Orthopaedic knee scooter-related injury: prevalence and patient safety perception in a prospective cohort with exploratory risk factor analysis.

BACKGROUND: There is a paucity of research investigating the harms associated with orthopaedic knee scooter (OKS) use and patient safety perceptions. This prospective study aimed to define the prevalence of OKS-related injuries, describe the patient perceptions of OKS safety, and identify potential risk factors. METHODS: This study was conducted at a single foot and ankle fellowship-trained surgeon's community-based clinic from 6/2020 to 4/2021 and enrolled 134 patients. Our primary outcome was an OKS-related event (injury or fall) and informed an a priori power analysis. Point estimate of association magnitude was calculated as an odds ratio (OR) for statistically and clinically significant associations. RESULTS: There were 118 (88%) patients eligible for analysis; fourteen enrolled patients did not use OKS, and two withdrew. The prevalence of patient falls was 37% (44/118), and the prevalence of patient injury was 15% (18/118). Four percent of patients would not recommend OKS and 8% would not use an OKS again. Sedentary lifestyle increased risk (OR = 4.67, 1.52-14.35 95 CI) for OKS-related injury. CONCLUSIONS: Despite a high prevalence of patient falls (37%), there is a low prevalence of injury (15%) and a favorable perception of OKS safety. Sedentary lifestyles may be a risk factor for OKS-related injury and should be considered in the development of a risk model.

Atmospheric Microplastics: Perspectives on Origin, Abundances, Ecological and Health Risks.

Microplastic (MP) pollution has aroused a tremendous amount of public and scientific interest worldwide. MPs are found widely ranging from terrestrial to aquatic ecosystems primarily due to the over-exploitation of plastic products and unscientific disposal of plastic waste. There is a large availability of scientific literature on MP pollution in the terrestrial and aquatic ecosystems, especially the marine environments; however, only recently has greater scientific attention been focused on the presence of MPs in the air and its retrospective health implications. Besides, atmospheric transport has been reported to be an important pathway of transport of MPs to the pristine regions of the world. From a health perspective, existing studies suggest that airborne MPs are priority pollutant vectors, that may penetrate deep into the body through inhalation leading to adverse health impacts such as neurotoxicity, cancer, respiratory problems, cytotoxicity, and many more. However, their effects on indoor and outdoor air quality, and on human health are not yet clearly understood due to the lack of enough research studies on that and the non-availability of established scientific protocols for their characterization. This scientific review entails important information concerning the abundance of atmospheric MPs worldwide within the existing literature. A thorough comparison of existing sampling and analytical protocols has been presented. Besides, this review has unveiled the areas of scientific concern especially air quality monitoring which requires immediate attention, with the information gaps to be filled have been addressed.

Revision Achilles Tendon Repair Using Posterior Tibial Tendon Allograft and Flexor Hallucis Longus Transfer.

Achilles tendon ruptures are common injuries seen by orthopaedic surgeons. A myriad of surgical options have been used in the management of Achilles tendon ruptures, but currently no gold standard exists. Re-rupture of Achilles tendon injuries occurs 1.7% to 5.6% of the time, and there has been no direct relationship demonstrated between complications and repair techniques used. The aim of this technique is to provide a method of fixation for the patient with an Achilles tendon re-rupture that provides a stable repair construct and mitigates the potential sequela of re-rupture. We describe the treatment of an Achilles tendon re-rupture with the use of a flexor hallucis longus tendon transfer and posterior tibial tendon allograft for repair of an 8.5 cm tendon gap.

Clinical outcomes in the surgical management of ankle fractures: A systematic review and meta-analysis of fibular intramedullary nail fixation vs. open reduction and internal fixation in randomized controlled trials.

BACKGROUND: What level I evidence exists to support the use of FNF for surgical management of ankle fractures in high risk patients? The purpose of this study was to compare clinical outcomes following fibular intramedullary nail fixation (FNF) and open reduction and internal fixation (ORIF) of ankle fractures. METHODS: A systematic review of the current literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines. Certainty of evidence reported according to GRADE (Grading of Recommendations Assessment, Development, and Evaluation). Our primary hypothesis was that patients undergoing FNF procedures to manage an ankle fracture would have significantly higher patient reported outcome scores (PROs) than patients undergoing ORIF. Primary study outcome measures were validated PROs. Secondary outcome measures included complication rate, secondary surgery rate, and bony union. RESULTS: The primary outcome analysis revealed no evidence of a significant effect difference on Olerud and Molander Ankle Score (OMAS) PRO and no evidence of statistical heterogeneity. Secondary outcome analysis revealed a significant 0.30 (0.12-0.74 95CI) relative risk reduction for complications in FNF (P = 0.008). No evidence of an effect difference for bony union. The GRADE certainty of the evidence was rated as low for bone union. No evidence of reporting bias was appreciated. Sensitivity analyses did not significantly alter effect estimates. CONCLUSION: This systematic review and meta-analysis restricted to evidence derived from RCTs revealed that the quality of evidence is reasonably strong and likely sufficient to conclude: (1) there is likely no clinically important difference between FNF and ORIF up to 12 months post-operatively, as defined by OMS (moderate certainty); (2) surgeons may reasonably expect reduced complications in 14 out of every 100 patients treated with FNF (moderate certainty); (3) there is likely no difference in bony union (low certainty). Future studies should investigate more patient-centered outcomes and if short-term findings are durable over time if these findings apply to lower risk populations. LEVEL OF EVIDENCE: Systematic review and meta-analysis of level I evidence.

Achilles Tendon Rip-Stop SpeedBridge Repair.

Achilles tendon injuries have been on the rise secondary to our increased participation in sports, increase in societal obesity rates, and the growing elderly population. There has been disagreement in recent years about whether to treat injuries such as Achilles tendon ruptures operatively or nonoperatively with aggressive functional rehabilitation. For those opting to surgically manage Achilles tendon ruptures, insertional Achilles tendonitis, or augment the described SpeedBridge Achilles tendon repair, we propose a modified rip-stop technique. The goal of this technique is to provide a biomechanical advantage to our current operative interventions for these injuries, a greater load-to-failure and a speedier, more reliable return to sport in our athletic populations.

Treatment of Severe Ankle and Hindfoot Deformity: Technique Using Femoral Head Allograft for Tibiotalocalcaneal Fusion Using a Cup-and-Cone Reamer.

Limb shortening due to structural bone loss in tibiotalocalcaneal arthrodesis is a concern that can negatively impact the patient's gait and weight-bearing. To aid in preventing the risk of limb shortening, the use of a femoral head allograft and intramedullary nail in tibiotalocalcaneal arthrodesis has been shown to successfully preserve limb length in patients with structural bone deficits. We present our technique using a femoral head allograft with a cup-and-cone reamer for the treatment of severe ankle and hindfoot deformity.

Ulnar Collateral Ligament Reconstruction Technique Utilizing Suture Tape Augmentation.

The prevalence of ulnar collateral ligament injuries and reconstructions among overhead throwing athletes has significantly risen in recent years. Surgical reconstruction has become the main treatment modality for athletes who have failed conservative treatment and wish to return to their sport. There has been an increased interest in graft augmentation in ligament reconstruction surgeries as surgeons search for ways to decrease the chance of graft failure. Augmented graft techniques have been described for other procedures. We present a technique that incorporates a cross-linked suture tape into either a palmaris longus or gracilis tendon autograft or allograft for ulnar collateral ligament reconstruction. This may allow for a biomechanically stronger construct because it appears this is the case in other settings. The goal is that this would lead to decreased rates of failure or possibly allow athletes to return at an accelerated rate.

TcG2/TcG4 DNA Vaccine Induces Th1 Immunity Against Acute Trypanosoma cruzi Infection: Adjuvant and Antigenic Effects of Heterologous T. rangeli Booster Immunization.

Background: Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). Two antigenic candidates, TcG2 and TcG4, are recognized by antibodies in naturally infected dogs and humans; and these vaccine candidates provided protection from Tc infection in mice and dogs. Trypanosoma rangeli (Tr) is non-pathogenic to mammals and shown to elicit cross-reactive anti-Tc antibodies. In this study, we investigated if fixed Tr (fTr) can further enhance the efficacy of the TcG2/TcG4 DNA vaccine. Methods and Results: C57BL/6 mice were immunized with TcG2/TcG4 DNA vaccine and fTr (delivered as an adjuvant or in prime-boost approach), and challenged with Tc. Serology studies showed that fTr (±quil-A) elicited Tc- and Tr-reactive IgGs that otherwise were not stimulated by TcG2/TcG4 vaccine only, and quil-A had suppressive effects on fTr-induced IgGs. After challenge infection, TcG2/TcG4-vaccinated mice exhibited potent expansion of antigen- and Tc-specific IgGs that were not boosted by fTr±quil-A. Flow cytometry analysis showed that TcG2/TcG4-induced dendritic cells (DC) and macrophages (Mφ) responded to challenge infection by expression of markers of antigen uptake, processing, and presentation, and production of pro-inflammatory cytokines. TcG2/TcG4-induced CD4+T cells acquired Th1 phenotype and expressed markers that orchestrate adaptive immunity. A fraction of vaccine-induced CD4+T cells exhibited iTreg phenotype responsible for aversion of self-injurious immune responses. Further, TcG2/TcG4-vaccinated mice exhibited potent expansion of poly-functional CD8+T cells with TNF-α/IFN-γ production and cytolytic phenotype post-infection. Subsequently, tissue parasites and pathology were hardly detectable in TcG2/TcG4-vaccinated/infected mice. Inclusion of fTr±quil-A had no clear additive effects in improving the Tc-specific adaptive immunity and parasite control than was noted in mice vaccinated with TcG2/TcG4 alone. Non-vaccinated mice lacked sufficient activation of Th1 CD4+/CD8+T cells, and exhibited >10-fold higher levels of tissue parasite burden than was noted in vaccinated/infected mice. Conclusion:TcG2/TcG4 vaccine elicits highly effective immunity, and inclusion of fTr is not required to improve the efficacy of DNA vaccine against acute Tc infection in mice.

Gene Expression Profiling and Functional Characterization of Macrophages in Response to Circulatory Microparticles Produced during Trypanosoma cruzi Infection and Chagas Disease.

BACKGROUND: Chronic inflammation and oxidative stress are hallmarks of chagasic cardiomyopathy (CCM). In this study, we determined if microparticles (MPs) generated during Trypanosoma cruzi (Tc) infection carry the host's signature of the inflammatory/oxidative state and provide information regarding the progression of clinical disease. METHODS: MPs were harvested from supernatants of human peripheral blood mononuclear cells in vitro incubated with Tc (control: LPS treated), plasma of seropositive humans with a clinically asymptomatic (CA) or symptomatic (CS) disease state (vs. normal/healthy [NH] controls), and plasma of mice immunized with a protective vaccine before challenge infection (control: unvaccinated/infected). Macrophages (mφs) were incubated with MPs, and we probed the gene expression profile using the inflammatory signaling cascade and cytokine/chemokine arrays, phenotypic markers of mφ activation by flow cytometry, cytokine profile by means of an ELISA and Bioplex assay, and oxidative/nitrosative stress and mitotoxicity by means of colorimetric and fluorometric assays. RESULTS: Tc- and LPS-induced MPs stimulated proliferation, inflammatory gene expression profile, and nitric oxide (∙NO) release in human THP-1 mφs. LPS-MPs were more immunostimulatory than Tc-MPs. Endothelial cells, T lymphocytes, and mφs were the major source of MPs shed in the plasma of chagasic humans and experimentally infected mice. The CS and CA (vs. NH) MPs elicited >2-fold increase in NO and mitochondrial oxidative stress in THP-1 mφs; however, CS (vs. CA) MPs elicited a more pronounced and disease-state-specific inflammatory gene expression profile (IKBKB, NR3C1, and TIRAP vs. CCR4, EGR2, and CCL3), cytokine release (IL-2 + IFN-γ > GCSF), and surface markers of mφ activation (CD14 and CD16). The circulatory MPs of nonvaccinated/infected mice induced 7.5-fold and 40% increases in ∙NO and IFN-γ production, respectively, while these responses were abolished when RAW264.7 mφs were incubated with circulatory MPs of vaccinated/infected mice. CONCLUSION: Circulating MPs reflect in vivo levels of an oxidative, nitrosative, and inflammatory state, and have potential utility in evaluating disease severity and the efficacy of vaccines and drug therapies against CCM.

S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure.

Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

Changes in Proteome Profile of Peripheral Blood Mononuclear Cells in Chronic Chagas Disease.

Trypanosoma cruzi (Tc) infection causes chagasic cardiomyopathy; however, why 30-40% of the patients develop clinical disease is not known. To discover the pathomechanisms in disease progression, we obtained the proteome signature of peripheral blood mononuclear cells (PBMCs) of normal healthy controls (N/H, n = 30) and subjects that were seropositive for Tc-specific antibodies, but were clinically asymptomatic (C/A, n = 25) or clinically symptomatic (C/S, n = 28) with cardiac involvement and left ventricular dysfunction. Protein samples were labeled with BODIPY FL-maleimide (dynamic range: > 4 orders of magnitude, detection limit: 5 f-mol) and resolved by two-dimensional gel electrophoresis (2D-GE). After normalizing the gel images, protein spots that exhibited differential abundance in any of the two groups were analyzed by mass spectrometry, and searched against UniProt human database for protein identification. We found 213 and 199 protein spots (fold change: |≥ 1.5|, p< 0.05) were differentially abundant in C/A and C/S individuals, respectively, with respect to N/H controls. Ingenuity Pathway Analysis (IPA) of PBMCs proteome dataset identified an increase in disorganization of cytoskeletal assembly and recruitment/activation and migration of immune cells in all chagasic subjects, though the invasion capacity of cells was decreased in C/S individuals. IPA predicted with high probability a decline in cell survival and free radical scavenging capacity in C/S (but not C/A) subjects. The MYC/SP1 transcription factors that regulate hypoxia and oxidative/inflammatory stress were predicted to be key targets in the context of control of Chagas disease severity. Further, MARS-modeling identified a panel of proteins that had >93% prediction success in classifying infected individuals with no disease and those with cardiac involvement and LV dysfunction. In conclusion, we have identified molecular pathways and a panel of proteins that could aid in detecting seropositive individuals at risk of developing cardiomyopathy.

Therapeutic Efficacy of a Subunit Vaccine in Controlling Chronic Trypanosoma cruzi Infection and Chagas Disease Is Enhanced by Glutathione Peroxidase Over-Expression.

Trypanosoma cruzi-induced oxidative and inflammatory responses are implicated in chagasic cardiomyopathy. In this study, we examined the therapeutic utility of a subunit vaccine against T. cruzi and determined if glutathione peroxidase (GPx1, antioxidant) protects the heart from chagasic pathogenesis. C57BL/6 mice (wild-type (WT) and GPx1 transgenic (GPxtg) were infected with T. cruzi and at 45 days post-infection (dpi), immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach. The plasma and tissue-sections were analyzed on 150 dpi for parasite burden, inflammatory and oxidative stress markers, inflammatory infiltrate and fibrosis. WT mice infected with T. cruzi had significantly more blood and tissue parasite burden compared with infected/GPxtg mice (n = 5-8, p<0.01). Therapeutic vaccination provided >15-fold reduction in blood and tissue parasites in both WT and GPxtg mice. The increase in plasma levels of myeloperoxidase (MPO, 24.7%) and nitrite (iNOS activity, 45%) was associated with myocardial increase in oxidant levels (3-4-fold) and non-responsive antioxidant status in chagasic/WT mice; and these responses were not controlled after vaccination (n = 5-7). The GPxtg mice were better equipped than the WT mice in controlling T. cruzi-induced inflammatory and oxidative stress markers. Extensive myocardial and skeletal tissue inflammation noted in chagasic/WT mice, was significantly more compared with chagasic/GPxtg mice (n = 4-6, p<0.05). Vaccination was equally effective in reducing the chronic inflammatory infiltrate in the heart and skeletal tissue of infected WT and GPxtg mice (n = 6, p<0.05). Hypertrophy (increased BNP and ANP mRNA) and fibrosis (increased collagen) of the heart were extensively present in chronically-infected WT and GPxtg mice and notably decreased after therapeutic vaccination. We conclude the therapeutic delivery of D/P vaccine was effective in arresting the chronic parasite persistence and chagasic pathology; and GPx1 over-expression provided additive benefits in reducing the parasite burden, inflammatory/oxidative stress and cardiac remodeling in Chagas disease.

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi.

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ+ and TFNα+) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4+ and CD8+ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8+T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease.

Immune protection against Trypanosoma cruzi induced by TcVac4 in a canine model.

Chagas disease, caused by Trypanosoma cruzi, is endemic in southern parts of the American continent. Herein, we have tested the protective efficacy of a DNA-prime/T. rangeli-boost (TcVac4) vaccine in a dog (Canis familiaris) model. Dogs were immunized with two-doses of DNA vaccine (pcDNA3.1 encoding TcG1, TcG2, and TcG4 antigens plus IL-12- and GM-CSF-encoding plasmids) followed by two doses of glutaraldehyde-inactivated T. rangeli epimastigotes (TrIE); and challenged with highly pathogenic T. cruzi (SylvioX10/4) isolate. Dogs given TrIE or empty pcDNA3.1 were used as controls. We monitored post-vaccination and post-challenge infection antibody response by an ELISA, parasitemia by blood analysis and xenodiagnosis, and heart function by electrocardiography. Post-mortem anatomic and pathologic evaluation of the heart was conducted. TcVac4 induced a strong IgG response (IgG2>IgG1) that was significantly expanded post-infection, and moved to a nearly balanced IgG2/IgG1 response in chronic phase. In comparison, dogs given TrIE or empty plasmid DNA only developed high IgG titers with IgG2 predominance in response to T. cruzi infection. Blood parasitemia, tissue parasite foci, parasite transmission to triatomines, electrocardiographic abnormalities were significantly lower in TcVac4-vaccinated dogs than was observed in dogs given TrIE or empty plasmid DNA only. Macroscopic and microscopic alterations, the hallmarks of chronic Chagas disease, were significantly decreased in the myocardium of TcVac4-vaccinated dogs. We conclude that TcVac4 induced immunity was beneficial in providing resistance to T. cruzi infection, evidenced by control of chronic pathology of the heart and preservation of cardiac function in dogs. Additionally, TcVac4 vaccination decreased the transmission of parasites from vaccinated/infected animals to triatomines.

Caspase-1/ASC inflammasome-mediated activation of IL-1ß-ROS-NF-κB pathway for control of Trypanosoma cruzi replication and survival is dispensable in NLRP3-/- macrophages.

In this study, we have utilized wild-type (WT), ASC-/-, and NLRP3-/- macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1ß production in mφs in comparison to LPS-treated controls. When WT and ASC-/- macrophages were treated with inhibitors of caspase-1, IL-1ß, or NADPH oxidase, we found that IL-1ß production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1ß regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3-/- macrophages, despite an inability to elicit IL-1ß activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3-/- macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1ß-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1ß/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1ß/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.

Serum-mediated activation of macrophages reflects TcVac2 vaccine efficacy against Chagas disease.

Chagas disease is endemic in Latin America and an emerging infectious disease in the United States. No effective treatments are available. The TcG1, TcG2, and TcG4 antigens are highly conserved in clinically relevant Trypanosoma cruzi isolates and are recognized by B and T cells in infected hosts. Delivery of these antigens as a DNA prime/protein boost vaccine (TcVac2) elicited lytic antibodies and type 1 CD8(+) T cells that expanded upon challenge infection and provided >90% control of parasite burden and myocarditis in chagasic mice. Here we determined if peripheral blood can be utilized to capture the TcVac2-induced protection from Chagas disease. We evaluated the serum levels of T. cruzi kinetoplast DNA (TckDNA), T. cruzi 18S ribosomal DNA (Tc18SrDNA), and murine mitochondrial DNA (mtDNA) as indicators of parasite persistence and tissue damage and monitored the effect of sera on macrophage phenotype. Circulating TckDNA/Tc18SrDNA and mtDNA were decreased by >3- to 5-fold and 2-fold, respectively, in vaccinated infected mice compared to nonvaccinated infected mice. Macrophages incubated with sera from vaccinated infected mice exhibited M2 surface markers (CD16, CD32, CD200, and CD206), moderate proliferation, a low oxidative/nitrosative burst, and a regulatory/anti-inflammatory cytokine response (interleukin-4 [IL-4] plus IL-10 > tumor necrosis factor alpha [TNF-α]). In comparison, macrophages incubated with sera from nonvaccinated infected mice exhibited M1 surface markers, vigorous proliferation, a substantial oxidative/nitrosative burst, and a proinflammatory cytokine response (TNF-α ≫ IL-4 plus IL-10). Cardiac infiltration of macrophages and TNF-α and oxidant levels were significantly reduced in TcVac2-immunized chagasic mice. We conclude that circulating TcDNA and mtDNA levels and macrophage phenotype mediated by serum constituents reflect in vivo levels of parasite persistence, tissue damage, and inflammatory/anti-inflammatory state and have potential utility in evaluating disease severity and efficacy of vaccines and drug therapies.

TcVac3 induced control of Trypanosoma cruzi infection and chronic myocarditis in mice.

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8(+)T cell response with type-1 cytokine (IFN-γ(+)TNF-α>IL-4(+)IL-10) and cytolytic effector (CD8(+)CD107a(+)IFN-γ(+)Perforin(+)) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn't enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ(+)CD8(+)T cells, a predominance of immunoregulatory IL-10(+)/CD4(+)T and IL10(+)/CD8(+)T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8(+)T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.

Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

BACKGROUND: Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease. METHODS AND RESULTS: Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st)-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd)-phase for antibody response to the recombinant antigens (individually or mixed) by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175). The IgG antibodies to TcG1, TcG2, and TcG4 (individually) and TcG(mix) were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%), TcG2- (96%), TcG4- (94.6%) and TcG(mix)- (98%) based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8%) in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects. CONCLUSIONS: Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

Delivery of antigenic candidates by a DNA/MVA heterologous approach elicits effector CD8(+)T cell mediated immunity against Trypanosoma cruzi.

In this study, we have characterized the immune mechanisms elicited by antigenic candidates, TcG2 and TcG4, delivered by a DNA-prime/MVA-boost approach, and evaluated the host responses to Trypanosoma cruzi infection in C57BL/6 mice. Immunization of mice with antigenic candidates elicited antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1) and CD8(+)T cells that exhibited type-1 cytolytic effector (CD8(+)CD107a(+)IFN-γ(+)Perforin(+)) phenotype. The extent of TcG2-dependent type 1 B and T cell immunity was higher than that noted in TcG4-immunized mice, and expanded accordingly in response to challenge infection with T. cruzi. The progression of chronic phase in immunized mice was associated with persistence of IgGs, 55-90% reduction in the frequency of proinflammatory (IFN-γ(+) or TNF-α(+)) CD8(+)T cells, and an increase or emergence of immunoregulatory (IL-10(+)) CD4/CD8 T cells. The tissue parasitism, infiltration of inflammatory infiltrate, parasite persistence, and fibrosis were decreased by 82-92% in heart and skeletal muscle of immunized/chronically infected mice. Control mice exhibited a significantly low antibody response, consistent activation of effector CD8(+)T cells dominated by pro-inflammatory phenotype and mixed cytokine profile (IFN-γ+TNF-α>IL-4+IL-10), parasite persistence and pathologic damage in chagasic hearts. We conclude that delivery of TcG2 or TcG4 by DNA-rMVA approach elicits effective antibody and CD8(+)T cell mediated immunity against T. cruzi and Chagas disease. The emergence of type 2 cytokine and T cell response in chronic phase was indicative of prevention of clinical disease.

Serum proteomic signature of human chagasic patients for the identification of novel potential protein biomarkers of disease.

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.