Surface activation markers of T lymphocytes: role in the detection of infection in neonates

Diagnosis of perinatal infection in the newborn is difficult; there may be few clinical signs and current tests are slow or non-specific. Detection of organisms, antigen or specific antibody to common pathogens often requires repeat samples and does not give immediate results. Haematological parameters, although relied upon frequently to diagnose infection in the neonate prior to a positive bacterial isolation, are unreliable and insensitive. Indicators such as an increase in neutrophil band cell counts are highly variable between morphologists. Infection induces the expression of a number of T lymphocyte surface markers, including CD45RA/CD45RO and CD45RO. The use of changed expression of surface markers as a laboratory test for detection of infection in neonates was evaluated. We used multiparameter flow cytometry to detect expression of early (CD45RA/CD45RO) and late (CD45RO) activation markers. In the respective groups of 50 full term (including 25 normal vaginal deliveries and 25 caesarean deliveries) and 30 premature, i.e. < 36 weeks gestation (born by either normal vaginal delivery or caesarean delivery) the CD45RA isoform was brightly expressed on newborn ‘naive’ CD4⁺ T cells, whereas the CD45RO isoform (including both ‘bright’ and ‘dim’ populations) was present on < 19% of CD4⁺ T cells from these newborn infants. In a group of 37 infants, tested to evaluate possible effects of non-infective parameters such as respiratory distress and iso-immunization, no significant changes in surface marker expression were found and specificity of the test was confirmed. In 14 neonates with documented sepsis, up-regulation of dual staining CD45RA/CD45RO isoforms on CD4⁺ T cells was detected early in the infection. In addition, we found that CD45RO expression persisted for several weeks after bacterial infection, and up to several months in viral infection. In conclusion, detection of T cell activation by flow cytometry for the early diagnosis of neonatal infection is an easy test to carry out on small volumes of blood, is inexpensive, and may be a specific indicator of infection.     

Immunoreactivity of sinusoids in hepatoblastoma: an immunohistochemical study using lectin UEA-1 and antibodies against endothelium-associated antigens, including CD34

Sinusoids are found not only in the normal liver but also in certain liver tumours, including hepatoblastoma, the most common malignant liver tumour in childhood. In this study, sinusoids in 12 hepatoblastomas, of various subtypes, and in normal liver were investigated with UEA-1 and antibodies against von Willebrand's factor, CD31 and CD34 to detect differences of possible diagnostic significance. In the normal liver, staining of sinusoids was seen with all these markers, but it was focal and confined to a few sinusoids near the portal tracts. In hepatoblastoma, the endothelial markers reacted with the sinusoids to varying extents. UEA-1 and anti-CD34 usually stained the majority of these vessels, anti-CD34 staining greater numbers of sinusoids and with greater intensity. Immunostaining revealed that both number and spatial organization of sinusoids in hepatoblastoma are dependent on the subtype. In addition to staining of endothelium, one of the two small cell hepatoblastomas exhibited strong immunoreactivity of the tumour cells for CD34. These findings show that the marked difference in sinusoidal immunoreactivity for CD34 between normal liver and hepatoblastoma could be useful for discriminating between non-neoplastic liver tissue and highly differentiated fetal hepatoblastoma. Our findings also show that small cell hepatoblastoma, in addition to acute leukaemia, should be considered when immunoreactivity for CD34 is found in small round and blue cell tumours in childhood.     

Reizhusten, Belastungsdyspnoe und zystische Lungenveränderungen bei einem 28jährigen Patienten

Zusammenfassung Eine neu aufgetretene Dyspnoesymptomatik und eine symmetrische, apikal betonte retikulonodul?re Zeichnungsvermehrung im R?ntgen-Thorax bei jungen rauchenden Erwachsenen müssen an das seltene Krankheitsbild der pulmonalen Histiocytosis X denken lassen. Klinischer Befund, laborchemische- und Lungenfunktionsuntersuchungen zeigen unspezifische Befunde. Neue radiologische Verfahren wie die hochaufl?sende Computertomographie (HRCT) leisten bei gezielter Indikationsstellung eine entscheidende differentialdiagnostische Hilfestellung. Dieser Fall verdeutlicht die M?glichkeit einer Diagnosesicherung durch transbronchiale Biopsien unter Verzicht auf offene Lungenbiopsien. Eine ambulante Diagnostik war m?glich, das h?here Risiko eines operativen Eingriffes konnte vermieden werden. Die Indikation zur Therapie ist nicht gesichert und wird daher durch den Grad der subjektiven bzw. funktionellen Einschr?nkung sowie den Verlauf bestimmt.     

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

Elevation of plasma thioredoxin levels in HIV-infected individuals

Thioredoxin (Trx), a ubiquitous protein intimately involvedin redox and protein disulfide reductions, has been shown tobe released from cells and to have cytokine-like activities.In addition, Trx has been implicated in the redox regulationof immunological responses and shown to be deficient in tissuesfrom AIDS patients. In studies presented here, plasma Trx levelswere measured by ELISA in plasma samples from HIV-infected individuals(n = 136) and HIV-negative controls (n = 47). To account forthe release of Trx into plasma due to hemolysis, the Trx measurementswere corrected according to the level of hemoglobin in the plasmasample. Data presented show that, in contrast to tissue Trxlevels, corrected plasma Trx levels are significantly higherin HIV-infected individuals than in controls (P < 0.0001).Furthermore, {small tilde}25% of the HIV-infected individualsstudied have plasma Trx levels greater than the highest levelfound in controls (37 ng/ml). Detailed multiparameter FACS analysisof peripheral blood mononuclear cells (PBMC) from the infectedindividuals demonstrates that those with higher plasma Trx levels(37 ng/ml or greater) tend to have lower overall CD4 counts.In addition, increases in plasma Trx levels correlate with decreasesin monochlorobimane staining (indicative of lower intracellularglutathione levels in PBMC) and with changes in surface antigenexpression (CD62L, CD38 and CD20) that occur in the later stagesof HIV infection. These correlations suggest that elevationof plasma Trx levels may be an important component of advancedHIV disease, perhaps related to the oxidative stress that oftenoccurs at this stage.     

脐血CD34~+细胞体外短期培养扩增研究

为寻找更有效的体外扩增脐血CD34 + 细胞的造血细胞因子组合 ,采集健康产妇脐带血 ,用免疫磁珠法分选CD34 + 细胞。采用SCF、FLT3 L、TPO和IL 34种具有早期作用的细胞因子的不同组合进行脐血CD34 + 细胞短期无血清液体培养 ,观察培养前后有核细胞、CD34 + 细胞、CD34 + /CD38- 细胞、CFU GEMM、CFU GM和BFU E数量的变化。结果在 3种不同的细胞因子组合中 ,同时应用SCF、FLT3 L、TPO和IL 34种细胞因子培养 7d的扩增效果最好。突出的发现是在这种条件下CD34 + /CD38- 细胞亚群达到平均 1 97.9倍的扩增效果。提示 :SCF、FLT3 L、TPO和IL 34种细胞因子是脐血CD34 + 细胞体外扩增理想的细胞因子组合     

Lectin-induced increase in clonogenic growth of haematopoietic progenitor cells

Abstract: The galactoside-specific plant lectin, Viscum album agglutinin (VAA-I) increases cellular parameters of natural host defence. It also binds to a variety of haematopoietic cells, including progenitors. We investigated whether VAA-I has a stimulatory effect on haematopoietic progenitor cells. Peripheral blood progenitor cells from 7 healthy volunteers were cultured in a colony assay with VAA-I plus erythropoietin (EPO) and stem cell factor (SCF). At 50 pg/ml VAA-I induced a significant increase in the cytokine-dependent clonogenic growth (52% in median, p<0.05). In another set of experiments purified CD34+ cells were isolated from the bone marrow aspirate of 4 patients with non-metastatic breast cancer using fluorescence-activated cell sorting. Binding to CD34+ cells was demonstrated by using directly fluorescence-conjugated VAA-I. Co-incubation with d -galactose significantly abrogated this effect. CD34+ cells were cultured in the presence of EPO, SCF, interleukin-3, granulocyte/monocyte colony-stimulating factor and granulocyte colony-stimulating factor. VAA-I alone had no measurable effect on the clonogenic growth of the isolated cells. However, at concentrations of 100 and 250 pg/ml VAA-I increased the cytokine-dependent proliferation and differentiation of CD34+ cells by a median of 75 and 85%, respectively. The results show that VAA-I binds to haematopoietic progenitor cells and has a co-stimulatory effect on their proliferation.     

Interactions between peripheral blood CD8 T lymphocytes and intestinal epithelial cells (iEC)

Intestinal intraepithelial lymphocytes (iIEL) are primarily CD8 cells and most of them have a CD28^? phenotype, the phenotype of effector cytotoxic T cells. We asked whether the predominance of CD8⁺ CD28^? T cells in the gut may result from peripheral blood T cells preferentially migrating to the iIEL compartment and adhering to iEC. Compared with CD4 cells, adhesion of resting CD8⁺ T cells to iEC cell lines was significantly higher. Adhesion could be blocked with a MoAb to gp180, a molecule expressed on iEC which is known to interact with CD8/lck. No significant difference in the level of adhesion was observed between CD8⁺ CD28⁺ and CD8⁺ CD28^? T cells. Thus CD8 cells may preferentially migrate to the iIEL compartment, but loss of CD28 expression could occur in situ after migration. Consistent with this hypothesis, the CD8⁺ CD28^? cells became enriched after co-culturing T cells with iEC cell lines and primary iEC. Induction of the CD8⁺ CD28^? phenotype in cord blood and adult T cells was observed in co-cultures with iEC and also with mitogens and superantigens. In the latter case, CD28 down-modulation was seen specifically in the Vβ subset targeted by the superantigen, indicating that loss of CD28 expression is a direct result of T cell receptor (TCR)-mediated stimulation. The combined results suggest that CD8⁺ CD28^? T cells are antigen experienced T cells, and that they may have a survival advantage in the presence of gut epithelial cells in vitro. This may contribute to the predominance of CD8⁺ CD28^? T cells in the iIEL compartment.