Nephrotoxicity Evaluation and Proteomic Analysis in Kidneys of Rats Exposed to Thioacetamide

Thioacetamide (TAA) was administered orally at 0, 10, and 30 mg/kg bw daily to Sprague-Dawley rats aged 6-7 weeks for 28 consecutive days. The bodyweight decreased, however, the relative organ weights including the liver and kidneys increased. No significant histopathologic abnormalities were found in the kidneys. The numbers of monocytes and platelets were significantly increased, however, the mean corpuscular volume and hematocrit values were decreased significantly in rats exposed to 30 mg/kg bw TAA. The expression levels of Kim-1 and NGAL were increased 4 to 5-fold in the kidney tissue of rats exposed to TAA, resulting in significant nephrotoxicity. Proteomic analysis was conducted and a total of 5221 proteins spots were resolved. Of these, three and 21 protein spots were up- and downregulated in a dose-dependent manner, respectively. The validation of seven proteins was performed by Western blot analysis. The expression level of ASAP2 was significantly upregulated, whereas RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were reduced. Sixteen isoforms of MUP were found by the 2DE immunoblot assay and were significantly downregulated with increasing exposure to TAA. MUP isoforms were compared in the liver, kidneys, and urine of untreated rats and a total of 43 isoforms were found.

Elevated gaseous luminal nitric oxide and circulating IL-8 as features of Helicobacter pylori-induced gastric inflammation

Background: Gastric nitric oxide (NO) production in response to Helicobacter pylori via inducible nitric oxide synthase (iNOS) is suggested as a biomarker of inflammation and cytotoxicity. The aim of this study was to investigate relationships between gastric [NO], immunological biomarkers and histopathology. Materials and methods: Esophagogastroduodenoscopy was done in 96 dyspepsia patients. Luminal [NO] was measured by chemiluminescence. Biopsies were taken from gastric antrum and corpus for culture and histopathology. H. pylori IgG was detected by immunoblot assay. Biobanked plasma from 76 dyspepsia patients (11 H. pylori positives) was analyzed for 39 cytokines by multiplexed ELISA. Results: H. pylori-positive patients had higher [NO] (336 ± 26 ppb, mean ± 95% CI, n = 77) than H. pylori-negative patients (128 ± 47 ppb, n = 19) (P < 0.0001). Histopathological changes were found in 99% of H. pylori-positive and 37% of H. pylori-negative patients. Histopathological concordance was 78–100% between corpus and antrum. Correlations were found between gastric [NO] and severity of acute, but not chronic, inflammation. Plasma IL-8 (increased in H. pylori positives) had greatest difference between positive and negative groups, with eotaxin, MIP-1β, MCP-4, VEGF-A, and VEGF-C also higher (P < 0.004 to P < 0.032). Diagnostic odds ratios using 75% cut-off concentration were 7.53 for IL-8, 1.15 for CRP, and 2.88 for gastric NO. Conclusions: Of the parameters tested, increased gastric [NO] and circulating IL-8 align most consistently and selectively in H. pylori-infected patients. Severity of mucosal inflammatory changes is proportional to luminal [NO], which might be tied to IL-8 production. It is proposed that IL-8 be further investigated as a blood biomarker of treatment outcomes.

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

Mycobacterium tuberculosis (Mtb) infects one-quarter of the world’s population. Mtb and HIV coinfections enhance the comorbidity of tuberculosis (TB) and AIDS, accounting for one-third of all AIDS-associated mortalities. Humoral antibody to Mtb correlates with TB susceptibility, and engineering of Mtb antibodies may lead to new diagnostics and therapeutics. The characterization and validation of functional immunoglobulin (Ig) variable chain (IgV) sequences provide a necessary first step towards developing therapeutic antibodies against pathogens. The virulence-associated Mtb antigens SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) are potential therapeutic targets but lacked IgV sequence characterization. Putative IgV sequences were identified from the mRNA of hybridomas targeting these antigens and isotype-switched into a common immunoglobulin fragment crystallizable region (Fc region) backbone, subclass IgG2aκ. Antibodies were validated by demonstrating recombinant Ig assembly and secretion, followed by the determination of antigen-binding specificity using ELISA and immunoblot assay.

Multiple Allergen Simultaneous Test-Immunoblot Assay for Immunoglobulin E Detection in Patients with Isolated Allergic Conjunctivitis

We aimed to investigate the immunoglobulin E (IgE) detection rate and allergen patterns in patients with isolated allergic conjunctivitis using the multiple allergen simultaneous test (MAST)-immunoblot assay. A total of 120 patients with allergic conjunctivitis and no associated rhinitis, asthma, or dermatitis underwent the MAST-immunoblot assay to measure serum total IgE (TIgE) and serum specific IgE (SIgE) against 57 allergens. Patients were classified into subgroups based on the season when the eye symptoms were exacerbated, and TIgE and SIgE positivity. Differences between sex and age groups were also analyzed. Of the 120 patients, 57.5% (69 patients) and 69.2% (83 patients) were positive for TIgE (≥100 IU/mL) and SIgE (≥0.7 IU/mL), respectively. The allergens that most frequently triggered sensitization in the study population were Dermatophagoides farinae, Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, Alternaria, and house dust. House dust mites, such as D. farinae and D. pteronyssinus, showed the highest detection rates regardless of the season. Men had a higher positive rate for TIgE than women, whereas a higher rate of sensitization, detected as SIgE positivity, was seen in younger patients. In conclusion, MAST-immunoblot assay can detect sensitizing allergens in patients with isolated allergic conjunctivitis.

Anti-TIF-1γ Antibody Detection Using a Commercial Kit vs In-House Immunoblot: Usefulness in Clinical Practice

ObjectivesAnti-TIF-1γ autoantibody detection is important for cancer screening in patients with dermatomyositis. The gold standard for anti-TIF-1γ detection, immunoprecipitation, is only available from a few specialized laboratories worldwide, so commercial ELISA/immunoblot tests have emerged in recent years. To analyze their usefulness in diagnosing cancer-associated dermatomyositis, we compared Euroimmun Euroline profile with our previously validated in-house immunoblot assay with human recombinant TIF-1γ.MethodsWe included 308 adult patients from Hospital de la Santa Creu I Sant Pau and Vall Hebrón Hospital (Barcelona, Spain) tested for anti-TIF-1γ autoantibodies using the Euroline profile and an in-house immunoblot assay.ResultsA total of 27 anti-TIF-1γ were detected by the Euroline and 12 by the in-house assay. Fair agreement was observed between Euroline and the in-house immunoblot Cohen’s kappa 0.3163. Expected prevalence of anti-TIF-1γ autoantibodies was observed for the two methods for dermatomyositis and undifferentiated connective tissue diseases, but unexpectedly high prevalence of anti-TIF-1γ autoantibodies was detected by Euroline compared to the in-house immunoblot for other diseases (16.5% Euroline vs 0.8% in-house immunoblot, p&lt;0.01). The in-house IB compared to Euroline more reliably detected cancer in patients with DM with anti-TIF-1γ antibodies (p=0.0014 vs p=0.0502 for in-house immunoblot vs Euroline).ConclusionWe recommend using a second validated method to confirm Euroline-detected anti-TIF-1γ antibodies when the dermatomyositis diagnosis is not definitive. Furthermore, in the context of definite DM diagnosis with negative anti-TIF-1γ antibodies by Euroline and no other myositis specific antibody, is also recommendable to confirm by a second validated method.

Chicoric acid alleviates LPS-induced inflammatory response through miR-130a-3p/IGF-1pathway in human lung A549 epithelial cells

To investigate the effects and potential mechanisms of chicoric acid (CA) on LPS-induced inflammatory response in A549 cells. 0–800 μM CA was added to A549 cells to determine the toxicity of CA using MTT assay. Then, 2 μg/mL LPS and 50 μM CA were simultaneously added to A549 cells to investigate the effects of CA. In order to investigate the effects of miR-130a-3p and IGF-1 on LPS-induced A549 cells, cells were infected with inhibitor of miR-130a-3p and si RNA IGF-1. The levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α were measured by real-time RT-PCR and enzyme-linked immunosorbent (ELISA) assay. The IGF-1 pathway and NF-κB expression were measured using immunoblot assay. Moreover, a luciferase activity assay was used to indicate the binding site of miR-130a-3p on the 3′UTR of IGF-1. 0–50 μM CA had no toxicity on A549 cells. Thus, we chose 50 μM CA for the following study. CA attenuated the inflammatory response by LPS through decreasing IL-1β, IL-6, and TNF-α levels and increasing IGF-1/IGF-1R expression. Inhibition of miR-130a-3p reduced the inflammatory response and restored IGF-1/IGF-1R pathway induced by LPS. Furthermore, luciferase activity results indicated that miR-130a-3p directly targeted IGF-1 to regulate inflammatory response. CA alleviates LPS-induced inflammatory response through miR-130a-3p/IGF-1pathway in A549 cells.

Chrysotoxine attenuates sevoflurane-induced neurotoxicity in vitro via regulating PI3K/AKT/GSK pathway

Objective: The aim of this study is to investigate the neuroprotective effect of chrysotoxine (CTX) on sevoflurane-treated nerve cells and uncover the potential regulation mechanism. Methods: Nerve cells treated with sevoflurane and CTX were subjected to MTT and apoptotic detection. Cell apoptosis and oxidative stress were detected by flow cytometry (FCM) and ELISA assays. In addition, immunoblot assay was performed to study the signaling pathway affected by CTX treatment. Results: CTX treatment promoted the cell viability and suppressed the apoptosis of sevoflurane-treated SH-SY5Y cells. In addition, CTX inhibited the sevoflurane-induced oxidative stress response and inflammatory response in nerve cells. Mechanically, CTX ameliorated neurotoxicity through activating the PI3K/AKT/GSK signaling pathway. Conclusion: Therefore, CTX can serve as a promising drug target for treating anesthetics-induced neurotoxicity.

Evaluation of HCV RNA by PCR and Signal-to-Cutoff Ratios of HCV Antibody Assays for Diagnosis of HCV Infection

Objective In this study, we assessed whether a hepatitis C virus (HCV) RNA test could replace recombinant immunoblot assay (RIBA) and reduce unnecessary supplemental tests as the signal-to-cutoff (S/Co) ratio from anti-HCV antibody (Ab) tests. Methods Anti-HCV Ab tests were performed to screen for HCV infections, and RIBA and real-time polymerase chain reaction were performed for HCV RNA to confirm HCV infection. Receiver operating characteristic curves were evaluated to determine the optimal S/Co ratios for predicting HCV infection. Results The cutoff value for the S/Co ratio was 3.63 for predicting RIBA results and 10.6 for predicting HCV RNA results. Our data suggested that an S/Co ratio ≥10.6 indicated a high risk of active HCV infection. An S/Co ratio of 3.63 to 10.6 needed further evaluation and repeat HCV RNA testing. No further testing was required for S/Co ratios &lt;3.63 or ≥10.6. Conclusion We determined that the S/Co ratio of the anti-HCV Ab test provides useful information to confirm HCV infections, including the need for further laboratory testing or clinical follow-up.