Biomedical subjects
Publications and source records attributed to S H Chen.
The syntheses and preliminary investigation of antifungal activities of two dehydro PSB derivatives 8 and 10 as well as one 3-imido-9-dechloro PSB analogue 13 are described.
Severe infantile Gaucher disease associated with ichthyosis and neonatal death is a rare subgroup of Type II Gaucher disease. This group of infants has little, if any, detectable beta-glucocerebrosidase activity, and prior genetic analyses have been limited in detecting the mutations responsible for this phenotype. We document an Hispanic infant succumbing with arthrogryposis and collodion membrane covering the skin who had no detectable beta-glucocerebrosidase activity in tissue samples and who was homozygous for a rare recombinant allele, RecNciI. Microscopic evaluation demonstrated accumulation of Gaucher cells in visceral organs and extensive loss of neurons in the anterior horns, brainstem, and cortex of the nervous system. The apoptosis of neuronal cells from the anterior horns and brainstem are a reasonable explanation for the arthrogryposis and neonatal death, respectively.
In vivo microdialysis coupled with liquid chromatography was developed for the continuous monitoring of brain neurochemicals during cerebral ischemia in awake, free moving gerbils. The dead volume of the microdialysis system was estimated to be less than 30 microl. The detection limits of the present assay were 0.2 to 2.0 microM for analytes at a signal to noise ratio of five. To validate this assay, a focal cerebral ischemia was produced by occlusion of one common carotid artery for 60 min and then reperfusion for additional 3 h in awake gerbils. A microdialysis probe was inserted into the striatum of the gerbil. Dialysates were autoinjected and analyzed extracellular pyruvate, lactate, and ascorbic acid by liquid chromatography with a UV detector during cerebral ischemia. Significant changes of pyruvate and the lactate/pyruvate ratio were observed. Biphasic and dynamic changes in ascorbic acid and lactate were proposed to correlate a secondary damage. This assay can be used as a tool to study dynamic changes of brain neurochemicals in awake animals.
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Microfluidic devices were fabricated on poly(methyl methacrylate) (PMMA) substrate using two small-diameter (79 microm) wires to create a cross impression in plastics softened by low-temperature heating. The resulting channels had a rounded shape and 75 microm in depth. The variability of the channel dimensions was found to be less than 6% from different locations of the same channel and less than 10% between chips. Moreover, the fabricated PMMA chip appeared to sustain an electric field strength up to 300 V/cm without significant Joule heating. The function of resulting devices for electrophoretic injection and separation of a DNA size marker, HaeIII digest of (phiX174, was also characterized. Results indicated that all of the 11 DNA fragments of the size marker could be identified in less than 3 min with relative standard deviations less than 0.4% and 8% for migration time and peak area, respectively. Moreover, with the use of near infrared (IR) dye, fluorescence signals of the higher molecular weight fragments (> 603 bp in length) could be detected at total DNA concentrations as low as 0.1 microg/mL (S/N = 4.2). In conclusion, the performance of wire-imprinted devices on PMMA substrate were comparable to those fabricated by other professional means.
The chelating and antioxidant effects of pyrrolidine dithiocarbamate (PDTC) have been investigated extensively for preventing cell death induced by different insults. However, the toxic effects of PDTC have been studied only recently and fewer studies on the toxic effects on astrocytes have been reported. In our study, we demonstrated that both PDTC and Cu(2+) alone were rated as only weakly toxic in inducing cell death in cortical astrocytes with IC(50) of 300 microM and 180 microM, respectively. However, PDTC and Cu(2+) in the complex form markedly potentiated with each other by about 1,000-fold with IC(50) of 0.3 microM PDTC plus 10 microM Cu(2+). Other metals at concentrations of 3-10 microM (VO(4)(5+), Cr(6+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Zn(2+), Pb(2+), Bi(2+), Ba(2+), UO(2+), Cs(+), SeO(4)(2-), La(3+)) had no such potentiating effects on PDTC. Changes in morphology (nuclear condensation), apoptotic body formation, and hypodiploidity of DNA suggested that the PDTC-Cu(2+) complex induced cell death through an apoptotic process. Further studies showed that the PDTC-Cu(2+) complex decreased mitochondrial membrane potential, increased hydrogen peroxide production, and depleted GSH contents. After the increased oxidative stress, PDTC-Cu(2+) complex differentially activated JNKs, ERK, p38 and caspase 3, which caused PARP degradation in a time-dependent manner. All these effects were consistent with the increased cellular Cu contents. The nonpermeable copper-specific chelator bathocuproine disulfonate (BCPS), but not the permeable Cu(2+) chelator neocuproine, abolished all the observed effects. Antioxidants (N-acetylcysteine [NAC], vitamin C), catalase, and Cu(2+)-binding proteins (albumin, hemoglobin, and higher serum) reduced the cytotoxic effects of PDTC-Cu(2+) complex. We concluded that the death signaling pathway of PDTC-Cu(2+) complex was mediated by oxidative stress and subsequent JNK activation. These findings imply that PDTC, a widely used pesticide and medicine that is capable of penetrating the blood-brain barrier, may cause neurotoxicity through astrocyte dysfunction.
Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 x 10(9), 2 x 10(10), 2 x 10(11), or 2 x 10(12) vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses <==2 x 10(11) VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 x 10(12) VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.
In an orthotopic model of metastatic colon carcinoma established in the liver of mice, we have previously shown that the natural killer (NK) cells were the major effectors after intratumoral delivery of a recombinant adenovirus expressing the murine IL-12 gene. However, tumor cure and long-term survival were achieved only in a minority of animals. In the present study, we generated an effective antitumoral CD8(+ ) T-cell response by the combination of IL-12 gene therapy and systemic delivery of an agonistic monoclonal antibody against 4-1BB, a costimulatory molecule expressed on activated T cells. In the IL-12 plus anti-4-1BB combination treatment, the effective dose of IL-12 could even be reduced even up to 18-fold and still achieved a better efficacy than the maximal dose of either treatment alone. We further demonstrate that the innate and the adaptive antitumoral immune responses were synergistic, as animals bearing hepatic as well as multiple pulmonary metastases were quantitatively cured of their diseases after IL-12 gene therapy + anti-4-1BB combination treatment. Both NK and CD8(+) T cells were necessary in maintaining the long-term antitumor immunity, as depletion of either cell type in the cured animals abolished their abilities to reject tumor cells implanted at distal sites. These results indicate that synergism between innate and adaptive immune responses may be effectively exploited to treat patients with metastatic diseases.
This paper summarizes data from a comprehensive workplace exposure monitoring program for refractory ceramic fiber (RCF) conducted since 1990, including data collected under a 5-year consent agreement (1993-1998) between the U.S. Environmental Protection Agency and the Refractory Ceramic Fibers Coalition. It presents additional data and analyses, which complement an earlier article published in this journal (L. D. Maxim et al., 1997, Regul. Toxicol. Pharmacol. 26, 156-171). As part of this exposure monitoring program, data were collected at plants operated by RCF manufacturers and at customer facilities. Beginning in 1993, at least 720 samples (each sample consisting of one or more cassettes) were collected annually, distributed according to a stratified random sampling plan. The strata consisted of workers in eight functional job categories (FJCs) at manufacturing and customer plants. Time-weighted average (TWA) and task length average data were gathered and analyzed using phase contrast optical microscopy and (for a subset of samples) transmission electron microscopy methods. Data on respirator usage (by type) were also collected. Statistical analysis indicates that there are significant differences in TWA fiber concentrations (exposure) among FJCs (installation, finishing, and removal categories have the highest average concentrations), that workplace concentration data are approximately lognormally distributed, and that weighted average fiber concentrations decreased over the period from 1990 to 1998-although trend curves leveled out as exposures have decreased and further improvements became more difficult. Material differences exist in exposure between RCF manufacturers and their customers (largely because the mix of jobs differ), but these differences have narrowed over the years. Respirator usage varies with exposure. Respirator data are used to derive improved estimates of actual worker exposure. Alternative criteria for selecting FJCs for control efforts are defined and illustrated. Lessons learned for future monitoring efforts are summarized.
In completed and ongoing clinical trials, adenovirus-mediated (Ad.) expression of herpes-simplex-virus thymidine-kinase (HSV-tk) gene transduction followed by ganciclovir (GCV) therapy has produced limited toxicity and evidence of antitumor activity following injection of the prostate. Furthermore, this system has been shown to direct systemic antitumor activity in several experimental cancer models, including that of prostate cancer, which may serve as the basis for in-situ immunomodulatory gene therapy. In a mouse model of prostate cancer, natural killer (NK) cells have been identified as the mediator of antimetastatic activity following Ad.HSV-tk + GCV, resulting in the combination of Ad.HSV-tk and adenovirus-mediated expression of interleukin 12 (Ad.IL-12) to exploit this cytokine's ability to enhance NK proliferation and cytotoxicity. Combination therapy demonstrated superior local and systemic growth suppression over that obtained with either therapy alone. Importantly, when the metastatic tumor burden was increased to an extent that negated the growth-suppressive activity directed by Ad.HSV-tk + GCV or Ad.IL-12 alone, combination therapy continued to demonstrate significant growth suppression. Examination of tumor-infiltrating lymphocytes documented enhanced NK lytic activity following combination therapy. Therefore, it appears that the combination of Ad.HSV-tk and Ad.IL-12 should be validated in a clinical trial for the treatment of prostate cancer.
OBJECTIVES: This study was conducted to test hypotheses stating that: 1) altered signaling for mitochondrial membrane proteins occurs during postinfarction remodeling, and 2) successful myocardial adaptation relates to promotion of specific mitochondrial membrane components. BACKGROUND: Abnormalities in high-energy phosphate content and limitations in adenosine 5'-triphosphate (ATP) synthesis rate occur during the transition to contractile failure from compensatory remodeling after left ventricular infarction. The adenine nucleotide translocator (ANT) and F1-ATPase respectively regulate mitochondrial adenosine 5'-diphosphate (ADP)/ATP exchange and ADP-phosphorylation, which are key components of high-energy phosphate metabolism. METHODS: Steady-state mRNA and protein expression for ANT isoform1 and the beta subunit of the F1-ATPase (betaF1) were analyzed in myocardium remote from the infarction zone eight weeks after left circumflex coronary artery ligation in pigs, demonstrating either successful left ventricular remodeling (LVR, n = 8) or congestive heart failure (CHF, n = 4) as determined by clinical and contractile performance parameters. RESULTS: Substantial reductions in steady-state mRNA expression for ANT1 and betaF1 relative to normal (n = 8) occur in CHF, p < 0.01, but not in LVR. Relative expression for both proteins coordinated with their respective steady-state mRNA levels; CHF at 40% normal, p < 0.05 for ANT and 70% normal for betaF1, p < 0.05. CONCLUSIONS: Maintained signaling for major mitochondrial membrane proteins occurs in association with successful remodeling and adaptation after infarction. Reduced expression of these proteins relates to limited ATP synthesis capacity and high energy phosphate kinetic abnormalities previously demonstrated in CHF. These findings imply that mitochondrial processes participate in myocardial remodeling after infarction.
An intrahepatic tumor model for metastatic breast cancer was generated in syngeneic mice by direct inoculation of JC cells, a murine mammary adenocarcinoma cell line. Intratumoral administration of a recombinant adenoviral vector expressing the murine Interleukin-12 (ADV-mIL-12) resulted in significant reduction in the tumor volume compared to control vector. Tumor regression was also evident on histopathologic analysis of the liver, where inflammatory changes as opposed to nuclear atypia predominated after IL-12 vector treatment. There was a significant prolongation in the long term survival of IL-12 treated animals, with complete tumor rejection in 40% of the animals. In vivo depletion studies using specific monoclonal antibodies against the various lymphocyte subsets showed a significant reduction in long term survival after natural killer (NK) cell depletion. This suggests that the NK cell is a critical effector in the antitumor effect mediated by IL-12. These results strongly support the potential role of gene mediated cytokine therapy for the treatment of metastatic breast cancer.
Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.
Psoriasis is a T cell-mediated inflammatory disease characterized by hyperproliferation and by aberrant differentiation. We found cathepsin D and zinc-alpha(2)-glycoprotein, two catalytic enzymes associated with apoptosis and desquamation, to be present in the stratum corneum of the normal epidermis but absent from the psoriatic plaque. Psoriasis is characterized by an altered response to interferon-gamma (IFN-gamma), including the induction of apoptosis in normal but not in psoriatic keratinocytes, often with opposite effects on gene expression of suprabasal proteins. We found that IFN-gamma binding and signaling were attenuated in psoriasis: The IFN-gamma receptor, the signal transducer and activator of transcription STAT-1, and the interferon regulatory factor IRF-1 were strongly up-regulated by IFN-gamma in normal keratinocytes, but not in psoriatic ones. IFN-gamma strongly up-regulated the expression of the catalytic enzymes cathepsin D and zinc-alpha(2)-glycoprotein in normal keratinocytes but down-regulated them in psoriatic ones; the reverse was true of the apoptotic suppressor bcl-2. We believe that the aberrant response to IFN-gamma plays a central role in the pathophysiology of psoriasis, particularly the disruption of apoptosis and desquamation.
The literature includes no studies on the use of video-assisted thoracoscopic surgery in the management of tuberculous spondylitis, and its role in the management of tuberculosis involving the thoracic spine remains unclear. The authors experience with 10 consecutive patients (six women, four men) who underwent video-assisted thoracoscopic surgery for the treatment of spinal tuberculosis involving levels from T5 to T11, from January 1996 to December 1997, was analyzed. Using the extended manipulating channel method (2.5-3.5 cm portal incisions), video-assisted thoracoscopic surgery was performed with a three-portal technique (seven patients) or a modified two-portal minithoracotomy technique that required a small incision for the thoracoscope and a larger incision, measuring 5 to 6 cm, for the procedures in three patients. All the patients were studied prospectively. The followup ranged from 17 to 42 months (mean, 24 months). Postoperative complications included one lung atelectasis. Pleural adhesions, owing to local inflammation or paravertebral abscess, were seen in four patients and one patient with severe pleurodesis needed an open technique for treatment. Postoperative air leaks were seen in four (40%) of 10 patients but all were transient. The average neurologic recovery was 1.1 grades on the Frankel's scale. The data from this series of patients with tuberculous spondylitis show that video-assisted thoracoscopic surgery has diagnostic and therapeutic roles in the management of tuberculous spondylitis. Technically, a combination of thoracoscopy and conventional spinal instruments to perform video-assisted thoracoscopic surgery through the extended manipulating channels, which were placed slightly more posterior than usual, was effective and safe.
A method is proposed to determine the spectral function of the clipped-random-wave (CRW) model directly from scattering data. The spectral function f(k) (k is a wave number) gives the distribution of the magnitude of wave vectors of the sinusoidal waves that describes the essential features of the two-phase morphology. The proposed method involves "inverse clipping" of a correlation function to obtain f(k) and does not require any a priori assumptions for f(k). A critical test of the applicability of the inverse-clipping method was carried out by using three-component bicontinuous microemulsions. The method was then used to determine f(k) of the bicontinuous structure of a phase-separating polymer blend. f(k) for the polymer blend turned out to be a multipeaked function, while f(k) for the microemulsions exhibits a single broad maximum representing periodicity of the morphology. These results indicate the presence of the long-range regularity in the morphology of the polymer blend. Three-dimensional (3D) morphology corresponding to the scattering data of the polymer blend was generated using the CRW model together with the multipeaked f(k). Interface curvatures of the 3D morphology calculated from f(k) were measured and compared with those experimentally determined directly from the laser scanning confocal microscopy in the same blend.
X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.
A novel L-nucleoside analog of deoxycytidine, 2',3'-dideoxy-2', 3'-didehydro-beta-L-5-fluorocytidine (beta-L-Fd4C), was recently shown to strongly inhibit hepatitis B virus (HBV) replication in the 2.2.15 cell line. Therefore, its antiviral activity was evaluated in the duck HBV (DHBV) infection model. Using a cell-free system for the expression of the DHBV polymerase, beta-L-Fd4C-TP exhibited a concentration-dependent inhibition of dCTP incorporation into viral minus-strand DNA with a 50% inhibitory concentration of 0.2 microM which was lower than that of other tested deoxycytidine analogs, i.e. , lamivudine-TP, ddC-TP, and beta-L-FddC-TP. Further analysis showed that beta-L-Fd4C-TP is likely to be a competitive inhibitor of dCTP incorporation and to cause premature DNA chain termination. In primary duck hepatocyte cultures infected in vitro, beta-L-Fd4C administration exhibited a long-lasting inhibitory effect on viral DNA synthesis but could not clear viral covalently closed circular DNA (CCC DNA). Results of short-term antiviral treatment in experimentally infected ducklings showed that beta-L-Fd4C exhibited the most potent antiviral effect, followed by beta-L-FddC, lamivudine, and ddC. Longer administration of beta-L-Fd4C induced a sustained suppression of viremia (>95% of controls) and of viral DNA synthesis within the liver. However, the persistence of trace amounts of viral CCC DNA detected only by PCR was associated with a recurrence of viral replication after drug withdrawal. In parallel, beta-L-Fd4C treatment suppressed viral antigen expression within the liver and decreased intrahepatic inflammation and was not associated with any sign of toxicity. Our data, therefore, demonstrate that in the duck model of HBV infection, beta-L-Fd4C is a potent inhibitor of DHBV reverse transcriptase activity in vitro and suppresses viral replication in the liver in vivo.