Lymph Node Germinal Centers Research Articles

Tartrate-Resistant Acid Phosphatase as an Immunohistochemical Marker for Inflammatory Macrophages

Human serum contains 2 isoforms of type-5 tartrate-resistant acid phosphatase (TRACP): 5a and 5b. TRACP-5b is osteoclastic. Our goal was to determine if serum TRACP-5a could originate from inflammatory macrophages (MPhi). We stained 246 paraffin-embedded tissue samples for TRACP using monoclonal antibody 9C5 (mab9C5) to isoforms 5a and 5b and a novel mab220 specific to isoform 5a. CD68 and lysozyme were also stained. MPhi of chronic and granulomatous inflammation and in tissues that undergo strong antigenic stimulation were strongly positive for TRACP, more so with mab220 than with mab9C5. Noninflammatory MPhi in lymph node sinuses or germinal centers and red pulp MPhi of spleen were weak or negative for TRACP. Marginal zone lymphocytes and sebaceous glands of skin were weakly positive for TRACP. Tissue mast cells displayed strong TRACP staining. Neuroendocrine cells of gastrointestinal tissues were strongly immunoreactive with mab9C5 but negative with mab220. Restricted expression of TRACP primarily in inflammatory MPhi supports our hypothesis that circulating TRACP-5a could be a biomarker of chronic inflammatory disease activity.

Human Follicular Lymphoma Cells Contain Oligomannose Glycans in the Antigen-binding Site of the B-cell Receptor

Expression of surface immunoglobulin appears critical for the growth and survival of B-cell lymphomas. In follicular lymphoma, we found previously that the Ig variable (V) regions in the B-cell receptor express a strikingly high incidence of N-glycosylation sequons, NX(S/T). These potential glycosylation sites are introduced by somatic mutation and are lymphoma-specific, pointing to their involvement in tumor pathogenesis. Analysis of the V region sugars from lymphoma-derived IgG/IgM reveals that they are mostly oligomannose and, remarkably, are located in the antigen-binding site, possibly precluding conventional antigen binding. The Fc region contains complex glycans, confirming that the normal glycan processing pathway is intact. Binding studies indicate that the oligomannose glycans occupying the V regions are accessible to mannose-binding lectin. These findings suggest a potential contribution to lymphoma pathogenesis involving antigen-independent interaction of surface immunoglobulin of the B-cell receptor with mannose-binding molecules of innate immunity in the germinal center.

In vivo imaging of germinal centres reveals a dynamic open structure

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.

Mutations in the gene encoding the PML nuclear body protein Sp110 are associated with immunodeficiency and hepatic veno-occlusive disease

We describe mutations in the PML nuclear body protein Sp110 in the syndrome veno-occlusive disease with immunodeficiency, an autosomal recessive disorder of severe hypogammaglobulinemia, combined T and B cell immunodeficiency, absent lymph node germinal centers, absent tissue plasma cells and hepatic veno-occlusive disease. This is the first report of the involvement of a nuclear body protein in a human primary immunodeficiency and of high-penetrance genetic mutations in hepatic veno-occlusive disease.

Diffuse Alveolar Lesion in BALB/c Mice Induced with Human Reovirus BYD1 Strain and its Potential Relation with SARS

The objective of this study was to investigate the pathogenicity and associated lesions of a new reovirus (ReoV) isolated from patients with Severe Acute Respiratory Syndrome (SARS) in China. Twenty-five four-week-old BALB/c female mice inoculated intranasally with either ReoV (strain BYD1) alone, or ReoV combined with SARS-CoV (strain BJF) displayed ejecting fur and loss of body weight compared with control animals. ReoV and SARS-CoV were isolated from most postmortem tissues. The histopathological features of ReoV infected animals consisted of diffuse alveolar damage, with scattered hemorrhage, hyaline membrane formation and interstitial pneumonia. A typical type II pneumocyte hyperplasia and fibrogranulomatous tissue formation in the alveolar septae were observed both in the animals inoculated simultaneously with these two viruses and in the animals inoculated firstly with SARS-CoV, followed by ReoV. The animals inoculated firstly with ReoV, followed with SARS-CoV displayed scattered hemorrhage in the alveolar septa. Furthermore, other lesions in above two combination groups included depletion of lymphocytes in the germinal center of lymph nodes in the lung hilus and the spleen, hemorrhagic necrosis in white pulp of spleen, hydroid degeneration, and fatty degeneration in the liver and kidney. Mice induced with SARS-CoV alone did not display clinical signs, characteristically hyaline membrane formation, hemorrhage and early pulmonary fibrosis in lung tissue. This study demonstrated that the newly isolated ReoV might be a virulent pathogen for BALB/c mice. Mice infected firstly with SARS-CoV, followed with ReoV developed a typical diffuse alveolar lesion.

Eine unzureichende Erkennung und Beseitigung von sterbenden Zellen kann zur Entstehung von chronischer Autoimmunität führen

Not properly cleared dead cells are dangerous for the body. The dead cells accumulate, lose their membrane integrity, danger signals are released, and nuclear antigens get accessible in an inflammatory context. In times of increased apoptosis, tolerance can be broken, a chronic inflammation results which then can lead to an autoimmune reaction against nuclear constituents. An impaired clearance of dying cells represents a central pathogenic process in the development of chronic autoimmune diseases like in systemic lupus erythematosus (SLE). Many adaptor molecules and receptors are involved in the clearance of dying cells. Complement components, serum DNase I, phosphatidylserine, and modified glycoproteins participate crucially in the clearance of apoptotic and necrotic cells. We further observed intrinsic defects of macrophages of some SLE patients. Macrophages as well as granulocytes of some SLE patients showed heterogeneous clearance defects. Furthermore, we observed an accumulation of nuclear material in germinal centres of lymph nodes of some SLE patients. The non-ingested nuclear material may provide survival signals for autoreactive B cells and consecutively antinuclear autoantibodies (ANA) will be produced. We therefore conclude that drugs promoting the phagocytosis are important candidates of specific therapies in the future which expect a more gentle and purposive treatment of patients with SLE.

Expression of p63 in Diffuse Large B-Cell Lymphoma

The p63 gene, a homolog of the tumor suppressor gene TP53, maps to chromosome 3q27-28, a region frequently displaying genomic amplification in squamous cell carcinomas. p63 is expressed in a variety of epithelial tissues and has been reported to be critical for the normal development of stratified epithelia, including skin epidermis. In a previous study, the authors reported the expression of p63 in occasional cells in the germinal center of lymph nodes and also observed p63 expression in B-cell lymphomas, among other tumor types surveyed in that analysis. The present study was conducted to further analyze the potential clinical significance of identifying p63 expression, assessing a larger cohort of well-characterized patients with diffuse large B-cell lymphoma (DLBCL) (n = 172 cases) and a panel of established lymphoma cell lines. p63 expression at the microanatomic detail was examined by immunohistochemistry using a monoclonal antibody (clone 4A4), while distinction of p63 isoforms was analyzed by Western blotting and reverse transcription-polymerase chain reaction using isoform-specific primers. The authors found that a subset of DLBCL (32% of cases) expressed p63 in the nuclei of neoplastic lymphocytes. Examination of the different p63 isoforms revealed that the DeltaNp63 species was expressed by only one cell line, while the other p63 isoforms were found in most cell lines analyzed. The authors also observed that p63 expression correlated with high proliferative index, as assessed by Ki-67 immunostaining. Even though in univariate analysis p63 expression did not correlate with overall survival, the association of p63 with increased proliferative index suggests its involvement in DLBCL tumor progression.

Platelet-Activating Factor Antagonists Decrease Follicular Dendritic-Cell Stimulation of Human B Lymphocytes

Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF). In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs), which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA) gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig) G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA). WEB 2170 (10-6 to 10-8 M) inhibited [3H]-thymidine incorporation by up to 35% ± 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA) from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Galbeta 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA(high) lymphocytes) were made up of the great majority of CD5(+), CD4(+) and CD8(+) cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA(high) B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5(+), CD4(+) and CD8(+) cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed.

Susceptibility of TNF-α-deficient mice to Trypanosoma congolense is not due to a defective antibody response

C57BL/6 mice deficient in one or two copies of the gene for tumor necrosis factor alpha (TNF-α) were more susceptible to Trypanosoma congolense infection than their resistant, wild-type counterparts. The number of TNF-α genes was correlated with the capacity to control parasitaemia and with survival time. Absence of TNF-α resulted in a diminished capacity to form germinal centres in lymph nodes and spleen. Since germinal centres are involved in antibody production and affinity maturation, the susceptibility of the TNF-α-deficient mice could have been due to this secondary defect. Despite the lack of the germinal centres, the antibody responses to internal and exposed trypanosome antigens and to non-trypanosome antigens were not significantly different. Also the relative avidities measured in infected sera did not significantly differ between the two mouse strains. These data suggest that the role of TNF-α in control of T. congolense was not due to its role in the development of an antibody response.

Characterization of tissue specific expression of Notch-1 in human tissues

Signaling through the Notch cell surface receptors is a highly conserved mechanism of cell fate specification. Notch signaling regulates proliferation, differentiation and cell death. In vertebrates, putative gene duplication has originated four Notch genes, Notch-1, -2, -3 and -4. They have been implicated in neurogenesis, hematopoiesis, T-cell development, vasculogenesis and brain cortical growth. We have investigated Notch-1 distribution in normal human tissues by immunohistochemistry and immunoblot. We detected widespread expression of Notch-1 cytoplasmatic staining, with different tissue distributions in the different organs examined. In particular, high expression of Notch-1 was detected in the intermediate suprabasal layers, but not in the dead cells at the extreme periphery of stratified epithelia. Moreover, a low/intermediate level of Notch-1 was observed in lymphocytes in several peripheral lymphoid tissues; in particular the germinal centers of lymph nodes showed the most abundant number of positive cells, which appeared to be centroblasts/immunoblasts based on nuclear morphology. Notch-1 participates in keratinocytes differentiation. We showed by Western blot analysis that Notch-1 level was clearly increased in HaCaT cells after Ca ++ addition and remained substantially elevated until late differentiation stages. These results suggest that Notch-1 may function in numerous cell types in processes beyond cell fate determination, such as neuronal plasticity, muscle hypertrophy, liver regeneration, and germinal center lymphopoiesis during the immune response.

Extended histopathology in immunotoxicity testing: interlaboratory validation studies.

There has been considerable interest in the use of expanded histopathology as a primary screen for immunotoxicity assessment. To determine the utility of a semiquantitative histopathology approach for examining specific structural and architectural changes in lymphoid tissues, a validation effort was initiated. This study addresses the interlaboratory reproducibility of extended histopathology, using tissues from studies of ten test chemicals and both negative and positive controls from the National Toxicology Program's immunotoxicology testing program. We examined the consistency between experienced toxicologic pathologists, who had varied expertise in immunohistopathology in identifying lesions in immune tissues, and in the sensitivity of the individual and combined histopathological endpoints to detect chemical effects and dose response. Factor analysis was used to estimate the association of each pathologist with a so-called "common factor" and analysis-of-variance methods were used to evaluate biases. Agreement between pathologists was highest in the thymus, in particular, when evaluating cortical cellularity of the thymus; good in spleen follicular cellularity and in spleen and lymph node-germinal center development; and poorest in spleen red-pulp changes. In addition, the ability to identify histopathological change in lymphoid tissues was dependent upon the experience/training that the individual pathologist possessed in examining lymphoid tissue and the apparent severity of the specific lesion.

The impact of early immune destruction on the kinetics of postacute viral replication in rhesus monkey infected with the simian-human immunodeficiency virus 89.6P

Set-point viral load is positively correlated with the extent of initial viral replication in pathogenic simian-human immunodeficiency virus (SHIV) infection. To elucidate the mechanisms underlying the correlation, we conducted a systematic investigation in rhesus monkeys infected with the highly pathogenic SHIV 89.6P. This model is widely used in the preclinical evaluation of AIDS vaccine candidates and a thorough understanding of the model's biology is important to the proper interpretation of these evaluations. We found that the levels of peak viremia were positively correlated not only with the levels of set-point viremia but, importantly, with the extent of initial overall immune destruction as indicated by the degree of CD4 + T cell depletion and lymph node germinal center (GC) formation. The extent of initial overall immune destruction was inversely correlated with subsequent development and maintenance of virus-specific cellular and humoral immune responses. Thus, these data suggest that the extent of early immune damage determines the development and durability of virus-specific immunity, thereby playing a critical role in establishing the levels of set-point viral replication in SHIV infection. Vaccines that limit both the initial viral replication and the extent of early immune damage will therefore mediate long-term virus replication control and mitigation of long-term immune destruction in this model of immunodeficiency virus infection.

Estrogen Receptor α Is a Novel Marker Expressed by Follicular Dendritic Cells in Lymph Nodes and Tumor-Associated Lymphoid Infiltrates

During routine assessment of the hormonal phenotype of breast carcinomas, we detected expression of the estrogen receptor (ER) in the germinal centers of reactive lymphoid follicles surrounding malignant foci. To confirm and extend this finding, we compared ER-alpha, progesterone receptor (PR), and androgen receptor (AR) immunostaining in hyperplastic or metastatic lymph nodes obtained from patients with various pathology, disease location, gender and age. Irrespective of these parameters, we found that: 1) ER-alpha-positive cells were located prevalently in germinal centers, 2) the PR was weakly expressed by cells within and surrounding germinal centers, and 3) the androgen receptor was undetectable. Transcripts for ER-alpha and PR were also detected by reverse transcription-polymerase chain reaction on laser-microdissected lymph node germinal centers. Morphologically, the ER-positive cells resemble dendritic cells and by double immunostaining were found to express both CD21 and CD23, which is characteristic of follicular dendritic cells. Finally, we assessed the effects of Tamoxifen treatment by comparing the numbers of ER-positive follicular dendritic cells in lymph nodes obtained from breast cancer patients before and after treatment. The results show that Tamoxifen treatment generated larger germinal centers with more abundant ER(+)/CD21(+)/CD23(+) cells. Taken together, these results open new perspectives on the effects of sex steroids and their antagonists on the human response in cancer and inflammation.

Melano-macrophage centres and their role in fish pathology.

Melano-macrophage centres, also known as macrophage aggregates, are distinctive groupings of pigment-containing cells within the tissues of heterothermic vertebrates. In fish they are normally located in the stroma of the haemopoietic tissue of the spleen and the kidney, although in amphibians and reptiles, and some fish, they are also found in the liver. They may also develop in association with chronic inflammatory lesions elsewhere in the body and during ovarian atresia. In higher teleosts, they often exist as complex discrete centres, containing lymphocytes and macrophages, and may be primitive analogues of the germinal centres of lymph nodes. Melano-macrophage centres usually contain a variety of pigments, including melanins, and these increase in range and volume in older fish or in the presence of cachectic disease. Melano-macrophage centres act as focal depositories for resistant intracellular bacteria, from which chronic infections may develop. Iron capture and storage in haemolytic diseases appears to be a primary function, but antigen trapping and presentation to lymphocytes, sequestration of products of cellular degradation and potentially toxic tissue materials, such as melanins, free radicals and catabolic breakdown products are among other functions that have been ascribed. Recent work suggests that they are a site of primary melanogenesis rather than mere storage. Melano-macrophage centres increase in size or frequency in conditions of environmental stress and have been suggested as reliable biomarkers for water quality in terms of both deoxygenation and iatragenic chemical pollution.

Pathology of Inhalation Anthrax in Cynomolgus Monkeys (Macaca fascicularis)

Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure. The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose (LD50) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain). The LD50 and 95% confidence intervals were 61,800 (34,000 to 110,000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the Ames strain of B. anthracis is lethal by the inhalation route in the cynomolgus macaque. The LD50 of the Ames strain of B. anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans. The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.

Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray.

p63, cytokeratin (CK) 5/6 and CK 14 have been employed in diagnostic pathology as markers of basal, squamous and myoepithelial differentiation in several types of human neoplasms; however, there is scant data on the concurrent expression of these markers in large series of human neoplasms. We analyzed the distribution of these three immunohistochemical markers in 51 normal human tissue samples, 350 carcinomas, 25 malignant melanomas (MMs), and 25 glioblastomas using three serial sections of tissue array research program (TARP)-4 multi-tumor tissue microarray. Also, we performed double immunostainings to characterize the differential distribution of p63/CK 5/6 and p63/CK 14 in normal breast, salivary gland and skin. p63, CK 5/6 and CK 14 were expressed in basal cells of the prostate and respiratory epithelia and in breast and bronchial myoepithelial cells. p63 was also expressed in cytotrophoblast cells of human placenta and in scattered cells of lymph node germinal center. CK 5/6 and CK 14 also stained the cytoplasm of basal cells of esophageal stratified squamous epithelium and transitional epithelial cells of the bladder. No mesenchymal, neural, endothelial, smooth muscle or adipose cells were stained by any of the markers. p63, CK 5/6, and CK 14 were respectively expressed in 92.6%, 75.0%, and 52.9% of the squamous cell carcinomas of the lung, 10.2%, 20.0%, and 7.4% of the ductal carcinomas of the breast, 12.9%, 34.4%, and 11.8% of the serous and 25.0%, 0%, and 0% of the endometrioid carcinomas of the ovary. Lung, prostate and colonic adenocarcinomas, as well as MMs and glioblastomas were only rarely decorated by one of the markers. Only matched samples of 16 squamous cell carcinomas and two ductal carcinomas of the breast co-expressed these three markers. In double immunostainings, p63-CK 5/6, as well as p63-CK 14 were co-expressed by basal/myoepithelial cells of the salivary glands and basal cells of the epidermis. Our results demonstrate that p63, CK 5/6 and CK 14 may be used together in immunohistochemical panels to characterize squamous differentiation in poorly differentiated carcinomas or carcinomas of unknown origin.

HnRNP B1 expression in benign and malignant lung disease.

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non-neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non-small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non-specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis-interstitial lung disease, mineral dust disease, Sjögren's syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fisher's exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified.

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal – implications for antiphospholipid syndrome and systemic lupus erythematosus

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.

B cell activation in peripheral blood and lymph nodes during HIV infection.

The spontaneous in-vitro antibody synthesis observed in unstimulated lymphocyte cultures from HIV-infected patients closely reflects the in-vivo activation of the B cell compartment; however, the mechanisms underlying this phenomenon are far from clear. We compared the ability of peripheral blood mononuclear cells (PBMC) and lymph-node cells (LNC) from 10 HIV-infected patients to produce in vitro HIV-specific and total Ig spontaneously, and we correlated these parameters with tumour necrosis factor alpha (TNF-alpha) expression by CD4 T cells, viral dissemination in the organism, and the extent of HIV spread into lymph-node germinal centres, measured by in-situ hybridization (ISH). In-vitro spontaneous synthesis of both HIV-specific and total antibody was significantly higher in PBMC than in LNC; the two variables showed a good correlation in LNC, but not in PBMC. In both compartments, no correlation was found between B cell activation and the percentage of CD4 T cells expressing TNF-alpha, which was increased compared with seronegative donors. Furthermore, no correlation was found between in-vitro spontaneous antibody synthesis and the number of T cells containing proviral HIV in PBMC and LNC, or the plasma levels of HIV RNA. On the contrary, a good correlation was found between HIV-specific B cell activation and the extent of viral spread into lymph-node germinal centres, evaluated by ISH. These data suggest that the adhesion of HIV virions to the follicular dendritic cell network in lymph-node germinal centres may primarily contribute to sustaining the steady B cell activation observed in HIV-infected patients.