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Background: Tripartite motif-containing 27 (TRIM27) has been implicated in the progression of various cancers. However, the role of TRIM27 in sinonasal mucosal melanoma (SNMM) remains poorly understood. Materials and Methods: We retrospectively examined 28 patients with SNMM treated with between 2003 and 2021. We undertook immunohistochemical analysis of TRIM27, Ki-67, and p-Akt1 expression in SNMM tissues. We also investigated the relationship between TRIM27 expression and clinical characteristics, prognosis, Ki-67 as a tumor growth potential marker, and p-Akt1 as one of the prognostic factors in mucosal melanoma. Results: TRIM27 expression was significantly higher in T4 disease than in T3 disease and was higher in stage IV than in stage III. Patients with high-TRIM27 SNMM had a significantly poorer prognosis in terms of overall survival (OS) and disease-free survival.There was also a significantly higher rate of distant metastasis. Univariate analysis for OS revealed that TRIM27 and T classification were significant poor prognostic factors. In addition, the Ki-67 positive score and the p-Akt1 total staining score were significantly higher in the high-TRIM27 group than in the low-TRIM27 group. Conclusions: High TRIM27 expression in SNMM was associated with advanced T classification, poor prognosis and distant metastasis. We suggest that TRIM27 has potential as a novel biomarker for prognosis in SNMM.

Rapid electrical analysis of chemical liquids is a promising technique for on-site evaluation. In this study, the electrical impedance response of insulator nanoparticle films with condensed chemical vapors was investigated in structural isomers and polar aprotic chemical liquids. Headspace vapor was condensed in the nanoscale void between the nanoparticles, and ionic conduction subsequently occurred under an AC voltage. The transient electrical impedance response depends on the vapor pressure and conductivity of the liquid isomers. A chemical liquid of the structural isomers was identified by monitoring the impedance during exposure to its headspace vapor. The response time of the film impedance was 10.6, 4.7, 7.5, and 2.4 s for 1-butanol, 2-butanol, 2-methyl-1-propanol, and tert-butyl alcohol, respectively. Furthermore, the current conduction mechanism in the polar aprotic chemicals was discussed. Although these chemicals did not form molecular networks with the hydrogen bonds, the electrical current flowed in the system. We proposed that hydrogen bonds mediated by water molecules were formed and proton hopping through the condensed polar aprotic liquid occurred. This proposed method has the potential to detect protic and aprotic polar chemical vapors.

Plasmodium vivax blood-stage invasion into reticulocyte is critical for parasite development. Thus, validation of novel parasite invasion ligands is essential for malaria vaccine development. Recently, we demonstrated that EBP2, a Duffy binding protein (DBP) paralog, is antigenically distinct from DBP and could not be functionally inhibited by anti-DBP antibodies. Here, we took advantage of a small outbreak of P.vivax malaria, located in a non-malarious area of Brazil, to investigate for the first time IgM/IgG antibodies against EBP2 and DEKnull-2 (an engineering DBPII vaccine) among individuals who had their first and brief exposure to P.vivax (16 cases and 22 non-cases). Our experimental approach included 4 cross sectional surveys at 3-month interval (12-month follow-up). The results demonstrated that while a brief initial P.vivax infection was not efficient to induce IgM/ IgG antibodies to either EBP2 or DEKnull-2, IgG antibodies against DEKnull-2 (but not EBP2) were boosted by recurrent blood-stage infections following treatment. Of interest, in most recurrent P. vivax infections (4 out of 6 patients) DEKnull-2 IgG antibodies were sustained for 6 to 12 months. Polymorphisms in the ebp2 gene does not seem to explain EBP2 low immunogenicity as the ebp2 allele associated with the P.vivax outbreak presented high identity to the original EBP2 isolate used as recombinant protein. Although EBP2 antibodies were barely detectable after a primary episode of P.vivax infection, EBP2 was highly recognized by serum IgG from long-term malaria-exposed Amazonians (range from 35 to 92% according to previous malaria episodes). Taken together, the results showed that individuals with a single and brief exposure to P.vivax infection develop very low anti-EBP2 antibodies, which tend to increase after long-term malaria exposure. Finally, the findings highlighted the potential of DEKnull-2 as a vaccine candidate, as in non-immune individuals anti-DEKnull-2 IgG antibodies were boosted even after a brief exposure to P.vivax blood stages.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII–DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Individuals with asymptomatic infection due to Plasmodium vivax are posited to be important reservoirs of malaria transmission in endemic regions. Here we studied a cohort of P. vivax malaria patients in a suburban area in the Brazilian Amazon. Overall 1,120 individuals were screened for P. vivax infection and 108 (9.6%) had parasitemia detected by qPCR but not by microscopy. Asymptomatic individuals had higher levels of antibodies against P. vivax and similar hematological and biochemical parameters compared to uninfected controls. Blood from asymptomatic individuals with very low parasitemia transmitted P. vivax to the main local vector, Nyssorhynchus darlingi. Lower mosquito infectivity rates were observed when blood from asymptomatic individuals was used in the membrane feeding assay. While blood from symptomatic patients infected 43.4% (199/458) of the mosquitoes, blood from asymptomatic infected 2.5% (43/1,719). However, several asymptomatic individuals maintained parasitemia for several weeks indicating their potential role as an infectious reservoir. These results suggest that asymptomatic individuals are an important source of malaria parasites and Science and Technology for Vaccines granted by Conselho Nacional de may contribute to the transmission of P. vivax in low-endemicity areas of malaria., Author summary Malaria still poses as one of the most important parasitic diseases in the world. The advance of molecular diagnosis brought to light the existence of asymptomatic infections, which may represent most of the infections in some areas. Importantly, the role of asymptomatic carriers in the natural history of malaria is not completely understood. Herein we describe the general characteristics of asymptomatic individuals infected with Plasmodium vivax, and provide evidence of their potential as parasitic reservoirs, even when molecular methods fail to detect the infection. Our findings reinforce the need for better diagnostic tests and open a new window of complexity to be considered in control programs.

Malaria remains a major public health problem worldwide, and Plasmodium vivax is the most widely distributed malaria parasite. Naturally acquired binding inhibitory antibodies (BIAbs) to region II of the Duffy binding protein (DBPII), a P. vivax ligand that is critical for reticulocyte invasion, are associated with a reduced risk of clinical malaria. Owing to methodological issues in evaluating antibodies that inhibit the DBPII–DARC interaction, a limited number of studies have investigated DBPII BIAbs in P. vivax-exposed populations. Based on the assumption that individuals with a consistent BIAb response are characterized by strain-transcending immune responses, we hypothesized that detecting broadly reactive DBPII antibodies would indicate the presence of BIAb response. By taking advantage of an engineered DBPII immunogen targeting conserved DBPII neutralizing epitopes (DEKnull-2), we standardized a multiplex flow cytometry-based serological assay to detect broadly neutralizing IgG antibodies. For this study, a standard in vitro cytoadherence assay with COS-7 cells expressing DBPII was used to test for DBPII BIAb response in long-term P. vivax-exposed Amazonian individuals. Taken together, the results demonstrate that this DBPII-based multiplex assay facilitates identifying DBPII BIAb carriers. Of relevance, the ability of the multiplex assay to identify BIAb responders was highly accurate when the positivity for all antigens was considered. In conclusion, the standardized DBPII-based flow cytometric assay confirmed that DBPII-BIAb activity was associated with the breadth rather than the magnitude of anti-DBPII antibodies. Altogether, our results suggest that multiplex detection of broadly DBPII-reactive antibodies facilitates preliminary screening of BIAb responders.

Density functional theory (TDDFT) in real-time, which is expected to be a key technique to calculate optical spectra. We solve the time-dependent equation, keeping track of the dipole moment as time-series data. In the traditional optical analysis, the oscillator strength distribution is related to the imaginary part of the polarizability, which is usually calculated by the Fourier transform (FT) of the dipole moment. Theoretically, it works only if the time-series data is quite large. To obtain spectra of fairly high resolution with a relatively small number of time-series data, we have recognized that, from our analysis, even simple MEM provides the oscillator strength distribution at high resolution even with a half of the evolution time of a simple FT. In the practical optical analysis, we are much interested in the lower energy region near the band gap to obtain photo absorption and emission spectra. However, long enough time-evolution is still required in the calculation. We propose that, as a further improved MEM analysis, we use the concatenated data set made from the several-times repeated raw data. In this process, we also introduce an appropriate phase for the target peak frequency to reduce the side effect of the artificial periodicity. In this procedure, we can effectively take into account the information from large lags of autocorrelation, which represent the interesting signal in the lower energy region. We have compared the result of our improved MEM and that of FT for the analysis applied to the time-series data in the spectrum analysis of small-to-medium size molecules. As a result, we can observe the clear spectrum of MEM. Our new technique provides higher resolution in fewer time steps, compared to that of FT.

BackgroundPatients (pts) with rheumatoid arthritis (RA) and poor prognostic factors1 (PPF) are at risk for progression without adequate treatment. Filgotinib (FIL) is a once daily Janus kinase 1 preferential inhibitor. In FINCH 1 (NCT02889796), FIL 200 mg (FIL200) was effective vs placebo (PBO) and noninferior to adalimumab (ADA) in pts with RA and inadequate response to methotrexate (MTX-IR); FIL200 and FIL 100 mg (FIL100) were well tolerated.2ObjectivesThis post hoc, exploratory analysis examined efficacy and safety of FIL in MTX-IR pts with 4 or MethodsThe FINCH 1 52-week (W), double-blind trial randomised MTX-IR pts with moderate–severe RA to FIL200 or FIL100, ADA, or PBO; all received background MTX. PBO pts were rerandomised, blinded, at W24 to FIL200 or FIL100. We examined pts with 4 PPF at baseline (BL): erosions on X-ray, seropositivity for rheumatoid factor or anticyclic citrullinated peptide, high-sensitivity C-reactive protein (hsCRP) ≥6 mg/L, and disease activity score in 28 joints with CRP (DAS28[CRP]) >5.1, along with those with P values were nominal, not adjusted for multiplicity.ResultsAt BL, of 1755 randomized, treated pts, 687 had 4 PPF, and 1068 had 5.1). Pts with 4 vs P P P >.05). At W52, mTSS CFB in 4 PPF pts was 0.29, 0.84, and 0.80 for FIL200, FIL100, and ADA, respectively, and 0.14, 0.25, and 0.53 in Table 1.AEs and AEs of interest in BL 4 PPF and 4 PPFFIL200FIL100ADAPBO beforeFIL200FIL100ADAPBO(n = 191)(n = 189)(n = 126)W24 switch (n = 181)(n = 284)(n = 291)(n = 199)before W24 switch (n = 294)All AEs146 (76.4)136 (72.0)85 (67.5)90 (49.7)206 (72.5)214 (73.5)154 (77.4)164 (55.8)AEs of interestSerious infectious AE5 (2.6)4 (2.1)6 (4.8)2 (1.1)8 (2.8)9 (3.1)4 (2.0)2 (0.7)Opportunistic infections001 (0.8)0001 (0.5)0Active tuberculosis0000001 (0.5)0Herpes zoster1 (0.5)1 (0.5)1 (0.8)05 (1.8)3 (1.0)1 (0.5)2 (0.7)Hepatitis B or C01 (0.5)001 (0.5)01 (0.5)0MACE01 (0.5)01 (0.6)01 (0.3)1 (0.5)1 (0.3)VTE001 (0.8)1 (0.6)1 (0.4)001 (0.3)DVT001 (0.8)1 (0.6)0001 (0.3)PE00001 (0.4)000Malignancy0003 (1.7)2 (0.7)2 (0.7)2 (1.0)0(non-NMSC)GI perforation00001 (0.4)000Values are n (%). ADA, adalimumab; AE, adverse event; BL, baseline; DVT, deep vein thrombosis; FIL100, filgotinib 100 mg; FIL200; filgotinib 200 mg; GI, gastrointestinal; MACE, major adverse cardiac event; NMSC, nonmelanoma skin cancer; PBO, placebo; PE, pulmonary embolism; PPF, poor prognostic factor; VTE, venous thromboembolism; W, week.ConclusionIn high-risk (4 PPF) pts with MTX-IR RA, FIL200 and FIL100 showed similar reductions in disease activity vs PBO at W12 as in pts with References[1]Smolen JS et al. Ann Rheum Dis. 2020;79:685–99.[2]Combe B et al. Ann Rheum Dis. 2021;80:848–58.AcknowledgementsThis study was funded by Gilead Sciences, Inc., Foster City, CA. Medical writing support was provided by Rob Coover, MPH, of AlphaScientia, LLC, San Francisco, CA, and was funded by Gilead Sciences, Inc., Foster City, CA. Funding for this analysis was provided by Gilead Sciences, Inc. The sponsors participated in the planning, execution, and interpretation of the research.Disclosure of InterestsBernard Combe Speakers bureau: AbbVie, Bristol-Myers Squibb, Celltrion, Eli Lilly, Gilead-Galapagos, Janssen, Merck Sharp & Dohme, Novartis, Pfizer, and Roche-Chugai, Consultant of: AbbVie, Bristol-Myers Squibb, Celltrion, Eli Lilly, Gilead-Galapagos, Janssen, Merck Sharp & Dohme, Novartis, Pfizer, and Roche-Chugai, Grant/research support from: Pfizer and Roche-Chugai, Yoshiya Tanaka Speakers bureau: AbbVie, Amgen, Astellas, AstraZeneca, Behringer-Ingelheim, Bristol-Myers, Chugai, Eisai, Eli Lilly, Gilead Sciences, Inc., Mitsubishi-Tanabe, and YL Biologics, Paid instructor for: AbbVie, Amgen, Astellas, AstraZeneca, Behringer-Ingelheim, Bristol-Myers, Chugai, Eisai, Eli Lilly, Gilead Sciences, Inc., Mitsubishi-Tanabe, and YL Biologics, Grant/research support from: AbbVie, Asahi-Kasei, Boehringer-Ingelheim, Chugai, Corrona, Daiichi-Sankyo, Eisai, Kowa, Mitsubishi-Tanabe, and Takeda, Maya H Buch Speakers bureau: AbbVie, Eli Lilly, Gilead Sciences, Inc., Merck-Serono, Pfizer, Roche, Sanofi, and UCB, Paid instructor for: AbbVie, Eli Lilly, Gilead Sciences, Inc., Merck-Serono, Pfizer, Roche, Sanofi, and UCB, Consultant of: AbbVie, Eli Lilly, Gilead Sciences, Inc., Merck-Serono, Pfizer, Roche, Sanofi, and UCB, Grant/research support from: AbbVie, Eli Lilly, Gilead Sciences, Inc., Merck-Serono, Pfizer, Roche, Sanofi, and UCB, Gerd Rüdiger Burmester Speakers bureau: AbbVie, Eli Lilly, Pfizer, and Gilead Sciences, Inc., Consultant of: AbbVie, Eli Lilly, Pfizer, and Gilead Sciences, Inc., Beatrix Bartok Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Alena Pechonkina Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Ling Han Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Kahaku Emoto Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences K.K., Shungo Kano Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences K.K., Thijs Hendrikx Employee of: Galapagos BV, Daniel Aletaha Speakers bureau: Bristol-Myers Squibb, Merck Sharp & Dohme, and UCB; AbbVie, Amgen, Celgene, Eli Lilly, Medac, Merck, Novartis, Pfizer, Roche, Sandoz, and Sanofi/Genzyme., Consultant of: AbbVie, Amgen, Celgene, Eli Lilly, Medac, Merck, Novartis, Pfizer, Roche, Sandoz, and Sanofi/Genzyme; Janssen, Grant/research support from: from AbbVie, Merck Sharp & Dohme, Novartis, and Roche

Plasmodium vivax invasion into human reticulocytes is a complex process. The Duffy binding protein (DBP) dimerization with its cognate receptor is vital for junction formation in the invasion process. Due to its functional importance, DBP is considered a prime vaccine candidate, but variation in B-cell epitopes at the dimer interface of DBP leads to induction of strain-limited immunity. We believe that the polymorphic residues tend to divert immune responses away from functionally conserved epitopes important for receptor binding or DBP dimerization. As a proof of concept, we engineered the vaccine DEKnull to ablate the dominant Bc epitope to partially overcome strain-specific immune antibody responses. Additional surface engineering on the next generation immunogen, DEKnull-2, provides an immunogenicity breakthrough to conserved protective epitopes. DEKnull-2 elicits a stronger broadly neutralizing response and reactivity with long-term persistent antibody responses of acquired natural immunity. By using novel engineered DBP immunogens, we validate that the prime targets of protective immunity are conformational epitopes at the dimer interface. These successful results indicate a potential approach that can be used generally to improve efficacy of other malaria vaccine candidates.

Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro. The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes. IMPORTANCE In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium. Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains.

Plasmodium vivax relapse is one of the major causes of sustained global malaria transmission. Primaquine (PQ) is the only commercial drug available to prevent relapses, and its efficacy is dependent on metabolic activation by cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 function, caused by allelic polymorphisms, leads to the therapeutic failure of PQ as a radical cure for P. vivax malaria. Here, we hypothesized that the host immune response to malaria parasites modulates susceptibility to P. vivax recurrences in association with CYP2D6 activity. We performed a 10-year retrospective study by genotyping CYP2D6 polymorphisms in 261 malaria-exposed individuals from the Brazilian Amazon. The immune responses against a panel of P. vivax blood-stage antigens were evaluated by serological assays. We confirmed our previous findings, which indicated an association between impaired CYP2D6 activity and a higher risk of multiple episodes of P. vivax recurrence (risk ratio, 1.75; 95% confidence interval [CI], 1.2 to 2.6; P = 0.0035). An important finding was a reduction of 3% in the risk of recurrence (risk ratio, 0.97; 95% CI, 0.96 to 0.98; P < 0.0001) per year of malaria exposure, which was observed for individuals with both reduced and normal CYP2D6 activity. Accordingly, subjects with long-term malaria exposure and persistent antibody responses to various antigens showed fewer episodes of malaria recurrence. Our findings have direct implications for malaria control, since it was shown that nonimmune individuals who do not respond adequately to treatment due to reduced CYP2D6 activity may present a significant challenge for sustainable progress toward P. vivax malaria elimination.

In this work, we describe a molecular mechanism of heterologous immunity between two distant species of Plasmodium. Our results suggest a mechanism that subverts the classic parasite strategy of presenting highly polymorphic epitopes in surface antigens to evade immunity to that parasite. This alternative immune pathway can be exploited to protect pregnant women from falciparum placental malaria by designing vaccines to cryptic epitopes that elicit broadly inhibitory antibodies against variant parasite strains., Many pathogens evolve extensive genetic variation in virulence proteins as a strategy to evade host immunity. This poses a significant challenge for the host to develop broadly neutralizing antibodies. In Plasmodium falciparum, we show that a mechanism to circumvent this challenge is to elicit antibodies to cryptic epitopes that are not under immune pressure. We previously discovered that antibodies to the Plasmodium vivax invasion protein, PvDBP, cross-react with P. falciparum VAR2CSA, a distantly related virulence factor that mediates placental malaria. Here, we describe the molecular mechanism underlying this cross-species immunity. We identified an epitope in subdomain 1 (SD1) within the Duffy binding-like (DBL) domain of PvDBP that gives rise to cross-reactive antibodies to VAR2CSA and show that human antibodies affinity purified against a synthetic SD1 peptide block parasite adhesion to chondroitin sulfate A (CSA) in vitro. The epitope in SD1 is subdominant and highly conserved in PvDBP, and in turn, SD1 antibodies target cryptic epitopes in P. falciparum VAR2CSA. The epitopes in VAR2CSA recognized by vivax-derived SD1 antibodies (of human and mouse origin) are distinct from those recognized by VAR2CSA immune serum. We mapped two peptides in the DBL5ε domain of VAR2CSA that are recognized by SD1 antibodies. Both peptides map to regions outside the immunodominant sites, and antibodies to these peptides are not elicited following immunization with VAR2CSA or natural infection with P. falciparum in pregnancy, consistent with the cryptic nature of these target epitopes.

Background For recanalization of coronary chronic total occlusion (CTO) lesions, subintimal guidewire tracking in both antegrade and retrograde approaches are commonly used. Purpose This study aimed to assess the impact of subintimal tracking on long-term clinical outcomes after recanalization of CTO lesions. Methods Between January 2009 and December 2016, 474 CTO lesions (434patients) were successfully recanalized in our center. After guidewire crossing in a CTO lesion, those lesions were divided into intimal tracking group (84.6%, n=401) and subintimal tracking group (15.4%, n=73) according to intravascular ultrasound (IVUS) findings. Long-term clinical outcomes including death, target lesion revascularization (TLR), target vessel revascularization (TVR) were compared between the two groups. In addition, the rate of re-occlusion after successful revascularization was also evaluated. Results The median follow-up period was 4.7 years (interquartile range, 2.8–6.1). There was no significant difference of the rate of cardiac death between the two groups (intimal tracking vs. subintimal tracking: 7.0% vs. 4.1%; hazard ratio, 0.61; 95% confidence interval [CI], 0.19 to 2.00; p=0.41), TLR (14.3% vs. 16.2%; hazard ratio, 1.34; 95% CI, 0.71 to 2.53; p=0.37), and TVR (17.5% vs. 20.3%; hazard ratio, 1.27; 95% CI, 0.72 to 2.23; p=0.42). However, the rate of re-occlusion was significantly higher in the subintimal tracking group than intimal tracking group at 3-years re-occlusion (4.2% vs. 14.5%; log-rank test, p=0.002, Figure). In the multivariate COX regression, subintimal guidewire tracking was an independent predictor of re-occlusion after CTO recanalization (HR: 5.40; 95% CI: 2.11–13.80; p Figure 1 Conclusions Subintimal guidewire tracking for recanalization of coronary CTO was associated with significantly higher incidence of target lesion re-occlusion during long-term follow-up period.

Background The unexpected high proportion of submicroscopic malaria infections in areas with low transmission intensity challenges the control and elimination of malaria in the Americas. The current PCR-based assays present limitations as most protocols still rely on amplification of few-copies target gene. Here, the hypothesis was that amplification of different plasmodial targets—ribosomal (18S rRNA) and non-ribosomal multi-copy sequences (Pvr47 for Plasmodium vivax and Pfr364 for Plasmodium falciparum)—could increase the chances of detecting submicroscopic malaria infection. Methods A non-ribosomal real-time PCR assay targeting Pvr47/Pfr364 (NR-qPCR) was established and compared with three additional PCR protocols, two of them based on 18S rRNA gene amplification (Nested-PCR and R-qPCR) and one based on Pvr47/Pfr364 targets (NR-cPCR). The limit of detection of each PCR protocol, at single and artificial mixed P. vivax/P. falciparum infections, was determined by end-point titration curves. Field samples from clinical (n = 110) and subclinical (n = 324) malaria infections were used to evaluate the impact of using multiple molecular targets to detect malaria infections. Results The results demonstrated that an association of ribosomal and non-ribosomal targets did not increase sensitivity to detect submicroscopic malaria infections. Despite of that, artificial mixed-malaria infections demonstrated that the NR-qPCR was the most sensitive protocol to detect low-levels of P. vivax/P. falciparum co-infections. Field studies confirmed that submicroscopic malaria represented a large proportion (up to 77%) of infections among asymptomatic Amazonian residents, with a high proportion of infections (~ 20%) identified only by the NR-qPCR. Conclusions This study presents a new species-specific non-ribosomal PCR assay with potential to identify low-density P. vivax and P. falciparum infections. As the majority of subclinical infections was caused by P. vivax, the commonest form of malaria in the Amazon area, future studies should investigate the potential of Pvr47/Pfr364 to detect mixed-malaria infections in the field. Electronic supplementary material The online version of this article (10.1186/s12936-019-2781-3) contains supplementary material, which is available to authorized users.

Background. In pregnancy, Plasmodium falciparum parasites express the surface antigen VAR2CSA, which mediates adherence of red blood cells to chondroitin sulfate A (CSA) in the placenta. VAR2CSA antibodies are generally acquired during infection in pregnancy and are associated with protection from placental malaria. We observed previously that men and children in Colombia also had antibodies to VAR2CSA, but the origin of these antibodies was unknown. Here, we tested whether infection with Plasmodium vivax is an alternative mechanism of acquisition of VAR2CSA antibodies. Methods. We analyzed sera from nonpregnant Colombians and Brazilians exposed to P. vivax and monoclonal antibodies raised against P. vivax Duffy binding protein (PvDBP). Cross-reactivity to VAR2CSA was characterized by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry, and antibodies were tested for inhibition of parasite binding to CSA. Results. Over 50% of individuals had antibodies that recognized VAR2CSA. Affinity-purified PvDBP human antibodies and a PvDBP monoclonal antibody recognized VAR2CSA, showing that PvDBP can give rise to cross-reactive antibodies. Importantly, the monoclonal antibody inhibited parasite binding to CSA, which is the primary in vitro correlate of protection from placental malaria. Conclusions. These data suggest that PvDBP induces antibodies that functionally recognize VAR2CSA, revealing a novel mechanism of cross-species immune recognition to falciparum malaria. 0047449

Plasmodium falciparum and Plasmodium vivax have evolved with host switches between non-human primates (NHPs) and humans. Studies on the infection dynamics of Plasmodium species in NHPs will improve our understanding of the evolution of these parasites; however, such studies are hampered by the difficulty of handling animals in the field. The aim of this study was to detect genomic DNA of Plasmodium species from the faeces of New World monkeys. Faecal samples from 23 Alouatta clamitans from the Centre for Biological Research of Indaial (Santa Catarina, Brazil) were collected. Extracted DNA from faecal samples was used for molecular diagnosis of malaria by nested polymerase chain reaction. One natural infection with Plasmodium simium was identified by amplification of DNA extracted from the faeces of A. clamitans. Extracted DNA from a captive NHP was also used for parasite genotyping. The detection limit of the technique was evaluated in vitro using an artificial mixture of cultured P. falciparum in NHP faeces and determined to be 6.5 parasites/µL. Faecal samples of New World primates can be used to detect malaria infections in field surveys and also to monitor the genetic variability of parasites and dynamics of infection.

BackgroundA low proportion of P. vivax-exposed individuals acquire protective strain-transcending neutralizing IgG antibodies that are able to block the interaction between the Duffy binding protein II (DBPII) and its erythrocyte-specific invasion receptor. In a recent study, a novel surface-engineered DBPII-based vaccine termed DEKnull-2, whose antibody response target conserved DBPII epitopes, was able to induce broadly binding-inhibitory IgG antibodies (BIAbs) that inhibit P. vivax reticulocyte invasion. Toward the development of DEKnull-2 as an effective P. vivax blood-stage vaccine, we investigate the relationship between naturally acquired DBPII-specific IgM response and the profile of IgG antibodies/BIAbs activity over time.Methodology/principal findingsA nine-year follow-up study was carried-out among long-term P. vivax-exposed Amazonian individuals and included six cross-sectional surveys at periods of high and low malaria transmission. DBPII immune responses associated with either strain-specific (Sal1, natural DBPII variant circulating in the study area) or conserved epitopes (DEKnull-2) were monitored by conventional serology (ELISA-detected IgM and IgG antibodies), with IgG BIAbs activity evaluated by functional assays (in vitro inhibition of DBPII-erythrocyte binding). The results showed a tendency of IgM antibodies toward Sal1-specific response; the profile of Sal1 over DEKnull-2 was not associated with acute malaria and sustained throughout the observation period. The low malaria incidence in two consecutive years allowed us to demonstrate that variant-specific IgG (but not IgM) antibodies waned over time, which resulted in IgG skewed to the DEKnull-2 response. A persistent DBPII-specific IgM response was not associated with the presence (or absence) of broadly neutralizing IgG antibody response.Conclusions/significanceThe current study demonstrates that long-term exposure to low and unstable levels of P. vivax transmission led to a sustained DBPII-specific IgM response against variant-specific epitopes, while sustained IgG responses are skewed to conserved epitopes. Further studies should investigate on the role of a stable and persistent IgM antibody response in the immune response mediated by DBPII.

Malaria has provided a major selective pressure and has modulated the genetic diversity of the human genome. The variants of the Duffy Antigen/Receptor for Chemokines (DARC) gene have probably been selected by malaria parasites, particularly the FY*O allele, which is fixed in sub-Saharan Africa and confers resistance to Plasmodium vivax infection. Here, we showed the influence of genomic ancestry on the distribution of DARC genotypes in a highly admixed Brazilian population and confirmed the decreased susceptibility of the FY*A/FY*O genotype to clinical P. vivax malaria. FY*B/FY*O individuals were associated with a greater risk of developing clinical malaria. A remarkable difference among DARC variants concerning the susceptibility to clinical malaria was more evident for individuals who were less exposed to malaria, as measured by the time of residence in the endemic area. Additionally, we found that DARC-negative and FY*A/FY*O individuals had a greater chance of acquiring high levels of antibodies against the 19-kDa C-terminal region of the P. vivax merozoite surface protein-1. Altogether, our results provide evidence that DARC polymorphisms modulate the susceptibility to clinical P. vivax malaria and influence the naturally-acquired humoral immune response to malaria blood antigens, which may interfere with the efficacy of a future vaccine against malaria.

Plasmodium vivax remains a global health problem and its ability to cause relapses and subpatent infections challenge control and elimination strategies. Even in low malaria transmission settings, such as the Amazon basin, where progress in malaria control has caused a remarkable reduction in case incidence, a recent increase in P. vivax transmission demonstrates the continued vulnerability of P.vivax-exposed populations. As part of a search for complementary approaches to P.vivax surveillance in areas in which adults are the majority of the exposed-population, here we evaluated the potential of serological markers covering a wide range of immunogenicity to estimate malaria transmission trends. For this, antibodies against leading P. vivax blood-stage vaccine candidates were assessed during a 9 year follow-up study among adults exposed to unstable malaria transmission in the Amazon rainforest. Circulating antibody levels against immunogenic P. vivax proteins, such as the Apical Membrane Antigen-1, were a sensitive measure of recent P. vivax exposure, while antibodies against less immunogenic proteins were indicative of naturally-acquired immunity, including the novel engineered Duffy binding protein II immunogen (DEKnull-2). Our results suggest that the robustness of serology to estimate trends in P.vivax malaria transmission will depend on the immunological background of the study population, and that for adult populations exposed to unstable P.vivax malaria transmission, the local heterogeneity of antibody responses should be considered when considering use of serological surveillance.

Although the importance of humoral immunity to malaria has been established, factors that control antibody production are poorly understood. Follicular helper T cells (Tfh cells) are pivotal for generating high-affinity, long-lived antibody responses. While it has been proposed that expansion of antigen-specific Tfh cells, interleukin (IL) 21 production and robust germinal center formation are associated with protection against malaria in mice, whether Tfh cells are found during Plasmodium vivax (P. vivax) infection and if they play a role during disease remains unknown. Our goal was to define the role of Tfh cells during P. vivax malaria. We demonstrate that P. vivax infection triggers IL-21 production and an increase in Tfh cells (PD-1+ICOS+CXCR5+CD45RO+CD4+CD3+). As expected, FACS-sorted Tfh cells, the primary source of IL-21, induced immunoglobulin production by purified naïve B cells. Furthermore, we found that P. vivax infection alters the B cell compartment and these alterations were dependent on the number of previous infections. First exposure leads to increased proportions of activated and atypical memory B cells and decreased frequencies of classical memory B cells, whereas patients that experienced multiple episodes displayed lower proportions of atypical B cells and higher frequencies of classical memory B cells. Despite the limited sample size, but consistent with the latter finding, the data suggest that patients who had more than five infections harbored more Tfh cells and produce more specific antibodies. P. vivax infection triggers IL-21 production by Tfh that impact B cell responses in humans., Author summary Plasmodium vivax is the most widely spread malaria parasite species and represents a significant impediment to social and economic development in endemic countries. Our goal was to assess the importance of T follicular helper cells in the development of the immune response during malaria. We found that P. vivax infection promotes expansion of circulating Tfh cells that secrete IL-21 to boost immunoglobulin production by B-cells. Accordingly, malaria infection led to marked changes in B cell subpopulations, including expansion of plasma cells and increased production of antigen-specific IgG1 and IgG3. Re-exposure to P. vivax led to amplified Tfh cells cell responses that were concomitantly associated with increased frequencies of classical memory B cells. Thus, Tfh cells that are induced during P. vivax infection could impact the efficiency of humoral immune responses that underlie protective immunity.

Blood infection by the simian parasite, Plasmodium simium, was identified in captive (n = 45, 4.4%) and in wild Alouatta clamitans monkeys (n = 20, 35%) from the Atlantic Forest of southern Brazil. A single malaria infection was symptomatic and the monkey presented clinical and haematological alterations. A high frequency of Plasmodium vivax-specific antibodies was detected among these monkeys, with 87% of the monkeys testing positive against P. vivax antigens. These findings highlight the possibility of malaria as a zoonosis in the remaining Atlantic Forest and its impact on the epidemiology of the disease.

Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania.

It is shown in this work that the presence of an ultrathin (~ 1.5 nm) SiO 2 interfacial layer reactively formed in between a thick (~ 20 nm) high-permittivity CeO x film and a metal NiSi 2 bottom electrode in a metal–insulator–metal structure has remarkable consequences for the hysteretic current–voltage (I–V) characteristic of the device. Conductance values in the low resistance state (LRS) close to integer and half-integer values of the quantum conductance unit G 0 = 2 e 2 / h , where e is the electron charge and h the Planck constant, reveal the formation of filamentary structures across the stack with bottlenecks of atomic dimensions. Even though the hysteretic behavior partially remains when the bottom electrode (Ti, TiN, or W) is changed, the LRS I–V no longer exhibits so well defined conductance levels as in the NiSi 2 case. This distinctive behavior is attributed to the presence of the SiO 2 switching layer. Supplementary post-breakdown I–V data obtained from Al/SiO 2 /Si capacitors supports this hypothesis.

Background The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. Methodology/Principal Findings A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. Conclusions/Significance The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII., Author Summary Vaccines are a crucial component of the current efforts to eliminate malaria, and much of the vaccine-related research on P. vivax has been focused on the Duffy binding protein II (DBPII), a ligand for human blood stage infection. A high proportion of individuals who are naturally exposed to P. vivax fail to develop neutralizing antibodies, but the host genetic factors modulating this immune response are poorly characterized. We investigated whether DBPII responsiveness was dependent on the variability of human leucocyte antigen (HLA) class II cell surface proteins involved in the regulation of immune responses. To obtain a reliable estimate of DBPII antibodies, we carried out a longitudinal study, collecting serum from the same individuals over a period of 12-months. The results confirmed the heritability of the DBPII immune response, with genetic variation in HLA class II genes influencing both the development and persistence of the antibody response. HLA class II genotype also influenced the ability of DBPII antibodies to block the ligand-receptor interaction in vitro. Computational approaches identified structural specificity between HLA variants, which we propose as an explanation for differences between a good or poor antibody responder. These results may have implications for vaccine development, and might lead to strategies for controlling the type of immune response activated in response to DBPII.

S?o Paulo State University. Department of Biology. S?o Jos? do Rio Preto, SP, Brazil. College of Medicine of S?o Jos? do Rio Preto. Department of Dermatologic, Infectious, and Parasitic Diseases. S?o Jos? do Rio Preto, SP, Brazil. S?o Paulo State University. Department of Biology. S?o Jos? do Rio Preto, SP, Brazil. Federal University of Sergipe. Department of Biology. Aracaju, SE, Brazil. Federal University of Par?. Institute of Biological Sciences. Laborat?rio of Microbiology and Immunology. Bel?m, PA, Brazil. Oswaldo Cruz Foundation. Laboratory of Malaria, Ren? Rachou Research Center. Belo Horizonte, MG, Brazil. Oswaldo Cruz Foundation. Laboratory of Malaria, Ren? Rachou Research Center. Belo Horizonte, MG, Brazil. University of S?o Paulo. Faculty of Pharmaceutical Sciences. Department of Clinical and Toxicological Analyses. S?o Paulo, SP, Brazil. Federal University of Par?. Laboratory of Human and Medical Genetics. Bel?m, PA, Brazil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Pesquisa B?sica em Mal?ria. Ananindeua, PA, Brasil. Minist?rio da Sa?de. Secretaria de Vigil?ncia em Sa?de. Instituto Evandro Chagas. Laborat?rio de Pesquisa B?sica em Mal?ria. Ananindeua, PA, Brasil / S?o Paulo State University. Department of Biology. S?o Jos? do Rio Preto, SP, Brazil. The development of an effective immune response can help decrease mortality from malaria and its clinical symptoms. However, this mechanism is complex and has significant inter-individual variation, most likely owing to the genetic contribution of the human host. Therefore, this study aimed to investigate the influence of polymorphisms in genes involved in the costimulation of B-lymphocytes in the naturally acquired humoral immune response against proteins of the asexual stage of Plasmodium vivax. A total of 319 individuals living in an area of malaria transmission in the Brazilian Amazon were genotyped for four SNPs in the genes CD40, CD40L, BLYS and CD86. In addition, IgG antibodies against P. vivax apical membrane antigen 1 (PvAMA-1), Duffy binding protein (PvDBP) and merozoite surface protein 1 (PvMSP-119) were detected by ELISA. The SNP BLYS -871CT was associated with the frequency of IgG responders to PvAMA-1 and PvMSP-119. The SNP CD40 -1CT was associated with the IgG response against PvDBP, whereas IgG antibody titers against PvMSP-119 were influenced by the polymorphism CD86 +1057GA. These data may help to elucidate the immunological aspects of vivax malaria and consequently assist in the design of malaria vaccines.