 18, and 78), highlighting different evolutionary trajectories for BAPS 2-1 (lineage 78) and 3-3 (lineage 17 and lineage 18) isolates. Phylogenomic analysis of 29 E. faecium isolates showed agreement between BAPS assignment of STs and their relative positions in the phylogenetic tree. Odds ratio calculation confirmed the significant association between hospital isolates with BAPS 3-3 and lineages 17, 18, and 78. Admixture analysis showed a scarce number of recombination events between the different BAPS groups. For the E. faecium hospital population, we propose an evolutionary model in which strains with a high propensity to colonize and infect hospitalized patients arise through horizontal gene transfer. Once adapted to the distinct hospital niche, this subpopulation becomes isolated, and recombination with other populations declines., Importance: Multiresistant Enterococcus faecium has become one of the most important nosocomial pathogens, causing increasing numbers of nosocomial infections worldwide. Here, we used Bayesian population genetic analysis to identify groups of related E. faecium strains and show a significant association of hospital and farm animal isolates to different genetic groups. We also found that hospital isolates could be divided into three lineages originating from sequence types (STs) 17, 18, and 78. We propose that, driven by the selective pressure in hospitals, the three hospital lineages have arisen through horizontal gene transfer, but once adapted to the distinct pathogenic niche, this population has become isolated and recombination with other populations declines. Elucidation of the population structure is a prerequisite for effective control of multiresistant E. faecium since it provides insight into the processes that have led to the progressive change of E. faecium from an innocent commensal to a multiresistant hospital-adapted pathogen.

Pathology in the craniocervical junction is a serious complication of osteogenesis imperfecta (OI). Our aim was to analyze the prevalence and natural course of craniocervical junction anomalies in patients with OI during growth. In a one-center retrospective study, we analyzed lateral skull radiographs and midsagittal magnetic resonance images of 76 patients with either type I, III, or IV OI. The material included longitudinal series of 31 patients. In total, 150 patient images taken at ages 0 to 39 years were analyzed and compared with age-matched control data. Craniocervical anomalies were observed in 37% of patients and in all OI types studied. Of the three types of anomalies, basilar invagination was seen in 13%, basilar impression in 15%, and platybasia in 29% of the patients. From those with an abnormal finding, 44% displayed more than one type of anomaly. At a group level, we found no evidence of progression of craniocervical junction pathology with age. We provide longitudinal and cross-sectional data on craniocervical junction dimensions in growing patients with OI and, based on those, suggest a radiological management strategy for diagnosis of cranial base pathology. A higher risk of having any of the pathological conditions was associated with a lower height Z-score. Careful follow-up of cranial base anomalies particularly in subjects with OI and severe growth failure is warranted., (Copyright © 2012 American Society for Bone and Mineral Research.)

Turner syndrome (TS) that is due to a total or partial lack of an X chromosome affects about 1 in 2000 girls. The syndrome is characterized by short stature and gonadal dysgenesis. Its documented craniofacial features include retrognathic jaws, a short mandible, and a large cranial base angle. Our aim was to find out whether the syndrome also has an effect on the pharyngeal airway space. We retrospectively analysed lateral cephalograms of 35 TS subjects whose age ranged from 6.5 to 21 years and of 35 healthy female controls matched for age. On those, we did 7 linear and 10 angular cephalometric measurements and 9 pharyngeal measurements. Differences between the subjects with TS and their controls were assessed by paired two-tailed T-test. In the girls with TS, both the maxilla and the mandible were more retrognathic (SNA, P = 0.015 and SNB, P < 0.001), the mandible was shorter (TM-Pgn, P = 0.016), and the cranial base angle was larger (SNBa, P = 0.025) than in the controls, confirming the results of earlier studies. Notably, all six pharyngeal airway measurements were smaller in girls with TS. Two of them, PNS-ad2 and PAS, were statistically significantly smaller (P = 0.019 and P = 0.012, respectively). Thus, a narrow pharynx, either as a primary finding or as a consequence of the maxillo-mandibular retrognathism, further delineates the phenotype. This may imply an elevated risk of sleep apnoea in females with TS.

Cranial base abnormalities are an important complication of osteogenesis imperfecta (OI), a hereditary bone fragility disorder that in most patients is caused by mutations affecting collagen type I. To elucidate which clinical characteristics are associated with the occurrence of cranial base abnormalities in OI, we compared cephalometric results of 187 OI patients (median age 12.0 years, range 3.4 to 47 years; 96 female) with those of 191 healthy subjects and related findings to clinical descriptors of the disease. Overall, 41 patients (22%) had at least one unambiguously abnormal skull base measure. Multivariate logistic regression analysis in patients with OI types I, III, and IV (n = 169) revealed that height Z-score [odds ratio (OR) = 0.53, 95% confidence interval (CI) 0.43-0.66, p < .001]--but not age, gender, scleral hue, lumbar spine areal bone mineral density, or a history of bisphosphonate treatment--was a significant independent determinant of skull base abnormalities. Among patients with a height Z-score below -3, 48% had a skull base abnormality regardless of whether they had received bisphosphonate treatment in the first year of life or not. Genotype-phenotype correlations were evaluated in patients with detectable mutations in COL1A1 or COL1A2, the genes coding for collagen type I (n = 140). Skull base abnormalities were present in 6% of patients with haploinsufficiency (frameshift or nonsense) mutations, in 43% of patients with helical glycine substitutions caused by COL1A1 mutations, in 32% of patients with helical glycine substitutions owing to COL1A2 mutations, and in 17% of patients with splice-site mutations affecting either COL1A1 or COL1A2. However, multivariate logistic regression analysis showed that height Z-score but not the type of collagen type I mutation was independently associated with the prevalence of skull base abnormalities. In conclusion, this study shows that clinical severity of OI, as expressed by the height Z-score, was the strongest predictor of skull base abnormalities. We did not find evidence for the hypothesis that bisphosphonate treatment protects against skull base abnormalities., (Copyright © 2011 American Society for Bone and Mineral Research.)

Population genetics encompasses a strong theoretical and applied research tradition on the multiple demographic processes that shape genetic variation present within a species. When several distinct populations exist in the current generation, it is often natural to consider the pattern of their divergence from a single ancestral population in terms of a binary tree structure. Inference about such population histories based on molecular data has been an intensive research topic in the recent years. The most common approach uses coalescent theory to model genealogies of individuals sampled from the current populations. Such methods are able to compare several different evolutionary scenarios and to estimate demographic parameters. However, their major limitation is the enormous computational complexity associated with the indirect modeling of the demographies, which limits the application to small data sets. Here, we propose a novel Bayesian method for inferring population histories from unlinked single nucleotide polymorphisms, which is applicable also to data sets harboring large numbers of individuals from distinct populations. We use an approximation to the neutral Wright-Fisher diffusion to model random fluctuations in allele frequencies. The population histories are modeled as binary rooted trees that represent the historical order of divergence of the different populations. A combination of analytical, numerical, and Monte Carlo integration techniques are utilized for the inferences. A particularly important feature of our approach is that it provides intuitive measures of statistical uncertainty related with the estimates computed, which may be entirely lacking for the alternative methods in this context. The potential of our approach is illustrated by analyses of both simulated and real data sets.

Introduction: Abnormal configuration of the craniocervical junction in the form of basilar impression or invagination, with often-associated platybasia, is a clinically significant cause of neurological symptoms particularly in patients with inherited diseases affecting the connective tissue. To better understand the course of development of these basilar abnormalities and further define their diagnostic criteria in children, we analysed longitudinally changes in the vertical dimensions of the craniocervical junction and in the flexion of the anterior skull base in normal growing individuals., Methods: The distance of the odontoid process to four reference lines and the anterior skull base angle was measured in consecutive series of at least five lateral skull radiographs of 30 females and 23 males. Their mean age was 6.4 years at the beginning and 22.4 years at the end of the observation period., Results: In young children, the odontoid process was situated in a caudal relation to the skull base structures and reached a level similar to that of adults approximately at the age of 7 years in both males and females. Cross-sectional observation of the results camouflages, however, how the intra-individual changes were markedly non-linear. Changes in the anterior skull base angle remained nonsignificant., Conclusions: Normal values for McRae's, Chamberlain's and McGregor's measurements and the more recently documented D-M measurement are age dependent. We provide reference values specific for ages from 4 years. A notable deviation from the documented values indicates a need of further examination.

A repetitive sequence collection is a set of sequences which are small variations of each other. A prominent example are genome sequences of individuals of the same or close species, where the differences can be expressed by short lists of basic edit operations. Flexible and efficient data analysis on such a typically huge collection is plausible using suffix trees. However, the suffix tree occupies much space, which very soon inhibits in-memory analyses. Recent advances in full-text indexing reduce the space of the suffix tree to, essentially, that of the compressed sequences, while retaining its functionality with only a polylogarithmic slowdown. However, the underlying compression model considers only the predictability of the next sequence symbol given the k previous ones, where k is a small integer. This is unable to capture longer-term repetitiveness. For example, r identical copies of an incompressible sequence will be incompressible under this model. We develop new static and dynamic full-text indexes that are able of capturing the fact that a collection is highly repetitive, and require space basically proportional to the length of one typical sequence plus the total number of edit operations. The new indexes can be plugged into a recent dynamic fully-compressed suffix tree, achieving full functionality for sequence analysis, while retaining the reduced space and the polylogarithmic slowdown. Our experimental results confirm the practicality of our proposal.

Objective: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) causes multiple endocrine deficiencies, oral candidiasis and different forms of ectodermal dystrophy including enamel hypoplasia, documented in permanent teeth. Our purpose was to examine dental aberrations associated with APECED, including possible manifestations in primary teeth., Design: We studied clinically, radiographically, and by scanning electron microscopy (SEM) teeth of children belonging to two APECED families with different mutations in the AIRE gene., Results: In addition to enamel defects in the permanent teeth we observed hypoplastic pits and hypomaturated patches in the deciduous teeth with underlying changes in the prismatic ultrastructure. The enamel of the permanent molars exhibited a layered arrangement with included whirl-like formations., Conclusions: Our findings confirm that APECED causes enamel defects that are individually but chronologically distributed, and can alter enamel development early enough to affect deciduous teeth.

Objective: The objective of this study is to quantitate expression of genes possibly contributing to insulin resistance and fat deposition in the human liver., Research Design and Methods: A total of 24 subjects who had varying amounts of histologically determined fat in the liver ranging from normal (n = 8) to steatosis due to a nonalcoholic fatty liver (NAFL) (n = 16) were studied. The mRNA concentrations of 21 candidate genes associated with fatty acid metabolism, inflammation, and insulin sensitivity were quantitated in liver biopsies using real-time PCR. In addition, the subjects were characterized with respect to body composition and circulating markers of insulin sensitivity., Results: The following genes were significantly upregulated in NAFL: peroxisome proliferator-activated receptor (PPAR) gamma 2 (2.8-fold), the monocyte-attracting chemokine CCL2 (monocyte chemoattractant protein [MCP]-1, 1.8-fold), and four genes associated with fatty acid metabolism (acyl-CoA synthetase long-chain family member 4 [ACSL4] [2.8-fold], fatty acid binding protein [FABP]4 [3.9-fold], FABP5 [2.5-fold], and lipoprotein lipase [LPL] [3.6-fold]). PPARgamma coactivator 1 (PGC1) was significantly lower in subjects with NAFL than in those without. Genes significantly associated with obesity included nine genes: plasminogen activator inhibitor 1, PPARgamma, PPARdelta, MCP-1, CCL3 (macrophage inflammatory protein [MIP]-1 alpha), PPAR gamma 2, carnitine palmitoyltransferase (CPT1A), FABP4, and FABP5. The following parameters were associated with liver fat independent of obesity: serum adiponectin, insulin, C-peptide, and HDL cholesterol concentrations and the mRNA concentrations of MCP-1, MIP-1 alpha, ACSL4, FABP4, FABP5, and LPL., Conclusions: Genes involved in fatty acid partitioning and binding, lipolysis, and monocyte/macrophage recruitment and inflammation are overexpressed in the human fatty liver.

IL-27 is a novel member of the IL-12 cytokine family. IL-27 has pro- and anti-inflammatory properties, and it controls the responses of adaptive immunity. It promotes the differentiation of naïve Th cells and suppresses the effector functions of Th17 cells. Biologically active IL-27 is a heterodimer composed of EBV-induced gene 3 (EBI3) and p28 proteins. We report that TLR-dependent expression of IL-27 in human macrophages is mediated by IFN-alpha. Stimulation of macrophages with agonists for TLR3 {polyinosinic:polycytidylic acid [poly(I:C)]}, TLR4 (LPS), or TLR7/8 (R848) results in concurrent expression of EBI3 and p28. The p28 expression is inhibited with neutralizing anti-IFN-alpha antibodies. Unlike poly(I:C), LPS, and R848, TLR2 agonist (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride does not stimulate macrophages to produce IFN-alpha, and therefore, it is not able to turn on the expression of p28. There is an IFN-stimulated response element (ISRE) in the p28 gene promoter. IFN-alpha enhances the expression of IFN regulatory factor 1 (IRF-1) in macrophages and induces binding of IRF-1 to the p28 ISRE site. The data provide a mechanistic basis for the IFN-alpha-mediated activation of IL-27. The data emphasize a role of IFN-alpha in immune responses, which rely on the recognition of pathogens by TLRs.

Object: Osteogenesis imperfecta (OI), which usually results from mutations in type I collagen genes, causes bone fragility and deformities. The head is often abnormally shaped, and changes in skull base anatomy in the form of basilar impression and basilar invagination have been reported. The authors analyzed the skull base anatomy on standardized lateral cephalograms from 54 patients with OI (Types I, III, and IV) and 108 control volunteers. They were surprised to find that the previously used diagnostic measures for basilar abnormality in patients with OI were exceeded in 6.5 to 7.4% of the controls, and hence needed to be reevaluated., Methods: The authors calculated the distance from the odontoid process to four reference lines, including a novel one, in the controls. The normal mean distances were exceeded by more than two standard deviations (SDs) in 28.3 to 35.2%, and by more than three SDs in 13.2 to 16.6% of the patients with OI. The latter figures reliably reflect the prevalence of basilar impression. As a sign of basilar invagination the odontoid process protruded into the foramen magnum or reached the foramen magnum level in 22.2% of the patients with OI, whereas none of the controls showed this feature. Platybasia (an anterior cranial base angle > 146 degrees) was present in 11.1% of the patients but in none of the controls., Conclusions: Platybasia, basilar impression, and basilar invagination were often coexpressed, but each was also present as an isolated abnormality. These three abnormalities and wormian bones were predominantly found in OI Types III and IV as well as in patients exhibiting dentinal abnormality.

Activation of host cell antiviral responses is mediated by pattern recognition receptors. Cytoplasmic RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (mda-5) have been identified to function as receptors for double-stranded RNA. Here we show that interferon (IFN)-alpha pretreatment enhances influenza A virus-induced expression of IFN-alpha, IFN-beta, interleukin (IL)-28 and IL-29 genes in human dendritic cells and epithelial cell lines. Both IFN-alpha and IFN-beta strongly enhanced RIG-I and mda-5 mRNA and protein expression in these cell types. Expression of RIG-I and mda-5 gene constructs, but not that of TLR3, lead to a dramatic enhancement of IFN-beta promoter driven transcription in influenza A virus-infected epithelial cells. Furthermore, dominant negative RIG-I gene construct inhibited influenza A virus-induced IFN-beta promoter activity. In conclusion, our results show that in epithelial cells influenza A virus-induced antiviral cytokine gene expression is triggered by RIG-I and mda-5, whose expression is positively regulated by IFN-alpha.

Activation of host cell antiviral responses is mediated by receptors detecting the presence of viruses. Here we have studied the role of double-stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and Toll-like receptor 3 (TLR3) in measles virus (MV)-induced expression of antiviral cytokines and chemokines in human A549 lung epithelial cells and human umbilical vein endothelial cells (HUVECs). We show that MV infection results in the activation of mda-5, RIG-I, and TLR3 gene expression that is followed by high expression of interferon (IFN)-beta, interleukin (IL)-28 and IL-29, CCL5, and CXCL10 genes. We also demonstrate that IFN-alpha and IFN-beta upregulate mda-5, RIG-I, and TLR3 gene expression in epithelial and endothelial cell lines. Forced expression of mda-5, but not that of RIG-I or TLR3, leads to enhanced IFN-beta promoter activity in MV-infected A549 cells. Our results suggest that IFN-inducible mda-5 is involved in MV-induced expression of antiviral cytokines.

Macrophages and dendritic cells (DCs) play essential roles in host defence against microbial infections. In the present study, it is shown that human monocyte-derived macrophages and DCs express both type I and type III interferons (IFNs) [IFN-alpha, IFN-beta and interleukin 28 (IL-28), IL-29, respectively], tumour necrosis factor alpha and the chemokines CCL5 and CXCL10 after herpes simplex virus 1 (HSV-1) infection. The cytokine-inducing activity of HSV-1 was dependent on viability of the virus, because UV-inactivated virus did not induce a cytokine response. Pretreatment of the cells with IFN-alpha or IL-29 strongly enhanced the HSV-1-induced cytokine response. Both IFN-alpha and IL-29 decreased viral immediate-early (IE) gene infected-cell protein 27 (ICP27) transcription, suggesting that IL-29 possesses antiviral activity against HSV-1 comparable to that of IFN-alpha. Macrophage infection with HSV-1 lacking functional ICP27 (d27-1 virus) resulted in strongly enhanced cytokine mRNA expression and protein production. In contrast, viruses lacking functional IE genes ICP0 and ICP4 induced cytokine responses comparable to those of the wild-type viruses. The activation of transcription factors IRF-3 and NF-kappaB was strongly augmented when macrophages were infected with the ICP27 mutant virus. Altogether, the results demonstrate that HSV-1 both induces and inhibits the antiviral response in human cells and that the type III IFN IL-29, together with IFN-alpha, amplifies the antiviral response against the virus. It is further identified that viral IE-gene expression interferes with the antiviral response in human macrophages and ICP27 is identified as an important viral protein counteracting the early innate immune response.

Epithelial cells of the lung are the primary targets for respiratory viruses. Virus-carried single-stranded RNA (ssRNA) can activate Toll-like receptors (TLRs) 7 and 8, whereas dsRNA is bound by TLR3 and a cytoplasmic RNA helicase, retinoic acid-inducible protein I (RIG-I). This recognition leads to the activation of host cell cytokine gene expression. Here we have studied the regulation of influenza A and Sendai virus-induced alpha interferon (IFN-alpha), IFN-beta, interleukin-28 (IL-28), and IL-29 gene expression in human lung A549 epithelial cells. Sendai virus infection readily activated the expression of the IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas influenza A virus-induced activation of these genes was mainly dependent on pretreatment of A549 cells with IFN-alpha or tumor necrosis factor alpha (TNF-alpha). IFN-alpha and TNF-alpha induced the expression of the RIG-I, TLR3, MyD88, TRIF, and IRF7 genes, whereas no detectable TLR7 and TLR8 was seen in A549 cells. TNF-alpha also strongly enhanced IKK epsilon mRNA and protein expression. Ectopic expression of a constitutively active form of RIG-I (deltaRIG-I) or IKK epsilon, but not that of TLR3, enhanced the expression of the IFN-beta, IL-28, and IL-29 genes. Furthermore, a dominant-negative form of RIG-I inhibited influenza A virus-induced IFN-beta promoter activity in TNF-alpha-pretreated cells. In conclusion, IFN-alpha and TNF-alpha enhanced the expression of the components of TLR and RIG-I signaling pathways, but RIG-I was identified as the central regulator of influenza A virus-induced expression of antiviral cytokines in human lung epithelial cells.

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.

Facial and calvarial bones form intramembranously without a cartilagenous model; however, cultured chick calvarial mesenchyme cells may differentiate into both osteoblasts and chondroblasts and, in rodents, small cartilages occasionally form at the sutures in vivo. Therefore, we wanted to investigate what factors regulate normal differentiation of calvarial mesenchymal cells directly into osteoblasts. In embryonic mouse heads and in cultured tissue explants, we analyzed the expression of selected transcription factors and extracellular matrix molecules associated with bone and cartilage development. Cartilage markers Sox9 and type II collagen were expressed in all craniofacial cartilages. In addition, Msx2 and type I collagen were expressed in sense capsule cartilages. We also observed that the undifferentiated calvarial mesenchyme and the osteogenic fronts in the jaw expressed Col2A1. Moreover, we found that cultured mouse calvarial mesenchyme could develop into cartilage. Of the 49 explants that contained mesenchyme, intramembranous ossification occurred in 35%. Only cartilage formed in 4%, and both cartilage and bone formed in 4%. Our study confirms that calvarial mesenchyme, which normally gives rise to intramembranous bone, also has chondrogenic potential.

TLRs play a critical role in early innate immune response to virus infection. TLR3 together with TLR7 and TLR8 constitute a powerful system to detect genetic material of RNA viruses. TLR3 has been shown to bind viral dsRNA whereas TLR7 and TLR8 are receptors for viral single-stranded RNA. In this report we show that TLR7 or TLR8 are not expressed in human epithelial A549 cells or in HUVECs. Accordingly, A549 cells and HUVECs were unresponsive to TLR7/8 ligand R848. TLR3 was expressed at a higher level in HUVECs than in A549 cells. The TLR3 ligand poly(I:C) up-regulated IFN-beta, IL-28, IL-29, STAT1, and TLR3 expression in HUVECs but not in A549 cells. An enhanced TLR3 expression by transfection or by IFN-alpha stimulation conferred poly(I:C) responsiveness in A549 cells. Similarly, IFN-alpha pretreatment strongly enhanced poly(I:C)-induced activation of IFN-beta, IL-28, and IL-29 genes also in HUVECs. In conclusion, our results suggest that IFN-alpha-induced up-regulation of TLR3 expression is involved in dsRNA activated antiviral response in human epithelial and endothelial cells.

Osteogenesis imperfecta (OI) is a heterogeneous group of connective tissue diseases that mainly manifest as bone fragility and skeletal deformity. In most families it segregates as a dominant trait and results from mutations in type I collagen genes. In this study we analyzed the size and form of the bony structures in heads of 59 consecutive patients with OI types I, III, or IV (Sillence classification), using lateral radiographs. Paired controls were matched for gender and age. The purpose was to obtain baseline information of craniofacial development in OI patients that have not received bisphosphonate treatment. In OI type I we found smaller than normal linear measurements, indicating a general growth deficiency, but no remarkable craniofacial deformity. In OI types III and IV, the growth impairment was pronounced, and the craniofacial form was altered as a result of differential growth deficiency and bending of the skeletal head structures. We found strong support both for an abnormally ventral position of the sella region due to bending of the cranial base, and for a closing mandibular growth rotation. Vertical underdevelopment of the dentoalveolar structures and the condylar process were identified as the main reasons for the relative mandibular prognathism in OI. Despite of the widespread intervention with bisphosphonates, the facial growth impairment will probably remain characteristic for many OI patients, and their orthodontic treatment should be further developed., ((c) 2005 Wiley-Liss, Inc.)

Toll-like receptors (TLRs) mediate host cell activation by various microbial components. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are the receptors that have been associated with virus-induced immune response. We have previously reported that all these TLRs, except TLR9, are expressed at mRNA levels in human monocyte-derived macrophages. Here we have studied TLR2, TLR3, TLR4, and TLR7/8 ligand-induced IFN-alpha, IFN-beta, IL-28, and IL-29 expression in human macrophages. IFN-alpha pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-alpha, IFN-beta, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-alpha, IFN-beta, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-alpha enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-alpha also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-beta, IkappaB kinase-epsilon, receptor interacting protein 1, and IFN regulatory factor 7. Furthermore, the activation of transcription factor IFN regulatory factor 3 by TLR3 and TLR4 agonists was dependent on IFN-alpha pretreatment. In conclusion, our results suggest that IFN-alpha sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.

NK cells participate in innate immune responses by secreting gamma interferon (IFN-gamma) and by destroying virus-infected cells. Here the interaction between influenza A or Sendai virus-infected macrophages and NK cells has been studied. A rapid, cell-cell contact-dependent production of IFN-gamma from NK cells cultured with virus-infected macrophages was observed. Expression of the MHC class I-related chain B (MICB) gene, a ligand for NK cell-activating receptor NKG2D, was upregulated in virus-infected macrophages suggesting a role for MICB in the activation of the IFN-gamma gene in NK cells. IL12Rbeta2, IL18R and T-bet mRNA synthesis was enhanced in NK cells cultured with virus-infected macrophages. Upregulation of these genes was dependent on macrophage-derived IFN-alpha. In contrast to IL12Rbeta2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-alpha. In conclusion, these results show that virus-infected macrophages activate NK cells via cytokines and direct cellular interactions and further emphasize the role of IFN-alpha in the activation of innate immunity.

Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels. In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL-2 or IL-12 family cytokines was detected. Bacteria-induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated. Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria. Although pathogenic S. pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.

Genetic and molecular studies in humans and mice indicate that Runx2 (Cbfa1) is a critical transcriptional regulator of bone and tooth formation. Heterozygous mutations in Runx2 cause cleidocranial dysplasia (CCD), an inherited disorder in humans and mice characterized by skeletal defects, supernumerary teeth, and delayed eruption. Mice lacking the Runx2 gene die at birth and lack bone and tooth development. Our extended phenotypic studies of Runx2 mutants showed that developing teeth fail to advance beyond the bud stage and that mandibular molar organs were more severely affected than maxillary molar organs. Runx2 (-/-) tooth organs, when transplanted beneath the kidney capsules of nude mice, failed to progress in development. Tooth epithelial-mesenchymal recombinations using Runx2 (+/+) and (-/-) tissues indicate that the defect in mesenchyme cannot be rescued by normal dental epithelium. Finally, our molecular analyses showed differential effects of the absence of Runx2 on tooth extracellular matrix (ECM) gene expression. These data support the hypothesis that Runx2 is one of the key mesenchymal factors that influences tooth morphogenesis and the subsequent differentiation of ameloblasts and odontoblasts.

NK and T cell-derived IFN-gamma is a key cytokine that stimulates innate immune responses and directs adaptive T cell response toward Th1 type. IL-15, IL-18, and IL-21 have significant roles as activators of NK and T cell functions. We have previously shown that IL-15 and IL-21 induce the expression of IFN-gamma, T-bet, IL-12R beta 2, and IL-18R genes both in NK and T cells. Now we have studied the effect of IL-15, IL-18, and IL-21 on IFN-gamma gene expression in more detail in human NK and T cells. IL-15 clearly activated IFN-gamma mRNA expression and protein production in both cell types. IL-18 and IL-21 enhanced IL-15-induced IFN-gamma gene expression. IL-18 or IL-21 alone induced a modest expression of the IFN-gamma gene but a combination of IL-21 and IL-18 efficiently up-regulated IFN-gamma production. We also show that IL-15 activated the binding of STAT1, STAT3, STAT4, and STAT5 to the regulatory sites of the IFN-gamma gene. Similarly, IL-21 induced the binding of STAT1, STAT3, and STAT4 to these elements. IL-15- and IL-21-induced STAT1 and STAT4 activation was verified by immunoprecipitation with anti-phosphotyrosine Abs followed by Western blotting with anti-STAT1 and anti-STAT4 Abs. IL-18 was not able to induce the binding of STATs to IFN-gamma gene regulatory sites. IL-18, however, activated the binding of NF-kappa B to the IFN-gamma promoter NF-kappa B site. Our results suggest that both IL-15 and IL-21 have an important role in activating the NK cell-associated innate immune response.

Background: The management of gastrointestinal-cutaneous fistulas may be complicated by the difficulty in obtaining adequate control of the fistula tract. This study describes a new method to obtain better fistula control utilizing a semi-rigid stent in the form of a gastrostomy tube., Methods: Consecutive patients with intestinal-cutaneous fistulas of at least 3 weeks duration and treated by the new technique were analyzed. The technique involved the insertion of a guide wire into the fistula tract from the luminal side using an endoscope, snaring the wire with a Dormia basket inserted into the fistula tract from the cutaneous side and then exteriorized. The gastrostomy tube was then pulled with the guide wire from the lumen along the fistula tract and out through the skin., Results: Five patients had had fistulas for a median duration of 42 (range 26-140) days before insertion of the gastrostomy tube. The gastrostomy tube was replaced with a smaller diameter tube in 4 of the patients (range 1-3 changes). The patients were discharged from the hospital at a median of 14 (range 12-23) days after the tube insertion but with the tube in situ. The median time from the insertion of the tube to its removal was 42 (range 32-108) days., Conclusions: Gastrostomy tube insertion using minimally invasive techniques may improve fistula control enabling patients to be discharged home sooner than otherwise and improve the rate of healing., (Copyright 2003 S. Karger AG, Basel)

Laparoscopic adrenalectomy is gaining widespread acceptance. To evaluate this new approach, the authors evaluated 40 laparoscopic adrenalectomies. Between June 1995 and February 1999, 40 lateral transperitoneal laparoscopic adrenalectomies were performed in 38 patients. The clinical diagnoses were primary aldosteronism (20 patients), Cushing adenoma (2 patients), cortical hyperplasia with hypercortisolism (2 patients), pheochromocytoma (8 patients), and other conditions (6 patients). There were no deaths or subsequent procedures. The mean operative time was 121 minutes. One procedure performed for hypercortisolism was converted to open adrenalectomy because of hepatomegaly and postoperative adhesions. Seven patients had complications: one patient with small pulmonary embolus with transient dyspnea, one patient with pneumothorax, two patients with postoperative bleeding, two patients with prolonged pain at a trocar wound, and one patient with a urinary tract infection. Lateral transperitoneal laparoscopic adrenalectomy seems to be a safe and effective minimally invasive approach for adrenal surgery, and the authors consider it to be the standard surgical procedure for benign adrenal tumors.

Most adrenal incidentalomas are nonfunctioning cortical adenomas that do not require surgery. Operative treatment is indicated if the incidentaloma has malignant features as seen during the radiologic workup, is hormonally active, or grows during follow-up. A conservative attitude is justified in asymptomatic patients if we know that the natural course of these tumors is not disadvantageous. We followed a group of patients treated by observation to determine the natural course of adrenal incidentalomas. Altogether 30 incidentalomas in 27 patients were detected in the Helsinki University Central Hospital from June 1981 through December 1992 and were re-examined during 1997. The patients were evaluated clinically, and hormonal testing was done by performing a 1-mg overnight dexamethasone suppression test; the next day we measured the 24-hour urinary excretion of vanillylmandelic acid, metanephrines and normetanephrines, and serum potassium. Magnetic resonance imaging (MRI) was used to determine the size of the tumor. If the patient had died during the follow-up period, the causes of death from the death certificates and autopsy reports were reviewed. The mean follow-up was 7 years. Nine patients had died, with none of deaths related to the incidentaloma. Sixteen patients were evaluated clinically and by the basic hormonal tests and MRI; none showed any signs of hormonal activity or features of malignancy. Two patients did not want to be reexamined but stated that they were asymptomatic. This follow-up study supports the conclusion that conservative therapy is justified because the growth tendency of adrenal incidentalomas is slow and they seem to remain hormonally inactive.

This case report describes superior mesenteric and portal vein thrombosis after laparoscopic Nissen fundoplication. As a thromboembolic prophylaxis, 2,500 IU of dalteparin was given preoperatively. After postoperative day 19, the patient experienced gradually increasing abdominal pain, mostly related to meals. Physical examination and laboratory tests were normal. CT scan revealed a portal and superior mesenteric vein thrombosis. Dalteparin and warfarin treatment was started, and symptoms relieved rapidly. In a control Doppler ultrasound 1 month after the onset of the treatment, a good flow in the portal and superior mesenteric vein was seen. Possible mechanisms are discussed., (Copyright 2000 S. Karger AG, Basel)

Laparoscopic adrenalectomy has become a viable option for removal of small adrenal neoplasms. We present our initial experience of this operation for primary aldosteronism. In this prospective study, 12 consecutive patients with primary aldosteronism were operated on laparoscopically by one surgeon. Operative time, blood loss, postoperative pain, complications, hospital stay, convalescence time, and outcome were analyzed. Five right-sided and seven left-sided adrenalectomies were performed in six female and six male patients with a mean age of 51 years. The mean operative time was 126 minutes. All procedures were successfully carried out laparoscopically. No major complications occurred. All patients turned normokalemic and the medication for hypertension could be stopped or diminished in all cases. The mean hospital stay was 3.4 days, and the mean sick leave was 13 days. Laparoscopic adrenalectomy seems to be a safe and effective treatment for primary aldosteronism and should be considered the operation of choice.

EEG has been used widely in diagnosing encephalitis, as it demonstrates rather typical abnormalities, especially in herpes simplex virus encephalitis (HSVE). We analysed 204 EEG recordings from 98 consecutive acyclovir-treated patients with acute encephalitis between 1984 and 1994. Periodic complexes (PC) in the acute phase predicted poor outcome (Kendall tau 0.40, P < 0.001). However, unlike in many other diseases, e.g. stroke and intracerebral haemorrhage, the diffuse slowing of the background activity at acute phase did not predict outcome (Kendall tau -0.6, P = 0.35). At follow-up, the emergence of diffuse slow background activity was significantly associated with a less favourable outcome (Kendall tau 0.33, P = 0.0016). Among clinical variables, only epileptic seizures early during the course of the disease correlated significantly with outcome. EEG does have value as a prognostic indicator in acute encephalitides, but it seems that diffuse slowing of background activity or irritative features acutely are not as important as previously thought, based on the experiences of the pre-acyclovir era.

Primary aldosteronism consists of a mixture of subgroups. The operative treatment is successful only in cases of aldosterone-producing neoplasia (and in rare cases of primary unilateral hyperplasia); all other cases should be treated medically. The aim of this study was to determine if aldosterone-producing neoplasia had been successfully differentiated from the other subgroups and the outcome of operative treatment. Altogether 29 patients with primary aldosteronism were operated on between January 1, 1979 and December 31, 1993. Patient charts were reviewed retrospectively. The follow-up data were collected from the patients' charts, and all patients were contacted to obtain recent blood pressure and serum potassium values. The patients were asked about symptoms related to hyperaldosteronism. If any suspicion of recidive aldosteronism was present, patients were carefully reexamined by hormonal tests and computed tomography (CT). A total of 27 patients had unilateral adenoma, 1 patient had hyperplasia, and 1 patient had an aldosterone-producing cortical carcinoma. There was no operative mortality or morbidity. The serum potassium level had normalized in all patients. Mean follow-up time was 76 months. One patient died during the follow-up from cholangiocarcinoma; 11 patients (41%) were cured by the operation, 10 patients (37%) have a mild but medicated hypertension, and in the remaining 22% the hypertension persisted but was well controlled by the medication. Of the 29 patients, 28 were correctly diagnosed as having an aldosterone-producing neoplasm. Basic hormonal studies and CT can be used effectively to differentiate aldosterone-producing neoplasia from hyperplasia in most cases.

Cerebrospinal fluid (CSF) amino acid neurotransmitter concentrations in 23 patients with acute encephalitis were compared with those in patients with acute brain infarction, multiple sclerosis and controls. The concentration of glutamate was significantly higher in encephalitis (5.2+/-6.7 micromol/l) and stroke patients (9.6+/-14.2 micromol/l) than in MS patients (1.6+/-0.9 micromol/l) and controls (1.7+/-0.8 micromol/l; p < 0.001). The concentration of glycine was significantly higher in encephalitis (11.0+/-4.7 micromol/l) than in stroke (7.6+/-3.2 micromol/l) and MS patients (6.3+/-2.1 micromol/l) or controls (5.6+/-1.8 micromol/l; p < 0.002). Taurine levels were significantly lower in encephalitis patients than in the other groups (p = 0.04). The correlation of high glutamate levels with poor outcome was almost significant (Kendall tau 0.63, p = 0.06). Our observations suggest that exicitotoxic neurotransmission may play an important role in the series of events that lead to neuronal damage in encephalitis.

Background: Laparoscopic cholecystectomy (LC) has become the treatment of choice for elective cholecystectomy, but controversy persists over use of this approach in the treatment of acute cholecystitis. We undertook a randomised comparison of the safety and outcome of LC and open cholecystectomy (OC) in patients with acute cholecystitis., Methods: 63 of 68 consecutive patients who met criteria for acute cholecystitis were randomly assigned OC (31 patients) or LC (32 patients). The primary endpoints were hospital mortality and morbidity, length of hospital stay, and length of sick leave from work. Analysis was by intention to treat. Suspected bile-duct stones were investigated by preoperative endoscopic retrograde cholangiography (LC group) or intraoperative cholangiography (OC group)., Findings: The two randomised groups were similar in demographic, physical, and clinical characteristics. 48% of the patients in the OC group and 59% in the LC group were older than 60 years. 13 patients in each group had gangrene or empyema, and one in each group had perforation of the gallbladder causing diffuse peritonitis. Five (16%) patients in the LC group required conversion to OC, in most because severe inflammation distorted the anatomy of Calot's triangle. There were no deaths or bile-duct lesions in either group, but the postoperative complication rate was significantly (p=0.0048) higher in the OC than in the LC group: seven (23%) patients had major and six (19%) minor complications after OC, whereas only one (3%) minor complication occurred after LC. The postoperative hospital stay was significantly shorter in the LC than the OC group (median 4 [IQR 2-5] vs 6 [5-8] days; p=0.0063). Mean length of sick leave was shorter in the LC group (13.9 vs 30.1 days; 95% CI for difference 10.9-21.7)., Interpretation: Even though LC for acute and gangrenous cholecystitis is technically demanding, in experienced hands it is safe and effective. It does not increase the mortality rate, and the morbidity rate seems to be even lower than that in OC. However, a moderately high conversion rate must be accepted.

Three cases of intrahepatic biliary cystadenoma with mesenchymal stroma and one case of biliary cystadenocarcinoma are presented. Their immunohistochemical features and the surgical treatment are discussed together with a brief review of the literature. The benign cystadenomas stained positive for cytokeratin and CA 19-9 in the epithelium of the cyst wall. Mesenchymal stromal cells were strongly positive for a-SMA and moderately positive for desmin. The epithelium of the cystadenocarcinoma, however, was positive only for cytokeratin and the stroma only for a-SMA. Our findings indicate that biliary cystadenomas seem to be of primitive hepatobiliary origin. Furthermore, the malignant variant cystadenocarcinoma may loose its immunoreactivity for CA 19-9 and desmin.

We studied 88 patients with acute encephalitis using hexamethylpropyleneamine oxime and single photon emission computed tomography (SPECT). All patients had been initially treated with intravenous acyclovir. The etiology could be disclosed in 37 patients (42%), which included 15 patients with herpes simplex encephalitis, 7 with varicella-zoster encephalitis, and 29 with other encephalitides (Mycoplasma, adenovirus, influenza, rotavirus, rubella, Epstein-Barr, arbovirus, syphilis, and tuberculosis). Unilateral hyperperfusion in SPECT was an independent predictor of poor prognosis, whereas neither clinical outcome variables, such as seizures, state of consciousness, and focal neurologic findings, nor CSF or EEG findings were not. Focal unilateral hyperperfusion is an indicator of severe inflammation of the brain tissue and predicts a poor outcome as assessed in terms of activities of daily living after recovery.

Nine immunocompetent patients with acute herpes zoster encephalitis (HZE) were studied with the help of neurological investigations. All patients were treated with acyclovir. Neuropsychological performance was compared with that of a group of 16 healthy controls. Computed tomography of the head showed infarct-like hypodense lesions in two patients, involving the internal capsule in one case and the temporoparietal cortex and white matter in another. Hypoperfusion shown by single photon emission computed tomography, mostly involving the frontal areas bilaterally, was seen in six of the seven patients examined. Hyperperfusion as seen in herpes simplex encephalitis was not encountered. One patient remained mildly demented, but all the other patients recovered relatively well. Neuropsychological examination after acyclovir treatment showed a decline in memory and speed of cognitive processes, without circumscribed neuropsychological deficits. Six of the nine patients showed behavioural disinhibition, and mood changes were also observed. Memory impairment in HZE was not as global or as severe as is described after encephalitis due to herpes simplex virus. In HZE both the brain perfusion pattern and the neuropsychological test profile showed features compatible with subcortical dysfunction.

We studied two patients with herpes encephalitis (HSE) by [99mTc]HMPAO and [123I]iomazenil single photon emission computed tomography. Increased uptake of HMPAO was seen for up to 63 days in the HSE affected brain area. Iomazenil binds to benzodiazepine receptors and can measure neurone loss. Decreased iomazenil uptake was observed a few days after onset, at a time when hyperfixation of HMPAO occurred. Because in HSE neurone loss occurs simultaneously with hyperfixation of HMPAO, it is unlikely that this hyperfixation is caused by increased neuronal activity, as in epilepsy. This suggests that the hyperfixation of HMPAO in HSE occurs in glia and is sustained by inflammation-related hypermetabolism and acidity. The early neurone loss in HSE stresses the importance of immediate antiviral treatment.

Incidental discovery of an adrenal mass during radiologic examinations is common. Several recommendations have been made for the management of so-called incidentalomas. It has become clear that not all incidentalomas should be operated, but the criteria for nonoperative treatment have been under continuous debate. In this study 36 operated incidentalomas are presented, and the indications for operative treatment are discussed with a review of the recent literature on the subject. Four pheochromocytomas and three hormonally active cortical adenomas, two producing cortisol and one androgens, were found. In this series there were no malignancies. The operation could have been avoided in most cases, and patients could have been followed up with repeated radiologic examinations. It is suggested that masses smaller than 6 cm in diameter be followed radiologically after 3, 9, and 18 months. Masses between 3 and 6 cm could be further examined using magnetic resonance imaging and fine needle aspiration and then operated if features suggestive of malignancy are found. Masses larger than 6 cm in diameter should be treated operatively.