Infection-induced IL-10 and Jak-Stat: A review of the molecular circuitry controlling immune hyperactivity in response to pathogenic microbes

Generation of effective immune responses against pathogenic microbes depends on a fine balance between pro- and anti-inflammatory responses. Interleukin-10 (IL-10) is essential in regulating this balance and has garnered renewed interest recently as a modulator of the response to infection at the JAK-STAT signaling axis of host responses. Here, we examine how IL-10 functions as the “master regulator” of immune responses through JAK-STAT, and provide a perspective from recent insights on bacterial, protozoan, and viral infection model systems. Pattern recognition and subsequent molecular events that drive activation of IL-10-associated JAK-STAT circuitry are reviewed and the implications for microbial pathogenesis are discussed.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix.

Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and E.necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P < 0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P < 0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

In vitro infection of sheep lice (Bovicola ovis Schrank) by Steinernematid and Heterorhabditid nematodes.

Control of sheep lice with conventional pesticides can be compromised by difficulty in contacting lice in the dense water repellent fleeces of sheep, particularly when sheep have not been recently shorn. Entomopathogenic nematodes (ENs) are motile and are able to actively seek out insect hosts. They have particular advantages for the control of pests in cryptic habitats, such as the fleeces of sheep and avoid many of the problems frequently associated with chemical controls. This study investigated whether ENs were able infect and kill Bovicola ovis and compared the effectiveness of different species at different temperatures and when applied to wool. Four species of nematodes, Steinernema carpocapsae, Steinernema riobrave, Steinernema feltiae and Heterorhabditis bacteriophora were tested. All were shown to infect and kill lice in Petri dish assays at 30C. At 35C, the percent infection for S. carpocapsae and S. riobrave was significantly higher than for the other two species and percent infection by S. feltiae was significantly greater than for H. bacteriophora (P<0.05). At 37C the percent mortality induced by S. riobrave was significantly greater than for S. carpocapsae (P<0.05). All species were able to locate and infect lice in wool when formulated in water with 8% Tween 80. In wool assays the percent lice infected with nematodes was significantly greater for S. riobrave than H.bacteriophora at 25C, but there were no other differences between species (P=0.05). S. carpocapsae, S. riobrave and S. feltiae caused significantly higher lice mortality than H. bacteriophora at both 25 and 35C in wool assays, but mortality induced by the three steinernematid species did not differ significantly (P>0.05). It is concluded that of the ENs studied S. riobrave is likely to be most effective against B. ovis when applied to live sheep because of its greater tolerance to high temperatures and 'cruiser' foraging strategy .

The influence of HIV infection to the periodontium; a clinical, microbiological, and enzymological study

The main targets of human immunodeficiency virus (HIV) are CD4 receptors of CD4+ lymphocytes and many other cells such as monocytes/macrophages, megakaryocytes, peripheral blood dendritic cells, follicular dendritic cells (DC), epidermal Langerhans cells, and astrocytes. Infection and killing of CD4+ lymphocytes or false reaction of the body to HIV infection and the spontaneous apoptosis of CD4+ lymphocytes decrease CD4+ lymphocyte counts leading to immunosuppression, further disease progression, and appearance of opportunistic infections and malignancies. Oral manifestations are considered to be among the first signs of HIV infection. Enhanced degradation of extracellular matrix and basement membrane components in oral diseases including periodontitis is caused by Zn-dependent enzymes called matrix metalloproteinases (MMPs). The levels and degrees of activation of MMP-1, -2, -3, -7, -8, -9, -25, -26, tissue inhibitors of MMPs (TIMP)-1 and -2, and myeloperoxidase (MPO) and collagenolytic/gelatinolytic activities, and also Ig A, -G, and -M, total protein, and albumin levels in a two-year follow-up were studied from salivary samples. The expression of MMP-7, -8, -9, -25, and -26 immunoreactivities in gingival tissue specimens were studied. Healthy HIV-negative subjects served as controls. All studied clinical periodontal parameters and microbiological evaluation of the periodontopathogens showed that periodontal health of the HIV-positive patients was moderately decreased in comparison to the healthy controls. The levels of Candida in the periodontal pockets and salivary MPO increased with the severity of HIV infection. Immunoreactivities and levels of MMPs and TIMPs, and MMP activities (collagenase, gelatinase) were enhanced in the HIV-positive patient salivary samples relative to the healthy controls regardless of the phase of HIV infection. However, these parameters did not reflect periodontal status in a similar way as in the generally healthy periodontitis patients. Salivary total protein, albumin, IgA, -G, and -M levels were significantly higher in all phases of HIV infection compared to the controls, and salivary total protein, IgG and IgM levels remained higher after two years follow-up, partly correlating with the disease progression and which may reflect the leakage of serum components into the mouth and thus a decreased mucosal barrier. Salivary analyses of MMPs and TIMPs with immunohistochemical analyses showed that HIV infection could predispose to periodontal destruction when compared with healthy controls or the body s defence reactions associated with HIV infection may have been reflected or mediated by MMPs.

A native Wolbachia Endosymbiont does not limit Dengue virus infection in the mosquito Aedes notoscriptus (Diptera: Culicidae)

The endosymbiotic bacterium Wolbachia pipientis infects many species of insects and has been transinfected into the mosquito Aedes aegypti (L.), the primary vector of dengue virus (DENV). Recently, it has been shown that Wolbachia blocks the replication and transmission of RNA viruses, such as DENV, in a number of mosquito species including Ae. aegypti and Aedes albopictus (Skuse), which is naturally infected with Wolbachia and considered a secondary vector for DENV. The mosquito species Aedes notoscriptus (Skuse) is highly prevalent in Australia, including in areas where DENV outbreaks have been recorded. The mosquito has been implicated in the transmission of Ross River and Barmah Forest viruses, but not DENV. We investigated whether Wolbachia naturally infects this mosquito species and whether it has an impact on the ability of Ae. notoscriptus to transmit DENV. We show, for the first time, that Ae. notoscriptus is naturally infected with a strain of Wolbachia that belongs to supergroup B and is localized only in the ovaries. However, Wolbachia infection in Ae. notoscriptus did not induce resistance to DENV and had no effect on overall DENV infection rate or titer. The presence of a native Wolbachia in Ae. notoscriptus cannot explain why this mosquito is an ineffective vector of DENV.

Molecular pathogenesis of human parechovirus 1 infection

The Parechoviruses (HPEV) belong to the family Picornaviridae of positive-stranded RNA viruses. Although the parechovirus genome shares the general properties of other picornaviruses, the genus has several unique features when compared to other family members. We found that HPEV1 attaches to αv integrins on the cell surface and is internalized through the clathrin-mediated endocytic pathway. During he course of the infection, the Golgi was found to disintegrate and the ER membranes to swell and loose their ribosomes. The replication of HPEV1 was found to take place on small clusters of vesicles which contained the trans-Golgi marker GalT as well as the viral non-structural 2C protein. 2C was additionally found on stretches of modified ER-membranes, seemingly not involved in RNA replication. The viral non-structural 2A and 2C proteins were studied in further detail and were found to display several interesting features. The 2A protein was found to be a RNA-binding protein that preferably binds to positive sense 3 UTR RNA. It was found to bind also duplex RNA containing 3 UTR(+)-3 UTR(-), but not other dsRNA molecules studied. Mutagenesis revealed that the N-terminal basic-rich region as well as the C-terminus, are important for RNA-binding. The 2C protein on the other hand, was found to have both ATP-diphosphohydrolase and AMP kinase activities. Neither dATP nor other NTP:s were suitable substrates. Furthermore, we found that as a result of theses activities the protein is autophosphorylated. The intracellular changes brought about by the individual HPEV1 non-structural proteins were studied through the expression of fusion proteins. None of the proteins expressed were able to induce membrane changes similar to those seen during HPEV1 infection. However, the 2C protein, which could be found on the surface of lipid droplets but also on diverse intracellular membranes, was partly relocated to viral replication complexes in transfected, superinfected cells. Although Golgi to ER traffic was arrested in HPEV1-infected cells, none of the individually expressed non-structural proteins had any visible effect on the anterograde membrane traffic. Our results suggest that the HPEV1 replication strategy is different from that of many other picornaviruses. Furthermore, this study shows how relatively small differences in genome sequence result in very different intracellular pathology.

New aspects of the diagnosis of Helicobacter pylori infection

Aims: Helicobacter pylori infection, although the prevalence is declining in Western world, is still responsible for several clinically important diseases. None of the diagnostic tests is perfect and in this study, the performance of three stool antigen tests was assessed. In areas of high H. pylori prevalence, the definition of patients with the greatest benefit from eradication therapy may be a problem; the role of duodenal gastric metaplasia in categorizing patients at risk for duodenal ulcer was evaluated in this respect. Whether persistent chronic inflammation and elevated H. pylori antibodies after successful eradication are associated with each other or with atrophic gastritis, a long term sequelae of H. pylori infection, were also studied. Patients and methods: The three stool antigen tests were assessed in pre- and post-eradication settings among 364 subjects in two studies as compared to the rapid urease test (RUT), histology, culture, the 13C-urea breath test (UBT) and enzyme immunoassay (EIA) based H. pylori serology. The association between duodenal gastric metaplasia with duodenal ulcer was evaluated in a retrospective study including 1054 patients gastroscopied due to clinical indications and 154 patients previously operated for duodenal ulcer. The extent of duodenal gastric metaplasia was assessed from histological specimens in different patient groups formed on the basis of gastroscopy findings and H. pylori infection. Chronic gastric inflammation (108 patients) and H. pylori antibodies and serum markers for atrophy (77 patients) were assessed in patients earlier treated for H. pylori. Results: Of the stool antigen tests studied, the monoclonal antibody-based EIA-test showed the highest sensitivity and specificity both in the pre-treatment setting (96.9% and 95.9%) and after therapy (96.9% and 97.8%). The polyclonal stool antigen test and the in-office test had at baseline a sensitivity of 91% and 94%, and a specificity of 96% and 89%, respectively and in a post-treatment setting, a sensitivity of 78% and 91%, and a specificity of 97%, respectively. Duodenal gastric metaplasia was strongly associated with H. pylori positive duodenal ulcer (odds ratio 42). Although common still five years after eradication, persistent chronic gastric inflammation (21%) and elevated H. pylori antibodies (33%) were neither associated with each other nor with atrophic gastritis. Conclusions: Current H. pylori infection can feasibly be diagnosed by a monoclonal antibody-based EIA test with the accuracy comparable to that of reference methods. The performance of the polyclonal test as compared to the monoclonal test was inferior especially in the post-treatment setting. The in-office test had a low specificity for primary diagnosis and hence positive test results should probably be confirmed with another test before eradication therapy is prescribed. The presence of widespread duodenal gastric metaplasia showed promising results in detecting patients who should be treated for H. pylori due to an increased risk of duodenal ulcer. If serology is used later on in patients with earlier successfully treated for H. pylori, it should be taken into account that H. pylori antibodies may persist elevated for years for unknown reason. However, this phenomenon was not found to be associated with persistent chronic inflammation or atrophic changes.

Markers of systemic inflammation in diagnostics and in prediction of outcome of community-acquired infection

Sepsis is associated with a systemic inflammatory response. It is characterised by an early proinflammatory response and followed by a state of immunosuppression. In order to improve the outcome of patients with infection and sepsis, novel therapies that influence the systemic inflammatory response are being developed and utilised. Thus, an accurate and early diagnosis of infection and evaluation of immune state are crucial. In this thesis, various markers of systemic inflammation were studied with respect to enhancing the diagnostics of infection and of predicting outcome in patients with suspected community-acquired infection. A total of 1092 acutely ill patients admitted to a university hospital medical emergency department were evaluated, and 531 patients with a suspicion of community-acquired infection were included for the analysis. Markers of systemic inflammation were determined from a blood sample obtained simultaneously with a blood culture sample on admission to hospital. Levels of phagocyte CD11b/CD18 and CD14 expression were measured by whole blood flow cytometry. Concentrations of soluble CD14, interleukin (IL)-8, and soluble IL-2 receptor α (sIL-2Rα) were determined by ELISA, those of sIL-2R, IL-6, and IL-8 by a chemiluminescent immunoassay, that of procalcitonin by immunoluminometric assay, and that of C-reactive protein by immunoturbidimetric assay. Clinical data were collected retrospectively from the medical records. No marker of systemic inflammation, neither CRP, PCT, IL-6, IL-8, nor sIL-2R predicted bacteraemia better than did the clinical signs of infection, i.e., the presence of infectious focus or fever or both. IL-6 and PCT had the highest positive likelihood ratios to identify patients with hidden community-acquired infection. However, the use of a single marker failed to detect all patients with infection. A combination of markers including a fast-responding reactant (CD11b expression), a later-peaking reactant (CRP), and a reactant originating from inflamed tissues (IL-8) detected all patients with infection. The majority of patients (86.5%) with possible but not verified infection showed levels exceeding at least one cut-off limit of combination, supporting the view that infection was the cause of their acute illness. The 28-day mortality of patients with community-acquired infection was low (3.4%). On admission to hospital, the low expression of cell-associated lipopolysaccharide receptor CD14 (mCD14) was predictive for 28-day mortality. In the patients with severe forms of community-acquired infection, namely pneumonia and sepsis, high levels of soluble CD14 alone did not predict mortality, but a high sCD14 level measured simultaneously with a low mCD14 raised the possibility of poor prognosis. In conclusion, to further enhance the diagnostics of hidden community-acquired infection, a combination of inflammatory markers is useful; 28-day mortality is associated with low levels of mCD14 expression at an early phase of the disease.

Human papillomavirus infection and vaccination: Awareness and knowledge of HPV and acceptability of HPV vaccine among mothers of teenage daughters in Weihai, Shandong, China

In preparation for the introduction of human papillomavirus (HPV) vaccine, we investigated awareness and knowledge of HPV/HPV vaccine and potential acceptability to HPV vaccine among mothers with a teenage daughter in Weihai, Shandong, China. A cross-sectional survey was conducted in 2013 with a sample of 1850 mothers who had a daughter (aged 9–17 years) attending primary, junior and senior high schools. In the final sample (N = 1578, response rate 85.30%), awareness of HPV was reported by 305 (19.32%) mothers. Awareness varied significantly by daughter’s age (P<0.01), mother’s education level (P<0.01), mother’s occupation (P<0.01), household income (P<0.01) and residence type (P<0.01). Knowledge about HPV/HPV vaccine was poor with a mean total score of 3.56 (SD = 2.40) out of a possible score of 13. Mothers with a higher education level reported higher levels of knowledge (P = 0.02). Slightly more than one-fourth (26.49%) of mothers expressed their potential acceptability of HPV vaccine for their daughters. Acceptability increased along with increased daughters’ age (P<0.01), household income (P<0.01) and knowledge level (P<0.01). House wives and unemployed mothers had the highest acceptability (P<0.01). The most common reasons for not accepting HPV vaccination were “My daughter is too young to have risk of cervical cancer (30.95%)”, “The vaccine has not been widely used, and the decision will be made after it is widely used (24.91%)”, “Worry about the safety of the vaccine (22.85%)”. Awareness and knowledge of HPV/HPV vaccines are poor and HPV vaccine acceptability is low among these Chinese mothers. These results may help inform appropriate health education programs in this population.

Dengue virus infection : Diagnostics and molecular epidemiology

Dengue is a mosquito-borne viral disease caused by the four dengue virus serotypes (DENV-1-4) and is currently considered as the most important arthropod-borne viral disease in the world. Nearly half of the human population lives in risk areas, and 50-100 million infections occur yearly according to World Health Organization. The disease can vary from a mild febrile disease to severe haemorrhagic fever and shock. A secondary infection with heterologous serotype increases the risk for severe disease outcome. During the last three decades the impact of dengue has dramatically increased in the endemic areas including the tropics and subtropics of the world. The current situation with massive epidemics of severe disease forms has been associated with socio-ecological changes that have increased the transmission and enabled the co-circulation of different serotypes. Consequently, an increase of dengue has also been observed in travelers visiting these areas. Currently approximately 30 cases are diagnosed yearly in Finnish travelers. In travelers dengue is rarely a life-threatening disease, however in the current study, a fatality was documented in a young Finnish patient who experienced a prolonged primary dengue infection. To improve particularly early laboratory diagnostics, a novel real-time RT-PCR method was developed for the detection of DENV-1-4 RNA based on TaqMan chemistry. The method was shown to be sensitive and specific for detecting DENV RNA and suitable for diagnostic use. The newly developed real-time RT-PCR was compared to other available early diagnostic methods including IgM and NS1 antigen detection using a panel of selected patient samples. The results suggest that the best diagnostic rates are achieved by a combination of IgM with RNA or NS1 detection. The dengue virus strains studied here included the first DENV strains isolated from serum samples of Finnish travelers collected in 2000-2005. The results of sequence analysis demonstrated that the 11 isolates included all four DENV serotypes and presented a global sample of DENV strains from different geographical areas including Asia, Africa and South America. In the present study sequence analysis was also carried out for a collection of 23 novel DENV-2 isolates from Venezuelan patients collected in 1999-2005. The Venezuelan DENV-2 exclusively represented the American-Asian genotype, suggesting that no foreign DENV-2 lineages have recently been introduced to the country. The results also suggest that the DENV-2 viruses detected earlier from Venezuela have been maintained in the area where they have evolved into several lineages. This is in contrast to the pattern observed in some other dengue endemic areas, where introductions of novel virus types and lineages are frequently detected.

Infection by Alternaria alternata causes discolouration of Backhousia myrtifolia foliage and flowers.

The pathogenicity of three isolates of Alternaria alternata from Backhousia myrtifolia leaves was characterised and compared. Isolate BRIP 52222 was virulent compared to isolates BRIP 52223 and BRIP 52221. A comparison of inoculation methods showed that abrasion was more effective at establishing an infection than puncture wounding. Koch's postulates were assessed to confirm the pathogenicity of A. alternata on B. myrtifolia foliage and floral tissues using a conidial suspension of the most virulent isolate. Sporulation was triggered by incubating A. alternata (BRIP 52222) at 28 degrees C for 10 d under alternating 12 h black-light/12 h dark conditions on half-strength potato dextrose agar (PDA). In contrast, incubation of A. alternata under continuous black-light on either half- or full-strength PDA did not yield conidia. Host symptoms caused by inoculation with the pathogen included a brown-black discolouration of both foliage and floral tissues. Microscopic examination of cellular structures suggested that perturbation of oil glands may contribute to the tissue discolouration in B. myrtifolia caused by A. alternata infection. Oil gland structures can be disrupted during an active A. alternata infection, causing the leakage of essential oil followed by discolouration.

Antibody Responses to Sarcoptes scabiei Apolipoprotein in a Porcine Model: Relevance to Immunodiagnosis of Recent Infection

No commercial immunodiagnostic tests for human scabies are currently available, and existing animal tests are not sufficiently sensitive. The recombinant Sarcoptes scabiei apolipoprotein antigen Sar s 14.3 is a promising immunodiagnostic, eliciting high levels of IgE and IgG in infected people. Limited data are available regarding the temporal development of antibodies to Sar s 14.3, an issue of relevance in terms of immunodiagnosis. We utilised a porcine model to prospectively compare specific antibody responses to a primary infestation by ELISA, to Sar s 14.3 and to S. scabiei whole mite antigen extract (WMA). Differences in the antibody profile between antigens were apparent, with Sar s 14.3 responses detected earlier, and declining significantly after peak infestation compared to WMA. Both antigens resulted in >90% diagnostic sensitivity from weeks 8–16 post infestation. These data provide important information on the temporal development of humoral immune responses in scabies and further supports the development of recombinant antigen based immunodiagnostic tests for recent scabies infestations.