Epstein-Barr virus (EBV)-specific cell-mediated and humoral immune responses in ataxia-telangectasia patients

As a part of studies on cell-mediated immune (CMI) responses of immunocompromised, Epstein-Barr virus (EBV)-infected patients who can or cannot restrict the proliferation of EBV-transformed B cells, we have studied 16 Turkish patients with ataxia-telangectasia (AT). Fifteen were EBV seropositive; one was seronegative. Among the seropositives, eight had no or only low anti-EBV-determined nuclear antigen (EBNA) antibody titers, while seven had normal anti-EBNA levels. EBV-seropositive and -seronegative healthy Turkish children were used as controls. We have particularly asked the question whether low EBNA antibody titers can be correlated with the level of EBV-specific and -nonspecific cell-mediated immunity. Non-EBV-specific tests included cell count, phenotypical characterization with monoclonal antibodies, assessment of natural killer (NK)-cell activity, and ability to suppress mitogen-induced immunoglobulin production. Two EBV-specific CMI tests were used: outgrowth inhibition (OI) and leukocyte migration inhibition (LMI). The majority of the patients of the low-EBNA antibody group was IgA deficient and had high levels of -fetoprotein (a-FP). Cells reacting with OKT8 monoclonal antibody predominated in both AT patient groups. In contrast, the suppressor activity was present in only a few patients and NK and interferon-activated killing (IAK) activities were normal. EBV-specific cell-mediated responses were defective in seven of eight patients in the low-anti-EBNA group and five of seven patients in the group with normal anti-EBNA titers. It is concluded that AT patients are often defective in their EBV-specific cell-mediated immune responses and with regard to their EBNA antibody levels. These defects are associated with a predominance of T cells reacting with OKT8 monoclonal antibody.     

Vasoactive intestinal polypeptide enhances oral tolerance by regulating both cellular and humoral immune responses

Vasoactive intestinal polypeptide (VIP) is an important signal molecule of the neuroendocrine-immune network. In the immune system, VIP has been found to act as an endogenous anti-inflammatory mediator. In the current study, it was found that VIP administration regulated oral tolerance by inhibiting both cellular and humoral responses. Compared with vehicle-treated mice, mice treated with VIP during the development of ovalbumin (OVA)-induced oral tolerance exhibited the least delayed-type hypersensitivity (DTH), showed profoundly reduced proliferative capacity and produced less interferon (IFN)-gamma, interleukin (IL)-6, IL-5, IL-10 and interferon-inducible protein (IP-10). IgA-secreting cells in the gut as well as OVA-specific IgG and other isotypes levels in plasma were inhibited significantly after VIP-treatment. The VPAC2 receptor may be involved in VIP-mediated oral tolerance enhancement. Taken together, these results suggest that VIP enhanced oral tolerance via regulating both cellular and humoral responses.     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Immune responses and pathogenesis in immunocompromised chickens in response to infection with the H9N2 low pathogenic avian influenza virus

The H9N2 low pathogenic avian influenza (LPAI) viruses have often caused moderate mortality with severe clinical signs in domestic poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans, but the basis for their pathogenesis remains unclear especially in chickens. To better understand the effect of immunosuppression on the risk of H9N2 viral infection, the pathogenesis and host immune responses of the H9N2 LPAI virus in a T-cell-suppressed chicken model were investigated. Cyclosporin A (CsA) treatment led to suppression of cell-mediated immunity such as CD8+ T-cells and reduced expression of IFN-gamma mRNA. T-cell suppression correlated with high viral load in the oropharynx and cloaca of H9N2 LPAI virus-infected specific pathogen free (SPF) chickens. Elevated level of viral RNA in the peripheral blood lymphocytes was found only in immunocompromised chickens. Viral protein and associated cellular apoptosis were observed only in the kidney of the immunocompromised chickens, particularly in those that had died. Our findings suggest that T-cell-mediated responses are important in influenza viral clearance and may help to explain in part the reasons for the increased mortality in chickens infected with H9N2 LPAI viruses in domestic poultry farms.     

Equine neonates have attenuated humoral and cell-mediated immune responses to a killed adjuvanted vaccine compared to adult horses

The objectives of this study were to compare relative vaccine-specific serum immunoglobulin concentrations, vaccine-specific lymphoproliferative responses, and cytokine profiles of proliferating lymphocytes between 3-day-old foals, 3-month-old foals, and adult horses after vaccination with a killed adjuvanted vaccine. Horses were vaccinated intramuscularly twice at 3-week intervals with a vaccine containing antigens from bovine viral respiratory pathogens to avoid interference from maternal antibody. Both groups of foals and adult horses responded to the vaccine with a significant increase in vaccine-specific IgGa and IgG(T) concentrations. In contrast, only adult horses and 3-month-old foals mounted significant vaccine-specific total IgG, IgGb, and IgM responses. Vaccine-specific concentrations of IgM and IgG(T) were significantly different between all groups, with the highest concentrations occurring in adult horses, followed by 3-month-old foals and, finally, 3-day-old foals. Only the adult horses mounted significant vaccine-specific lymphoproliferative responses. Baseline gamma interferon (IFN-γ) and interleukin-4 (IL-4) concentrations were significantly lower in 3-day-old foals than in adult horses. Vaccination resulted in a significant decrease in IFN-γ concentrations in adult horses and a significant decrease in IL-4 concentrations in 3-day-old foals. After vaccination, the ratio of IFN-γ/IL-4 in both groups of foals was significantly higher than that in adult horses. The results of this study indicate that the humoral and lymphoproliferative immune responses to this killed adjuvanted vaccine are modest in newborn foals. Although immune responses improve with age, 3-month-old foals do not respond with the same magnitude as adult horses.     

Human and rodent humoral immune responses to Andes virus structural proteins

In the present work we identified B-cell epitopes recognized by sera of humans and rodents naturally infected with Andes virus, a hantavirus present in Chile and Argentina. Analysis of patient and rodent sera with overlapping peptides revealed 21 human and rodent epitopes on the three structural proteins. Whereas in the nucleoprotein the region comprising aa 248-260 was shown to be the key determinant of human sera, the major antigenic site of rodent antibody reactivity is located at aa 326-338. In G1, the main epitope recognized by human sera was mapped to aa 14-26, while rodent antibodies bound predominantly to aa 599-611. In contrast, humans and mice had strong responses to three regions in G2 (aa 691-703, aa 918-930, aa 955-967), of which the last two are associated with neutralization of Hantaan virus. This insight affords important information for the development of immunotherapies for the acute phase of hantavirus cardiopulmonary syndrome.     

In vivo studies of cell-mediated and humoral immune responses to adenovirus 12-induxced tumour cells

Summary Mice immunized with 3 or 5 doses of cell-free extract from adenovirus 12-induced tumour cells were relatively immune to subsequent challenge with 5 × 10⁵ viable adenovirus 12-induced tumour cells. Using spleen cell transfer experiments, this immunity was shown to be cell mediated. Thus, subcutaneous inoculation with spleen cells from mice immunized with tumour extract together with tumour cells in a 151 ratio, respectively, inhibited tumour growth, as shown by a reduction in tumour incidence and the size of tumours, compared to the control mice inoculated with tumour cells and normal mouse spleen cells. When the tumour cells and spleen cells were given by different routes of inoculation, inhibition was not as marked as when the two were given together. Mice inoculated with spleen cells, from mice immune to challenge with live tumour cells (tumour immune mice) or spleen cells from mice bearing large transplantable tumours, were shown to be relatively immune to transplanted tumour cells. Transfer of serum from mice immunized with tumour extract to normal mice did not confer any immunity to transplanted tumour cell challenge; tumour growth was neither retarded or enhanced, compared to control mice given normal CBA mouse serum. Transfer of serum from mice immune to tumour cell challenge also did not affect the growth of tumours. However, transfer of serum from tumour-bearing mice resulted in an apparent earlier development of tumours in two distinct experiments; however, in neither experiment was the difference statistically significant compared to the tumour incidence in control mice.     

Time course of humoral and cell-mediated immune responses to human papillomavirus type 16 in infected women

The time course of cell-mediated and humoral immune responses was elucidated in eight women with human papillomavirus type 16 (HPV-16) infection by performing serial HPV-16 E6 and E7 cytotoxic T-lymphocyte (CTL) assays and HPV-16 virus-like particle (VLP) antibody analyses. Four subjects had a single incident of HPV-16 DNA detection, and four subjects had two periods of HPV-16 DNA detection. In two of the women in the latter group, the second episode of HPV-16 detection occurred in the presence of high titers of HPV-16 VLP antibody, bringing into question the protective role of humoral immunity in preventing repeated infection. However, all four subjects rapidly became HPV-16 DNA negative following the second detection of HPV-16 DNA, suggesting the presence of immunological memory. In addition, one subject rapidly became negative for HPV-16 DNA despite having no evidence of CTL or VLP antibody response prior to the second HPV-16 DNA detection, suggesting the presence of immunological responses at an undetectable level. Overall, seven of eight subjects (88%) had detectable HPV-16 E6 and/or E7 CTL responses and seven of eight women (88%) had detectable HPV-16 VLP antibody responses.     

Biological and molecular characterization of Avian influenza virus (H9N2) isolates from Iran

Three Influenza A virus (H9N2) isolates obtained from three separate broiler flocks with variable mortality rates were cloned twice in embryonated SPF chicken eggs by limiting dilution. Biological properties of these isolates were examined in 4-week-old SPF chickens and chick embryo fibroblast (CEF) cultures. The isolates neither caused mortality in the inoculated chickens nor produced CPE in cell cultures, indicating low pathogenicity. PCR products of 486 bp containing the sequences for hemagglutinin (HA) cleavage site, which were generated from the isolates, were subjected to nucleotide sequencing. Sequence analysis of the HA region containing the cleavage site of the isolates showed a similar sequence motif (PARSSRG) but different flanking regions. Phylogenetic analysis of deduced amino acid sequences revealed that the isolates were closely related to those isolated earlier, indicating a common source. Moreover. the amino acid sequences of the recent isolates were very similar to those from Saudi Arabia, Germany and Pakistan. It is postulated that, except for some Chinese isolates, the pathogenicity of Iranian isolates seems to be similar to that of other Eurasian isolates. It is possible that an elevation in mortality rate under field condition could be caused by co-infection of recent isolates with the bacteria such as mycoplasma, Escherichia coli, and Ornithobacterium rhinotracheale rather than by an emerging a pathogenic H9N2 subtype of the virus.     

Timing of Toll-like receptor 9 agonist administration in pneumococcal vaccination impacts both humoral and cellular immune responses as well as nasopharyngeal colonization in mice

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs, CpG ODN, are Toll-like receptor 9 agonists (TLR9a), which have been used as adjuvants in pneumococcal vaccines to improve antibody responses in immunodeficient patients. Here, we examined whether the coadministration of TLR9a with pneumococcal CRM(197)-conjugate vaccine enhances protection against pneumococcal colonization, the levels of antipolysaccharide antibodies, and the CD4(+) T-cell responses. Wild-type BALB/c mice and B-cell-deficient BALB/c Igh-J(tm1Dhu) mice were immunized twice with the following: (i) PCV alone; (ii) simultaneous PCV and TLR9a; (iii) PCV and then TLR9a, after a 48-h delay; (iv) TLR9a alone; and (v) phosphate-buffered saline. Nasopharyngeal protection, serum antibodies, CD4(+) T-cell responses, and clearance of bacteremia after intraperitoneal challenge with Streptococcus pneumoniae 6B were evaluated. We found decreased nasopharyngeal protection against S. pneumoniae 6B colonization after simultaneous immunization with PCV and TLR9a compared to immunization with PCV alone in wild-type BALB/c mice (P = 0.037). A similar trend was observed in B-cell-deficient BALB/c Igh-J(tm1Dhu) mice. Simultaneous administration did not enhance antibody levels and lowered the CRM(197)-specific cytokine release of gamma interferon, interleukin-2 (IL-2), IL-5 and IL-13. Immunization with PCV and then TLR9a, after a 48-h delay, significantly improved nasopharyngeal protection compared to simultaneous administration (P = 0.011). Furthermore, delaying TLR9a delivery increased antibody titers compared to both simultaneous administration (P = 0.001) and PCV immunization alone (P = 0.026). In conclusion, the immunological and clinical impact of adjuvanting a pneumococcal conjugate vaccine (Prevnar; Pfizer) with a TLR9a is highly depended on timing of the adjuvant administration. Thus, careful timing of adjuvant administration may improve novel vaccine formulations.     

Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (10¹–10⁶ EID₅₀). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.     

Comparison of different inoculation methods of H9N2 subtype avian influenza virus in broiler chickens

H9N2 of avian influenza (AI) subtype has been reported from commercial poultry farms in many countries. This virus has been circulating in poultry industry of different parts of Iran for the last decade. To study the infection of avian influenza H9N2 in chicken of Kerman, one of the characterized H9N2 subtype of Iranian isolate was inoculated by intravenous (IV), intratrachea (IT), and intranasal-ocular (INO) routs in 6-week-old broiler chickens. The trachea, lung, kidney, and fabriciuos bursa tissues were taken at 1–10?days post-inoculation (PI). Each of the samples was divided into two parts. One part was kept in ?70°C for virus isolation. The second part was kept in formalin buffer for preparing paraffin embedded tissue sections. The tissue sections were subjected to indirect immunofluorescence assay (IIF) assay using a monoclonal antibody against N2 influenza antigen and goat-anti-mouse fluorescein isothiocyanate (FITC) conjugated antibody. The results showed that the inoculated virus isolated from lung and kidney by IV, trachea and lung by IT, and trachea by INO methods. The sensitivity and specificity were 88% and 60% for IIF assay, respectively. In addition, the positive and negative prediction values were 64% and 86%, respectively. The accuracy IIF assay compared with virus isolation was 73.3%. It could be suggested that specificity and positive and negative prediction values for tissue samples testing were better in IIF assay using monoclonal antibodies than the virus isolation test.     

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D¹⁴⁴ mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers DQ408506-DQ408529.     

Family Clusters of Avian Influenza A H7N9 Virus Infection in Guangdong Province,China

Since its first identification, the epizootic avian influenza A H7N9 virus has continued to cause infections in China. Two waves were observed during this outbreak. No cases were reported from Guangdong Province during the first wave, but this province became one of the prime outbreak sites during the second wave. In order to identify the transmission potential of this continuously evolving infectious virus, our research group monitored all clusters of H7N9 infections during the second wave of the epidemic in Guangdong Province. Epidemiological, clinical, and virological data on these patients were collected and analyzed. Three family clusters including six cases of H7N9 infection were recorded. The virus caused severe disease in two adult patients but only mild symptoms for all four pediatric patients. All patients reported direct poultry or poultry market exposure history. Relevant environment samples collected according to their reported exposures tested H7N9 positive. Virus isolates from patients in the same cluster shared high sequence similarities. In conclusion, although continually evolving, the currently circulating H7N9 viruses in Guangdong Province have not yet demonstrated the capacity for efficient and sustained person-to-person transmission.     

Phleum pratense pollen starch granules induce humoral and cell-mediated immune responses in a rat model of allergy

BACKGROUND: Timothy grass (Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. OBJECTIVE: We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. METHODS: Brown-Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 microm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. RESULTS: An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. CONCLUSIONS: It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma.