Lok Ting Lau

H5N1 infection of the respiratory tract and beyond: a molecular pathology study

Summary Background Human infection with avian influenza H5N1 is an emerging infectious disease characterised by respiratory symptoms and a high fatality rate. Previous studies have shown that the human infection with avian influenza H5N1 could also target organs apart from the lungs. Methods We studied post-mortem tissues of two adults (one man and one pregnant woman) infected with H5N1 influenza virus, and a fetus carried by the woman. In-situ hybridisation (with sense and antisense probes to haemagglutinin and nucleoprotein) and immunohistochemistry (with monoclonal antibodies to haemagglutinin and nucleoprotein) were done on selected tissues. Reverse-transcriptase (RT) PCR, real-time RT-PCR, strand-specific RT-PCR, and nucleic acid sequence-based amplification (NASBA) detection assays were also undertaken to detect viral RNA in organ tissue samples. Findings We detected viral genomic sequences and antigens in type II epithelial cells of the lungs, ciliated and non-ciliated epithelial cells of the trachea, T cells of the lymph node, neurons of the brain, and Hofbauer cells and cytotrophoblasts of the placenta. Viral genomic sequences (but no viral antigens) were detected in the intestinal mucosa. In the fetus, we found viral sequences and antigens in the lungs, circulating mononuclear cells, and macrophages of the liver. The presence of viral sequences in the organs and the fetus was also confirmed by RT-PCR, strand-specific RT-PCR, real-time RT-PCR, and NASBA. Interpretation In addition to the lungs, H5N1 influenza virus infects the trachea and disseminates to other organs including the brain. The virus could also be transmitted from mother to fetus across the placenta.

Astrocytes Produce and Release Interleukin-1, Interleukin-6, Tumor Necrosis Factor Alpha and Interferon-Gamma Following Traumatic and Metabolic Injury

The brain is no longer considered immune-privileged due to its capability of producing cytokines in response to neurotrauma; however, the cellular sources of cytokines have not been defined. This study focused on the production of four inflammatory cytokines, interleukin-1 (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFN-gamma) in primary culture of astrocytes under two different injury models which simulated in vivo mechanical trauma (scratch injury) and ischemia. Results demonstrated that astrocytes after scratch injury were positively immunostained with IL-1alpha, IL-6, and TNFalpha. A slot-blot study of culture media showed that the release of IL-1alpha, IL-6, TNFalpha, and IFN-gamma by astrocytes subsequent to scratch and ischemic injury reached approximately twice the control values. The temporal expression of these cytokines was different for the two models. All four cytokines began to increase 1 h postscratch and remained at high levels throughout the experiment. In the ischemic model, however, the increase of cytokine expression was delayed until 4-8 h of ischemia, when sharp increases were seen in all four cytokines. In this culture system, the exogenous influence of blood-borne factors and leukocytes, which occur with in vivo trauma and ischemia, was eliminated. Accordingly, the cytokines detected in the culture media were derived from astrocytes. This study provides the first evidence that astrocytes, without the influence from other cell types, can produce and release cytokines following mechanical and ischemic injury.

Astrocytes: Implications for Neuroinflammatory Pathogenesis of Alzheimers Disease

Alzheimers disease (AD) is a neurodegenerative disease with major clinical hallmarks of memory loss, dementia, and cognitive impairment. Neuroinflammation is involved in the onset of several neurodegenerative disorders. Astrocyte is the most abundant type of glial cells in the central nervous system (CNS) and appears to be involved in the induction of neuroinflammation. Under stress and injury, astrocytes become astrogliotic leading to an upregulation of the expression of proinflammatory cytokines and chemokines, which are associated with the pathogenesis of AD. Cytokines and related molecules play roles in both neuroprotection and neurodegeneration in the CNS. During early AD pathogenesis, amyloid beta (Aβ), S100B and IL-1β could bring about a vicious cycle of Aβ generation between astrocytes and neurons leading to chronic, sustained and progressive neuroinflammation. In advanced stages of AD, TRAIL secreted from astrocytes have been shown to bind to death receptor 5 (DR5) on neurons to trigger apoptosis in a caspase-8-dependent manner. Furthermore, astrocytes could be reactivated by TGFβ1 to generate more Aβ and to undergo the aggravating astrogliosis. TGFβ2 was also observed to cooperate with Aβ to cause neuronal demise by destroying the stability of lysosomes in neurons. Inflammatory molecules can be either potential biomarkers for diagnosis or target molecules for therapeutic intervention. Understanding their roles and their relationship with activated astrocytes is particularly important for attenuating neuroinflammation in the early stage of AD. The main purpose of this review is to provide a comprehensive insight into the role of astrocytes in the neuroinflammatory pathogenesis of AD.

Minocycline inhibits LPS-induced retinal microglia activation.

Retinal neurodegenerative disease involves an inflammatory response in the retina characterized by an increase in inflammatory cytokines and activation of microglia. The degree of microglia activation may influence the extent of retinal injury following an inflammatory stimulus. Cytokines released by activated microglia regulate the influx of inflammatory cells to the damaged area. Thus, a therapeutic strategy to reduce cytokine expression in microglia would be neuroprotective. Minocycline, a semisynthetic tetracycline derivative, is known to protect rodent brain from ischemia and to inhibit microglial activation. In this study, we activated retinal microglia in culture with lipopolysaccharide (LPS) and attempted to determine whether minocycline could reduce the production of cytokines from activated microglia at both gene and protein levels. Changes in inflammatory cytokines, TNF-alpha and IL-1beta, were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) in the presence or absence of LPS. We also measured the levels of nitric oxide (NO) by the nitrate reductase method under similar conditions. LPS treatment induced a significant upregulation of the mRNA and release of TNF-alpha, IL-1beta, and NO from retinal microglia. Minocycline inhibited these releases. Thus, minocycline might exert its antiinflammatory effect on microglia by inhibiting the expression and release of TNF-alpha, IL-1beta, and NO.

A real-time PCR for SARS-coronavirus incorporating target gene pre-amplification

Abstract An enhanced polymerase chain reaction (PCR) assay to detect the coronavirus associated with severe acute respiratory syndrome (SARS-CoV) was developed in which a target gene pre-amplification step preceded TaqMan real-time fluorescent PCR. Clinical samples were collected from 120 patients diagnosed as suspected or probable SARS cases and analyzed by conventional PCR followed by agarose gel electrophoresis, conventional TaqMan real-time PCR, and our enhanced TaqMan real-time PCR assays. An amplicon of the size expected from SARS-CoV was obtained from 28/120 samples using the enhanced real-time PCR method. Conventional PCR and real-time PCR alone identified fewer SARS-CoV positive cases. Results were confirmed by viral culture in 3/28 cases. The limit of detection of the enhanced real-time PCR method was 102-fold higher than the standard real-time PCR assay and 107-fold higher than conventional PCR methods. The increased sensitivity of the assay may help control the spread of the disease during future SARS outbreaks.

Presence of Neuroglobin in Cultured Astrocytes

Neuroglobin (Ngb), a recently discovered intracellular respiratory globin in neurons, may play a crucial role in oxygen homeostasis in the brain. We report preliminary findings indicating the presence of functional neuroglobin in primary cultures of cerebral cortical astrocytes. Reverse transcription real‐time polymerase chain reaction (RRT–PCR) and immunostaining confirmed such presence in cultured astrocytes isolated from newborn mouse brain. Ngb antisense treatment increased apoptosis in ischemic astrocytes. The discovery of Ngb in astrocytes may provide some insight into how oxygen homeostasis is regulated in the brain.

Neutrophils May Be a Vehicle for Viral Replication and Dissemination in Human H5N1 Avian Influenza

Abstract The mechanism of systemic spread of H5N1 virus in patients with avian influenza is unknown. Here, H5N1 nucleoprotein and hemagglutinin were identified by immunohistochemistry in the nucleus and cytoplasm of neutrophils in the placental blood of a pregnant woman. Viral RNA was detected in neutrophils by in situ hybridization and enhanced real-time polymerase chain reaction. Therefore, neutrophils may serve as a vehicle for viral replication and transportation in avian influenza.

Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3γ in astrocyte

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14‐3‐3γ could be up‐regulated by ischemia in astrocyte to protect cells from ischemia‐induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14‐3‐3γ up‐regulation in primary culture of astrocytes under ischemic‐like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia‐induced 14‐3‐3γ up‐regulation in astrocytes. At the same time, we observed an ischemia‐induced nuclear translocation of p‐c‐Jun, a major downstream component of JNK. Inhibition of AP‐1 with curcumin also inhibited 14‐3‐3γ up‐regulation indicating that ischemia‐induced up‐regulation of 14‐3‐3γ in astrocyte involves activation of the JNK/p‐c‐Jun/AP‐1 pathway.

Development of multiplex nucleic acid sequence-based amplification for detection of human respiratory tract viruses.

A group of common lower respiratory tract infections, influenza A, influenza B, human parainfluenza virus 1-4 (HPIV1-4), respiratory syncytial virus (RSV), rubella virus (RV) and Coxsackie virus (CSV), were selected for the development of a multiplex nucleic acid sequence-based amplification (NASBA) assay. Quantifiable measurement utilizing an enzyme-linked oligonucleotide capture (EOC) optical detection method, which was described previously, alleviated the requirement of specialized instrumentation that is commonly used in other molecular techniques. Multiplex NASBA-EOC provided rapid and specific detection of a single virus from a multiplexed group, reducing laboratory testing time and enabling high throughput screening. The uniquely designed primers and probes proved to be highly sensitive and specific, exemplifying the robustness of the multiplex NASBA-EOC technique.

Glial Cell Line-Derived Neurotrophic Factor Protects Astrocytes From Staurosporine- and Ischemia- Induced Apoptosis

Glial cell line‐derived neurotrophic factor (GDNF) promotes the survival and functions of neurons. It has been shown to be a promising candidate in the treatment of ischemia and other neurodegenerative diseases. We transfected mouse astrocytes in primary cultures with a human GDNF gene and found that their conditioned medium could not only support the growth and survival of cultured dopaminergic neurons but also protect astrocytes from staurosporine‐ and ischemia‐induced apoptosis. This indicated that these transfected astrocytes could release GDNF. A similar protective effect on astrocytes against apoptosis was evident when recombinant human GDNF was used. Moreover, GDNF reduced caspase‐3 activity but not that of caspase‐1 in cultured astrocytes after ischemia treatment. Thus, GDNF protects astrocytes from apoptosis by inhibiting the activation of caspase‐3.