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Adipose tissue macrophage (ATM) inflammation is involved with meta-inflammation and pathology of metabolic complications. Here we report that in adipocytes, elevated lactate production, previously regarded as the waste product of glycolysis, serves as a danger signal to promote ATM polarization to an inflammatory state in the context of obesity. Adipocyte-selective deletion of lactate dehydrogenase A (Ldha), the enzyme converting pyruvate to lactate, protects mice from obesity-associated glucose intolerance and insulin resistance, accompanied by a lower percentage of inflammatory ATM and reduced production of pro-inflammatory cytokines such as interleukin 1β (IL-1β). Mechanistically, lactate, at its physiological concentration, fosters the activation of inflammatory macrophages by directly binding to the catalytic domain of prolyl hydroxylase domain-containing 2 (PHD2) in a competitive manner with α-ketoglutarate and stabilizes hypoxia inducible factor (HIF-1α). Lactate-induced IL-1β was abolished in PHD2-deficient macrophages. Human adipose lactate level is positively linked with local inflammatory features and insulin resistance index independent of the body mass index (BMI). Our study shows a critical function of adipocyte-derived lactate in promoting the pro-inflammatory microenvironment in adipose and identifies PHD2 as a direct sensor of lactate, which functions to connect chronic inflammation and energy metabolism., (© 2022. The Author(s).)

A novel double-resonant plasmonic substrate for fluorescence amplification in a chip-based apta-immunoassay is herein reported. The amplification mechanism relies on plasmon-enhanced fluorescence (PEF) effect. The substrate consists of an assembly of plasmon-coupled and plasmon-uncoupled gold nanoparticles (AuNPs) immobilized onto a glass slide. Plasmon-coupled AuNPs are hexagonally arranged along branch patterns whose resonance lies in the red band (∼675 nm). Plasmon-uncoupled AuNPs are sprinkled onto the substrate, and they exhibit a narrow resonance at 524 nm. Numerical simulations of the plasmonic response of the substrate through the finite-difference time-domain (FDTD) method reveal the presence of electromagnetic hot spots mainly confined in the interparticle junctions. In order to realize a PEF-based device for potential multiplexing applications, the plasmon resonances are coupled with the emission peak of 5-carboxyfluorescein (5-FAM) fluorophore and with the excitation/emission peaks of cyanine 5 (Cy5). The substrate is implemented in a malaria apta-immunoassay to detect Plasmodium falciparum lactate dehydrogenase ( Pf LDH) in human whole blood. Antibodies against Plasmodium biomarkers constitute the capture layer, whereas fluorescently labeled aptamers recognizing Pf LDH are adopted as the top layer. The fluorescence emitted by 5-FAM and Cy5 fluorophores are linearly correlated (logarithm scale) to the Pf LDH concentration over five decades. The limits of detection are 50 pM (1.6 ng/mL) with the 5-FAM probe and 260 fM (8.6 pg./mL) with the Cy5 probe. No sample preconcentration and complex pretreatments are required. Average fluorescence amplifications of 160 and 4500 are measured in the 5-FAM and Cy5 channel, respectively. These results are reasonably consistent with those worked out by FDTD simulations. The implementation of the proposed approach in multiwell-plate-based bioassays would lead to either signal redundancy (two dyes for a single analyte) or to a simultaneous detection of two analytes by different dyes, the latter being a key step toward high-throughput analysis.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii , two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii , suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Neves, Wong, Aimanianda, Simenel, Guijarro, Walls, Willment, Gow, Munro, Brown and Lopes-Bezerra.)

Multiplex assays for malaria antigen detection can gather data from large sample sets, but considerations for the consistency and quality assurance (QA) of mass testing lack evaluation. We present a QA framework for a study occurring November 2019 to March 2020 involving 504 assay plates detecting four Plasmodium antigens: pan-Plasmodium aldolase and lactate dehydrogenase (LDH), histidine-rich protein 2 (HRP2), P. vivax LDH (PvLDH). Controls on each plate included buffer blank, antigen negative blood, and 4-point positive dilution curve. The blank and negative blood provided consistently low signal for all targets except for pAldolase, which showed variability. Positive curve signals decreased throughout the 5-month study duration but retained a coefficient of variation (CV) of < 5%, with the exception of HRP2 in month 5 (CV of 11%). Regression fittings for inter-plate control signals provided mean and standard deviations (SDs), and of 504 assay plates, 6 (1.2%) violated the acceptable deviation limits and were repeated. For the 40,272 human blood samples assayed in this study, of 161,088 potential data points (each sample × 4 antigens), 160,641 (99.7%) successfully passed quality checks. The QA framework presented here can be utilized to ensure quality of laboratory antigen detection for large sample sets.

A plasmon-enhanced fluorescence-based antibody-aptamer biosensor - consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly - is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies - covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) - and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10-15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays.

In malaria-endemic countries, rapid diagnostic tests (RDTs) targeting Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and lactate dehydrogenase (PfLDH) have been widely used. However, little is known regarding the diagnostic performances of these RDTs in the Assosa zone of northwest Ethiopia. The objective of this study was to determine the diagnostic performances of PfHRP2 and PfLDH RDTs using microscopy and quantitative PCR (qPCR) as a reference test. A health facility-based cross-sectional study design was conducted from malaria-suspected study participants at selected health centers from November to December 2018. Finger-prick blood samples were collected for microscopy, RDTs, and qPCR method. The prevalence of P. falciparum was 26.4%, 30.3%, and 24.1% as determined by microscopy, PfHRP2 RDT, and PfLDH RDT, respectively. Compared with microscopy, the sensitivity and specificity of the PfHRP2 RDT were 96% and 93%, respectively, and those of the PfLDH RDT were 89% and 99%, respectively. Compared with qPCR, the specificity of the PfHRP2 RDT (93%) and PfLDH RDT (98%) was high, but the sensitivity of the PfHRP2 RDT (77%) and PfLDH RDT (70%) was relatively low. These malaria RDTs and reference microscopy methods showed reasonable agreement with a kappa value above 0.85 and provided accurate diagnosis of P. falciparum malaria. Thus, the current malaria RDT in the Ministry of Health program can be used in the Assosa zone of Ethiopia. However, continuous monitoring of the performance of PfHRP2 RDT is important to support control and elimination of malaria in Ethiopia.

Sterol regulatory element binding protein-2 (SREBP-2) is activated by cytokines or pathogen, such as virus or bacteria, but its association with diminished cholesterol levels in COVID-19 patients is unknown. Here, we evaluated SREBP-2 activation in peripheral blood mononuclear cells of COVID-19 patients and verified the function of SREBP-2 in COVID-19. Intriguingly, we report the first observation of SREBP-2 C-terminal fragment in COVID-19 patients' blood and propose SREBP-2 C-terminal fragment as an indicator for determining severity. We confirmed that SREBP-2-induced cholesterol biosynthesis was suppressed by Sestrin-1 and PCSK9 expression, while the SREBP-2-induced inflammatory responses was upregulated in COVID-19 ICU patients. Using an infectious disease mouse model, inhibitors of SREBP-2 and NF-κB suppressed cytokine storms caused by viral infection and prevented pulmonary damages. These results collectively suggest that SREBP-2 can serve as an indicator for severity diagnosis and therapeutic target for preventing cytokine storm and lung damage in severe COVID-19 patients.

Laboratory detection of malaria antigens has proved valuable for research and epidemiological purposes. We recently developed a bead-based multiplex antigen assay for pan- Plasmodium and Plasmodium falciparum targets. Here, we report integration of a Plasmodium vivax -specific target to this multiplex panel: P. vivax lactate dehydrogenase (PvLDH). Within the multiplex panel, assay signal for purified PvLDH antigen titrated into the single-digit picogram range. Against a panel of polymerase chain reaction (PCR)-confirmed samples from acute P. vivax infections ( n = 36), sensitivity was 91.7% in using PvLDH detection for identifying the presence of parasites. Specificity against a panel of persons with no Plasmodium infection ( n = 44) was 100%, and specificity against a panel of PCR-confirmed P. falciparum, Plasmodium malariae, or Plasmodium ovale infections ( n = 164) was 90.2%. Addition of this PvLDH capture and detection system into the multiplex antigen panel will now allow for sensitive screening for species identification of both P. falciparum and P. vivax in the laboratory.

This study aimed to investigate the renal protective effect of atorvastatin (ATV) on the kidney inflammation induced by calcium oxalate (CaOx) crystals. A cell model of cell-crystal interactions and a rat model of CaOx kidney stone were established. The expressions of TLR4, NF-κB, NLRP3, and cleaved caspase-1 in cells and rat kidney tissues were detected using Western blot, immunohistochemical, and/or immunofluorescence. The concentrations of malondialdehyde (MDA), superoxide dismutase (SOD), reactive oxygen species (ROS) in cells, and lactic acid dehydrogenase (LDH) in the culture medium were measured. The secreted levels of interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) were examined by ELISA. The serum levels of creatinine (CRE) and blood urea nitrogen (BUN) were measured. von Kossa staining was used for the evaluation of renal lens deposition. The CaOx model group showed significantly decreased SOD level; increased concentrations of MDA; ROS and LDH; elevated expressions of TLR4, NF-κB, NLRP3, and cleaved caspase-1; and the elevated release of IL-1β, IL-18, IL-6, and TNF- α as compared to the control group. The treatment with ATV significantly inhibited the formation of CaOx kidney stone by increasing the level of SOD; downregulating MDA, ROS, and LDH; inhibiting the expressions of TLR4, NF-κB, NLRP3 and cleaved caspase-1; and blocking the secretion of inflammatory cytokines. In addition, the serum levels of CRE and BUN, and the intrarenal crystal deposition were also significantly decreased in ATV-treated rats. In summary, oxidative stress, TLR4/NF-κB, and NLRP3 inflammasome pathways are involved in renal inflammatory responses induced by CaOx crystals. ATV treatment significantly suppressed oxidative stress, inhibited the activation of TLR4/NF-κB and NLRP3 inflammasome pathways, and decreased the release of inflammatory mediators, thereby ameliorating CaOx crystal-induced damage and crystal deposition in HK-2 cells and rat kidney tissues., (© 2020 International Union of Biochemistry and Molecular Biology.)

Lactate dehydrogenase C (LDHC) is an archetypical cancer testis antigen with limited expression in adult tissues and re-expression in tumors. This restricted expression pattern together with the important role of LDHC in cancer metabolism renders LDHC a potential target for immunotherapy. This study is the first to investigate the immunogenicity of LDHC using T cells from healthy individuals. LDHC-specific T cell responses were induced by in vitro stimulation with synthetic peptides, or by priming with autologous peptide-pulsed dendritic cells. We evaluated T cell activation by IFN-γ ELISpot and determined cytolytic activity of HLA-A*0201-restricted T cells in breast cancer cell co-cultures. In vitro T cell stimulation induced IFN-γ secretion in response to numerous LDHC-derived peptides. Analysis of HLA-A*0201 responses revealed a significant T cell activation after stimulation with peptide pools 2 (PP2) and 8 (PP8). The PP2- and PP8-specific T cells displayed cytolytic activity against breast cancer cells with endogenous LDHC expression within a HLA-A*0201 context. We identified peptides LDHC 41-55 and LDHC 288-303 from PP2 and PP8 to elicit a functional cellular immune response. More specifically, we found an increase in IFN-γ secretion by CD8 + T cells and cancer-cell-killing of HLA-A*0201/LDHC positive breast cancer cells by LDHC 41-55 - and LDHC 288-303 -induced T cells, albeit with a possible antigen recognition threshold. The majority of induced T cells displayed an effector memory phenotype. To conclude, our findings support the rationale to assess LDHC as a targetable cancer testis antigen for immunotherapy, and in particular the HLA-A*0201 restricted LDHC 41-55 and LDHC 288-303 peptides within LDHC.

The impact of malaria on global health has continually prompted the need to develop more effective diagnostic strategies that could overcome deficiencies in accurate and early detection. In this review, we examine the various biosensor-based methods for malaria diagnostic biomarkers, namely; Plasmodium falciparum histidine-rich protein 2 (PfHRP-2), parasite lactate dehydrogenase (pLDH), aldolase, glutamate dehydrogenase (GDH), and the biocrystal hemozoin. The models that demonstrate a potential for field application have been discussed, looking at the fabrication and analytical performance characteristics, including (but not exclusively limited to): response time, sensitivity, detection limit, linear range, and storage stability, which are first summarized in a tabular form and then described in detail. The conclusion summarizes the state-of-the-art technologies applied in the field, the current challenges and the emerging prospects for malaria biosensors.

High-altitude deacclimatization syndrome (HADAS) is involved in hypoxia-reoxygenation injury and inflammatory response, induced a series of symptoms, and has emerged as a severe public health issue. Here, we investigated the mechanism as well as potential means to prevent HADAS using Shenqi pollen capsules (SPCs) in subjects with HADAS in a multicenter, double-blinded, randomized, placebo-controlled study. All subjects were at the same high altitude (3650 m) for 4-8 months before returning to lower altitudes. Subjects ( n = 288) in 20 clusters were diagnosed with mild or moderate HADAS on the third day of the study. We randomly allocated 20 clusters of subjects (1 : 1) to receive SPCs or a placebo for 7 weeks, and they were then followed up to the 14 th week. The primary endpoints were subjects' HADAS scores recorded during the 14 weeks of follow-up. Compared with the placebo, SPC treatment significantly decreased the subjects' HADAS scores and reduced the incidence of symptom persistence. SPC therapy also reduced the serum levels of CK, CK-MB, LDH, IL-17A, TNF- α , and miR-155 and elevated IL-10 and miR-21 levels. We thus demonstrate that SPCs effectively ameliorated HADAS symptoms in these subjects via suppression of the hypoxia-reoxygenation injury and inflammatory response., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Binfeng He et al.)

Current malaria rapid diagnostic tests (RDTs) contain antibodies against Plasmodium falciparum -specific histidine-rich protein 2 (PfHRP2), Plasmodium lactate dehydrogenase (pLDH), and aldolase in various combinations. Low or high parasite densities/target antigen concentrations may influence the accuracy and sensitivity of PfHRP2-detecting RDTs. We analyzed the SD Bioline Malaria Ag P.f/Pan RDT performance in relation to P. falciparum parasitemia in Madagascar, where clinical Plasmodium vivax malaria exists alongside P. falciparum . Nine hundred sixty-three samples from patients seeking care for suspected malaria infection were analyzed by RDT, microscopy, and Plasmodium species-specific, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Plasmodium infection positivity by these diagnostics was 47.9%, 46.9%, and 58%, respectively. Plasmodium falciparum -only infections were predominant (microscopy, 45.7%; LDR-FMA, 52.3%). In all, 16.3% of P. falciparum , 70% of P. vivax , and all of Plasmodium malariae , Plasmodium ovale , and mixed-species infections were submicroscopic. In 423 P. falciparum mono-infections, confirmed by microscopy and LDR-FMA, the parasitemia in those who were positive for both the PfHRP2 and pan-pLDH test bands was significantly higher than that in those who were positive only for the PfHRP2 band ( P < 0.0001). Plasmodium falciparum parasitemia in those that were detected as P. falciparum -only infections by microscopy but P. falciparum mixed infections by LDR-FMA also showed similar outcome by the RDT band positivity. In addition, we used varying parasitemia (3-0.0001%) of the laboratory-maintained 3D7 strain to validate this observation. A positive pLDH band in high P. falciparum -parasitemic individuals may complicate diagnosis and treatment, particularly when the microscopy is inconclusive for P. vivax , and the two infections require different treatments.

Background: Detection of Plasmodium antigens provides evidence of malaria infection status and is the basis for most malaria diagnosis., Methods: We developed a sensitive bead-based multiplex assay for laboratory use, which simultaneously detects pan-Plasmodium aldolase (pAldo), pan-Plasmodium lactate dehydrogenase (pLDH), and P. falciparum histidine-rich protein 2 (PfHRP2) antigens. The assay was validated against purified recombinant antigens, monospecies malaria infections, and noninfected blood samples. To test against samples collected in an endemic setting, Angolan outpatient samples (n = 1267) were assayed., Results: Of 466 Angolan samples positive for at least 1 antigen, the most common antigen profiles were PfHRP2+/pAldo+/pLDH+ (167, 36%), PfHRP2+/pAldo-/pLDH- (163, 35%), and PfHRP2+/pAldo+/pLDH- (129, 28%). Antigen profile was predictive of polymerase chain reaction (PCR) positivity and parasite density. Eight Angolan samples (1.7%) had no or very low PfHRP2 but were positive for 1 or both of the other antigens. PCR analysis confirmed 3 (0.6%) were P. ovale infections and 2 (0.4%) represented P. falciparum parasites lacking Pfhrp2 and/or Pfhrp3., Conclusions: These are the first reports of Pfhrp2/3 deletion mutants in Angola. High-throughput multiplex antigen detection can inexpensively screen for low-density P. falciparum, non-falciparum, and Pfhrp2/3-deleted parasites to provide population-level antigen estimates and identify specimens requiring further molecular characterization.

Objective: Rapid diagnostic tests have been of tremendous help in malaria control in endemic areas, helping in diagnosis and treatment of malaria cases. It is heavily relied upon in many endemic areas where microscopy cannot be obtained. However, caution should be taken in the interpretation of its result in clinical setting due to its limitations and inherent weakness. This paper seeks to present the varying malaria RDT test results, the possible interpretations and explanation of these results common in endemic regions. Published works on malaria RDT studies were identified using the following search terms "malaria RDT in endemic areas", "Plasmodium falciparum and bacterial coinfection" "Plasmodium falciparum RDT test results in children in endemic areas" in Google Scholar and PubMed., Results: The review results show that RDT positive results in febrile patients can either be true or false positive. True positive, representing either a possible single infection of Plasmodium or a co-infection of bacteria and P. falciparum. False RDT negative results can be seen in febrile patient with P. falciparum infection in prozone effect, Histidine rich protein 2 (HRP2) gene deletion and faulty RDT kits. Hence, a scale up of laboratory facilities especially expert microscopy and other diagnostic tools is imperative.

The role of resveratrol (trans-3,5,4'-trihydroxystilbene; RES) in lysophosphatidylcholine (LPC)‑induced injury and inflammation in endothelial cells (regarded as an early event in arteriosclerosis) is unclear. The present study investigated whether RES reduces lactate dehydrogenase (LDH) activity and secretion of inflammatory cytokines such asinterleukin‑6 and tumor necrosis factor‑α, via the Toll‑like receptor (TLR)‑4/myeloid differentiation primary response gene 88 (MyD88)/nuclear factor (NF)‑κB signal transduction pathway in LPC‑induced damage and inflammation in human umbilical vein endothelial‑12 (HUVE‑12) cells. Using an ELISA and western blotting, the present study investigated the effects of RES on LDH activity and cytokine secretion. The effects of TLR‑4 short hairpin (sh)RNA and TLR‑4 cDNA transfection on NF‑κB activation during LPC‑induced damage and inflammation was also investigated in HUVE‑12 cells. The results demonstrated that RES significantly inhibited the effect of LPC on enzyme activity, pro‑inflammatory cytokine secretion, and expression of TLR‑4, MyD88 and NF‑κBp65 expression. In addition, RES and TLR‑4 shRNA transfection suppressed LPC‑induced injury and inflammation by blocking the TLR‑4/MyD88/NF‑κB signaling pathway Conversely, transfection with TLR‑4 cDNA enhanced LPC‑induced injury and inflammation, which abrogated the protective effects of RES. These data suggested that RES significantly suppressed LPC‑induced damage and inflammation, via suppression of the TLR‑4/MyD88/NF‑κB signaling pathway, which may provide a new mechanistic evidence for the treatment of arteriosclerosis by RES.

Lactate dehydrogenase (LDH) is key for anaerobic glycolysis. LDH is induced by the hypoxia inducible factor -1 (HIF-1). HIF-1 induces genes involved in glucose metabolism and regulates cellular oxygen homeostasis. HIF-1 is formed by a regulatory α-subunit (HIF-1α) and a constitutive β-subunit (HIF-1β). The white spot syndrome virus (WSSV) induces anaerobic glycolysis in shrimp hemocytes, associated with lactate accumulation. Although infection and lactate production are associated, the LDH role in WSSV-infected shrimp has not been examined. In this work, the effects of HIF-1 silencing on the expression of two LDH subunits (LDHvan-1 and LDHvan-2) in shrimp infected with the WSSV were studied. HIF-1α transcripts increased in gills, hepatopancreas, and muscle after WSSV infection, while HIF-1β remained constitutively expressed. The expression for both LDH subunits increased in each tissue evaluated during the WSSV infection, translating into increased enzyme activity. Glucose concentration increased in each tissue evaluated, while lactate increased in gills and hepatopancreas, but not in muscle. Silencing of HIF-1α blocked the increase of LDH expression and enzyme activity, along with glucose (all tissues) and lactate (gills and hepatopancreas) concentrations produced by WSSV infection. These results demonstrate that HIF-1 up regulates the expression of LDH subunits during WSSV infection, and that this induction contributes to substrate metabolism in energetically active tissues of infected shrimp., (Copyright © 2018. Published by Elsevier Ltd.)

Along with the determination of malaria infection rate among suspected patients attending hospitals in Hodeidah governorate, the present study evaluated the accuracy of Plasmodium falciparum histidine-rich protein-2 (PfHRP-2)/parasite-specific lactate dehydrogenase (pLDH)-based rapid diagnostic test (RDT) for the diagnosis of microscopy-confirmed falciparum malaria. An overall malaria infection rate of 19.3% (57/295) among suspected patients attending hospitals was microscopically confirmed. The sensitivity of thin blood films for the detection of malaria parasites was 79.0% compared to thick films and was greatly affected by the parasite density, being 65.0% or less at parasite densities of ≤1000 parasites/μl of blood. Compared to light microscopy, the present study revealed sensitivity levels of 100.0% (95% CI: 92.0-100.0) vs. 94.7% (95% CI: 84.2-98.6), specificity levels of 97.3% (95% CI: 89.8-99.5) vs. 100.0% (95% CI: 93.9-100.0), positive predictive values of 89.9% (95% CI: 88.3-99.0) vs. 100.0 (95% CI: 91.6-100.0) and negative predictive values of 100.0% (95% CI: 93.9-100.0) vs. 98.7% (95% CI: 89.3-98.7) for the PfHRP-2 and pLDH components of SD BIOLINE ® RDT, respectively, for falciparum malaria diagnosis. Therefore, the overall accuracy levels of the PfHRP-2 and pLDH components of the investigated RDT for the diagnosis of microscopy-confirmed falciparum malaria are 98.5% (95% CI: 94.6-99.6) and 97.7% (95% CI: 93.5-99.2), respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time., Methods: This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks., Results: Stability of HRP2 at ambient temperature was reported at 21-24 weeks while that of PAN antigens (pLDH and aldolase) was 2-18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever's solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min)., Conclusions: These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.

Cryptosporidium parvum is a zoonotic protozoan that can cause a life-threatening gastrointestinal syndrome in children and in immunocompromised adults. Currently, the only approved drug for treatment of Cryptosporidium infections in humans is nitazoxanide, but it is not effective in immunocompromised individuals or in children with malnutrition. This is compounded by the lack of genetic methods for studying and validating potential drug targets in the parasite. Therefore, in this study, we endeavoured to adapt the use of a phosphorodiamidate morpholino oligomer (morpholino) antisense approach to develop a targeted gene knockdown assay for use in C. parvum. We show that morpholinos, at non-toxic concentrations, are rapidly internalised by both C. parvum and host cells (HCT-8), and distribute diffusely throughout the cytosol. Using morpholinos to separately target C. parvum lactate dehydrogenase and putative arginine n-methyltransferase genes, within 36h of in vitro culture, we achieved over 10-fold down-regulation of the respective encoded proteins in C. parvum. Pursuant to this, we observed that knockdown of C. parvum lactate dehydrogenase produced a dramatic reduction in intracellular growth and development of C. parvum by 56h of culture. On the other hand, C. parvum putative arginine n-methyltransferase knockdown did not appear to have any effect on parasite growth, but nevertheless provided the proof-of-principle that the morpholino knockdown assay in C. parvum was consistent. Together, our findings present a gene regulation approach for interrogating gene function in C. parvum in vitro, and further provide genetic evidence for the essential role of C. parvum lactate dehydrogenase in fueling the growth and development of intracellular C. parvum., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The aim of the present study was to investigate the effect of chitosan (a naturally derived polymer) on the immune responses and glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactate dehydrogenase (LDH) levels in WEHI‑3 cell‑generated leukemia mice. Mice were divided into control, WEHI‑3 control, acetic acid (vehicle)‑treated, and 5 and 20 mg/kg chitosan‑treated groups. Mice were subsequently weighed, blood was collected, and liver and spleen samples were isolated and weighed. Blood samples were measured for cell markers, the spleen underwent phagocytosis and natural killer (NK) cell activity examination, and cell proliferation was analyzed by flow cytometry. Chitosan did not significantly affect the weights of body, liver and spleen at 5 and 20 mg/kg treatment. Chitosan increased the percentage of CD3 (T cells marker), decreased the levels of CD19 (B‑cell marker) and CD11b at 5 mg/kg treatment, and decreased the levels of Mac‑3 at 5 and 20 mg/kg treatment. Chitosan significantly increased macrophage phagocytosis of PBMCs, but did not significantly affect macrophage phagocytosis in the peritoneal cavity. Chitosan treatment did not significantly affect the cytotoxic activity of NK cells, and also did not affect T- and B-cell proliferation. Chitosan significantly increased total white blood cell numbers, and GOT and GPT activities were both significantly increased. However, chitosan did not significantly affect LDH activity in leukemia mice. Chitosan may aid in future studies on improving immune responses in the treatment of leukemia.

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Metabolism of neoplastic cells is shifted toward high glucose uptake and enhanced lactate production. Lactate dehydrogenase (LDH), which is comprised of two major subunits, LDH-A and LDH-B, reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate. LDH-A has a higher affinity for pyruvate and is a key enzyme in the glycolytic pathway. Elevated LDH is a negative prognostic biomarker not only because it is a key enzyme involved in cancer metabolism, but also because it allows neoplastic cells to suppress and evade the immune system by altering the tumor microenvironment. LDH-A alters the tumor microenvironment via increased production of lactate. This leads to enhancement of immune-suppressive cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and dendritic cells (DCs); and inhibition of cytolytic cells, such as natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs). By promoting immune-suppression in the tumor microenvironment, LDH-A is able to promote resistance to chemo/radio/targeted therapy. Here we discuss the evidence that LDH is both a metabolic and an immune surveillance prognostic biomarker and its elevation is harbinger of negative outcome in both solid and hematologic neoplasms.

Immunometabolism is emerging as a critical determinant of cancer pathophysiology. In this study, we explored the contributions of macrophage-expressed lactate dehydrogenase-A (LDH-A) to tumor formation in a K-Ras murine model of lung carcinoma. Myeloid-specific deletion of LDH-A promoted accumulation of macrophages with a CD86 high and MCP-1 high M1-like phenotype that suppressed tumor growth. This phenotypic effect was accompanied by reduced VEGF expression and angiogenesis, diminished numbers of PD-L1 + cancer cells, increased numbers of CD3 + T cells, and activation status of CD8 + T cells. Furthermore, it was associated with more pronounced antitumor T-cell immunity via induction of IL17 and IFNγ-producing CD8 + T (Tc17 and Tc1) cells, likely via suppression of lactate-driven PD-L1 expression. Our results suggest that expressions of LDH-A and lactate by macrophage in the tumor microenvironment are major drivers of T-cell immunosuppression, strongly supporting the concept of targeting stromal LDH-A as an effective strategy to blunt tumoral immune escape. Cancer Res; 77(13); 3632-43. ©2017 AACR ., (©2017 American Association for Cancer Research.)

We have developed a rapid magnetic microparticle-based detection strategy for malarial biomarkers Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein II (PfHRPII). In this assay, magnetic particles functionalized with antibodies specific for pLDH and PfHRPII as well as detection antibodies with distinct enzymes for each biomarker are added to parasitized lysed blood samples. Sandwich complexes for pLDH and PfHRPII form on the surface of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. The developed simultaneous capture and sequential detection (SCSD) assay detects both biomarkers in samples as low as 2.0parasites/µl, an order of magnitude below commercially available ELISA kits, has a total incubation time of 35min, and was found to be reproducible between users over time. This assay provides a simple and efficient alternative to traditional 96-well plate ELISAs, which take 5-8h to complete and are limited to one analyte. Further, the modularity of the magnetic bead-based SCSD ELISA format could serve as a platform for application to other diseases for which multi-biomarker detection is advantageous., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Hyperlipidemia aggravates myocardial ischemia/reperfusion (MI/R) injury through stimulating excessive inflammatory response. Therefore, blockade of inflammatory signal is a potential therapeutic management for MI/R complicated with hyperlipidemia. Hydroxysafflor yellow A (HSYA, a monomer extracted from Carthamus tinctorius L.), was studied in this article to address that the regulation of inflammatory signal would alleviate MI/R combined with hyperlipidemia injury. High-fat diet induced hyperlipidemia worsened MI/R mediated heart injury (elevation of infarct size, CK-MB and LDH activity), activated TLR4 over-expression in hearts, released inflammatory cytokines (LPS, TNF-α and IL-1β) excessively. HSYA administration suppressed the over-expression of TLR4 and alleviated heart damage caused by MI/R complicated with hyperlipidemia. Furthermore, HSYA had little influence on MI/R injury in TLR4-knockout mice, which indicated that HSYA protected MI/R through TLR4 inhibition. In vitro, hypoxia/reoxygenation (H/R) coexisting with LPS model in neonatal rat ventricular myocytes (NRVMs) induced serious damage compared with H/R injury to NRVMs. HSYA decreased excessive secretion of inflammatory cytokines, down-regulated over-expression of TLR4 and NF-κB in H/R + LPS injured NRVMs. In conclusion, HSYA alleviated myocardial inflammatory injury through suppressing TLR4, offering an alternative medication for MI/R associated with hyperlipidemia.

Okadaic acid (OA) is produced by dinoflagellates during harmful algal blooms and is a diarrhetic shellfish poisoning toxin. This toxin is particularly problematic for bivalves that are cultured for human consumption. This study aimed to reveal the effects of exposure to OA on the immune responses of bay scallop, Argopecten irradians. Various immunological parameters were assessed (total hemocyte counts (THC), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH), and nitric oxide (NO) in the hemolymph of scallops at 3, 6, 12, 24, and 48 h post-exposure (hpe) to different concentrations of OA (50, 100, and 500 nM). Moreover, the expression of immune-system-related genes (CLT-6, FREP, HSP90, MT, and Cu/ZnSOD) was also measured. Results showed that ROS, MDA, and NO levels and LDH activity were enhanced after exposure to different concentrations of OA; however, both THC and GSH decreased between 24-48 hpe. The expression of immune-system-related genes was also assessed at different time points during the exposure period. Overall, our results suggest that exposure to OA had negative effects on immune system function, increased oxygenic stress, and disrupted metabolism of bay scallops.

Multiplex assays detect the presence of more than one analyte in a sample. For diagnostic applications, multiplexed tests save healthcare providers time and resources by performing many assays in parallel, minimizing the amount of sample needed and improving the quality of information acquired regarding the health status of a patient. These advantages are of particular importance for those diseases that present with general, overlapping symptoms, which makes presumptive treatments inaccurate and may put the patient at risk. For example, malaria and dengue fever are febrile illnesses transmitted through mosquito bites, and these common features make it difficult to obtain an accurate diagnosis by symptoms alone. In this manuscript, we describe the development of a multiplexed, patterned paper immunoassay for the detection of biomarkers of malaria and dengue fever: malaria HRP2, malaria pLDH, and dengue NS1 type 2. In areas coendemic for malaria and dengue fever, this assay could be used as a rapid, point-of-care diagnostic to determine the cause of a fever of unknown origin. The reagents required for each paper-based immunoassay are separated spatially within a three-dimensional device architecture, which allows the experimental conditions to be adjusted independently for each assay. We demonstrate the analytical performances of paper-based assays for each biomarker and we show that there is no significant difference in performance between the multiplexed immunoassay and those immunoassays performed in singleplex. Additionally, we spiked individual analytes into lysed human blood to demonstrate specificity in a clinically relevant sample matrix. Our results suggest multiplex paper-based devices can be an essential component of diagnostic assays used at the point-of-care.

Lactate dehydrogenase (LDH) is a terminal enzyme in anaerobic glycolytic pathway. It widely exists in various organisms and is in charge of converting the glycolysis product pyruvic acid to lactic acid. Most parasites, including Clonorchis sinensis, predominantly depend on glycolysis to provide energy. Bioinformatic analysis predicts that the LDHs from many species have more than one transmembrane region, suggesting that it may be a membrane protein. C. sinensis LDH (CsLDH) has been confirmed as a transmembrane protein mainly located in the tegument. The antibodies against CsLDH can inhibit the worm's energy metabolism, kill the worm, and may have the same effects on human cancer cells. In this study, we cloned and characterized human LDHA (HsLDHA), HsLDHB, and CsLDH. Semi-quantitative real-time RCP showed that HsLDHB only existed in hepatocarcinoma cell SMMC-7721. Confocal microscopy and Western blot experiments revealed that HsLDHB was localized in the plasma membrane of SMMC-7721 cells, and the antibodies against CsLDH could cross-react with it. This cross-reaction could inhibit the enzymatic activity of HsLDHB. The cancer cells co-cultured with anti-CsLDH sera showed a significant decrease in cell proliferation rate and increases in caspase 9 and reactive oxygen species (ROS) levels. Therefore, anti-CsLDH antibodies can induce the apoptosis of cancer cells SMMC-7721 and may serve as a new tool to inhibit tumor.

The effect of whole body gamma irradiation (WBI) in single fraction was studied, as well as its influence on the secretion of various biochemical markers and cellular component that could be used as acute radiation lung injury marker. Sprague dawley rats were treated with WBI (60Co) of radiation dose from 1 Gy to 5 Gy (dose rate - 0.95 Gy/min). Bronchoalveolar lavage fluid was retrieved from all animals in control and radiation treated groups up to 72 h post radiation. Bronchoalveolar lavage fluid (BALF) was analyzed for lactate dehydrogenase (LDH ), acid phosphatase (AP ), alkaline phosphatase (ALP ), cell count and total protein. Intragroup and intergroup comparison of BALF parameters at different radiation doses showed significant difference. LDH was significantly increased as the dose increased from 1Gy to 5Gy (P = 0.00) after 2 h with effect size of difference (r > 0.3). ALP was significantly altered after 3Gy and 4Gy (P < 0.05). AP was significantly altered at 2Gy-5Gy (p < 0.05). Total protein level changed significantly from 1Gy to 5Gy (P < 0.00). Cellular content of BALF showed significant changes after radiation exposure. BALF parameters like LDH, AP, ALP, neutrophils, lymphocytes, total leukocyte count and total protein were sensitive to radiation exposure and their levels vary significantly up to 72 h after single whole body radiation exposure in Sprague dawley rats. It can be concluded that the biochemical indices in BALF have more wide application in evaluation of acute radiation induced lung injury.

Avian coccidiosis is mostly caused by mixed infection of several Eimeria species under natural conditions and immunity to avian coccidiosis is largely dependent on T-cell immune response. In this study, 14 T-cell epitope fragments from eight antigens of Eimeria tenella (E. tenella), Eimeria necatrix (E. necatrix), Eimeria maxima (E. maxima) and Eimeria acervulina (E. acervulina) were ligated with pVAX1 producing 14 monovalent DNA vaccines, respectively. Protective immunity of the monovalent DNA vaccines was assessed by in vivo challenge experiments and then four most protective fragments of each species were chosen to construct multivalent epitope DNA vaccines with or without chicken IL-2 as genetic adjuvant. Protective efficacies of the epitope DNA vaccines on chickens against E. tenella, E. necatrix, E. maxima and E. acervulina were evaluated. The results showed that the constructed multivalent epitope DNA vaccines significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds. Especially, the multivalent epitope DNA vaccines of pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1 and pVAX1-NA4-1-TA4-1-LDH-2-EMCDPK-1-IL-2 not only significantly increased body weight gain, alleviated enteric lesions and reduced oocyst output of the infected birds, but also resulted in anti-coccidial index (ACI) more than 170 against E. tenella, E. necatrix, E. maxima and E. acervulina, which indicated they could induce protective immunity against E. tenella, E. necatrix, E. maxima and E. acervulina. Our findings suggest the constructed multivalent epitope DNA vaccines are the potential candidate multivalent vaccines against mixed infection of Eimeria., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pheroid® technology was assessed as an alternative to Freund's adjuvant to raise antibodies in experimental animals. Chickens were immunized with two recombinantly expressed Plasmodium falciparum proteins, lactate dehydrogenase (PfLDH) and glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), alone or in combination with Freund's adjuvant or Pheroid®. Chicken egg yolk antibodies (IgY) were isolated and compared for specificity, sensitivity and yield. Freund's adjuvant and Pheroid® stimulated prolonged antibody responses in chickens against both antigens. Affinity purified antibodies had specificity for the recombinant and the native proteins on Western blots. Antibodies generated in the presence of Freund's adjuvant had high sensitivity for both antigens. Pheroid® generated antibodies that detected the lowest concentration of recombinant PfLDH. Freund's adjuvant and Pheroid® both improved chicken IgY yields, with Pheroid® showing a 2-fold increase relative to controls. Pheroid® was well-tolerated in chickens and has potential for development as a safe adjuvant for testing alternative stimulatory factors to improve adjuvant formulations.

Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80 kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80 kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80 kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80 kDA HAS suggest that the synthetic Peptide-1 of 80 kDa HSA is the promising candidate for development of male contraceptive vaccine.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.

Background: Due to limitation of conventional malaria diagnostics, including microscopy, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), alternative accurate diagnostics have been demanded for improvement of sensitivity and specificity., Methods: Serially diluted Plasmodium LDH antigens, Plasmodium falciparum-infected human red blood cells (RBC) derived from in vitro culture or patient's samples were used for evaluation of the performance of fluorescence-linked immunosorbent assay (FLISA). Microscopic examination was used to determine parasite density and the performance of FLISA was compared to ELISA. Finally, sensitivity and specificity of FLISA was determined by human specimens infected with P. falciparum, Plasmodium vivax, Toxoplasma gondii, and amoebae., Results: As a result of FLISA, the fluorescent intensity was highly correlated with antigen amount and FLISA was more sensitive than ELISA. FLISA detected at least 0.01 ng/ml of pLDH antigen, which showed 1,000-fold higher sensitivity than ELISA. In vitro-cultured P. falciparum was detected up to 20 parasite number/μL in FLISA but 5120 parasite number/μLin sandwich ELISA. In vitro P. falciparum-infected RBC number was highly correlated with fluorescent intensity (R2 = 0.979), showing that FLISA was reliable for detection of P. falciparum and available for quantification of parasite numbers. Furthermore, eighteen patient samples infected with P. falciparum (n = 9) and P. vivax (n = 9) showed 100% of sensitivity (18/18). FLISA showed 96.3% of specificity (26/27) because one sample of patient blood infected with T. gondii gave a false positive reactivity among healthy donors (n = 9), T. gondii-infected patients (n = 9), and amoeba-infected patients (n = 9)., Conclusion: FLISA has a keen and high performance to detect malaria antigen, suggesting a potential assay as malaria immunodiagnostic.

Bivalent rapid diagnostic tests are promising diagnostic tools for Plasmodium falciparum and P. vivax. Their diagnostic performance was evaluated against thick blood smear to assist national malaria control programs. A cross-sectional study was conducted to evaluate the performance of CareStart against thick blood smears among 398 acute febrile patients visiting the Felegeselam Health Center in December of 2011. Thick blood smears were examined under 100× objectives to diagnose Plasmodium species. Similarly, CareStart Malaria Pf/Pv Combo Test was performed as per the manufacturer's instruction. The ability of CareStart Malaria Pf/Pv Combo Test to diagnose Plasmodium malaria was very good, with 99.8% (95% confidence interval = 97.7-100%) sensitivity and 97.7% (95% confidence interval = 94.6-99.1%) specificity. The sensitivity and specificity of the CareStart Test is comparable with the thick blood smear in diagnosing malaria. Hence, it is preferable to use the CareStart Malaria Pf/Pv Combo Test instead of microscopy in areas where microscopic diagnosis is limited., (© The American Society of Tropical Medicine and Hygiene.)

Lactobacillus coryniformis is known to produce d-lactic acid as a dominant fermentation product at a cultivation temperature of approximately 30°C. However, the considerable production of l-lactic acid is observed when the fermentation temperature is greater than 40°C. Because optically pure lactates are synthesized from pyruvate by the catalysis of chiral-specific d- or l-lactate dehydrogenase, the higher thermostability of l-LDHs is assumed to be one of the key factors decreasing the optical purity of d-lactic acid produced from L. coryniformis at high temperature. To verify this hypothesis, two types of d-ldh genes and six types of l-ldh genes based on the genomic information of L. coryniformis were synthesized and expressed in Escherichia coli. Among the LDHs tested, five LDHs showed activity and were used to construct polyclonal antibodies. d-LDH1, l-LDH2, and l-LDH3 were found to be expressed in L. coryniformis by Western blotting analysis. The half-life values (t1/2) of the LDHs at 40°C were estimated to be 10.50, 41.76, and 2311min, and the T50(10) values were 39.50, 39.90, and 58.60°C, respectively. In addition, the Tm values were 36.0, 41.0, and 62.4°C, respectively, which indicates that l-LDH has greater thermostability than d-LDH. The higher thermostability of l-LDHs compared with that of d-LDH1 may be a major reason why the enantiopurity of d-lactic acid is decreased at high fermentation temperatures. The key enzymes characterized will suggest a direction for the design of genetically modified lactic acid bacteria to produce optically pure d-lactic acid., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum., Methods: Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies ("Test 1") and another which uses anti-LDH 35-305aa PAbs ("Test 2") as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests., Results: "Test 2" performed better at detecting microscopy-positive blood samples when compared to "Test 1", identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using "test 1". "Test 1" produced one false positive sample (from the 20 malaria-free control) blood samples; "test 2" produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with "test 1", but 0.734 when microscope-positive blood smears were compared with the results from "test 2". Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for "Test 1", and 99% and 45%, for "test 2". No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay., Conclusion: Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.

Plasmodial lactate dehydrogenase, terminal enzyme of the glycolytic pathway, has been shown to be biochemically, immunologically and structurally different from the mammalian enzyme. The substrate specific loop region of plasmodial lactate dehydrogenase (pLDH) has 5 amino acids insert (DKEWN) important for anti-malarial drug targeting. In the present study, we have produced six monoclonal antibodies, which are against three different epitopes of Plasmodium falciparum LDH (PfLDH). Two of these monoclonal antibodies (10C4D5 and 10D3G2) are against the substrate specific loop region of PfLDH (residues 98-109, AGFTKAPGKSDKEWNR). The 10C4D5 and 10D3G2 monoclonals bind to substrate specific loop region resulting in inhibition of PfLDH activity. A Microplate Sandwich ELISA was developed employing high affinity non-inhibitory (10A5H5, Kaff 1.272 ± 0.057 nM) and inhibitory (10C4D5, Kaff 0.306 ± 0.011 nM) monoclonal antibodies and evaluated using gossypol, a well known inhibitor of pLDH. The binding of gossypol to substrate specific loop region resulted in inhibition of binding of 10C4D5 monoclonal. This Microplate Sandwich ELISA can be utilized for identification of compounds inhibitory to PfLDH (binding to substrate specific loop region of parasite LDH) from combinatory chemical libraries or medicinal plants extracts. The Microplate Sandwich ELISA has also shown potential for specific diagnosis of malaria using finger prick blood samples.

Context: Cadmium oxide nanoparticles (CdO NPs) are employed in optoelectronic devices and as a starting material for generating quantum dots as well as for medical imaging and targeting of pharmaceutical agents to disease sites. However, there are lack of data concerning short- and long-term effects of CdO NPs on the lungs., Objective: To determine the effects of inhaled CdO NPs at an occupationally relevant concentration on pulmonary injury and repair, and on systemic immunity in adult male mice., Methods: Mice were exposed to 240 μg CdO NPs/m(3) for seven days (3 h/d) and lavage levels of pulmonary injury/inflammatory markers, bacterial uptake by circulating phagocytes, and lung histology examined either one or seven days following the final exposure., Results: Levels of total protein, lactate dehydrogenase activity, cytokine markers of inflammation (i.e. interleukin-1β, tumor necrosis factor-α, and interferon-γ), tissue remodeling matrix metalloproteinases (MMP)-2 and -9 activity, and phagocytic activity of circulating phagocytes were significantly increased one day after the final exposure. By seven days post-exposure, MMP-2 activity decreased to control levels, while MMP-9 activity remained significantly above control values, although dropping by about half from day one., Conclusions: This study demonstrates that short-term inhalation exposure to CdO NPs can stimulate pathways in the lungs associated with inflammation, cell injury, and tissue remodeling as well as alter immune function. Findings here demonstrate that even short-term inhalation exposure to CdO NPs in the workplace could lead to deleterious pulmonary effects in exposed workers.

Background: Malaria is a leading cause of mortality in southern Benin. The main causative agent, Plasmodium falciparum, poses a threat on critical transfusions in pregnant women and children. This study's objective was to compare the performance of different malaria screening methods in blood donors in southern Benin, a malaria-endemic country., Methods: Blood from 2,515 voluntary blood donors in Benin was collected over a period of 10 months in ethylenediaminetetraacetic acid (EDTA) tubes, which were then classified according to extraction time: long rainy season, short dry season, short rainy season, and long dry season. Microscopic examination was used to count parasites. Parasite density (PD) was expressed as the number of parasites per μL of blood. Pan Plasmodium pLDH detection was assessed by an ELISA-malaria antigen test. Using crude soluble P. falciparum antigens, an ELISA-malaria antibody test detected anti-Plasmodium antibodies., Results: Among the 2,515 blood donors (2,025 males and 488 females) screened, the rate of asymptomatic Plasmodium carriage was 295/2,515 (11.72%, 95% CI: 10.5-13.1%). Males had a higher infection rate (12.4%) than did females (8.8%). Parasite density was very low: between seven and100 parasites per μL of blood was reported in 80% of donors with parasitaemia. Three Plasmodium species were diagnosed: P. falciparum in 280/295 patients (95.0%), Plasmodium malariae in 14/295 (5.0%), and Plasmodium ovale in 1/295 (0.34%). Malaria prevalence in donors was higher during the rainy seasons (13.7%) compared with the dry seasons (9.9%). The use of a highly sensitive assay enabled pan Plasmodium pLDH detection in 966/2,515 (38.4%, 95% CI: 36.5%-40.3%). Malaria antibody prevalence was 1,859/2,515 (73.9%, 95% CI: 72.16-75.6%). Donors' antigenaemia and antibody levels varied significantly (P <0.05) over the course of the four seasons. The highest antigenaemia rate 323/630 (51.3%), was observed during the short rainy season, while the highest antibody prevalence, 751/886 (84.7%), was recorded during the long dry season., Conclusion: Blood donations infected with Plasmodium can transmit malaria to donation recipients. Malaria diagnostic methods are currently available, but the feasibility criteria for mass screening in endemic areas become preponderant. Detection of the pLDH antigen seems to be an adequate screening tool in endemic areas, for this antigen indicates parasite presence. Routine screening of all donated blood would prevent infected blood donations and reduce P. falciparum transmission in critical patients, such as children and pregnant women. This tool would also decrease medical prophylaxis in donation recipients and contribute to lower Plasmodium resistance.

Autism spectrum disorders (ASDs) are neurodevelopmental in origin, affecting an estimated 1 in 88 children in the United States. We previously described ASD-specific maternal autoantibodies that recognize fetal brain antigens. Herein, we demonstrate that lactate dehydrogenase A and B (LDH), cypin, stress-induced phosphoprotein 1 (STIP1), collapsin response mediator proteins 1 and 2 (CRMP1, CRMP2) and Y-box-binding protein to comprise the seven primary antigens of maternal autoantibody-related (MAR) autism. Exclusive reactivity to specific antigen combinations was noted in 23% of mothers of ASD children and only 1% of controls. ASD children from mothers with specific reactivity to LDH, STIP1 and CRMP1 and/or cypin (7% vs 0% in controls; P<0.0002; odds ratios of 24.2 (95% confidence interval: 1.45-405)) had elevated stereotypical behaviors compared with ASD children from mothers lacking these antibodies. We describe the first panel of clinically significant biomarkers with over 99% specificity for autism risk thereby advancing our understanding of the etiologic mechanisms and therapeutic possibilities for MAR autism.

In this study, changes in physical fatigue and biological functions of Japanese female soccer players were investigated by determining changes in neutrophil and lymphocyte functions. Study subjects included 18 female soccer players. Body composition, serum myogenic enzymes, neutrophil function, including reactive oxygen species (ROS) production capability, phagocytic activity (PA) and serum opsonic activity, as well as lymphocyte subpopulation were measured before and after a soccer match. Levels of myogenic enzymes (AST, ALT, CK and LDH) and immunoglobulins (IgG and IgA) and complements (C3) increased significantly after the match. In addition, leukocyte, neutrophils and lymphocyte counts increased whereas total PA decreased significantly. The number of T and Th1 cells (subsets of T helper cells) decreased whereas Th2 increased significantly. In addition, the number of B cells increased and NK cells decreased significantly after the match. The match was found to result in degenerative changes in and damage to athlete muscle tissues together with damage- and change-mediated stress. These data also suggest a post-match accelerated inflammatory reaction and potential immunosuppression as indicated by reductions in neutrophil PA and lymphocyte functions., (Copyright © 2012 John Wiley & Sons, Ltd.)

Predicting poor stem cell mobilization may prevent the costs and complications associated with remobilization. We retrospectively reviewed the relationship between serum lactate dehydrogenase (sLDH) activity and peripheral blood CD34 (PBCD34) cell counts in 46 granulocyte colony stimulating factor (G-CSF) induced mobilization cycles with or without chemotherapy. A significant correlation between post-mobilization (pre-apheresis) sLDH activity and PBCD34 count was found (r(s)=0.43, p=0.007). A strong correlation was shown between the pre- and post-mobilization sLDH activity difference and PBCD34 count (r(s)=0.55, p=0.001). With respect to sLDH activity differences; (a) all patients with a difference of >300 U/L, (b) 71% of patients with a difference of 100-300 U/L, and (c) 44% of patients with a difference of <100 U/L were considered to have good mobilizations. The sLDH activity difference between pre- and post-mobilization is a good indicator of successful mobilization. According to our results, the success of mobilization can be predicted based on sLDH activity differences; (i) good mobilization if the difference is >300 U/L, (ii) intermediate risk if the difference is 100-300U/L, and (iii) high risk if the difference is <100 U/L., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells., Methods: γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells., Results: After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35μg/ml to 18.75μg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group., Conclusion: Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.