Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.
Animal ; Calcineurin/antagonists & inhibitors* ; Cell Line, Transformed ; Cell Transformation, Viral ; Cyclosporine/pharmacology* ; Enzyme Inhibitors/pharmacology ; Fibroblasts ; Fibronectins/metabolism ; Fibronectins/genetics* ; Gene Expression Regulation* ; Human ; Lung/cytology ; Mice ; Osteoblasts ; Tacrolimus/pharmacology ; Tacrolimus/analogs & derivatives*

OBJECTIVETo explore the feasibility of regulated expansion and committed differentiation potential of JAK2 gene modified hematopoietic stem/progenitor cells in vitro.

METHODSA murine stem cell virus (MSCV) based retroviral vector MGI-F2Jak2, which encodes a green fluorescent protein (GFP) and a fusion protein containing two copies of modified FK506 binding protein (F36v) linked tyrosine kinase JAK2 was cloned. F36v served as a high-affinity binding site for dimerizer AP20187. GpE + 86 packaging cell was transfected with this vector. Bone marrow cells from C57BL/6 mice were transduced by co-cultured with irradiated (1500 cGy) GpE + 86 producer clone for 48 h. Transduced marrow cells were expanded in X-VIVO 15 and divided into four groups as follows: (1) control group; (2) AP20187 alone group; (3) SCF alone group and (4) AP20187 + SCF group. The phenotypes of the expanded cells were analyzed by directly phycoerythrin-labeled anti-Sca1, c-kit, CD(34), Gr1, CD(11b), TER119, CD(41), B220 and CD(3) monoclonal antibodies for flow cytometry. Committed differentiation, progenitor colony assay and spleen colony forming units (CFU-S) were further evaluated.

RESULTSA significant sustained outgrowth of transduced marrow cells was obtained only in the AP20187 + SCF group. Cells expanded up to 10(14)-fold after 80 days culture. The doubling time was about 30 hs. The phenotypes of the expanded cells were homogeneously strong positive for CD(34), c-kit and Sca1, while were almost negative for Gr1, CD(11b), TER119, CD(41), B220 and CD(3). Functional assay demonstrated that the expanded cells had multipotential to differentiate into granulocyte, macrophage, erythrocyte, or B-cells under different cytokines combinations. A prominent megakaryocytic differentiation was observed when cultured with SCF/Tpo/IL-11 combination. The expanded cells were also capable of forming BFU-E, CFU-GM and CFU-Mix in methylcellulose colony assay. The expanded cells over three months could still form CFU-S.

CONCLUSIONSAP20187 combined SCF mediated activation of JAK2 signaling domain can dramatically expand hematopoietic stem/progenitor cells, and the expanded cells can be regulated and committed to differentiate into multilineage cells. This system may provide important insights into stem cell biology and may be promising for gene and cell therapy.

Animals ; Cell Differentiation ; Female ; Hematopoietic Stem Cells ; cytology ; Janus Kinase 2 ; Mice ; Mice, Inbred C57BL ; Protein-Tyrosine Kinases ; genetics ; physiology ; Proto-Oncogene Proteins ; Tacrolimus ; analogs & derivatives ; pharmacology ; Transfection

OBJECTIVETo investigate the antipruritic mechanisms of pimecrolimus cream for women facial dermatitis.

METHODSTopical pimecrolimus cream 1% was applied in 52 women patients with facial dermatitis. The Investigators Global Assessment (IGA) score, severity of pruritus (SP) scores, and a basic syntax and molecular substrate (molecular psychophysics) of nociception and pruriception established by temperature-sensitive transient receptor potential (TRP) channels were used to evaluate the clinical signs, severity of pruritus, and skin sensory phenomenon.

RESULTSThe IGA scores at day 1 and 4 of treatment and the SP score at day 1, 4, and 11 of treatment were significantly lower than the baseline scores before treatment (P < 0.05). Among these 52 patients, 28 (53.8%) showed positive capsaicin-like response (i.e., burning with consequent rapid amelioration of pruritus) at the application sites, 12 (23.1%) showed camphor-like response (i.e., warming with consequent rapid amelioration of pruritus), and 12 (23.1%) showed negative capsaicin-like response or negative camphor-like response.

CONCLUSIONSTreatment with pimecrolimus cream 1% can rapidly and effectively improve the signs and symptoms of facial dermatitis in adult women patients. Pimecrolimus cream 1% may act on the transient potential vanilloid 1 (TRPV1) receptor in the skin sensory afferents to induce capsaicin-like response or camphor-like response and then desensitizes TRPV1 and rapidly inhibits or alleviate itching.

Administration, Topical ; Adolescent ; Adult ; Antipruritics ; administration & dosage ; Dermatitis ; complications ; drug therapy ; Face ; Female ; Humans ; Middle Aged ; Pruritus ; drug therapy ; etiology ; Tacrolimus ; administration & dosage ; analogs & derivatives ; Young Adult

The purpose of this study is to investigate the effects of multiple-trough sampling design and nonlinear mixed effect modeling (NONMEM) algorithm on the estimation of population and individual pharmacokinetic parameters. Oxcarbazepine and tacrolimus were used as one-compartment and two-compartment model drugs, respectively. Seven sampling designs were investigated using various number of trough concentrations per individual ranging from 1-4. Monte Carlo simulations were performed to produce state-steady trough concentrations. One-compartment model was used to fit simulated data from oxcarbazepine and tacrolimus. The accuracy and precision of the estimated parameters were evaluated using the median prediction error (PE), the median absolute PE and boxplot. The results indicated that trough concentrations could yield reliable estimates of apparent clearance (CL/F). For oxcarbazepine, as the number of trough concentrations per subject increased, the accuracy and precision of CL/F, between-subject variability (BSV) of CL/F and residual variability (RUV) tended to be improved. For tacrolimus, however, although no improvement were observed in the accuracy of CL/F and BSV of CL/F, the PE distribution ranges were significantly narrowed and the RUV estimates were less bias and imprecise. In terms of algorithm, Monte Carlo importance sampling (IMP) and IMP assisted by mode a posteriori estimation (IMPMAP) were consistently better than other methods. Additionally, the sampling design had no significant effects on the individual parameter estimates, which were only depended on the interaction between BSV and RUV in various algorithms. Decreased in BSV and RUV levels can improve the accuracy and precision of the estimation for both population and individual pharmacokinetic parameter estimates.
Algorithms ; Bayes Theorem ; Carbamazepine ; analogs & derivatives ; pharmacokinetics ; Humans ; Immunosuppressive Agents ; pharmacokinetics ; Models, Biological ; Monte Carlo Method ; Nonlinear Dynamics ; Regression Analysis ; Tacrolimus ; pharmacokinetics

To assess the influence of cyclosporin A (CsA) and tacrolimus (FK506) on mycophenolic acid (MPA) and correlation analysis of the pharmacokinetic parameters and patient characteristics, clinical outcome in Chinese kidney transplant recipients, the pharmacokinetics of 1000 mg mycophenolate mofetil (MMF) twice daily was measured by high-performance liquid chromatography (HPLC). PKS (Pharmaceutical Kinetics Software) 1.0.2 software package was used for the calculation of pharmacokinetic parameters. The mean C(max), t(max), and AUC((0-12))were (21.88+/-10.52) microg/ml, (1.20+/-0.95) h, and (52.546+/-13.215) microg.h/ml, respectively. The level of AUC((0-12)) in the FK506 group was significantly higher than that in the CsA group. MPA appeared not to be affected by renal function. MPA AUC((0-12)) showed statistically significant difference according to the patient's gender.
Adult ; Cyclosporine ; administration & dosage ; Female ; Humans ; Immunosuppressive Agents ; administration & dosage ; pharmacokinetics ; Kidney Transplantation ; physiology ; Male ; Middle Aged ; Mycophenolic Acid ; administration & dosage ; analogs & derivatives ; pharmacokinetics ; Tacrolimus ; administration & dosage

Pimecrolimus cream 1% has shown to be effective in patients with a variety of inflammatory cutaneous disorders. And it might be a useful modality in the treatment of seborrheic dermatitis. This prospective study was aimed at assessing the efficacy and tolerability of pimecrolimus cream 1% in the treatment of facial seborrheic dermatitis. Twenty patients were instructed to apply pimecrolimus cream 1% for 4 consecutive weeks. Assessment of the disease severity was performed at baseline and at week 1, 2, and 4. Clinical assessments of erythema, scaling, and pruritus were measured using a 4-point scale (0-3). Global assessments of the disease severity by patients and investigators were performed at each visit. Mean clinical scores of erythema, scaling, and pruritus significantly improved by 87.4%, 91.9%, and 91.5% respectively at week 4 (p<0.001). Improvements in the global assessment of disease severity determined by patients and investigators also showed excellent results. No specific adverse events other than transient burning and tingling sensations were noted. The relapse of facial seborrheic dermatitis was mostly observed between 3 to 8 weeks after the discontinuation of pimecrolimus. We suggest that the topical application of pimecrolimus cream 1% can be an effective and safe alternative for treatment of facial seborrheic dermatitis.
Adult ; Aged ; Dermatitis, Seborrheic/*drug therapy ; Erythema/drug therapy ; Face ; Female ; Follow-Up Studies ; Humans ; Korea ; Male ; Middle Aged ; Severity of Illness Index ; Tacrolimus/*analogs & derivatives/therapeutic use ; Time Factors ; Treatment Outcome

OBJECTIVETo study the effects of different immunodepressants on the sperm parameters of kidney transplant recipients.

METHODSIn 15 healthy fertile men and 37 kidney transplant recipients, ejaculates were aseptically obtained by masturbation. Thirty-seven patients were divided into two groups, 20 patients were treated with Prograf (FK506) combination with mycophenolate mofetil (MMF) and prednisone; 17 patients were treated with cyclosporine (CsA) combination with azathioprine with prednisone. The sperm viability, mobility parameters such as prorsad percentage motility, straight line velocity (VSL), curve line velocity (VCL), velocity of average path (VAP) and morph were estimated with a computer-assisted sperm analyzer (CASA) provided with a multiple-exposure photography system.

RESULTSThere were no significant difference in sperm viability rate [(81.7 +/- 5.7)%, (79.4 +/- 6.8)% and (83.8 +/- 6.0)%], VCL [(24.1 +/- 8.6)%, (23.9 +/- 4.4)%, (24.8 +/- 4.2)% ] and VAP [(19.7 +/- 6.6)%, (18.6 +/- 2.9)%, (21.0 +/- 4.0)%] among groups of FK506, CsA and control, respectively (P > 0.05). The rate of anomaly [(67.8 +/- 5.7)%], the prorsad percentage motility [(46.4 +/- 8.1)%] and VSL [(15.4 +/- 4.6)%] in the group of FK506 were respectively significantly lower and higher than those in the group of CsA [(80.1 +/- 5.6%, (33.3 +/- 6.4)%, (10.2 +/- 2.4)%] (P < 0.05).

CONCLUSIONThe application of FK506 combined with MMF could help recover the mobility and morphology of the sperm in kidney transplantation recipients.

Adolescent ; Adult ; Case-Control Studies ; Cyclosporine ; pharmacology ; Drug Therapy, Combination ; Humans ; Immunosuppressive Agents ; pharmacology ; Kidney Transplantation ; Male ; Mycophenolic Acid ; analogs & derivatives ; pharmacology ; Prednisone ; pharmacology ; Sperm Motility ; drug effects ; Tacrolimus ; pharmacology

OBJECTIVETo investigate the effectiveness of pimecrolimus cream 1% for sensitive skin in adult women and its underlying mechanisms.

METHODSThe changes of subjective symptoms and signs were evaluated before and after the application of pimecrolimus cream 1% based on the severity of pruritus (SP) and severity of burning sensation (SB) scores, and on a basic syntax and molecular substrate (molecular psychophysics) of nociception and proprioception established by temperature-sensitive transient receptor potential (TRP) channels.

RESULTSThe SP and SB scores were significantly decreased in 32 patients with sensitive skin after using topical pimecrolimus cream 1% (P<0.05). Twenty (62.5%) patients showed positive capsaicin-like response (i.e. burning with consequent rapid amelioration of pruritus or burning sensation) and 6 (18.8%) showed positive camphor-like response (i.e. warming with consequent rapid amelioration of pruritus) on application sites after using the topical pimecrolimus cream 1%, and 6 (18.8%) showed negative capsaicin-like response and/or negative camphor-like response.

CONCLUSIONSPimecrolimus may rapidly inhibit or alleviate itch or burning sensation of patients with sensitive skin. The therapeutic effect of pimecrolimus is relevant to the mechanisms that activate or sensitize transient receptor potential vanilloid 1 (TRPV1) and desensitizes TRPV1 in the skin sensory afferents.

Adolescent ; Adult ; Dermatitis, Atopic ; drug therapy ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Pruritus ; drug therapy ; Skin ; drug effects ; TRPV Cation Channels ; metabolism ; Tacrolimus ; analogs & derivatives ; therapeutic use ; Young Adult

Adjuvants, Immunologic ; adverse effects ; therapeutic use ; Azathioprine ; adverse effects ; therapeutic use ; Cyclosporine ; adverse effects ; therapeutic use ; Glucocorticoids ; adverse effects ; therapeutic use ; Humans ; Mycophenolic Acid ; adverse effects ; analogs & derivatives ; therapeutic use ; Tacrolimus ; adverse effects ; therapeutic use

OBJECTIVEChronic graft versus host disease (cGVHD) is the most common late complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and it represents the major cause of mortality in long-term survivors. Over the past decade, although conventional therapy has achieved complete responses in approximately 50% of patients, the prophylaxis and treatment of cGVHD are still not satisfactory. In the late years, utilization of new immunosuppressant such as tacrolimus (FK506), mycophenolate mofetil (MMF) on cGVHD improved the curative effects. This study tried to analyze the results of combination of methylprednisolone (MP), MMF and FK506 or cyclosporine A (CSA) as immunosuppressive therapies for cGVHD and to explore the effective regimen for children.

METHODSForty-five patients received allo-HSCT. Among them 32 received UCBT and 13 received PBSCT. The conditional regimen mainly consisted of busalphan, cyclophosphamide, antihuman thymocyte globulin, fludarabin, melphalan, thiotepa and total lymph node irradiation. Prophylaxis of GVHD consisted of CSA, MP and MMF. Patients with cGVHD received a regimen with combination of MP, MMF and FK506 or CSA.

RESULTSSeventeen out of 32 patients who received UCBT were engrafted. while 9 out of 13 patients who received PBSCT were engrafted. Nine cases of the 30 engrafted patients developed cGVHD (morbidity 30%). Among the 17 patients who received UCBT, 3 developed cGVHD (18%). Among the 13 patients who received PBSCT, 6 developed cGVHD (46%). Six cGVHD continued from aGVHD (6/9). One patient was given CSA plus MMF, and 8 were given three-drug regimen with MP, MMF and FK506. The overall response rate was 100%. Two patients died of CMV-IP or septicemia (mortality 20%). Seven (78%) patients survived (event free survival, EFS) longer than 3 years. The side effects included hepatotoxicity, nephrotoxicity, hypertension, articular capsulitis and arrhythmia. The main complication and the major causes of death were infection.

CONCLUSIONThe incidence of cGVHD is low in children. The incidence of cGVHD after PBSCT is higher than that after UCBT. aGVHD is a highly dangerous factor. Combined therapy of MP plus MMF and FK506 or CSA is safe and effective for the treatment of cGVHD in children.

Child ; Child, Preschool ; Chronic Disease ; Drug Therapy, Combination ; Female ; Graft vs Host Disease ; drug therapy ; epidemiology ; prevention & control ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Incidence ; Male ; Methylprednisolone ; administration & dosage ; Mycophenolic Acid ; administration & dosage ; analogs & derivatives ; Tacrolimus ; administration & dosage