Modification of the ferret model for pneumonia from seasonal human influenza A virus infection.

The primary complication of seasonal influenza in humans is viral pneumonia. A conventional animal model--intranasal inoculation of ferrets with 10(6) median tissue culture infectious dose of virus--results in disease that is neither consistent nor comparable with severe viral pneumonia in humans. Therefore, the authors modified the experimental procedures by increasing the median tissue culture infectious dose to 10(9) and by inoculating via the intratracheal route, testing these procedures with H1N1 strains (A/Bilthoven/3075/1978 and A/Netherlands/26/2007) and H3N2 strains (A/Bilthoven/16190/1968 and A/Netherlands/177/2008) of seasonal influenza virus. The ferrets of all groups (n = 3 per virus strain) had clinical signs, increased body temperature, virus excretion from day 1, loss of body weight, and increased relative lung weight at 4 days postinoculation. All ferrets had severe pulmonary consolidation, and histologic examination revealed moderate to severe necrotizing bronchointerstitial pneumonia with severe edema, necrosis of alveolar epithelium, inflammatory infiltrates in alveolar septa and lumina, epithelial regeneration, and perivascular and peribronchiolar inflammatory infiltrates. The lesions were associated with the presence of influenza virus antigen in respiratory epithelium by immunohistochemistry. Although all 4 virus strains caused pulmonary lesions of comparable severity, virus isolation in the lungs, trachea, nasal concha, and tonsils showed higher mean virus titers in the H1/07 and H3/68 groups than in the H1/78 and H3/08 groups. In conclusion, the above H1N1 and H3N2 strains cause severe pneumonia in ferrets by use of the modified experimental procedures and provide a good model for pneumonia caused by seasonal influenza A virus infection in humans.

A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets.

Highly pathogenic avian influenza A viruses of the H5N1 subtype continue to circulate in poultry, and zoonotic transmissions are reported frequently. Since a pandemic caused by these highly pathogenic viruses is still feared, there is interest in the development of influenza A/H5N1 virus vaccines that can protect humans against infection, preferably after a single vaccination with a low dose of antigen. Here we describe the induction of humoral and cellular immune responses in ferrets after vaccination with a cell culture-derived whole inactivated influenza A virus vaccine in combination with the novel adjuvant CoVaccine HT. The addition of CoVaccine HT to the influenza A virus vaccine increased antibody responses to homologous and heterologous influenza A/H5N1 viruses and increased virus-specific cell-mediated immune responses. Ferrets vaccinated once with a whole-virus equivalent of 3.8 microg hemagglutinin (HA) and CoVaccine HT were protected against homologous challenge infection with influenza virus A/VN/1194/04. Furthermore, ferrets vaccinated once with the same vaccine/adjuvant combination were partially protected against infection with a heterologous virus derived from clade 2.1 of H5N1 influenza viruses. Thus, the use of the novel adjuvant CoVaccine HT with cell culture-derived inactivated influenza A/H5N1 virus antigen is a promising and dose-sparing vaccine approach warranting further clinical evaluation.

Pandemic 2009 H1N1 influenza virus causes diffuse alveolar damage in cynomolgus macaques.

The pathogenesis of lower respiratory tract disease from the pandemic 2009 H1N1 (H1N1v) influenza A virus is poorly understood. Therefore, either H1N1v virus or a seasonal human H1N1 influenza A virus was inoculated into cynomolgus macaques as a nonhuman primate model of influenza pneumonia, and virological, pathological, and microarray analyses were performed. Macaques in the H1N1v group had virus-associated diffuse alveolar damage involving both type I and type II alveolar epithelial cells and affecting an average of 16% of the lung area. In comparison, macaques in the seasonal H1N1 group had milder pulmonary lesions. H1N1v virus tended to be reisolated from more locations in the respiratory tract and at higher titers than seasonal H1N1 virus. In contrast, differential expression of messenger RNA transcripts between H1N1v and seasonal H1N1 groups did not show significant differences. The most upregulated genes in H1N1v lung samples with lesions belonged to the innate immune response and proinflammatory pathways and correlated with histopathological results. Our results demonstrate that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a nonhuman primate model. Its higher pathogenicity compared with a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract.

Caste-specific compounds in male carpenter ants.

Three caste-specific substances new to arthropod glandular secretions occur in the mandibular glands of male ants of five species in the genus Camponotus. These volatile compounds, which are not found in alate females or workers, have been identified as methyl 6-methyl salicylate, 2,4-dimethyl-2-hexenoic acid, and methyl anthranilate. The free acid has not been described previously.

Pathology and virus distribution in chickens naturally infected with highly pathogenic avian influenza A virus (H7N7) During the 2003 outbreak in The Netherlands.

The largest recorded outbreak of highly pathogenic avian influenza virus of the subtype H7N7 occurred in The Netherlands in 2003. We describe the immunohistochemical and histopathologic findings of 3 chickens naturally infected during this outbreak. Influenza virus antigen occurred in endothelial cells and mononuclear cells of all tissues examined and occurred in parenchymal cells of heart, lung, kidney, pancreas, and trachea, often associated with multifocal inflammation and necrosis. These findings are consistent with the acute stage of highly pathogenic avian influenza from other subtypes. In the severely edematous wattle skin, most endothelial cells contained virus antigen, while in all other tissues virus antigen was only detected in a few endothelial cells. Virus histochemistry showed that this H7N7 virus attached to more endothelial cells in wattle skin than in other vascular beds. This might explain, at least partly, the tropism of the virus and the associated severity of lesions in this tissue.

Renal cysts, nephrolithiasis, and ovarian cysts in a donkey suspected of having been sexually abused.

A female donkey was thought by its owner to have been sexually abused because it had severe perineal swelling. Besides the perineal swelling and a very small vaginal erosion, there were no other abnormalities at clinical examination. Haematology and blood biochemistry revealed an increased leukocyte count, an elevated blood lactate concentration, and a low ionized calcium concentration. During night the donkey's condition deteriorated and it was euthanized in the morning. At necropsy severe haemorrhages were found within the subserosa of the caudal abdomen. Both kidneys were polycystic, and multiple calculi were found in the right kidney. Both ovaries had multiple cysts. Lesions (fibrosis and mineralization) were found in the liver, lungs, and mesenteric artery and were suggestive of an earlier parasitic infection. There was no evidence of sexual abuse.

Use of the intramuscular relative-dose-response test to predict bronchopulmonary dysplasia in premature infants.

We tested the hypothesis that an intramuscular relative dose response (IM-RDR) on day 1 of life would more accurately predict which premature infants would develop bronchopulmonary dysplasia (BPD) than single measurements of retinol, retinyl palmitate (RP), or retinol binding protein (RBP). Seventy-five premature infants < or = 32 wk gestation had the IM-RDR on day 1 of life. An RDR > or = 25% occurred in 6 of 37 infants who did not develop BPD compared with 15 of 38 infants who developed BPD (P = 0.025). Retinol, RP, and RBP on day 1 were not different between the groups. BPD infants who received postnatal dexamethasone during their hospital course had a higher day-28 baseline retinol concentration (1.19 +/- 0.15 mumol/L) than did the group with no BPD (0.82 +/- 0.06 mumol/L) (P = 0.03). However, the effect of postnatal dexamethasone on serum retinol was biphasic, rising initially, and then declining after 8-12 d. RP values at time 0 and 5 h on day 28 were higher than day 1 values in both infants without BPD and infants with BPD who did not receive dexamethasone. Retrospective analysis also revealed a significant correlation between a day-1 RDR > or = 25% and the incidence of intraventricular hemorrhage in these premature infants. Because the IM-RDR is more helpful in predicting the development of BPD than single serum retinol and RP analyses, this test could be useful in determining which premature infants might benefit from supplemental vitamin A for BPD.

Upregulation of IFN-gamma and soluble interleukin-2 receptor release and altered serum cortisol and prolactin concentration during general anesthesia.

The effects of surgery, surgical stress, and anesthesia compromise the optimal function of the immune system. Recent studies demonstrate the influence of anesthesia on the immune response by modulation of neural-immune interactions. To evaluate the immunologic effects of general anesthesia with the hypnotic agent propofol and the opioid fentanyl, two drugs used frequently in anesthesia, we studied 30 patients undergoing elective orthopedic surgery before and during narcosis. We found a significant enhancement of interferon-gamma (IFN-gamma) and soluble interleukin-2 receptor (sIL-2R) release in lipopolysaccharide (LPS)-stimulated whole blood cultures after induction of anesthesia. Similar results were observed in cultures stimulated with polyclonal T cell activators, such as staphylococcal enterotoxin B (SEB) and phytohemagglutinin (PHA). IL-1beta and IL-8 release was not affected, but the anti-inflammatory cytokine IL-10 decreased after skin incision. Serum prolactin significantly increased immediately after induction of anesthesia, whereas serum cortisol levels declined. Our results point to enhanced proinflammatory T lymphocyte and natural killer (NK) cell activity, probably caused by prolactin and cortisol modulation in the serum. This may disturb the balance of human proinflammatory and anti-inflammatory pathways during surgery and general anesthesia.

Kinetics and organ distribution of allogeneic natural killer lymphocytes transfused into patients suffering from renal cell carcinoma.

The transfusion of natural killer (NK) lymphocytes into patients suffering from malignant diseases is an approach of current interest in the field of immunotherapy. Little is known about the organ distribution, survival, and clearance of donor immune effector cells in cellular therapy, and no reports exist on these important parameters considering NK cells in particular or any other type of allogeneic lymphocytes in humans. In the context of a clinical Phase I/II study we examined the distribution of transfused allogeneic NK cells in patients suffering from renal cell carcinoma. The NK cells were ex vivo cultivated and activated before transfusion. To assess the circulation of the transfused cells in the peripheral blood, we used a nested PCR technique to detect HLA DRB1 alleles of the NK cell donors. Post-transfusion, all patients showed evidence of circulating donor cells for up to 3 days. After 7 days, all donor cells were cleared from the blood to undetectable levels. To assess organ distribution, (111)In-labeled NK cells were injected and monitored by whole-body scintiscans. A distribution to the whole body, with preference for liver, spleen, and bone marrow, was observed after a short initial uptake in the lungs. No activity was observed in lymphatic nodes. A total of 2/4 evaluable metastases showed a clear accumulation of transfused NK cells. The half-life corrected activity in all body compartments remained almost constant over the 6-day observation period in concordance with the absence of any excretion of radioactivity. This may indicate an extended survival of the transfused cells, despite their foreign nature, in the host organism.

Cytokine production of the human monocytic cell line Mono Mac 6 in comparison to mature monocytes in peripheral blood mononuclear cells.

Mono Mac 6 is a human monocytic cell line with several features of mature blood monocytes such as CD 14 antigen expression, phagocytotic ability, and the functional ability to produce cytokines. This line is often used as an in vitro model to demonstrate the actions of monocytes. In our study, the production of cytokines by Mono Mac 6 cells in response to various stimulants was analyzed and compared to that of mature monocytes in peripheral blood mononuclear cells (PBMC). Interestingly, the Mono Mac 6 cells produced IL-1 alpha/beta, IL-6, and TNF-alpha after induction with the lectin phytohaemagglutinin A (PHA), mainly known as a T cell activator. The amount of cytokine release did not decrease in the presence of polymyxin B (Pmb), an inhibitor of LPS-induced effects. Kinetic studies revealed maximum cytokine levels 24h after stimulation, whereas human PBMC produced higher yields of all cytokines and enhancement was observed up to 48 hours after induction. Stimulation with the superantigen derived from the supernatant of mycoplasma arthritidis (MAS) induced expression of IL-1 beta, IL-6, and TNF-alpha, whereas staphylococcus enterotoxin B (SEB) did not induce any cytokine release. Further experiments analyzed the ability of Mono Mac 6 cells to produce IFN-alpha which is an important characteristic of mature monocytes. The cells were induced either with inactivated Newcastle Disease Virus (NDV), Sendai Virus, or the synthetic stimulus poly I:C IFN-alpha expression was not detected on the transcriptional or the protein level. In addition, no co-expression of IL-1 and IL-6 was observed in response to these stimuli. Since NDV, Sendai Virus, and poly I:C represent strong IFN-alpha inducers in peripheral blood monocytes, these data indicate that Mono Mac 6 cells lack the ability to express IFN-alpha. In conclusion, our findings show that this cell line is a potent cytokine producer, but the capacity to produce IFN is apparently deficient.

Induction of cytokines in human whole blood cultures by a mitogen derived from Mycoplasma arthritidis and by staphylococcal enterotoxin B.

Mycoplasma arthritidis produces a so-far only partially characterized soluble material (MAS) that has a potent mitogenic effect on T lymphocytes of several species. Similar to staphylococcal enterotoxins and a number of related toxins secreted by other species of bacteria, nanogram quantities of these so-called superantigens are sufficient to induce significant amounts of cytokines in the supernatant of lymphocyte cultures. Induction of interleukin-6 (IL-6) by MAS in murine bone marrow-derived macrophages has recently been described. In our study, we examined the differential effects of MAS and Staphylococcus aureus enterotoxin B (SEB) on human blood cells. When compared to MAS, SEB induced a higher proliferative response and, accordingly, a higher release of IFN-gamma. In contrast, large amounts of the macrophage products IL-1, IL-6 and tumor necrosis factor alpha (TNF-alpha) were observed in supernatants of cell cultures stimulated with MAS, whereas only small amounts were induced by SEB. Staphylococci and mycoplasmas are responsible for a number of diseases with various symptoms in man and animals. Our results suggest that SEB and MAS show different qualities in lymphocyte and macrophage stimulation which may be relevant in the pathogenesis of diseases.

Trimethylamine: the substance mainly responsible for the fishy odor often associated with bacterial vaginosis.

The vaginal discharge of women with bacterial vaginosis often has a prominent fishy odor. Intensification of this fishy odor by the addition of strong base to the vaginal discharge suggests that it could be due to trimethylamine, the substance responsible for the characteristic odor of spoiling fish. Samples were collected from 11 women with a vaginal discharge having a fishy odor and from 10 women with no detectable odor. Gas chromatographic analysis of headspace samples of alkalinized vaginal discharges indicated the presence of trimethylamine in all 11 samples with the fishy odor but not in the other samples. The chemical identity of trimethylamine was confirmed by gas chromatography-mass spectrometry of headspace samples from two vaginal discharge samples. It is concluded that trimethylamine is the primary cause of the fishy odor associated with bacterial vaginosis.

Structure of an extracellular polysaccharide produced by Erwinia chrysanthemi.

Erwinia chrysanthemi pv zeae strain SR260, a phytopathogen of corn, produced from lactose an acidic extracellular polysaccharide which was purified and found to consist of L-rhamnose, D-mannose, D-glucose, and D-glucuronic acid in the ratio of 3:1:1:1. A combination of chemical (carboxyl-group reduction, methylation analysis, periodate oxidation, Smith degradation, and lithium-ethylenediamine degradation) and physical (1 and 2D NMR spectroscopy) methods revealed that the polysaccharide is composed of a hexasaccharide repeating unit 1: [formula: see text]

Extracellular polysaccharides of modified strains of Erwinia spp.

The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain A2148 has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl-linkage analysis, and 1D 1H NMR spectroscopy. The polysaccharide is structurally similar, if not identical, to the EPS produced by E. chrysanthemi strain A350. A streptomycin-resistant strain of E. chrysanthemi Ech6 (Ech6S(+)) has been generated and has an elevated production of EPS, as does a streptomycin-resistant strain (Ech9Sm6) of E. chrysanthemi Ech9. These modified E. chrysanthemi spp. have been ribotyped and found to be closely related to their parent strains.

Spontaneous regression of a primary cardiac tumor presenting as fetal tachyarrhythmias.

Spontaneous regression of a primary intramyocardial tumor is reported. The infant with the tumor had tachycardia in utero and this presented in the newborn period. The presence and later complete regression of the tumor was documented by two-dimensional echocardiography.

Avoiding the risk of aspiration in intubated infants.

Aspiration is a potential problem in intubated infants who are fed enterally. In this prospective study, intubated preterm infants were fed orogastrically or oroduodenally. Aspiration was assessed by examining endotracheal aspirates for evidence of blue-colored dye. None of the babies had blue-colored endotracheal secretions when fed by either route. Heart and respiratory rates, blood pressure, and transcutaneous oxygen and carbon dioxide measurements were recorded at each interval of the study. There were no significant differences from baseline for these measurements. These findings indicate that there is no significant aspiration in intubated preterm infants who are fed by the orogastric or the oroduodenal route.

The pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models.

Respiratory viruses that emerge in the human population may cause high morbidity and mortality, as well as concern about pandemic spread. Examples are severe acute respiratory syndrome coronavirus (SARS-CoV) and novel variants of influenza A virus, such as H5N1 and pandemic H1N1. Different animal models are used to develop therapeutic and preventive measures against such viruses, but it is not clear which are most suitable. Therefore, this review compares animal models of SARS and influenza, with an emphasis on non-human primates, ferrets and cats. Firstly, the pathology and pathogenesis of SARS and influenza are compared. Both diseases are similar in that they affect mainly the respiratory tract and cause inflammation and necrosis centred on the pulmonary alveoli and bronchioles. Important differences are the presence of multinucleated giant cells and intra-alveolar fibrosis in SARS and more fulminant necrotizing and haemorrhagic pneumonia in H5N1 influenza. Secondly, the pathology and pathogenesis of SARS and influenza in man and experimental animals are compared. Host species, host age, route of inoculation, location of sampling and timing of sampling are important to design an animal model that most closely mimics human disease. The design of appropriate animal models requires an accurate pathological description of human cases, as well as a good understanding of the effect of experimental variables on disease outcome.