Constitutive interaction of the P2Y2 receptor with the hematopoietic cell-specific G protein G(alpha16) and evidence for receptor oligomers

Hematopoietic cells uniquely express G(alpha16), a G protein alpha-subunit of the G(q)-type. G(alpha16) is obligatory for P2Y2 receptor-dependent Ca2+-mobilization in human erythroleukemia cells and induces hematopoietic cell differentiation. We tested whether P2Y2 receptors physically interact with G(alpha16). Receptor and G protein were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein (GFP), respectively. When expressed in K562 leukemia cells, the fusion proteins were capable of triggering a Ca2+-signal upon receptor stimulation, demonstrating their functional integrity. In fluorescence resonance energy transfer (FRET) measurements using confocal microscopy, a strong FRET signal from the plasma membrane region of fixed, resting cells was detected when the receptor was co-expressed with the G protein as the FRET acceptor, as well as when the CFP-tagged receptor was co-expressed with receptor fused to YFP. We conclude that, under resting conditions, G(alpha16) and P2Y2 receptors form constitutive complexes, and that the P2Y2 receptor is present as an oligomer.

Mammary epithelial-specific knockout of the ephrin-B2 gene leads to precocious epithelial cell death at lactation

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor-ligand interaction requires direct cell-cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy-lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo-alveolar structures. Accordingly, massive epithelial cell death and expression of involution-specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.

The kinetics of feline leukaemia virus shedding in experimentally infected cats are associated with infection outcome

Feline leukaemia virus (FeLV) infection in felids results mainly from oronasal exposure to infectious saliva and nasal secretions, but the potential for viral transmission through faeces and urine has not been completely characterized. In order to assess and compare potential FeLV transmission routes, we determined the viral kinetics in plasma, saliva, faeces and urine during early experimental FeLV infection (up to week 15 post-exposure) in specific pathogen-free cats. In addition to monitoring p27 antigen levels measured by ELISA, we evaluated the presence of infectious particles by cell culture assays and quantified viral RNA loads by a quantitative real-time TaqMan polymerase chain reaction. RNA load was associated with infection outcome (high load-progressive infection; low load-regressive infection) not only in plasma, but also in saliva, faeces and urine. Infectious virus was isolated from the saliva, faeces and urine of infected cats with progressive infection as early as 3-6 weeks post-infection, but usually not in cats with regressive infection. In cats with progressive infection, therefore, not only saliva but also faeces and to some extent urine might represent potential FeLV transmission routes. These results should be taken into account when modelling FeLV-host interactions and assessing FeLV transmission risk. Moreover, during early FeLV infection, detection of viral RNA in saliva may be used as an indicator of recent virus exposure, even in cats without detectable antigenaemia/viraemia. To determine the clinically relevant outcome of FeLV infection in exposed cats, however, p27 antigen levels in the peripheral blood should be measured.

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.

Molecular Determinants Defining the Triggering Range of Prefusion F Complexes of Canine Distemper Virus

The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this "pocket'" appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE: With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering "stimulus" (attachment protein and receptor types) define a "triggering range," which governs the initiation of the membrane fusion process. A central "pocket" microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency.

Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Cell-type specific expression and regulation of apolipoprotein D and E in human endometrium.

OBJECTIVE To assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. STUDY DESIGN Endometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45(+) leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. RESULTS ApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. CONCLUSION We demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.

Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n=16), whereas that of Staph. aureus non-GTB herds was 38% (n=38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9-56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6-58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3-27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.

Impact of synchronous metastasis distribution on cancer specific survival in renal cell carcinoma after radical nephrectomy with tumor thrombectomy

PURPOSE Metastatic renal cell carcinoma can be clinically diverse in terms of the pattern of metastatic disease and response to treatment. We studied the impact of metastasis and location on cancer specific survival. MATERIALS AND METHODS The records of 2,017 patients with renal cell cancer and tumor thrombus who underwent radical nephrectomy and tumor thrombectomy from 1971 to 2012 at 22 centers in the United States and Europe were analyzed. Number and location of synchronous metastases were compared with respect to patient cancer specific survival. Multivariable Cox regression models were used to quantify the impact of covariates. RESULTS Lymph node metastasis (155) or distant metastasis (725) was present in 880 (44%) patients. Of the patients with distant disease 385 (53%) had an isolated metastasis. The 5-year cancer specific survival was 51.3% (95% CI 48.6-53.9) for the entire group. On univariable analysis patients with isolated lymph node metastasis had a significantly worse cancer specific survival than those with a solitary distant metastasis. The location of distant metastasis did not have any significant effect on cancer specific survival. On multivariable analysis the presence of lymph node metastasis, isolated distant metastasis and multiple distant metastases were independently associated with cancer specific survival. Moreover higher tumor thrombus level, papillary histology and the use of postoperative systemic therapy were independently associated with worse cancer specific survival. CONCLUSIONS In our multi-institutional series of patients with renal cell cancer who underwent radical nephrectomy and tumor thrombectomy, almost half of the patients had synchronous lymph node or distant organ metastasis. Survival was superior in patients with solitary distant metastasis compared to isolated lymph node disease.

Combination of expression levels of miR-21 and miR-126 is associated with cancer-specific survival in clear-cell renal cell carcinoma

BACKGROUND Renal cell carcinoma (RCC) is marked by high mortality rate. To date, no robust risk stratification by clinical or molecular prognosticators of cancer-specific survival (CSS) has been established for early stages. Transcriptional profiling of small non-coding RNA gene products (miRNAs) seems promising for prognostic stratification. The expression of miR-21 and miR-126 was analysed in a large cohort of RCC patients; a combined risk score (CRS)-model was constructed based on expression levels of both miRNAs. METHODS Expression of miR-21 and miR-126 was evaluated by qRT-PCR in tumour and adjacent non-neoplastic tissue in n = 139 clear cell RCC patients. Relation of miR-21 and miR-126 expression with various clinical parameters was assessed. Parameters were analysed by uni- and multivariate COX regression. A factor derived from the z-score resulting from the COX model was determined for both miRs separately and a combined risk score (CRS) was calculated multiplying the relative expression of miR-21 and miR-126 by this factor. The best fitting COX model was selected by relative goodness-of-fit with the Akaike information criterion (AIC). RESULTS RCC with and without miR-21 up- and miR-126 downregulation differed significantly in synchronous metastatic status and CSS. Upregulation of miR-21 and downregulation of miR-126 were independently prognostic. A combined risk score (CRS) based on the expression of both miRs showed high sensitivity and specificity in predicting CSS and prediction was independent from any other clinico-pathological parameter. Association of CRS with CSS was successfully validated in a testing cohort containing patients with high and low risk for progressive disease. CONCLUSIONS A combined expression level of miR-21 and miR-126 accurately predicted CSS in two independent RCC cohorts and seems feasible for clinical application in assessing prognosis.