Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

[Analysis of chloramphenicol in propolis extract by LC/MS/MS].

A quantitative analysis method using LC/MS/MS of chloramphenicol (CAP) in propolis extract (ethanol extract) has been established. Extraction of CAP from propolis extract was performed by adding water, followed by salting-out with sodium chloride. Through this procedure, the wax components of propolis extract could be effectively removed. LC separation was performed with a reverse-phase column (Mightysil RP-18 GP Aqua, 2.0 mm x 150 mm, 5 microm), using 10 mmol/L of ammonium acetate-acetonitrile (75 : 25) as a mobile phase. The flow rate was 0.2 mL/min. Ionization was performed by electrospray ionization (ESI) in the negative mode. The detection and quantification limits of CAP in propolis extract were 0.05 and 0.15 ng/g, respectively, and the recovery rate (spiked CAP level was 0.5 ng/g) was 111.2%. When eight samples of propolis extract products on the market were analyzed using this method, CAP was not detected (N.D.) in any of the samples.

Foot shock stress prolongs the telogen stage of the spontaneous hair cycle in a non-depilated mouse model.

BACKGROUND: There is an increasing evidence to indicate that stress can influence skin disease and cutaneous functions. Previous studies have shown that stress alters the murine hair cycle; however, these studies have been carried out by using mouse models in which the hair cycle is forcibly synchronized after depilation. OBJECTIVE: To examine whether foot shock stress (FS) changes the spontaneous hair cycle in a non-depilated animal model, and to evaluate the role of mast cells and substance P (SP) in the influence of stress on the hair cycle. METHODS: Changes in the spontaneous hair cycle and the inhibitory effects of a specific SP NK1 receptor antagonist were examined in non-depilated mice during 3-4 weeks of FS. RESULTS: Foot shock stress prolonged the telogen stage of the hair cycle and delayed the induction of the subsequent anagen stage in the animal model. FS caused an increase in the ratio of de-granulated mast cells in the skin, an increase in the number of TUNEL-positive cells, and a decrease in the number of Ki67-positive cells. The NK1 receptor antagonist, WIN 62577, inhibited these stress responses. CONCLUSION: Our results strongly support previous work, demonstrating that stress alters active hair-cycling in vivo through the action of SP.

Dynamic changes in nerve growth factor and substance P in the murine hair cycle induced by depilation.

Increasing evidence suggests that various neurotrophins and neuropeptides play an important role in the progression of hair follicle cycling. Among them, nerve growth factor (NGF) and substance P (SP) have attracted special interest recently. However, the interaction between these factors during hair cycling has not yet been systematically studied. We therefore investigated the mutual relationships between NGF and SP and the mechanism by which the anagen stage of the hair cycle is initiated. Fluctuations in numbers of SP-positive nerve fibers and variations in amounts of SP, NGF, and another neurotrophic factor, glial cell-derived neurotrophic factor, in skin in the C57BL/6 mouse depilation-induced hair cycle model, together with the spatiotemporal expression patterns of each of these factors, were followed simultaneously by enzyme-linked immunosorbent assay and immunohistochemistry. The main finding was that a surge in NGF expression and a rapid increase in NGF content in skin is an initial event within 1 day after depilation, followed by elevation of SP content and numbers of SP-containing fibers 2 days after the increase in NGF. Our findings suggest that a rapid and abundant increase in NGF plays a key role in the induction and progression of anagen hair cycling through keratinocyte growth promotion. NGF may also induce plastic changes such as sprouting and hyperplasia in dermal nerve fibers and enhance their SP production. Elevated levels of SP in skin may additionally contribute to the progression of consecutive anagen hair cycles.

Electroacupuncture and moxibustion influence the lipopolysaccharide-induced TNF-alpha production by macrophages.

The purpose of this study was to evaluate the modifying effects of electroacupuncture (EA) and moxibustion (Mox) through the communication networks of the neuroimmune system using the two-step bacterial stimulation method. When EA or Mox treatment was implemented immediately following injection of the Streptococcus pyogenes preparation OK-432, a significant suppression of the tumor necrosis factor (TNF)alpha production by the peritoneal macrophages induced by lipopolysaccharide (LPS 1 microg/ml) was observed. When the stimulation with EA or Mox was reduced in volume, the degree of suppression decreased correspondingly. Naloxone antagonized to some extent the suppression of TNFalpha induced by EA, but did not compete with the suppression of TNFalpha release induced by Mox. These results suggest that activated macrophages are an important target of the immuno-suppressive effects of EA and Mox and that mu-opioid receptor-mediated mechanisms are responsible, to some extent, for the suppressive effect of EA, although Mox may not be dependent on these mechanisms.

Intermittent foot shock stress prolongs the telogen stage in the hair cycle of mice.

Stress significantly influences skin diseases and cutaneous functions. Recently, interactions between stress and skin conditions have been studied in animal models using various systemic stressors. Here, we studied the effect of intermittent foot shock stress on the hair cycle of C57BL/6 mice. After a 2-week period of intermittent foot shock stress, we examined the changes in the depilation-synchronized hair cycle macroscopically and histologically and we also measured the plasma levels of corticosterone. We found that foot shock stress prolonged the telogen stage and delayed the subsequent anagen induction in the hair cycle. The distribution patterns of corticotrophin releasing factor or corticotrophin releasing factor receptor positive cells in the skin of stressed or of control mice were identical with those in the ordinal hair cycle. It is noteworthy that corticotrophin releasing factor positive keratinocytes were observed in the telogen follicles of the stressed mice but were negative in the telogen follicles of the non-stressed mice in this study. Plasma corticosterone levels were significantly higher in the stressed group than in the control group. These results suggest that increased levels of plasma corticosterone may be involved in the mechanism underlying the stress-induced delay of the hair cycle.

Two cases of lymphomatoid papulosis in children.

An 8-year-old Japanese boy had a 4-month history of self-healing crops of violaceous, scaling papules and several small indurated areas of erythema on his limbs, buttocks, and trunk. Histologically there was an infiltration of small lymphocytic cells with scattered large atypical cells expressing CD30. Characterization of T-cell receptor gene rearrangement showed monoclonality of the infiltrating cells. The second patient, a 15-year-old Japanese girl, had a 2-week history of self-healing papulovesicular eruptions on her face and limbs. Large CD30+ atypical cells were also noted in the perivascular lymphocytic infiltration. Immunohistochemical studies revealed CD8 expression on almost all CD30+ cells in the second case. In the literature, there have been two reports of children with large CD30+ atypical cells expressing CD8 and two cases expressing CD4, whereas all adult cases reported have had cells expressing only CD4.