Acetyl-l-carnitine and nucleoside reverse transcriptase inhibitor-associated neuropathy in HIV infection.

OBJECTIVES: Antiretroviral toxic neuropathy (ATN) is associated with dideoxynucleoside reverse transcriptase inhibitor use in patients infected with HIV, possibly as a result of mitochondrial toxicity. Acetyl-l-carnitine (ALC) has been linked to symptomatic improvement in ATN. We present an open-label single-arm pilot study to evaluate changes in intra-epidermal nerve fibre (IENF) density and mitochondrial DNA (mtDNA) copies/cell among subjects treated with 3000 mg ALC daily. METHODS: Punch skin biopsies were examined at baseline and after 24 weeks of therapy. Participants reported neuropathic symptoms using the Gracely Pain Intensity Score. Neurological examinations were completed. RESULTS: Twenty-one subjects completed the study. ALC was generally well tolerated. The IENF density did not change in cases completing 24 weeks of ALC therapy, with median (90% confidence interval) IENF changes of -1.70 (-3.50, infinity) (P=0.98) and 2.15 (-0.10, infinity) (P=0.11) for the distal leg and proximal thigh, respectively. Fat mtDNA copies/cell did not change with therapy. Improvements in neuropathic pain (P<0.01), paresthesias (P=0.01), and symptoms of numbness (P<0.01) were noted. Similarly, improvement was noted on the Gracely Pain Intensity Score. CONCLUSIONS: ALC therapy coincided with improvements in subjective measures of pain in this open-label single-arm study. However, changes were not observed in objective measures of IENF density or mtDNA levels, providing little objective support for use of ALC in this setting.

Peptide mimicrying between SARS coronavirus spike protein and human proteins reacts with SARS patient serum.

Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS) is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927-937) and D08 (residues 942-951) were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490-502) stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

The potential of soybean foods as a chemoprevention approach for human urinary tract cancer.

Isoflavones are excreted in human urine and can be modulated by soy-rich diets. Recently, isoflavones were suggested to have protective effects against bladder cancer cells. We sought to determine the efficacy of the antitumorigenic effects of isoflavones at concentrations found in the range of human urine excretion and compare normal urothelium and bladder cancer cells for differential cytotoxicity. A total of seven human bladder cancer cell lines and an immortalized uroepithelial cell line were used to examine the effects of genistein, daidzein, and biochanin-A, either individually or as an equal-proportion mixture regimen, on cell growth, DNA synthesis, alterations of cell cycle distribution, and induction of apoptosis. The role of cyclin B1 and cdc2 kinase in cell cycle arrest was analyzed. In addition, severe combined immunodeficient mice were used to confirm the anti-cancer effects of isoflavones in vivo. Cooperative action of isoflavones was more effective in growth inhibition and apoptosis induction than any single compound. Genistein tends to cause a dose-dependent induction of G2-M cell cycle arrest and an inhibition of cdc2 kinase activity. However, both daidzein and biochanin-A directly induced apoptosis without altering cell cycle distribution. The IC50 values in non-transformed cells were higher than those in most cancer cell lines, and the IC50 of the mixture regimen was within reach of the levels observed in urine after a soy challenge. Furthermore, both genistein and combined isoflavones exhibited a significant tumor suppressor effect in vivo (P < 0.05). The results justify the potential use of soybean foods as a practical chemoprevention approach for patients with urinary tract cancer.

Evaluating the effectiveness of implementing quality management practices in the medical industry.

OBJECTIVE: To discuss the effectiveness of 30 quality management practices (QMP) including Strategic Management, Balanced ScoreCard, Knowledge Management, and Total Quality Management in the medical industry. DESIGN: A V-shaped performance evaluation matrix is applied to identify the top ten practices that are important but not easy to use or implement. Quality Function Deployment (QFD) is then utilized to find key factors to improve the implementation of the top ten tools. SETTING AND PARTICIPANTS: The questionnaires were sent to the nursing staff and administrators in a hospital through e-mail and posts. A total of 250 copies were distributed and 217 copies were valid. MEASUREMENTS: The importance, easiness, and achievement (i.e., implementation level) of 30 quality management practices were used. RESULTS: Key factors for QMP implementation were sequenced in order of importance as top management involvement, inter-department communication and coordination, teamwork, hospital-wide participation, education and training, consultant professionalism, continuous internal auditing, computerized process, and incentive compensation. CONCLUSIONS: Top management can implement the V-shaped performance matrix to determine whether quality management practices need improvement and if so, utilize QFD to find the key factors for improvement.

Macrophage migration inhibitory factor has a permissive role in concanavalin A-induced cell death of human hepatoma cells through autophagy.

Concanavalin A (ConA) is a lectin and T-cell mitogen that can activate immune responses. In recent times, ConA-induced cell death of hepatoma cells through autophagy has been reported and its therapeutic effect was confirmed in a murine in situ hepatoma model. However, the molecular mechanism of ConA-induced autophagy is still unclear. As macrophage migration inhibitory factor (MIF), which is a proinflammatory cytokine, can trigger autophagy in human hepatoma cells, the possible involvement of MIF in ConA-induced autophagy was investigated in this study. We demonstrated that cell death is followed by an increment in MIF expression and secretion in the ConA-stimulated human hepatoma cell lines, HuH-7 and Hep G2. In addition, ConA-induced autophagy and cell death of hepatoma cells were blocked in the presence of an MIF inhibitor. Knockdown of endogenous MIF by small hairpin RNA confirmed that MIF is required for both ConA-induced autophagy and death of hepatoma cells. Furthermore, signal pathway studies demonstrated that ConA induces signal transducer and activator of transcription 3 (STAT3) phosphorylation to trigger MIF upregulation, which in turn promotes Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)-dependent autophagy. By using a murine in situ hepatoma model, we further demonstrated that MIF contributes to anti-hepatoma activity of ConA by regulating STAT3-MIF-BNIP3-dependent autophagy. In summary, our findings uncover a novel role of MIF in lectin-mediated anti-hepatoma activities by regulating autophagy.

Antibody-mediated endothelial cell damage via nitric oxide.

Vascular disorders, resulting from endothelial cell dysfunction, may be caused by various stimuli, including infectious pathogens, cytotoxic reagents, and pathophysiological mechanisms mediated by immune responses. Endothelial cell dysfunction characterized by apoptosis and abnormal immune activation is, at least in part, induced by anti-endothelial cell antibody (AECA) in some cases of autoimmune disease. However, the molecular mechanisms of AECA-mediated pathogenetic damage to host vascular system remain unclear. The dual role of nitric oxide (NO) both in endothelial cell apoptosis and survival has been described. In this paper, endothelial cell apoptosis caused by the presence of cross-reactive AECA via a NO-mediated mechanism is demonstrated in dengue virus infection. Endothelial cells undergo apoptosis via the mitochondria-dependent pathway that is regulated by NO production. NO-regulated endothelial cell injury thus may play a role in the disruption of vessel endothelium and contribute to the AECA-induced pathogenesis of vasculopathy. The modulation of NO may provide the therapeutic strategies for autoimmune diseases by preventing the AECA-mediated endothelial cell damage.

Clonotypic analysis of anti-acetylcholine receptor antibodies produced against native and denatured antigen.

Rats were immunized with either conformationally intact acetylcholine receptor (AChR) or reduced and sodium dodecyl sulfate (SDS)-denatured AChR. As expected, challenge with native AChR (nAChR) resulted in the production of serum antibodies reactive with native AChR; these antibodies were, as observed in earlier studies, oligoclonal, dominated by the rat IgG2a subclass, heterogeneous with respect to binding avidity, and importantly, able to interfere with normal AChR-dependent muscle contraction. Antibodies produced as a result of immunization with denatured AChR (dAChR) that were crossreactive with nAChR were similar but not identical to those produced directly against nAChR; in contrast, however, dAChR-stimulated antibodies were clearly incapable of causing detectable impairment of AChR-dependent muscle contractile function. This difference in disease-causing potential was demonstrated in spite of the observation that either nAChR or dAChR could generate similar levels of circulating antibodies that reacted with the native form of AChR. Moreover, the same difference in disease-causing potential was again observed upon passive intravenous administration of anti-AChR antibodies into naive recipient rats. IEF analyses demonstrated that antibodies stimulated by dAChR immunization expressed the same clonotypic heterogeneity and isotype distribution as those stimulated by nAChR immunization. However, an apparent shift was observed in the preferred clonotypes expressed in rats immunized with dAChR; antibodies with relatively lower binding avidities were more markedly represented than the higher avidity antibodies commonly accompanying immunization with nAChR. Furthermore, a distinct subset of high avidity clonotypes was expressed following immunization with dAChR not associated with nAChR immunization.

Influence of T cell specificity on the heterogeneity and disease-causing capability of antibody against the acetylcholine receptor.

Adoptive secondary anti-acetylcholine receptor (AChR) antibody responses were examined in rats to evaluate the influence of helper T cell specificity on the nature and disease-causing potential of antibody produced. Mixtures of B cells reactive with the intact AChR plus T cells reactive with purified AChR subunits (alpha, beta, gamma, delta) were transferred and antigen-challenged in immunologically naive recipient rats; the serum anti-AChR antibody produced was assessed by radioimmunoassay for differences in titers and by isoelectric focusing for differences in clonal heterogeneity as a function of the subunit specificity of T cells transferred. In addition, rats receiving different sources of AChR or AChR subunit-reactive T cells were examined for AChR-dependent muscle dysfunction. The results indicated a clear reduction in anti-AChR antibody concentrations and clonal heterogeneity in recipient rats receiving T cells of specificities restricted to individual subunits. However, except for a clear relationship between serum anti-AChR antibody concentration and disease induction, no particular AChR subunit-reactive helper T cell specificity appeared to preferentially cause muscle dysfunction. We conclude that if such relationships exists, T cells with specificities more restricted than those described here will have to be used.

Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production.

In this study dengue virus (DV) was found to infect primary endothelial cells derived from human umbilical cord veins (HUVEC) and alter their cytokine production. Dengue virus infection of HUVEC was confirmed by an increase in plaque-forming units in the culture supernatant and by immunofluorescence assay. HUVEC produced large amounts of interleukin (IL)-6 and IL-8 but not IL-1beta after DV infection. Both the replication of DV and the production of IL-6 and IL-8 by HUVEC after DV infection were inhibited by ribavirin, an antiviral synthetic guanosine analogue. Additionally, increased serum levels of IL-6 and IL-8 were observed in patients with dengue hemorrhagic fever but not dengue fever. Therefore, our results suggest that endothelial cells can be a target for DV infection, and that DV-induced IL-6 and IL-8 production by endothelial cells may contribute to the pathogenesis of dengue hemorrhagic fever.

The dynamic responses of pro-inflammatory and anti-inflammatory cytokines of human mononuclear cells induced by uromodulin.

This study was undertaken to examine the dynamic response of human peripheral blood mononuclear cells (PBMC) in the secretion of proinflammatory and anti-inflammatory cytokines induced by uromodulin (URO). Levels of tumor necrosis factor-alpha (TNFalpha), TNF soluble receptor (sTNFRI and II), interleukin 1-beta (IL-1beta), and IL-1 receptor antagonist (IL-1Ra) in the supernatants of URO-stimulated PBMC were measured by ELISA. URO stimulated the secretion of all these cytokines in a dose dependent manner except sTNFRI. Peak levels of TNFalpha and IL-1beta were reached at 6-12 h, while 5-10 fold higher in sTNFR II and IL-1Ra levels were observed at 24-48 h after URO stimulation. URO-induced secretion of TNFalpha, IL-1beta, sTNFRII and IL-1Ra could be enhanced by human plasma. Specifically, serum proteins including C3, sCD14 and IgG not only bound to URO but also enhanced URO-induced TNFalpha secretion of PBMC. Collectively, our data suggest that URO might have dual immunomodulating effect through regulating the secretion of proinflammatory and anti-inflammatory cytokines, and that serum binding proteins might enhance this activity.

Effects of active HCV replication on neurologic status in HIV RNA virally suppressed patients.

BACKGROUND: Hepatitis C virus (HCV) is a frequent copathogen with HIV. Both viruses appear to replicate in the brain and both are implicated in neurocognitive and peripheral neuropathy syndromes. Interaction of the viruses is likely to be complicated and better understanding of the contributions of each virus will be necessary to make evidence-based therapeutic decisions. METHODS: This study was designed to determine if active HCV infection, identified by quantitative HCV RNA determination, is associated with increased neurocognitive deficits or excess development of distal sensory peripheral neuropathy in HIV coinfected patients with stable HIV viral suppression. The AIDS Clinical Trials Group Longitudinal Linked Randomized Trials (ALLRT) study was the source of subjects with known HIV treatment status, neurocognitive and neuropathy evaluations, and HCV status. Subjects were selected based on HCV antibody status (249 positive; 310 negative). RESULTS: HCV RNA viral loads were detectable in 172 participants with controlled HIV infection and available neurologic evaluations in the ALLRT. These participants were compared with 345 participants with undetectable HCV viral load and the same inclusion criteria from the same cohort. Neurocognitive performance measured by Trail-Making A or B and digit symbol testing was not dissimilar between the 2 groups. In addition, there was no significant association between active HCV replication and distal sensory neuropathy. CONCLUSION: Clinically significant neurocognitive dysfunction and peripheral neuropathy were not exacerbated by active hepatitis C virus infection in the setting of optimally treated HIV infection.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Effects of alpha 1-acid glycoprotein on tissue factor expression and tumor necrosis factor secretion in human monocytes.

Activated monocytes express tissue factor (TF) and secrete tumor necrosis factor alpha (TNF alpha), which are important in the initiation of blood coagulation and inflammation. We investigated the effect of alpha 1-acid glycoprotein (alpha 1-AGP), an acute phase protein, on the induction of the expression of TF and the secretion of TNF alpha in human monocytes in vitro. The TF activity of both fresh human monocytes and human monocytic cell line U937 significantly increased in a dose-dependent manner after a 6 h incubation with human or bovine alpha 1-AGP. The activity of TF gradually tailed off after 24 h. RT-PCR and Southern blot analysis revealed that TF mRNA synthesis was induced in monocytes. Inhibition of alpha 1-AGP induced TF expression by actinomycin D (ActD) further support that de novo TF mRNA synthesis was required. The specificity of the alpha 1-AGP-induced TF activity was demonstrated by anti-alpha 1-AGP antibody inhibition. TNF alpha secretion in alpha 1-AGP stimulated monocytes was also increased; this could be blocked by pentoxifylline (PTX). The possible contamination of lipopolysaccharide (LPS) in the alpha 1-AGP was excluded by limulus amoebocyte lysate. Therefore, these results indicate that alpha 1-AGP may contribute to the cellular initiation of coagulation and inflammation by increasing TF expression and TNF alpha secretion of monocytes.

T cells reactive with a small synthetic peptide of the acetylcholine receptor can provide help for a clonotypically heterogeneous antibody response and subsequently impaired muscle function.

The influence of T cell specificity was evaluated with regard to its role in the antibody response against the acetylcholine receptor (AChR) and resulting AChR-dependent muscle dysfunction. The reactivity of immune Th cells was restricted to a small region of the AChR alpha-subunit (amino acid residues 100-116) reported to be highly immunogenic. T cells primed to this peptide were found to demonstrate significant proliferation when challenged in vitro with either the homologous peptide or the intact AChR. Adoptive transfer of the peptide-immune T cells into immunologically naive recipient rats followed by AChR challenge resulted in the production of anti-AChR antibodies very similar to those produced under the regulation of T cells immune to the entire intact AChR with regard to overall clonotypic heterogeneity (measured by IEF) and their ability to interfere with AChR-dependent muscle contraction. Interestingly, when the threonine at position 106 was substituted with a proline, the resulting peptide continued to be equally, if not exceedingly, capable of stimulating T cell-proliferative responses, but was found to be ineffective at stimulating the levels of anti-AChR antibodies necessary for producing neuromuscular dysfunction.

Clonotypic analysis of anti-acetylcholine receptor antibodies from experimental autoimmune myasthenia gravis-sensitive Lewis rats and experimental autoimmune myasthenia gravis-resistant Wistar Furth rats.

A single immunization of Lewis rats with purified acetylcholine receptor (AChR) emulsified in adjuvant typically stimulates the production of oligoclonal AChR-reactive antibodies (as demonstrated by IEF) dominated by the IgG2a subclass, of moderate but clonotypically heterogeneous relative Ag-binding avidity, and capable of inducing symptoms of experimental autoimmune myasthenia gravis. Although similar immunization of Wistar Furth rats produces AChR-reactive antibodies with similar characteristics of clonotypic heterogeneity, avidity, and isotype expression, no detectable signs of AChR-dependent muscle impairment is observed. This contrasts the ability to induce impaired AChR function upon the passive transfer of pre-formed Lewis anti-AChR antibodies into naive Wistar Furth rats, suggesting that disease resistance in this model is not conferred at the level of the AChR itself. Moreover, if more aggressive immunization protocols are used (i.e., multiple injections of AChR), a transient breakthrough of AChR-dependent muscle dysfunction can be induced directly in the Wistar Furth strain indicating that the potential for the production of disease-causing antibodies does exist in the Wistar Furth repertoire. IEF analysis of Wistar Furth anti-AChR antibodies has revealed that hyperimmunization results in modified antibody clonotype expression that might explain changing expression of disease symptoms; however, explanations for the apparent "resistance" of Wistar Furth rats to disease induction are likely to be complex.

Skewed B cell VH family repertoire in Bcl-2-Ig transgenic mice.

The proto-oncogene Bcl-2 is normally expressed in B lineage cells in a stage specific manner and extends cell survival. Deregulated Bcl-2 expression has been shown to cause a major expansion in surface IgM and IgD positive B cells. In this report, the influence of deregulated expression of Bcl-2 on the VH repertoire of B cells was studied. This was accomplished by stimulating B cells from both adult and fetal Bcl-2-Ig transgenic mice and their normal littermates using the polyclonal activator lipopolysaccharide. Activated cells were then analyzed by in situ hybridization using radiolabeled C mu and VH gene probes. The D-proximal VH families 7183 and Q52 were preferentially expressed in the adult transgenic mice compared to their normal littermates. VH 7183 and Q52 were also over-represented in fetal transgenic mice but not to a greater extent than that observed with normal fetuses. These results demonstrate that the overproduction of Bcl-2, which prolongs cell survival independent of affecting proliferation, substantially alters the VH gene repertoire.

Exacerbated muscle dysfunction by procainamide in rats with experimental myasthenia gravis.

The induction of experimental autoimmune myasthenia gravis (EAMG) has long been shown to result in inefficient function of the acetylcholine receptor (AChR) and concomitant impairment of AChR-dependent neuromuscular communication. As an animal model of human myasthenia gravis, AChR-immunized rats demonstrate symptoms of MG very similar to those observed in human patients resulting from the presence of circulating anti-AChR antibodies which interfere with the normal function of the receptor. In addition to antibody antagonists of neuromuscular function, a variety of drugs have been observed to be associated with possible exacerbations of impaired neuromuscular function leading to myasthenic crisis in some MG patients. One drug, the cardiac anti-arrhythmic agent, procainamide, has been reported to cause both pre-synaptic and post-synaptic electrophysiologic effects at the neuromuscular junction. The study described below extends these observations to include the demonstration of perturbed AChR-dependent contractile muscle function in a rat model of MG.

Lewis rats given antibodies against denatured acetylcholine receptor become resistant to induction of experimental autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) in rats can be produced as the result of immunization with purified acetylcholine receptor (AChR). However, antibodies produced against an irreversibly denatured AChR were not capable of producing detectable AChR-dependent neuromuscular impairment such as that seen following immunization with AChR of intact conformation. This immunopathological difference was observed despite the fact that both immunizations resulted in the production of clonotypically heterogeneous antibodies with similar titers, isotype distribution, and relative binding avidities for conformationally intact AChR. Although they had no apparent disease-causing potential of their own, antibodies produced against denatured AChR could, however, bind AChR at the neuromuscular junction and mediate in vivo AChR-dependent neuromuscular impairment if a second anti-antibody was provided. Finally, immunization against denatured AChR, or administration to naive rats of antibodies obtained by immunization against denatured AChR, resulted in the recipient rats becoming resistant to the usual pathological effects of antibodies produced against intact AChR (either induced by active immunization or following passive antibody transfer). These observations suggest that disease severity in this system may be influenced by relationships between disease-causing and disease-abrogating antibodies.

Lipopolysaccharide binding and antibacterial activities of a synthetic peptide representing amino acids 90-101 of bactericidal/permeability-increasing protein.

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes is a potent antibacterial agent specific for gram-negative bacteria. BPI can bind to lipopolysaccharide (LPS) and neutralize its toxicity. However, little is known about the specific site and mechanisms of the BPI involved in this LPS binding and antibacterial activities. This study compared the amino acid sequences among BPI, cecropin A, magainin 2, and polymyxin B, and identified a common structure among these four bactericidal agents. They share a basic amphipathic alpha helix motif (Baah). A short peptide that represents amino acids 90-101 of BPI was then synthesized to test if it possessed any LPS binding and antibacterial activities. Results from in vitro lymphocyte culture indicated this peptide was able to inhibit LPS-induced lymphocyte proliferation, suggesting that it may interact with LPS. This LPS binding ability of BPI peptide 90-101 was further supported by the results from HPLC assays which showed the mobility of the peptide shifted in the presence of LPS. Furthermore, the antibacterial spectra of this peptide and cecropin peptide 1-11 were very similar to that of polymyxin B, even though the antibacterial activities of these two peptides were less potent than that of polymyxin B. In addition, the antibacterial activities of these two peptides and polymyxin B were inhibited by free LPS or a high concentration of MgCl2. These results thus suggest that a common structure (Baah) and antibacterial mechanism may be involved in these antibacterial agents.