Wk Luk

Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

BACKGROUND In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. METHODS We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response. FINDINGS The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta. The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus. INTERPRETATION The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha. This characteristic may contribute to the unusual severity of human H5N1 disease.

Rapid Diagnosis of a Coronavirus Associated with Severe Acute Respiratory Syndrome (SARS)

Severe acute respiratory syndrome (SARS) is a recently emerged disease associated with pneumonia in infected patients (1). The disease is unusual in its severity, and patients suffering from this disease do not respond to empirical antimicrobial treatment for acute community-acquired typical or atypical pneumonia (2). By the end of March 2003, a cumulative total of 1622 cases and 58 deaths had been reported from 13 countries (3). The disease is highly infectious, and attach rates >56% have been reported in healthcare workers caring for SARS patients (2). Recently, we identified a novel virus in the family Coronaviridae in SARS patients (4). Of patients from whom paired acute and convalescent sera were available, all had seroconverted or had a greater than fourfold increase in antibody titer to this novel virus (4), suggesting that it plays an important role in the etiology of SARS. Thus, the establishment of a rapid noninvasive test for this virus is a high priority for monitoring and control of this disease. Here, we report a real-time quantitative PCR assay to detect this virus in clinical specimens. Patients with a clinical diagnosis of SARS and admitted in two hospitals in Hong Kong between February 26, 2003, and March 26, 2003, were considered for this study. Inclusion criteria were a fever of 38 °C or higher, cough or shortness of breath, new pulmonary infiltrate(s) on chest radiographs, and either a history of exposure to a patient with SARS or absence of response to empirical antimicrobial coverage for typical and atypical pneumonia. Samples were collected with informed consent. In total, 29 SARS patients with paired sera and nasopharyngeal aspirate (NPA) samples were available for the study. The diagnosis of SARS was confirmed in all patients by the presence of antibodies against the novel coronavirus in the serum (4). The …

Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus-Infected Macrophages In Vitro: Possible Relevance to Pathogenesis

ABSTRACT The pathogenesis of severe acute respiratory syndrome (SARS) remains unclear. Macrophages are key sentinel cells in the respiratory system, and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus (SARS-CoV) and other respiratory viruses. Primary human monocyte-derived macrophages were infected with SARS-CoV in vitro. Virus replication was monitored by measuring the levels of positive- and negative-strand RNA, by immunofluorescence detection of the SARS-CoV nucleoprotein, and by titration of the infectious virus. The gene expression profiles of macrophages infected with SARS-CoV, human coronavirus 229E, and influenza A (H1N1) virus were compared by using microarrays and real-time quantitative reverse transcriptase PCR. Secreted cytokines were measured with an enzyme-linked immunosorbent assay. SARS-CoV initiated viral gene transcription and protein synthesis in macrophages, but replication was abortive and no infectious virus was produced. In contrast to the case with human coronavirus 229E and influenza A virus, there was little or no induction of beta interferon (IFN-β) in SARS-CoV-infected macrophages. Furthermore, SARS-CoV induced the expression of chemokines such as CXCL10/IFN-γ-inducible protein 10 and CCL2/monocyte chemotactic protein 1. The poor induction of IFN-β, a key component of innate immunity, and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS.

Adoptive transfer of autologous Epstein‐Barr virus–specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV‐specific CTLp in peripheral blood from NPC patients, long‐term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3– 3,000/106 PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3–1,500/106 PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 × 107–3 × 108 autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC.

EBV SPECIFIC ANTIBODY-BASED AND DNA-BASED ASSAYS IN SEROLOGIC DIAGNOSIS OF NASOPHARYNGEAL CARCINOMA

We assessed 5 EBV specific assays for their capacity to effect serologic diagnosis of suspected NPC. The assays were the immunofluorescent assays, VCA IgA and EA IgA, the enzyme‐linked immunosorbent assays specific for EBNA 1 IgA or zta IgG and an EBV DNA assay. Serum samples were taken from 218 symptomatic NPC patients presenting consecutively at a public hospital in Hong Kong, 51 of whom were subsequently diagnosed as having NPC; 4 had EBV‐associated lung cancer with similar serology as NPC. The remaining patients included 23 who had other cancers and 140 who had other diseases. Objectives of serodiagnosis under such clinical settings, therefore, are to both exclude and predict a diagnosis of NPC. None of the assays individually can meet both requirements adequately, however. The difficulty was best overcome by combining EBNA 1 IgA and zta IgG. It was shown that 68.3% of the patients gave a confirmed test results, negative or positive, by both tests. A confirmed negative result was associated with a negative predictive value of 99.1%, providing a clear indication to exclude a diagnosis of NPC; a confirmed positive result was associated with a positive predictive value of 86.8%, providing a clear indication to proceed with diagnostic work‐up of NPC. The remaining patients gave equivocal test results, being positive for one or the other test, which were associated with a positive predictive value of 43.3% and 24.2%, respectively.

Unique risk factors for bacteraemia in allogeneic bone marrow transplant recipients before and after engraftment

A study of the risk factors associated with bacteraemia in 191 allogeneic bone marrow transplant (BMT) recipients (1991–1996) was performed. In contrast to risk factors commonly cited for cancer chemotherapy, mucositis, degree of conditioning toxicity of the gut and lungs, duration of neutropenia, and severity of neutropenia and monocytopenia were not associated with bacteraemia in the pre-engraftment period, during which the only significant risk factor was late stage underlying disease (P < 0.05). after engraftment, hickman catheter infection, and severe acute and chronic graft-versus-host disease (gvhd) were found to be independently associated with bacteraemia by multivariate analysis (P < 0.001, <0.05 and <0.05, respectively). this might be explained by intense antimicrobial prophylaxis, early empirical treatment, and non-routine use of haemopoietic growth factors. no significant difference in mortality was detected between bacteraemic and non-bacteraemic patients in both periods. allogeneic bmt recipients are therefore a group of patients distinct from other cancer patients receiving chemotherapy at risk of developing bacteraemia. the study findings prompt consideration of a management protocol incorporating early and routine use of haemopoietic growth factors before engraftment in high-risk patients with late stage underlying malignancies, routine antimicrobial prophylaxis for acute gvhd with intense immunosuppression, and intravenous immunoglobulin therapy for chronic gvhd. further cost-benefit analyses are warranted.

Assessing the risk of nasopharyngeal carcinoma on the basis of EBV antibody spectrum.

We have evaluated the performance of 3 new EBV ELISA for the diagnosis of nasopharyngeal carcinoma (NPC). The tests were specific for EBNA 1 IgA, EBNA 1 IgG and zta IgG, respectively. Their distinct antigenic specificity permits these assays to be used in concert in an approach that differentiates patients and apparently healthy subjects on the basis of their antibody spectrum. By so exploiting a distinguishing feature of NPC first described by the late Werner Henle that the patients sustain high levels of a broad spectrum of serum EBV antibodies, this approach achieved a sensitivity of 92% and a specificity of 93%, surpassing the performance of each of these assays individually. The enhanced performance is especially useful in population screening. It was shown that relative risk of NPC sustained by apparently healthy subjects residing in a high incidence area for NPC in the Pearl River estuary in Southern China may vary according to EBV antibody spectrum. The risk of the cancer was markedly reduced with odds ratios of 0.009 for 59% of those who had low level of all 3 antibodies. The risk was increased as antibody spectrum broadens and the risk was the highest with an odds ratio of 138 for 0.4% of those who had high levels of all 3 antibodies. Thus, EBV antibody spectrum may serve to guide follow‐up measures for early detection of the cancer and/or risk counseling according to level of the risk of the cancer sustained by the screened individuals.

The epidemiology of acinetobacter infections in Hong Kong

A retrospective survey was conducted of the characteristics of acinetobacter infections in Hong Kong--seasonal and geographic distributions, frequency of isolation from various body sites, antimicrobial susceptibility and molecular epidemiology. Most (80%) isolates of Acinetobacter spp. belonged to DNA groups 2 (A. baumannii) or 13, as defined by growth at 44 degrees C. An increased isolation rate in summer was related to higher ambient temperatures. The notion that acinetobacters are opportunist nosocomial pathogens was supported by the body site- and ward-specific distributions, which were similar to those of Pseudomonas aeruginosa and in marked contrast to those of coagulase-negative staphylococci and Escherichia coli. Typing of Acinetobacter isolates by arbitrary-primed polymerase chain reaction revealed extensive genotypic polymorphism, suggesting that numerous unrelated strains were circulating between patients. In view of the association with a high incidence of polymicrobial bacteraemia and multiresistance to antibiotics, a careful selection of appropriate antibiotics in combination is necessary for empirical therapy of infections caused by Acinetobacter spp.

Distinct Tumour Specificity and IL-7 Requirements of CD56−and CD56+ Subsets of Human γδ T Cells

γδ T cells are believed to recognize tissue injury caused by infections, tumours, as well as chemical and physical agents. The present study was carried out to study the feasibility of the ex vivo expansion of γδ T cells from healthy individuals, and to determine their functional capacity against tumours. We selectively expanded the peripheral γδ T cells of five donors against a myeloma cell line, XG‐7. Under optimal conditions, the resulting bulk cultures comprised about 82% of the γδ T cells, more than 90% of which showed the T‐cell receptor (TCR)‐Vγ9δ2 rearrangement. These γδ T‐cell cultures exhibited TCR‐γδ dependent cytotoxicity against different tumour cell lines including Molt‐4, BJAB, Epstein–Barr virus (EBV) transformed lymphoid cell lines (LCL), and the nasopharyngeal carcinoma (NPC) cell lines, CNE2 and 915, in addition to the stimulator XG‐7. By competitive cytotoxicity assays, the γδ T cells demonstrated recognition of at least three distinct target specificities expressed by Molt‐4, CNE2 and LCL, respectively, which were related to that expressed by the stimulator XG‐7 cells. The recognition of the specificity expressed by XG‐7 and Molt‐4 was further shown to require the participation of heat shock protein (HSP). The specificity expressed by CNE2 and 915 was preferentially recognized by the CD56 subset of γδ T cells, which could be sustained in the presence of interleukin (IL)‐7. These results suggested that γδ T‐cell immunity against tumour cell lines may be acquired in response to other types of tissue injury and, hence, implicates a role for their use in the prevention and treatment of tumours.