Enumeration of haemagglutinin-specific CD8+ T cells after influenza vaccination using MHC class I peptide tetramers.

With emergence of MHC class I tetramers loaded with CD8+ T-cell viral epitopes, it is possible to study virus-specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)-specific T cells in 26 healthy influenza-vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four-colour flow cytometry was used for monitoring of vaccine induced T-cell response. In 15 donors, two- to fivefold increase in frequency of HA-specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA-specific CD8+/IFN-gamma+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA-specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA-specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.

Outcome of influenza vaccination in combat-related post-traumatic stress disorder (PTSD) patients.

Post-traumatic stress disorder (PTSD) is an anxiety disorder that can occur after exposure to extreme traumatic experience such as war trauma, and is accompanied by fear, helplessness or horror. Exposure to trauma can result in immune dysregulation and influence susceptibility to infectious disease as well as vaccine efficacy. The aim of the study was to determine the relation of psychological stress and the immune response to influenza vaccination in combat-related PTSD patients (n = 28). Detection of anti-viral antibody titre was performed by inhibition of haemagglutination assay. Ex vivo tetramer staining of CD8(+) T lymphocytes was used to monitor T cells specific for human leucocyte antigen (HLA)-A*0201-restricted influenza A haemagglutinin antigens before and after vaccination. Twenty patients showed a fourfold antibody titre increase to one or both influenza A viral strains, and 18 of them showed the same response for both influenza B viral strains. Ten of 15 healthy controls showed a fourfold rise in antibody titre to both influenza A viral strains and eight of them showed the same response for both influenza B viral strains. HLA-A*0201(+) PTSD patients (n = 10) showed a significant increase of influenza-specific CD8 T cells after vaccination. Although those PTSD patients had a lower number of influenza-specific CD8(+) T cells before vaccination compared to HLA-A*0201(+) healthy controls (n = 6), there was no difference in influenza A antibody titre between PTSD patients and control subjects before vaccination. The generated humoral and cellular immune response in PTSD patients argues against the hypothesis that combat-related PTSD in war veterans might affect protection following influenza vaccination.

Increased Toll-like receptor 4 expression in infants with respiratory syncytial virus bronchiolitis.

The fusion protein of the respiratory syncytial virus (RSV) binds to the pattern recognition receptors, TLR4 and CD14, and initiates innate immunity response to the virus. The aim of the study was to investigate the expression of TLR4 on peripheral blood lymphocytes and monocytes in peripheral blood of infants in both acute and convalescent phase of RSV bronchiolitis (n = 26). In addition, TNF-alpha expression in lipopolysaccharide-stimulated monocytes was also assessed. The results showed TLR4 to be expressed predominantly by monocytes in both sick infants and controls. During the acute phase of infection monocytes up-regulated TLR4 in eight infants, which returned to the levels recorded in controls 4-6 weeks from infection. There was no difference in the percentage of TNF-alpha secreting monocytes. Of the clinical parameters tested, minimal oxygen saturation was found to correlate negatively with this expression in the group of infants with increased TLR4. Additional studies are under way to correlate this finding with the outcome of the immune response to RSV.

Non-radioactive digoxigenin DNA labeling and immunologic detection of HSV PCR products.

BACKGROUND: Herpes simplex virus (HSV) is a common cause of human skin and mucous membrane infections, and also causes sporadic meningoencephalitis. As a new method for rapid HSV diagnostics, polymerase chain reaction (PCR) has been introduced in clinical laboratories. Radioactive labeling of DNA probes has become a common practice in experimental laboratories. To avoid radioactive labeling of HSV oligonucleotide probes or PCR products, non-radioactive compounds, which are easily detected by enzyme or immunoassay techniques, are introduced. OBJECTIVES: The aim of our study was (1) to introduce non-radioactive labeling of HSV DNA probe by digoxigenin-labeled dUTP; (2) to establish a rapid and reliable laboratory method for rapid HSV diagnostics; (3) to compare the PCR method with the standard virology techniques, such as cell culture virus isolation and HSV direct fluorescent antibody test (DFA). STUDY DESIGN: We have tested the efficiency of PCR method and non-radioactive labeling of HSV DNA probe for detection of HSV from 30 clinical specimens (skin and mucous membrane swabs). HSV was detected in the specimens by standard virology techniques and PCR. Replicated HSV DNA was non-radioactively labeled by random incorporation of digoxigenin-labeled deoxyuridine triphosphate (DIG-dUTP), and the hybrids were detected by the antibody conjugates and the appropriate enzyme-mediated staining reaction (DIG DNA labeling and detection kit non-radioactive, Boehringer Mannheim GmbH). RESULTS: Non-radioactive labeling of hybridization DNA probes with digoxigenin-dUTP was obtained. HSV DNA was successfully multiplied and detected in the HSV-infected cell culture supernatant; however, it was not detected in the clinical specimen supernatant or sediment. HSV DNA was detected by direct PCR method in non-centrifugated clinical specimens. CONCLUSIONS: The PCR method could be successfully used for diagnoses of HSV infections. Since the sensitivity of this method is partly limited by the virus quantity in the specimen, we recommend cultivating the virus in the cell culture at least 24 h prior to PCR. The use of non-radioactive labeling of hybridization DNA probes, such as random primed DNA labeling with digoxigenin-dUTP, has proven both sensitive and specific, and more appropriate for diagnostic purposes than radioactive DNA labeling to be used until standardized commercial tests appear.

Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry.

Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type-2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV-infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL-4 and interferon-gamma (IFN-gamma) were measured by flow cytometry. The cells from RSV-infected infants produced more IL-4 and less IFN-gamma than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-gamma production increased with disease severity. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.

Clinical and epidemiological aspects of respiratory syncytial virus lower respiratory tract infections.

This paper has analyzed respiratory syncytial virus lower respiratory tract infections in 201 hospitalized children. In children with wheezing, erythrocyte sedimentation rate (ESR) was significantly higher in those with pneumonia than with syndroma pertussis, while the white blood cell (WBC) count was significantly lower in patients with bronchitis than in those with bronchiolitis and syndroma pertussis. Bronchodilatators were applied in 75.6% and corticosteroids in 20% of patients. Ten patients were ventilated. Fatal disease outcome was observed in one infant. Twelve consecutive-year study of respiratory syncytial virus (RSV) infections showed that 27.3% of these diseases were bronchiolitis and pneumonia.

Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus.

Infection with respiratory syncytial virus (RSV) may induce asthma-like symptoms and RSV-specific IgE in infected infants as a result of Th2-like response to RSV. The effect of RSV infection on the expression of B cell antigens CD21 and CD23, putative participants in Th2 responses, was investigated. Samples from bronchiolitic infants (n = 19) were tested by three-color immunofluorescence flow cytometry during the acute phase of infection and 4-6 weeks later. In 6 of 10 RSV-positive infants, the percentage of CD23+ B cells was higher than in 9 RSV-negative children and in controls. Both CD21+ and CD21- B cells exhibited a higher percentage of CD23. The group with increased expression of CD23 antigen had RSV-specific IgE and IgG4 antibodies. These findings corroborate the hypothesis that RSV could provoke a Th2-type response, but the relationship between CD23 antigen and RSV infection must be determined.

Pityriasis rosea Gibert: detection of Legionella micdadei antibodies in patients.

Some epidemiological and clinical characteristics of Pityriasis rosea Gibert has led us to hypothesize that this disease may be the clinical manifestation of an infection caused by legionellas. We have thus tested the sera of 36 patients ill with Pityriasis rosea and 19 controls for Legionella pneumophila serogroup 1-6 and Legionella micdadei antibodies. These, who had the same age and sex distribution as study patients, were receiving treatment for other diseases in the same ward. Also tested were 200 sera from the voluntary blood donors from the same region as study patients. Legionella micdadei antibodies were detected in 12 (33.3%) Pityriasis rosea cases and in one (5.2%) control. They were significantly more common in Pityriasis rosea cases than in either controls or voluntary blood donor population. The findings to date encourage continued research into the causative relationship between the Legionella micddadei infection and the onset of Pityriasis rosea Gibert.

[Legionnaires' disease in Yugoslavia (an epicritical review of patients)]. / Legionarska bolest u nas (epikriticki pregled bolesnika).

A review of patients with Legionnaires' disease hospitalized in our Clinic from the first cases recorded in 1978 up to now has been given. The diagnosis was confirmed by indirect immunofluorescent method in 23 patients with pneumonia, using antigen and method of the Center for Disease Control Biological Products Division, Atlanta, Georgia, USA (Dr Wilkinson). Most of the patients (16) were sporadic cases and twice as less cases were from two epidemics (7). Two patients among sporadic cases developed a very severe form of the disease during the immunosuppressive therapy after kidney transplantation. Clinical, epidemiological and laboratory data of 10 patients hospitalized and treated during the last three years (1986-1988) were analyzed in details. Two thirds of these patients were above 40 years of age and the youngest one was 29 years old. Except the advanced age no other firm disposable factor was found in patients belonging to sporadic cases. In cases from epidemics the source of infection was probably water from water-pipes i.e. hotel devices (bathrooms, douches, drinking water, air-conditioning units, water used in kitchen for cooking etc). All patients recovered after a longer symptomatic and antibiotic treatment. Erythromycin and rifampicin were applied the most frequently. In some patients treatment with chloramphenicol was effective.

[An epidemic of legionnaires' disease in a hotel]. / Epidemija legionarske bolest u jednom hotelu.

An outbreak of legionnaires' disease and epidemic control measures taken at one of our hotels are described. Twenty (1% morbidity) hotel guests were affected with one dying. As a result of field investigations and survey the hot water supply system has been identified as the source of infection. Control measures which created conditions unfavourable to Legionella growth and replication in the hot water system stopped the outbreak.