Validation of a multiplex microsphere immunoassay for detection of antibodies to Trypanosoma cruzi in dogs.

The vector-borne protozoan parasite Trypanosoma cruzi causes Chagas disease in humans, dogs, and many other mammalian hosts. Canine Chagas disease is increasingly diagnosed in dogs of the southern United States where triatomine insect vectors occur, and there are limited veterinary testing options; only the indirect fluorescent antibody (IFA) test is offered at a single accredited diagnostic laboratory. We evaluated a multiplex microsphere immunoassay (MIA) for the detection of antibodies against T. cruzi in dogs and compared it with existing serologic methods to establish cutoff values and relative sensitivity and specificity. We tested 135 canine sera that had been characterized using the IFA and off-label use of 2 commercial rapid assays with our multiplex MIA against 12 antigens: 9 T. cruzi antigens, a negative control recombinant protein (green fluorescent protein, GFP), a Leishmania antigen, and a canine parvovirus antigen (used as an antibody control given near-ubiquitous parvoviral vaccination). The median fluorescence intensity (MFI) ratio between each T. cruzi antigen and GFP was calculated for every sample. Samples with an antigen:GFP MFI ratio > 4 SDs above the mean of 25 known-negative sera were considered positive to that antigen. Samples testing positive to ≥ 2 antigens were considered positive for T. cruzi antibodies. Compared to the IFA, our multiplex MIA had a relative sensitivity of 100% and specificity of 97.0%. Given its precision, high-throughput format, potential for automation, and lack of subjective interpretation, our multiplex MIA should be considered a valid and improved assay for T. cruzi antibodies in dogs.

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates.

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease.

Application of SARS-CoV-2 Serology to Address Public Health Priorities.

Background: Antibodies against SARS-CoV-2 can be detected by various testing platforms, but a detailed understanding of assay performance is critical. Methods: We developed and validated a simple enzyme-linked immunosorbent assay (ELISA) to detect IgG binding to the receptor-binding domain (RBD) of SARS-CoV-2, which was then applied for surveillance. ELISA results were compared to a set of complimentary serologic assays using a large panel of clinical research samples. Results: The RBD ELISA exhibited robust performance in ROC curve analysis (AUC> 0.99; Se = 89%, Sp = 99.3%). Antibodies were detected in 23/353 (6.5%) healthcare workers, 6/9 RT-PCR-confirmed mild COVID-19 cases, and 0/30 non-COVID-19 cases from an ambulatory site. RBD ELISA showed a positive correlation with neutralizing activity (p = <0.0001, R2 = 0.26). Conclusions: We applied a validated SARS-CoV-2-specific IgG ELISA in multiple contexts and performed orthogonal testing on samples. This study demonstrates the utility of a simple serologic assay for detecting prior SARS-CoV-2 infection, particularly as a tool for efficiently testing large numbers of samples as in population surveillance. Our work also highlights that precise understanding of SARS-CoV-2 infection and immunity at the individual level, particularly with wide availability of vaccination, may be improved by orthogonal testing and/or more complex assays such as multiplex bead assays.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease.

BACKGROUND: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease. METHODS: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months. RESULTS: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption. CONCLUSIONS: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

A flexible, pan-species, multi-antigen platform for the detection and monitoring of SARS-CoV-2-specific antibody responses.

The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines (e.g. Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific responses. All of the antigens utilized in our study can be made in-house, many in E. coli using readily available plasmids. Commercially sourced antigens may also be incorporated and newly available antigen variants can be rapidly produced and integrated, making the platform adaptable to the evolving viral strains in this pandemic.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA-CCR7-CD62L- T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

Stromal Gli signaling regulates the activity and differentiation of prostate stem and progenitor cells.

Interactions between cells in the stroma and epithelium facilitate prostate stem cell activity and tissue regeneration capacity. Numerous molecular signal transduction pathways, including the induction of sonic hedgehog (Shh) to activate the Gli transcription factors, are known to mediate the cross-talk of these two cellular compartments. However, the details of how these signaling pathways regulate prostate stem and progenitor cell activity remain elusive. Here we demonstrate that, although cell-autonomous epithelial Shh-Gli signaling is essential to determine the expression levels of basal cell markers and the renewal potential of epithelial stem and progenitor cells, stromal Gli signaling regulates prostate stem and progenitor cell activity by increasing the number and size of prostate spheroids in vitro Blockade of stromal Gli signaling also inhibited prostate tissue regeneration in vivo The inhibition of stromal Gli signaling suppressed the differentiation of basal and progenitor cells to luminal cells and limited prostate tubule secretory capability. Additionally, stromal cells were able to compensate for the deficiency of epithelial Shh signaling in prostate tissue regeneration. Mechanistically, suppression of Gli signaling increased the signaling factor transforming growth factor ß (TGFß) in stromal cells. Elevation of exogenous TGFß1 levels inhibited prostate spheroid formation, suggesting that a stromal Gli-TGFß signaling axis regulates the activity of epithelial progenitor cells. Our study illustrates that Gli signaling regulates epithelial stem cell activity and renewal potential in both epithelial and stromal compartments.

Fatty Acid Oxidation Is Required for Myxococcus xanthus Development.

Myxococcus xanthus cells produce lipid bodies containing triacylglycerides during fruiting body development. Fatty acid ß-oxidation is the most energy-efficient pathway for lipid body catabolism. In this study, we used mutants in fadJ (MXAN_5371 and MXAN_6987) and fadI (MXAN_5372) homologs to examine whether ß-oxidation serves an essential developmental function. These mutants contained more lipid bodies than the wild-type strain DK1622 and 2-fold more flavin adenine dinucleotide (FAD), consistent with the reduced consumption of fatty acids by ß-oxidation. The ß-oxidation pathway mutants exhibited differences in fruiting body morphogenesis and produced spores with thinner coats and a greater susceptibility to thermal stress and UV radiation. The MXAN_5372/5371 operon is upregulated in sporulating cells, and its expression could not be detected in csgA, fruA, or mrpC mutants. Lipid bodies were found to persist in mature spores of DK1622 and wild strain DK851, suggesting that the roles of lipid bodies and ß-oxidation may extend to spore germination.IMPORTANCE Lipid bodies act as a reserve of triacylglycerides for use when other sources of carbon and energy become scarce. ß-Oxidation is essential for the efficient metabolism of fatty acids associated with triacylglycerides. Indeed, the disruption of genes in this pathway has been associated with severe disorders in animals and plants. Myxococcus xanthus, a model organism for the study of development, is ideal for investigating the complex effects of altered lipid metabolism on cell physiology. Here, we show that ß-oxidation is used to consume fatty acids associated with lipid bodies and that the disruption of the ß-oxidation pathway is detrimental to multicellular morphogenesis and spore formation.

The phzA2-G2 transcript exhibits direct RsmA-mediated activation in Pseudomonas aeruginosa M18.

In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz) gene clusters, phzA1-G1 (phz1) and phzA2-G2 (phz2), each of which is responsible for phenazine-1-carboxylic acid (PCA) biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5'-untranslated region (UTR) of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3), located 21 nucleotides upstream of the Shine-Dalgarno (SD) sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes.

E3 ubiquitin ligase Cbl-b suppresses proallergic T cell development and allergic airway inflammation.

E3 ubiquitin ligase Cbl-b has emerged as a gatekeeper that controls the activation threshold of the T cell antigen receptor and maintains the balance between tolerance and autoimmunity. Here, we report that the loss of Cbl-b facilitates T helper 2 (Th2) and Th9 cell differentiation in vitro. In a mouse model of asthma, the absence of Cbl-b results in severe airway inflammation and stronger Th2 and Th9 responses. Mechanistically, Cbl-b selectively associates with Stat6 upon IL-4 ligation and targets Stat6 for ubiquitination and degradation. These processes are heightened in the presence of T cell receptor (TCR)/CD28 costimulation. Furthermore, we identify K108 and K398 as Stat6 ubiquitination sites. Intriguingly, introducing Stat6 deficiency into Cblb(-/-) mice abrogates hyper-Th2 responses but only partially attenuates Th9 responses. Therefore, our data reveal a function for Cbl-b in the regulation of Th2 and Th9 cell differentiation.

Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.

Preparation and evaluation of a novel delayed-onset sustained-release system of propranolol hydrochloride.

The objective of this work was to prepare and evaluate a new delayed-onset sustained-release system, comprising a sustained-release core tablet with hydroxypropyl methylcellulose as polymer matrix and an ethylcellulose/Eudragit L coating capable of delaying the drug release. The sustained core containing propranolol hydrochloride as the model drug was prepared by granulate tableting and the polymer coating was applied in a computer-controlled coating pan. The dissolution tests demonstrated that the in-vitro drug release was pH-dependent with sufficient gastric resistance, and the lag time (t(10%)) could be controlled by adjusting the coating level. Three dosage forms including commercial tablet, sustained-release tablet and the delayed-onset sustained-release tablet were administrated to six beagle dogs and the plasma levels of propranolol hydrochloride were measured with high-performance liquid chromatography. The delayed-onset sustained-release tablet had a lag time of 3.0 h in-vitro and 3.5 h in-vivo, and a t(max) of 7.0 h. The relative bioavailability for delayed-onset sustained-release tablet was 96.98% compared with commercial tablets. The results indicate that the new propranolol delayed-onset sustained-release system could achieve a relatively constant drug release followed by a programmed lag time, and this may provide a promising drug delivery form for chronopharmacotherapy of certain cardiovascular diseases.

[Expression of eosinophil major basic protein and neutrophil elastase in nasal polyp tissue and secretion].

OBJECTIVE: To explore the role in the pathogenesis of nasal polyp (NP) by comparison of eosinophil major basic protein (MBP), and neutrophil elastase (NE) in nasal polyps (NP) epithelium, stroma and the secretion of expression. METHOD: Immunohistochemical detection of 30 cases of patients with chronic sinusitis (CRS) NP epithelium NE stroma and the expression and secretion of MBP. RESULT: 1. There were significant differences of the expression of NE and MBP in epithelial tissue, stroma and secretion compared with the control group (P < 0.05); 2. There was not significant difference of the expression of NE and MBP between epithelial tissue and stroma (P > 0.05), while there was significant difference between epithelial tissue and the secretion (P < 0.05); 3. There were significant differences of the average positive expression of MBP and NE among epithelial tissue, stroma and secretion (P < 0.05); 4. MBP and NE were usually degranulated in secretion, while usually located in eosinophils (Eos) and neutrophils (Neu) in epithelial and mesenchymal; 5. There were abundant expression of MBP and NE in epithelial shedding regional, while small amounts of expression in stroma and integrated epithelial; 6. Electron microscopy could show the characteristics of electron density of MBP and NE particles. CONCLUSION: MBP and NE collaborated to cause pathological effects on the occurrence of NP.

[Introduction of Plasmodium falciparum C-terminal region of the merozoite surface protein gene into the chloroplast of tobacco].

OBJECTIVE: To construct chloroplast expression vector, and introduce the C-terminal region of the merozoite surface protein 1 gene (msp1-42) of Plasmodium falciparum 3D7 strain into the chloroplast genome of tobacco for expression of the recombinant protein MSP1-42. METHODS: Forward and reverse primers, adjusted to tobacco chloroplast codon preferences, were used for generation of msp1-42 gene from pBluntmsp plasmid which contains msp1-42 gene. A chloroplast expression vector LRrrmsp was constructed and bombarded into leaves of tobacco by a biolistic He particle delivery system. Media containing 500 mg/L spectinomycin were used for selection of spectinomycin resistant plant. PCR was carried out to check the introduction of the msp1-42 and aadA genes into the chloroplast genome. The transgenic plants with msp1-42 and aadA gene insertion were cut and cultured on the generation MS media containing 500 mg/L spectinomycin for at least 3 turns, and multiple PCR were applied to analyse their homogenization. RESULTS: A chloroplast expression vector LRrrmsp was constructed and confirmed with PCR and enzyme digestion analysis. Six transformants were obtained with a transformation rate 0.6/gun. The msp1-42 and aadA genes were amplified from spectinomycin resistant plants by PCR detection. Wild type chloroplast gene was detected by multiple-PCR analysis. CONCLUSION: A chloroplast expression vector containing msp1-42 gene was constructed. The msp1-42 gene was introduced into chloroplast genome of tobacco and heterogeneous transgenic tobacco was obtained.

Immunogenicity of recombinant hepatitis B virus surface antigen fused with preS1 epitopes expressed in rice seeds.

To test the possibility of producing a novel hepatitis B vaccine in plants, the modified hepatitis B virus (HBV) surface antigen (HBsAg) gene SS1 was expressed in rice under the control of the seed-specific Glub-4 promoter. The SS1 gene encodes a fusion protein consisting of amino acids 21-47 of the hepatocyte receptor-binding presurface 1 region (preS1) fused to the truncated C-terminus of the major HBV surface (S) protein. The production of antibodies against the preS1 region acts to protect humans against HBV infection by preventing HBV from binding to hepatocytes. The presence of SS1 in the genome of transgenic rice was confirmed by PCR and Southern blot analysis, and RNA dot blot analysis indicated that the fused SS1 gene was specifically expressed in rice seeds, with the highest expression level being about 31.5 ng/g dry weight grain. Western blot analysis revealed that the recombinant SS1 protein could be specifically recognized by both an anti-S protein antibody and an anti-preS1 antibody. The recombinant SS1 protein was also observed to form virus-like particles with a diameter of about 22 nm and a density of 1.25 g cm(-3). Furthermore, immunological responses against both the S and preS1 epitopes were induced in BALB/c mice immunized with the recombinant SS1 protein, indicating that this rice-derived SS1 protein could be a promising candidate as an alternative HBV vaccine for preventing hepatitis B.

[An experimental serum pharmacological study on an application method in traditional Chinese medicine for treatment and prevention of asthma].

OBJECTIVE: To study the mechanism of herbal application along meridians for treatment and prevention of asthma by using serum pharmacological test to observe the effects of serum containing herbs against the constriction of tracheal spiral strips induced by acetylcholine chloride (Ach). METHODS: Guinea pigs were randomly divided into normal control group, normal saline (NS) application group, aminophylline application group, aminophylline injection group, 1-day herb application group, 7-day herb application group and 14-day herb application group. Asthma was induced by Hutson's method in guinea pigs except the normal control group. Guinea pigs in herb application groups were treated by external application of a compound herbal medicine 60 min once every day. Guinea pigs in NS application group were treated by external application of NS. Guinea pigs in the two aminophylline-treated groups were treated by external application and intraperitoneal injection of aminophylline at a dose of 400 mg/kg, respectively. The guinea pigs were killed and the sera were obtained after 1-day, 7-day and 14-day treatment in the three herb application groups, 7-day treatment in the NS application group, the aminophylline application and injection groups, respectively. Serum pharmacological method was used to do the experiment, the effects of different sera on the constriction of tracheal strips were observed, and the constriction rates were calculated. RESULT: The serum containing herbs had an effect in reducing the constriction of tracheal spiral strips induced by Ach, and the effect was similar to that of the serum obtained from the aminophylline injection group. The constriction rate of the tracheal spiral strips was decreased when herbal application treatment was prolonged within a period of time, and it became stable when herbal application treatment was between 7-14 days. The constriction of tracheal spiral strips induced by Ach could be reduced remarkably when it was previously treated by serum containing herbs. CONCLUSION: The anti-acetylcholine function with a time-dependent effect is one of the mechanisms of herbal application treatment along meridians for asthma, and furthermore, herbal application treatment along meridians might be useful for preventing asthma.

Expression and purification of the synthetic preS1 gene of Hepatitis B Virus with preferred Escherichia coli codon preference.

To produce high levels of hepatitis B virus (HBV) preS1 protein at low cost, a DNA fragment encoding the preS1 region, residues 1-119, of HBV adr subtype was synthesized by overlapping-PCR according to Escherichia coli (E. coli) B preferred codon usage. The synthetic preS1 gene (spreS1) was cloned into the bacterial expression vector pET-30a and transferred into the expression strain E. coli BL21(DE3). Recombinant preS1 protein with an N-terminal His6 tag was expressed at high levels in soluble form, yielding about 44% of the total cellular protein. This technique overcomes problems that existed in previously reported expression systems of preS1 or its epitope, i.e., low-level expression or expression in inclusion bodies. Using this His-tagged preS1 expression system, recombinant protein was purified by single-step affinity chromatography on a Ni-NTA column resulting in a yield was about 28 mg recombinant protein per liter culture. Furthermore, Western blotting and indirect ELISA analysis demonstrate that the reactivity of preS1-specific antibody is comparable between the recombinant and commercialized preS1 protein. Thus, our improved expression system could be used for practical, low-cost large-scale production of recombinant preS1 without refolding steps.