Biomedical subjects
Publications and source records attributed to M C Yeung.
Tolosa-Hunt syndrome is characterized by a dull, persistent pain around the affected eye, ophthalmoplegia and, sometimes, involvement of other cranial nerves passing through the cavernous sinus. Corticosteroid administration is valuable in the treatment and frequently has a dramatic effect. We report a boy with Tolosa-Hunt syndrome who fails to respond to the initial steroid treatment. The role of the MRI in the management of this condition is discussed.
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Persistent infections by viruses such as HIV-1 and hepatitis B virus can pose long-term health hazards. Because establishment of persistent infections involves close interactions and adjustments in both host and virus, it would be informative to establish a paradigm with which a normally cytolytic viral infection can be easily converted to persistent infection, so that the different stages in developing persistent infection can be examined. Such a model system is described in this paper. Highly cytolytic encephalomyocarditis virus (EMCV) infection was shifted to persistent infection as a result of repressed expression of the double-stranded RNA-dependent protein kinase (PKR) in the promonocytic U937 cells. Because of the apoptogenic potential of PKR, a deficiency of PKR resulted in a delay in virus-induced apoptosis in EMCV-infected U937 cells, allowing the eventual establishment of persistent EMCV infection in these cells (U9K-AV2). That this was a bona fide persistent infection was demonstrated by the ability of infected cells to propagate as long-term virus-shedding cultures; electron microscopy studies showing presence of intracellular EMCV virions and chromatin condensation; detection of virus-induced chromosomal DNA fragmentation and sustained expression of apoptogenic p53 and IL-1beta converting enzyme; and demonstration of active EMCV transcription by reverse transcription-PCR. In addition, a host-virus coevolution was observed in U9K-AV2 cultures over time: U9K-AV2 cells exhibited slower growth rates, resistance to viral super-infection, and cessation of IFN-alpha synthesis, whereas the infectivity of EMCV was drastically attenuated. Finally, data are presented on the suitability of this model to study establishment of persistent infection by other viruses such as Sendai virus and reovirus.
Table bombardment by automated biopsy needle is an occasional technical problem during stereotactic core biopsy of the breast. It is most commonly encountered in small breasts and when lesions are close to the biopsy table. A reference chart is devised for stereotactic core biopsy using an add-on erect stereotactic biopsy table and automated spring-loaded biopsy gun. The chart serves as a reference for pre-biopsy assessment of the feasibility of core biopsy in the vertical approach due to the constraints of lesion depth and breast thickness. It allows easy and quick reference for the required needle length input for the stereotactic system to prevent table bombardment.
Despite what is known about the early signaling events in tumor necrosis factor (TNF) alpha-induced apoptosis, characterization of the downstream events remains largely undefined. It is now known that a cross-talk exists between the interferon and TNF-alpha pathways. This linkage allows recruitment of the cell proliferation suppressor PKR (dsRNA-dependent protein kinase) from the interferon pathway to play a pivotal role in TNF-alpha-induced apoptosis. In this study, we took advantage of the differential TNF-alpha susceptibilities of human promonocytic U937 subclones, deficient in or overexpressing PKR, to further characterize the role of PKR in apoptosis. By reverse transcription-polymerase chain reaction, we demonstrated that TNF-alpha transiently induces the tumor suppressor p53 in U937 cells. This p53 induction lags behind the TNF-alpha induction of PKR by 1 h. By cell viability determination, ultrastructural studies, apoptotic DNA laddering, and antisense techniques, it was shown that inhibition of p53 expression in PKR-overexpressing U937 cells abrogates the TNF-alpha-induced apoptosis in these cells. Conversely, overexpressing wild type p53 in PKR-deficient U937 cells confers the susceptibility of these cells to TNF-alpha-induced apoptosis. This latter result indicates that p53 induction is an event downstream of TNF-alpha-induced up-regulation of PKR, thereby further establishing the critical role of p53 in TNF-alpha-induced apoptosis in U937 cells. PKR-overexpressing U937 cells were found to possess a constitutively higher level of p53, which partly explains why these cells spontaneously undergo apoptosis even without TNF-alpha treatment. Finally, a model is presented on the interplay between PKR and p53 in effecting TNF-alpha-induced apoptosis in U937 cells.
OBJECTIVES: This study examines the cytotoxicity potential and the mechanism of toxicity of the HIV-1 gp120 on human neuroblastoma cells. DESIGN: Previous data from our group have suggested that the HIV-1 envelope protein gp120 promotes the secretion of tumor necrosis factor-alpha and other factors by astrocytes and microglial cells present in primary human brain cell cultures, thereby contributing to the injury of neurons in these cultures. This study investigates the cytotoxicity potential and the mechanism of toxicity of gp120 on human neuroblastoma cells. METHODS: SK-N-SH cells were treated with HIV-1 gp120, and was followed by in situ DNA fragmentation staining and small molecular weight DNA extraction studies to ascertain the induction of apoptosis by gp120 in these cells. To evaluate a potential role of the growth suppressor gene p53, gp120-treated SK-N-SH cells were subjected to reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses for the induction of p53. An antisense oligodeoxynucleotide against p53 was used to investigate the role of p53 in the gp120-induced apoptosis in these cells. RESULTS: Data from T7 DNA polymerase staining and small molecular weight DNA extraction studies demonstrated that gp120-induced DNA breakage in SK-N-SH cells with fragmentation patterns characteristic of apoptosis. RT-PCR and Western blot analyses revealed that the gp120-mediated induction of apoptosis was dependent on a gp120-induced and gp120-sustained upregulation of p53. The induction of p53 by gp120 was specific, since an antibody against gp120 prevented both the induction of p53 and subsequent apoptosis in SK-N-SH cells. The critical role of p53 was further illustrated by the effectiveness of a p53 antisense oligodeoxynucleotide to inhibit the gp120-induced apoptosis. As a control, the apoptosis-inducing potential of gp120 on SK-N-SH cells was not seen in the HIV-1 Gag proteins even when used at up to 5 nM. CONCLUSIONS: These results established that HIV-1 gp120 is potentially cytotoxic to human neuronal cells through the induction of p53, which may eventually lead to induction of apoptosis.
Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.
Recent advances in basic science and clinical trials have demonstrated that IFNs and myeloid hematopoietins play crucial roles in host defense against pathogens and immune surveillance. Here we have reviewed the biologic functions of GM-CSF, G-CSF, IFN-alpha and IFN-gamma. For patients with neutropenia resulting from cytotoxic chemotherapy, bone marrow transplantation, congenital agranulocytosis and cyclic neutropenia, therapeutic uses of GM-CSF and G-CSF were reviewed. Application of these growth factors to patient management represents a major contribution of biotechnology to a difficult area of therapeutics in febrile, neutropenic patients. Because IFN-alpha plays crucial roles in antiviral responses, its clinical applications in hepatitis B and C, human papilloma virus, HIV infection and malignancy were discussed. The use of IFN-gamma in bacterial prophylaxis in patients with chronic granulomatous disease was also presented. Advances in clinical applications of IFNs and hematopoietic growth factors serve as a paradigm for further development to investigate the use of other important cytokines in modern therapeutics.
Severe viral infection in newborns has been attributed to immaturity of the immune system including a defect in natural killer cytotoxicity (NKC) and decreased production of cytokines that are important for natural killer (NK) function. We investigated the induction of interferon (IFN)-gamma and activation of NK activity in adult and cord blood mononuclear cells (BMC) after IL-12 treatment. The levels of mRNA in these BMC were measured by Northern blot and reverse transcription-polymerase chain reactions using primers specific for IFN-gamma. The levels of IFN-gamma protein were measured by ELISA. In the absence of IL-12, only adult BMC spontaneously produced low levels of IFN-gamma. After IL-12 treatment, induction of IFN-gamma expression was detected as early as 4 h in both cord and adult BMC. Both cord and adult cells showed similar levels of IFN-gamma mRNA and protein expression in response to IL-12 at a concentration as low as 10 U/mL. In contrast, upon phorbol ester and ionomycin treatment, adult BMC produced more IFN-gamma mRNA than cord BMC. In a 51Cr release assay with human immunodeficiency-infected H9 cells as indicators, both cord and adult cells responded to IL-12 induction of NKC. Our findings demonstrate that cord BMC are capable of responding to IL-12 stimulation, competent in synthesizing IFN-gamma, and able to mount NKC. Thus, it appears that the deficiency in IFN-gamma production or NKC in cord cells is not due to an inherent defect in IL-12 response of the cord cells.
OBJECTIVE: To investigate the induction of cytokines as a possible mechanism for the neurotoxicity of the HIV-1 envelope protein gp120. DESIGN: The gp120 protein was tested directly on primary human brain cultures to examine its ability to induce cytokines and its neurotoxicity on human neural cells because gp120 is known to be toxic to rodent ganglion cultures, and neural cells such as astrocytes and microglia produce cytokines when stimulated. METHODS: Primary cultures of human brain cell aggregates, astrocytes and macrophages were exposed to HIV-1 recombinant (r) gp120SF2. Induction of cytokines was assayed by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR); neurotoxicity of rgp120SF2 and interleukin (IL)-6 on human brain cultures was examined by electron microscopy. RESULTS: ELISA and RT-PCR studies revealed that rgp120SF2 induced IL-6 and tumor necrosis factor (TNF)-alpha in brain cultures; IL-6 could also be induced by TNF-alpha added to brain cultures. Both IL-6 and TNF-alpha were upregulated in astrocytes and macrophage cultures on rgp120SF2 treatment. Ultrastructural studies demonstrated that IL-6 treatment for 72 h induced large cytoplasmic vacuoles in neural cells with morphology consistent with neurons; rgp120SF2 treatment for 7 days resulted in chromatin condensation along the inner margins of nuclear envelopes of neural cells. CONCLUSIONS: Our results demonstrated that HIV-1 rgp120SF2 can upregulate at least two known neurotoxic cytokines, IL-6 and TNF-alpha, which may injure neural cells and contribute to the neuropathology observed in AIDS dementia patients.
This report highlights the clinical features of two patients who presented with severe neuropathic chest wall pain caused by herniated C6-7 disc, and speculates on the pathophysiology of this syndrome. Worsening of symptoms with neck movement helped localize the process as cervical spine rather than plexus in origin. Both patients had herniated C6-7 disc material compressing the spinal cord and C7 nerve root, and neurological symptoms resolved promptly following surgery. Neuropathic chest wall pain should alert the clinician to consider the diagnosis of cervical disc herniation and prompt definitive imaging of the cervical spine by myelography or magnetic resonance imaging (MRI).
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Mice carrying an H-2K-v-jun transgene develop malignant sarcomas by a multistage mechanism following wounding. Here we show that these malignancies are often heterogeneous in composition, containing both undifferentiated mesenchymal cells as well as focal areas of skeletal muscle. Such myogenic areas are not detectable in premalignant precursor lesions, suggesting that cells competent for muscle differentiation arise at a late stage of tumorigenesis. Immunohistochemical staining of transgenic sarcomas reveals that levels of v-Jun correlate inversely with muscle-specific gene expression, suggesting that high levels may be inhibitory to myogenesis. Consistent with this idea, we demonstrate that whereas high levels of v-Jun are able to block MyoD-dependent gene expression in vitro, the levels of v-Jun in sarcoma-derived myogenic cells are below the threshold required to produce this effect. The cell of origin of v-jun wound sarcomas, as well as the relationship between myogenic determination and multistage tumorigenesis, are discussed in the light of these results.
We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.
Purified native sigma 1 proteins from [35S]methionine-labeled reovirions [serotypes 1 (T1) and 3 (T3)] were subjected to limited trypsin and chymotrypsin digestion. It was found that T1 sigma 1 was resistant to both trypsin and chymotrypsin, whereas T3 sigma 1 (49K molecular weight) was cleaved by trypsin to yield a 24K and a 25K fragment, and by chymotrypsin to yield a 42K fragment. The 24K tryptic fragment, but not the 25K tryptic fragment, was shown to possess L-cell binding capacity, and represents the carboxy-terminal half of T3 sigma 1 since it contains the single cysteine residue (amino acid 351) as revealed by tryptic analysis of [35S]cysteine-labeled sigma 1. Neither tryptic fragment was able to bind to glycophorin, the reovirus receptor on human erythrocytes. Thus, the mechanism of reovirus host cell attachment is distinct from that of reovirus hemagglutination. The two tryptic fragments were recognized by different neutralizing monoclonal anti-sigma 1 antibodies, indicating that neutralizing and cell attachment sites are not necessarily equivalent. The 42K chymotryptic fragment of T3 sigma 1 was shown to be generated by a cleavage proximal to the carboxy-terminus. Like intact T3 sigma 1, the 42K protein retained its capacity to bind to both L cells and glycophorin, and was recognized by all the neutralizing monoclonal anti-sigma 1 antibodies tested. Thus, the host cell receptor binding site on T3 sigma 1 is located between the trypsin-sensitive and the chymotrypsin-sensitive sites.
An evaluation of 6-[2-(di-n-propylamino)ethyl]indole (4), its rigid analogue N,N-di-n-propyl-5,6,7,8-tetrahydrobenz[f]indol-7-amine (5), and some related congeners, for ability to suppress serum prolactin in reserpinized rats, revealed modest biological activity in this in vivo model of dopaminergic activity. Although the indole N-H in these compounds can be considered to be oriented "meta" with respect to the ethylamine side chain, compounds with the indole N-H located in the other "meta" position (i.e. 4-[2-(di-n-propylamino)ethyl]indole (2) or its rigid benz[e]indole analogue 3) were much more potent dopamine agonists. The results argue for a particular orientation of the indole N-H vector. In addition, relatively potent dopamine agonists also resulted when the pyrrole portion of the indole ring was replaced by a methanesulfonamido function, supporting the idea that the indole N-H serves as a hydrogen-bond donor.
It has previously been shown that of all the soluble reovirus-specified proteins present in the infected cell lysate, protein sigma 1 alone possesses the capacity to bind to host cells (P.W.K. Lee, E.C. Hayes, and W.K. Joklik, 1981, Virology 108, 156-163). We found that sigma 1 from urea-disrupted reovirus particles was also capable of such specific binding. Reovirions were therefore used as a source of functional sigma 1. Accordingly, a simple procedure has been developed to purify sigma 1 by subjecting urea-disrupted reovirions to DEAE ion-exchange chromatography. Protein sigma 1 thus isolated was electrophoretically homogeneous and the recovery was estimated to be 50 to 60% of the theoretical yield. The purified protein presumably maintained its native conformation since it was recognized by a panel of monoclonal anti-sigma 1 antibodies previously isolated, and was capable of specifically binding to host cell receptors, agglutinating human erythrocytes and inducing neutralization and hemagglutination-inhibition antibodies. Subsequent chemical crosslinking studies revealed the presence of oligomeric (mostly dimeric) sigma 1 forms in the preparation. The amino acid composition of the purified sigma 1 was found to closely match that inferred from the S1 gene sequence. However, attempts to determine its amino-terminal sequence have not been successful. The p/ of the purified protein was determined to be 6.8. Circular dichroic measurements of the purified sigma 1 indicated that 54 and 19% of its residues were arranged in alpha-helical and beta-sheet secondary structures, respectively.
A series of three isomeric 2,4,5-substituted monoethoxy dimethoxy phenylisopropylamines were compared for their contractile effect in the rat fundus as potential antagonists to the effect of serotonin in the fundus. The three isomers were also evaluated for their discriminative stimulus properties in rats that had been trained to discriminate injections of saline from LSD tartrate (0.08 mg/kg). The drug discrimination studies revealed that the 2,5-dimethoxy-4-ethoxy substitution was most potent in rats, consistent with the reported clinical activity of this isomer in man. By contrast, of the three isomers examined, this was the weakest in eliciting a contraction in the fundus. None of the compounds antagonized serotonin induced contractions, and it was not possible to determine pA2 values. Questions are raised about the determination of pA2 values for partial agonists and it is concluded that the fundus is not a reliable model for prediction of hallucinogenic activity of phenethylamines.