Advanced microinjection protocol for gene manipulation using the model newt Pleurodeles waltl.

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

Characterization of pathogenic T cells and autoantibodies in C-protein-induced autoimmune polymyositis.

Although autoimmune processes may take place in human polymyositis, little is known with regard to its pathogenesis due to the lack of appropriate animal models. In the present study, we developed experimental autoimmune myositis (EAM) in Lewis rats by immunization with recombinant skeletal C-protein and examined the role of pathogenic T cells and autoantibodies. Using recombinant proteins and synthetic peptides, we demonstrated that skeletal C-protein Fragment 2 (SC2) has the strongest myositis-inducing ability and that myositis-inducing epitope(s) reside within the residues 334-363 of SC2 (SC2P3). However, immunization with SC2P3 induced only mild EAM compared with SC2 immunization. Characterization of T cells and antisera revealed that SC2P3 and SC2P7 contain the B cell epitope, while the T cell epitope resides in SC2P5. Furthermore, anti-SC2, but not anti-SC2P3, antisera contained antibodies against the conformational epitope(s) in the SC2 molecule. However, SC2P3 or SC2P5 immunization plus anti-SC2 antibody transfer aggravated the disease only slightly. These findings suggest that C-protein-induced EAM is formed by activation of C-protein-specific T cells along with antibodies against conformational epitopes in C-protein but that there are undetermined factors related to the disease progression. Further analysis of C-protein-induced EAM will provide useful information to elucidate the pathomechanisms of human polymyositis.

Versatility of the Limberg flap and the V-Y flap (based on a distal perforator) for covering sacral ulcers.

Our simple criteria for selection of two efficient flaps achieves consistently good results for most sacral ulcers. One hundred and ten patients had their sacral ulcers reconstructed with the Limberg flap (n = 48) or the distal-perforator-based V-Y (DPVY) flap (n = 62). The criteria for selection were based on pinching of the donor skin to estimate the feasibility of the Limberg flap. Overall, 101/110 (92%) of the flaps healed primarily, 43/48 (90%) in the Limberg flap group, and 58/62 (94%) in the DPVY flap group. The advantages of reconstruction using our two flaps include simple and consistent design of, and procedure for, both flaps, wide excursion of the DPVY flap, and consistency of the surgeons' skill because they used only two flaps.

Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine.

Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.

Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice.

Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.