Characterization of cytokine production in infectious mononucleosis studied at a single-cell level in tonsil and peripheral blood.

Cytokine profile and production was studied at a single-cell level in cells obtained from 14 patients with acute infectious mononucleosis (IM), with less than 7 days of symptomatic disease, by use of cytokine-specific MoAbs and indirect immunofluorescence technique. In producer cells, all the studied cytokines, except IL-1, accumulated in the Golgi system, which resulted in a characteristic morphology of the staining. Less than one in a thousand mononuclear cells obtained directly from IM blood and stained within 2 h of sampling produced IL-2, interferon-gamma (IFN-gamma), IL-4, IL-5, IL-6, IL-10, GM-CSF, tumour necrosis factor-alpha (TNF-alpha) or TNF-beta, spontaneously. However, these cells were induced to cytokine synthesis by T cell receptor ligation in vitro using immobilized anti-CD3 MoAbs for 2-3 h restimulation under conditions which did not activate normal cells. By this approach 168 +/- 120 cells/10,000 peripheral blood mononuclear cells produced IFN-gamma as compared with 10 +/- 8 cells/10,000 non-stimulated cultured cells obtained from IM patients (P < 0.001) and 1/10,000 cells obtained from healthy controls, respectively. No induced production of IL-2, IL-3, IL-4, IL-5, IL-10, GM-CSF or TNF-beta was detected in IM cells obtained from peripheral blood by this restimulation. In contrast, a spontaneous cytokine production was evident in tonsil material obtained from four IM patients tonsilectomized because of respiratory obstruction. From this site 160 +/- 40 cells/10,000 cells produced IL-2, 40 +/- 30 cells IL-6, 30 +/- 30 cells TNF-beta and 35 +/- 25 cells IFN-gamma, respectively. No such spontaneous IL-2, IL-6, TNF-beta or IFN-gamma production was evident in control cells obtained from patients tonsilectomized because of chronic tonsil hyperplasia.     

Absence of Epstein-Barr virus-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes in infectious mononucleosis.

Cytotoxic T lymphocytes (CTL) with the CD4+ phenotype that recognize major histocompatibility complex (MHC) class II antigens are detectable very frequently in cultures of human alloreactive or virus-specific T cells. The significance of these CD4+ CTL for an immune reaction in vivo is not clear. Since Epstein-Barr virus (EBV) transformed B cells express HLA-class I and class II antigens equally well both CD8+ and CD4+ CTL should be stimulated during an acute EBV infection. We analysed the MHC specificity and the phenotype of EBV-specific CTL from patients with infectious mononucleosis (IM). When tested directly without any previous culture, T cells from patients in the acute phase of IM showed specific MHC-restricted cytotoxicity against the autologous B cell line. Addition of a HLA class I specific monoclonal antibody (MoAb) but not of a HLA class II specific MoAb resulted in a complete blocking of the lytic activity. Cell sorting revealed that the entire cytotoxic activity was present in the CD8+ fraction whereas no specific CTL were detectable in the CD4+ fraction. The absence of cytotoxicity in CD4+ cells was not due to a lack of activation of these cells since both CD8+ and CD4+ cells were activated in situ, showing spontaneous growth in interleukin-2 (IL-2) and expressing the activation marker TP103. Frequency estimation revealed that 1/300-1/600 CD8+ but only 1/2000-1/4000 CD4+ T cells gave rise to a specific CTL colony after 10 days. If CD4+ colonies were tested repeatedly for cytotoxicity we found that CD4+ CTL acquired their cytotoxicity during in vitro culture. In addition, we isolated EBV-specific CD4+ T cell clones able to lyse their stimulator cells in the presence but not in the absence of lectin, even after a long period of culture. Taken together our results show that cytotoxicity mediated by CD4+ T cells does not play a role in an anti-viral immune response.     

Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response

Repeated challenge with antigen is involved in the pathogenesis of a variety of pulmonary diseases. Patients with cystic fibrosis (CF) experience recurrent pulmonary colonization with Pseudomonas aeruginosa before establishment of chronic lung infection. To mimic recurrent lung infections in CF patients, the lungs of susceptible BALB/c mice were re-infected with P. aeruginosa 14 days after the initial infection. Singly-infected BALB/c mice, as well as non-infected mice, were used as controls. Decreased mortality and milder lung inflammation in re-infected BALB/c mice, as well as a tendency for improved clearance of bacteria, was observed when compared with singly-infected mice. The improved outcome in re-infected mice correlated with changes in CD4 cell numbers. Surface expression of LFA-1 on pulmonary CD4 cells was increased in re-infected compared with singly-infected mice. Moreover, resistance to re-infection was paralleled by a shift towards a Th1-dominated response and increased IL-12 production. No significant increase in serum IgG was observed in the re-infected mice. In conclusion, these results indicate a protective role for a Th1-dominated response, independent of antibody production, in chronic P. aeruginosa lung infection in CF.     

EIAV减毒疫苗诱导马外周血单个核细胞Th1型细胞因子的转录

目的：探讨马传染性贫血病毒驴白细胞减毒疫苗免疫马后,外周血单个核细胞中Th1型细胞因子转录水平与免疫保护应答的关系,揭示DLV的免疫保护机制。方法：应用分子克隆及实时定量RT-PCR技术,建立了马PBMC中IFNγ-、IL-2、IL-12 mRNA转录水平的定量检测方法,定期观察4组（疫苗免疫组、阴性对照组、强毒株阳性对照组、自然感染组）12匹马外周血PBMC中细胞因子的转录水平及分布特征,同时监测体温变化等指标。疫苗株免疫动物8个月后,用EIAV强毒株攻击,观察攻击前后细胞因子转录水平的变化。结果：DLV免疫马,在免疫后3周外周血PBMC中IFN-γ、IL-2转录量显著高于阴性对照组及自然感染组（P〈0.01）;免疫后用EIAV强毒株攻击,IFNγ-、IL-2和IL-12转录的量明显升高,免疫马获得完全保护;强毒株攻击阳性对照马IFN-γ、IL-2转录量随疾病进展波动,发热期下降。结论：本研究首次证明EIAV减毒疫苗可诱导马外周血PBMC中IFN-γ、IL-2、IL-12基因高效转录,其转录水平与DLV的免疫保护密切相关,此结果在分子水平为阐明DLV的免疫保护机制提供了新的实验依据。     

传染性单核细胞增多症的临床病理及EB病毒定位性研究

目的 研究传染性单核细胞增多症(IM)的临床特征、病理特点、免疫表型和EB病毒原位感染特征,以提高对IM的认识和诊断水平.方法 采用HE染色以及免疫组织化学、原位杂交技术,结合临床资料分析,对15例IM进行了临床病理、免疫表型和EB病毒感染的研究.结果 (1)IM多见于儿童和青年人(中位年龄18岁),起病急,常有发热(12例),伴浅表淋巴结肿大,多数在短期内痊愈.(2)病变以T区增生为主,斑驳状改变常见,细胞混杂,种类多样,可见B细胞分化谱(活化淋巴样母细胞、免疫母细胞、浆样细胞、浆细胞),包膜不厚,间质不多.(3)病变中以CD3阳性的小T淋巴细胞为主,部分活化的淋巴样母细胞和免疫母细胞表达CD20和CD30,信号强弱不等,散在分布.(4)所有病例都有EB病毒编码的小RNA(EBER)阳性细胞,数量多少不一(10～100个/HPF),大中小淋巴细胞均可阳性,主要分布在T区,也见于套区、初级滤泡和生发中心内.结论 进一步确认了IM是EB病毒引起的一种急性自限性淋巴组织增生性疾病.IM在临床、病理、免疫表型和EB病毒感染方面都具有特点,只有综合考虑这4方面的信息才能减少错误,做出更准确的诊断.     

Influence of epidemiological factors on blood transfusion

The prevalence, incidence and risk factors of infectious diseases observed in the general population have been described to directly influence transfusion medicine, especially the blood selection. The objective is to ensure the blood safety. The characterization of modes of transmission influences the donor selection: the risk factors of the main blood‐borne infections have permitted to adapt the pre‐donation questionnaire in order to exclude at‐risk donors. The prevalence of infections also has an impact on the blood screening strategy. For example, anti‐HBc antibody (Ab) screening is currently performed only in countries where the HBV prevalence is compatible with a reasonable number of donor exclusions. HTLV Ab screening is implemented in countries in which the rate of donors originating from endemic areas could represent a risk for blood components. Measurement of incidence which contributes to the residual risk has led to the introduction of nucleic acid testing (NAT) for HIV, HCV and in some cases for HBV in viral screening strategy in many countries worldwide. The observed NAT yield differs according to the incidence of the infection and according to the country. Finally, the putative blood transmission of new and emerging pathogens has led to implement specific and non‐specific measures in order to enhance blood safety. Conversely, although the blood donor population is selected, the data observed in this population have also contributed to better understand epidemiology and pathogenesis of infection. Moreover, owing to the recent progress in developing modelling approaches for estimating risk, we are able to anticipate a transfusion transmission threat by introducing, when necessary, specific measures intended for reduce this risk.     

Optimizing vaccine design for cellular processing,MHC binding and TCR recognition

In this review we describe the methods and processes that our group have developed while aiming to test and design multiepitope vaccines for infectious diseases and cancer. Testing the performance of vaccines composed of epitopes restricted by human leukocyte antigen (HLA) molecules is accomplished by in vitro antigenicity assays, as well as in vivo immunogenicity assays in HLA transgenics. The efficiency by which multiepitope vaccines are processed is optimized by spacer residues, which are designed to facilitate generation by natural processing of the various class I- and class II-restricted epitopes. Methods and strategies to test and optimize HLA binding affinity, patient coverage from the vaccine construct, and TCR recognition of HLA/epitope complexes are also discussed.     

Acute optic neuritis: a virological study in relation to multiple sclerosis.

Previous ultrastructural examination of peripheral blood lymphocytes revealed the presence of intranuclear filamentous structures in multiple sclerosis (MS) and in some optic neuritis (ON) patients. The present investigation was undertaken in the attempt to correlate the presence of such structures with the etiology of ON and MS and possibly to demonstrate the viral origin of the filaments. Suitable virological and serological techniques were used to detect and isolate infectious agents from peripheral blood samples and body excretions of 12 monosymptomatic ON patients at their first acute attack. Nevertheless, any efforts to demonstrate the presence of a virus in these patients have been unsuccessful: no evidence of active viral infection was obtained by serological studies of serum and cerebrospinal fluid samples, nor could viral antigens or inclusions be observed by immunofluorescence and cytochemical analysis. Negative results were also obtained from studies performed in parallel on MS patients and various controls. The significance of the failure to isolate infectious agents from either ON and MS patients is discussed.     

T lymphocyte anergy during acute infectious mononucleosis is restricted to the clonotypic receptor activation pathway.

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by the Epstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclear cells (PBMC) obtained from IM patients showed a significant specific impairment in their proliferative response to both phytohaemagglutinin (PHA; P less than 0.05) and to an anti-CD3 MoAb (P less than 0.001), although both responses reached normal control levels by addition of a submitogenic dose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activation signals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways (PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC. In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA was impaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 or ionophore. Lastly, PHA failed to induce IL-2 alpha receptor (IL-2R alpha) expression in IM PBMC from four tested patients, but the presence of PMA completely corrected this defect. Taken together, these results strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor (TCR)-specific impairment in the induction of genes involved in T cell proliferation (including those coding for IL-2 and IL-2R alpha) upon membrane signalling to otherwise normal T lymphocytes, since CD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in these particular pathological conditions (and perhaps in most in vivo situations). This and other similar experimental approaches to transient secondary immunodeficiencies may help to unravel the physiopathological role of different surface molecules in T cell activation.     

Sequence Comparison of Avian Infectious Bronchitis Virus S1 Glycoproteins of the Florida Serotype and Five Variant Isolates from Georgia and California

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6–99.3% similar to one another and only 76.6%–76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55–96, 115–149, 255–309, and 378–395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas. This revised version was published online in July 2006 with corrections to the Cover Date.