Intensified NK cell therapy in combination with low-dose chemoradiotherapy against human colorectal cancer.

The therapeutic potential of adoptive natural Killer (NK) cells immunotherapy in combination with chemoradiotherapy, the main treatment modality for colorectal cancer (CRC), has not yet been explored. Here, we aimed to investigate the efficacy of NK cells to potentiate primary tumor control and improve survival outcomes, especially in combination with low-dose chemoradiotherapy. Ex vivo activated NK cells (> 90% purity) from healthy donors were obtained. NK cells were administered intravenously to the CRC-bearing mice and intensified in vivo in combination with low-dose 5-fluorouracil (0.5 mg/kg or 1 mg/Kg) and irradiated tumors with low doses (2 Gy or 4 Gy). Real-time NK cell cytotoxicity demonstrated a synergistic killing effect of a combination of low-dose chemoradiotherapy, mainly through NKp30 and NKG2D, showing a decrease in NK cell degranulation after blocking NKG2D and NKp30. In vivo tumor characteristics after combination treatment showed decreased CD112, CD155, MICA, and MICB expression. Under the combination strategy, 70% of the mice had free lung metastasis and 90% without secondary gross tumors, indicating suppressed distant metastasis to lung and axillary regions. This combination therapy resulted in significantly synergistic antitumor activity against primary solid tumors compared to chemoradiotherapy only. Furthermore, the intensified NK cell administration showed significantly better primary tumor control and survival outcomes than the non-intensified NK cell administration in a human colorectal HT-29 model treated with low-dose chemoradiotherapy. Optimized NK cell therapy combined with low-dose chemoradiotherapy can provide effective therapeutic potential for intractable cold human colorectal cancer.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Chrysin Induces Apoptosis via the MAPK Pathway and Regulates ERK/mTOR-Mediated Autophagy in MC-3 Cells.

Chrysin is a flavonoid found abundantly in substances, such as honey and phytochemicals, and is known to exhibit anticancer effects against various cancer cells. Nevertheless, the anticancer effect of chrysin against oral cancer has not yet been verified. Furthermore, the mechanism underlying autophagy is yet to be clearly elucidated. Thus, this study investigated chrysin-mediated apoptosis and autophagy in human mucoepidermoid carcinoma (MC-3) cells. The change in MC-3 cell viability was examined using a 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide cell viability assay, as well as 40,6-diamidino-2-phenylindole, annexin V, and propidium iodide staining. Western blotting was used to analyze the proteins related to apoptosis and the mitogen-activated protein kinase (MAPK) pathway. In addition, the presence or absence of autophagy and changes in the expression of related proteins were investigated using acridine orange staining and Western blot. The results suggested that chrysin induced apoptosis and autophagy in MC-3 oral cancer cells via the MAPK/extracellular signal-regulated kinase pathway. Moreover, the induced autophagy exerted a cytoprotective effect against apoptosis. Thus, the further reduced cell viability due to autophagy as well as apoptosis induction highlight therapeutic potential of chrysin for oral cancer.

Safety and immunological effects of recombinant canine IL-15 in dogs.

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays pivotal roles in innate and adaptive immunity. It is also a promising cytokine for treating cancer. Despite growing interest in its use as an immunotherapeutic, its safety and immunological effects in dogs have not been reported. In this study, healthy dogs were given recombinant canine IL-15 (rcIL-15) intravenously at a daily dose of 20 µg/kg for 8 days and monitored for 32 days to determine the safety and immunological effects of rcIL-15. The repeated administration of rcIL-15 was well tolerated, did not cause any serious side effects, and promoted the selective proliferation and activation of canine anti-cancer effector cells, including CD3+CD8+ cytotoxic T lymphocytes, CD3+CD5dimCD21-, and non-B/non-T NK cell populations, without stimulating Treg lymphocytes. The rcIL-15 injections also stimulated the expression of molecules and transcription factors associated with the activation and effector functions of NK cells, including CD16, NKG2D, NKp30, NKp44, NKp46, perforin, granzyme B, Ly49, T-bet, and Eomes. These results suggest that rcIL-15 might be a valuable therapeutic adjuvant to improve immunity against cancer in dogs.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo.

BACKGROUND AIMS: Tracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. METHODS: Ex vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1'-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. RESULTS: The fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3- NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. CONCLUSIONS: Administered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3- NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.

Canine non-B, non-T NK lymphocytes have a potential antibody-dependent cellular cytotoxicity function against antibody-coated tumor cells.

BACKGROUND: The antibody-dependent cellular cytotoxicity (ADCC) is a cell-mediated immune defense mechanism in which effector immune cells actively lyse antibody-coated target cells. The ADCC of tumor cells is employed in the treatment of various cancers overexpressing unique antigens, and only natural killer (NK) cells are known to be major effectors of antibody mediated ADCC activity. Canine NK cells are still defined as non-B, non-T large granular lymphocytes because of the lack of information regarding the NK cell-restricted specific marker in dogs, and it has never been demonstrated that canine NK cells have ADCC ability against tumor cells. In the present study, we investigated whether canine non-B, non-T NK cells have ADCC ability against target antibody-coated tumor cells, using cetuximab and trastuzumab, the only human antibodies reported binding to canine cancer cells. RESULTS: Activated canine non-B, non-T NK cells (CD3-CD21-CD5-TCRαß-TCRγδ-) for 13~17 days ex vivo showed ADCC ability against trastuzumab- or cetuximab-coated target tumor cells expressing various levels of human epidermal growth factor receptor 2 (HER-2) and epidermal growth factor receptor (EGFR). Trastuzumab and cetuximab induced significant ADCC responses of canine NK cells even in CMT-U334 and CF41.Mg cells expressing low levels of HER-2 and/or EGFR, as well as in SKBR3 and DU145 cells overexpressing HER-2 and/or EGFR. The trastuzumab-mediated ADCC activity of NK cells was significantly enhanced by treatment with rcIL-21. CONCLUSIONS: The results of this study suggest that canine non-B, non-T NK lymphocytes have a potential ADCC function and that combinational strategies of monoclonal antibodies with either cytokines, which activate NK cells in vivo, or adoptive transfer of NK cells may be a feasible method for amplifying the efficacy of immunotherapy against malignant cancers even with very low expression of target molecules in dogs.

Effect of irradiation-induced intercellular adhesion molecule-1 expression on natural killer cell-mediated cytotoxicity toward human cancer cells.

BACKGROUND AIMS: Irradiation enhances the adhesion between natural killer (NK) cells and target cells by up-regulating intercellular adhesion molecule-1 (ICAM-1) on target cells. Therefore, we investigated the effect of irradiation-induced ICAM-1 expression on human cancer cells on NK cell-mediated cytotoxicity. METHODS: Expression levels of ICAM-1 on the target cell surface before and after irradiation of six human cancer cell lines (HL60, SKBR-3, T47D, HCT-116, U937 and U251) were analyzed by flow cytometry. Ex vivo expansion of NK cells from human peripheral blood mononuclear cells was performed by co-culture with irradiated K562 cells. The related adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) on NK cells was analyzed by flow cytometry. An enzyme-linked immunosorbent assay was used to detect interferon-γ (IFN-γ), and WST-8 assays were performed to check NK cell cytotoxicity. Finally, blocking assays were performed using monoclonal antibodies against ICAM-1 or LFA-1. RESULTS: LFA-1 expression increased on NK cells after expansion (P <0.001). The expression of ICAM-1 was significantly upregulated by irradiation after 24 h in various cell lines, including HL60 (P <0.001), SKBR-3 (P <0.001), T47D (P <0.001) and U937 (P <0.001), although the level of expression depended on the cell line. ICAM-1 expression was extremely low before and after irradiation in U251 cells. NK cell-mediated cytotoxicity increased after irradiation of HL60 (P <0.001), SKBR-3 (P <0.001), T47D (P = 0.003), and U937 (P = 0.004) cells, in which ICAM-1 expression was significantly increased after irradiation. IFN-γ production by NK cells in response to HL60 (P <0.001) and T47D (P = 0.011) cells significantly increased after irradiation. NK cell-mediated cytotoxicity against irradiated SKBR-3 (P <0.001) and irradiated T47D cells (P = 0.035) significantly decreased after blocking of ICAM-1. Blocking of LFA-1 on NK cells resulted in reduced cytotoxicity against irradiated HL60 (P <0.001) and irradiated SKBR-3 (P <0.001). CONCLUSIONS: Irradiation upregulates ICAM-1 expression on the surface of human cancer cells and enhances activated NK cell-mediated cytotoxicity. Therefore, irradiation combined with NK cell therapy may improve the antitumor effects of NK cells.

Bioequivalence of a fixed-dose repaglinide/metformin combination tablet and equivalent doses of repaglinide and metformin tablets
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OBJECTIVE: This study was conducted to determine whether a fixed-dose combination (FDC) tablet of repaglinide/metformin (2/500 mg) is equivalent to coadministration of equivalent doses of individual (EDI) tablets of repaglinide (2 mg) and metformin (500 mg) in healthy Korean male subjects. MATERIALS AND METHODS: This study was conducted as an open-label, randomized, single-dose, two-period, two-sequence crossover design in 50 healthy Korean male subjects who received an FDC tablet or EDI tablets. Plasma concentrations of repaglinide and metformin were determined for up to 24 hours using a validated UPLC-MS/MS method. Bioequivalence was assessed according to current guidelines issued by the U.S. Food and Drug Administration (FDA) and Korean legislation. Tolerability was also evaluated throughout the study via subject interview, vital signs, and blood sampling. RESULTS: Point estimates (90% CIs) for AUC0-t, AUC0-∞, and Cmax based on EDI tablets were 110.07 (102.25 - 118.49), 109.90 (101.70 - 118.39), and 112.60 (101.49 - 124.85), respectively, for repaglinide. They were 95.18 (89.62 - 101.05), 95.00 (89.74 - 100.65), and 98.44 (92.72 - 104.50), respectively, for metformin. These results satisfied the bioequivalence criteria of 80.00 - 125.00% proposed by the FDA and Korean legislation. CONCLUSION: Results of pharmacokinetic analysis suggested that repaglinide and metformin in FDC tablets were bioequivalent to EDI tablets of repaglinide (2 mg) and metformin (500 mg) in healthy Korean male subjects. Both formulations appeared to be well tolerated.
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The Anti-Stress Effect of Mentha arvensis in Immobilized Rats.

Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and rats were immobilized for 2 h per day for 14 days using a restraining cage. MA (100 mg/kg) and FMA (100 mg/kg) were orally administered to rats 1 h prior to immobilization. Using high-performance liquid chromatography (HPLC) analysis, we determined the rosmarinic acid content of MA and FMA. The generation of malondialdehyde (MDA) and nitric oxide (NO) in RAW 246.7 cells were suppressed by both MA and FMA. In rats, MA and FMA notably improved the body weight, daily food intake, and duodenum histology. MDA and NO level were gradually decreased by MA and FMA treatment. MA and FMA significantly controlled the stress-related hormones by decreasing corticosterone and ß-endorphin and increasing serotonin level. Moreover, protein expression levels of mitogen activated protein kinases (MAPK) and cyclooxygenase-2 (COX-2) were markedly downregulated by MA and FMA. Taken together, MA and FMA could ameliorate immobilized-stress by reducing oxidative stress, regulating stress-related hormones, and MAPK/COX-2 signaling pathways in rats. Particularly, FMA has shown greater anti-stress activities than MA.

Irradiation of breast cancer cells enhances CXCL16 ligand expression and induces the migration of natural killer cells expressing the CXCR6 receptor.

BACKGROUND AIMS: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. METHODS: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA). The cell-free culture supernatant was collected 96 h after irradiation of breast cancer cell lines for migration and blocking assays. RESULTS: The activated NK cells expressed CXCR6. Expression of the CXCR6 ligand CXCL16 increased in a time- and dose-dependent manner in all analyzed cancer cell lines. CXCL16 expression was statistically significantly enhanced in all breast cancer cell lines on day 3 after 20 Gy irradiation. Activated NK cells migration correlated with CXCL16 concentration (R2 = 0.91; P <0.0001). Significantly enhanced migration of NK cells to irradiated cancer cells was observed for a dose of 20 Gy in MCF7 (P = 0.043) and SKBR3 (P = 0.043) cells, but not in MDA-MB231 (P = 0.225) cells. A blocking assay using a CXCR6 antibody showed a significant decrease in the migration of activated NK cells in all cancer cell lines. CONCLUSIONS: Our data indicate that irradiation induces CXCL16 chemokine expression in cancer cells and enhances the migration of activated NK cells expressing CXCR6 to irradiated breast cancer cells. These results suggest that radiation would improve the anti-tumor effect of NK cells through enhanced migration of NK cells to tumor site for the treatment of patients with breast cancer.

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture.

BACKGROUND AIMS: Interleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture. METHODS: IL-2/IL-15-activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry. RESULTS: Compared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the "first week" group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the "first week" group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of "first week" NK cells that received IL-21 treatment for an additional 2 days compared with the "first week" NK cells (P < 0.05). CONCLUSIONS: These data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.

Kaempferol Inhibits Cervical Cancer Cells by Inducing Apoptosis and Autophagy via Inactivation of the PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND/AIM: Kaempferol, a natural flavonoid, occurs abundantly in fruits and vegetables. It has various bioactivities, with antioxidant, anti-inflammatory, and other beneficial properties. The aim of this study was to investigate the in vitro effects of kaempferol on the proliferation, apoptosis, and autophagy of KB cells, a human cervical cancer cell line, and the corresponding action mechanisms. MATERIALS AND METHODS: The inhibitory efficacy of kaempferol on KB cervical cancer cells was investigated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration assay, 4',6-diamidino-2-phenylindole staining, flow cytometry, acridine orange staining and western blotting. RESULTS: Kaempferol reduced KB cell viability and migration in a dose-dependent manner. Additionally, kaempferol-induced apoptosis was confirmed, and kaempferol treatment influenced levels of apoptotic proteins. Autophagy was detected upon visualization of characteristic autophagic vacuoles and acidic vesicular organelles, and verified using western blotting, which revealed elevated levels of autophagy-related proteins. Kaempferol-mediated apoptosis and autophagy were evidently attributable to reduced phosphorylation in the phosphoinositide 3-kinase (PI3K)/serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) pathway. This finding was validated using a pharmacological inhibition assay with the PI3K pathway inhibitor LY294002, which promoted KB cell apoptosis and autophagy. CONCLUSION: Our results suggest that kaempferol induces apoptosis and autophagy by inhibiting the PI3K/AKT/mTOR pathway in human cervical cancer cells, empirically showing the anticancer effects of kaempferol, and thereby presenting it as a potential anticancer therapeutic agent.

Evaluating a combination treatment of NK cells and reovirus against bladder cancer cells using an in vitro assay to simulate intravesical therapy.

Intravesical treatment using either reovirus or natural killer (NK) cells serves as an efficient strategy for the treatment of bladder cancer cells (BCCs); however, corresponding monotherapies have often shown modest cytotoxicity. The potential of a locoregional combination using high-dose reovirus and NK cell therapy in an intravesical approach has not yet been studied. In this study, we evaluated the effectiveness of reoviruses and expanded NK cells (eNK) as potential strategies for the treatment of bladder cancer. The anti-tumor effects of mono-treatment with reovirus type 3 Dearing strain (RC402 and RP116) and in combination with interleukin (IL)-18/-21-pretreated eNK cells were investigated on BCC lines (5637, HT-1376, and 253J-BV) using intravesical therapy to simulate in vitro model. RP116 and IL-18/-21-pretreated eNK cells exhibited effective cytotoxicity against grade 1 carcinoma (5637 cells) when used alone, but not against HT-1376 (grade 2 carcinoma) and 253J-BV cells (derived from a metastatic site). Notably, combining RP116 with IL-18/-21-pretreated eNK cells displayed effective cytotoxicity against both HT-1376 and 253J-BV cells. Our findings underscore the potential of a combination therapy using reoviruses and NK cells as a promising strategy for treating bladder cancer.

Platycodin D induces apoptosis via regulating MAPK pathway and promotes autophagy in colon cancer cell.

Platycodin D (PD) is the main component of triterpene saponins found in Platycodi radix. In this study, we observed a decrease in cell viability, an increase in apoptotic bodies, and an increase in the rate of apoptosis. Also, we observed an increase in cleaved PARP and Bax, a decrease in Bcl-2, and p-ERK, and an increase in p-p38 and p-JNK. Furthermore, a change in cell viability and the expression of p-p38, Bax, and Bcl-2 using the p38 inhibitor revealed a decrease in p-p38 and Bax and an increase in Bcl-2 in the inhibitor treatment group. In addition, we observed an increase in vacuole formation through morphological changes and an increase in acidic vesicular organelles (AVOs). We also observed an increase in the expression of beclin 1, LC 3-I, and -II. There was no significant decrease in cell viability in the group treated with 3-MA, but a decrease in cell viability was noted in the group treated with HCQ. HCQ treatment resulted in an increase in Bax and a decrease in Bcl-2. These findings reveal that in HT-29 colon cancer cells, PD induces apoptosis through the MAPK pathway, thereby exerting anticancer effects. Moreover, autophagy caused by PD inhibits apoptosis by protecting the cells.

Exploring cellular immunotherapy platforms in multiple myeloma.

Despite major advances in therapeutic platforms, most patients with multiple myeloma (MM) eventually relapse and succumb to the disease. Among the novel therapeutic options developed over the past decade, genetically engineered T cells have a great deal of potential. Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, are rapidly becoming an effective therapeutic modality for MM. Marrow-infiltrating lymphocytes (MILs) derived from the bone marrow of patients with MM are a novel source of T cells for adoptive T-cell therapy, which robustly and specifically target myeloma cells. In this review, we examine the recent innovations in cellular immunotherapies, including the use of dendritic cells, and cellular tools based on MILs, natural killer (NK) cells, and CAR T cells, which hold promise for improving the efficacy and/or reducing the toxicity of treatment in patients with MM.

Development of microsatellite markers in Pungtungia herzi using next-generation sequencing and cross-species amplification in the genus Pseudopungtungia.

Nuclear microsatellite markers for Pungtungia herzi were developed using a combination of next-generation sequencing and Sanger sequencing. One hundred primer sets in the flanking region of dinucleotide and trinucleotide repeat motifs were designed and tested for efficiency in polymerase chain reaction amplification. Of these primer sets, 16 new markers (16%) were successfully amplified with unambiguous polymorphic alleles in 16 individuals of Pungtungia herzi. Cross-species amplification with these markers was then examined in two related species, Pseudopungtungia nigra and Pseudopungtungia tenuicorpa. Fifteen and 11 primer pairs resulted in successful amplification in Pseudopungtungia nigra and Pseudopungtungia tenuicorpa, respectively, with various polymorphisms, ranging from one allele (monomorphic) to 11 alleles per marker. These results indicated that developing microsatellite markers for cross-amplification from a species that is abundant and phylogenetically close to the species of interest is a good alternative when tissue samples of an endangered species are insufficient to develop microsatellites.

Comparative analysis of the aberrant immunophenotype and clinical characteristics in dogs with lymphoma: a study of 27 cases.

Introduction: Aberrant lymphoma phenotypes are frequently found in dogs, but the clinical implications are sparse. Methods: Twenty-seven dogs with aberrant lymphoma diagnosed using flow cytometry between 2017 and 2023 were analyzed. Major paraneoplastic syndromes, prognostic factors, and clinical features of lymphoma were compared to their immunophenotypes. Results: Twenty-seven dogs had aberrant immunophenotypes, with MHCII- (48%) and CD3+/CD21+ (44%) being the most commonly identified aberrancies. In B-cell lymphoma, the most frequent aberrancies were MHC II- (53%), CD3+/CD21+ (41%), CD34+ (24%), and CD79a- (24%). Meanwhile, in T-cell lymphoma, CD3+/CD21+ (63%), CD4-/CD8-(50%), CD5- (50%), and CD45- (50%) were the most common. The platelet-neutrophil ratio was significantly higher in the CD3+/CD21+ group than in the other groups, where either one or both markers were not expressed (55.23 ± 39.64; 18.72 ± 14.95, respectively; p = 0.001). Serum albumin concentration was significantly lower in the MHCII-group (2.59 g/dL, 95% CI 2.31-2.87) than in the MHCII+ group (3.06 g/dL, 95% CI 2.88-3.23; p = 0.009). CD34 expression showed significant correlations with cranial mediastinal mass, WHO clinical substage, and fever (p = 0.028, p = 0.041, and p = 0.047, respectively). MHCII expression was correlated with adverse reactions to chemotherapy, cranial mediastinal masses, and fever (p = 0.009, p = 0.023, and p < 0.001, respectively). No statistically significant differences in the survival period were observed for any of the phenotypic aberrancies. Conclusion: Aberrant lymphomas are common in dogs. Some clinical prognostic factors that significantly correlate with aberrant immunophenotypes have been identified and can be applied clinically.

Genetic Structure and Genetic Diversity of the Endemic Korean Aucha Perch, Coreoperca herzi (Centropomidae), in Korea.

The Korean endemic aucha perch, Coreoperca herzi, belongs to the family Centropomidae. Thus far, studies on C. herzi have focused on mitochondrial genomes, egg development, and early life history, while studies on their genetic diversity or genetic structure are lacking. We investigated these aspects in this study using mitochondrial DNA data. Haplotypes were divided into the Hangang River, Nakdonggang River, Geumgang River, and southwest region water system populations. A translocated population, the Yangyang Namdaechun Stream, was confirmed to have originated from the Hangang River water system population based on haplotype distribution and genetic structure results. The FST of the mitochondrial DNA indicated distinct genetic differentiation in the Hangang, Nakdonggang, Geumgang, and southwest regions. According to COI and analyses, the analysis of molecular variance revealed a higher variance in the four water system groups (98.41%) than in the southwest region water system versus the Hangang River water system (80.27%) groups. This study presents basic data for conservation by providing extensive information on the genetic diversity, genetic structure, and translocation population of C. herzi.

Bio-conjugated nanoarchitectonics with dual-labeled nanoparticles for a colorimetric and fluorescent dual-mode serological lateral flow immunoassay sensor in detection of SARS-CoV-2 in clinical samples.

Serological detection of antibodies for diagnosing infectious diseases has advantages in facile diagnostic procedures, thereby contributing to controlling the spread of the pathogen, such as in the recent SARS-CoV-2 pandemic. Lateral flow immunoassay (LFIA) is a representative serological antibody detection method suitable for on-site applications but suffers from low clinical accuracy. To achieve a simple and rapid serological screening as well as the sensitive quantification of antibodies against SARS-CoV-2, a colorimetric and fluorescent dual-mode serological LFIA sensor incorporating metal-enhanced fluorescence (MEF) was developed. For the strong fluorescence signal amplification, fluorophore Cy3 was immobilized onto gold nanoparticles (AuNPs) with size-controllable spacer polyethyleneglycol (PEG) to maintain an optimal distance to induce MEF. The sensor detects the target IgG with a concentration as low as 1 ng mL-1 within 8 minutes. The employment of the MEF into the dual-mode serological LFIA sensor shows a 1000-fold sensitivity improvement compared with that of colorimetric LFIAs. The proposed serological LFIA sensor was tested with 73 clinical samples, showing sensitivity, specificity, and accuracy of 95%, 100%, and 97%, respectively. In conclusion, the dual-mode serological LFIA has great potential for application in diagnosis and an epidemiological survey of vaccine efficacy and immunity status of individuals.

Helixor-M Suppresses Immunostimulatory Activity through TLR4-Dependent NF-κB Pathway in RAW 264.7 Cells.

Inflammation causes a protective immune response, which can be observed by examining the inflammatory responses of macrophages. Macrophages release various immunostimulatory factors when destroying external pathogens. We induced lipopolysaccharides (LPS) in RAW 264.7 cells, a macrophage cell line, to determine whether Helixor-M can cause immuno-suppression. Helixor-M is known to have anticancer and immune effects. However, an indicator that regulates immunity has not been clearly confirmed. To this end, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to confirm Helixor-M was not cytotoxic. Western blotting and real-time polymerase chain reaction (RT-PCR) confirmed the anti-inflammatory effects. Additionally, immunofluorescence assay confirmed the translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, a representative inflammatory pathway. Helixor-M was found to be non-cytotoxic, induce the NF-κB pathway, and reduce the levels of pro-inflammatory cytokine and mitogen-activated protein kinase (MAPK). We found Helixor-M affected the PI3K/AKT/JNK pathway. Therefore, we confirmed Helixor-M acts as an anti-inflammatory agent through NF-κB, TLR4 and PI3K inhibition and that it could be an effective immunosuppressive drug.