Bone marrow-derived mesenchymal stem cells inhibit CD8+ T cell immune responses via PD-1/PD-L1 pathway in multiple myeloma.

High expression of the inhibitory receptor programmed cell death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found to play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the programmed cell death 1 (PD-1)/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in multiple myeloma (MM) remains poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSC-mediated regulation of CD8+ T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased compared to that in normal controls (NC) (18·81 ± 1·61 versus 2·78± 0·70%; P < 0·001). Furthermore, the PD-1 expression on CD8+ T cells with NDMM patients was significantly higher than that in normal controls (43·22 ± 2·98 versus 20·71 ± 1·08%; P < 0·001). However, there was no significant difference in PD-1 expression of CD4+ T cells and natural killer (NK) cells between the NDMM and NC groups. Additionally, the co-culture assays revealed that BMSCs significantly suppressed CD8+ T cell function. However, the PD-L1 inhibitor effectively reversed BMSC-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+ T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells to promote the immune escape of MM.

Milk protein responses to balanced amino acid and removal of Leucine and Arginine supplied from jugular-infused amino acid mixture in lactating dairy cows.

This study was undertaken to evaluate the milk protein response when cows were supplied a balanced AA profile and to determine whether a deficiency of Leucine (Leu) or Arginine (Arg) had a negative effect on milk protein. Eight mid-lactation Holstein cows were randomly assigned to 5-day continuous jugular infusions of saline (CTL), EAA mixture prepared on the profile of casein and supplied (in % of lysine (Lys)) 100% of Lys, 33.3% of methionine (Met), 110.2% of Leu, 43.6% of Arg, 50.8% of threonine (Thr), 81.6% of valine (Val), 69.7% of isoleucine (Ile), 61.4% of phenylalanine (Phe) and 34.2% of histidine (His) (Casein, 160 g/d), EAA mixture excluding Leu (-Leu, 163 g/d) or EAA mixture excluding Arg (-Arg, 158 g/d) in a duplicated 4 × 4 Latin square design with four infusion periods separated by 7-day interval period. The basal diet supplied 1.6 Mcal NEL and 94.4 g MP per 1 kg DM to meet requirements for lactation. The Casein treatment provided a balanced supply (in % of MP) of 10.3% Leu and 5.3% Arg, whereas in the two subsequent -Leu and -Arg treatments, the concentration of Leu and Arg was reduced to 8.4 and 4.6% respectively. Dry matter intake (15.4 kg/day) was not affected by treatments. The Casein treatment increased milk yield (14.9%, p < 0.001), milk protein yield (120 g, p < 0.001) and milk protein efficiency (0.03, p = 0.099) than CTL treatment. However, the -Leu treatment decreased the responses of above-measured parameters by 6.25%, 70 g, 0.05 (p < 0.06) (compared with Casein). These effects of Leu were related to decreased Leu concentration and improved concentration of Ile and Val in plasma. The -Arg treatment decreased the plasma Arg concentration than the Casein treatment, whereby resulted in the decrease of milk yield (5.7%, p = 0.073), milk protein yield (60 g, p = 0.011) and milk protein efficiency (0.04, p = 0.037). In conclusion, supply of EAA profile of casein can increase the lactation production in dairy cows, and 8.6% of Leu in MP partly limits the milk protein response when the requirements of Lys, Met and His were met. The level of Arg at 4.6% MP is not deemed to an ideal profile, as evidenced by decreased milk protein efficiency.

Combined effects of oleic, linoleic and linolenic acids on lactation performance and the milk fatty acid profile in lactating dairy cows.

The potential combined effects of oleic, linoleic and linolenic acids supplementation on lactation performance and the milk fatty acid (FA) profile in dairy cows have not been well investigated. Our objective was to examine the effects of supplementation with a combination of these FA as well as the effects of removing each from the combination on lactation performance and the milk FA profile in dairy cows. Eight Holstein cows (101±11 days in milk) received four intravenously infused treatments in a 4×4 Latin square design, and each period lasted for 12 days which consisted of 5 days of infusion and 7 days of recovery. The control treatment (CTL) contained 58.30, 58.17 and 39.96 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively. The other three treatments were designated --C18 : 1 (20.68, 61.17 and 41.72 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively), -C18 : 2 (61.49, 19.55 and 42.13 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively) and -C18 : 3 (60.89, 60.16 and 1.53 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively). Dry matter intake and lactose content were not affected by the treatments, but the milk protein content was lower in cows treated with -C18 : 2 than that in CTL-treated cows. Milk yield as well as milk fat, protein and lactose yields were higher in cows treated with -C18 : 3 than the yields in CTL-treated cows, and these yields increased linearly as the unsaturation degree of the supplemental FA decreased. Compared with the CTL treatment, the -C18 : 2 treatment decreased milk C18 : 2 cis-9 content (by 2.80%) and yield (by 22.12 g/day), and the -C18 : 3 treatment decreased milk C18 : 3 cis-9, cis-12, cis-15 content (by 2.72%) and yield (by 22.33 g/day). In contrast, removing C18 : 1 cis-9 did not affect the milk content or yield of C18 : 1 cis-9. The -C18 : 2-treated cows had a higher C18 : 1 cis-9 content and tended to have a higher C18 : 1 cis-9 yield than CTL-treated cows. The yields of C8 : 0, C14 : 0 and C16 : 0 as well as

Protective effects of fentanyl preconditioning on cardiomyocyte apoptosis induced by ischemia-reperfusion in rats.

We aimed to study the effect of fentanyl (Fen) preconditioning on cardiomyocyte apoptosis induced by ischemia-reperfusion (I/R) in rats. A total of 120 Sprague Dawley male rats (age: 3 months) were randomly divided into: sham operation group (S group), I/R group, normal saline I/R group (NS group), and fentanyl low, middle, and high dose groups (Fen1: 2 µg/kg; Fen2: 4 µg/kg; Fen3: 6 µg/kg). Heart rate (HR), mean arterial pressure (MAP), left ventricular developed pressure (LVDP), ±dp/dtmax, malondialdehyde (MDA), superoxide dismutase (SOD) activity, creatine phosphokinase-MB (CK-MB), and cardiac troponin-I (cTnI) were measured. Myocardial ischemic (MI) area, total apoptotic myocardial cells, and protein and mRNA expressions of B-cell lymphoma 2 (Bcl-2) and Bax were detected. HR and MAP were higher, while LVDP and ±dp/dtmax were close to the base value in the Fen groups compared to those in the I/R group. Decreased MDA concentration and CK-MB value and increased SOD activity were found in the Fen groups compared to the I/R group, while cTnI concentration was significantly lower in the Fen1 and Fen2 groups (all P<0.05). Myocardial damage was less in the Fen groups compared to the I/R group and the MI areas and apoptotic indexes were significantly lower in the Fen1 and Fen2 groups (all P<0.05). Furthermore, significantly increased protein and mRNA expressions of Bcl-2, and decreased protein and mRNA expressions of Bax were found in the Fen groups compared to the I/R group (all P<0.05). Fentanyl preconditioning may suppress cardiomyocyte apoptosis induced by I/R in rats by regulating Bcl-2 and Bax.

Molecular and Physiological Analysis of a Thylakoid K+ Channel Protein.

Transport studies identified a K+ channel protein in preparations of purified spinach (Spinacea oleracea) thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33-kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. This identification was made using an antibody raised against a synthetic peptide representing a highly conserved region of K+ channel proteins. K+ channel activity co-migrated with the immunoreactive 33-kD polypeptide when solubilized thylakoid membrane protein was fractionated on a Suc density gradient. The antibody was used to immunoprecipitate the 33-kD polypeptide. Physiological function of this thylakoid membrane protein was elucidated by measuring photosynthetic electron transport of thylakoid preparations in the presence and absence of a K+ channel blocker. Results indicated that K+ efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity. The effect this protein has on photosynthetic capacity is likely due to the requirement for K+ efflux from the thylakoid lumen to charge-balance light-induced proton pumping across this membrane.

Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release.

Porous chitosan microspheres suitable for the delivery of antigen were prepared using a wet phase-inversion method. The pore structure of the chitosan microsphere could be modified by the change of pH value of the coagulation medium, which is the aqueous tripolyphosphate (TPP) solution. High porosity of chitosan microsphere with an open porous structure on its surface was prepared by coagulation in TPP aq. solution of pH 8.9. The porous chitosan microspheres were modified chemically with reagents to introduce three types of functional groups; carboxyl, hydrophobic acyl and quaternary ammonium groups. Antigen of ND vaccine was immobilized into the pores of porous chitosan microspheres and the adsorbed antigen was assayed by the Hemoglobin Aggregation (HA) analytical method. Sustained-release of ND vaccine's antigen could be achieved through an adsorption-desorption release test. The chemical modifications of the porous chitosan microspheres have a strong large influence on the adsorption efficiency or release rates of the antigen investigated. The porous microspheres have a higher adsorption efficiency and the slower release rate of antigen when modified chemically with 3-chloro-2-hydroypropyltrimethylamonium chloride.

Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing.

A novel asymmetric chitosan membrane has been prepared by immersion-precipitation phase-inversion method and evaluated as wound covering. This new type of chitosan wound dressing which consists of skin surface on top-layer supported by a macroporous sponge-like sublayer was designed. The thickness of the dense skin surface and porosity of sponge-like sublayer could be controlled by the modification of phase-separation process using per-evaporation method. The asymmetric chitosan membrane showed controlled evaporative water loss, excellent oxygen permeability and promoted fluid drainage ability but could inhibit exogenous microorganisms invasion due to the dense skin layer and inherent antimicrobial property of chitosan. Wound covered with the asymmetric chitosan membrane was hemostatic and healed quickly. Histological examination confirmed that epithelialization rate was increased and the deposition of collagen in the dermis was well organized by covering the wound with this asymmetric chitosan membrane. The results in this study indicate that the asymmetric chitosan membrane thus prepared could be adequately employed in the future as a wound dressing.

[Ultra-weak luminescence of blood in cancer patients].

Ultra-weak luminescence of blood in 67 normal blood donors and 57 lung cancer patients was studied using the sensitive single photon counting system manufactured in China. The results showed that the blood emission intensity of lung cancer patients was higher than that of the normal blood donors with significant statistical difference (P less than 0.05). The characteristic peak of blue light ranging from 426.8 nm to 446.8 nm in lung cancer patients may imply that ultra-weak luminescence test be of value in the diagnosis of lung cancer.

[Synergetic radiosensitizing effect of isomers S-8932 and S-8933 in vitro].

The synergetic radiosensitizing effect of isomers S-8932 and S-8933 on HeLa-S3 cell-line was investigated in vitro using cell colony forming assay. The results suggested that the combined use of the two isomers showed a lower cytotoxicity than their single use. The ID50 was 55.3 mM. Their synergetic radiosensitizing effect on HeLa-S3 cells under hypoxic condition was higher than either one used alone. The SER of the combined one at 4mM was 1.98 (D0 ratio) and 1.79 (D9 ratio) indicating a sensitizing effect at both low and high radiation doses. The type of the synergetic radiosensitization of the two compounds indicates that they are radiosensitizers of "alpha-beta" type according to the analysis of their molecular models. If the two isomers are used in combination in fractionation radiotherapy, less number of fraction and larger dose per fraction should be selected in the scheme of treatment.

[Establishment of a human colonic carcinoma xenograft in nude mice and its chief biological characteristics].

A human colonic cancer specimen cut into 1-2 mm3 pieces under aseptic condition was heterotransplanted hypodermically by trocar to the inguinal region of BALB/cATcL nude mice bred in our laboratory. 53 days later, a 10 X 9 X 11 mm3 tumor was obtained. The take rate was 100% (48/48). The take rate of liquid nitrogen frozen and recovered tissue pieces was also 100% (13/13). Grossly, the tumor was rich in blood supply and well encapsulated. Within the tumor, the tissue was cream-colored. G-banded chromosome analysis revealed a human chromosome pattern with the mode 70-90. Pathologic diagnosis was signet-ring cell cancer, which was identical to the surgical specimen. No change was found after maintaining 13 passages in nude mice. It was named the human colonic carcinoma xenograft XHCn/w. It provides an ideal tumor model for in vivo or in vitro experiments.

Design and synthesis of N-(pyridylacryl) amino acid derivatives as potential radiosensitizers against HeLa-S3 cells in vitro.

A new series of N-(pyridylacryl) amino acid derivatives have been designed and synthesized as potential radiosensitizers in an effort to increase therapeutic efficacy with less toxicity. Radiosensitization and cytotoxicity of the newly synthesized compounds upon HeLa-S3 cells were measured. The main effects of the reduction of the shoulder width and Do value of the survival curve by 3-pyridylacrylsarcosine (3A) and 4-pyridylacrylsarcosine (4A) were observed. This work has demonstrated that this series of compounds, especially 3A and 4A, or their structurally related compounds, showed great clinical potential as radiosensitizers if significant radiosensitizing activity in vivo could be achieved.

[Synthesis and radiosensitizing activity of nitro-arylimino-diethyl-sodium thiosulfate and nitro phenylalanine derivatives].

A series of compounds was synthesized, these compounds were tested for Hela-S3 cells in vitro for radiosensitizing activity. Five of them are 2,2'-(arylimino)-diethyl-sodium thiosulfate and two of them are phenylalanine derivatives. Most of them showed various degrees of radiosensitizing activity. Among them, SER of L07 was 1.89 at 3 mmol, and had low cytotoxicity to Hela-S3 cells, ID50 was 18.8 mmol. The relationship between radiosensitizing effects and chemical structure was discussed. It offers a base for further exploration of selectively hypoxic cell radiosensitizers.

[Investigation of plasma and urine concentration of Cholecystokinin and Somatostatin in pilots and ground crew].

Concentration of Cholecystokinin(CCK) and Somatostantin(SS) in Plasma, 24 h urine and morning urine were observed in 57 pilots (22-45 years, mean 27.9) and 60 ground crew (23-47 years, mean 30.7). The results showed that CCK level in plasma, 24 h urine and morning urine (20.14 +/- 4.15, 10.08 +/- 2.29 and 11.29 +/- 2.18 pmol/L respectively) in pilots were significantly lower than those in ground crew (22.89 +/- 3.76, 7.04 +/- 1.03, 8.10 +/- 1.94 pmol/ L respectively) and those of SS were distinctly higher than in ground crew (12.38 +/- 2.34, 6.75 +/- 1.18, 7.41 +/- 1.27pmol/L respectively). Concentration of CCK and SS in plasma were positively related to those in 24 h urine and morning urine both in pilots and ground crew. It suggests that these changes might play potential role in the pathogenesis of some diseases of digestive system in pilots.

Development of a K(+)-channel probe and its use for identification of an intracellular plant membrane K+ channel.

Polyclonal antibodies were generated against a 9-amino acid, synthetic peptide corresponding to the selectivity filter in the pore region of K(+)-channel proteins. The sequence of amino acids in the ion-conducting pore region of K+ channels is the only highly conserved region of members of this protein family. The objectives of the present work were (i) to determine whether the anti-channel pore peptide antibody was immunoreactive with known K(+)-channel proteins and (ii) to demonstrate the usefulness of the antibody by employing it to identify a newly discovered K(+)-channel protein. Anti-channel pore peptide was immunoreactive with various K(+)-channel subtypes native to a number of different species. Immunoblot analysis demonstrated affinity of the antibody for the drk1, maxi-K, and KAT1 K(+)-channel proteins. Studies also suggested that the anti-channel pore peptide antibody did not immunoreact with membrane proteins other than K+ channels. The anti-channel pore peptide antibody was used to establish the identity of a 62-kDa chloroplast inner envelope polypeptide as a putative component of a K(+)-channel protein. It was concluded that an antibody generated against the conserved pore region/selectivity filter of K+ channels has broad but selective affinity for this class of proteins. This K(+)-channel probe may be a useful tool for identification of K(+)-channel proteins in native membranes.

Sustained-release of oxytetracycline from chitosan microspheres prepared by interfacial acylation and spray hardening methods.

Chitosan microspheres containing oxytetracycline (OTC), an antibiotic agent, were prepared by spray hardening and interfacial acylation methods. The object of this study was to prepare oxytetracycline-containing microspheres for oral administration and injection using different molecular weight (Mw 70,000 approximately 2,000,000) of chitosan. By the spray hardening method, microspheres with particle sizes between 5 and 30 microns could be obtained and might be suitable for intramuscular injection. On the other hand, chitosan microspheres with the ability to extend the dissolution period of oxytetracycline in low pH medium were also prepared by the interfacial acylation method. The result indicated that the releasing of oxytetracycline from various acylated chitosan microspheres was decreased with increasing the molecular weight of chitosan and would show well sustained-release property. Besides, the morphology of various microspheres and crystalline form transition of oxytetracycline were also studied using electron scanning microscope and X-ray analysis.

Characterization of a chloroplast inner envelope K+ channel.

A K(+)-conducting protein of the chloroplast inner envelope was characterized as a K+ channel. Studies of this transport protein in the native membrane documented its sensitivity to K+ channel blockers. Further studies of native membranes demonstrated a sensitivity of K+ conductance to divalent cations such as Mg2+, which modulate ion conduction through interaction with negative surface charges on the inner-envelope membrane. Purified chloroplast inner-envelope vesicles were fused into an artificial planar lipid bilayer to facilitate recording of single-channel K+ currents. These single-channel K+ currents had a slope conductance of 160 picosiemens. Antibodies generated against the conserved amino acid sequence that serves as a selectivity filter in the pore of K+ channels immunoreacted with a 62-kD polypeptide derived from the chloroplast inner envelope. This polypeptide was fractionated using density gradient centrifugation. Comigration of this immunoreactive polypeptide and K+ channel activity in sucrose density gradients further suggested that this polypeptide is the protein facilitating K+ conductance across the chloroplast inner envelope.

[Isolation, purification and activity measurement of proteolytic enzymes in dental plaque].

The purpose of this study was to investigate the types and activities of proteolytic enzymes in human root surface plaque. Sephadex G-200 gel filtration, fluorometry and spectrometry were used to isolate, purify and demonstrate the activities of proteolytic enzymes. The results indicated that three enzymes were present in the root surface plaque, namely leucine amino peptidase, dipeptidyl peptidase i.v. and trypsin-like proteinase, and their activities were different.

Evaluation of various types of new hypoxic cell sensitizers using the EMT6 single cell-spheroid-solid tumour system.

Eleven new hypoxic cell sensitizers representative of those developed in Japan between 1980 and 1985 were evaluated in vitro and in vivo in comparison with misonidazole (MISO), SR-2508, Ro 03-8799, and ANT (2-amino-5-nitrothiazole). The new compounds included 2-nitroimidazole nucleoside analogues, nitrotriazoles and other nitroaromatics, non-nitro compounds, and electron-affinic compounds that readily intercalate DNA. The sensitizing activity in the EMT6 single cells correlated not only with the reduction potential but, for some compounds, also with the reactivity with non-protein sulphydryls. The sensitizers were also tested using the EMT6 spheroids and solid tumours. The patterns of changes in sensitizer enhancement ratios (SERs) for single cells, spheroids, and solid tumours were classified into two types: (1) SERs for the three testing systems were similar; and (2) SERs decreased in the order of: single cells, spheroids, and solid tumours. Only nitroimidazole and nitrotriazole derivatives belonged to the former type. RK-28 and RK-29, 2-nitroimidazoles with sugar analogue components, had in vivo effects almost equal to those of MISO. Also 3- and 4-nitrotriazole derivatives had definite in vivo effects.

Preparation and release properties of biodegradable chitin microcapsules: II. Sustained release of 6-mercaptopurine from chitin microcapsules.

Chitin microcapsules are prepared using a simple desolvation or nonsolvent addition phase separation method with 6-mercaptopurine (6-MP) as a reference core. Chitin with a molecular weight about 400,000 is used to prepare different core loaded microcapsules. The drug release rates of chitin microcapsules prepared by simple desolvation or nonsolvent addition method have different release profiles which are related to the rate of phase separation. With respect to the solubility parameter difference (delta delta) value between solvent and nonsolvent, the release rate of 6-MP from microcapsules decreases with increasing delta delta of the preparative system. The chitin beads show poor swelling properties and their release rates are pH-dependent. Sustained release of 6-MP from chitin microcapsules in low pH and neutral medium can be accomplished. To determine if the drug release from the polymer matrix is via a diffusion controlled or by an erosion controlled process, 6-MP release profiles of various chitin microcapsules degraded by lysozyme are investigated. The drug-release patterns of the chitin microcapsules prepared by nonsolvent addition (acetone, n-propanol, n-butanol) and simple desolvation in acetone are not only diffusion but also lysozyme digestion influenced. Whereas, by using water or ethanol as nonsolvent or desolvating agent, release profiles of the microcapsules prepared by nonsolvent addition and the simple desolvation method seem to be little affected by enzyme degradation. These results indicate that chitin might prove useful as a polymer carrier for the sustained release of drugs.