Study On Influence Of Immunity in Mice

To learn more info plz contact david.wan@wecistanche.com

Two of thirty male DBA/2NCrl (D2N)mice used as a control strain in background data analysis of DBA/2N-MDX mice, presented with abnormal findings suggested of infection. Case 1: A 4-week-old male D2N mouse showed a white plaque lesion in the left kidney (no clinical symptoms), and a pathological examination of the kidney was performed. Inflammatory cell infiltration, mainly neutrophils, was observed in the tubules and interstitium of the left kidney, and Staphylococcus aureus (S.aureus)was detected from the left renal segment. Therefore, this lesion on the left kidney was diagnosed as suppurative tubulointerstitial nephritis caused by S. aureus infection. Case 2: Left torticollis with weight loss was detected in a 9-week-old, male D2N mouse. Histologic evaluation revealed exudative inflammation around the pharyngeal orifice of eustachian tubes, and purulent inflammation of both middle ears and left inner ear with abscess formation and clusters as gram-positive microcolonies. S. aureus was identified from swab cultures of both external ear canals. As a result, this case of torticollis was caused by S. aureus-suppurative otitis media and the internet. S. aureus is well known as an opportunistic pathogen with the potential to frequently cause suppurative diseases to immunocompetent laboratory animals. However, to our knowledge, this case of S. aureus infection presented with torticollis in a D2N mouse is unreported.

Click here to learn more about Cistanche

A methicillin-resistant Staphylococcus aureus(MRSA) isolates emerged in a laboratory mouse at the Center for Laboratory Animal Science of the National Defense Medical College in 2018 and MRSA isolates were recovered from mice also in the other mouse rooms thereafter. On these MRSA isolates, molecular typing such as Multi-locus sequence typing(MLST), Staphylococcus aureus Protein A(spa)typing, SCCmec typing, and coagulase typing were performed for the determination of their genotypes. All isolates revealed identical genotypes (ST1, spa t1784, SCCmec type IV, and coagulase type VII), suggesting that the isolates were identical clones. Before the emergence of MRSA in 2018, no isolates with such genotypes had not been isolated at this facility, it was assumed that the MRSA clone was brought into the facility via human or laboratory animals. STl1 is the first community-acquired MRSA recovered from children in the United States, however, ST1 is recovered from humans and animals worldwide currently. In Japan, ST1 and SCCmec type IV strains have been isolated from humans and cats, suggesting that ST1 is transmitted between humans and animals. An MRSA strain with these genotypes but also with spa tl784 has been recovered from humans. Currently, the distinction between hospital-acquired MRSA, community-acquired MRSA, and livestock-related MRSA is more ambiguous. These results suggest that mice may act as reservoirs of MRSA.

In recent cancer immunotherapy, the activity of molecules such as PD-1 is inhibited to increase the activity of immune cells against cancer. Despite the high therapeutic effect, there are cases in which autoimmune disease develops in cancer immunotherapy. Here, we established a system that can degrade PD-1 in a time-specific manner in cultured cells and mice using the SMASh degron system. PD-1-SMASh fusion protein in Jurkat cells and CD3+ splenocytes was reduced by 1 day after administration of Asunaprevir(ASV) or Grazoprevir(GRV) which are NS3/4A protease inhibitors. Growth of MC-38 adenocarcinoma cells inoculated in PD-1-cherry-SMASh knockin(KI) mice with ASV was repressed compared to wild-type(WT) and untreated KI mice. Moreover, the WT mice transplanted with KI bone marrow cells after being irradiated with lethal radiation could reject MC-38 cells with GRV. KI mice appeared healthy and showed no signs of autoimmune disease. We suggest that the use of the degron system in living organisms is expected to advance not only various biological studies but the treatment of diseases.

Progesterone (P4)is secreted from the placenta to maintain gestation and functions as an immune regulator. Glucocorticoid is another immune-regulatory steroid to regulate inflammation. In this study, human peripheral blood mononuclear cells(PBMCs) were transplanted into NOG mice to induce graft-versus-host-disease (GVHD)and used for the comparison between P4 and cortisol (COR)effects. PBMCs were stimulated in the presence of various concentrations of P4 or COR and the cells were analyzed by flow cytometry (FCM). Aliquots of PBMCs were transplanted into NOG mice, and P4 or COR was injected into the mice twice a week. After 28 days, FCM and immunohistochemical analyses were performed. As a result, a high concentration of P4 inhibited T cell activation in vitro, while the inhibition did not continue after the removal. The suppression of T cell activation was less in COR treatment compared to P4, and T cell exhaustion was more prominent. P4-injected mice carried more splenocytes, especially CD8 T cells, compared to control mice. However, the lower expression of CD25, less infiltration of human T cells into the lung and liver, and fewer GVHD symptoms were observed. On the other hand, the number of splenocytes, especially T cells, in COR-injected mice was significantly lower, and activated/exhausted cells were abundant. These results suggest that P4 prefers to maintain T cells compared to COR in vitro.

Helicobacter hepaticus and Helicobacter bills are known as pathogens that cause hepatitis and colitis in mice. The existence of several positive cases is still confirmed annually in experimental animal facilities in Japan. It is best to use cecal contents or fresh feces as a sample for PCR testing of H. hepaticus, and H. bilis.

Recently, DNA and RNA transport and storage reagents have been manufactured by various manufacturers. It is expected that these reagents will be the problems in transporting samples used for PCR tests will be cleared. Therefore, in this study, the usefulness of the nucleic acid storage reagent was examined by performing a PCR test of Helicobacter using the contents of the cecum preserved using the nucleic acid preservation reagent. As a result, samples stored for 7 days using the preservation solution could be detected Helicobacter at the same level as the fresh sample. From the above, it was suggested that in the PCR test of Helicobacter, Even if the transportation takes days, it is possible to carry out the test with high accuracy by transporting the sample immersed in the nucleic acid storage reagent by refrigerated flight.

We have previously developed E.coli mutant strains with significantly enhanced adherence to in vitro solid surfaces by modifying the genes that respond to nutrient starvation. To evaluate the interaction between these adhesion-enhanced E. coli strains(AEEs) and in vivo intestinal environment, we assessed their colonization ability to the intestinal tract by inoculating mice.

Before the evaluation experiment, we transformed AEEs with a plasmid expressing luciferase for in vivo visualizing. as were orally inoculated into fasted mice and collected feces every few days. The number of live AEEs was estimated by measuring the CFU from the collected feces.

As a result, compared to the control strain, AEEs were more abundant in the feces for more than one week, which indicates enhanced colonization in the intestinal tract. By observing the localization of luciferase luminescence by non-invasive imaging using IVIS, it was also found that AEEs tended to colonize at the cecum and colon.

[Objective] Currently, [Pasteurella] pneumotropica has been reclassified as a new genus Rodentibacter. Of these, R.pneumotropicus and R. Healy are the main targets for microbiological monitoring in rodents. We have isolated a number of Rodentibacter sp. that could not be identified as species from rodents. In this study, we explore the characteristics of Rodentibacter sp. isolate, focusing on bacterial virulence. [Methods] Rodentibacter sp. that was isolated from rat trachea were used for the study. Draft genome sequencing was performed on the next-generation sequencer, and then, the virulence-associated genes were predicted. In animal experiments, Rag2 mice were used for Rodentibacter sp. infection.|Results| By the draft genome sequencing, a unique RTX toxin-coding gene had been identified in Rodentibacter sp. The protein sequence of RTX toxin was more homologous to those produced by Enterobacteriaceae than by Pasteurellaceae. In an animal experiment, almost all infected Rag 2 mice were confirmed fatal pneumonia or septicemia. Although the isolate could not be identified as R. pneumotropicus and R. heylii, this closely related species can be lethal to immunocompromised animals in the presence of virulence factors such as RTX toxin.

In the NBRP Basic Technology Development Program (2018-2019), we sequenced the genomes of murine opportunistic pathogenic bacteria, and performed RNAseq of protozoa in mice, in order to develop PCR assays for these organisms. We used these results to analyze Rodentibacter spp. and protozoa in rats in collaboration with Kyoto University (NBRP Rat). Here, we report the test results in rat samples. [Methods] The 16S rRNA gene sequences of about 1.5 kbp were read by PCR for six rat isolates of Rodentibacter spp., and the draft genome sequence of one isolate(#25)was determined using Illumina HiSeq 2500. PCR of rat cecal contents was performed based on RNAseq results of mouse protozoa. [Results] The 16S rRNA gene sequences of all isolates were identical, and they were identified as Rodentibacter Ratti by OTU analysis. Resequencing of isolate #25 also revealed this strain was R. Ratti.PCR for murine protozoa gave positive results for rat amoeba. No positive results were obtained for Trichomonas PCR on rat specimens. [Discussion] The rat isolates of Rodentibacter spp. examined in this report were all R. Ratti. It is important to be aware of the presence of R. Ratti when performing Rodentibacter tests in rats. Some rat fecal samples showed positive for mouse amoeba PCR. This is the first step in the development of PCR for the detection of protozoa in rats.

Rodentibacter organisms 'Pasteurella pneumotropica'are opportunistic pathogenic bacterium that causes respiratory diseases in mice and rats. They were formerly classified in the genus Pasteurella, and several strains were tentatively proposed as biotypes. In 2017, they were collectively reorganized into the genus Rodentibacter. MaM and MaR strains were isolated from mice and rats, respectively, around the 1980s. In this study, we determined the genome sequences of both strains and compared them with those of the genus Rodentibacter, materials and MethodsIThe MaM and MaR strains were obtained from the National Institute of Infectious Diseases (NIAID)

(Manabu Saito --> Yasushi Ami -->Fumio Ike). Both strains were grown by liquid culture to extract genomic DNA, and the genomic sequences were determined using Illumina Hiseq 2500 and PacBio Sequel.[Results]Based on the 16S rRNA gene sequence, the MaM strain was identified as R.pneumotropicus and the MaR strain as R. Ratti. Furthermore, the complete genome sequence(2,213,961 bp)of the MaR strain was determined (the number of rRNA operons was 6 and the number of genes was estimated to be 2,063). [Discussionl The MaM and MaR strains have been used as standards for identifying'Pasteurella pneumotropica'in Japan. From this analysis, we determined the species of both strains as follows: the MaM strain is R.pneumotropicus and MaR strain is R.ratti. Remarkably, these results show R. Ratti can be isolated from rats in Japan.

Clostridium pili for me(CP) is susceptible to infectious in various laboratory animals including rats and mice and shows asymptomatic infection except in immunocompromised animals. It is considered a major differential diagnosis to maintain animal facilities and is well-known to affect experimental data from animal studies. Serodiagnosis (ELISA, IFA)and PCR are commonly used for the diagnosis of CP because the isolation in the artificial nutrient medium is difficult for CP. However, the existence of the non-specific antibody indicating the cross-reactivity with CP using the serodiagnosis is reported and is recognized often in a similar case in our facilities. Moreover, the PCR method has limits as a reliable diagnostic method with periodical monitoring, because CP is undetectable in feces after the increasing antibody titer. Recently, we examined a different approach, preserving feces from animals every month. We adopted the PCR test for CP not affected by the influence of the antibodies as more reliable diagnostics using the feces continuously preserved. In this report, we show a recent instance. (non-specific antibody) to lead to inspection of the flow developing in our company.