Continuous large-scale production of the cytokine CXCL8 from a novel porcine cell line

Cytokine production from two unstimulated porcine cell lines (SL-24 and SK-L) was examined using porcine cytokine detection ELISA kits and RT-PCR. Porcine IL-1 alpha, IL-6, and CXCL8 were detected in all samples examined. In particular, the SL-24 cell line (derived from bone marrow cells of a malignant lymphoma-affected pig), produced large amounts of porcine CXCL8. Flow cytometer analysis showed the cell line to be strongly CD44 positive, and was therefore considered to be of monocyte or macrophage origin. Porcine CXCL8 production was greatest (83.86 +/- 32.33 ng/mL) at six days post-cultivation. The SK-L cell line (derived from porcine kidney) also produced CXCL8, but production was less than 1.5 ng/mL. Porcine CXCL8 from the SL-24 cell line, induced chemotactic activity in porcine neutrophils, while the production of CXCL8 from the SL-24 cell line was inhibited by dexamethasone, which suggests that the mechanism of CXCL8 production is related to an NF-kappaB binding site. The production of CXCL8 from the SL-24 cell line was enhanced by the addition of recombinant porcine IL-15, which is the first reported observation of such CXCL8 production. Cloning of the SL-24 cell line by limited dilution revealed two types of cells present in the starting population. One cell type, designated as long-form cells (LC), produced large amounts of CXCL8, while the other, designated short-form cells (SC), produced small amounts of the cytokine. The LC cells were adapted to grow in serum-free medium in which they produced large amounts of CXCL8. The large-scale production of porcine CXCL8 from the SL-24 cell line will be of value in determining the mechanism of cytokine production and as a source of naturally produced porcine CXCL8.     

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23^ahl) and ahl8 (Fscn2^ahl8) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2^ahl8 allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3–54.5, 64.6–119.9, and 119.9–137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80–3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F₁ × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2^ahl8 to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23^ahl allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.     

Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation

Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17–20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2–4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.     

Development of a novel pink-eyed dilution mouse model showing progressive darkening of the eyes and coat hair with aging

Oca2^p-cas (oculocutaneous albinism II; pink-eyed dilution castaneus) is a coat color mutant gene on mouse chromosome 7 that arose spontaneously in wild Mus musculus castaneus mice. Mice homozygous for Oca2^p-cas usually exhibit pink eyes and gray coat hair on the non-agouti genetic background, and this ordinary phenotype remains unchanged throughout life. During breeding of a mixed strain carrying this gene on the C57BL/6J background, we discovered a novel spontaneous mutation that causes darkening of the eyes and coat hair with aging. In this study, we developed a novel mouse model showing this unique phenotype. Gross observations revealed that the pink eyes and gray coat hair of the novel mutant young mice became progressively darker in color by approximately 3 months after birth. Light and transmission-electron microscopic observations revealed a marked increase in melanin pigmentation of coat hair shafts and choroid of the eye in the novel mice compared to that in the ordinary mice. Sequence analysis of Oca2^p-cas revealed a 4.1-kb deletion involving exons 15 and 16 of its wild-type gene. However, there was no sequence difference between the two types of mutant mice. Mating experiments suggested that the novel mutant phenotype was not inherited in a simple fashion, due to incomplete penetrance. The novel spontaneous mutant mouse is the first example of progressive hair darkening animals and is an essential animal model for understanding of the regulation mechanisms of melanin biosynthesis with aging.     

Effect of new training technique on affinity of cynomolgus monkeys for animal care personnel

To confirm our hypothesis that the sex and age of cynomolgus monkeys influences the effect of training, we employed a new training technique designed to increase the animal’s affinity for animal care personnel. During 151 days of training, monkeys aged 2 to 10 years accepted each 3 raisins/3 times/day, and communicated with animal care personnel (5 times/day). Behavior was scored using integers between −1 and 5. Before training, 35 of the 61 monkeys refused raisins offered directly by animal care personnel (Score −1, 0 and 1). After training, 28 of these 35 monkeys (80%) accepted raisins offered directly by animal care personnel (>Score 2). The mean score of monkeys increased from 1.2 ± 0.1 to 4.3 ± 0.2. The minimum training period required for monkeys to reach Score 2 was longer for females than for males. After 151 days, 6 of the 31 females and 1 of the 30 males still refused raisins offered directly by animal care personnel. Beneficial effects of training were obtained in both young and adult monkeys. These results indicate that our new training technique markedly improves the affinity of monkeys for animal care personnel, and that these effects tend to vary by sex but not age. In addition, abnormal behavior and symptoms of monkeys were improved by this training.     

A Soluble Form of LMIR5/CD300b Amplifies Lipopolysaccharide-Induced Lethal Inflammation in Sepsis

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (M) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal M via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal M. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.     

The development of colitogenic CD4(+) T cells is regulated by IL-7 in collaboration with NK cell function in a murine model of colitis

We previously reported that IL-7(-/-)RAG(-/-) mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4(+) T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4(+) T cells. To further investigate these roles of NK cells, RAG(-/-) and IL-7(-/-)RAG(-/-) mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (T(EM)) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44(+)CD62L(-) T(EM) and unique CD44(-)CD62L(-) T cell subsets were observed in the T cell-reconstituted RAG(-/-) recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44(+)CD62L(-) T(EM) subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG(-/-) and IL-7(-/-)RAG(-/-) recipient mice through targeting of colitogenic CD4(+)CD44(+)CD62L(-) T(EM) and, possibly, of the newly observed CD4(+)CD44(-)CD62L(-) subset present at the early stage of T cell development.     

ICOS-Expressing CD4 T Cells Induced via TLR4 in the Nasal Mucosa Are Capable of Inhibiting Experimental Allergic Asthma

Modulation of adaptive immune responses via the innate immune pattern recognition receptors, such as the TLRs, is an emerging strategy for vaccine development. We investigated whether nasal rather than intrapulmonary application of Protollin, a mucosal adjuvant composed of TLR2 and TLR4 ligands, is sufficient to elicit protection against murine allergic lower airway disease. Wild-type, Tlr2(-/-), or Tlr4(-/-) BALB/c mice were sensitized to a birch pollen allergen extract (BPEx), then received either intranasal or intrapulmonary administrations of Protollin or Protollin admixed with BPEx, followed by consecutive daily BPEx challenges. Nasal application of Protollin or Protollin admixed with BPEx was sufficient to inhibit allergic lower airway disease with minimal collateral lung inflammation. Inhibition was dependent on TLR4 and was associated with the induction of ICOS in cells of the nasal mucosa and on both CD4(+)Foxp3(+) and CD4(+)Foxp3(-) T cells of the draining lymph nodes (LNs), as well as their recruitment to the lungs. Adoptive transfer of cervical LN CD4(+)ICOS(+), but not CD4(+)ICOS(-), cells inhibited BPEx-induced airway hyperresponsiveness and bronchoalveolar lavage eosinophilia. Thus, our data indicate that expansion of resident ICOS-expressing CD4(+) T cells of the cervical LNs by nasal mucosal TLR4 stimulation may inhibit the development of allergic lower airway disease in mice.     

Interplay between minor and major groove-binding transcription factors Sox2 and Oct1 in translocation on DNA studied by paramagnetic and diamagnetic NMR

The pathways whereby Sox2 scans DNA to locate its specific binding site are investigated by NMR in specific and nonspecific Sox2·DNA complexes and in a specific ternary complex with Oct1 on the Hoxb1 regulatory element. Direct transfer of Sox2 between nonspecific sites on different DNA molecules occurs without dissociation into free solution at a rate of ～10(6) M(-1) s(-1), whereas one-dimensional sliding proceeds with a diffusion constant of ≥0.1 μm(2)·s(-1). Translocation of Sox2 from one specific DNA site to another occurs via jumping, involving complete dissociation into free solution (k(d) ～5-6 s(-1)) followed by reassociation (k(a) ～5 × 10(8) M(-1) s(-1)). In the presence of Oct1 bound to an adjacent specific site, k(d) is reduced by more than 10-fold. Paramagnetic relaxation measurements, however, demonstrate that sparsely populated (<1%), transient states involving nonspecifically bound Sox2 in rapid exchange with specifically bound Sox2 are sampled in both binary Sox2·DNA- and ternary Oct1·Sox2·Hoxb1-DNA-specific complexes. Moreover, Sox2 modulates the mechanism of translocation of Oct1. Both Sox2 and the Oct1 POU(HD) domain are transiently released from the specific ternary complex by sliding to an adjacent nonspecific site, followed by direct transfer to another DNA molecule, whereas the Oct1 POU(S) domain is fixed to its specific site through direct interactions with Sox2. Intermolecular translocation of POU(HD) results in the formation of a bridged intermediate spanning two DNA molecules, enhancing the probability of complete intermolecular translocation of Oct1. By way of contrast, in the specific Oct1·DNA binary complex, POU(S) undergoes direct intermolecular translocation, whereas POU(HD) scans the DNA by sliding.