Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming
Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde-3-phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic-co-glycolic acid) – Chitosan (PLGA-Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated.
Two treatment groups [T1 – (PLGA-Chit-NPs-pDNA), T2 – (PLGA-NPs-pDNA) and one control group (T0 – 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT-PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish.
Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.
Influence of recombinant human-soluble thrombomodulin on extracorporeal circuit clotting in septic patients undergoing blood purification: a propensity-matched cohort study
Blood purification has been widely performed for critically ill patients, even in cases without renal failure. Effective anticoagulation of the extracorporeal circuit is necessary to prevent circuit clotting. We hypothesized that administration of recombinant human-soluble thrombomodulin (rhsTM) to septic patients undergoing blood purification may prevent circuit clotting, because this agent regulates coagulation. We performed a retrospective, single-center, propensity-matched cohort study in the intensive care unit of Nishichita General Hospital.
We included septic patients admitted to the intensive care unit from May 2015 to August 2020 who underwent blood purification. Patients who received rhsTM during intensive care unit admission to the end of the first blood purification (rhsTM group) were matched 1:1 with other patients (control group). The primary outcome was the occurrence of circuit clotting during the first blood purification. A total of 138 patients were included in the study [43 patients (31%) in the rhsTM group and 95 patients (69%) in the control group].
After propensity score matching, 42 pairs of patients were selected, and patients in the rhsTM group had a lower incidence of circuit clotting (21 vs. 55%, P = 0.003). One case of major bleeding occurred in the rhsTM group, but there was no difference in the incidence of major bleeding between groups (2 vs. 0%, P = 1.0). In conclusion, this propensity-matched cohort study indicated that the administration of rhsTM to septic patients undergoing blood purification may prevent extracorporeal circuit clotting.
Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer
Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently.
Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety.
Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs. Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.
The insecticidal activity of recombinant nemertide toxin α-1 from Lineus longissimus towards pests and beneficial species
The nemertide toxins from the phylum Nemertea are a little researched family of neurotoxins with potential for development as biopesticides. Here we report the recombinant production of nemertide α-1 (α-1), a 65-residue inhibitor cystine knot (ICK) peptide from Lineus longissimus, known to target insect voltage-gated sodium channels. The insecticidal activity of α-1 was assessed and compared with the well characterised ICK venom peptide, ω-atracotoxin/hexatoxin-Hv1a (Hv1a). α-1 elicited potent spastic paralysis when injected into cabbage moth (Mamestra brassicae) larvae; conferring an ED50 3.90 μg / larva (10.30 nmol / g larva), followed by mortality (60 % within 48 hours after 10 μg injection).
By comparison, injection of M. brassicae larvae with recombinant Hv1a produced short-lived flaccid paralysis with an ED50 over 6 times greater than that of α-1 at 26.20 μg / larva (64.70 nmol / g larva). Oral toxicity of α-1 was demonstrated against two aphid species (Myzus persicae and Acyrthosiphon pisum), with respective LC50 values of 0.35 and 0.14 mg / mL, some 6-fold lower than those derived for recombinant Hv1a. When delivered orally to M. brassicae larvae, α-1 caused both paralysis (ED50 11.93 μg / larva, 31.5 nmol / g larva) and mortality.
This contrasts with the lack of oral activity of Hv1a, which when fed to M. brassicae larvae had no effect on feeding or survival. Hv1a has previously been shown to be non-toxic by injection to the beneficial honeybee (Apis mellifera). By contrast, rapid paralysis and 100 % mortality was observed following injection of α-1 (31.6 nmol / g insect). These results demonstrate the great potential of naturally occurring non-venomous peptides, such as α-1, for development as novel effective biopesticides, but equally highlights the importance of understanding the phyletic specificity of a given toxin at an early stage in the quest to discover and develop safe and sustainable pesticides.