Cryosolvent interaction with cellular actin using 3T3-L1cells as a model system

Previous immunolocalisation studies using intact cells have identified modification of the cytoskeleton by cryoprotectants. In the present study we have used a proteomics approach to directly resolve the interactive effects of 3T3-L1cells exposed to two cryoprotectants, dimethyl sulphoxide (Me(2)SO) and 1,2-propanediol (PROH) in 5,10, 20 and 50(v/v) percent solutions, respectively. Two-dimensional protein electrophoresis and Western blot analysis of the cell extracts identified a range of immunoreactive actin fragments with varying molecular weights and isoelectric points at all cryoprotectant concentrations. The addition of either 10mM l-cysteine or reduced glutathione to the cells prior to cryprotectant exposure modified the actin fragmentation. In this preliminary report, we have provided direct evidence of actin fragmentation when exposed to cryoprotectants and have demonstrated that the use of redox agents can modify the cryprotectant action.     

Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions.     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll + 0.6 M sucrose (10⁻³ M Ficoll + 0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS_basic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive.Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.     

Nitric oxide enhances osteoclastogenesis possibly by mediating cell fusion

Osteoclasts are multinucleated bone resorbing cells which form by fusion of pre-osteoclasts. Here, we investigate how nitric oxide (NO) affects osteoclastogenesis. Time lapse photomicrography, using the fluorescent NO indicator dye, 4,5-diaminofluorescein diacetate, revealed an intense NO signal in pre-osteoclasts preceding cell fusion. Osteoclastogenesis in RAW264.7 cells increased when exposed to the NO synthase inhibitor, l-NMMA (0.25 μM), for the initial 48 h. In contrast, pre-osteoclast fusion decreased when RAW264.7 cells were exposed to l-NMMA from 48 to 96 h. Both NO synthase inhibitors, l-NMMA and l-NAME, decreased osteoclast formation during this time period. The inhibitory effect of l-NMMA on osteoclast formation was abolished with increasing concentrations (25–200 ng/ml) of sRANKL suggesting signaling cross talk. NO donors increased osteoclast formation in a dose-dependent manner, with greatest stimulation at 15 μM NOC-12 (2.3 fold) and 5 μM NOC-18 (2.4 fold). Measuring nitrite (NO end product) daily from culture media of RAW264.7 cells undergoing osteoclastogenesis revealed that an increase in NO production coincided with the fusion of pre-osteoclasts (day 4). Inhibiting fusion by plating cells on polystyrene dishes pre-coated with poly-(l-lysine) decreased both osteoclast formation and NO production. To address if NO mediates fusion through the actin cytoskeleton, actin free barbed ends were measured. 0.25 μM l-NMMA decreased, while 15 μM NOC-12 and 5 μM NOC-18 increased actin free barbed ends. We hypothesize that while NO initially negatively regulates pre-osteoclast differentiation; it later facilitates the fusion of mononuclear pre-osteoclasts, possibly by up regulating actin remodeling.     

Use of hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 expressed thermophilic alcohol dehydrogenases as whole-cell catalyst in solvent-free organic media

The hydrophobic bacterium Rhodococcus rhodochrous NBRC15564 was employed as a whole-cell biocatalyst to examine its potential for bioconversion in solvent-free organic media. The genes encoding two different thermostable alcohol dehydrogenases (ADH_Tt1 and ADH_Tt2) of Thermus thermophilus HB27 were expressed in R. rhodochrous cells. To inactivate indigenous mesophilic enzymes in R. rhodochrous, transformant cells were heated at 70 °C for 10 min. Heat-treated hydrophobic wet cells were used for the bioconversion of 2,2,2-trifluoroacetophenone (TFAP) to α-(trifluoromethyl) benzyl alcohol (TFMBA) as a model reaction with ADH_Tt1. NADH, which was supplied in aqueous solution, was regenerated by converting cyclohexanol to cyclohexanone by ADH_Tt2. All reactions were performed by suspending heat-treated cells in solvent-free organic media consisting of 3.7 M TFAP and 4.8 M cyclohexanol (1:1, v/v ratio) at 60 °C. When 800 mg heat-treated R. rhodochrous cells were dispersed in 2 mL of solvent-free organic media (400 mg cells/mL), the product concentration reached about 3.6 M TFMBA by 48 h with a total NADH turnover number of approximately 900. The overall productivity was 190 mol TFMBA/kg cells/h.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D

There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0–1.5 mg/ml, 124±8 to 202±27 Pa) and stiff (3.0 mg/ml, 502±48 Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and ～1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700 Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000 Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.     

Casearin X exhibits cytotoxic effects in leukemia cells triggered by apoptosis

Clerodane diterpenes have demonstrated cytotoxic, antiplasmodial and anti-ulcer properties. In the present work, we determined the cytotoxic effect of casearin L (Cas L), O (Cas O) and X (Cas X) and (?)-hardwickiic acid isolated from Casearia sylvestris leaves, and investigated the underlying mechanisms involved in in vitro cell death induced by Cas X in HL-60 leukemia cells (0.7, 1.5 and 3.0 μM). Cytotoxicity tests demonstrated that Cas X was the most active compound studied, showing greater cytotoxic effects against CEM and HL-60 lines (IC₅₀ of 0.4 μM) and human peripheral blood mononuclear cells (PBMC, IC₅₀ of 1.2 μM). After 24 h exposure, Cas X caused a decrease in 5-bromo-20-deoxyuridine (BrdU) incorporation (36.6 and 24.5% labeling at 0.7 and 1.5 μM, respectively), reduction in viability, and increase in apoptotic and necrotic leukemia cells in a dose-dependent manner evidenced by the trypan blue and AO/EB (acridine orange/ethidium bromide) assays. Moreover, Cas X-treated cells exhibited nuclear fragmentation and cytoplasmic vacuolization depending on the concentration tested. These characteristics of apoptosis or secondary necrosis were confirmed by flow cytometry which revealed DNA fragmentation, phosphatidylserine externalization, activation of the effector caspases 3/7 and mitochondrial depolarization. We then found evidence that Cas X causes cell death via apoptotic pathways, corroborating the potential of casearins as compounds with promising antitumor-related properties.     

Indirubin derivatives as potent FLT3 inhibitors with anti-proliferative activity of acute myeloid leukemic cells

Indirubin derivatives were identified as potent FLT3 tyrosine kinase inhibitors with anti-proliferative activity at acute myeloid leukemic cell lines, RS4;11 and MV4;11 which express FLT3-WT and FLT3-ITD mutation, respectively. Among several 5 and 5′-substituted indirubin derivatives, 5-fluoro analog, 13 exhibited potent inhibitory activity at FLT3 (IC₅₀ = 15 nM) with more than 100-fold selectivity versus 6 other kinases and potent anti-proliferative effect for MV4;11 cells (IC₅₀ = 72 nM) with 30-fold selectivity versus RS4;11 cells. Cell cycle analysis indicated that compound 13 induced cell cycle arrest at G₀/G₁ phase in MV4;11 cells.     

4-(3-Aryloxyaryl)quinoline alcohols are liver X receptor agonists

A series of 4-(3-aryloxyaryl)quinolines with alcohol substituents on the terminal aryl ring was prepared as potential LXR agonists, in which an alcohol group replaced an amide in previously reported amide analogs. High affinity LXR ligands with excellent agonist potency and efficacy in a functional model of LXR activity were identified, demonstrating that alcohols can substitute for amides while retaining LXR activity. The most potent compound was 5b which had an IC₅₀ = 3.3 nM for LXRβ binding and EC₅₀ = 12 nM (122% efficacy relative to T0901317) in an ABCA1 mRNA induction assay in J774 mouse cells.     

Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H₂O₂, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN + HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN + HP-induced fragmentation only ∼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ∼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Discovery of (E)-5-(benzylideneamino)-1H-benzo[d]imidazol-2(3H)-one derivatives as inhibitors for PTK6

A lead compound 1, which inhibits the catalytic activity of PTK6, was selected from a chemical library. Derivatives of compound 1 were synthesized and analyzed for inhibitory activity against PTK6 in vitro and at the cellular level. Selected compounds were analyzed for cytotoxicity in human foreskin fibroblasts using MTT assays and for selectivity towards PTK members in HEK 293 cells. Compounds 20 (in vitro IC₅₀ = 0.12 μM) and 21 (in vitro IC₅₀ = 0.52 μM) showed little cytotoxicity, excellent inhibition of PTK6 in vitro and at the cellular level, and selectivity for PTK6. Compounds 20 and 21 inhibited phosphorylation of specific PTK6 substrates in HEK293 cells. Thus, we have identified novel PTK6 inhibitors that may be used as treatments for PTK6-positive carcinomas, including breast cancer.     

Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL⁻¹) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.     

A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The purified “disintegrin protein” (64 kDa) from the venom of the Indian cobra snake (Naja naja) exhibited cytotoxic effects of various types of human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549) and liver cancer (HepG2). In vitro cytotoxicity, DNA fragmentation, an apoptotic assay and a cell cycle analysis were performed to evaluate the anticancer activity of disintegrin against the above cell lines. The IC₅₀ value of disintegrin was determined to be 2.5 ± 0.5 μg/mL, 3.5 ± 0.5 μg/mL, and 3 ± 0.5 μg/mL for the MCF-7, A549 and HepG2 cell lines respectively. Moreover, the increased distribution of G0/G1 and S phase led to decreased populations of cells in the G2/M phase of MCF-7, HepG2 and A549 cells.     

Synthesis and biological evaluation of novel 2-(substituted phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones as potent HIV-1 NNRTIs

A series of novel 2-(phenylaminocarbonylmethylthio)-6-(2,6-dichlorobenzyl)-pyrimidin-4(3H)-ones have been designed and synthesized. All of the new compounds were evaluated for their anti-HIV activities in MT-4 cells. Most of these new compounds showed moderate to potent activities against wild-type HIV-1 with an EC₅₀ ranging from 4.48 μM to 0.18 μM. Among them, 2-[(4-bromophenylamino)carbonylmethylthio]-6-(2,6-dichlorobenzyl)-5-methylpyrimidin-4(3H)-one 4b3 was identified as the most promising compound (EC₅₀ = 0.18 ± 0.06 μM, CC₅₀ >243.56 μM, SI >1326). The structure–activity relationship (SAR) of these new congeners is discussed.     

Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry

A quantitative assay for simultaneous measurement of individual human neutrophil peptide-1, -2 and -3 concentrations will aid in exploring the potential of these antimicrobial peptides as biomarkers for various diseases. Therefore, a liquid chromatography–tandem mass spectrometry method has been developed and validated to allow separate quantification of the three human neutrophil peptides in human plasma and serum. Plasma and serum samples (100 μl) were deproteinized by precipitation, followed by chromatographic separation on a Symmetry 300 C₁₈ column (50 mm × 2.1 mm I.D., particle size 3.5 μm), using a water–methanol gradient containing 0.25% (v/v) formic acid and human alpha-defensin 5 as internal standard. Tandem mass spectrometric detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization. Despite low fragmentation efficiency of the antimicrobial peptides, multiple reaction monitoring was used for detection, though selecting the quaternary charged ions as both precursor and product. The method was linear for concentrations between 5 and 1000 ng/ml with a limit of detection around 3 ng/ml for all peptides. Intra- and inter-assay precisions were 14.8 and 19.1%, respectively, at the lowest measured endogenous concentration (6.4 ng/ml of HNP-1 in plasma), representing the lower limit of quantification of the assay. Recoveries of HNP-1, -2 and -3 from plasma and serum ranged between 85 and 128%. Analysis of serum samples from intensive care patients showed average concentrations of 362, 570 and 143 ng/ml for HNP-1, -2 and -3, respectively.     

One-step dilution of open-pulled-straw (OPS)-vitrified mouse blastocysts in sucrose-free medium

Embryos vitrified by the open-pulled-straw (OPS) method are only briefly exposed to cryoprotectants and not fully equilibrated with the cryoprotectant. That being the case, conceivably the post-thawing de- and rehydration processes may be omitted. This would render thawing and dilution in a single step and direct transfer to recipients possible without the need for a microscope and other laboratory equipment. Morphologically intact mouse blastocysts from superovulated 5- to 8-week-old virgin female NMRI mice were vitrified according to a protocol [6] slightly modified from the classical OPS-procedure of Vajta et al. [29] consisting of exposure to 10% dimethyl-sulfoxide (Me₂SO) + 10% ethylene glycol (EG) for 1 min, followed by 20% Me₂SO + 20% EG for 20 s before loading into straws that are plunged into liquid nitrogen. In Group 1, 75 blastocysts were exposed to the standard thawing and dilution regimen involving exposure to three solutions of decreasing sucrose content (Control). In Groups 2, 3 and 4, 75 blastocysts each were transferred, in a single step, to medium at 37 °C containing 0.66, 0.33 or 0 M sucrose, respectively. After 48 h of in vitro culture the proportion of hatched blastocysts was determined. In Group 1, this proportion amounted to 82.7%, in Groups 2, 3 and 4 to 76.0%, 73.3% and 78.7%, respectively (P > 0.05). To examine their potential to continue development in vivo, OPS-vitrified blastocysts thawed according to the regimens of Groups 1 and 4 were transferred to recipients (10 embryos/recipient). In Group 1, 9/10 recipients got pregnant with 4.7 ± 0.6 (mean ± SEM) fetuses, in Group 4, 8/10 recipients with 5.0 ± 0.5 fetuses. The overall embryo survival rate per group was 42% for Group 1 and 40% for Group 4. All fetuses were normally developed and viable and there were no significant differences between groups (P > 0.05). It may be concluded that warming and transfer of OPS-vitrified mouse embryos in a single step in medium devoid of sucrose is feasible, which is tantamount to a substantial simplification of embryo transfer operations.     

Discovery of substituted benzyl tetrazoles as histamine H3 receptor antagonists

A series of potent and subtype selective H3 receptor antagonists containing a novel tetrazole core and diamine motif is reported. A one-pot multi-component Ugi reaction was utilised to rapidly develop the structure–activity relationships (SAR) of these compounds. Optimisation for liver microsome stability (t_1/2 >60 min), minimal CYP inhibition (IC₅₀ >50 μM) and high cell permeability (Caco-2 P_app >20 × 10^?6 cm/s) identified several compounds with drug-like properties.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38α, p38β, p38γ, and p38δ MAP kinases: IC₅₀ = 0.034, 0.572, >10, and >10 μM, respectively; (ii) inhibition of TNFα, IL-1β, IL-6, and IL-8 production in human whole blood: IC₅₀ = 0.026, 0.020, 0.88, and 0.016 μM, respectively; (iii) inhibition of LPS induced TNFα, IL-1β and IL-6 production in mice: ID₅₀ = 0.93, 8.63, and 0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID₅₀ = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID₅₀ = 2.38 mg/kg, po; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID₅₀ = 3.1 mg/kg, po; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID₅₀ = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID₅₀ = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.