U. C. Chaturvedi

Elevated levels of IL-8 in dengue hemorrhagic fever.

Dengue virus causes dengue fever, a mild febrile illness, and at times dengue hemorrhagic fever (DHF), a severe illness the pathogenesis of which is not fully understood. Given the crucial roles played by interleukin‐8 (IL‐8) as a chemoattractant cytokine and in inflammatory processes, levels of circulating IL‐8 in the sera and IL‐8 mRNA in the peripheral blood mononuclear cells (PBMC) were measured in 99 patients of a recent dengue epidemic that occurred in India in 1996 and in 21 normal healthy controls. Twenty‐six of the patients had dengue fever (DF) and the remaining 73 were diagnosed as having different grades of DHF. All the control normal sera were negative for IL‐8, so were their PBMC for IL‐8 mRNA. Increased levels of IL‐8 in the sera and IL‐8 mRNA in their PBMC were observed in patients with severe illness of DHF grades III and IV. Only two out of 26 patients of DF and one out of 10 DHF grade I patient were positive for IL‐8 and all three deteriorated to DHF grade IV within 24 hr. All six patients of DHF grade IV who died had higher serum level of IL‐8 above 200 pg/ml, the highest being 5,568 pg/ml in one patient; the presence of mRNA for IL‐8 was very high in all patients. A striking correlation was observed between increased levels of IL‐8 and severe DHF, with greater levels in patients with increased grade of the disease and death. These results suggest that IL‐8 may have an important role and may be an indicator of increasing severity of the disease and death. J. Med. Virol. 56:280–285, 1998.

Vascular endothelium: the battlefield of dengue viruses

Abstract Increased vascular permeability without morphological damage to the capillary endothelium is the cardinal feature of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS). Extensive plasma leakage in various tissue spaces and serous cavities of the body, including the pleural, pericardial and peritoneal cavities in patients with DHF, may result in profound shock. Among various mechanisms that have been considered include immune complex disease, T‐cell‐mediated, antibodies cross‐reacting with vascular endothelium, enhancing antibodies, complement and its products, various soluble mediators including cytokines, selection of virulent strains and virus virulence, but the most favoured are enhancing antibodies and memory T cells in a secondary infection resulting in cytokine tsunami. Whatever the mechanism, it ultimately targets vascular endothelium (making it a battlefield) leading to severe dengue disease. Extensive recent work has been done in vitro on endothelial cell monolayer models to understand the pathophysiology of vascular endothelium during dengue virus (DV) infection that may be translated to help understand the pathogenesis of DHF/DSS. The present review provides a broad overview of the effects of DV infection and the associated host responses contributing towards alterations in vascular endothelial cell physiology and damage that may be responsible for the DHF/DSS.

Breakdown of the blood-brain barrier during dengue virus infection of mice.

A breakdown of the blood-brain barrier occurred in mice inoculated intracerebrally (i.c.) or intraperitoneally (i.p.) with dengue virus type 2 (DEN2). This resulted in leakage of protein-bound Evans blue dye and 51Cr-labelled erythrocytes into the brain tissue. The leakage increased with time after infection and coincided with an increase of a DEN2-induced cytokine, the cytotoxic factor (CF), in the spleens of such mice. The titres of virus in the brain increased exponentially in i.c. inoculated mice but the virus was not detected in brains of mice given DEN2 by the i.p. route. Similar breakdown of the blood-brain barrier also occurred in mice inoculated intravenously with CF; the damage was dose-dependent and the vascular integrity was restored during the 3 h period after inoculation. Treatment of mice with antihistamine drugs, blocking H1 or H2 receptors, decreased the DEN2-induced protein leakage by up to 50% in i.c. inoculated mice and up to 92% in those inoculated i.p. Indomethacin, a prostaglandin synthetase inhibitor, had no effect. In i.c. inoculated mice protein leakage was inhibited by about 60% by treatment with CF-specific (CFA) or DEN2-specific antisera (DEN2A) whereas protection was complete with the combined treatment with both antisera. On the other hand, in i.p. inoculated mice the inhibition of protein leakage was 80 to 89% with CFA. These findings show a breakdown of the blood-brain barrier leading to cerebral oedema during DEN2 infection which is mediated via the release of histamine by a virus-induced cytokine.

Sequential production of cytokines by dengue virus-infected human peripheral blood leukocyte cultures

The study was undertaken to elucidate the sequence of appearance of T helper (Th)1‐ and Th2‐type cytokines in human peripheral blood leucocyte cultures infected in vitro with dengue type 2 virus. Commercial sandwich enzyme‐linked immunosorbent assay kits were used to assay the levels of tumour necrosis factor‐alpha (TNF‐α), interferon‐gamma (IFN‐γ), interleukin (IL)‐2, IL‐4, IL‐5, IL‐6, and IL‐10 in culture supernatants. Culture supernatants were also screened for the cytotoxic factor and the dengue virus titres determined. The cytokines that appeared in the culture supernatants on the first day post‐infection (p.i.) were cytotoxic factor, TNF‐α, IL‐2, and IL‐6; their levels were highest on the second day p.i. IFN‐γ appeared on the second day with a peak on the third day p.i. The levels of these cytokines declined quickly, except for human cytotoxic factor (hCF) and IL‐2. The cytokines that appeared later were IL‐10 and IL‐5 on the fourth day and IL‐4 on the sixth day p.i. Dengue virus replicated in the peripheral blood leucocyte (PBL) cultures and was present throughout the course of the study. The findings of the present study show that dengue virus induced a predominant Th1‐type cytokine response during the first 3 days of infection of PBL cultures that was replaced by a Th2‐type response later. J. Med. Virol. 59:335–340, 1999.

Suboptimal chlorine treatment of drinking water leads to selection of multidrug-resistant Pseudomonas aeruginosa

The present study was undertaken to investigate the spectrum of bacteria present in the River Gomti water before and after chlorination for drinking purposes. We observed that the strains of Pseudomonas aeruginosa that survived chlorination on three out of seven occasions were resistant to almost all the antibiotics tested. The chlorine-resistant bacteria had mucoid colonies and grew better at 24 degrees C. All attempts to isolate the plasmid responsible for chlorine resistance were unsuccessful. Laboratory experiments using different strains of the P. aeruginosa in distilled water showed that only the resistant strain survived chlorine treatment at a dose of < or =500 microg/L. Similar results were obtained when water collected from seven different sites on the River Gomti was treated with graded doses of chlorine. At the higher dose of chlorine, all the bacteria died in 30 min, whereas with lower doses all the bacteria survived. The present study underscores the importance of measuring water chlorine concentrations to assure they are sufficiently high to remove pathogenic bacteria from drinking water. To our knowledge, this is the first report in the literature of the selection of multidrug-resistant bacteria by suboptimal chlorine treatment of water.

JAPANESE ENCEPHALITIS VIRUS LATENCY IN PERIPHERAL BLOOD LYMPHOCYTES AND RECURRENCE OF INFECTION IN CHILDREN

In a study group of 40 children who had been admitted to hospital with acute encephalitis, the disease was due to infection with Japanese encephalitis virus (JEV). Three children developed recurrence of disease 8‐9 months later. No virus had been isolated from these three patients during the acute stage of their illness, but virus was recovered from all during the recurrence phase by co‐cultivation of their peripheral blood mononuclear cells in primary mouse embryo fibroblast cultures. Virus was also recovered by co‐cultivation of peripheral blood mononuclear cells collected 8 months after their acute disease from three out of eight randomly selected asymptomatic children within the study group but not from similar cultures set up from JEV‐seronegative children used as controls. Virus was also isolated by co‐cultivation of T lymphocytes of asymptomatic children as detected by indirect immunofluorescence or by inoculation in mice.

Host Defence Mechanisms Against Japanese Encephalitis Virus Infection in Mice

The role of antibody and cell-mediated immunity in the resistance to Japanese encephalitis virus (JEV) infection was studied in adult mice. Passively transferred antibodies obtained up to 2 weeks after primary infection protected the recipient mice against a challenge infection with JEV. Antibody obtained at 4 or 5 weeks failed to protect despite the presence of high titres of neutralizing antibody. Protection was abrogated by pretreatment of the early serum with 2-mercaptoethanol to remove IgM. Similarly, adoptive transfer of immune spleen cells obtained up to 2 weeks after immunization provided protection. The protective effect was abolished by pretreatment of the immune spleen cells with anti-Thy 1.2 antiserum and complement. These findings suggest a role of T lymphocytes and IgM antibody in recovery from JEV infection.

Dengue and dengue haemorrhagic fever: Indian perspective

The relationship of this country with dengue has been long and intense. The first recorded epidemic of clinically dengue-like illness occurred at Madras in 1780 and the dengue virus was isolated for the first time almost simultaneously in Japan and Calcutta in 1943–1944. After the first virologically proved epidemic of dengue fever along the East Coast of India in 1963–1964, it spread to allover the country. The first full-blown epidemic of the severe form of the illness, the dengue haemorrhagic fever/dengue shock syndrome occurred in North India in 1996. Aedes aegypti is the vector for transmission of the disease. Vaccines or antiviral drugs are not available for dengue viruses; the only effective way to prevent epidemic degure fever/dengue haemorrhagic fever (DF/DHF) is to control the mosquito vector, Aedes aegypti and prevent its bite. This country has few virus laboratories and some of them have done excellent work in the area of molecular epidemiology, immunopathology and vaccine development. Selected work done in this country on the problems of dengue is presented here.

Persistence, latency and reactivation of Japanese encephalitis virus infection in mice

Persistent and latent Japanese encephalitis virus (JEV) infection was studied in pregnant and non-pregnant mice. Following intraperitoneal inoculation into pregnant mice JEV persisted for 16 weeks in contrast to 4 weeks in non-pregnant mice. This was followed by a higher frequency of latent infection in pregnant mice. The virus could be reactivated during pregnancy or by cyclophosphamide treatment, the latter being more effective.

Various cells of the immune system and intestine differ in their capacity to reduce hexavalent chromium

The cells of the immune system form a strong line of defence against foreign substances. The present study was undertaken to investigate the capacity of different cells of Wistar rats to reduce potentially carcinogenic hexavalent chromium (Cr-VI) into less toxic trivalent chromium in vitro. 5 x 10(6) cells were incubated with 10 or 25 microg ml(-1) of Cr (VI) in the form of K2Cr2O7 at 37 degrees C in the presence of 5% CO2 in air. At various time periods the remaining amount of Cr (VI) was measured and the percentage of Cr (VI) reduced was calculated. Among the single cell suspensions from the splenic cells a peak reduction of 55% was observed with the total spleen cells, 40% with the B-lymphocyte-enriched subpopulation, 10% with T-lymphocytes and 24% with the macrophages. The reduction by splenic and peritoneal macrophages was similar. Total thymocytes reduced 54% of the Cr (VI). Since the most common route of entry of chromium is through drinking water and food, intestinal cells were also investigated. Among the intestinal cells the maximum reduction of 100% (of 10 microg ml(-1)) was observed with the upper villus cells and 72% with the middle villus cells while reduction was the least (4%) with the crypt cells. The reduction in the intestinal loop in situ was 100%. The time taken by each cell type for the peak reduction to Cr (VI) was markedly different. The findings thus show that the capacity of different cells in the body differs vastly in their capacity and time taken to reduce hexavalent chromium. The most efficient handling of Cr (VI) by the intestine, due to the presence of a variety of cells and bacteria, protects the body from its adverse effects.