Study on antigenic protein Omp2b in combination with Omp31 and BP26 for serological detection of human brucellosis.

BACKGROUND: Brucellosis is a very common zoonosis in certain localized areas worldwide, with a high prevalence in most developing countries. The detection of brucellosis still faces many challenges such as the need for more sensitive and specific diagnostic antigens. METHODS: To evaluate the efficacy of Brucella outer membrane proteins (Omps) Omp2b in combination with omp31 and BP26 as diagnostic antigens for the serological detection of human brucellosis, these proteins were prepared by a prokaryotic expression system. Human brucellosis-positive and-negative sera were collected, and the detection effects of the diagnostic antigens were evaluated using an established indirect ELISA (iELISA) method. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC), true positives, true negatives, false positives, false negatives, accuracy, positive predictive value, negative predictive value, analytical specificity, and sensitivity were obtained to evaluate the effectiveness of Omp2b and antigen combinations. RESULTS: The iELISA results showed that the AUC of the antigenic proteins was 0.9100, 0.9387, 0.9343, and 0.9448, respectively, and that the combination of Omp31 and BP26 improved the accuracy and was superior to that of Omp2b alone. Analysis at the determined cut-off values showed that the analytical sensitivity of the assay was 0.8739 (95% CI:0.7974-0.9293) and the analytical specificity was 0.8539 (95% CI:0.7632-0.9199) when using Omp2b alone and 0.8649 when using the combination of Omp2b + BP26 (95% CI:0.7869-0.9223) with an analytical specificity of 0.9213 (95% CI:0.8446-0.9678) and 0.8468 (95% CI:0.7662-0.9082) and an analytical sensitivity of 0.9101 (95% CI:0.8305-0.9604). When Omp2b + Omp31 + BP26 was combined, the analytical sensitivity and specificity were 0.8559 (95% CI:0.7765-0.9153) and 0.9326 (95% CI:0.8590-0.9749), respectively. Protein antigens, including antigen combinations, did not cross-react with Yersinia enterocolitica O9 and E. coli O157: H7, indicating that their specificity was better than that of lipopolysaccharide (LPS). CONCLUSIONS: Compared with individual Omp2b, antigen combinations improved the effectiveness in detecting brucellosis, but were still not as effective as LPS antigen. Omp2b, combined with Omp31 and BP26 as diagnostic antigens, can be used to detect human brucellosis.

Comparison of BP26, Omp25 and Omp31 and a Multiepitope-Based Fusion Protein in the Serological Detection of Canine Brucellosis.

Background: Brucellosis is one of the most important zoonotic diseases in the world. Canine brucellosis, caused mainly by Brucella canis, is seriously neglected, and there is a lack of accurate diagnostic tools. Methods: In this study, to compare BP26, Omp25, Omp31 and a multiepitope-based fusion protein in the serological detection of canine brucellosis, using 34 brucellosis-positive dog sera and 62 negative control sera, the Brucella outer membrane proteins Omp31, BP26, Omp25 and a multiepitope-based fusion protein were evaluated by iELISA for their potential use as antigens in the serological diagnosis of canine brucellosis. Results: The results showed that the multiepitope-based fusion protein performed best in distinguishing brucellosis-positive and brucellosis-negative dog sera, with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 98.41%. BP26 and Omp31 showed excellent sensitivity in detecting brucellosis-positive dog sera, but their cross reaction to sera infected with Vibrio parahaemolyticus and Listeria monocytogenes may hinder their application as diagnostic reagents. Omp25 lacked sufficient sensitivity and showed limited ability in distinguishing positive and negative dog sera. Conclusion: The multiepitope-based fusion protein can be used as an ideal antigen for serologically diagnosing canine brucellosis currently prevalent worldwide.

Evaluation of the Combined Use of Major Outer Membrane Proteins in the Serodiagnosis of Brucellosis.

Background: Brucellosis is a zoonotic disease that causes substantial public health problems and endangers the development of animal husbandry in endemic areas. Early diagnosis of infected animals and humans is a crucial step in reducing the incidence of brucellosis. In this study, we designed different combinations of Brucella major outer membrane proteins (omps) including omp10, omp16, omp19, omp25, omp31 and BP26 as antigens and evaluated their efficiency in serodiagnosis for brucellosis. The efficiency assay was conducted using the method of indirect enzyme-linked immunosorbent assay (iELISA) together with a collection of brucellosis-positive sera and healthy sera from multiple species (161 from human, 120 from goat and 144 from cattle). The diagnostic effectiveness of each omp combination was analyzed by receiver operating characteristic (ROC) curve with the software GraphPad Prism version 6.05. Results: The omp25/omp31/BP26 combination showed the best efficiency in diagnosis for human brucellosis. The area under the ROC curve (AUC) was 0.995 and, compared with the serum tube agglutination test (SAT) and the Rose Bengal plate agglutination test (RBPT), the positive and negative diagnostic accuracies of iELISA were 94.59% (105/111) and 100.0% (50/50), respectively. Evaluation of the 120 goat and 144 cattle serum samples showed that the best combination for diagnosing both omp31/BP26, the AUC was 0.9262 in goat and 0.9344 in cattle, and compared with those of SAT and RBPT, the positive and negative diagnostic accuracies in goat were 72.73% (48/66) and 100.0% (54/54), respectively. The positive and negative diagnostic accuracies in cattle were 79.79% (75/94) and 100.0% (50/50), respectively. Cross-reaction assays showed that omp25/omp31/BP26 and omp31/BP26 do not cross with other common pathogens. Conclusion: The results indicated that combinations of omps, as protein antigens, can be used to diagnose brucellosis with high accuracy in human, goat and cattle.

Estimation of Plasmodium falciparum transmission using multiepitope chimeric antigen in the postelimination phase in Yunnan, China.

BACKGROUND: The continuous monitoring of malaria transmission intensity is still required to maintain elimination status after reaching the malaria elimination stage. In this study, serological surveillance with multiepitope artificial antigen was used to assess the transmission of Plasmodium falciparum in Yunnan, China, where malaria elimination has just been achieved, to provide data to support malaria control in the postelimination period. METHODS: Samples were collected in three border counties and one inland county in Yunnan Province in 2016 using a stratified whole-group sampling method. Fingerstick blood was collected from all participants, and antibodies to Malaria Random Constructed Antigen-1 (M. RCAg-1) were detected by indirect ELISA. The transmission intensity of P. falciparum malaria was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). RESULTS: A total of 5566 samples were collected. There was no statistically significant difference in antibody level between the inland county and the nonendemic area, but the antibody level in border counties was significantly higher than those in the inland county and the nonendemic control area. No seropositive cases were found in Yanjin County, and the seropositivity rate increased with age in the three border counties. The highest intensity of P. falciparum malaria transmission was in Zhenkang County (SCR = 0.0030, CI: 0.0029, 0.0031), followed by Gengma County (SCR = 0.0013, CI: 0.0012, 0.0015) and Yingjiang County (SCR = 0.00088, CI: 0.00083, 0.00090). CONCLUSION: The transmission intensity of P. falciparum malaria in Yunnan Province has obviously decreased in recent years, but for the border areas where malaria has just been eliminated, the transmission intensity will not immediately drop to zero, and it still needs to be monitored for a period of time to maintain malaria elimination status.

Surveillance of Plasmodium vivax transmission using serological models in the border areas of China-Myanmar.

BACKGROUND: To understand the Plasmodium vivax malaria transmission intensity and to assess the effectiveness of prevention and control measures taken along the China-Myanmar border, a catalytic model was used to calculate the seroconversion rate, an important indicator of malaria transmission intensity with high sensitivity, which is particularly useful in areas of low transmission. METHODS: Five counties in Yunnan Province bordering Myanmar were selected as survey sites, and subjects were obtained in each county by stratified random sampling in 2013-2014. Fingerstick blood was collected from each subject and tested for antibodies to P. vivax Merozoite Surface Protein 1-19 (PvMSP1-19) using indirect ELISA. A catalytic conversion model was used to assess the transmission intensity of P. vivax malaria based on the maximum likelihood of generating a community seroconversion rate. RESULTS: A total of 3064 valid blood samples were collected. Antibody levels were positively correlated with age. The seroconversion rate (SCR) values for each village were Luoping (0.0054), Jingqiao (0.0061), Longpen (0.0087), Eluo (0.0079), Banwang (0.0042) and Banbie (0.0046), respectively. CONCLUSION: Overall, the intensity of P. vivax malaria transmission in the border areas of Yunnan Province is low and not entirely consistent across counties. Consecutive serological surveys are needed to provide a sensitive evaluation of transmission dynamics and can help to confirm areas where infection is no longer present.

Nicotine-derived NNK induces the stemness enrichment of CRC cells through regulating the balance of DUSP4-ERK1/2 feedback loop.

Cigarette smoking has been considered as an independent risk factor for colorectal cancer (CRC) initiation and progression. In this study, we found that cigarette smoking was significantly associated with poor CRC differentiation (P = 0.040). Since studies have indicated that poorly differentiated tumors are more aggressive and metastasize earlier, leading to poorer prognosis; and cancer stem cells (CSCs) are largely responsible for tumor differentiation state, here we observed that the exposure of nicotine-derived 4-(methylnitrosamino)- 1-(3-pyridyl)- 1-butanone (NNK) promoted cell sphere formation and the expression of the stem cell markers, CD44, OCT4, C-MYC and NANOG in HCT8 and DLD-1 cells. Further colony formation assay, CCK-8 assay and tumor-bearing experiment showed that NNK exposure significantly increased the proliferative and growth ability of CRC cells. In mechanism, we found that NNK-activated ERK1/2 played an important role in enrichment of CRC stem cells and the up-regulation of DUSP4, a major negative regulator of ERK1/2. Moreover, DUSP4 up-regulation was essential for maintaining NNK-activated ERK1/2 in an appropriate level, which was an required event for NNK-induced stemness enrichment of CRC cells. Taken together, our findings provided a possible mechanistic insight into cigarette smoking-induced CRC progression.

Phase II trial of co-administration of CD19- and CD20-targeted chimeric antigen receptor T cells for relapsed and refractory diffuse large B cell lymphoma.

PURPOSE: Anti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has demonstrated remarkable efficacy for refractory and relapsed diffuse large B cell lymphoma (R/R DLBCL). However, this therapy failed in nearly 25% patients mainly due to antigen loss. The authors performed a phase Ⅱ trial by coadministration of anti-CD19 and anti-CD20 CAR-T cells treatment for R/R DLBCL and evaluated its efficacy and toxicity. METHODS: Totally 21 patients with DLBCL were enrolled in this study. The patients were conditioned with fludarabine and cyclophosphamide before the infusion of anti-CD19 and anti-CD20 CAR-T cells. Treatment response, toxicity, and persistence were monitored continuously. RESULTS: Of the 21 patients received the treatment, the objective response rate (ORR) is 81.0% (95% confidence interval [CI], 58.1%-94.6%) with four cases of bulk (4/5) and one case of testis involvement; 52.4% (95% CI, 29.8%-74.3%) had a complete response (CR). Peak levels of anti-CD19 and anti-CD20 CAR cells were associated with response (P = .007 and .002). Grade 3-4 cytokine release syndrome (CRS) and neurologic events occurred in 28.5% and 9.5% patients, respectively. Median overall survival (OS) and progression-free survival (PFS) were 8.1 and 5.0 months, respectively. The maximum standard uptake value (SUVmax) of CD4/CD8 ratio before and after infusion were associated with responses, and the total lesion glycolysis (TLG) before infusion correlates with cytokines level. CONCLUSIONS: Coadministration of anti-CD19 and CD20 CAR-T cells therapy for DLBCL is feasible with manageable toxicity. Cytokine markers are related to toxicity and SUVmax could predict efficacy. This trial was registered at www.clinicaltrials.gov as NCT03207178.

Multi-epitope chimeric antigen used as a serological marker to estimate Plasmodium falciparum transmission intensity in the border area of China-Myanmar.

BACKGROUND: Following the decline of malaria transmission in many countries and regions, serological parameters have become particularly useful for estimating malaria transmission in low-intensity areas. This study evaluated a novel serological marker, Malaria Random Constructed Antigen-1 (M.RCAg-1), which contains 11 epitopes from eight Plasmodium falciparum antigens, as a tool for assessing malaria transmission intensity along the border area of China-Myanmar. METHOD: Serum from Plasmodium falciparum and P. vivax patients was used to detect the properties of M.RCAg-1 and antibody responses. Cross-sectional surveys were conducted at the China-Myanmar border and in Hainan province in 2012 and 2013 using cluster sampling. Filter blood spot papers were collected from all participants. Antibodies against M.RCAg-1 were detected using indirect ELISA. The Mann-Whitney test and Spearman's rank correlation test were performed to analyze antibody data. P. falciparum malaria transmission intensity was estimated using a catalytic conversion model based on the maximum likelihood of generating a community seroconversion rate (SCR). RESULTS: M.RCAg-1 was well-recognized by the naturally acquired anti-malaria antibodies in P. falciparum patients and had very limited cross-reactivity with P. vivax infection. The total amount of IgG antibodies was decreased with the decrease in parasitemia after taking medication and lasted several weeks. In a population survey, the antibody levels were higher in residents living close to the China-Myanmar border than those living in non-epidemic areas (P < 0.0001), but no significant difference was observed between residents from Hainan and non-epidemic areas. The calculated SCR was 0.0128 for Jieyangka, 0.004 for Susuzhai, 0.0047 for Qiushan, and 0.043 for Kayahe. The estimated exposure rate obtained from the anti-M.RCAg-1 antibody level correlated with traditional measures of transmission intensity derived from altitude. CONCLUSION: Our study demonstrates that M.RCAg-1 is potentially useful as a serological indicator of exposure to P. falciparum malaria, especially for malaria surveillance in low transmission areas.

[Hemozoin detection in malaria diagnosis].

In malaria parasites, hemozion is often called malaria pigment. Plasmodium spp. digest hemoglobin and release high quantity of free heme, which is a non-protein component of hemoglobin. Until recent years, researchers have found that hemozoin has many effects on malaria parasites. This paper reviews the hemozoin detection apllied in malaria diagnosis.

Deep profiling of the novel intermediate-size noncoding RNAs in intraerythrocytic Plasmodium falciparum.

Intermediate-size noncoding RNAs (is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in Plasmodium falciparum, which is the most virulent malaria parasite infecting human being. By using Illumina/Solexa paired-end sequencing of an is-ncRNA-specific library, we performed a systematic identification of novel is-ncRNAs in intraerythrocytic P. falciparum, strain 3D7. A total of 1,198 novel is-ncRNA candidates, including antisense, intergenic, and intronic is-ncRNAs, were identified. Bioinformatics analyses showed that the intergenic is-ncRNAs were the least conserved among different Plasmodium species, and antisense is-ncRNAs were more conserved than their sense counterparts. Twenty-two novel snoRNAs were identified, and eight potential novel classes of P. falciparum is-ncRNAs were revealed by clustering analysis. The expression of randomly selected novel is-ncRNAs was confirmed by RT-PCR and northern blotting assays. An obvious different expressional profile of the novel is-ncRNA between the early and late intraerythrocytic developmental stages of the parasite was observed. The expression levels of the antisense RNAs correlated with those of their cis-encoded sense RNA counterparts, suggesting that these is-ncRNAs are involved in the regulation of gene expression of the parasite. In conclusion, we accomplished a deep profiling analysis of novel is-ncRNAs in P. falciparum, analysed the conservation and structural features of these novel is-ncRNAs, and revealed their differential expression patterns during the development of the parasite. These findings provide important information for further functional characterisation of novel is-ncRNAs during the development of P. falciparum.