Proliferation Of Peripheral Blood Lymphocytes Research Articles

Molecular characterization, antibacterial and immunoregulatory activities of liver-expressed antimicrobial peptide 2 in black rockfish, Sebastes schlegelii

LEAP2 (liver expression antimicrobial peptide 2), is an antimicrobial peptide widely found in vertebrates and mainly expressed in liver. LEAP2 plays a vital role in host innate immunity. In teleosts, a number of LEAP2 homologs have been reported, but their in vivo effects on host defense are still limited. In this study, a LEAP2 homolog (SsLEAP2) was identified from black rockfish, Sebastes schlegelii, and its structure, expression as well as biological functions were analyzed. The results showed that the open reading frame of SsLEAP2 is 300 bp, with a 5′- untranslated region (UTR) of 375 bp and a 3’ - UTR of 238 bp. The deduced amino acid sequence of SsLEAP2 shares the highest overall identity (96.97%) with LEAP2 of Sebastes umbrosus. SsLEAP2 possesses conserved LEAP2 features, including a signal peptide sequence, a prodomain and a mature peptide, in which four well-conserved cysteines formed two intrachain disulphide domain. The expression of SsLEAP2 was highest in liver and could be induced by experimental infection with Listonella anguillarum, Edwardsiealla piscicida and Rock bream iridovirus C1 (RBIV-C1). Recombinant SsLEAP2 (rSsLEAP2) purified from Escherichia coli was able to bind with various Gram-positive and Gram-negative bacteria. Further analysis showed that rSsLEAP2 could enhance the respiratory burst activity, and induce the expression of immune genes including interleukin 1-β (IL-1β) and serum amyloid A (SAA) in macrophages; additionally, rSsLEAP2 could also promote the proliferation and chemotactic of peripheral blood lymphocytes (PBLs). In vivo experiments indicated that overexpression of SsLEAP2 could inhibit bacterial infection, and increase the expression level of immune genes including IL-1β, tumor necrosis factor ligand superfamily member 13B (TNF13B) and haptoglobin (HP); conversely, knock down of SsLEAP2 promoted bacterial infection and decreased the expression level of above genes. Taken together, these results suggest that SsLEAP2 is a novel LEAP2 homolog that possesses apparent antibacterial activity and immunoregulatory property, thus plays a critical role in host defense against pathogens invasion.

Impact of apitherapy on canine, equine, and chicken lymphocytes, in vitro

Apitherapy is a form of alternative medicine that utilizes products from the western honeybee (Apis mellifera), including honey, propolis, and honeybee venom, to improve the health status of human patients by altering host immunity. An added benefit of these products is that they are nutraceuticals and relatively inexpensive to aquire. Currently, little is known about the use of honeybee products in veterinary species, as well as their impact on host immunity. In the present in vitro study, honey, propolis, and honeybee venom were co-cultured with enriched canine, equine, and chicken peripheral blood lymphocytes (PBLs) with cell proliferation, cell viability/apoptosis, and cellular morphology evaluated. Concanavalin A (Con A) and dexamethasone were used as stimulatory and suppressive controls, respectively. Honeybee products’ effects on the three veterinary species varied by product and the species. Honey stimulated the PBLs proliferation in all three species but also displayed some increased cytotoxicity. Propolis stimulated proliferation in canine and equine PBLs, however, it suppressed proliferation in the chicken PBLs. Honeybee venom was the strongest PBL stimulant for all three species and in the equine, surpassed the stimulant response of Con A and yet, enhanced PBL cell viability post culture. In summary, the results of this preliminary in vitro study show that these three honeybee products do impact lymphocyte proliferation and viability in dogs, horses, and chickens, and that more research both in vitro and in vivo will be necessary to draw conclusions regarding their future use as immune stimulants or inhibitors.

Evaluation of the protective efficacy of six major immunogenic proteins of Mycoplasma Synoviae.

Mycoplasma synoviae (MS) is a primary avian pathogen prevalent worldwide that causes airsacculitis and synovitis in birds. Vaccination is recommended as the most cost-effective strategy in the control of MS infection. Novel alternative vaccines are needed for eradicating and controlling MS infection in flocks. DnaK, enolase, elongation factor Tu (EF-Tu), MSPB, NADH oxidase and LP78 are the major immunogenic antigens of MS and are promising targets for subunit vaccine candidates. In the present study, genes encoding DnaK, enolase, EF-Tu, MSPB, LP78, and NADH oxidase were cloned and expressed in Escherichia coli. Enzyme-linked immunosorbent assay showed that the six recombinant proteins were recognized by convalescent sera, indicating that they were expressed during infection. Two injections of the six subunit vaccines induced a robust antibody response and increased the concentrations of IFN-γ and IL-4, especially rEnolase and rEF-Tu. The proliferation of peripheral blood lymphocytes was enhanced in all of the immunized groups. Chickens immunized with rEnolase, rEF-Tu, rLP78, and rMSPB conferred significant protection against MS infection, as indicated by significantly lower DNA copies in the trachea, lower scores of air sac lesions, and lesser tracheal mucosal thickness than that in the challenge control. Especially, rEnolase provided the best protective efficacy, followed by rEF-Tu, rMSPB, and rLP78. Our finds demonstrate that the subunit vaccines and bacterin can only reduce the lesions caused by MS infection, but not prevent colonization of the organism. Our findings may contribute to the development of novel vaccine agents against MS infection.

Probiotic Enterococcus faecalis surface-delivering key domain of EtMIC3 proteins: immunoprotective efficacies against Eimeria tenella infection in chickens.

Enterococcus faecalis MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the Eimeria tenella microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant E. faecalis were recorded. The immunized chickens were challenged with 4.0 × 104 E. tenella-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of E. faecalis, respectively. Oral immunizations with recombinant E. faecalis, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (P < 0.01). The groups immunized with four recombinant E. faecalis showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic E. faecalis delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of Eimeria infection. IMPORTANCE Avian coccidiosis caused by Eimeria brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time, Eimeria infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot. Eimeria parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens. Eimeria tenella microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the Eimeria invasion. Enterococcus faecalis is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by E. faecalis, respectively. Oral immunizations were performed to investigate immune protective effects against Eimeria infection.

Self-assembled nanoparticle with E protein domain III of DTMUV based on ferritin as carrier can induce a more comprehensive immune response and against DTMUV challenge in duck

Duck Tembusu virus (DTMUV) causes severe reduction in egg production and neurological symptoms in ducklings. Vaccination is the primary measure used to prevent DTMUV infections. In this study, self-assembled nanoparticles with the E protein domain III of DTMUV, using ferritin as a carrier (EDⅢ-RFNp), were prepared using a prokaryotic expression system. Ducks were intramuscularly vaccinated with EDⅢ-RFNp, EDⅢ protein, an inactivated vaccine HB strain (InV-HB), and PBS. At 0, 4, and 6 weeks post-primary vaccination, the EDIII protein-specific antibody titre, IL-4, and IFN-γ concentrations in serum were determined by ELISA, and neutralising antibodies titres in sera were determined by virus neutralising assay. Peripheral blood lymphocytes proliferation was determined by CCK-8 kit. Following challenge with the virulent DTMUV strain, the clinical signals and survival rate of the vaccinated ducks were recorded, and DTMUV RNA levels in the blood and tissues of the surviving ducks were determined by real-time quantitative RT-PCR. The near-spherical EDⅢ-RFNp nanoparticles with 13.29 ± 1.43 nm diameter were observed by transmission electron microscope. At 4 and 6 weeks post-primary vaccination, special and Virus neutralisation (VN) antibodies, lymphocyte proliferation (stimulator index, SI), and concentrations of IL-4 and IFN-γ in the EDⅢ-RFNp group were significantly higher than in the EDⅢ and PBS groups. In the DTMUV virulent strain challenge test, the EDⅢ-RFNp-vaccinated ducks showed milder clinical signs and higher survival rates than EDⅢ- and PBS-vaccinated ducks. The DTMUV RNA levels in the blood and tissues of EDⅢ-RFNp-vaccinated ducks were significantly lower than those in EDⅢ- and PBS-vaccinated ducks. Additionally, the EDⅢ protein-special and VN antibodies, SI value, and concentration of IL-4 and IFN-γ in the InV-HB group was significantly higher than that of the PBS group at 4 and 6 weeks post-primary vaccination. InV-HB provided more efficient protection than PBS based on a higher survival rate, milder signals, and lower levels of the DTMUV virus in the blood and tissues. These results indicated that EDⅢ-RFNp effectively protected ducks against DTMUV challenge and could be a vaccine candidate to prevent DTMUV infection.

Activation of T Lymphocytes with Anti-PDL1-BiTE in the Presence of Adipose-Derived Mesenchymal Stem Cells (ASCs).

Due to their ability to recruit immune cells to kill tumor cells directly, bispecific T cell engager antibodies (BiTE) hold great potential in T cell redirecting therapies. BiTE is able to activate T cells through CD3 and target them to tumor-expressed antigens. However, there are many components in the tumor microenvironment (TME) such as mesenchymal stem cells (MSCs) that may interfere with BiTE function. Herein, we designed an anti-PDL1-BiTE that targets programmed death ligand 1 (PDL1) and CD3 and investigated its effect on PDL1pos cancer cells in the presence or absence of adipose-derived MSCs (ASCs). Our anti-PDL1-BiTE comprises of VL and VH chains of anti-CD3 monoclonal antibody (mAb) linked to the VL and VH chains of anti-PDL1 mAb, which simultaneously bind to the CD3ε subunit on T cells and PDL1 on tumor cells. Flow cytometry was employed to assess the strength of binding of anti-PDL1-BiTE to tumor cells and T cells. Cytotoxicity, proliferation, and activation of peripheral blood lymphocyte (PBLs) were evaluated by CFSE assay and flow cytometry after using anti-PDL1-BiTE in the presence or absence of ASCs and their conditioned media (C.M.). Anti-PDL1-BiTE had the ability to induce selective lysis of PDL1pos U251-MG cancer cells while PDL1neg cells were not affected. Also, anti-PDL1-BiTE significantly stimulated peripheral blood lymphocyte (PBL) proliferation and CD69 expression. ASCs/C.M. did not show a significant effect on the biological activity of anti-PDL1-BiTE. Overall, anti-PDL1-BiTE selectively depletes PDL1pos cells and represents a new immunotherapeutic approach. It would increase the accumulation of T cells and can improve the prognosis of PDL1pos cancers in spite of the immunomodulatory effects of ASCs and C.M.

Performance evaluation of two antigen-extracted xenogeneic ostein and experimental study on repairing skull defects in rats

To evaluate the physical and chemical properties, immunogenicity, and osteogenesis of two antigen-extracted xenogeneic bone scaffolds-decalcified bone matrix (DBM) and calcined bone. By removing the inorganic and organic components of adult pig femus, xenogeneic DBM and calcined bone were prepared respectively. The density and pH value of the two materials were measured and calculated, the material morphology and pore diameter were observed by scanning electron microscope, and the surface contact angle was measured by automatic contact angle measuring instrument. The safety, osteogenic activity, and immunogenicity of the two materials were evaluated by cytotoxicity test, osteoblast proliferation test, DNA residue test, and human peripheral blood lymphocyte proliferation test. The two materials were implanted into the 5 mm full-thickness skull defect of 6-week-old male Sprague Dawley rats (the blank control group was not implanted with materials). The materials were taken at 4 and 8 weeks after operation, the repair effect of the materials on the rat skull was observed and evaluated by gross observation, Micro-CT scanning, and HE staining observation. Compared with calcined bone, DBM has lower density and poor hydrophilicity; the pH value of the two materials was 5.5-6.1, and the pore diameter was 160-800 μm. The two materials were non-cytotoxic and could promote the proliferation of osteoblasts. The absorbance ( A) values of osteoblast proliferation at 1, 4, and 7 days in the DBM group were significantly higher than those in the calcined bone group ( P<0.05). The DNA residues of the two materials were much lower than 50 ng/mg dry weight, and neither of them could stimulate the proliferation and differentiation of human peripheral blood lymphocytes. The results of animal experiments in vivo showed that the bone volume/total volume (BV/TV) in DBM group and calcined bone group were significantly higher than that in blank control group at 4 weeks after operation ( P<0.05), and that in calcined bone group was significantly higher than that in DBM group ( P<0.05); at 8 weeks after operation, there was no significant difference in BV/TV between groups ( P>0.05). HE staining showed that at 4 and 8 weeks after operation, the defect in the blank control group was filled with fibrous connective tissue, the defect was obvious, and no bone growth was found; the defect in DBM group and calcined bone group had been repaired to varying degrees, and a large number of new bone formation could be seen. The material degradability of DBM group was better than that of calcined bone group. The physical and chemical properties and degradability of the two kinds of xenogeneic bone scaffolds were slightly different, both of them have no immunogenicity and can promote the repair and reconstruction of skull defects in rats.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy—RC) and nine cervical cancer patients (treatment with radiotherapy alone—R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

PD-1 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c Mouse Model of CP BVDV Acute Infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

Oral Immunization of Recombinant Lactococcus lactis and Enterococcus faecalis Expressing Dendritic Cell Targeting Peptide and Hexon Protein of Fowl Adenovirus 4 Induces Protective Immunity Against Homologous Infection

Hepatitis-hydropericardium syndrome (HPS) causes severe economic losses in the global poultry industry. The present study aims to explore oral immunization of recombinant Lactococcus lactis and Enterococcus faecalis expressing Hexon protein of fowl adenovirus 4 (FAdV-4). The bacteria L. lactis NZ9000 and E. faecalis MDXEF-1 were, respectively, modified as host strain to deliver truncated Hexon protein (ΔHexon) or ΔHexon protein fusing with dendritic cell (DC) targeting peptide (DC-ΔHexon) on the surface of bacteria. The expression of target protein in L. lactis NZ9000 and E. faecalis MDXEF-1 were detected by western blot. To evaluate the immune responses and protective efficacies provided by the live recombinant bacteria, chickens were immunized with the constructed ΔHexon-expressing bacteria three times at 2-week intervals, then experimentally challenged with hypervirulent FAdV-4/GX01. The results showed that oral immunizations with the four ΔHexon-expressing bacteria (NZ9000/ΔHexon-CWA, NZ9000/DC-ΔHexon-CWA, MDXEF-1/ΔHexon-CWA, and MDXEF-1/DC-ΔHexon-CWA), especially the two bacteria carrying DC-targeting peptide, stimulated higher levels of ΔHexon-specific sera IgG and secretory IgA (sIgA) in jejunal lavage fluid, higher proliferation of peripheral blood lymphocytes (PBLs) and higher levels of Th1/Th2-type cytokines, along with significantly decreased virus loads in liver and more offered protective efficacies against FAdV infection compared with PBS and empty vector control groups (p < 0.01). For chickens in the group MDXEF-1/DC-ΔHexon-CWA, the levels of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in sera, and the virus loads in livers were significantly decreased vs. the other three ΔHexon-expressing bacteria (p < 0.01). The pathological changes in the hearts, livers, spleens and kidneys of chickens in MDXEF-1/DC-ΔHexon-CWA group were relatively slight compared to infection control group and other three ΔHexon-expressing bacteria groups. The rate of protection in MDXEF-1/DC-ΔHexon-CWA group was 90%. The present work demonstrated that cell surface-displayed target protein and immune enhancers in L. lactis and E. faecalis might be a promising approach to enhance immunity and immune efficacy against pathogen FAdV-4 infection.

Effective Activation and Expansion of Canine Lymphocytes Using a Novel Nano-Sized Magnetic Beads Approach.

Expansion protocols for human T lymphocytes using magnetic beads, which serve as artificial antigen presenting cells (aAPCs), is well-studied. Yet, the efficacy of magnetic beads for propagation and functionality of peripheral blood lymphocytes (PBLs) isolated from companion dogs still remains limited. Domestic dog models are important in immuno-oncology field. Thus, we built the platform for induction of canine PBLs function, proliferation and biological activity using nano-sized magnetic beads (termed as MicroBeads) coated with anti-canine CD3 and CD28 antibodies. Herein we reveal that activation of canine PBLs via MicroBeads induces a range of genes involved in immediate-early response to T cell activation in dogs. Furthermore, canine T lymphocytes are effectively activated by MicroBeads, as measured by cluster formation and induction of activation marker CD25 on canine T cells as quickly as 24 h post stimulation. Similar to human T cells, canine PBLs require lower activation signal strength for efficient proliferation and expansion, as revealed by titration studies using a range of MicroBeads in the culture. Additionally, the impact of temperature was assessed in multiple stimulation settings, showing that both 37°C and 38.5°C are optimal for the expansion of canine T cells. In contrast to stimulation using plant mitogen Concanavalin A (ConA), MicroBead-based activation did not increase activation-induced cell death. In turn, MicroBeads supported the propagation of T cells with an effector memory phenotype that secreted substantial IL-2 and IFN-γ. Thus, MicroBeads represent an accessible and affordable tool for conducting immunological studies on domestic dog models. Similarities in inducing intracellular signaling pathways further underscore the importance of this model in comparative medicine. Presented herein MicroBead-based expansion platforms for canine PBLs may benefit adoptive immunotherapy in dogs and facilitate the design of next-generation clinical trials in humans.

An OMV-Based Nanovaccine Confers Safety and Protection against Pathogenic Escherichia coli via Both Humoral and Predominantly Th1 Immune Responses in Poultry.

Avian pathogenic Escherichia coli (APEC) infection in poultry causes enormous economic losses and public health risks. Bacterial outer membrane vesicles (OMVs) and nano-sized proteolipids enriched with various immunogenic molecules have gained extensive interest as novel nanovaccines against bacterial infections. In this study, after the preparation of APEC O2-derived OMVs (APEC_OMVs) using the ultracentrifugation method and characterization of them using electron microscopy and nanoparticle tracking analyses, we examined the safety and vaccination effect of APEC_OMVs in broiler chicks and investigated the underlying immunological mechanism of protection. The results showed that APEC_OMVs had membrane-enclosed structures with an average diameter of 89 nm. Vaccination with 50 μg of APEC_OMVs had no side effects and efficiently protected chicks against homologous infection. APEC_OMVs could be effectively taken up by chicken macrophages and activated innate immune responses in macrophages in vitro. APEC_OMV vaccination significantly improved activities of serum non-specific immune factors, enhanced the specific antibody response and promoted the proliferation of splenic and peripheral blood lymphocytes in response to mitogen. Furthermore, APEC_OMVs also elicited a predominantly IFN-γ-mediated Th1 response in splenic lymphocytes. Our data revealed the involvement of both non-specific immune responses and specific antibody and cytokine responses in the APEC_OMV-mediated protection, providing broader knowledge for the development of multivalent APEC_OMV-based nanovaccine with high safety and efficacy in the future.

Hepatoprotective Activities of Colocasia esculenta (L.) Schottin Mice Model with Liver Injury Induced by Paracetamol

This paper uses the model of experimental mouse liver damage with paracetamol to evaluate the liver protective effects of the methanol extract (Colocasia esculenta (L.) Schott) (MCE). After 8 days, the hepatoprotective activities of MCE in mice (damaged by paracetamol) at doses of 500, 1,000 and 2,000 mg/kg/day proved effective as the AST enzyme content decreased by 6.98, 22.84 and 26.59%, respectively; and ALT decreased by 53.17, 56.46 and 57.93%, respectively. MCE at a dose of 1,000 mg/kg/day had a protective effect equivalent to that of silymarin (used in liver treatment) at a dose of 50 mg/kg/day and MCE at a dose of 2,000 mg/kg/day had better effect. Observation of the microscopic liver tissue cross section also revealed that the mice treated with MCE of C. esculenta at doses of 1,000 and 2,000 mg/kg/day showed significantly improvement in their liver tissues compared to the non-treated control group.&#x0D; Keywords:&#x0D; Colocasia esculenta, hepatoprotective, paracetamol.&#x0D; References&#x0D; [1] S. Shahani. Evaluation of hepatoprotective efficacy of APCL-A poly herbal formulation in vivo in rats, Indian Drugs. 36(1999) 628-631.[2] Y. Cui, X. Yang, X. Lu, J. Chen, Y. Zhao. Protective effects of polyphenols-enriched extract from Huangshan Maofeng green tea against CCl4-induced liver injury in mice, Chemico- Biological Interactions 220 (5) (2014) 75-83. https://doi.org/10.1016/j.cbi.2014.06.018 [3] K.C. Choi, W.T. Chung, J.K. Kwon, J.Y. Yu, Y.S. Jang, S.M. Park, S.Y. Lee, J.C. Lee. Inhibitory effects of quercetin on aflatoxin B1 induced hepatic damage in mice, Food Chemical Toxicology, 48 (10) (2010) 2747-2753 . https://doi.org/10.1016/j.fct.2010.07.001[4] E.S. Sabina, J. Samue, S.R. Ramya, S. Patel, N. Mandal, P. Preety, P.P. Mishra, M.K. Rasool. Hepatoprotective and antioxidant potential of Spirulina fusiformis on acetaminophen induced hepatotoxicity in mice, International Journal of Integrative Biology 6 (1) (2009) 1-5. http://ijib.classicrus.com/.../1501.pdf[5] Pham Hoang Ho. Vietnamese plants, episode III. Young Publishing House, (1999), pp. 353 (in Vietnamese).[6] C.O. Eleazu, M. Iroaganachi, K.C. Eleazu. Ameliorative potentials of cocoyam (Colocasia esculenta L.) and unripe plantain (Musa paradisiaca L.) on the relative tissue weights ofstreptozotocin-induced diabetic rats. Journal of Diabetes Research, 197 (2013), https://doi.org/10.1155/2013/160964.[7] R.C. Cambie, L.R. Ferguson. Potential functional foods in the traditional Maori diet. Mutation Research Letters 523–524 (2003) 109-117. https://doi.org/10.1016/s0027-5107(02)00344-5[8] C.L. Abraham, K. Yoshinori, T. Masakuni, I. Hironori, O. Hirosuke, T. Hajime. Flavonoid glycosides in the shoot system of Okinawa Taumu (Colocasia esculenta S). Food Chemistry 119 (2010) 630- 635. https://doi.org/10.1016/j.foodchem.2009.07.004[9] P.J. Bouic, D. Etsebeth, R.W. Liebenberg, C.F. Albrecht, K. Pegel, J.P.P. Van. Beta sitosterol and beta sitosterol glycoside stimulate human peripheral blood lymphocyte proliferation: Implications for their use as an immunomodulatory vitamin combination. International Journal of Immuno-pharmacology 18 (12) (1996) 693-700. https://doi.org/10.1016/S0192-0561(97)85551-8.[10] Vo Van Chi. Dictionary of Vietnamese medicinal plants. Medical Publishing House (1997), pp. 871-873 (in Vietnamese).[11] Do Trung Dam. Method of determining drug toxicity. Medical Publishing House, Hanoi (2014), pp. 11-137 (in Vietnamese). [12] J.T. Litchfield, F. Wilcoxon. A simplified method of evaluating dose-effect experiments, Journal of Pharmcology and Experimental Therapeutics, 96 (2) (1949) 99-113. PMID: 18152921 [13] Doan Thi Nhu, Do Trung Dam, Pham Duy Mai. Method research of the pharmacological effect of drugs from medicinal herbs, Science and Technics Publishing House, Hanoi (2006), pp. 142 (in Vietnamese).[14] A. Godwin. Histochemical uses of haematoxylin - A Review. JPCS 1 (2011) 24-34. [15] Ho Thi Thanh Huyen, Thai Nguyen Hung Thu, Nguyen Thai An. Some results of plan microscopic studies and chemical composition of Bombax malabaricum DC., Bombacaceae, Proceeding Pharma Indochina VII, 10/2011, The 7th Indochina Conference on Pharmaceutical science, Bangkok, Thai Lan (2011) 270-274.[16] A.N. Chinonyelum, A.P. Uwadiegwu, O.C. Nwachukwu, O. Emmanuel. Evaluation of hepatoprotective activity of Colocasia esculenta (L. Schott) leaves on thioacetamide-induced hepatotoxicity in rats. Pakistan Journal Pharmaceutical Sciences, 28 (6S) (2015) 2237-2241. [17] O.F. Dayo, B.C. Eluke, E.O. Ukaejiofo, O.L. Olayinka, C.I. Johnpaul, O.A.K .Tajudeen, I. Chinwe, L.S. Adetona. Hepatoproactive and haematopoietic modulatory efficacy of leaf extract of Colocasia esculenta in Albino wistar rats. International Journal of Tropical Medicine 12 (3-6) (2017) 35-41. https://doi.org/3923/ijtmed.2017.35.41.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D;

Synthesis and Biological Activity of New 7-Amino-oxazolo[5,4-d]Pyrimidine Derivatives.

The synthesis of a series of novel 7-aminooxazolo[5,4-d]pyrimidines 5, transformations during their synthesis and their physicochemical characteristics have been described. Complete detailed spectral analysis of the intermediates 2–4, the N′-cyanooxazolylacetamidine by-products 7 and final compounds 5 has been carried out using MS, IR, 1D and 2D NMR spectroscopy. Theoretical research was carried out to explain the privileged formation of 7-aminooxazolo[5,4-d]pyrimidines in relation to the possibility of their isomer formation and the related thermodynamic aspects. Additionally, the single-crystal X-ray diffraction analysis for 5h was reported. Ten 7-aminooxazolo[5,4-d]pyrimidines 5 (SCM1–10) were biologically tested in vitro to preliminarily evaluate their immunological, antiviral and anticancer activity. Compounds SCM5 and SCM9 showed the best immunoregulatory profile. The compounds displayed low-toxicity and strongly inhibited phytohemagglutinin A-induced proliferation of human peripheral blood lymphocytes and lipopolysaccharide-induced proliferation of mouse splenocytes. Compound SCM9 caused also a moderate suppression of tumor necrosis factor α (TNF-α) production in a human whole blood culture. Of note, the compounds also inhibited the growth of selected tumor cell lines and inhibited replication of human herpes virus type-1 (HHV-1) virus in A-549 cell line. Molecular investigations showed that the compounds exerted differential changes in expression of signaling proteins in Jurkat and WEHI-231 cell lines. The activity of SCM5 is likely associated with elicitation of cell signaling pathways leading to cell apoptosis. The compounds may be of interest in terms of therapeutic utility as inhibitors of autoimmune disorders, virus replication and antitumor agents.

Preliminary safety and efficacy of GX-I7, a long-acting interleukin-7, in combination with pembrolizumab in patients with refractory or recurrent metastatic triple negative breast cancer (mTNBC): Dose escalation period of Phase Ib/II study (KEYNOTE-899).

1072 Background: Pembrolizumab monotherapy did not significantly improve OS as 2nd and 3rd-lines treatment for mTNBC compared to standard chemotherapy in phase III study (KEYNOTE-119) leading to high unmet needs of effective treatment. Recent studies showed that higher lymphocyte count is an independent factor which correlates with better response to checkpoint blockade in cancer patients. GX-I7, a long-acting interleukin-7, could potentially provide synergistic anti-tumor efficacy with pembrolizumab by increasing number of T cells both in tumor microenvironment (TME) and peripheral blood (PB). Methods: This is an open-label, phase Ib/II study in patients with refractory or recurrent TNBC who failed from standard chemotherapy in the metastatic setting, with ≤3rd-lines of previous chemotherapy. The dose escalation phase adopted the 3+3 design. The GX-I7 doses were administered IM q9w or q12w, with or without cyclophosphamide pre-conditioning depending on the allocation, and in combination with pembrolizumab 200 mg IV q3w. The objectives were dose limiting toxicities (DLTs), safety, pharmacodynamic markers including lymphocyte increase and RP2D. Results: As of January 30, 2020, GX-I7 and pembrolizumab were exposed to 24 patients (median age 46.0 years [29-75], ECOG PS 1 [58.3%], median cycle no. 3 [1-9]). Treatment was discontinued in 13 (54.2%), majority due to PD and 11 patients are ongoing. No DLTs were reported in all dose groups. Treatment related AEs occurred in 91.7% of patients with grade 1-2 and 9.1% with grade 3 (no grade 4). Common AEs were injection site reaction (39.0%) and fever (13.0%), which were easily managed. Grade 3 toxicity were AST/ALT elevation and infusion related reaction, reported from 1 patient each (4.2%). GX-I7 induced dose-dependent lymphocyte proliferation in PB, with approximately 4-folds increase in high doses. 17 patients were evaluable; confirmed objective responses from ongoing patients included one partial response (5.9%), 2 stable disease (11.8%) and 1 durable unconfirmed PD (5.9%). Conclusions: GX-I7 in combination with pembrolizumab was well tolerated, with no DLTs reported. There was no apparent increase of immune-related AEs with the addition of GX-I7. Pharmacodynamics data support proof of mechanism and it warrants further clinical studies. Clinical trial information: NCT03752723 .

Effects of beta-endorphin and dynorphin A on in vitro apoptosis in human peripheral blood lymphocytes

Along with functioning in the nerve system, the endogenous opioid peptides as the ligands of mu-, delta-, and kappa-opioid receptors exert multiple effects on immune cells. Moreover, experimental evidence showed that morphine as an exogenous agonist of mu-opioid receptors affects immune cell viability. Such effects were discovered in experiments with cultured cells and laboratory animals. Hence, we studied effects of endogenous opioid peptides dynorphin A and beta-endorphin on viability of human peripheral blood mononuclear cells in vitro. For this, we used samples of peripheral blood cells collected from the fourteen healthy volunteers, who provided with signed informed consent and might request any information regarding the research. Mononuclear cells were collected from the heparinized blood samples according to standard protocol and cultivated in the humid atmosphere for 72 hours. Two μCi 3H-Methyl-thymidine was added into each test tube at 18 hours before the end of the cultivation period. Scintillation counting was performed by using Guardian liquid scintillation analyzer (Wallac, Finland) expressing the data as count per minute. To assess apoptosis, the cells were cultured for 24 hours in similar conditions except for adding radioactive probe. Next, the cells were stained with Annexin V-FITC/7-AAD kit (Beckman Coulter, USA) according to the manufacturer’s instructions for further recording apoptotic cells in flow cytometers BD FACSCalibur (Becton Dickinson, USA) or CytoFLEX S (Beckman Coulter, USA). The lymphocyte gate set by light scatter parameters was shown in typical Annexin V-FITC vs 7-AAD plot followed by counting Annexin V+/7-AADcells. All data were expressed as means ± S.E. Statistical significance was assessed by using Student’s t-test. It was found that physiologic concentrations of mu- and kappa-opioid receptor agonists beta-endorphin and dynorphin A exerted multidirectional effects on proliferation of human peripheral blood lymphocytes. In particular, dynorphin A increased basal proliferation and proliferation in response to suboptimal mitogen stimulation. Moreover, beta-endorphin enhanced effects of mitogen stimulation at suboptimal concentration but profoundly suppressed proliferation in maximally activated cells. The modulating effects of beta-endorphin and dynorphin A on in vitro proliferation are not associated with augmented cell apoptosis.

Intracellular K+ and water content in human blood lymphocytes during transition from quiescence to proliferation

Many evidence shows that K+ ions are required for cell proliferation, however, changes in intracellular K+ concentration during transition of cells from quiescence to cycling are insufficiently studied. Here, we show using flame emission assay that a long-term increase in cell K+ content per g cell protein is a mandatory factor for transition of quiescent human peripheral blood lymphocytes (PBL) to proliferation induced by phytohemagglutinin, phorbol ester with ionomycin, and anti-CD3 antibodies with interleukin-2 (IL-2). The long-term increase in K+ content is associated with IL-2-dependent stage of PBL activation and accompanies the growth of small lymphocytes and their transformation into blasts. Inhibition of PBL proliferation with drugs specific for different steps of G0/G1/S transit prevented both blast-transformation and an increase in K+ content per cell protein. Determination of the water content in cells by measuring the density of cells in the Percoll gradient showed that, unlike the K+ content, the concentration of K+ in cell water remains unchanged, since water and K+ change in parallel. Correlation of proliferation with high cell K+ and water content has been confirmed by the data obtained in comparative study of PBL and permanently cycling Jurkat cells. Our data suggest that K+ is important for successful proliferation as the main intracellular ion that participates in regulation of cell water content during cell transition from quiescence to proliferation. We concluded that high K+ content in cells and the associated high water content is a characteristic feature of proliferating cells.

Abstract CT082: Phase (Ph) I/Ib study of NIZ985 with and without spartalizumab (PDR001) in patients (pts) with metastatic/unresectable solid tumors

Abstract Background: NIZ985 is a soluble IL-15/IL-15 receptor α heterodimer (hetIL-15) that binds IL-2/IL-15 receptor β/γ and promotes effector T-cell and NK cell expansion and activation. Spartalizumab is a humanized IgG4 mAb that blocks binding of programmed death 1 (PD-1) to programmed death ligand 1/2 (PD-L1/2). In preclinical studies, IL-15 and anti-PD-1/PD-L1 combinations demonstrated synergistic antitumor activity. Here we report dose escalation results from a Ph I/Ib study of NIZ985 as a single agent (SA) and in combination with spartalizumab (NCT02452268). Methods: Pts with metastatic/unresectable solid tumors received subcutaneous NIZ985 TIW (2 weeks on/2 weeks off); 0.25-4 μg/kg NIZ985 SA or 1 μg/kg NIZ985 with IV spartalizumab (400 mg, Q4W). Ph I endpoints: DLTs, safety, PK, pharmacodynamics (PD), preliminary efficacy, and recommended dose for expansion (RDE), statistically guided by a 3+3 study design. Results: As of Oct 9, 2018, 14 pts (median age 56.5 y [range 42.0-73.0]) received NIZ985 SA and 11 pts (median age 61 y [range 34.0-75.0]) received NIZ985 + spartalizumab. Treatment was discontinued in 13 (93%) and 10 (91%) pts receiving NIZ985 SA and NIZ985 + spartalizumab, respectively, mainly due to disease progression (57% and 82%); 1 pt in each group is ongoing. No DLTs were reported in the first cycle. AEs suspected to be drug related were reported in 100% of pts (36% grade 3/4) for NIZ985 SA and 100% of pts treated with the combination (27% grade 3/4). Common AEs [all grades; grade 3/4], defined as ≥50% of pts for any grade, were injection site reaction [100%; 7%], chills [64%; 0%], and fatigue [57%; 0%] in NIZ985 SA; and injection site reaction [100%; 9%], fatigue [82%; 0%], diarrhea [64%; 9%], and nausea [55%; 0%] in NIZ985 + spartalizumab. Median duration of NIZ985 exposure (range) across doses was 7.6 weeks (2.6-15.6) in NIZ985 SA and 21.6 weeks (5.6-53.6) in NIZ985 + spartalizumab. In 13 evaluable NIZ985 SA pts, confirmed best overall responses (BORs) were stable disease (SD; n=3, 23%), progressive disease (n=8, 62%), and unknown (n=2, 15%); SD is ongoing in 1 pt with spindle cell ocular melanoma. Confirmed BORs in 11 evaluable NIZ985 + spartalizumab pts were SD (n=5, 45%) and progressive disease (n=6, 55%); SD is ongoing in 1 pt with gastric adenocarcinoma. RDE was declared as 1 μg/kg NIZ985 (TIW; 2 weeks on/2 weeks off) for SA and with spartalizumab (400 mg, Q4W). PD analyses showed NIZ985 induces lymphocyte proliferation in peripheral blood. Preliminary antitumor activity, PK, and PD data from tumors and peripheral blood will be presented. Conclusions: NIZ985 SA and with spartalizumab was well tolerated in pts with metastatic/unresectable solid tumors, with no DLTs reported. RDEs were NIZ985 1 μg/kg (TIW, 2 weeks on/2 weeks off) for SA and with spartalizumab (400 mg, Q4W). PD data support proof of mechanism. Dose escalation with an alternate NIZ985 schedule is ongoing. Citation Format: Kevin Conlon, Rom Leidner, Douglas McNeel, Sumati Gupta, Andrea Wang-Gillam, Thomas A. Waldmann, George Pavlakis, Mirek Dostalek, Sema Kurtulus, Niladri Roy Chowdhury, Jessica O'Keeffe, Nehal S. Parikh, John A. Thompson. Phase (Ph) I/Ib study of NIZ985 with and without spartalizumab (PDR001) in patients (pts) with metastatic/unresectable solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT082.

Immunophenotype and Immune-Modulatory Activities of Human Fetal Cartilage-Derived Progenitor Cells

We have previously reported human fetal cartilage progenitor cells (hFCPCs) as a novel source of therapeutic cells showing high proliferation and stem cell properties superior to those of adult mesenchymal stem cells (MSCs). In this study, we investigated the immunophenotype and immune-modulatory activities of hFCPCs. With institutional review board approval, hFCPCs were isolated from fetuses at 11–13 weeks of gestation. hFCPCs showed strong expression of HLA class I molecules but low or no expression of HLA class II and co-stimulatory molecules, which was not changed significantly after 4 days of IFN-γ treatment. In a mixed lymphocyte reaction (MLR), hFCPCs showed no allogeneic immune response to peripheral blood lymphocytes (PBLs) and suppressed concanavalin A (Con A)-mediated proliferation of PBLs in a dose-dependent manner. In addition, hFCPCs inhibited Con A-induced secretion of pro-inflammatory cytokines TNF-α and IFN-γ from PBLs but showed no significant decrease of secretion of IL-10, anti-inflammatory cytokine. Co-culture of hFCPCs with stimulated PBLs for 4 days resulted in a significant increase in CD4+CD25+FoxP3+ T regulatory cells (Tregs). hFCPCs expressed LIF, TGF-β1, TSG-6, and sHLA-G5 but did not express IDO and HGF. Stimulation of hFCPCs with TNF-α for 12 h showed slight induction in the expression of LIF, TSG-6, IDO, and HGF, whereas stimulation with IFN-γ did not affect expression of any of these factors. These results suggest that hFCPCs have low allogeneic immunogenicity and immune-modulatory activity in vitro, comparable to those of MSCs. However, compared with MSCs, hFCPCs were less responsive to TNF-α and IFN-γ, and the mechanisms underlying responses to these two cell types appeared distinct.

Immunomodulatory and immunogenic properties of mesenchymal stem cells derived from bovine fetal bone marrow and adipose tissue

Little information is currently available on therapeutic features of bovine mesenchymal stem cells (MSCs), despite the development of large animal experimental models including cattle may open alternative strategies for investigating MSC physiology and eventual applications for regenerative therapy. The aim of the present study was to compare in vitro immunomodulatory and immunogenic potentials of bovine fetal MSCs (bfMSCs) derived from bovine fetal bone marrow (BM-MSCs) and adipose tissue (AT-MSCs). Immunomodulatory analyses in bfMSCs were performed by determination of the effect of interferon-γ (IFNγ) on mRNA levels of indoleamine 2, 3-dioxygenase (IDO), transforming growth factor β1 (TGFβ1), prostaglandin E receptor 2 (PTGER2), interleukin-6 and -10 (IL-6 and IL-10), and IDO enzymatic activity. The effect of conditioned medium from IFNγ-stimulated bfMSCs on the proliferation of alloantigen-activated peripheral blood lymphocytes (PBLs) was assessed. Immunogenicity of bfMSCs was determined by quantification of mRNA levels of major histocompatibility complex I and II (MHC-I and -II), CD80 and CD86, and the proportion of cells positive for MHC-I and -II by flowcytometry (FACS) analyses. IFNγ treatment increased IL-6, PTGER2 and IDO gene expression and activity in bfMSCs but did not affect suppressive effect on proliferation of PBLs. Lower proportion of AT-MSCs expressed MHC-I and MHC-II in comparison to BM-MSCs. In conclusion, BM-MSCs and AT-MSCs upregulated expression of immunomodulatory genes in a similar way after IFNγ stimuli. BM-MSCs and AT-MSCs in basal condition and treated with IFNγ displayed similar in vitro immunomodulatory ability. Lower expression of MHC-I and MHC-II suggest that AT-MSCs might be less immunogenic compared to BM-MSCs.