Detection of tuberculosis in cynomolgus macaques (Macaca fascicularis) using a supplementary Monkey Interferon Gamma Releasing Assay (mIGRA).

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.

Antigen host response differences between the animal-type strain and human-clinical Pythium insidiosum isolates used for serological diagnosis in Thailand.

The detection of Pythium insidiosum-specific-immunoglobulin-G antibody (Pi-Ab) with enzyme-linked immunosorbent assay (ELISA) test depends on the source of antigen. In this study, the Pi-Ab levels in 140 serum samples from patients with pythiosis were evaluated by ELISA using antigens from 10 P. insidiosum clinical isolates in comparison with antigen from the equine-standard-type strain. The ELISA values (EVs), calculated from antibody levels from serum of patients with pythiosis or other infections versus healthy controls, were significantly higher in the test with clinical-isolates antigen than the standard-equine-type strain (6.0 ± 2.6 vs 4.0 ± 1.7, respectively; P < .0001). ELISA with antigen from human source might be more proper diagnosis test.

Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155).

Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZαA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

Inhibition of nitric oxide production in the macrophage-like RAW 264.7 cell line by protein from the rhizomes of Zingiberaceae plants.

Nitric oxide (NO) plays a key role in the pathogenesis of inflammation and has been implicated in endotoxin-induced tissue injury. Zingiberaceae is a family of indigenous plants of tropical regions, many of which have traditionally been used as anti-inflammatory agents. Here, the ability of crude protein extracts from the rhizomes of 15 Zingiberaceae species to inhibit NO production in the RAW 264.7 cell line after co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) was evaluated. The crude protein extract of Zingiber ottensii Valeton exhibited the highest inhibitory activity, with an IC(50) value of 38.6 ± 0.34 µg protein/mL, and also suppressed the LPS- and rm-interferon (IFN)-γ-mediated increase in the inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and tumor necrosis factor (TNF)-α mRNA transcript expression levels, suggesting the interference was mediated at the transcriptional level. This strong anti-inflammatory activity may have the potential to be developed as a therapeutic compound. Analytical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry revealed four main protein bands, including a likely lectin, superoxide dismutase, and cysteine protease, in the fractions related to the antioxidant activity.

Defects in Notch1 upregulation upon activation of T Cells from patients with systemic lupus erythematosus are related to lupus disease activity.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of autoantibodies and deposition of immune complexes in various organs. T cells play a central role in driving disease progression, and multiple defects in T cells from patients with SLE have been uncovered. Notch signalling is an evolutionarily well-conserved signalling cascade involved in the proliferation, differentiation and apoptosis of T lymphocytes during development and peripheral effector functions. In this study, we investigated the correlation between expression of Notch receptor and the severity of SLE disease. On the contrary to T lymphocytes from healthy controls (n=11), Tlymphocytes from patients with active SLE (n=12) failed to upregulate Notch1 upon in-vitro stimulation as quantified by quantitative real time RT-PCR (P

Suppression of apoptotic cell death of IL-3-dependent cell lines by ER/SR Ca2+-ATPase inhibitors upon IL-3 deprivation.

An ER/SR Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA), was found to suppress apoptotic cell death of IL-3-dependent cell lines, FDC.P2, IC-2, and Ba/F3, upon IL-3 deprivation. Structurally unrelated ER/SR Ca2+-ATPase inhibitors, thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone also maintained cell viability in the absence of IL-3. In the Ca2+-free medium CPA failed to suppress apoptosis, suggesting that the anti-apoptotic activity of CPA is dependent on extracellular calcium. The culture supernatant of CPA-treated cells was able to prolong cell survival of FDC.P2 in the absence of IL-3. An anti-IL-4 antibody almost completely eliminated the anti-apoptotic activity of CPA. Indeed, a significant amount of IL-4 was detected in the supernatant of cells treated with ER/SR Ca2+-ATPase inhibitors. Thus, our present data clearly demonstrate not only that ER/SR Ca2+-ATPase inhibitors induce the secretion of IL-4 in IL-3-dependent cell lines, but also that IL-4 can replace IL-3 in protecting these cells from apoptotic cell death in an autocrine manner.