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Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi-organ failure, and death. convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID-19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS-CoV-2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS-CoV-2 antibody profiles of pooled lyophilized pathogen-reduced CCP from COVID-19-recovered blood donors retains virus-neutralizing efficacy as reported for frozen pathogen-reduced CCP., Methods: Pooled lyophilized pathogen-reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS-CoV-2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared., Results: The mean ratio for antibody binding to SARS-CoV-2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment., Conclusions: The antibody activity in pooled pathogen-reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization., (© 2023 AABB.)

Background: While others have reported severe acute respiratory syndrome-related coronavirus 2(SARS-CoV-2) seroprevalence studies in health care workers (HCWs), we leverage the use of a highly sensitive coronavirus antigen microarray to identify a group of seropositive health care workers who were missed by daily symptom screening that was instituted prior to any epidemiologically significant local outbreak. Given that most health care facilities rely on daily symptom screening as the primary method to identify SARS-CoV-2 among health care workers, here, we aim to determine how demographic, occupational, and clinical variables influence SARS-CoV-2 seropositivity among health care workers., Methods: We designed a cross-sectional survey of HCWs for SARS-CoV-2 seropositivity conducted from May 15th to June 30th 2020 at a 418-bed academic hospital in Orange County, California. From an eligible population of 5,349 HCWs, study participants were recruited in two ways: an open cohort, and a targeted cohort. The open cohort was open to anyone, whereas the targeted cohort that recruited HCWs previously screened for COVID-19 or work in high-risk units. A total of 1,557 HCWs completed the survey and provided specimens, including 1,044 in the open cohort and 513 in the targeted cohort. Demographic, occupational, and clinical variables were surveyed electronically. SARS-CoV-2 seropositivity was assessed using a coronavirus antigen microarray (CoVAM), which measures antibodies against eleven viral antigens to identify prior infection with 98% specificity and 93% sensitivity., Results: Among tested HCWs (n = 1,557), SARS-CoV-2 seropositivity was 10.8%, and risk factors included male gender (OR 1.48, 95% CI 1.05-2.06), exposure to COVID-19 outside of work (2.29, 1.14-4.29), working in food or environmental services (4.85, 1.51-14.85), and working in COVID-19 units (ICU: 2.28, 1.29-3.96; ward: 1.59, 1.01-2.48). Amongst 1,103 HCWs not previously screened, seropositivity was 8.0%, and additional risk factors included younger age (1.57, 1.00-2.45) and working in administration (2.69, 1.10-7.10)., Conclusion: SARS-CoV-2 seropositivity is significantly higher than reported case counts even among HCWs who are meticulously screened. Seropositive HCWs missed by screening were more likely to be younger, work outside direct patient care, or have exposure outside of work., (© 2023. The Author(s).)

Background: The authors conducted a 2-year retrospective follow-up to investigate the efficiency of an extraforaminal full-endoscopic approach with foraminoplasty used to treat lateral compressive diseases of the lumbar spine in 247 patients., Methods: The visual analogue scale (VAS), Oswestry disability index (ODI), and MacNab scale were used to analyze the results collected during the preoperative and postoperative periods., Results: The most common diagnosis was disk herniation with lateral recess stenosis, and the most common surgical level among patients was between L4 and L5 on the left side. Pain decreased over time, as determined during sessions held to evaluate pain in the lumbar, gluteal, led, and foot regions. The ODI demonstrated significant enhancement over the evaluation period and the MacNab scale classified the surgery as good or excellent. The most common complication was dysesthesia., Conclusions: An extraforaminal full-endoscopic approach with foraminoplasty can be recommended in cases of lateral herniation or stenosis for patients with symptoms of radiculopathy, and for those who have not responded to conventional rehabilitation treatment or chronic pain management. Few complications arose as a result of this approach, and most of them were treated clinically.

Flavivirus are the most alarming prevalent viruses worldwide due to its vast impact on public health. Most early symptoms of diseases caused by Flavivirus are similar among each other and to other febrile illnesses making the clinical differential diagnosis challenging. In addition, due to cross-reactivity and a relatively limited persistence of viral RNA in infected individuals, the current available diagnosis strategies fail to efficiently provide a differential viral identification. In this context, virus-specific tests are essential to improve patient care, as well as to facilitate disease surveillance and the effective control of transmission. Here, we describe the use of protein microarrays as an effective tool for screening peptides differentially recognized by anti-Yellow Fever virus antibodies induced by vaccination or by natural viral infection., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Efficacy of donated COVID-19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics., Study Design and Methods: In a single-center hypothesis-generating prospective case-control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID-19 pneumonia received 2 × 200 ml pathogen-reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in COVID-19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28-day mortality, and dCCP -related adverse events in recipients., Results: Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP-related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV-2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS-CoV-2 antigens over 14-21 days post dCCP in all except 4 immunosuppressed recipients., Discussion: PRT did not impact dCCP anti-virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post-transfusion antibody responses indicate the need for controlled trials using well-characterized dCCP with informative assays., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Many studies have emphasized the importance of interface contact between implants and the vertebral endplate (VE). The goal of this study was to analyze the shape and other specific parameters of the VE to provide reference data for better implant interface contact in intervertebral disc space procedures., Methods: Cervical, thoracic, and lumbar spine midsagittal plane magnetic resonance images of 100 adults (58 women) were analyzed. The morphology of the VEs was classified as concave, convex, flat, or irregular. Midsagittal endplate length (ML), endplate concavity depth (ECD), and endplate concavity axis (ECA) location were measured in the midsagittal plane. The parameters were compared between the cervical, thoracic, and lumbar spines and between the sexes., Results: The VE morphology, ML, ECD, and ECA showed variations along the spine, mainly in the cervical and lower lumbar spines. The sagittal geometry of the VE was not flat or uniform along the cervical, thoracic, and lumbar spines. Different morphological types were observed along different spinal segments and according to sex. In the cervical spine, the majority of cranial VEs were flat, while caudal VEs were mostly concave., Conclusion: Sagittal VE geometry should be taken into consideration during the use of intervertebral cages or disc arthroplasty., Competing Interests: Declaration of Conflicting Interests: The authors report no conflicts of interest in this work., (This manuscript is generously published free of charge by ISASS, the International Society for the Advancement of Spine Surgery. Copyright © 2022 ISASS. To see more or order reprints or permissions, see http://ijssurgery.com.)

Yellow Fever disease is caused by the Yellow Fever virus (YFV), an arbovirus from the Flaviviridae family. The re-emergence of Yellow Fever (YF) was facilitated by the increasing urbanization of sylvatic areas, the wide distribution of the mosquito vector, and the low percentage of people immunized in the Americas, which caused severe outbreaks in recent years, with a high mortality rate. Therefore, serological approaches capable of discerning antibodies generated from the wild-type (YFV-WT) strain between the vaccinal strain (YFV-17DD) could facilitate vaccine coverage surveillance, enabling the development of strategies to avoid new outbreaks. In this study, peptides were designed and subjected to microarray procedures with sera collected from individuals infected by WT-YFV and 17DD-YFV of YFV during the Brazilian outbreak of YFV in 2017/2018. From 222 screened peptides, around ten could potentially integrate serological approaches aiming to differentiate vaccinated individuals from naturally infected individuals. Among those peptides, one was synthesized and validated through ELISA.

Introduction: The objective of this study was to describe a surgical technique that uses transforaminal full-endoscopic access, which is different from the existing protocol, and to demonstrate another method of dural tear repair during endoscopic spine surgery., Background: Endoscopic spine surgery was initially described for lumbar disc pathologies. Technical advances and new materials have made it possible to treat cervical and thoracic spinal degenerative disorders. These advances have also made it possible to treat surgical complications, notably dural tears with CSF fistulas. The literature indicates that the incidence of these injuries ranges from 1% to 17%., Materials and Methods: Descriptive technical note of innovative and improved endoscopic surgical procedure exemplified with illustrative clinical case and comparative literature review., Results: There is only one report describing a full-endoscopic suture technique for dural sac repair. The gold standard for treatment of the most significant nonpunctate lesions continues to be a conversion to open surgery for lesion closure. Conversion can be problematic because most surgeries are performed under sedation and local anesthesia., Conclusions: In this case report and the new endoscopic suture technique described here, we show that primary correction of dural tears through endoscopy is possible. In addition to representing a paradigm break in solving one of the main complications of these procedures, it can expand the possibilities of spine endoscopy., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 João Paulo Machado Bergamaschi et al.)

Background: COVID-19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID-19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion-transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA-PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS-CoV-2 neutralizing antibodies., Study Design and Methods: This study assessed the impact of A/UVA-PRT on SARS-CoV-2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed., Results: A/UVA-PRT did not negatively impact antibodies to SARS-CoV-2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross-reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses., Conclusion: The findings of this study support the selection of effective CCP combined with the use of A/UVA-PRT in the production of CCP for patients with COVID-19., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Knowledge of the Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates., (© 2021. The Author(s).)

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT 50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses., Author Summary: The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoniae . B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae , but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity., Competing Interests: GW was employed by the company Novartis Institute for BioMedical Research. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ercoli, Ramos-Sevillano, Nakajima, de Assis, Jasinskas, Goldblatt, Felgner, Weckbecker and Brown.)

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes., Competing Interests: Competing Interests The coronavirus antigen microarray is intellectual property of the Regents of the University of California that is licensed for commercialization to Nanommune Inc. (Irvine, CA), a private company for which P. Felgner is the largest shareholder and several coauthors (R. de Assis, A. Jain, R. Nakajima, A. Jasinskas, J. Obiero, H. Davies, and S. Khan) also own shares. Nanommune Inc. has a business partnership with Sino Biological Inc. (Beijing, China) which expressed and purified the antigens used in this study. The convalescent plasma used in this study was collected for clinical use by independent blood centers using licensed plasma or platelet processing systems manufactured by Cerus Corporation, for which multiple authors (L. Corash, A. Bagri) are shareholders and employees. Convalescent sera were also provided by Ortho Clinical Diagnostics, which is using these specimens to validate a clinical diagnostic test, and P. Hosimer, C. Noesen, and P. Contestable are shareholders and employees of this company. M. Battegay, A. Buser and A. Holbro are employees of the University of Basel and have no conflicts of interest.

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

Dengue virus (DENV) is a Flavivirus estimated to cause 390 million infections/year. Currently, there is no anti-viral specific treatment for dengue, and efficient DENV vector control is still unfeasible. Here, we designed and produced chimeric proteins containing potential immunogenic epitopes from the four DENV serotypes in an attempt to further compose safer, balanced tetravalent dengue vaccines. For this, South American DENV isolate sequences were downloaded from the NCBI/Virus Variation/Dengue virus databases and intraserotype-aligned to generate four consensuses. Four homologous DENV sequences were retrieved using BLAST and then interserotype-aligned. In parallel, sequences were subjected to linear B epitope prediction analysis. Regions of the envelope and NS1 proteins that are highly homologous among the four DENV serotypes, non-conserved antigenic regions and the most antigenic epitopes found in the C, prM, E and NS1 DENV proteins were used to construct 11 chimeric peptides. Genes encoding the chimeric proteins were commercially synthesized, and proteins were expressed, purified by affinity chromatography and further subjected to ELISA assays using sera from individuals infected with DENVs 1, 2, 3 or 4. As a proof-of-concept, the chimeric EnvEpII protein was selected to immunize BALB/c and C57BL/6 mice strains. The immunization with EnvEpII protein associated with aluminum induced an increased number of T CD4 + and CD8 + cells, high production of IgG 1 and IgG 2 antibodies, and increased levels of IL-2 and IL-17 cytokines, in both mouse strains. Because the EnvEpII protein associated with aluminum induced an efficient cellular response by stimulating the production of IL-2, IL-4, IL-17 and induced a robust humoral response in mice, we conclude that it resembles an efficient specific response against DENV infection. Although further experiments are required, our results indicate that epitope selection by bioinformatic tools is efficient to create recombinant proteins that can be used as candidates for the development of vaccines against infectious diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

The inflammatory process plays a major role in the prognosis of dengue. In this context, the eicosanoids may have considerable influence on the regulation of the Dengue virus -induced inflammatory process. To quantify the molecules involved in the cyclooxygenase and lipoxygenase pathways during Dengue virus infection, plasma levels of thromboxane A2, prostaglandin E2 and leukotriene B4; mRNA levels of thromboxane A2 synthase, prostaglandin E2 synthase, leukotriene A4 hydrolase, cyclooxygenase-2 and 5-lipoxygenase; and the levels of lipid bodies in peripheral blood leukocytes collected from IgM-positive and IgM-negative volunteers with mild dengue, and non-infected volunteers, were evaluated. Dengue virus infection increases the levels of thromboxane A2 in IgM-positive individuals as well as the amount of lipid bodies in monocytes in IgM-negative individuals. We suggest that increased levels of thromboxane A2 in IgM-positive individuals plays a protective role against the development of severe symptoms of dengue, such as vascular leakage., Competing Interests: The authors declare no conflict of interest.

Background: Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes. Its species-specific polymorphisms are found mainly in the sugars that branch off the conserved Gal(β1,4)Man(α1)-PO 4 backbone of repeat units. Leishmania amazonensis is one of the most important species causing human cutaneous leishmaniasis in the New World. Here, we describe LPG intraspecific polymorphisms in two Le. amazonensis reference strains and their role during the development in three sand fly species., Results: Strains isolated from Lutzomyia flaviscutellata (PH8) and from a human patient (Josefa) displayed structural polymorphism in the LPG repeat units, possessing side chains with 1 and 2 β-glucose or 1 to 3 β-galactose, respectively. Both strains successfully infected permissive vectors Lutzomyia longipalpis and Lutzomyia migonei and could colonize their stomodeal valve and differentiate into metacyclic forms. Despite bearing terminal galactose residues on LPG, Josefa could not sustain infection in the restrictive vector Phlebotomus papatasi., Conclusions: LPG polymorphisms did not affect the ability of Le. amazonensis to develop late-stage infections in permissive vectors. However, the non-establishment of infection in Ph. papatasi by Josefa strain suggested other LPG-independent factors in this restrictive vector.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance (SmSULT-OR) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1-11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27-0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A proteome microarray consisting of 992 Schistosoma mansoni proteins was produced and screened with sera to determine antibody signatures indicative of the clinical stages of schistosomiasis and the identification of subunit vaccine candidates. Herein, we describe the methods used to derive the gene list for this array (representing approximately 10% of the predicted S. mansoni proteome). We also probed a pilot version of the microarray with sera from individuals either acutely or chronically infected with S. mansoni from endemic areas in Brazil and sera from individuals resident outside the endemic area (USA) to determine if the array is functional and informative., (Copyright © 2016. Published by Elsevier Ltd.)

In this work, some aspects of the glycobiology of Leishmania shawi were examined, as it is a causative agent of cutaneous leishmaniasis in the New World. Additionally, the interaction of L. shawi's main glycoconjugates [lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs)] with macrophages was evaluated in vitro. L. shawi LPG was devoid of side-chains in its repeat units, whereas monosaccharide analysis showed that GIPLs were suggestive of mannose-rich (type I or hybrid). In order to evaluate the biological roles of those molecules, BALB/c resident peritoneal macrophages were incubated with these glycoconjugates for 24h, and the levels of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-12p70 and IL-10, were determined. In general, the GIPLs exhibited a greater proinflammatory role than the LPGs did. However, for the first time, the GIPLs from this species were able to trigger the production of IL-10, an anti-inflammatory cytokine. In conclusion, L. shawi glycoconjugates were able to interact with the innate immune compartment. These data reinforce the role of parasite glycoconjugates during parasite and host cell interactions., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown., Methods: Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively., Results: LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO4. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF-α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE)., Conclusion: The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.

Background: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units., Methods: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated., Results: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB., Conclusion: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions.

Background: Protozoan parasites of the genus Leishmania cause a number of important diseases in humans and undergo a complex life cycle, alternating between a sand fly vector and vertebrate hosts. The parasites have a remarkable capacity to avoid destruction in which surface molecules are determinant for survival. Amongst the many surface molecules of Leishmania, the glycoconjugates are known to play a central role in host-parasite interactions and are the focus of this review., Scope of the Review: The most abundant and best studied glycoconjugates are the Lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs). This review summarizes the main studies on structure and biological functions of these molecules in New World Leishmania species., Major Conclusions: LPG and GIPLs are complex molecules that display inter- and intraspecies polymorphisms. They are key elements for survival inside the vector and to modulate the vertebrate immune response during infection., General Significance: Most of the studies on glycoconjugates focused on Old World Leishmania species. Here, it is reported some of the studies involving New World species and their biological significance on host-parasite interaction. This article is part of a Special Issue entitled Glycoproteomics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T. cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. The significance of these variations was assessed after analysis of the terminal α-galactosyl residues. Enzymatic treatment with α-galactosidase indicated a differential expression of O-linked α-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T. cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T. cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I).

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2-/- and TLR4-/-) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)