Heat-stable Antigen Research Articles

EnterotoxigenicEscherichia coliMultilocus Sequence Types in Guatemala and Mexico

The genetic backgrounds of 24 enterotoxigenic Escherichia coli (ETEC) strains from Mexico and Guatemala expressing heat-stable toxin (ST) and coli surface antigen 6 (CS6) were analyzed. US travelers to these countries and resident children in Guatemala were infected by ETEC strains of sequence type 398, expressing STp and carrying genetically identical CS6 sequences.

Characterisation of antibodies for the immunochemical detection of Enterobacter sakazakii

An indirect competitive enzyme immunoassay of Enterobacter sakazakii has been developed. The rabbit polyclonal antibodies to heat-labile or heat-stable antigen of the type strain E. sakazakii CNCTC 5739<sup>T</sup> were prepared for these purposes. The detection limits of enzyme immunoassays were within the range 0.6–14.4 × 10<sup>5</sup> cells/ml. Antibodies raised to heat-labile antigen were serotype-specific. Although they contain non-specific IgG fractions binding periplasmatic and cytosol proteins, the interactions of these immunoglobulins are not manifested under conditions of ELISA developed.

Efficient magnetic bead-based separation of HIV-1-infected cells using an improved reporter virus system reveals that p53 up-regulation occurs exclusively in the virus-expressing cell population

HIV-1 infection in cell lines is very efficient, since the target population is clonal and highly dividing. However, infection of primary cells such as CD4 T lymphocytes and monocyte-derived macrophages is much more difficult, resulting in a very small percentage of infected cells. In order to study events occurring in productively infected primary cells, we determined that a way to isolate this population from bystander cells was needed. We engineered a novel HIV-1-based reporter virus called NL4-3-IRES-HSA that allows for the magnetic separation of cells infected with fully competent virions. This X4-using virus encodes for the heat-stable antigen (HSA/murine CD24) without the deletion of any viral genes by introducing an IRES sequence between HSA and the auxiliary gene Nef. Using commercial magnetic beads, we achieved efficient purification of HIV-1-infected cells (i.e. purity >85% and recovery >90%) from diverse primary cell types at early time points following infection. We used this system to accurately quantify p53 protein levels in both virus-infected and uninfected bystander primary CD4(+) T cells. We show that p53 up-regulation occurs exclusively in the infected population. We devised a strategy that allows for an efficient separation of HIV-1 infected cells from bystanders. We believe that this new reporter virus system will be of great help to study in depth how HIV-1 interacts with its host in a primary cells context.

A rapid recombination assay of HIV-1 using murine CD52 as a novel biomarker

Biomarkers are commonly used for verification of infection in conjunction with the development of viral vectors or experiments involving virus infection. Leukocyte surface antigens (CDs) are a prime option for biomarkers since they can be easily visualized and analyzed by flow cytometry after indirect fluorescent staining. For analyses of human cells, murine CD24 (Heat Stable Antigen: HSA) and CD90.2 (Thy-1.2) are currently being used. In the study reported here, we attempted to develop a rapid system for measuring retroviral genome recombination efficiency. For this purpose, we looked for an alternative CD molecule which could be used as a marker on a viral vector concurrently with other markers. We found that murine CD52 is suitable for this purpose because of its small gene size, low inhibitory effect on virus production, and measurable level of surface expression. With this novel biomarker, we succeeded in developing a rapid viral recombination measuring system using a flow cytometer.

Comparative evaluation of parasitology and serological tests in the diagnosis of visceral leishmaniasis in India: a phase III diagnostic accuracy study

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.

Heterogeneity of Non-serotypableCampylobacter jejuniIsolates

Several phenotypic and genotypic methods are currently used to identify subtypes of Campylobacter jejuni isolates. Of the phenotypic methods one, i.e. serotyping based on the heat stable antigen, is often hindered by the relatively large number of un-typable (NT) strains. Little is known, however, about the heterogeneity of the group formed by these NT strains. Therefore we serotyped 92 Hungarian, non-outbreak C. jejuni isolates and subjected the 28 NT strains to molecular analysis using PCR-RFLP of the flaA gene and to pulsed-field gel electrophoresis. With both methods the NT strains were classified into several molecular types (17 and 25, respectively), while the number of subgroups based on the results of the two techniques combined was twenty-six. These results indicate that the NT group of strains is extremely heterogeneous in Hungary, and the epidemiological connection between two NT isolates cannot be established or excluded without the use of molecular typing techniques.

B7-1-HSA (CD80-CD24), a recombinant hybrid costimulatory molecule retains ligand binding and costimulatory functions

Optimal activation of naïve T lymphocyte requires two signals; an antigen-specific signal initiated by engagement of TCR with the antigen–MHC complex and a costimulatory signal independent of the antigen receptor complex. Without the costimulatory signal, T cells become anergic. Various adhesion molecules, such as B7-1 (CD80) and heat stable antigen (HSA, CD24), expressed on antigen presenting cells have been demonstrated to provide costimulatory signals to T cells. It was reported that the combinations of different adhesion molecules could induce even stronger immune response. In this study, we made a hybrid costimulatory molecule, B7-1-HSA, and tested its T cell stimulatory function. Chinese hamster ovary (CHO) cells expressing this hybrid molecule bound both anti-CD80 and anti-CD24 monoclonal antibodies, and induced stronger T cell proliferation than CHO cells expressing B7-1 or HSA alone. These results suggest that the B7-1-HSA hybrid molecule can deliver two costimulatory signals simultaneously that can synergize in inducing T cell proliferation. The purified B7-1-HSA protein reacted with both anti-B7-1 and anti-HSA mAbs in Western blotting and specifically mediated adhesion of Jurkat cells. Furthermore, purified B7-1-HSA molecule spontaneously incorporated onto cell membrane through its glycolipid anchor suggesting that this hybrid costimulatory molecule can be used in protein transfer to develop effective cancer vaccines.

The new portrait of mammary gland stem cells

Breast cancer occurs at rate of 1 out of 7. After so many years of research, even though treatment has been dramatically improved and survival rate and length significantly increased, there is still not cure for the disease. The possible existence of mammary gland stem cell and breast cancer stem cell 1, 2 lights the hope for finding a cure to this deadly disease. The rational behind is that once the stem cell can be isolated, the signalling pathways that regulate its proliferation, self-renewal, survival and differentiation will be illustrated, and this might shed light on the mechanisms of breast cancer formation, therefore leading to a better therapeutic treatment. Existence of stem cell in normal mammary gland has been convincingly demonstrated by Kordon and Smith 1. In their report, an entire mammary gland could be regenerated with the progeny of a single cell following transplantation into cleared mammary fat pads. By definition, mammary gland stem cells are those that rarely divide and persist throughout reproductive life. Classical markers 3 for identifying and purifying mammary stem cells are label retention (tritiated thymidine or BrdU), stem cell antigen Sca-1 expression, ability to exclude dyes such as Hoechst 33342 or rhodamine 123 (side population -- SP, e.g. Hoechst 33342 negative) because of elevated expression of membrane transporter proteins, such as P-glycoproteins, and “small light” cell by electronic microscopy. However, these profiling methods are controversial and confusing. Sometimes, for a layman, it is very difficult to handle. But this situation is about to change with two recent publications in Nature. In January, Shackleton et al published “Generation of a functional mammary gland from a single stem cell” 4. In this report, the authors cleared mammary gland mixture with CD31 (endothelial marker), CD45 and TER119 (haematopoietic antigens) by FACS sorting (Lin- population). Using repopulating cleared mammary fat pad (Mammary repopulating 'units'--MRUs) as criteria, they were able to increase the MRUs from 1/4900 to 1/64 just by applying two more markers on Lin- population -- CD29 (beta1-integrin) and CD24 (heat-stable antigen). Lin-CD29hiCd24+ cells have expended differentiation ability and colony-formation ability. A single Lin-CD29hiCD24+ cell can repopulate cleared mammary fat pad and develop into a fully functioning mammary gland, demonstrating its high proliferating and multi-lineage differentiation capacity in vivo. Lin-CD29hiCd24+ cells can self-renew. In mammary gland of MMTV-Wnt-1 transgenic mice, Lin-CD29hiCd24+ population are increased and mammary gland outgrowths of Lin-CD29hiCd24+ MMTV-Wnt-1 cells are profoundly hyperplastic. Lin-CD29hiCd24+ cells were enriched for long-term label-retaining cells, CD49f+ cells. However, neither high Sca-1 expression nor Hoechst33342 dye exclusion was enriched in this population. In the February publication “Purification and unique properties of mammary epithelial stem cells” 5, Stingl et al purified CD45-Ter119-CD31-CD49fhiCD24med cells and demonstrated that they were the mammary gland stem cells. In consistency, CD45-Ter119-CD31-CD49fhiCD24med cells were Sca-1 negative and only minority of these cells can efflux Hoechst 33342 and Rhodamine-123. Interestingly, the authors took one step further to illustrate that CD45-Ter119-CD31-CD49fhiCD24med cells are in G1 or S/G2/M fractions, indicating the stem cell population is a cycling population. Most notably, these two publications completely changed the old mammary gland stem cell picture -- Hoechst 33342 negative, slowly dividing and Sca-1 positive. They demonstrated that CD45-Ter119-CD31-CD49fhiCD24med and Lin-CD29hiCd24+ are the mammary stem cell populations, whereas previous SP and Sca-1+ cells only take very few percentage of these two populations if not at all. Since label retention coincides very well with Lin-CD29highCD24+ or CD45-Ter119-CD31-CD49fhiCD24med, it joins CD29, CD49f and CD24 as one of the most efficient 4 mammary gland stem cell markers. These new markers make it easier to isolate mammary gland stem cells, therefore open a door for further characterizing these cells. Importantly, with the same markers, cancer stem cells can be purified as well. This provides a new opportunity to develop new targeted therapies to killing cancer stem cells. Finally, the report proved that mammary gland stem cells were actually cycling within cell cycle. This observation lays an important foundation for testing new methods of chemoprevention and chemotherapy.

A novel small reporter gene and HIV-1 fitness assay

Most currently available HIV-1 reporter gene constructs are large and disrupt the nef reading frame. This report describes a novel reporter gene based on the small murine heat stable antigen (HSA) protein, which is expressed on the surface of infected cells. This HSA reporter can be inserted in the vpr reading frame, leaving nef intact. Nine amino acids from the extracellular domain of HSA are replaced with an influenza hemagglutinin (HA) antibody epitope (HSA-HA). Like the parental reporter protein, this novel reporter is expressed on the surface of infected cells. Antibodies for HSA and HA specifically detect reporter viruses with each construct, indicating disruption of the original HSA antibody epitope. Finally, a strategy is developed to detect each reporter virus by real-time PCR quantitation. The growth of viruses tagged with each reporter allows precise assessment of the relative growth of viruses differing in mutations of interest. Moreover, the availability of these reporters in either of two half-genome plasmids allows convenient production of reporter and non-reporter HIV-1 by co-transfection of appropriately paired plasmids. These paired reporter viruses offer a potentially useful standardized method for measurement of HIV-1 fitness in competition assays.

Rapid Size Dependent Deletion of Foreign Gene Sequences Inserted into Attenuated HIV-1 upon Infection In Vivo: Implications for Vaccine Development

Live attenuated HIV vaccines offer a means to introduce exogenous sequences into the viral genome to target the virus elimination in vivo. Foreign genes inserted into the nef region of HIV-1 NL4-3 were found to be rapidly deleted following virus infection and/or replication, in a size dependent manner, in the human fetal Thymus/Liver implants of severe combined immunodeficient mouse (SCID-hu) model. When the murine heat stable antigen (HSA) of 283 bp was substituted into HIV-1 nef region, the viral loads in vivo were comparable to the negative control nef attenuated HIV-1, and the reporter HSA gene was not deleted upon infection. However, the murine Thy1.2 gene (505 bp) substituted into the nef attenuated HIV-1, upon infection and replication, deleted 441 bp in vitro and 437 bp in vivo, of the inserted Thy1.2 gene. When the enhanced green fluorescence protein (eGFP) gene (720 bp) was substituted for nef, virus replication was aborted in vivo in the Thy/Liv implants, as seen by the background levels of viral loads, comparable to mock infected implants, and the eGFP gene was deleted. When the herpes simplex virus thymidine kinase gene, HSV-TK (1.15 kbp), or HSA gene, was substituted into the viral vpr gene, TK but not HSA gene was deleted, upon infection in vitro. Moreover, NL-TKI reporter virus with both intact nef and vpr genes shows deletion of TK gene both in vitro and in vivo. Excision of foreign genes occurred within the exogenous segments but not in the viral own regions. These results suggest that larger "suicide" genes introduced via HIV-1 can be deleted upon infection. However, smaller size nucleotide sequences or genes (approximately 300 bp) inserted in place of viral nef or vpr gene may be used to target the virus or its components, for attack and elimination in vivo, and thus have implications for the development of live attenuated HIV vaccines.

Characterization of the early stages of thymic NKT cell development

Upon reaching the mature heat stable antigen (HSA)low thymic developmental stage, CD1d-restricted Vα14-Jα18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-γ–producing NKT phenotype. However, their more immature HSAhigh precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4+CD8+ double-positive (DP) stage, and when the CD4+ and CD4−CD8− double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSAhigh precursors in thymuses of newborn mice, including an elusive DPlow stage and a CD4+ stage, which were present at a frequency of ∼10−6. These HSAhigh DP and CD4+ stages appeared to be nondividing, and already exhibited the same Vβ8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN γδ T cells, the DN NKT cells did not originate from a pTα-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSAlow NK1.1neg CD4 cells. These findings identify the HSAhigh CD4+ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.

Analysis of Human Immunodeficiency Virus Cytopathicity by Using a New Method for Quantitating Viral Dynamics in Cell Culture

Human immunodeficiency virus (HIV) causes complex metabolic changes in infected CD4(+) T cells that lead to cell cycle arrest and cell death by necrosis. To study the viral functions responsible for deleterious effects on the host cell, we quantitated the course of HIV type 1 infection in tissue cultures by using flow cytometry for a virally encoded marker protein, heat-stable antigen (HSA). We found that HSA appeared on the surface of the target cells in two phases: passive acquisition due to association and fusion of virions with target cells, followed by active protein expression from transcription of the integrated provirus. The latter event was necessary for decreased target cell viability. We developed a general mathematical model of viral dynamics in vitro in terms of three effective time-dependent rates: those of cell proliferation, infection, and death. Using this model we show that the predominant contribution to the depletion of viable target cells results from direct cell death rather than cell cycle blockade. This allows us to derive accurate bounds on the time-dependent death rates of infected cells. We infer that the death rate of HIV-infected cells is 80 times greater than that of uninfected cells and that the elimination of the vpr protein reduces the death rate by half. Our approach provides a general method for estimating time-dependent death rates that can be applied to study the dynamics of other viruses.

Genetic Recombination of Human Immunodeficiency Virus Type 1 in One Round of Viral Replication: Effects of Genetic Distance, Target Cells, Accessory Genes, and Lack of High Negative Interference in Crossover Events

Recombination is a major mechanism that generates variation in populations of human immunodeficiency virus type 1 (HIV-1). Mutations that confer replication advantages, such as drug resistance, often cluster within regions of the HIV-1 genome. To explore how efficiently HIV-1 can assort markers separated by short distances, we developed a flow cytometry-based system to study recombination. Two HIV-1-based vectors were generated, one encoding the mouse heat-stable antigen gene and green fluorescent protein gene (GFP), and the other encoding the mouse Thy-1 gene and GFP. We generated derivatives of both vectors that contained nonfunctional GFP inactivated by different mutations. Recombination in the region between the two inactivating mutations during reverse transcription could yield a functional GFP. With this system, we determined that the recombination rates of markers separated by 588, 300, 288, and 103 bp in one round of viral replication are 56, 38, 31, and 12%, respectively, of the theoretical maximum measurable recombination rate. Statistical analyses revealed that at these intervals, recombination rates and marker distances have a near-linear relationship that is part of an overall quadratic fit. Additionally, we examined the segregation of three markers within 600 bp and concluded that HIV-1 crossover events do not exhibit high negative interference. We also examined the effects of target cells and viral accessory proteins on recombination rate. Similar recombination rates were observed when human primary CD4(+) T cells and a human T-cell line were used as target cells. We also found equivalent recombination rates in the presence and absence of accessory genes vif, vpr, vpu, and nef. These results illustrate the power of recombination in generating viral population variation and predict the rapid assortment of mutations in the HIV-1 genome in infected individuals.

Characterization and Functional Analysis of a Murine Model for Chronic Myeloid Leukemia Stem Cells.

The purpose of this study was to characterize the phenotype and analyze the functional characteristics of CML stem cells (CML-SCs) in a mouse model system. A retrovirus transduction model was employed using a vector expressing p210/BCR/ABL and the green fluorescent protein (GFP). Transplantation studies of BCR/ABL infected cells in recipient mice demonstrated an aggressive myeloproliferative disease within approximately 3 weeks, as has previously been observed with this approach. Phenotypic analysis of bone marrow from BCR/ABL mice showed contribution by the transduced cells to multiple lineages, suggesting a primitive stem/progenitor cell gives rise to the disease. Subsequent analysis of primary cells that arise in vivo showed the leukemia-initiating activity resides in cells that are Sca-1+/c-kit+/Lin-. Cell cycle analyses (as detected by BrdU incorporation) of the Lin−/c-kit+/Sca-1+ compartment of BCR/ABL mice demonstrated an enhanced cycle rate (approximately 2-fold) in the leukemic population, signifying a possible increase in the self-renewal or proliferative properties of this primitive compartment that could lead to development/progression of the disease. Further, comparison of the BCR/ABL mice and GFP control mice revealed preferential over-expression of CD24 (heat-stable antigen) in the primitive leukemic cells (90% vs. 52% in normal counterparts) demonstrating a possible BCR/ABL induced activation of the antigen. Sorting and serial transplantation studies of CD24+ vs CD24− cells from BCR/ABL mice showed that the leukemia-initiating activity resides in the CD24+/Lin− population. Collectively, these data indicate that BCR/ABL expression increases proliferation of primitive CML cells and up-regulates the CD24 antigen. We suggest that the CD24 antigen represents a potential marker to distinguish normal from CML stem cells and that it may confer specific functional properties on the CML population.

The heat stable antigen (CD24) is not required for the generation of CD4 + effector and memory T cells by dendritic cells in vivo

Previous work has established that CD24 is a costimulatory molecule for T-cell clonal expansion. Studies using CD24 −/− mice demonstrated that CD24 plays a critical role in the CD28-independent immune response against virus and soluble antigens. The role of CD24 on dendritic cells (DCs) has not been reported. Here, we compare the CD24 +/+ and CD24 −/− DCs in the induction of initial clonal expansion and elicitation of memory CD4 + T cells in vivo. Our results demonstrate that the CD24 expressed on DCs is essential neither for the induction of initial T-cell clonal expansion nor for elicitation of memory activity of primed T cells in vivo.

Immunologic immaturity, but high IL-4 productivity, of murine neonatal thymic CD4 single-positive T cells in the last stage of maturation.

To determine the levels of maturation and differentiation of murine CD4 single-positive (SP) T cells, we compared the secondary responses of staphylococcal enterotoxin A (SEA)-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts prepared from whole or heat-stable antigen(low) CD4 SP T cells. Proliferative responses upon re-stimulation with SEA were strong in adult splenic CD4 SP T cell blasts, but quite weak in neonatal thymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2 production was weaker in neonatal thymic blasts than in the adult splenic CD4 SP T cell blasts. In contrast, SEA-induced IL-4 production was high in neonatal thymic CD4 SP T cell blasts, and low in adult splenic and thymic CD4 SP T cell blasts. Expression of GATA-3, that directs production of IL-4 in T cells, examined at protein and mRNA levels, was higher in neonatal thymic cells than in adult thymic and splenic cells. These results suggest that neonatal and adult thymic CD4 SP T cells in the final stage of maturation are relatively immature compared with adult splenic CD4 SP T cells. The cytokine production profile of neonatal thymic CD4 SP T cells suggests that they are inclined towards a T(h)2 response.

Role of cannabinoid receptors in inhibiting macrophage costimulatory activity

Delta 9-tetrahydrocannabinol (THC) inhibits several immunologic functions of macrophages. THC's impact on peritoneal macrophages to deliver costimulatory signals to a helper T cell hybridoma was investigated by T cell interleukin-2 production stimulated with immobilized anti-CD3 antibody. The drug's inhibition of costimulatory activity depended on the macrophages. THC decreased costimulation provided by peritoneal cells elicited with polystyrene beads and thioglycollate, but the drug had no influence with macrophages elicited with thioglycollate alone. Bead administration induced CB2 mRNA expression in macrophages, while CB1 mRNA was not detected. Although inhibition was associated with functional heat-stable antigen, a costimulatory molecule, on macrophages, THC exposure did not alter cell surface heat-stable antigen expression. Inhibition by THC and anti-heat-stable antigen antibody was not additive suggesting the inhibitory mechanisms may overlap. Cannabinoid suppression was stereoselective; low affinity synthetic isomer CP56,667 did not diminish the T cell response. CB1-selective antagonist SR141716A completely reversed, and CB2-selective antagonist SR144528 partially blocked THC's inhibition. Both antagonists appeared to behave as inverse agonists in a receptor-selective manner. Although T cells expressed a low level of CB2 mRNA, neither THC nor SR141716A affected T cell activation in a system independent of macrophages, while SR144528 was inhibitory. High affinity synthetic agonist CP55,940, but not partial agonist THC, impaired costimulation by macrophages from mice lacking CB2 receptor. Although CB1 mRNA was not detected in CB2 null macrophages, CP55,940 reversed the inverse agonist activity of SR141716A. Hence, CB2 and possibly another receptor subtype may be involved in mediating cannabinoid suppression of macrophage costimulation.

Establishment and characterization of cloned CD4- CD8- alphabeta-T cell receptor (TCR)-bearing autoreactive T cells from autoimmune NZB x NZW F1 mice.

Murine models such as NZB/W F1, NZB.H-2bm12 and MRL.lpr/lpr mice have provided greater insight into the pathogenic mechanisms of lupus. To understand further the roles of T cells and cytokines in the pathogenesis of murine lupus, 11 cloned anti-DNA antibodies augmenting autoreactive T cell lines were derived from NZB/W F1 mice. All these autoreactive cells responded to syngeneic splenic cells and helped syngeneic B cells to produce anti-DNA antibodies, especially the IgG antibody. Ten out of 11 autoreactive T cell lines expressed neither CD4 nor CD8 cell surface markers on their surface. In addition, the cytokine production pattern of these autoreactive T cell lines was predominantly of type 0 (Th0) or type 2 T helper cells (Th2). To further investigate the role of accessory molecules in the activation of these autoreactive T cell lines, expression of IL-2R and heat-stable antigen (HSA) on these autoreactive T cells was analysed. Results suggest that the HSA played a critical role in the activation and function of these double-negative cloned autoreactive T cells.

Enhanced infectivity of HIV-1 by X4 HIV-1 coinfection

Two strains of human immunodeficiency virus type 1 (HIV-1) expressing different reporters, human placental alkaline phosphatase (PLAP) and murine heat stable antigen (HSA, CD24), were used for dual infection. Flow cytometric analysis enabled us to distinguish cells not only infected with individual reporter virus but also superinfected with both reporter viruses. When the CD4 positive T cell line, PM1, was dually infected by both reporter viruses with different coreceptor utilization, coinfection with CXCR4-tropic HIV-1 (X4 HIV-1) expressing one reporter increased the rate of cells infected with HIV-1 expressing another reporter. This enhancement was accompanied by an increased level of p24 antigen Gag in culture supernatant, indicating that infectivity of HIV-1 was augmented by X4 HIV-1 coinfection. The CXCR4 antagonist, T140 eliminated this enhancement, suggesting the role of X4 envelope via CXCR4. These results imply the role of X4 HIV-1 at the late stage of infection.

Heat-stable antigen serotyping of Campylobacter jejuni strains isolated from hospitalized children in Athens, Greece.

A hundred and twentynine Campylobacter jejuni strains isolated from hospitalized children with gastroenteritis were serotyped by the heat-stable antigen scheme (HS, Penner's method). Isolates belonged to two different periods. Group A contained strains isolated in 1987-1988 and group B contained strains which were isolated in 1998-2000. A variety of serotypes was found. Serotype HS:2 was predominant, followed by the HS:4 complex and HS:1,44. Many clinically important Guillain-Barré Syndrome associated serotypes--like HS:19--were identified. There were no significant differences in the distribution of serotypes between the two periods. The present report provides reference data, as this is the first C. jejuni serotyping study ever made in Greece.