Evidence of Urtica dioica Agglutinin's Antiproliferative and Anti-migratory Potentials on the Hyaluronic Acid-Overexpressing Prostate Cancer Cells.

Hyaluronic acid is composed of repeating sugar units, glucuronic acid and N-acetylglucosamine, which are often associated with increased tumor progression. Urtica dioica agglutinin is a potential component that exhibits a high affinity for binding to N-acetylglucosamine. This study aimed to investigate U. dioica Agglutinin's potential to inhibit the proliferation and migration of prostate cancer cells with high expression of hyaluronic acid through molecular docking and in vitro studies. The expression of hyaluronan synthase genes in prostate tissue and cell lines was checked by an in silico study, and the interaction between hyaluronic acid with both CD44 transmembrane glycoprotein and U. dioica agglutinin was analyzed through molecular docking. U. dioica Agglutinin's effect on cell viability (neutral red uptake assay), migration (scratch wound healing assays), and both CD44 and Nanog expression (quantitative real-time polymerase chain reaction) were assessed in vitro. The results showed that in prostate cancer cell lines, the PC3 cell line has the highest expression of hyaluronan synthase genes. U. dioica agglutinin exhibits an interaction of six specific residues on CD44 compared to hyaluronic acid's singular residue. While U. dioica agglutinin alone effectively reduced cell viability and wound closer (≥ 150 µg/mL), combining it with hyaluronic acid significantly shifted the effective concentration to a higher dose (≥ 350 µg/mL). These results, together with low Nanog and high CD44 gene expression, suggest that U. dioica agglutinin may impair the CD44-HA pathway in PC3 cells. This possibility is supported by U. dioica Agglutinin's ability to compete with hyaluronic acid for binding to CD44. Based on this, U. dioica agglutinin as a plant lectin shows promise in inhibiting cancer proliferation and migration by targeting its dependence on hyaluronic acid.

Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers.

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

Refolding, Crystallization, and Crystal Structure Analysis of a Scavenger Receptor Cysteine-Rich Domain of Human Salivary Agglutinin Expressed in Escherichia coli.

Scavenger receptors are a protein superfamily that typically consists of one or more repeats of the scavenger receptor cysteine-rich structural domain (SRCRD), which is an ancient and highly conserved protein module. The expression and purification of eukaryotic proteins containing multiple disulfide bonds has always been challenging. The expression systems that are commonly used to express SRCRD proteins mainly consist of eukaryotic protein expression systems. Herein, we established a high-level expression strategy of a Type B SRCRD unit from human salivary agglutinin using the Escherichia coli expression system, followed by a refolding and purification process. The untagged recombinant SRCRD was expressed in E. coli using the pET-32a vector, which was followed by a refolding process using the GSH/GSSG redox system. The SRCRD expressed in E. coli SHuffle T7 showed better solubility after refolding than that expressed in E. coli BL21(DE3), suggesting the importance of the disulfide bond content prior to refolding. The quality of the refolded protein was finally assessed using crystallization and crystal structure analysis. As proteins refolded from inclusion bodies exhibit a high crystal quality and reproducibility, this method is considered a reliable strategy for SRCRD protein expression and purification. To further confirm the structural integrity of the refolded SRCRD protein, the purified protein was subjected to crystallization using sitting-drop vapor diffusion method. The obtained crystals of SRCRD diffracted X-rays to a resolution of 1.47 Å. The solved crystal structure appeared to be highly conserved, with four disulfide bonds appropriately formed. The surface charge distribution of homologous SRCRD proteins indicates that the negatively charged region at the surface is associated with their calcium-dependent ligand recognition. These results suggest that a high-quality SRCRD protein expressed by E. coli SHuffle T7 can be successfully folded and purified, providing new options for the expression of members of the scavenger receptor superfamily.

Salivary Th17 cytokine, human ß-defensin 1-3, and salivary scavenger and agglutinin levels in Crohn's disease.

OBJECTIVES: Crohn's disease patients, who are prone to develop periodontal diseases, may carry genetic defects in their Th17 cytokine, human beta-defensin (hBD) 1-3, and salivary and scavenger agglutinin (SALSA) expressions. Biochemical composition of saliva reflects the oral consequences of systemic immune response modifications. Our aim was to evaluate the salivary Th17 cytokine, epithelial hBD 1-3, and SALSA levels in relation to Crohn's disease. MATERIALS AND METHODS: This cross-sectional study included 42 Crohn's disease patients and 34 systemically healthy controls. Periodontal and dental indexes were measured, and stimulated saliva samples were collected. Salivary Th17 cytokine levels were analyzed by multiplex technique, and hBD 1-3 and SALSA levels by enzyme-linked immunosorbent assay. RESULTS: There were 19 gingivitis and 11 initial periodontitis patients in the Crohn's disease group, and 15 gingivitis and 4 initial periodontitis in the control group. In comparison to controls, higher salivary Th17 cytokine levels were observed in Crohn's disease patients. No statistical difference was observed between Crohn's disease and control groups in terms of their salivary hBD 1-3 and SALSA levels. Based on the regression analysis, there is no independent association between Crohn's disease and salivary Th17 cytokine levels. CONCLUSIONS: Crohn's disease does not relate to salivary antimicrobial hBD 1-3 or SALSA levels. While Crohn's disease patients have higher salivary Th17 cytokine levels in comparison to systemically healthy controls, an independent association between Crohn's disease and Th17 cytokine profile is still missing. CLINICAL RELEVANCE: Diminished Th17 cytokine response in Crohn's disease, which might be related to genetic susceptibility, can be also visualized in saliva.

Oral epithelial cell segmentation from fluorescent multichannel cytology images using deep learning.

BACKGROUND AND OBJECTIVES: Cytology is a proven, minimally-invasive cancer screening and surveillance strategy. Given the high incidence of oral cancer globally, there is a need to develop a point-of-care, automated, cytology-based screening tool. Oral cytology image analysis has multiple challenges such as, presence of debris, blood cells, artefacts, and clustered cells, which necessitate a skilled expertise for single-cell detection of atypical cells for diagnosis. The main objective of this study is to develop a semantic segmentation model for Single Epithelial Cell (SEC) separation from fluorescent, multichannel, microscopic oral cytology images and classify the segmented images. METHODS: We have used multi-channel, fluorescent, microscopic images (number of images; n = 2730), which were stained differentially for cytoplasm and nucleus. The cytoplasmic and cell membrane markers used in the study were Mackia Amurensis Agglutinin (MAA; n: 2364) and Sambucus Nigra Agglutinin-1 (SNA-1; n: 366) with a nuclear stain DAPI. The cytology images were labelled for SECs, cluster of cells, artefacts, and blood cells. In this study, we used encoder-decoder models based on the well-established U-Net architecture, modified U-Net and ResNet-34 for multi-class segmentation. The experiments were performed with different class combinations of data to reduce imbalance. The derived MAA dataset (n: 14,706) of SEC, cluster, and artefacts/blood cells were used for developing a classification model. InceptionV3 model and a new custom Convolutional-Neural-Network (CNN) model (Artefact-Net) were trained to classify SNA-1 marker stained segmented images (n:6101). For segmentation models, Intersection Over Union (IoU) and F1 score were used as the evaluation matrices, while the classification models were evaluated using the conventional classification metrics like precision, recall and F1-Score. RESULTS: The U-Net and the modified U-Net models gave the best IoU overall (0.73-0.76) as well as for SEC segmentation (079). The images segmented using the modified U-Net model were classified by Artefact-Net and Inception V3 model with F1 scores of 0.96 and 0.95 respectively. The Artefact-Net, when compared to InceptionV3, provided a better precision and F1 score in classifying clusters (Precision: 0.91 vs 0.80; F1: 0.91 vs 0.86). CONCLUSION: This study establishes a pipeline for SEC segmentation with the segmented component containing only single cells. The pipline will enable automated, cytology-based early detection with reduced bias.

Characteristics of Exosomes and the Vascular Landscape Regulate Exosome Sequestration by Peripheral Tissues and Brain.

Exosomes mediate intercellular communication, shuttling messages between cells and tissues. We explored whether exosome tissue sequestration is determined by the exosomes or the tissues using ten radiolabeled exosomes from human or murine, cancerous or noncancerous cell lines. We measured sequestration of these exosomes by the liver, kidney, spleen, and lung after intravenous injection into male CD-1 mice. Except for kidney sequestration of three exosomes, all exosomes were incorporated by all tissues, but sequestration levels varied greatly among exosomes and tissues. Species of origin (mouse vs. human) or source (cancerous vs. noncancerous cells) did not influence tissue sequestration. Sequestration of J774A.1 exosomes by liver involved the mannose-6 phosphate (M6P) receptor. Wheatgerm agglutinin (WGA) or lipopolysaccharide (LPS) treatments enhanced sequestration of exosomes by brain and lung but inhibited sequestration by liver and spleen. Response to LPS was not predictive of response to WGA. Path and heat map analyses included our published results for brain and found distinct clusters among the exosomes and the tissues. In conclusion, we found no evidence for a universal binding site controlling exosome-tissue interactions. Instead, sequestration of exosomes by tissues is differentially regulated by both exosomes and tissues and may be stimulated or inhibited by WGA and inflammation.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

The Association between Clusterin Sialylation Degree and Levels of Oxidative-Antioxidant Balance Markers in Seminal Plasmas and Blood Sera of Male Partners with Abnormal Sperm Parameters.

Nearly 30% of infertility cases are caused by male factor. This study aimed at checking the associations between the sialylation degree of glycoprotein clusterin (CLU) and levels of oxidative-antioxidant balance markers in infertile men. Using lectin-ELISA with biotinylated lectins specific to α2,6-linked (Sambucus nigra agglutinin, SNA) and α2,3-linked (Maackia amurensis agglutinin, MAA) sialic acid (SA), the CLU sialylation in 132 seminal plasmas (SP) and 91 blood sera (BS) were analyzed. Oxidative-antioxidant status was measured by determining Sirtuin-3 (SIRT3), Sirtuin-5 (SIRT5), total antioxidant status (TAS), and ferric reducing antioxidant power (FRAP) levels. We indicate that multiple sperm disorders are associated with decreased expression of MAA-reactive SA in SP. Decreased SP SIRT3 concentrations may be associated with teratozoospermia and oligoasthenoteratozoospermia. ROC curve and cluster analysis revealed that SP relative reactivity of CLU glycans with MAA, the value of MAA/SNA ratio, and SIRT3 and SIRT5 concentrations may constitute an additional set of markers differentiating infertile oligoasthenoteratozoospermic patients (OAT) from normozoospermic (N), asthenoteratozoospermic (AT) and teratozoospermic (T). The multinomial logistic regression analysis confirmed the potential utility of SIRT3 determinations for differentiation between N and OAT groups as well as between N and T groups for SIRT3 and SIRT5. For BS, based on ROC curve and cluster analysis, relative reactivities of CLU glycans with SNA, MAA, SIRT3 and FRAP concentrations may be useful in the differentiation of normozoospermic patients from those with sperm disorders. The multinomial logistic regression analysis showed that the SNA relative reactivity with CLU glycans significantly differentiated the N group from AT, OAT and T groups, and FRAP concentrations significantly differed between N and AT groups, which additionally confirms the potential utility of these biomarkers in the differentiation of infertile patients with abnormal sperm parameters. The knowledge about associations between examined parameters may also influence future research aimed at seeking new male infertility therapies.

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins.

BACKGROUND: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.

Candida albicans evades NK cell elimination via binding of Agglutinin-Like Sequence proteins to the checkpoint receptor TIGIT.

Candida albicans is the most common fungal pathogen and a prevalent cause of deadly bloodstream infections. Better understanding of the immune response against it, and the ways by which it evades immunity, are crucial for developing new therapeutics against it. Natural Killer (NK) cells are innate lymphocytes best known for their role against viruses and tumors. In recent years it became clear that NK cells also play an important role in anti-fungal immunity. Here we show that while NK cells recognize and eliminate C. albicans, the fungal cells inhibit NK cells by manipulating the immune checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) in both humans and mice. We identify the responsible fungal ligands as members of the Als (Agglutinin-Like Sequences) protein family. Furthermore, we show that blocking this interaction using immunotherapy with a TIGIT-blocking antibody can re-establish anti-Candida immunity and serve as a potential therapeutic tool.

Investigations on the Biological Activity of Allium sativum Agglutinin (ASA) Isolated from Garlic.

BACKGROUND: Garlic (Allium sativum) from the family Amaryllidaceae is widely used in culinary and is reported to have potential anticancer, anti-diabetic, antimicrobial, and cardioprotective activities. Allium sativum agglutinin (ASA) is a bulb-type lectin (BTL) domaincontaining lectin isolated from garlic and has been studied for its various biological functions. Previous studies have reported the anti-cancer effects of ASA on histiocytic lymphoma (U937), promyelocytic leukemia (HL60), and oral cancer (KB). METHODS: In this study, we have purified and characterized ASA and evaluated it for its anticancer effects on other cancer cell lines. MTT assay and FACS analysis was done to corroborate the anticancer findings against cervical (HeLa) and lung cancer (A549) cell lines. RESULTS: IC50 value of 37 µg/ml in HeLa and a weak activity (26.4 ± 1.9% cellular inhibition at 100µg/ml treatment) in A549 were found in the MTT assay. FACS analysis further corroborated these findings and showed the apoptotic effects of ASA in these cell lines. CONCLUSION: Anticancer activity for members of bulb-type lectin (BTL) domain-containing lectins has been widely reported, and we hope that our study forms a basis for the development of ASA as a therapeutic agent.

Changes in Gastric Mucosal Glycosylation Before and After Helicobacter pylori Eradication Using Lectin Microarray Analysis.

BACKGROUND: Glycosylation is a common post-translational modification, and it has been reported that alterations in the glycosylation patterns on cells are related to cell proliferation, differentiation, tissue adhesion, and carcinogenesis. This study aimed to investigate the relationship between Helicobacter pylori infection and gastric mucosal glycosylation using a lectin microarray system. METHODS: Gastric mucosal samples were obtained from 10 Helicobacter pylori-non-infected patients, 10 H. pylori-infected patients, and 10 after H. pylori-eradicated patients who underwent gastric mucosal biopsy by endoscopy in our institute. The gastric gland cells which were isolated from formalin-fixed, paraffin-embedded gastric mucosal biopsy samples using laser capture microdissection were used for lectin microarray to obtain lectin-glycan interaction values. RESULTS: Comparison of the lectin-glycan interaction values before and after eradication in the same patients showed significant increases for Ricinus communis agglutinin 120, Trichosanthes japonica agglutinin II, Euonymus europaeus lectin, jacalin, Amaranthus caudatus agglutinin, and Maclura pomifera agglutinin and significant decreases for Urtica dioica agglutinin, Lycopersicon esculentum lectin, Ulex europaeus agglutinin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I. Furthermore, jacalin and MPA in the gastric antrum were significantly decreased with H. pylori infection compared with the without infection group and improved to the levels seen without infection as a result of eradication. Lycopersicon esculentum lectin, Sambucus nigra agglutinin, Sambucus sieboldiana agglutinin, and Trichosanthes japonica agglutinin I in the gastric body were significantly increased with H. pylori infection and improved to the level seen without infection as a result of eradication. CONCLUSION: H. pylori infection changes the lectin binding state which is related to various cancers on the gastric mucosal cell. Furthermore, those changes are reversible by H. pylori eradication.

A Novel Serum Glycobiomarker for Diagnosis and Prognosis of Cholangiocarcinoma Detected by Butea monosperma Agglutinin.

Plant lectins are widely used in medical glycosciences and glycotechnology. Many lectin-based techniques have been applied for the detection of disease-associated glycans and glycoconjugates. In this study, Butea monosperma agglutinin (BMA), a lectin purified from seeds of the medicinal plant Butea monosperma, was used for the detection of cholangiocarcinoma (CCA)-associated glycans. Expression of BMA-binding N-acetyl galactosamine/galactose (GalNAc/Gal)-associated glycan (BMAG) in CCA tissues was determined using BMA lectin histochemistry; the results showed that BMAG was undetectable in normal bile ducts and drastically increased in preneoplastic bile ducts and CCA. The study in hamsters showed that an increase of BMAG was associated with carcinogenesis of CCA. Using an in-house double BMA sandwich enzyme-linked lectin assay, BMAG was highly detected in the sera of CCA patients. The level of serum BMAG in CCA patients (N = 83) was significantly higher than non-CCA controls (N = 287) and it was applicable for diagnosis of CCA with 55.4% sensitivity, 81.9% specificity, and 76.0% accuracy. A high level of serum BMAG (≥82.5 AU/mL) was associated with unfavorable survival of CCA patients; this information suggested the potential of serum BMAG as a poor prognostic indicator of CCA. In summary, BMAG was aberrantly expressed in preneoplastic bile ducts and CCA, it was also highly detected in patient serum which potentially used as a marker for diagnosis and prognostic prediction of CCA.

Use of the reticulocyte channel warmed to 41 °C of the XN-9000 analyzer in samples with the presence of cold agglutinins

ABSTRACT Objectives The purpose of this study was to compare data obtained from the reticulocyte channel (RET channel) heated to 41 °C with those obtained from impedance channel (I-Channel) at room temperature in the samples with the mean corpuscular hemoglobin concentration (MCHC) < 370 g/L and in samples with the MCHC > 370 g/L, in the presence of cold agglutinins. Methods In this study, 60 blood samples (group 1) with the MCHC < 370 g/L (without cold agglutinins) and 78 blood samples (group 2) with the MCHC > 370 g/L (with cold agglutinins) were used to compare the two analytical channels of the XN-9000 analyzer in different preanalytical conditions. The parameters evaluated in both groups were the following: red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), RBC-most frequent volume (R-MFV), mean hemoglobin concentration (MCH) and mean cellular hemoglobin concentration (MCHC). Results The results of this study showed an excellent correlation with both channels of the XN-9000 analyzer in samples with and without cold agglutinins, except for the MCHC. The bias between the values obtained in the I-channel and those obtained in the RET channel of both groups was insignificant and remained within the limits of acceptability, as reported by Ricos et al. for all considered parameters, except for MCHC. Conclusions The presence of cold agglutinins in blood samples can be detected by a spurious lowering of the RBC count and by a spurious increase in the MCHC. The RET channel represents a great opportunity to correct the RBC count in a rapid manner without preheating. However, neither methodology can completely solve the residual presence of cold agglutinins in all samples, despite the MCHC values being < 370 g/L.

The Agglutinin of Common Nettle (Urtica dioica L.) Plant Effects on Gene Expression Related to Apoptosis of Human Acute Myeloid Leukemia Cell Line.

Treatment of acute myeloid leukemia (AML) requires new drugs as result of a rise in new cases and high disease relapse. Plant lectins with the ability to bind carbohydrates on the cell surface have the potential to treat cancer. Urtica dioica L. agglutinin (UDA) is a low weight lectin with anti-benign prostatic hyperplasia (BPH) impact. Here, we examine the impact of UDA on HL-60 cell line. Cytotoxicity and cytostatic effects were assessed in HL-60 cells treated with UDA and vincristine (positive control). The effects of the lectin on cell cycle phases and cell death mechanism were surveyed by propidium iodide (PI) staining and annexin V/PI, respectively. The activation status of the apoptosis pathway was determined by western blotting. Finally, the expression levels of 84 genes were examined by the Human cancer drug target gene PCR array kit. The results indicated that the increase in UDA concentration inhibited the proliferation of HL-60 cells as well as apoptosis induction. Cell cycle analysis showed that the number of sub G1 cells increased essentially. Experimental observations showed that UDA can induce cell apoptosis through a caspase 9-dependent pathway. The expression changes of 21 genes confirmed the apoptotic events in HL-60 cells treated with UDA. In this, we have presented the first investigation on the cytotoxic and apoptotic effects of a lectin isolated from rhizomes and roots of Urtica dioica L. on human AML cells. Generally, the results suggest that UDA may have therapeutic value for leukemia and would be studied further as a new drug for AML later on.

Combined lectin- and immuno-histochemistry (CLIH) for applications in cell biology and cancer diagnosis: Analysis of human urothelial carcinomas.

Lectin histochemistry (LHC) and immunohistochemistry (IHC), which demonstrate the composition and localisation of sugar residues and proteins in cell membranes, respectively, are generally used separately. Using these two methods, we previously demonstrated that malignant transformation of urothelial cells results in the alterations of protein glycosylation and reduced expression of urothelium-specific integral membrane proteins uroplakins (UPs). However, the correlation between these changes was not studied yet. To evaluate this correlation, we developed innovative method, which we named combined lectin- and immuno- histochemistry (CLIH). We used human biopsies of 6 normal urothelia and 9 papillary urothelial carcinomas, i.e. 3 papillary urothelial neoplasms of low malignant potential (PUNLMP), 3 non-invasive papillary urothelial carcinomas of low grade (pTa, l.g.), and 3 invasive papillary urothelial carcinomas of high grade (pT1, h.g.). We tested five different protocols (numbered 1-5) of CLIH on paraffin and cryo-semithin sections and compared them with LHC and IHC performed separately. Additionally, we carried out western and lectin blotting with antibodies against UPs and lectins Amaranthus caudatus agglutinin (ACA), Datura stramonium agglutinin (DSA), and jacalin, respectively. We showed that incubation with primary antibodies first, followed by the mixture of secondary antibodies and lectins is the most efficient CLIH method (protocol number 5). Additionally, 300 nm thick cryo-semithin sections enabled better resolution of co-localisation between sugar residues and proteins than 5 µm thick paraffin sections. In the normal urothelium, CLIH showed co-localisation of lectins ACA and jacalin with UPs in the apical plasma membrane (PM) of superficial umbrella cells. In papillary urothelial carcinomas, all three lectins (ACA, DSA and jacalin) labelled regions of apical PM, where they occasionally co-localised with UPs. Western and lectin blotting confirmed the differences between normal urothelium and papillary urothelial carcinomas. Our results show that CLIH, when used with various sets of lectins and antigens, is a useful, quick, and reliable method that could be applied for basic cell biology research as well as detailed subtyping of human urothelial carcinomas.

H-type lectins - Structural characteristics and their applications in diagnostics, analytics and drug delivery.

Lectins are ubiquitous carbohydrate-binding proteins that interact with sugar moieties in a highly specific manner. H-type lectins represent a new group of lectins that were identified in invertebrates. These lectins share structural homology and bind mainly to N-acetylgalactosamine (GalNAc). Recent structural studies on the H-type lectins provided a detailed description of the GalNAc-lectin interaction that is already exploited in a number of biomedical applications. Two members of the H-type lectin family, Helix pomatia agglutinin (HPA) and Helix aspersa agglutinin (HAA), have already been extensively used in many diagnostic tests due their ability to specifically recognize GalNAc. This ability is especially important because aberrant glycosylation patterns of proteins expressed by cancer cells contain GalNAc. In addition, H-type lectins were utilized in diagnostics of other non-cancer diseases and represent great potential as components of drug delivery systems. Here, we present an overview of the H-type lectins and their applications in diagnostics, analytics and drug delivery.

Primera serie de casos reportada en Colombia de la enfermedad por aglutininas frías primaria y secundaria. Experiencia de un centro de referencia en Medellín entre 2010 y 2017 / First case series reported in Colombia of primary and secondary cold agglutinin disease. Experience of a reference center in Medellín between 2010 and 2017

RESUMEN Introducción: la enfermedad por aglutininas frías (EAF) es un trastorno hematológico primario o secundario, caracterizado por la anemia hemolítica autoinmune causada por los anticuerpos IgM a bajas temperaturas. Clínicamente, presenta parestesias y acrocianosis inducidos por frío y fiebre, aunque también puede ser asintomática y solo identificarse por alteraciones en el hemograma. Objetivo: describir las manifestaciones clínicas y de laboratorio, las causas primarias y secundarias de la EAF y compararlas con series de casos descritos en la literatura. Materiales y métodos: análisis retrospectivo de datos clínicos de pacientes del Hospital Universitario San Vicente Fundación de Medellín con resultados positivos para aglutininas frías. Dichos análisis se realizaron en el laboratorio de hematología de la Universidad de Antioquia, consideramos como positivo título ≥ 1: 64 o con la prueba de Coombs directa y positiva para anticuerpos fríos. Resultados: se incluyen los títulos de crioaglutininas de 23 casos con EAF: 6 formas primarias, 4 asociadas con los linfomas no Hodgkin (LNH), 8 secundarias a enfermedades infecciosas y autoinmunes y, 5 asociados con enfermedades misceláneas. Discusión y conclusiones: esta es la primera serie de casos en Colombia de EAF. La edad y género fueron similares a los datos reportados en la literatura. Observamos un mayor número de pacientes que presentaban anemia hemolítica y con síntomas asociados al frío. La relación hemoglobina hematocrito fue 1:2. Dentro de las causas secundarias destacamos las vasculitis, el lupus y la malaria. De las causas primarias las más frecuentes fueron los LNH, específicamente, el linfoplasmocítico. El tratamiento más utilizado para pacientes con EAF primaria incluyo rituximab.

SUMMARY Introduction: Cold agglutinin disease (CAD) is a primary hematologic disorder or can be secondary to another disease. CAD is characterized by autoimmune hemolytic anemia associated with IgM type antibodies, at low temperatures. Clinically CAD is associated with cryoparesthesia and acrocyanosis induced by cold and fever, or it can be asymptomatic and can be detected by abnormalities on cell blood counts. Objective: To describe the clinical and laboratory data and the etiology of CAD. Comparison between this case series and those described in the literature. Materials and Methods: Retrospectively, we analyzed clinical data of patients from Hospital Universitario San Vicente Fundación with positive results for cold agglutinin assays made in the hematology lab from Universidad de Antioquia. We consider patients with titers ≥ 1:64 or Coombs test positive for cold antibodies. Results: We describe clinical and laboratory findings included crioagglutinin titers of 23 cases with CAD: 6 of them with primary CAD, 4 with non-Hodgkin Lymphoma (NHL), 8 patients with CAD associated with infectious and autoimmune disease and 5 with CAD miscellaneous diseases. Discussion and Conclusions: This is the first CAD case series described in Colombia. Age and gender were like others case series. Most of patients presented with hemolityc anemia and cold related symptoms. The hemoglobin/ hematocrit ratio was 1:2. Secondary causes were vasculitis, lupus and malaria. Primary CAD were related to NHL, specifically limphoplasmocytic Most of the treatments of primary CAD included rituximab.