Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy

BackgroundEpstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen.MethodsPeripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool.ResultsAfter immunoselection, a mean of 0.53 ± 0.25 × 10⁶ cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection.ConclusionsOur results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.     

Microbial plankton across Drake Passage

We determined biomass and activity of microbial plankton across the Polar Front (PF) in Drake Passage during January 1994. Temperature was around 0°C south and between 3 and 5°C north of the PF. Both biomass and activities of microorganisms were significantly lower in the Antarctic waters south of the PF than in the sub-Antarctic waters north of it. Thus, values of chlorophyll a, integrated between 0 and 200 m, reached 150 mgm⁻² north, but only 25 mg m⁻² south of the PF. Likewise, bacteria varied between 10¹⁴ and 4×10¹³ cells m⁻². However, the abundance of heterotrophic nanoflagellates was extremely low throughout Drake Passage (around 3×10¹⁰ cells m⁻²). Bacterial doubling times were long (mean of 25 days). Bacterivory was estimated from the abundance of predators and prey and from temperature. The grazing impact on bacterioplankton biomass was insignificant (less that 0.05% per day) and low on bacterial heterotrophic production (15% per day). Neither biomass nor the activities of microorganisms were found to increase at the PF. The microbial food web was uncoupled and the bacteria did not seem to be controlled by predation.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

E-rosette formation at 37 °C: Analysis with monoclonal OKT antibodies

When cultured in the presence of PHA, a proportion of human peripheral blood mononuclear cells acquires the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37 °C. The nature of this 37 °C stable E-rosette formation was investigated using a panel of monoclonal OKT antibodies directed to human T-lymphocyte surface antigens. OKT11A antibody, at a concentration of 0.2–0.4 μg/ml, markedly blocked 37 °C E rosetting. OKT1, OKT3, OKT4, OKT6, and OKT8 antibodies, when tested at 10 μg/ml, show no such inhibiting activity. Quantitative studies with ¹²⁵I-labeled OKT11A indicated that the antibody interacted strongly with both 37 °C E-rosetting and nonrosetting cells, the association constant being 1.6–2.0 × 10⁹M^?1. However, on the average, a threefold higher concentration of OKT11A receptor sites was found on 37 °C E-rosette-forming cells (14.8 × 10⁴ sites/cell) than on nonrosetting cells (4.8 × 10⁴ sites/cell). Our data suggest that 37 °C E-rosette formation is governed by a lymphocyte surface determinant recognized by OKT11A antibody. “Overexpression” of OKT11A antigenic sites on a proportion of PHA-stimulated lymphocytes may explain their capacity to form 37 °C stable E-rosettes.     

A comparison of the 8-hydroxydeoxyguanosine,chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes

We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxydeoxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH₃HgCl) and mercuric chloride (HgCl₂) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH₃HgCl or HgCl₂ increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 × 10⁻⁵ M (HgCl₂) or 2 × 10⁻⁶ M (CH₃HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. CH₃HgCl was about 4-fold more potent than HgCl₂. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH₃HgCl at a concentration that was about 50% of the amount of HgCl₂ required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl₂- and/or CH₃HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.     

Retroviral-mediated gene transfer as an effective tool for the in vitro genetic transformation of chicken embryonic cells and production of transgenic chickens

The data on the in vitro and in vivo (into embryonic disk) retroviral-mediated transfer of genetic information into chicken embryonic cells are presented. The estimated transformation frequency of the cultured target cells constituted 8 × 10^?4 to 5 × 10^?3. A transgenic rooster, carrying recombinant DNA in blood, heart, liver, and intestine cells, was obtained.     

Peroxyoxalate chemiluminescent assay for oxidase activities based on detecting enzymatically formed hydrogen peroxide

A sensitive peroxyoxalate chemiluminescent (PO-CL) assay for activities of oxidases (uricase, choline oxidase, cholesterol oxidase and xanthine oxidase) which catalyse a formation of hydrogen peroxide was developed using 4,4′-oxalyl-bis[(trifluoromethylsulphonyl)imino]trimethylene-bis(4-methylmorpholinium)trifluoromethanesulphonate as a chemiluminogenic reagent and 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine as a fluorophore. The standard curve for hydrogen peroxide was linear over the range 1 × 10^?7-1 × 10^?4 mol/L. Relative standard deviations for oxidase assays were 5.1–12.7% (n = 10). Detection limits were 1 × 10^?3 U/mL for uricase, 5 × 10^?4 U/mL for choline oxidase, 5 × 10^?3 U/mL for cholesterol oxidase and 5 × 10^?4 U/mL xanthine oxidase (sample to blank ratio, 3).     

Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation

Background aimsHematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein–Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens.MethodsTo expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9–12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector.ResultsAfter 18 days in culture nine CTL lines expanded from less than 1.5 × 10⁷ PBMC to a mean of 6.1 × 10⁷ T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47–995] and IE-1 (median 154 SFC, range 11–505), the Ad antigens hexon (median 153 SFC, range 26–465) and penton (median 37 SFC, range 1–353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9–301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets.ConclusionsCTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.     

The interaction of albumin and concanavalin A with normal and sickle human erythrocytes.

The interaction of human albumin and concanavalin A with normal and sickle human red blood cells previously washed in phosphate buffer at pH = 7.4 was studied by titration calorimetry. The amount of albumin bound to normal cells was (6.8 ± 2.2) × 10⁵ molecules/cell. An equilibrium constant of 5 × 10¹⁰ and an enthalpy change of ?(280 ± 90) kcal/mol albumin was determined for albumin interaction with normal cells. The amount of albumin bound to sickle cells was (12.4 ± 1.0) × 10⁵ molecules/cell and the enthalpy change for albumin interaction with sickle cells was ?(390 ± 140) kcal/mol. Normal cells bound (5.7 ± 2.4) × 10⁵ concanavalin A molecules/cell with an enthalpy change of ?(840 ± 200) kcal/mol concanavalin. All experiments were conducted at 25°C.     

Quantitative assessment of the adherence of normal human blood mononuclear cells to vascular endothelial cell monolayers

We have developed a method to reliably quantitate the in vitro adherence of ⁵¹Cr-labeled blood mononuclear leukocytes to cultured monolayers of vascular endothelial cells from human umbilical veins. Normal mononuclear leukocytes adhered to endothelial cells more than to cover glass at all studied time periods over 4 hr with major differences seen at 2 hr (9.7 ± 1.2% vs 3.7 ± 1.1%; P < 0.01). Only a minority of cells adhering to endothelium were esterase positive. Similar patterns of binding were seen using varying concentrations of suspended mononuclear cells (1–4 × 10⁶/ml) simulating that occurring in vivo in different clinical states. This approach shows promise for in vitro approaches to lymphocyte-vascular endothelial interactions in human immune/inflammatory disorders.     

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

Animal cells can be cultured both in basal media supplemented with fetal bovine serum (FBS) and in serum-free media. In this work, the supplementation of Grace’s medium with a set of nutrients to reduce FBS requirements in Spodoptera frugiperda (Sf9) cell culture was evaluated, aiming the production of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) at a cost lower than those for the production using Sf900 II medium. In Grace’s medium supplemented with glucose, Pluronic F68 (PF68) and yeast extract (YE), the effects of FBS and milk whey ultrafiltrate (MWU) on cell concentration and viability during midexponential and stationary growth phase were evaluated. In spite of the fact that FBS presented higher statistical effects than MWU on all dependent variables in the first cell passage studies, after cell adaptation, AgMNPV polyhedra production was comparable to that in Sf900 II. Batch cultivation in Grace’s medium with 2.7 g l⁻¹ glucose, 8 g l⁻¹ YE and 0.1% (w/v) PF68 supplemented with 1% (w/v) MWU and 3% (v/v) FBS increased viable cell concentration to about 5-fold (4.7×10⁶ cells ml⁻¹) when compared to Grace’s containing 10% (v/v) FBS (9.5×10⁵ cells ml⁻¹). AgMNPV polyhedra (PIBs) production was around 3-fold higher in the MWU supplemented medium (1.6×10⁷ PIBs ml⁻¹) than in Grace’s medium with 10% FBS (0.6×10⁷ PIBs ml⁻¹). This study therefore shows a promising achievement to significantly reduce FBS concentration in Sf9 insect cell media, keeping high productivity in terms of cell concentration and final virus production at a cost almost 50% lower than that observed for Sf900 II medium. C.A. Pereira is recipient of a CNPq fellowship.     

Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan from Ganoderma lucidum

Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and ¹³C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight M_w and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The M_w value (2.4×10⁴) of sulfated glucan S-GL-1 was much lower than that (44.5×10⁴) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C_∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]=1.32×10^-3M_w^1.06 (cm³ g^-1) and 16, respectively, in the M_w range from 1.1×10⁴ to 2.4×10⁴. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

ESTIMATION OF EFFECTIVE EOSINOPOIESIS AND BONE MARROW EOSINOPHIL RESERVE CAPACITY IN NORMAL MAN

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [³H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 × 10⁹ cells/kg body weight per day (mean 0.22 × 10⁹ cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 × 10⁹ cells/kg body weight (mean 0.14 × 10⁹ cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.     

Identification of Anion-selective Channels in the Basolateral Membrane of Mitochondria-rich Epithelial Cells

Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl^- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl^- channels. The two major populations constituted Ohmic Cl^- channels with limiting conductance (γ_125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl^- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10^-14, 5.7 (±0.7) × 10^-14, and 32 (±2) × 10^-14 cm³/sec, for the 10, 30 and 150 pS Cl^- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl^- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E _Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl^- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl^- channels are organized in clusters. Received: 10 October 1996/Revised: 8 January 1997     

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device

Background aimsThe CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ⁺) and CD19 positive (CD19⁺) cells from apheresis products.MethodsInvestigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2–10.4) × 10¹⁰ mononuclear cells were used with a median 358 (92–1432) × 10⁶ CD34⁺ cells. There were 24.8% (6.1–45.7%) median TCRαβ⁺ cells and 4.4% (1.2–11.7%) median B cells in the apheresis product.ResultsAfter depletion, a median 0.00097% (0.00025–0.0048%) of TCRαβ⁺ cells could be detected, and B cells, as determined as CD20⁺ cells, were reduced to 0.0072% (0.0008–0.072%). TCRαβ⁺ cells were depleted by log 4.7 (3.8–5.5), and B cells were depleted by log 4.1 (3.0–4.7). Recovery of mononuclear cells was 55% (33–77%), and recovery of CD34⁺ cells was 73% (43–98%). Recovery of CD56⁺/3^? natural killer cells was 80% (35–142%), recovery of TCR gamma/delta positive (TCRγδ⁺) T cells was 83% (39–173%) and recovery of CD14⁺ cells was 79% (22–141%). Viability of cells was 98% (93–99%) after separation (all values median).ConclusionsProfound depletion of TCRαβ⁺ T cells can be achieved with the CliniMACS system. Recovery of CD34⁺ stem cells is in the same range than after CD34⁺ enrichment and CD3/CD19 depletion. Transplantation with >4 × 10⁶ CD34⁺ cells/kg can be performed for every patient with 1–5 × 10⁴ TCRαβ⁺ cells/kg and about 5–10 × 10⁶ TCRγδ⁺ cells/kg with two rounds of apheresis.     

Endogenous prostaglandin E2 enhances polyclonal immunoglobulin production by tonically inhibiting T suppressor cell activity

The immunoregulatory effect of endogenous prostaglandin E₂ (PGE₂) on immunoglobulin production was studied in an in vitro culture system of human peripheral blood mononuclear cells, stimulated with pokeweed mitogen (PWM). Three different cyclooxygenase inhibitors (indomethacin, carprofen, and piroxicam) suppressed Ig synthesis by ~50%. This inhibitory effect could be reversed by adding low doses of exogenous PGE₂ (3 × 10^?9 to 3 × 10^?8M). These doses are endogenously produced in PWM-stimulated cultures, and a concentration of 2 × 10^?8M is reached after 48 hr of culture. When B cells were directly stimulated with helper factor, PGE₂ did not enhance Ig production and doses of 3 × 10^?7M to 3 × 10^?6M were inhibitory. The effects of indomethacin and PGE₂ were eliminated when T cells were irradiated or treated with mitomycin prior to culture. The enhancing effects of PGE₂ were substantially reduced after OKT8(+) T cells were removed from the system. PWM-stimulated cultures of lymphocytes from healthy subjects over age 70 were more sensitive to inhibition by indomethacin and to stimulation by PGE₂ than were cultures of lymphocytes from young controls. Thus the major role of endogenous PGE in polyclonal Ig production in vitro is to tonically inhibit a radiosensitive, OKT8(+) suppressor T cell. This tonic inhibition is increased in subjects over 70, which provides one explanation for decreased suppressor cell function in elderly subjects.     

Aujeszky’s disease virus production in disposable bioreactor

A novel, disposable-bag bioreactor system that uses wave action for mixing and transferring oxygen was evaluated for BHK 21 C13 cell line growth and Aujeszky’s disease virus (ADV) production. Growth kinetics of BHK 21 C13 cells in the wave bioreactor during 3-day period were determined. At the end of the 3-day culture period and cell density of 1.82 × 10⁶ cells ml^-1, the reactor was inoculated with 9 ml of gE^- Bartha K-61 strain ADV suspension (10^5.9 TCID₅₀) with multiplicity of infection (MOI) of 0.01. After a 144 h incubation period, 400 ml of ADV harvest was obtained with titre of 10^7.0 TCID₅₀ ml⁻¹, which corresponds to 40,000 doses of vaccine against AD. In conclusion, the results obtained with the wave bioreactor using BHK 21 C13 cells showed that this system can be considered as suitable for ADV or BHK 21 C13 cell biomass production.     

Anti-coccidial and anti-apoptotic activities of palm pollen grains on Eimeria papillata-induced infection in mice

The present work aimed to study the effect of palm pollen extract (PPE) as an anticoccidial and anti-apoptotic modulator during the course of murine intestinal Eimeria papillata infection. The fact that PPE has an anticoccidial efficacy against intestinal E. papillata infection in mice has been clarified by the reduction of faecal output of oocysts on day five post infection from 49.5 × 10³ to 34 × 10³ oocyst/g. Moreover, the number of intracellular eimerian stages of zygots and developing oocysts decreased by about 89% and that of schizonts and gamonts to 42% and 72%, respectively. E. papillata infection also induced an increase in the number of apoptotic cells from 17.5 to 122.8 apoptotic nuclei/10 villous crypt units (VCU). In addition, it caused a state of systemic inflammatory response as revealed by an elevation in levels of the pro-inflammatory biomarkers, inducible nitric oxide synthase (iNOs) and tumor necrosis factor alpha (TNF-α) from 5.3 and 78.3 to 33 pmol ml^?1 and 96.3 pg ml^?1 in blood, respectively, with concurrent duplication in the total leucocytic number. Upon treatment of infected mice with the aqueous PPE, the activity of iNOs was reduced by 55% and the level of TNF-α was decreased by 30%. Moreover, the total leucocytic count was significantly reduced from 9.05 × 10³ to 7.8 × 10³ cells/mm³. Based on our results, PPE showed both anti-coccidial, anti-inflammatory and anti-apoptotic activities. So it can be used in developing new herbal medicine against animal coccidiosis and may be suitable agent for treating eimeriosis associated inflammatory response.     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

Inhibition of sterol synthesis in L cells by 14alpha-ethyl-5alpha-cholest-7-en-15alpha-ol-3-one at nanomolar concentrations

14α-Ethyl-5α-cholest-7-en-15α-ol-3-one was prepared in 85% yield by selective oxidation of the 3β-hydroxyl function of 14α-ethyl-5α-cholest-7-en-3β,15α-diol by cholesterol oxidase. 14α-Ethyl-5α-cholest-7-en-15α-ol-3-one caused a 50% inhibition of the incorporation of [1-¹⁴C]-acetate into digitonin-precipitable sterols at a concentration of 6 × 10^?9M in L cells and a 50% reduction in level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity in the same cells at a concentration of 4 × 10^?8 M.