Heparin modification of a biomimetic bone matrix modulates osteogenic and angiogenic cell response in vitro.

In this study, the effect of heparin-modified collagen type I/hydroxyapatite (HA) nanocomposites on key processes of bone regeneration - osteogenesis and angiogenesis - was characterised in vitro. Two approaches were applied for heparin modification: it was either integrated during material synthesis (in situ) or added to the porous scaffolds after their fabrication (post). Cultivation of human bone marrow-derived stromal cells (hBMSC), in heparin-modified versus heparin-free scaffolds, revealed a positive effect of the heparin modification on their proliferation and osteogenic differentiation. The amount of heparin rather than the method used for modification influenced the cell response favouring proliferation at smaller amount (30 mg/g collagen) and differentiation at larger amount (150 mg/g collagen). A co-culture of human umbilical vein endothelial cells (HUVEC) and osteogenically induced hBMSC was applied for in vitro angiogenesis studies. Pre-vascular networks have formed in the porous structure of scaffolds which were not modified with heparin or modified with a low amount of heparin (30 mg/g collagen). The modification with higher heparin quantities seemed to inhibit tubule formation. Pre-loading of the scaffolds with VEGF influenced formation and stability of the pre-vascular structures depending on the presence of heparin: In heparin-free scaffolds, induction of tubule formation and sprouting was more pronounced whereas heparin-modified scaffolds seemed to promote stabilisation of the pre-vascular structures. In conclusion, the modification of mineralised collagen with heparin by using both approaches was found to modulate cellular processes essential for bone regeneration; the amount of heparin has been identified to be crucial to direct cell responses.

Towards functional glycomics by lectin histochemistry: strategic probe selection to monitor core and branch-end substitutions and detection of cell-type and regional selectivity in adult mouse testis and epididymis.

The emerging insights into glycan functionality direct increasing attention to monitor core modifications of N-glycans and branch-end structures. To address this issue in histochemistry, a panel of lectins with respective specificities was devised. The selection of probes with overlapping specificities facilitated to relate staining profiles to likely target structures. The experiments on fixed sections of adult murine testis and epididymis were carried out at non-saturating lectin concentrations to visualize high-affinity sites with optimal signal-to-background ratio. They revealed selectivity in lectin reactivity for distinct cell types and segment-dependent staining in the epididymis. Leydig cells, for instance, were reactive with the Sambucus nigra agglutinin and human siglec-2 (CD22), two lectins also separating principal from basal and apical cells in the caput segments I-III of the epididymis. Apical cells were reactive with the Maackia amurensis agglutinin-I, and basal cells with the erythroagglutinin of Phaseolus vulgaris. The reported differences support the concept of lectin staining as cell marker. They thus intimate to study glycogene (genes for glycosyltransferases and lectins) expression and cellular reactivity with tissue lectins. These investigations will be instrumental to assign a role as biochemical signals to the detected staining properties.

Natural antibodies, intravenous immunoglobulin and their role in autoimmunity, cancer and inflammation.

Natural antibodies are produced by B lymphocytes in the absence of external antigen stimulation. With their ability to recognize self, altered self and foreign antigens, they comprise an important first-line defence against invading pathogens, but are also important for tissue homeostasis. By recognizing oligosaccharides expressed on tumour cells and modified cell surface structures accompanying necrosis, natural antibodies have an important anti-tumorigenic function. IVIg contains a wide spectrum of specificities presented in normal plasma including natural antibodies and has been shown to exert inhibitory effects on tumour cells through a subfraction of anti-vascular endothelial growth factor immunoglobulin (Ig)G antibodies with anti-angiogenic properties. IgA antibodies also have potent immunomodulatory properties, being able to both induce and suppress immune responses. IgA-mediated inhibitory function is able to inhibit several inflammatory diseases including asthma and glomerulonephritis. Autoantibodies of the IgM type, on the other hand, have shown promising results in the treatment of multiple sclerosis. These autoantibodies promote remyelination rather than modulating inflammation. Oxidation-specific epitopes, as found in atherosclerotic lesions and on apoptotic cells, comprise one important target of natural antibodies. By recognizing these epitopes, natural antibodies neutralize proinflammatory responses and mediate atheroprotection.

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum.

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.

CD antigens 2001.

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

The fucosylated histo-blood group antigens H type 2 (blood group O, CD173) and Lewis Y (CD174) are expressed on CD34+ hematopoietic progenitors but absent on mature lymphocytes.

The expression of LeY, H2, H3, and H4 on a broad variety of human leukemia cell lines and native lymphocytes as well as on CD34+ hematopoietic progenitor cells was examined by flow cytometry and immunocytochemistry. CD34+ leukemia cell lines (KG1, KG1a, and TF1) and native CD34+ hematopoietic progenitor cells expressed H2 (CD173) and LeY (CD174). In contrast, CD34(-) cell lines (HL-60, U937, JOK-1, Raji, Molt-3, Jurkat, and CEM-C7) and mature lymphocytes from peripheral blood and tonsils lacked CD173 and CD174. All cell lines and native lymphocytes as well as CD34+ precursor cells were negative for H3 and H4. Immunoprecipitation and consecutive Western blotting revealed a 170-kDa glycoprotein as the carrier molecule for the CD173 and CD174 oligosaccharide sequences on CD34+ hematopoietic precursors. The key enzyme for generating CD173 is the beta-D-galactoside 2-alpha-L-fucosyltransferase (FUT1). As shown by RT-PCR, FUT1 was expressed in immature hematopoietic cells but absent in mature lymphocytes, which indicates that expression of CD173 within the hematopoietic system is regulated at the transcriptional level by FUT1. Due to their exclusive presence on CD34+ hematopoietic progenitor cells, CD173 and CD174 represent novel markers of early hematopoiesis. The expression of the fucosylated histo-blood group antigens CD173 and CD174 in CD34+ hematopoietic progenitor cells and down-regulation of FUT1 in mature lymphocytes may be important factors influencing the homing process of hematopoietic stem cells to the bone marrow.

Breast cancer during the HIV epidemic in an African population.

Both human immunodeficiency virus (HIV) infection and certain malignancies including breast cancer occur predominantly in premenopausal women in an African population. Cancers that are associated with HIV infection are Kaposi's sarcoma (KS), non-Hodgkin's lymphoma (NHL) and invasive cervical carcinoma. Recently, cases of breast cancer have been reported in patients with HIV infection but an association between breast cancer and HIV infection has yet to be determined. The present study investigated for association between HIV infection and breast cancer. Among the 101 patients studied, 50 were cases with breast cancer while the remaining 51 were referents with conditions other than mammary cancer. Patients with breast cancer 30 years of age and below recorded in the Cancer registry during 1974-1987 constituted 8% while those recorded during the ongoing AIDS epidemic amounted to only 2%. When a similar comparison was undertaken among patients below 50 years there was also an overall decrease in the proportion of patients from 76.1 to 58.0%. Conversely, in the age groups above 50 years the breast cancer cases increased from 33.9 to 42% respectively (chi2=1.83 on 1df, p=0.18). The overall prevalence of HIV infection among the control group was 35.5% (95% CI=22.2-48.4) while among breast cancer patients it was 6% (95% CI=0.6-12.6). Women below 50 years of age with breast cancer were less likely to be HIV positive; OR=0.18: (95% CI=0.04-0.76) chi2=5.95; p=0.01. However, there is no basis to suggest that HIV infection is protective against this malignancy. AIDS associated mortality commonly occurs in the second and third decades of life and probably these deaths have changed the demographic of the disease in an African population. The impact of AIDS associated mortality on cancer registries needs attention.

CD antigens 2001.

The most recent Human Leucocyte Differentiation Antigen Workshop ("HLDA7") took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid "blind" screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new "intracellular CD" categorisation scheme is devised for such molecules).

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells.

In response to the chemoattractants interleukin 8, C5a, N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

Expression of estrogen and progesterone receptors in carcinomas of the female breast in Tanzania.

In Africa breast cancer has been reported to occur frequently in young females and to show an aggressive histological and clinical picture, suggesting that this malignancy might have a different biology from this disease in Western females. To investigate this, the present study assessed by immunohistochemistry the expression of estrogen receptors (ER) and progesterone receptors (PgR) in 60 fresh frozen breast cancer tissues from indigenous Tanzanian patients. This prospective study collected tissues from routine patients treated at the Muhimbili Medical Center, Dar es Salaam, Tanzania. These markers have not been previously investigated in indigenous sub-Saharan females with breast cancer. Patients in this study expressed lower frequencies of ER (33%) and PgR (18%) as compared to literature reports including those about African-Americans. Expression of these markers, however, correlated with the demographic, clinical and histological characteristics in a similar way as observed elsewhere. A compounding effect of younger patients' age, advanced disease or late stage at hospital presentation and race in this geographical region could be responsible for the poor expression of hormonal receptors in the majority of patients as observed in this study. A surprising finding was that the proportion of hormonal receptor positive tumors increased with disease duration. In view of the low frequency of expression of hormonal markers, only 26.7% of the patients would be expected to benefit from hormonal therapy based on their expression of the hormone receptors. There is great need to undertake an inter-African study that would evaluate the hormonal status of more African women with breast cancer in different geographical regions of sub-Saharan Africa and document the true picture of their hormonal status. The outcome of these results could be important for treatment strategies for the second most common cancer among African women.

UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.

Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.

Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.

Identification of an alpha2,6-sialyltransferase induced early after lymphocyte activation.

We have used mRNA differential display PCR to search for genes induced in activated T cells and we identified a gene encoding an alpha2,6-sialyltransferase (ST6GalNAc IV) that is rapidly induced in lymphocytes after antigen or mitogen stimulation. The 3.6 kb full-length cDNA clone (MK45) obtained contained a single open reading frame encoding a 302 amino acid protein and a 2.5 kb 3' untranslated region. MK45 expression in in vivo-activated CD8 T cells reached the highest level 4 h after antigen triggering and then declined rapidly to nearly base levels within 45 h. Northern blot analysis further revealed that MK45 expression was also induced in LPS-activated B cells and antigen-triggered CD4 T cells in vitro. MK45 expression was low or undetectable in most other mouse tissues examined, when compared to activated lymphocytes. Importantly, the mRNA expression level of other sialyltransferases remained largely unchanged during the early stage of lymphocyte activation. Finally, increased ecto-sialyltransferase activity and an altered sialylation pattern were demonstrated on the cell surface of early activated CD8 T cells. Our report identifies a candidate sialyltransferase gene that is involved in the early alteration of the sialylation pattern of cell surface molecules in activated lymphocytes.