ResearchPad - immunologic-techniques https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment]]> https://www.researchpad.co/article/elastic_article_13826 Murine gammaherpesvirus 68 is a rodent pathogen that is closely related to the human gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma-associated virus. All know gammaherpesviruses are associated with the development of lymphomas, as well as other cancers, in a small subset of infected individuals–particularly those with underlying defects in their immune system (i.e., transplant recipients and HIV infected patients). Because there are very limited small animal models for the human gammaherpesviruses, studies on murine gammaherepsviruses 68 can provide important insights into critical aspects of gammaherpesvirus infections and the association of these viruses with disease development. Another feature of all gammaherpesviruses is their ability to establish a chronic infection of their host–where the virus is maintained for the lifetime of the infected individual. The major target cell harboring chronic gammaherepsvirus infection are B lymphocytes–the cells in the immune system that produce antibodies in response to infections. Here we provide a detailed characterization of the populations of B lymphocytes that become infected by murine gammaherpesvirus 68. This has led to the identification of a specific population of B lymphocytes that is preferentially infected by the virus. This supports a model in which murine gammaherpesvirus infection of B lymphocytes is not random. However, it remains unclear why the virus targets this specific population of B cells for infection.

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<![CDATA[A systematic review of alternative surveillance approaches for lymphatic filariasis in low prevalence settings: Implications for post-validation settings]]> https://www.researchpad.co/article/elastic_article_13802 Lymphatic filariasis (LF) is a mosquito-borne disease, which can result in complications including swelling affecting the limbs (lymphoedema) or scrotum (hydrocele). LF can be eliminated by mass drug administration (MDA) which involves whole communities taking drug treatment at regular intervals. After MDA programmes, country programmes conduct the Transmission Assessment Survey (TAS), which tests school children for LF. It is important to continue testing for LF after elimination because there can be a 10-year period between becoming infected and developing symptoms, but it is thought that the use of TAS in such settings is likely to be too expensive and also not sensitive enough to detect low-level infections. Our study assesses the results from 44 studies in areas of low LF prevalence that have investigated methods of surveillance for LF which differ from the standardised TAS approach. These include both human and mosquito studies. Results show that there is currently no standardised approach to testing, but that surveillance can be made more sensitive through the use of new diagnostic tests, such as antibody testing, and also by targeting higher risk populations. However, further research is needed to understand whether these approaches work in a range of settings and whether they are affordable on the ground.

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<![CDATA[Potency and breadth of human primary ZIKV immune sera shows that Zika viruses cluster antigenically as a single serotype]]> https://www.researchpad.co/article/elastic_article_7747 The recent emergence of Zika virus as an important human pathogen has raised questions about the durability and breadth of Zika virus immunity following natural infection in humans. While global epidemic patterns suggest that Zika infection elicits a protective immune response that is likely to offer long-term protection against repeat infection by other Zika viruses, only one study to date has formally examined the ability of human Zika immune sera to neutralize different Zika viruses. That study was limited because it evaluated human immune sera no more than 13 weeks after Zika virus infection and tested a relatively small number of Zika viruses. In this study, we examine twelve human Zika immune sera as far as 3 years after infection and test the sera against a total of eleven Zika virus isolates. Our results confirm the earlier study and epidemic patterns that suggest Zika virus exists in nature as a single serotype, and infection with one Zika virus can be expected to elicit protective immunity against repeat infection by any Zika virus for years to decades after the first infection.

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<![CDATA[Clinical role, optimal timing and frequency of serum infliximab and anti-infliximab antibody level measurements in patients with inflammatory bowel disease]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdc1f4

Background

Serum infliximab (IFX) and antibody-to-infliximab (ATI) levels are objective parameters, that may have a great role in the therapeutic decisions during maintenance biological therapy.

Research design and methods

48 inflammatory bowel disease patients receiving maintenance IFX therapy were prospectively enrolled and divided into adequate (complete remission N = 20) and inadequate responder (partial response, loss of response, dose escalation; N = 28) groups. Blood samples were collected just before (trough level, TL) and two (W2aTL) and six weeks (W6aTL) after the administration of IFX.

Results

Single measurement of ATI titer was insufficient for predicting therapeutic response due to transient expression of ATI, however, using the three points’ measurements, significant difference has been detected between the adequate and inadequate responder group (5.0% vs 35.7%; p = 0.016). The mean value of TL was significantly higher in the adequate responder group (3.11±1.64 vs.1.19±1.11; p<0.001) without further difference on the second and sixth week. Sensitivity and specificity for predicting the therapeutic response were 85.0% and 71.4% based on the cut-off value of TL 2.0 μg/ml.

Conclusion

Simultaneous measurement of serum IFX level prior to administration of regular IFX infusion and ATI titers significantly increase the diagnostic accuracy for the therapeutic decision in patients uncertainly responding to the therapy. The measurement of W2aTL and W6aTL levels did not result in further improvement in the prediction of therapeutic response.

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<![CDATA[Constitutive hydrogen inhalation prevents vascular remodeling via reduction of oxidative stress]]> https://www.researchpad.co/article/Ne1330967-900e-43ee-b1f2-140543b0d511

Molecular hydrogen is thought to have an inhibitory effect on oxidative stress, thereby attenuating the onset and progression of various diseases including cardiovascular disease; however, few reports have assessed the preventive effect of constitutive inhalation of hydrogen gas on of vascular remodeling. Here, we investigated the effect of constitutive inhalation of hydrogen gas on vascular neointima formation using a cuff-induced vascular injury mouse model. After constitutive inhalation of compressed hydrogen gas (O2 21%, N2 77.7%, hydrogen 1.3%) or compressed air only (O2 21%, N2 79%) by C57BL/6 mice for 2 weeks from 8 weeks of age in a closed chamber, inflammatory cuff injury was induced by polyethylene cuff placement around the femoral artery under anesthesia, and hydrogen gas administration was continued until sampling of the femoral artery. Neointima formation, accompanied by an increase in cell proliferation, was significantly attenuated in the hydrogen group compared with the control group. NADPH oxidase NOX1 downregulation in response to cuff injury was shown in the hydrogen group, but the expression levels of NADPH oxidase subunits, p40phox and p47phox, did not differ significantly between the hydrogen and control groups. Although the increase in superoxide anion production did not significantly differ between the hydrogen and control groups, DNA damage was decreased as a result of reduction of reactive oxygen species such as hydroxyl radical (⋅OH) and peroxynitrite (ONOO-) in the hydrogen group. These results demonstrate that constitutive inhalation of hydrogen gas attenuates vascular remodeling partly via reduction of oxidative stress, suggesting that constitutive inhalation of hydrogen gas at a safe concentration in the living environment could be an effective strategy for prevention of vascular diseases such as atherosclerosis.

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<![CDATA[Risk factors associated to a high Mycobacterium tuberculosis complex seroprevalence in wild boar (Sus scrofa) from a low bovine tuberculosis prevalence area]]> https://www.researchpad.co/article/Nfbbd03ef-7cb8-4d82-b605-16cf8ee0d77e

Animal tuberculosis is a worldwide zoonotic disease caused principally by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). In southern Iberian Peninsula, wild reservoirs such as the wild boar, among other factors, have prevented the eradication of bovine tuberculosis. However, most of the studies have been focused on south-central Spain, where the prevalence of tuberculosis is high among wild ungulates and cattle herds. In northern regions, where wild boar density and bovine tuberculosis prevalence are lower, fewer studies have been carried out and the role of this species is still under debate. The aim of this study was to describe the temporal and spatial distribution of antibodies against MTC in wild boar from the Basque Country, northern Spain. Sera from 1902 animals were collected between 2010 and 2016. The seroprevalence was determined with an in house enzyme-linked immunosorbent assay and the search of risk factors was assessed by Generalized Linear Models. Overall, 17% of wild boars (326/1902; 95%CI, [15.5%–18.9%]) showed antibodies against MTC. Risk factors associated with seropositivity were the year and location of sampling, the number of MTC positive cattle, the distance to positive farms and the percentage of shrub cover. Younger age classes were associated with increased antibody titres among seropositive individuals. The seroprevalence detected was higher than those previously reported in neighbouring regions. Hence, further studies are needed to better understand the role of wild boar in the epidemiology of tuberculosis in low tuberculosis prevalence areas and consequently, its relevance when developing control strategies.

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<![CDATA[Lateral flow immunoassay (LFIA) for the detection of lethal amatoxins from mushrooms]]> https://www.researchpad.co/article/N089b971a-62b1-4256-a74f-acfba8aef66c

The mushroom poison that causes the most deaths is the class of toxins known as amatoxins. Current methods to sensitively and selectively detect these toxins are limited by the need for expensive equipment, or they lack accuracy due to cross-reactivity with other chemicals found in mushrooms. In this work, we report the development of a competition-based lateral flow immunoassay (LFIA) for the rapid, portable, selective, and sensitive detection of amatoxins. Our assay clearly indicates the presence of 10 ng/mL of α-AMA or γ-AMA and the method including extraction and detection can be completed in approximately 10 minutes. The test can be easily read by eye and has a presumed shelf-life of at least 1 year. From testing 110 wild mushrooms, the LFIA identified 6 out of 6 species that were known to contain amatoxins. Other poisonous mushrooms known not to contain amatoxins tested negative by LFIA. This LFIA can be used to quickly identify amatoxin-containing mushrooms.

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<![CDATA[Production of a rabbit monoclonal antibody for highly sensitive detection of citrus mosaic virus and related viruses]]> https://www.researchpad.co/article/Nfb5e596f-2980-40fa-8fa6-576f474cd99c

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10−10 to 5.5 × 10−11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.

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<![CDATA[Urea-mediated dissociation alleviate the false-positive Treponema pallidum-specific antibodies detected by ELISA]]> https://www.researchpad.co/article/5c8823e2d5eed0c484639234

The serological detection of antibodies to Treponema pallidum is essential to the diagnosis of syphilis. However, for the presence of cross-reaction, the specific antibody tests [e.g., enzyme-linked immunosorbent assay (ELISA)] always have false-positive results. In this study, we derived and validated the dissociation of urea in an attempt to alleviate the situation of false-positive antibodies to T. pallidum detected by ELISA. Six serum samples that were false-positive antibodies to T. pallidum detected by ELISA, and 16 control serum samples (8 sera positive for both specific IgG and IgM, and 8 IgG-positive and IgM-negative sera) were collected to select the appropriate dissociated concentration and time of urea. Our goal was to establish improved an ELISA method based on the original detection system of ELISA. The sensitivity of the improved ELISA was evaluated by 275 serum samples with class IgM-positive antibodies to T. pallidum. At 6 mol/L with 10 minutes dissociation of urea, 6 samples with false-positive antibodies to T. pallidum were converted to negative, and compared with true-positive antibodies to T. pallidum. The sensitivity of the improved ELISA was 100% by detecting the class IgM-positive antibodies to T. pallidum in sera of patients with syphilis. Considering the importance at the diagnosis of syphilis, antibodies to T. pallidum in serum samples should be retested by the improved ELISA method to avoid false-positive results.

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<![CDATA[Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen]]> https://www.researchpad.co/article/5c784fe8d5eed0c4840078de

Throughout the last several decades, vaccination has been key to prevent and eradicate infectious diseases. However, many pathogens (e.g., respiratory syncytial virus [RSV], influenza, dengue, and others) have resisted vaccine development efforts, largely because of the failure to induce potent antibody responses targeting conserved epitopes. Deep profiling of human B cells often reveals potent neutralizing antibodies that emerge from natural infection, but these specificities are generally subdominant (i.e., are present in low titers). A major challenge for next-generation vaccines is to overcome established immunodominance hierarchies and focus antibody responses on crucial neutralization epitopes. Here, we show that a computationally designed epitope-focused immunogen presenting a single RSV neutralization epitope elicits superior epitope-specific responses compared to the viral fusion protein. In addition, the epitope-focused immunogen efficiently boosts antibodies targeting the palivizumab epitope, resulting in enhanced neutralization. Overall, we show that epitope-focused immunogens can boost subdominant neutralizing antibody responses in vivo and reshape established antibody hierarchies.

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<![CDATA[Coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells]]> https://www.researchpad.co/article/5c8acc7ed5eed0c48498f892

Despite a broad cell-type tropism, cytomegalovirus (CMV) is an evidentially pulmonary pathogen. Predilection for the lungs is of medical relevance in immunocompromised recipients of hematopoietic cell transplantation, in whom interstitial CMV pneumonia is a frequent and, if left untreated, fatal clinical manifestation of human CMV infection. A conceivable contribution of CMV to airway diseases of other etiology is an issue that so far attracted little medical attention. As the route of primary CMV infection upon host-to-host transmission in early childhood involves airway mucosa, coincidence of CMV airway infection and exposure to airborne environmental antigens is almost unavoidable. For investigating possible consequences of such a coincidence, we established a mouse model of airway co-exposure to CMV and ovalbumin (OVA) representing a protein antigen of an inherently low allergenic potential. Accordingly, intratracheal OVA exposure alone failed to sensitize for allergic airway disease (AAD) upon OVA aerosol challenge. In contrast, airway infection at the time of OVA sensitization predisposed for AAD that was characterized by airway inflammation, IgE secretion, thickening of airway epithelia, and goblet cell hyperplasia. This AAD histopathology was associated with a T helper type 2 (Th2) transcription profile in the lungs, including IL-4, IL-5, IL-9, and IL-25, known inducers of Th2-driven AAD. These symptoms were all prevented by a pre-challenge depletion of CD4+ T cells, but not of CD8+ T cells. As to the underlying mechanism, murine CMV activated migratory CD11b+ as well as CD103+ conventional dendritic cells (cDCs), which have been associated with Th2 cytokine-driven AAD and with antigen cross-presentation, respectively. This resulted in an enhanced OVA uptake and recruitment of the OVA-laden cDCs selectively to the draining tracheal lymph nodes for antigen presentation. We thus propose that CMV, through activation of migratory cDCs in the airway mucosa, can enhance the allergenic potential of otherwise poorly allergenic environmental protein antigens.

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<![CDATA[Identification of Merkel cells associated with neurons in engineered skin substitutes after grafting to full thickness wounds]]> https://www.researchpad.co/article/5c8823d9d5eed0c484639153

Engineered skin substitutes (ESS), prepared using primary human fibroblasts and keratinocytes with a biopolymer scaffold, were shown to provide stable closure of excised burns, but relatively little is known about innervation of ESS after grafting. This study investigated innervation of ESS and, specifically, whether Merkel cells are present in healed grafts. Merkel cells are specialized neuroendocrine cells required for fine touch sensation in skin. We discovered cells positive for keratin 20 (KRT20), a general marker for Merkel cells, in the basal epidermis of ESS after transplantation to mice, suggesting the presence of Merkel cells. Cells expressing KRT20 were not observed in ESS in vitro. However, widely separated KRT20-positive cells were observed in basal epidermis of ESS by 2 weeks after grafting. By 4 weeks, these cells increased in number and expressed keratins 18 and 19, additional Merkel cells markers. Putative Merkel cell numbers increased further between weeks 6 and 14; their densities varied widely and no specific pattern of organization was observed, similar to Merkel cell localization in human skin. KRT20-positive cells co-expressed epidermal markers E-cadherin and keratin 15, suggesting derivation from the epidermal lineage, and neuroendocrine markers synaptophysin and chromogranin A, consistent with their identification as Merkel cells. By 4 weeks after grafting, some Merkel cells in engineered skin were associated with immature afferents expressing neurofilament-medium. By 8 weeks, Merkel cells were complexed with more mature neurons expressing neurofilament-heavy. Positive staining for human leukocyte antigen demonstrated that the Merkel cells in ESS were derived from grafted human cells. The results identify, for the first time, Merkel cell-neurite complexes in engineered skin in vivo. This suggests that fine touch sensation may be restored in ESS after grafting, although this must be confirmed with future functional studies.

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<![CDATA[Antibody responses to Plasmodium vivax Duffy binding and Erythrocyte binding proteins predict risk of infection and are associated with protection from clinical Malaria]]> https://www.researchpad.co/article/5c706793d5eed0c4847c7266

Background

The Plasmodium vivax Duffy Binding Protein (PvDBP) is a key target of naturally acquired immunity. However, region II of PvDBP, which contains the receptor-binding site, is highly polymorphic. The natural acquisition of antibodies to different variants of PvDBP region II (PvDBPII), including the AH, O, P and Sal1 alleles, the central region III-V (PvDBPIII-V), and P. vivax Erythrocyte Binding Protein region II (PvEBPII) and their associations with risk of clinical P. vivax malaria are not well understood.

Methodology

Total IgG and IgG subclasses 1, 2, and 3 that recognize four alleles of PvDBPII (AH, O, P, and Sal1), PvDBPIII-V and PvEBPII were measured in samples collected from a cohort of 1 to 3 year old Papua New Guinean (PNG) children living in a highly endemic area of PNG. The levels of binding inhibitory antibodies (BIAbs) to PvDBPII (AH, O, and Sal1) were also tested in a subset of children. The association of presence of IgG with age, cumulative exposure (measured as the product of age and malaria infections during follow-up) and prospective risk of clinical malaria were evaluated.

Results

The increase in antigen-specific total IgG, IgG1, and IgG3 with age and cumulative exposure was only observed for PvDBPII AH and PvEBPII. High levels of total IgG and predominant subclass IgG3 specific for PvDBPII AH were associated with decreased incidence of clinical P. vivax episodes (aIRR = 0.56–0.68, P≤0.001–0.021). High levels of total IgG and IgG1 to PvEBPII correlated strongly with protection against clinical vivax malaria compared with IgGs against all PvDBPII variants (aIRR = 0.38, P<0.001). Antibodies to PvDBPII AH and PvEBPII showed evidence of an additive effect, with a joint protective association of 70%.

Conclusion

Antibodies to the key parasite invasion ligands PvDBPII and PvEBPII are good correlates of protection against P. vivax malaria in PNG. This further strengthens the rationale for inclusion of PvDBPII in a recombinant subunit vaccine for P. vivax malaria and highlights the need for further functional studies to determine the potential of PvEBPII as a component of a subunit vaccine for P. vivax malaria.

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<![CDATA[Hospitalisations and outpatient visits for undifferentiated fever attributable to scrub typhus in rural South India: Retrospective cohort and nested case-control study]]> https://www.researchpad.co/article/5c7d95f2d5eed0c48473500b

Background

The burden of scrub typhus in endemic areas is poorly understood. This study aimed at estimating the proportion of hospitalisations and outpatient visits for undifferentiated fever in the community that may be attributable to scrub typhus.

Methodology and principal findings

The study was a retrospective cohort with a nested case-control study conducted in the South Indian state of Tamil Nadu. We conducted house-to-house screening in 48 villages (42965 people, 11964 households) to identify hospitalised or outpatient cases due to undifferentiated fever during the preceding scrub typhus season. We used scrub typhus IgG to determine past infection. We calculated adjusted odds ratios for the association between IgG positivity and case status. Odds ratios were used to estimate population attributable fractions (PAF) indicating the proportion of hospitalised and outpatient fever cases attributable to scrub typhus. We identified 58 cases of hospitalisation and 236 outpatient treatments. 562 people were enrolled as control group to estimate the background IgG sero-prevalence. IgG prevalence was 20.3% in controls, 26.3% in outpatient cases and 43.1% in hospitalised cases. The PAFs suggested that 29.5% of hospitalisations and 6.1% of outpatient cases may have been due to scrub typhus. In villages with a high IgG prevalence (defined as ≥15% among controls), the corresponding PAFs were 43.4% for hospitalisations and 5.6% for outpatients. The estimated annual incidence of scrub typhus was 0.8/1000 people (0.3/1000 in low, and 1.3/1000 in high prevalence villages). Evidence for recall error suggested that the true incidences may be about twice as high as these figures.

Conclusions

The study suggests scrub typhus as an important cause for febrile hospitalisations in the community. The results confirm the adequacy of empirical treatment for scrub typhus in hospitalised cases with undifferentiated fever. Since scrub typhus may be rare among stable outpatients, the use of empirical treatment remains doubtful in these.

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<![CDATA[Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation]]> https://www.researchpad.co/article/5c803c6cd5eed0c484ad893f

Hepatitis C virus (HCV) infection remains a major cause of hepatic inflammation and liver disease. HCV triggers NLRP3 inflammasome activation and interleukin-1β (IL-1β) production from hepatic macrophages, or Kupffer cells, to drive the hepatic inflammatory response. Here we examined HCV activation of the NLRP3 inflammasome signaling cascade in primary human monocyte derived macrophages and THP-1 cell models of hepatic macrophages to define the HCV-specific agonist and cellular processes of inflammasome activation. We identified the HCV core protein as a virion-specific factor of inflammasome activation. The core protein was both necessary and sufficient for IL-1β production from macrophages exposed to HCV or soluble core protein alone. NLRP3 inflammasome activation by the HCV core protein required calcium mobilization linked with phospholipase-C activation. Our findings reveal a molecular basis of hepatic inflammasome activation and IL-1β release triggered by HCV core protein.

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<![CDATA[Exposure of Candida albicans β (1,3)-glucan is promoted by activation of the Cek1 pathway]]> https://www.researchpad.co/article/5c5ca280d5eed0c48441e4da

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of β (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of β (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in β (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of β (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased β (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

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<![CDATA[Serum amyloid A and Janus kinase 2 in a mouse model of diabetic kidney disease]]> https://www.researchpad.co/article/5c6f149fd5eed0c48467a449

Background

Serum amyloid A (SAA), a potent inflammatory mediator, and Janus kinase 2 (JAK2), an intracellular signaling kinase, are increased by diabetes. The aims were to elucidate: 1) a JAK2-mediated pathway for increased SAA in the kidneys of diabetic mice; 2) a JAK2-SAA pathway for inflammation in podocytes.

Methods

Akita diabetic mice (129S6) with podocyte JAK2 overexpression and angiotensin II infusion (4 weeks) were given a JAK1,2 inhibitor (LY03103801, 3 mg/kg/day orally for the last two weeks). Kidneys were immunostained for SAA isoform 3 (SAA3). SAA3 knockout and control mouse podocytes were exposed to advanced glycation end products (AGE) or exogenous SAA with JAK2 inhibition (Tyrphostin AG 490, 50μM). JAK2 activity (phosphorylation, Western blot, 1 hour) and mRNA for SAA3 and associated inflammatory genes (Cxcl5, Ccl2, and Ccl5) were measured by RT-PCR (20 hours).

Results

SAA3 protein was present throughout the diabetic kidney, and podocyte JAK2 overexpression increased tubulointerstitial SAA3 compared to wild type diabetic controls, 43% versus 14% (p = 0.007); JAK1,2 inhibition attenuated the increase in SAA3 to 15% (p = 0.003). Urine albumin-to-creatinine ratio (r = 0.49, p = 0.03), mesangial index (r = 0.64, p = 0.001), and glomerulosclerosis score (r = 0.51, p = 0.02) were associated with SAA3 immunostaining scores across mouse groups. Exposing podocytes to AGE or exogenous SAA increased JAK2 activity within one hour and mRNA for associated inflammatory genes after 20 hours. JAK2 inhibition reduced SAA3 mRNA expression in podocytes exposed to AGE or SAA. SAA3 knockout podocytes had >85% lower AGE-induced inflammatory genes.

Conclusion

JAK1,2 inhibition reduced SAA and histological features of DKD in podocyte JAK2-overexpressing mice. In podocytes exposed to a diabetes-like condition, JAK2 inhibition reduced expression of SAA, while SAA knockout blocked expression of associated pro-inflammatory mediators. SAA may promote JAK2-dependent inflammation in the diabetic kidney.

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<![CDATA[Bivalent oral cholera vaccination induces a memory B cell response to the V. cholerae O1-polysaccharide antigen in Haitian adults]]> https://www.researchpad.co/article/5c5ca2c6d5eed0c48441eaae

The bivalent killed whole-cell oral cholera vaccine (BivWC) is being increasingly used to prevent cholera. The presence of O-antigen-specific memory B cells (MBC) has been associated with protective immunity against cholera, yet MBC responses have not been evaluated after BivWC vaccination. To address this knowledge gap, we measured V. cholerae O1-antigen MBC responses following BivWC vaccination. Adults in St. Marc, Haiti, received 2 doses of the BivWC vaccine, Shanchol, two weeks apart. Participants were invited to return at days 7, 21, 44, 90, 180 and 360 after the initial vaccination. Serum antibody and MBC responses were assessed at each time-point before and following vaccination. We observed that vaccination with BivWC resulted in significant O-antigen specific MBC responses to both Ogawa and Inaba serotypes that were detected by day 21 and remained significantly elevated over baseline for up to 12 months following vaccination. The BivWC oral cholera vaccine induces durable MBC responses to the V. cholerae O1-antigen. This suggests that long-term protection observed following vaccination with BivWC could be mediated or maintained by MBC responses.

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<![CDATA[Ornithine decarboxylase antizyme inhibitor 2 (AZIN2) is a signature of secretory phenotype and independent predictor of adverse prognosis in colorectal cancer]]> https://www.researchpad.co/article/5c70673fd5eed0c4847c6c9d

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. The two ODC antizyme inhibitors (AZIN1) and (AZIN2) are regulators of the catalytic activity of ODC. While AZIN1 is a regulator of cell proliferation, AZIN2 is involved in intracellular vesicle transport and secretion. There are no previous reports on the impact of AZIN2 expression in human cancer. We applied immunohistochemistry with antibodies to human AZIN2 on tissue micro- arrays of colorectal cancers (CRC) from 840 patients with a median follow-up of 5.1 years (range 0–25.8). The 5-year disease-specific survival rate was 58.9% (95% Cl 55.0–62.8%). High AZIN2 expression was associated with mucinous histology (p = 0.002) and location in the right hemicolon (p = 0.021). We found no association with age, gender, stage, or histological tumor grade. High tumor expression of AZIN2 predicted an unfavorable prognosis (p<0.0001, log-rank test), compared to low AZIN2 expression. Cox multivariable analysis identified AZIN2 as an independent factor of an unfavorable prognosis in CRC. The strongest AZIN2 expression was seen in invasive tumor cells having morphological features of epithelial-mesenchymal transition (EMT). Induction of EMT in HT-29 CRC cells lead to upregulated expression of endogenous AZIN2. Given that AZIN2 is a regulator of vesicle transport and secretion, we overexpressed human AZIN2 cDNA in T84 CRC cells, and found strongly enhanced accumulation of CD63-positive exosomes in the culture medium. These findings indicate that AZIN2 expression is a signature of EMT-associated secretory phenotype that is linked to an adverse prognosis in CRC.

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<![CDATA[Experimental Zika virus infection of Jamaican fruit bats (Artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells]]> https://www.researchpad.co/article/5c61e905d5eed0c48496f66d

The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1+ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations.

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