Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

In vitro infectivity and differential gene expression of Leishmania infantum metacyclic promastigotes: negative selection with peanut agglutinin in culture versus isolation from the stomodeal valve of Phlebotomus perniciosus.

BACKGROUND: Leishmania infantum is the protozoan parasite responsible for zoonotic visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been reported in this area. The life cycle of the parasite is digenetic. The promastigote stage develops within the gut of phlebotomine sand flies, whereas amastigotes survive and multiply within phagolysosomes of mammalian host phagocytes. The major vector of L. infantum in Spain is Phlebotomus perniciosus. The axenic culture model of promastigotes is generally used because it is able to mimic the conditions of the natural environment (i.e. the sand fly vector gut). However, infectivity decreases with culture passages and infection of laboratory animals is frequently required. Enrichment of the stationary phase population in highly infective metacyclic promastigotes is achieved by negative selection with peanut agglutinin (PNA), which is possible only in certain Leishmania species such as L. major and L. infantum. In this study, in vitro infectivity and differential gene expression of cultured PNA-negative promastigotes (Pro-PNA(-)) and metacyclic promastigotes isolated from the sand fly anterior thoracic midgut (Pro-Pper) have been compared. RESULTS: In vitro infectivity is about 30 % higher in terms of rate of infected cells and number of amastigotes per infected cell in Pro-Pper than in Pro-PNA(-). This finding is in agreement with up-regulation of a leishmanolysin gene (gp63) and genes involved in biosynthesis of glycosylinositolphospholipids (GIPL), lipophosphoglycan (LPG) and proteophosphoglycan (PPG) in Pro-Pper. In addition, differences between Pro-Pper and Pro-PNA(-) in genes involved in important cellular processes (e.g. signaling and regulation of gene expression) have been found. CONCLUSIONS: Pro-Pper are significantly more infective than peanut lectin non-agglutinating ones. Therefore, negative selection with PNA is an appropriate method for isolating metacyclic promastigotes in stationary phase of axenic culture but it does not allow reaching the in vitro infectivity levels of Pro-Pper. Indeed, GIPL, LPG and PPG biosynthetic genes together with a gp63 gene are up-regulated in Pro-Pper and interestingly, the correlation coefficient between both transcriptomes in terms of transcript abundance is R (2) = 0.68. This means that the correlation is sufficiently high to consider that both samples are physiologically comparable (i.e. the experiment was correctly designed and performed) and sufficiently low to conclude that important differences in transcript abundance have been found. Therefore, the implications of axenic culture should be evaluated case-by-case in each experimental design even when the stationary phase population in culture is enriched in metacyclic promastigotes by negative selection with PNA.

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

Comparison of Four Diagnostic Methods for Detection and Relative Quantification of Haemonchus contortus Eggs in Feces Samples.

We compared four methods for identification of Haemonchus contortus eggs. With increased trade in animals within and between countries and continents, it has become important to correctly identify H. contortus eggs in fecal samples. To validate the outcome of diagnostic tests, sheep feces (n = 38) were collected from naturally infected flocks in Sweden. Subsamples were analyzed with (a) McMaster egg counting; (b) differential counting of eggs after staining with peanut agglutinin (PNA); (c) detection of DNA following amplification by real-time quantitative polymerase chain reaction (qPCR); and (d) loop-mediated isothermal amplification (LAMP). Differences between similar tests (microscopic and molecular) and SD (±SD) were analyzed with Bland-Altman plots and Spearman rank correlation. Strongylid egg counts ranged from 200 to 12,100 eggs per gram (epg) (mean epg ± SD = 1,278 ± 2,049). Microscopy showed presence of H. contortus eggs in 27 (73%) unstained samples and in 28 (76%) samples stained with PNA, whereas 29 samples (78%) tested positive in LAMP and 34 (91%) in qPCR analysis. The cycle threshold (Ct) values with LAMP ranged between 13 and 38 (mean ± SD = 21 ± 7), and those in qPCR between 25 and 49 (mean ± SD = 33 ± 6). In the LAMP and qPCR analyses, seven (19%) and three (8%) samples, respectively, had a cycle threshold (Ct) >35, whereas no reactions were observed in eight (22%) and three (8%) samples, respectively. There was good agreement between the diagnostic tests based on microscopic examination and DNA detection, although the molecular tests were more sensitive. The bias between the microscopy methods (-4.2 ± 11) was smaller than for the molecular tests (-9.8 ± 10). The observed ranking in terms of test sensitivity was: McMaster counting by conventional microscopy < PNA < LAMP < qPCR. In conclusion, H. contortus can be identified by McMaster counting, without major mistakes regarding false positive results. However, molecular methods provide the capacity to diagnose H. contortus eggs with increased accuracy. This is essential when animals are investigated in quarantine or in studies evaluating anthelmintic treatment efficacy. These methods could also be applied to fecal samples from wildlife to investigate nematode transmission between wildlife and livestock.

A Case of Childhood Anemia Complicated by a Red Cell Membrane Sugar Chain Anomaly with Low Levels of Sialic Acid.

An 8-year-old girl presented with the pallor and purpura. She was diagnosed to suffer from anemia of an unknown cause with leukocytopenia and thrombocytopenia. The patient's red cells showed marked agglutination with peanut lectin (PNA), and erythroblasts forming CFU-E in the bone marrow reacted positively to avidin labeled PNA on enzyme immunohistochemistry, suggesting an abnormality of sugar chain on red cell and also erythroblast membranes. Membrane O-glycan sugar chains of her red cells showed a low level of sialic acid on high performance liquid chromatography (HPLC). Her anemia failed to respond to the corticosteroids, γ-globulin, recombinant erythropoietin (rhEPO), and recombinant granulocyte colony stimulating factor (G-CSF), and frequent red cell transfusion was required. After 6 years, the patient underwent an unrelated bone marrow transplantation (U-BMT). From 2 weeks after transplantation, the PNA reactivity of her red cells decreased and then disappeared and the red cell membrane antigens changed to the donor type after 4 weeks. These results suggested that the sugar chain abnormality causing low levels of sialic acid in the red cell membrane was already present on erythroid progenitor cells in the bone marrow.