Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys.

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease.

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.

Experimental hepatitis A virus (HAV) infection in Callithrix jacchus: early detection of HAV antigen and viral fate.

Common marmosets (Callithrixjacchus) were orally inoculated with a Brazilian strain (HAF-203) of hepatitis A virus (HAy). Three monkeys were euthanized at postinoculation hours 6, 12 and 24 to investigate the early events of HAV infection. Following others three inoculated and one control marmosets remained throughout the 46 day to evaluation of viral excretion. Different samples were collected to detect sequential presence of HAV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR) in liver, saliva, bile and stools at 6 hours to 461h days postinoculation. Liver tissues were examined by immunofluorescence assay in a confocal laser-scanning microscope for the presence of HAV antigen. HAV RNA was detected in saliva during the course of the study, in bile from 24 hours to 46 days. in stools from 7 to 46 days and liver at 12 hours postinfection. In immunofluorescence of liver stained preparations, viral antigen was present at six hours after inoculation throughout the remainder of the 46-day study. The animals developed histological and biochemical acute hepatitis after second week postinoculation. Spleen, duodenum, and mesenteric lymph nodes specimens were negative for HAV antigens. This study supports the possibility that in Callithrixjacchus orally inoculated with hepatitis A virus the saliva route may be additional way of viral elimination. The viral replication in the liver was responsible for biliary HAV presence and latter HAV detection in fecal samples.

Detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in Brazilian patients with dengue fever and dengue hemorrhagic fever.

Pro-inflammatory cytokines are believed to play an important role in the pathogenesis of dengue infection. This study reports cytokine levels in a total of 54 patients examined in Recife, State of Pernambuco, Brazil. Five out of eight patients who had hemorrhagic manifestations presented tumor necrosis factor-alpha (TNF-alpha) levels in sera which were statistically higher than those recorded for controls. In contrast, only one out of 16 patients with mild manifestations had elevated TNF-alpha levels. The levels of interleukin-6 (IL), IL-1beta tested in 24 samples and IL-12 in 30 samples were not significantly increased. Interferon-g was present in 10 out of 30 patients with dengue. The data support the concept that the increased level of TNF-alpha is related to the severity of the disease. Soluble TNF receptor p75 was found in most patients but it is unlikely to be related to severity since it was found with an equivalent frequency and levels in 15 patients with dengue fever and another 15 with dengue hemorrhagic fever.

Nitric oxide production by murine peritoneal macrophages in vitro and in vivo treated with Phyllanthus tenellus extracts.

Phyllanthus spp. are used traditionally for the treatment of viral, bacterial and parasitic infections. Macrophages may play a central role in innate and adaptive response against several infections. Nitric oxide (NO) can be induced during macrophage activation and may exert antimicrobial activity inhibiting the replication of several viruses or parasites. In the present study, we investigated the immunomodulatory role, both in vitro and in vivo, of aqueous extracts of fresh and dried Phyllanthus tenellus as well as an acetone/water extract of the dried plant. NO production by mouse peritoneal macrophages was detected in culture supernatants. Our results demonstrated that: (1) in vitro, a concentration of 100 microg/ml fresh extract stimulated significantly (P< or =0.05) NO production in all assays and the optimal production was achieved at 48-h incubation; (2) 10 and 50 mg/kg fresh extract injected twice intraperitonealy primed macrophages in vivo. Priming was detected by in vitro addition of a second stimulus with 100 microg/ml extract of the fresh plant. Thus, P. tenellus was able to pre-activate macrophages in vivo, and induce full activation in vitro. Further studies should be carried out to better evaluate the optimal dose schedules in terms of time/response for obtaining antiviral or other antimicrobial activity without host damage.

Characterisation of lymphocyte response and cytokine patterns in patients with dengue fever.

It is believed that the pathogenesis of dengue is generated by a deregulation of the immunological response. Dengue virus-infected monocytes/macrophages are likely to secrete monokines, which play a role in clinical features observed in patients with dengue haemorrhagic fever or dengue shock syndrome. This is a report on a study on 45 individuals presenting clinical and laboratory characteristics of dengue virus infection. During the acute phase of infection, immunophenotyping of peripheral mononuclear leukocytes was carried out in 19 patients and demonstrated a reduced frequency of CD2+ lymphocytes and their CD4+ and CD8+ subsets. Normal ratios were recovered during convalescence. Also, during the acute phase, mononuclear cells proliferated poorly in response to mitogens and dengue antigens as detected by incorporation of radiolabeled thymidine. During convalescence the lymphoproliferative response was re-established. In addition, the presence of circulating cytokines was investigated in the plasma of the same 45 patients. Concentrations of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and soluble tumor necrosis factor receptor (sTNF-Rp75) were found to be significantly elevated in patients when compared to normal controls. The increase in TNF-alpha was correlated with haemorrhagic manifestations and the increase in IL-10 with platelet decay. The data demonstrate that during the acute phase of dengue infection subsets of T lymphocytes are depressed in terms of both rate and function and provide evidence that circulating pro-inflammatory cytokines, such as TNF-alpha, are important in the pathogenesis and severity of dengue. IL-10 may be downregulating lymphocyte and platelet function.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen.

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Inducible nitric oxide synthase (iNOS) expression in liver and splenic T lymphocyte rise are associated with liver histological damage during experimental hepatitis A virus (HAV) infection in Callithrix jacchus.

Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.

Increased pro-inflammatory cytokines (TNF-alpha and IL-6) and anti-inflammatory compounds (sTNFRp55 and sTNFRp75) in Brazilian patients during exanthematic dengue fever.

Pro-inflammatory cytokines, tumor necrosis factor (TNF-alpha), interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) as well as anti-inflammatory compounds, soluble TNF-Receptor p55 (sTNFRp55), sTNFRp75 and IL-1 receptor antagonist (sIL-1Ra), were investigated in 34 Brazilian cases of dengue fever (DF) originated from a study of exanthematic virosis. The presence of pro-inflammatory cytokines was detected in sera from these patients by ELISA. TNF-alpha and IL-6 levels were significantly higher than control subjects in 32% and 52% patients, respectively. To our knowledge this was the first time a receptor antagonist and soluble receptors for cytokines were detected in sera obtained during exanthematic DF without hemorrhagic manifestations. Both sTNFRp55 and sTNFRp75 were consistently elevated in 42% and 84% patients, respectively. Most patients had IL-1beta levels not different from those of normal subjects, except for one case. Only 16% patients had altered levels of IL-1Ra. Previous studies in dengue hemorrhagic fever patients demonstrated production of these soluble factors; here we observed that they are found in absence of hemorrhagic manifestations. The possible role of these anti-inflammatory compounds in immune cell activation and in regulating cytokine-mediated pathogenesis during dengue infection is discussed.

Ultrastructural study on experimental infection of rotavirus in a murine heterologous model.

Viral replication, histopathological and ultrastructural changes were observed for a period of nine days in the small intestine of suckling mice infected with a simian rotavirus (SA11). Samples taken from duodenum, jejunum and ileum were prepared for light microscopy, transmission and scanning electron microscopy analysis. Histopathologic effect could be detected within 8 hr post-infection, when only a few altered cells were observed. Damage was extensive after 16 hr post-infection, showing swollen enterocytes and reduced and irregularly oriented microvilli at intestinal villi tips. Virus particles were detected at 16 and 48 hr post-infection, budding from the viroplasm into the rough endoplasmic reticulum cisternae in ileum enterocytes. Clear evidence of viral replication, observed by electron microscopy was not described before in heterologous murine models. Regeneration of the intestinal villi began at the third day post-infection. Despite some differences observed in clinical symptoms and microscopic analysis of homologous and heterologous rotavirus infections, we concluded that mechanisms of heterologous rotavirus infection in mice follow similar patterns to those observed in the homologous models.

Murine experimental infection with rotavirus SA-11: clinical and immunohistological characteristics.

Six- to eight-day old (lactent) mice were inoculated orally with simian SA-11 rotavirus. During the first hours after infection, the virus was already detected in villous apical enterocytes by immunohistologic reaction of paraffin sections of the duodenum and jejunum but not in the ileum. Late on the first day, some animals developed already diarrhoea and pathologic lesions were observed in the duodenum and jejunum. During the second day most mice developed diarrhoea; tissue lesions were intense and maximal from duodenum to ileum when compared to other days and some colon sections had mild pathological characteristics. At this point, the virus in the ileum was only detected by immunohistologic reaction. During the third day some animals still had diarrhoea but tissue histology was regenerated and no virus could be detected. We conclude that the SA-11 model follows an infection pattern similar to Epizootic Diarrhoea of Infant Mice (EDIM) and propose to study immunological parameters as young susceptible animals mature into adult resistant ones.

In vivo activation of macrophages by T cell-derived lymphokines: killing of tumor cells and schistosomula of Schistosoma mansoni.

We investigated the role of T cell-derived lymphokines for macrophage activation in vivo. We show for the first time that macrophages from casein-pretreated mice can be primed in vivo by intraperitoneal injection of immune interferon (IFN-gamma) and can be triggered by lipopolysaccharide (LPS) in vitro to kill schistosomula of S. mansoni. Similar results were obtained for the activation of tumoricidal macrophages. Injection of casein-pretreated mice with concanavalin A (Con A)-induced supernatant of a long-term T cell clone containing IFN-gamma and macrophage cytotoxicity inducing factor 2 (MCIF2), however, induced macrophage activation in vivo without further addition of LPS in vitro. These experiments show that macrophages can be activated by lymphokines in vivo. In addition, the data suggest that a combination of IFN-gamma with MCIF2 might be more effective than IFN-gamma alone. These data may be relevant for the strategy of treating cancer and infectious diseases with lymphokines.

Killing of schistosomula of Schistosoma mansoni by macrophages: induction by T-cell clone-derived lymphokines and interferon-gamma.

The induction of schistosomulicidal activity of peritoneal macrophages by concanavalin A-stimulated supernatants from long-term T-cell clones and by interferon-gamma (IFN-gamma) was investigated in detail. Optimal conditions of in vitro macrophage activation by T-cell clone supernatants were established. Macrophages from 13-week S. mansoni-infected mice responded to lymphokine activation as well as resident macrophages from uninfected mice. IFN-gamma was shown to play an essential role in induction of schistosomulicidal macrophage activity: recombinant IFN-gamma at high concentration could induce schistosomula killing, and an anti-IFN-gamma antiserum inhibited the induction of schistosomulicidal activity by T-cell clone supernatants. Our data also indicate that macrophage activation could be obtained by IFN-gamma in synergy with other lymphokines in the supernatant of long-term T-cell clones. Macrophages from mice injected with T-cell clone supernatants were primed in vivo and triggered to kill schistosomula in vitro in the presence of lipopolysaccharide (LPS). The data demonstrate that lymphokines produced by T-cell clones and, in particular, IFN-gamma can participate in the activation of schistosomulicidal macrophages.

Priming and triggering of tumoricidal and schistosomulicidal macrophages by two sequential lymphokine signals: interferon-gamma and macrophage cytotoxicity inducing factor 2.

We describe a new lymphokine activity, macrophage cytotoxicity inducing factor 2 (MCIF2), in the T cell mitogen-induced supernatant of a murine T cell clone in long-term culture. MCIF2 has the following properties: it elutes from a Sephadex G-100 column in three m.w. forms (10, 34, and 100 KD); it is acid labile (pH 2 to 4) and heat sensitive (80 min at 56 degrees C); it is not constitutively secreted, coexists in the same supernatant with immune interferon (IFN-gamma), and synergizes with IFN-gamma for induction of tumoricidal and schistosomulicidal resident peritoneal mouse macrophages. We uncoupled this synergy and show that IFN-gamma serves as the first ("priming") and MCIF2 as the second ("triggering") signal for macrophage activation. Application of the lymphokines in the reverse order was ineffective. These data demonstrate a two-step mechanism of macrophage activation.

Ingestion of yeast forms of Sporothrix schenckii by mouse peritoneal macrophages.

The ingestion by thioglycolate-elicited mouse peritoneal macrophages of yeast forms of two strains of Sporothrix schenckii was studied. Yeast forms opsonized with concanavalin A (ConA) were extensively phagocytized, and the phagocytic indexes depended on the concentration of ConA and apparently on the number of lectin receptors at the yeast surface as well. Neuraminidase treatment of S. schenckii increased the ingestion of unopsonized yeasts 7.7-fold. The addition of monosaccharides and derivatives partially inhibited phagocytosis. Mannose, rhamnose, and galactose, which are major constituents of S. schenckii surface antigens, reduced the phagocytic indexes by 40 to 50%. Glucosamine, N-acetylglucosamine, and N-acetylneuraminic acid were equally effective as inhibitors of phagocytosis. A mixture of five neutral sugars and glucosamine inhibited phagocytosis by 73%. The inhibitory effect of simple sugars could be amplified by using neuraminidase-treated yeast cells. Pentoses and glucose were inactive or slightly inhibitory. A purified rhamnomannan inhibited phagocytosis of the homologous strain, whereas partially purified peptidopolysaccharides were toxic to peritoneal macrophages. A partially purified galactomannan from S. schenckii was inhibitory (62% inhibition), and a peptidopolysaccharide fraction in which the O-linked carbohydrate chains had been removed neither was toxic to macrophages nor inhibited phagocytosis. Pretreatment of macrophages with simple sugars under conditions inhibiting ingestion or binding of S. schenckii did not affect phagocytosis of latex particles or sensitized sheep erythrocytes. The presence of receptors at the peritoneal macrophages which bind S. schenckii cell surface components is suggested.