Kuender D. Yang

Altered Cellular but Not Humoral Reactions in Children with Complicated Enterovirus 71 Infections in Taiwan

Enterovirus 71 (EV 71) infections have high neurovirulence and fatality. Immune responses were assessed in 78 patients with EV 71 infection. EV 71 meningoencephalitis occurred more frequently in younger children and in boys. C-reactive protein levels were not elevated, although total leukocyte counts were increased in these patients. The CD40-ligand expression on T cells significantly decreased in children with meningoencephalitis (P=.041). Polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4) at position 49 of exon 1 showed a higher frequency of G/G genotype in patients with EV 71 meningoencephalitis than in those without meningoencephalitis (18/31 vs. 14/47; P=.045) and in control subjects (18/31 vs. 25/93l; P=.007). Specific EV 71 neutralizing antibody titers were detectable but did not differ in children with and without meningoencephalitis in the acute and convalescent stages. Results from this study suggest that younger children with a certain CTLA-4 polymorphism and altered cellular but not humoral response may be linked to EV 71 meningoencephalitis.

Lymphocyte proliferation modulated by glutamine: involved in the endogenous redox reaction

Decreased glutamine concentrations are found during catabolic stress and are related to susceptibility to infections. However, little is known about the mechanism of glutamine modulation of lymphocyte functions. Glutamine is not only an important energy source in mitochondria, but is also a precursor of glutamate, which is used for cellular glutathione (GSH) biosynthesis in lymphocytes. In this study, we investigated the effects of glutamine on the redox reaction during lymphocyte proliferation. Peripheral blood mononuclear cells, obtained from healthy adult volunteers, were cultured and stimulated by phytohaemagglutinin (PHA) in the presence of different glutamine concentrations. Cells were harvested and prepared for analysis of lymphocyte proliferation, cell cycle propagation, intracellular glutathione levels and reactive oxygen species (ROS) production. We found that glutamine supplementation significantly enhanced PHA‐stimulated lymphocyte proliferation and propagation of the cell cycle from the G1 to S and G2/M phases. Glutamine also enhanced production of both intracellular ROS and GSH levels in PHA‐stimulated lymphocytes. Flow cytometric analysis by the mercury orange staining method showed that glutamine significantly enhanced intracellular non‐protein thiols in PHA‐stimulated CD4+, but not CD8+ lymphocyte subsets. Furthermore, intracellular GSH detected by monochlorobimane dye probe showed that glutamine enhanced GSH both in PHA‐stimulated CD4+ and CD8+ lymphocyte subsets. Inadequate glutamine supplementation resulted in decreased lymphocyte proliferation in association with decreased levels of intracellular GSH. Addition of exogenous GSH significantly enhanced lymphocyte proliferation, whereas blockade of GSH synthesis enhanced ROS production and suppressed lymphocyte proliferation. These results suggest that the modulation of PHA‐stimulated lymphocyte proliferation by glutamine is closely related to the maintenance of appropriate intracellular redox status.

Antibody‐dependent enhancement of heterotypic dengue infections involved in suppression of IFNγ production

Antibody‐dependent enhancement has been implicated in some outbreaks of epidemic dengue hemorrhagic fever, however, the mechanism of antibody‐dependent enhancement is not well known. This study was conducted to investigate the cross‐protection and cross‐enhancement of dengue‐2 virus infections by dengue‐1 immune sera. It was found that dengue‐1 immune sera at 1:5 dilution (n = 12) could neutralize dengue‐2 infections in BHK‐21 cells, as assessed by a standard plaque‐reduction neutralization assay. Two‐thirds of the dengue‐1 immune sera at 1:25 dilution demonstrated neutralizing effects for dengue‐2 infections, whereas, non‐immune sera revealed no neutralization for dengue‐2 infections in BHK‐21 cells. Human mononuclear leukocytes in response to dengue‐2 infections elicited a T cell helper 1 (Th1) response revealing induction of IFNγ but not IL‐4 production. Dengue‐1 immune sera did not neutralize dengue‐2 infections in mononuclear leukocytes. Subneutralizing titers of dengue‐1 immune sera at 1:250, but not at 1:10 dilution, enhanced dengue‐2 infections in mononuclear leukocytes (1.2 ± 0.7 × 104 vs. 2.8 ± 0.3 × 102 PFU/ml). The enhancement of dengue‐2 infections in mononuclear leukocytes by dengue‐1 immune sera at 1:250 was associated with an increase in the lymphocyte proliferation index, and a decrease in IFNγ production (56 ± 24 vs. 12 ± 3 pg/ml). The addition of IFNγ (0.1 μg/ml) suppressed significantly the antibody‐dependent enhancement induced by dengue‐1 immune sera, whereas the presence of anti‐IFNγ F(ab)2 antibody augmented the antibody‐dependent enhancement effect. Results from this study suggest that suppression of Th1 response may be involved in the antibody‐dependent enhancement of heterotypic dengue infections. Better regulation of Th1/Th2 reactions may be useful for prevention of heterotypic immune enhancement of dengue infections. J. Med. Virol. 63:150–157, 2001.

Clinical Manifestations and Laboratory Assessment in an Enterovirus 71 Outbreak in Southern Taiwan

An epidemic of enterovirus 71 (EV71) infection compatible with hand, foot and mouth disease and associated with high morbidity and mortality occurred in Taiwan in 1998. We recruited 90 patients (50 males, 40 females) with definite EV71 infections for clinical and laboratory analysis. The neurological signs and symptoms, all of which occurred during the febrile period, in patients with central nervous system (CNS) involvement (aseptic meningitis, encephalitis or myelitis) were myoclonic jerks (23/33), vomiting (10/33), ataxia (7/33), lethargy (6/33), seizure (4/33) and tremor (2/33). Patients with CNS involvement had longer durations of fever (4.6 ± 0.2 vs. 3.1 ± 0.3 d; p< 0.01) and a higher white blood cell count (12,512 ± 658 vs. 10,607 ± 409 cells/mm3; p= 0.01) than patients without CNS involvement. The case fatality rate in patients with CNS involvement was 4/33 (12%), whereas no fatalities (0/57) occurred in patients without CNS involvement. Six of 11 patients subjected to MRI showed a high intensity T2-weighted signal in the brainstem. A nested fluorescent RT-PCR for detection of virus in throat and stool specimens showed higher sensitivity than viral culture. Viremia was detectable using RT-PCR in 20% of cases (3/15), whereas no virus was isolated from culture or detected by RT-PCR in cerebrospinal fluid.

Combination of CTLA-4 and TGFβ1 gene polymorphisms associated with dengue hemorrhagic fever and virus load in a dengue-2 outbreak

The pathogenesis of dengue hemorrhagic fever (DHF) has been considered to be massive immune activation of T cells. Abnormal expression of the immune regulatory molecules, CTLA-4 and TGFbeta1, leads to disturbances of regulatory T cell immune response. We investigate the contribution of CTLA-4 and TGFbeta1 in DHF by analyzing them for association with virus load in blood and polymorphisms of CTLA-4 +49A/G, and TGFbeta1 -509C/T in a DEN-2 outbreak. The increased frequency of the TGFbeta1 -509 CC genotype in patients with DHF was compared to those with dengue fever (OR=1.9, p=0.034). Moreover, the presence of the CTLA-4 +49 G allele and TGFbeta1 -509 CC genotype increased the susceptibility to risk of DHF (OR=2.1, p=0.028) and significantly higher virus load (p=0.013). This finding suggests that a combination of CTLA-4 and TGFbeta1 polymorphisms is associated with the susceptibility of DHF and higher virus load.

Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation.

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15–30 μg/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 μg/ml, phenobarbital significantly suppressed formylmethionyl‐leucyl‐phenylalanine (fMLP)‐induced chemotaxis. Concentrations greater than 300 μg/ml also inhibited phorbol myristate acetate–stimulated membrane potential change. In contrast, 30 fig/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 μg/ml) was able to inhibit PHA‐induced interleukin‐2 (IL‐2) production and suppress the proliferation of PHA‐induced IL‐2 receptor–bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.

Effect of picibanil (OK432) on neutrophil-mediated antitumor activity: implication of monocyte-derived neutrophil-activating factors.

SummaryPicibanil (OK432), an extract from streptococci, has been widely utilized to treat malignant ascites and pleural effusions. The antitumor mechanism is believed to include complement-mediated neutrophil activation. Employing a flow-cytometric analysis of actin polymerization as an indicator of cell activation as well as a tumor proliferation assay, we have found that monocytederived neutrophil-activating factors were involved in OK432-induced neutrophil activation as well as antitumor activity. OK432-stimulated (0.1 KE/ml; 0.01 mg/ml) monocyte supernatants (OKMS) induced neutrophil actin polymerization and chemotaxis. OKMS were responsible for neutrophil-mediated inhibition of human leukemic (CEM) cell proliferation and stimulated neutrophils to produce superoxide in the presence of CEM leukemic cells at an effector/target ratio higher than 20/1. In contrast, OK432 alone, OK432-stimulated lymphocyte supernatants, or OK432-stimulated neutrophil supernatants had no effect on neutrophil activation or suppression of tumor cell proliferation. OK432 in combination with mononuclear cells also had no effect on the inhibition of CEM cell proliferation. Pretreatment of OKMS at 56°C for 30 min did not affect its ability to activate neutrophils, implying that complement activation is not responsible for the neutrophil activation. Supernatants from OK432-stimulated mononuclear cells, as determined by enzyme-linked immunosorbent assays and radioimmunoassays, contained high levels of interleukin-8 (IL-8; 1567±145 pg/ml) and tumor necrosis factor (TNFα; 2105±152 pg/ml), low levels of leukotriene B4 (800±45 pg/ml) and IL-1β (180±22 pg/ml), but interferon γ was not detectable. IL-1β, IL-8, and TNFα transcripts, undetectable in untreated monocytes, increased significantly after 30–60 min exposure to OK432. These results suggest that neutrophil-activating factors from monocytes or resident macrophages may play an important role in the OK432-induced neutrophil activation and antitumor activity.

Epidemiologic Study and Containment of a Nosocomial Outbreak of Severe Acute Respiratory Syndrome in a Medical Center in Kaohsiung, Taiwan

OBJECTIVE We conducted an epidemiologic investigation at the beginning of a nosocomial outbreak of severe acute respiratory syndrome (SARS) to clarify the dynamics of SARS transmission, the magnitude of the SARS outbreak, and the impact of the outbreak on the community. METHODS We identified all potential cases of nosocomially acquired SARS, linked them to the most likely infection source, and described the hospital containment measures. SETTING A 2,300-bed medical center in Kaohsiung, Taiwan. RESULTS A total of 55 cases of SARS were identified, and 227 hospital workers were quarantined. The index patient and neighboring patients were isolated. A chest physician team reviewed medical charts and chest radiographs and monitored the development of SARS in patients staying in the ward. The presence of underlying lung disease and immunocompromise in some patients made the diagnosis of SARS difficult. Some cases of SARS were diagnosed after the patients had died. Medical personnel were infected only if they cared for patients with unrecognized SARS, and caretakers played important roles in transmission of SARS to family members. As the number of cases of nosocomial SARS increased, the hospital closed the affected ward and expedited construction of negative-pressure rooms on other vacated floors for patient cohorting, and the last case in the hospital was identified 1 week later. CONCLUSIONS Timely recognition of SARS is extremely important. However, given the limitations of SARS testing, possible loss of epidemic links, and the nonspecific clinical presentations in hospitalized patients, it is very important to establish cohorts of persons with low, medium, and high likelihoods of SARS acquisition. Rapid closure of affected wards may minimize the impact on hospital operations. Establishment of hospitals dedicated to appropriate treatment of patients with SARS might minimize the impact of the disease in future epidemics.

A Model to Study Antioxidant Regulation of Endotoxemia-Modulated Neonatal Granulopoiesis and Granulocyte Apoptosis

Neonates with septicemia tend to develop granulocytopenia, which may, in part, be due to septic mediators such as oxygen free radicals and tumor necrosis factor alpha (TNF-α). Granulocytopenia may be caused by a decrease in granulocyte growth and/or an increase in granulocyte destruction. In the present study, we investigated antioxidant regulation of endotoxin-modulated neonatal granulopoiesis and granulocyte apoptosis. Using human umbilical cord blood (HUCB), we found that simulating endotoxemia in vitro elicited significant superoxide production within a few minutes. Endotoxin exposure suppressed colony-forming unit–granulocyte and monocyte formation in a dose-dependent fashion. Addition of antioxidants such as N-acetyl-cysteine could reverse the endotoxin suppression of colony-forming unit–granulocyte and monocyte formation (13 ± 5 versus 75 ± 5 colony-forming units/mL). Spontaneous in vitro granulocyte apoptosis in 6 h, as reflected by phosphatidylserine expression on the cell surface, was higher in granulocytes from HUCB than in those from adult blood (10.8 ± 1.0%versus 5.6 ± 1.2%). The addition of endotoxin or IL-8 to the cells in the in vitro model did not promote granulocyte apoptosis, but TNF-α, a major mediator of the effects of endotoxin, significantly induced granulocyte apoptosis in HUCB (control versus TNF-α: 8.9 ± 1.2%versus 35.9 ± 2.9%). Addition of the antioxidant N-acetyl-cysteine effectively blocked TNF-α-induced granulocyte apoptosis as demonstrated by DNA fragmentation. Results from these studies indicate that oxygen radicals are directly involved in endotoxin suppression of granulopoiesis, and indirectly promote granulocyte apoptosis, presumably through TNF-α-mediated action. Thus, under certain conditions, modulation of oxygen radical production in the blood may benefit neonates with granulocytopenia.

Sialic acid involved in hypermucoviscosity phenotype of Klebsiella pneumoniae and associated with resistance to neutrophil phagocytosis

Klebsiella pneumoniae (KP) with the hypermucoviscosity (HV) phenotype has abundant capsular polysaccharides (CPS) and usually causes an invasive syndrome. Sialic acid (Sia), a component of CPS in KP strains with the HV phenotype, may be anti-phagocytic. Sia-binding immunoglobulin-like lectin-9 (Siglec-9) act as an MHC class-I receptor on neutrophils that recognizes Sia and sends a signal to dampen inflammatory response. Three clinical KP strains with KP-M1 (HV-positive; capsular serotype K1), KP-14 (HV-negative; capsular serotype non-K1/K2), and DT-X (HV-negative; capsular serotype K1) were studied. We assessed total Sia in CPS extracts using enzymatic methods and phagocytosis by neutrophils of neuraminidase-treated bacteria using flow cytometry. Neutrophil killing was evaluated in the presence and absence of antibodies against Siglec-9. The concentration of Sia was significantly higher in the CPS extract of KP-M1 (56.75 ± 6.75 μmole/109 cfu) than in the CPS extract of KP-14 (0.02 ± 0.01 μmole/109 cfu) and DT-X (a negligible value). The KP-M1 (compared with the KP-14 and DT-X) was more resistant to neutrophil phagocytosis. Both the HV phenotype and resistance to phagocytosis of KP-M1 were significantly decreased after Sia removal with neuraminidase treatment. Fluorescence microscopy with an antibody against human Siglec-9 showed attachment of KP-M1 (but were absent of KP-14 and DT-X) to the surface of neutrophils and colocalization with human Siglec-9. Engagement of Siglec-9 via Sia enhanced neutrophils killing of KP-M1 by ex vivo human neutrophils bactericidal activity assay. The result showed that Sia might be a constituent of KP-M1 CPS responsible for HV, thereby contributing to anti-phagocytic activity of this pathogen.