Capsule and fimbriae modulate the invasion of Haemophilus influenzae in a human blood-cerebrospinal fluid barrier model

The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral “blood “side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.     

Minor genomic differences between related B6 and B10 mice affect severity of schistosome infection by governing the mode of dendritic cell activation

Infection with the helminth Schistosoma mansoni results in hepatointestinal granulomatous inflammation mediated by CD4 T cells directed against parasite eggs. The severity of disease varies greatly in humans and mice; however, the genetic basis of such a heterogenous immune response remains poorly understood.   Here we show that, despite their close genetic relationship, C57BL/10SnJ (B10) mice developed significantly more pronounced immunopathology and higher T helper 17 cell responses than C57BL/6J (B6) mice. Similarly, live egg‐stimulated B10‐derived dendritic cells (DCs) produced significantly more IL‐1β and IL‐23, resulting in higher IL‐17 production by CD4 T cells.  Gene expression analysis disclosed a heightened proinflammatory cytokine profile together with a strikingly lower expression of Ym1 in B10 versus B6 mice, consistent with failure of B10 DCs to attain alternative activation. To genetically dissect the differential response, we developed and analyzed congenic mouse strains that capture major regions of allelic variation, and found that the level of inflammation was controlled by a relatively small number of genes in a locus mapping to chromosome 4 117–143 MB. Our study has thus identified novel genomic regions that regulate the severity of the schistosome infection by way of controlling the mode of DC activation and consequent CD4 T‐cell subset development.     

Identification of genomic differences between Escherichia coli strains pathogenic for poultry and E. coli K-12 MG1655 using suppression subtractive hybridization analysis

Diseases of poultry caused by Escherichia coli result in significant economic loss every year. Specific virulence factors associated with E. coli strains pathogenic for poultry have been identified, but it is likely that others remain to be identified. To identify unique DNA fragments associated with avian strains we used suppression subtractive hybridization. The genome of E. coli K-12 strain MG1655 was subtracted from the genomes of two avian E. coli strains resulting in the identification of 62 fragments specific to the two avian strains. Sequence homology analysis was done and four types of fragments were identified: plasmid sequences, phage sequences, sequences with known function and sequences without any currently known function. Two E. coli collections, a reference collection of diverse strains (ECOR) and a collection of 41 avian isolates, were screened for the presence of 25 of the 62 fragments. We identified nine fragments present in significantly more of the avian strains than of the ECOR strains. Five fragments were in significantly more of the ECOR strains than the avian strains. These results suggested that the nine fragments could play a role in the pathogenesis of E. coli as it relates to diseases of poultry.     

Susceptibility to human cancer: From the perspective of a pathologist

The etiologies of human cancer can only be discerned when the genetic clustering of cancer occurs within a family or when cancer occurs endemically in a particular environment. The possible approaches to solving the nature/nurture problem, especially for human carcinogenesis, posit a fascinating challenge for pathologists. This perspective review presents some examples of how clues to human cancer etiologies and/or susceptibilities reside in the realm of pathology practice. These examples using various omics techniques including adductomics, which I would like to highlight in this article, show that the currently available concepts and methods in human pathology can open a path toward the brave new world of a post‐genomic era of medicine for young pathologists, whether their original intention was toward the pursuit of diagnostic or investigative knowledge.     

Escherichia coli O157: Insights into the adaptive stress physiology and the influence of stressors on epidemiology and ecology of this human pathogen

Abstract

Escherichia coli O157, a foodborne pathogen of major concern for public health, has been associated with numerous outbreaks of haemorrhagic colitis and hemolytic uremic syndrome worldwide. Human infection with E. coli O157 has been primarily associated with the food-chain transmission route. This transmission route commonly elicits a multi-faceted adaptive stress response of E. coli O157 for an extended period of time prior to human infection. Several recent research articles have indicated that E. coli O157:H7 has evolved unique survival characteristics which can affect the epidemiology and ecology of this zoonotic pathogen. This review article summarizes the recent knowledge of the molecular responses of E. coli O157 to the most common stressors found within the human food chain, and further emphasizes the influence of these stressors on the epidemiology and ecology of E. coli O157.     

Identification of antigenic differences between the phosphorylated and nonphosphorylated forms of the E7 protein of human papillomavirus type 16

To analyze the antigenic properties of the human papillomavirus type 16 E7 oncoprotein, two monoclonal antibodies, VD6 and IB10, that have different reactivities to the E7 protein were generated. While the VD6 antibody reacted strongly with E7 protein in CaSki cell extracts, the other antibody, IB10, showed much weaker reactivity with E7. This reactivity increased in a dose-dependent manner in the presence of the casein kinase II-specific inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). Antigenic site estimation and an in vitro phosphorylation assay, using bacterially expressed E7 protein, demonstrated that the weak reactivity of IB10 was related to the phosphorylation status of the E7 protein. Phosphorylation of E7 reduced considerably the reactivity of IB10 but did not affect the reactivity of VD6, which reacts with the N-terminal portion of E7. In immunoprecipitation (IP) assays, IB10 precipitated weakly the E7 protein from CaSki cell extracts. Together, these data suggest that unphosphorylated E7 protein shows distinct antigenic character compared to its phosphorylated form under denaturing conditions; however, under native conditions, the phosphorylated and nonphosphorylated E7 proteins have some antigenic cross-reactivity. J. Med. Virol. 54:129–134, 1998. © 1998 Wiley-Liss,Inc.     

Association between polymorphisms of the cytokine and cytokine receptor genes and immune response to hepatitis B vaccination in a Chinese Han population

The immune response to hepatitis B vaccination varies among individuals. It has been reported that polymorphisms in cytokine and cytokine receptor genes are associated with these individual differences. The aim of the current study was to investigate the association between polymorphisms of the Th1/Th2 cytokine and cytokine receptor genes and the response to hepatitis B vaccination in a Chinese Han population. A total of 10 single nucleotide polymorphisms distributed in 6 genes (TNFRSF1A, IL12A, IL12B, IFNG, IL4, and IL10) were genotyped in 214 high‐responders [hepatitis B surface antibody (anti‐HBs) ≥1,000 mIU/ml] and 107 low‐responders (anti‐HBs: 10–99 mIU/ml). The minor CTCTAA allele of rs17860508 in the IL12B gene was associated with a low response to hepatitis B vaccination (P = 0.039, odds ratio = 1.41, 95% confidence interval = 1.00–1.99). In addition, a significant gene–gene interaction was found: the frequency of the combined genotypes IL12A rs2243115 TT and IL12B rs17860508 CTCTAA/CTCTAA was significantly higher in the low‐response group than in the high‐response group (P = 0.008, odds ratio = 2.19, 95% confidence interval = 1.23–3.93). These findings suggest that polymorphisms in the IL12A and IL12B genes might play an important role jointly in determining the response to hepatitis B vaccination. J. Med. Virol. 84:26–33, 2011. © 2011 Wiley Periodicals, Inc.     

Coping with genetic diversity: the contribution of pathogen and human genomics to modern vaccinology

Vaccine development faces major difficulties partly because of genetic variation in both infectious organisms and humans. This causes antigenic variation in infectious agents and a high interindividual variability in the human response to the vaccine. The exponential growth of genome sequence information has induced a shift from conventional culture-based to genome-based vaccinology, and allows the tackling of challenges in vaccine development due to pathogen genetic variability. Additionally, recent advances in immunogenetics and genomics should help in the understanding of the influence of genetic factors on the interindividual and interpopulation variations in immune responses to vaccines, and could be useful for developing new vaccine strategies. Accumulating results provide evidence for the existence of a number of genes involved in protective immune responses that are induced either by natural infections or vaccines. Variation in immune responses could be viewed as the result of a perturbation of gene networks; this should help in understanding how a particular polymorphism or a combination thereof could affect protective immune responses. Here we will present: i) the first genome-based vaccines that served as proof of concept, and that provided new critical insights into vaccine development strategies; ii) an overview of genetic predisposition in infectious diseases and genetic control in responses to vaccines; iii) population genetic differences that are a rationale behind group-targeted vaccines; iv) an outlook for genetic control in infectious diseases, with special emphasis on the concept of molecular networks that will provide a structure to the huge amount of genomic data.     

Tuft-to-capsule adhesions and their precursors: differences between the vascular and tubular poles of the human glomerulus

Human glomerular capillary tufts were removed by microdissection and scanning electron microscopy was used to examine the surface of the capillary tuft and the interior of its Bowman's capsule in order to identify connections between the tuft and capsule. Glomeruli were examined in histologically normal renal cortex from 12 kidneys removed for tumour and 12 renal allografts removed for end-stage rejection. In normal kidney, the glomerular tuft was connected to Bowman's capsule by single podocytes and their processes. At the vascular pole, these were predominantly associated with parietal podocytes which lined Bowman's capsule. At the tubular pole, occasional podocytic processes derived from the capillary tuft bridged Bowman's space and connected to Bowman's capsule where there were no parietal podocytes. These podocytic connections were also found in all rejected transplants, but in addition adhesions were identified which consisted of thicker connections between the tuft and capsule. At the vascular pole, tuft-to-capsule adhesions were found in all 12 kidneys; these were always associated with parietal podocytes. Tubular pole adhesions were identified in ten of the 12 transplants. They were associated with abnormal squamous cells, but not with parietal podocytes. When the capillary tuft herniated into the proximal tubule, the tuft sometimes formed an adhesion with the origin of the proximal tubule. These observations suggest that podocyte connections between the glomerular tuft and Bowman's capsule may be precursors of glomerular adhesions at the vascular pole. Since tuft-to-capsule adhesions at the vascular pole differ morphologically from those at the tubular pole, this may reflect different pathogenetic mechanisms at the opposite poles of the glomerulus. © 1998 John Wiley & Sons, Ltd.     

Murine thymocytes with ability to inhibit Il-2 production: I. Genetic differences between mouse strains and characterization of the model system.

An experimental system has been established to understand the poor interleukin-2 (Il-2) production by activated thymocytes. This model system is further characterized here and studies were done on the possible mechanism(s) involved. Thymocytes activated by Concanavalin-A (Con-A), or through the CD3 complex of the T-cell receptor (TCR), inhibit 95-99% of the Il-2 production by spleen cells, while thymocytes stimulated by rIl-2 or lipopolysaccharides (LPS) do not. The mechanism of inhibition is not due to production of soluble factors, consumption of available interleukin-1 (Il-1) or Il-2, but is dependent on cell-to-cell contact. Although cellular contact is needed, cytotoxicity is not involved. Prostaglandin production is not required for the generation or exertion of the inhibitory activity. Protein and DNA synthesis are necessary for exertion of the suppressive effect. We also demonstrate a genetic difference between different mouse strains in the ability to generate the inhibitory thymocytes. Activated Balb/c thymocytes inhibit spleen cells' Il-2 production in a non-MHC-restricted manner. Our studies demonstrate a regulatory capacity of activated thymocytes in vitro. This ability of the postnatal cells could be of relevance for understanding the latter events in T-cell education in the thymus and the role of Il-2 during this process.     

Identification of three genomic haplotypes 5' to the human CD1D gene and their distribution in four ethnic groups

CD1d presents lipid antigen to a conserved population of natural killer (NK) T cells, which participate in host immune defense, tumor cell rejection and suppression of autoimmunity. The levels of human CD1d expression vary significantly between individuals. To understand such variation, we sequenced the region up to 1.7 kb 5' upstream of the translation start site and partially through exon 2 in 44 white Americans. We also studied two tagged single nucleotide polymorphisms (SNP) in 112 white Americans, 60 African-Americans, 88 Europeans, and 84 Chinese people from the region. Six SNP present in the region (-836C-->T, -773C-->T, -764C-->G, -713A-->T, -365A-->G and +363A-->G) were found to be in a complete linkage disequilibrium and comprised three haplotypes. Haplotype 1 had -836C, -773C, -764C, -713A, -365A and +363A. Haplotype 2 had -836C, -773T, -764C, -713A, -365A and +363A. Haplotype 3 had -836T, -773C, -764G, -713T, -365G and +363G. -773C-->T and -764C-->G can serve as the tagged SNP to differentiate the three haplotypes. The frequency of haplotype 1 was significantly higher in African Americans than in the other three ethnic groups, whereas the frequency of haplotype 3 was significantly higher in the Chinese people than those in the other three groups. The finding of the three haplotypes provides a genetic marker for CD1d and facilitates the study of the functional role of the genetic variations in human CD1d expression and regulation.     

Temporal association between food distribution and human caregiver presence and the development of affinity to humans in lambs

The presence of the caregiver around feeding favors the development of a human-animal relationship. To understand the underlying mechanism, we tested various temporal associations between food distribution and human presence: from an early age, a person was repeatedly present for 2 min just before milk distribution ("Forward"), during milk distribution ("Simultaneous"), and 20 min afterwards ("Delayed"). The "Control" group received no human contacts. During the treatments, "Forward" and "Delayed" lambs had more physical contacts with the person than "Simultaneous" lambs. When tested in unfamiliar environments, they stood longer near the person than did "Control" or "Simultaneous" lambs, which did not differ. Only "Forward" and "Delayed" lambs bleated when separated from the person. Fasting before testing had no effect. "Forward" and "Delayed" seemed to produce the same human-animal relationship, showing that this did not rely only on a conditioning process associating the caregiver with food. The caregiver may acquire properties for social support through other mechanisms (attachment and/or postingestive effects).     

From support to boundary: A qualitative study of the border between self-care and professional care

Objective

While healthcare systems globally are promoting self-care, patients’ perceptions of the policy shift remain relatively unexplored. Our qualitative study explored how organisation of care shaped patients’ perceptions and experiences of self-care in dealing with their respiratory illnesses.

Methods

We recruited 31 people, representing a range of ages and respiratory conditions and generated data using illness diaries, telephone interviews and focus group discussions. Data were transcribed, analysed and triangulated using the framework approach.

Results

Patients were aware of the increasing focus on self-care, but felt that the term was incongruous as it described what they were already doing. While many respondents appreciated increased clinical responsibility some felt ‘abandoned’ by professionals. To be active partners in care, they needed flexible access to trusted healthcare professionals who respected patients’ knowledge about their condition and preferences for management.

Conclusion

The shift to self-care needs to be underpinned by a whole system change. Professionals need communication skills to engage with patients as partners in care, working within systems that offer flexible access to supportive care.

Practice implications

Systems of care for people with long-term conditions need to ensure flexible access between self-care and professional support. Simplification of systems, clear sign-posting and co-ordination of individual patient care by a key trusted professional are essential.     

Genetically determined differences in noradrenergic input to the brain cortex: a histochemical and biochemical study in two inbred strains of mice.

The C57 BL/6 and Balb/c strains of mice are characterized by their opposite behavioral responses in several tests. Since several central catecholaminergic systems could be involved, we have analyzed particularly the noradrenergic input to the frontal, parietal, hippocampal and cerebellar cortex in the two strains, using three different approaches. The fluorescence histochemical study revealed the presence of more numerous noradrenergic terminals in all the areas examined (except in the deep layers of the parietal cortex) and a higher number of catecholaminergic cell bodies in the locus coeruleus of the C57 BL/6 mice. Higher levels of norepinephrine were also found in the same cortical areas, except the parietal cortex. However, as indicated by the estimations of the activity of the isoproterenol-sensitive adenylate cyclase and the specific binding of [³H]dihydroalprenolol, there was no difference in the number and the affinity of β-adrenergic receptors in the cerebral cortex of the two strains. The higher density of the cortical noradrenergic innervation observed in the C57 BL/6 mice, which persisted in older animals, seems to be genetically determined.The marked difference in the extent of the dorsal noradrenergic pathway seen between the C57 BL/6 and Balb/c strains could thus make them a valuable model to further define the functional role of the dorsal noradrenergic system.     

From Whole Gene Deletion to Point Mutations of EP300‐Positive Rubinstein–Taybi Patients: New Insights into the Mutational Spectrum and Peculiar Clinical Hallmarks

Rubinstein–Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by growth deficiency, skeletal abnormalities, dysmorphic features, and intellectual disability. Causative mutations in CREBBP and EP300 genes have been identified in ～55% and ～8% of affected individuals. To date, only 28 EP300 alterations in 29 RSTS clinically described patients have been reported. EP300 analysis of 22 CREBBP‐negative RSTS patients from our cohort led us to identify six novel mutations: a 376‐kb deletion depleting EP300 gene; an exons 17–19 deletion (c.(3141+1_3142‐1)_(3590+1_3591‐1)del/p.(Ile1047Serfs*30)); two stop mutations, (c.3829A>T/p.(Lys1277*) and c.4585C>T/p.(Arg1529*)); a splicing mutation (c.1878‐12A>G/p.(Ala627Glnfs*11)), and a duplication (c.4640dupA/p.(Asn1547Lysfs*3)). All EP300‐mutated individuals show a mild RSTS phenotype and peculiar findings including maternal gestosis, skin manifestation, especially nevi or keloids, back malformations, and a behavior predisposing to anxiety. Furthermore, the patient carrying the complete EP300 deletion does not show a markedly severe clinical picture, even if a more composite phenotype was noticed. By characterizing six novel EP300‐mutated patients, this study provides further insights into the EP300‐specific clinical presentation and expands the mutational repertoire including the first case of a whole gene deletion. These new data will enhance EP300‐mutated cases identification highlighting distinctive features and will improve the clinical practice allowing a better genotype–phenotype correlation.     

Insomnia related to postmenopausal syndrome and hormone replacement therapy: sleep laboratory studies on baseline differences between patients and controls and double-blind,placebo-controlled investigations on the effects of a novel estrogen-progestogen combination (Climodien,Lafamme) versus estrogen alone

Differences in sleep and awakening quality between 51 insomniac postmenopausal syndrome patients and normal controls were evaluated. In a subsequent double-blind, placebo-controlled, comparative, randomized, three-arm trial (Climodien 2/3 = estradiol valerate 2 mg + the progestogen dienogest 3 mg = regimen A, estradiol valerate 2 mg = regimen EV, and placebo = regimen P), the effects of 2 months of hormone replacement therapy were investigated, followed by a 2-month open-label phase in which all patients received Climodien 2/2 (EV 2 mg + dienogest 2 mg = regimen A*). Polysomnography at baseline demonstrated significantly deteriorated sleep initiation and maintenance, increased S1 and decreased S2 in patients. Subjective sleep and awakening quality, well-being, morning drive, wakefulness, memory and reaction time performance were deteriorated too. Treatment with both regimen A and regimen EV induced a moderate, although nonsignificant, improvement in the primary efficacy variable wakefulness during the total sleep period compared with baseline, while under placebo no changes occurred. Secondary efficacy variables concerning sleep initiation and maintenance, and sleep architecture showed similar findings. The apnea and apnea-hypopnea indices improved significantly under regimen A, compared with both baseline and placebo. Subjective sleep and awakening quality improved significantly after regimen A and EV compared with baseline, with the drug-induced changes being superior to those induced by placebo. In the open-label phase, subjective sleep quality improved further, significantly in the former regimen A group. Awakening quality, somatic complaints and morning thymopsyche did not yield any significant findings. Concerning morning noopsychic performance, memory improved significantly after regimen A compared with baseline, fine motor activity after regimen EV. Reaction time performance increased with all three compounds. In conclusion, Climodien significantly improved subjective sleep quality, the apnea and apnea-hypopnea indices of insomniac postmenopausal syndrome patients, while it only marginally improved variables concerning objective sleep and awakening quality.     

Venous drainage of the dorsal sector of the liver: differences between segments I and IX A study on corrrosion casts of the human liver

Summary The aim of this study was to determine the venous drainage of the dorsal sector of the liver in order to define the differences between segments I and IX and thier implications for sectorially and segmentally oriented hepatic surgery. The study was based on corrosion casts of 61 macroscopically healthy livers. The drainage pathways of veins at least 10 mm long and 1 mm wide were evaluated and statistically analysed. On average, 9 veins drained the two segments and three veins from both segments entered the inferior vena cava. In 95% of cases the veins from segment I drained predominantly into the inferior vena cava, whereas in segment IX this pathway was dominant in only 30% of cases. In 64% of cases a vein originating in segment IX entered the right hepatic v. The difference in the venous drainage of the two segments suggests that segment IX partly belongs to the neighbouring segments and may thus be only a paracaval region of the right liver.

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Drainage veineux du secteur dorsal du foie. Étude comparative des deux segments I et IX sur des foies humains injectés et corrodés

Résumé L'objet de cette étude concerne le drainage veineux du secteur dorsal du foie et plus précisément les segments I et IX avec des implications sur la chirurgie hépatique sectorielle et segmentaire. L'étude repose sur 61 injections — corrosions de foies non pathologiques prélevés selon les règles éthiques et légales. Les voies de drainage veineux d'un calibre d'1 mm et de 10 mm de longueur sont étudiées de façon statistique. Les deux segments sont drainés par 9 veines en moyenne, dont 3 gagnent directement la veine cave inférieure. Dans 95% des cas, les veines des segments I se drainent préférentiellement vers la veine cave inférieure, contre 30% pour les veines du segment IX. Dans 64% des cas, une veine du segment IX gagne la veine hépatique droite. Les différences de drainage entre les segments I et IX laissent entendre que le segment IX appartient au moins partiellement au segment voisin et de ce fait représente la partie latéro-cave du foie droit.