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Polycystic ovary syndrome (PCOS) is a major endocrine disorder strongly associated with androgen excess and frequently leading to female infertility. Although classically considered an ovarian disease, altered neuroendocrine control of gonadotropin-releasing hormone (GnRH) neurons in the brain and abnormal gonadotropin secretion may underpin PCOS presentation. Defective regulation of GnRH pulse generation in PCOS promotes high luteinizing hormone (LH) pulsatile secretion, which in turn overstimulates ovarian androgen production. Early and emerging evidence from preclinical models suggests that maternal androgen excess programs abnormalities in developing neuroendocrine circuits that are associated with PCOS pathology, and that these abnormalities are sustained by postpubertal elevation of endogenous androgen levels. This article will discuss experimental evidence, from the clinic and in preclinical animal models, that has significantly contributed to our understanding of how androgen excess influences the assembly and maintenance of neuroendocrine impairments in the female brain. Abnormal central gamma-aminobutyric acid (GABA) signaling has been identified in both patients and preclinical models as a possible link between androgen excess and elevated GnRH/LH secretion. Enhanced GABAergic innervation and drive to GnRH neurons is suspected to contribute to the pathogenesis and early manifestation of neuroendocrine derangement in PCOS. Accordingly, this article also provides an overview of GABA regulation of GnRH neuron function from prenatal development to adulthood to discuss possible avenues for future discovery research and therapeutic interventions. © 2022 American Physiological Society. Compr Physiol 12:3347-3369, 2022.

In this review, we explore the roles of divalent metal ions in structure and function within the extracellular matrix (ECM), specifically, their interaction with glycosaminoglycans (GAGs) during tumor progression. Metals and GAGs have been individually associated with physiological and pathological processes, however, their combined activities in regulating cell behavior and ECM remodeling have not been fully explored to date. During tumor progression, divalent metals and GAGs participate in central processes, such as cell migration and angiogenesis, either by modulating cell surface molecules, as well as soluble signaling factors. In addition, studies on metals and polysaccharides interactions have been of great value, as they provide structural information that can be correlated with function. Finally, we believe that understanding how metals are regulated in physiological and pathological conditions is paramount for the development of new treatment strategies, as well as diagnostic and exploratory tools.

Using a rat model of peritonitis, we herein report the inflammatory effect induced by the lectin isolated from Vatairea guianensis (VGL) seeds in the context of interactions between VGL and both toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1). Peritoneal macrophages were stimulated with VGL for dose-dependent gene expression and release of TNF-alpha. In vivo results showed that VGL (1 mg/kg; intraperitoneal) induced peritonitis in female Wistar rats. Leukocyte migration, macrophage activation, and protein leakage were measured 3 and 6 hours after induction. In vitro, peritoneal macrophages were stimulated with VGL for gene expression and TNF-alpha dosage (mean +/- SEM (n = 6), analysis of variance, and Bonferroni's test (P < .05)). In silico, VGL structure was applied in molecular docking with representative glycans. It was found that (a) VGL increases vascular permeability and stimulates leukocyte migration, both rolling and adhesion; (b) lectin-induced neutrophil migration occurs via macrophage stimulation, both in vitro and in vivo; (c) lectin interacts with TLR4 and TNFR1; and (d) stimulates TNF-alpha gene expression (RT-PCR) and release from peritoneal macrophages. Thus, upon lectin-glycan binding on the cell surface, our results suggest that VGL induces an acute inflammatory response, in turn activating the release of peritoneal macrophages via TNF-alpha and TLR and/or TNFR receptor pathways.

The development of the mammary gland involves formation of a branched arboreal structure resulting from the penetration and proliferation of epithelial cells into the fat pad. The mammary cells invade by remodeling their surrounding extracellular matrix (ECM), which are rich in proteins, and glycans such as heparan sulfate proteoglycans (HSPGs). There is increasing literature on how the interaction between signaling by ECM and matrix metalloproteinases (MMPs) is relevant to morphogenetic and physiological contexts. Here we sought to understand how heparanase, the sole mammalian heparan sulfate-degrading endoglycosidase may regulate mammary gland development. We found a robust localization of heparanase within growing end buds during branching in vivo. Using three-dimensional (3D) organotypic cultures, we showed that heparanase expression and activity are required for mammary epithelial invasion/branching within dense collagen I gels. Morphometric analysis of glands from both heparanase-overexpressing and knockout mice showed a direct correlation between degree of branching and the heparanase levels, confirming our 3D organotypic culture observations. Finally, we uncovered a reciprocal association between levels of heparanase and MMP14, a membrane-bound MMP, shedding further light on how branching occurs within developing mammary glands.

In yeast multicellular aggregates, such as biofilms and colonies, cells are supported by a yeast extracellular matrix (yECM) of glycosidic nature, the composition of which is mostly unknown. Saccharomyces cerevisiae ECM was produced, extracted and partitioned. An analytical-grade pure glycoside fraction was obtained, fractionated by anionic exchange liquid chromatography and analyzed by gas chromatography–mass spectrometry and polyacrylamide gel electrophoresis. Two different molecular weight polysaccharides were found, composed of glucose, mannose and small relative amounts of galactose. One of the polysaccharides had a low molecular weight, compatible with the association with glycoproteins abundantly occurring in yECM. In addition, these polysaccharide species were separated by diaminopropane agarose gel electrophoresis and induced metachromatic shift, suggesting chemical substitution, which was corroborated by anticoagulation activity. This was shown to be associated with the double deletion of the yeast homologues of the mammalian Hedgehog modulators Hhatl and Hhat, respectively yeast Gup1 and Gup2. These results pioneer the study of the molecular biology of the ECM supporting S. cerevisiae multicellular aggregates such as biofilms.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of s-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.

Viral infections have been associated with autoimmune connective tissue diseases. To evaluate whether active infection by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV)-6, -7, -8, as well as parvovirus B19 (B19V) occur in patients with autoimmune connective tissue diseases, viral DNA loads were assessed in paired samples of serum and peripheral blood mononuclear cells (PBMCs) of 115 patients affected by different disorders, including systemic sclerosis, systemic, and discoid lupus erythematosus, rheumatoid arthritis, and dermatomyositis. Two additional groups, patients affected by inflammatory diseases (n=51) and healthy subjects (n=58) were studied as controls. The titers of anti-HHV-6 and anti-EBV antibodies were also evaluated. Cell-free HHV-6 serum viremia was detected in a significantly higher proportion of connective tissue diseases patients compared to controls (P

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (γ = 0.62 and γ = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample

"Several lines of evidence suggest the involvement of infectious agents in the pathogenesis of atherosclerosis. Furthermore, a correlation between infection-driven inflammatory burden and acute manifestation of coronary artery disease has been hypothesized. The aim of this work was to assess whether human herpesvirus (HHV)-6 and HHV-8, two DNA viruses with a distinct tropism for endothelium and lymphocytes, may be associated with coronary instability. An age- and gender-matched cross-sectional study was undertaken in 70 patients with testing of plasma HHV-6 and HHV-8 DNA load in different cardiovascular clinical settings: 29 patients with acute myocardial infarction, 21 patients with stable coronary artery disease, and 20 patients without coronary and carotid artery atherosclerosis subjected to cardiac valve replacement. In all patients, HHV-6 and HHV-8 plasma DNA was tested by using highly sensitive, calibrated quantitative real-time PCR assays which employ a synthetic DNA calibrator to adjust for DNA extraction and amplification efficiency. HHV-8 viremia was undetectable in all three groups. HHV-6 viremia was detected in a substantial fraction of the samples examined (18.6%) without significant differences among the three groups (ST segment elevation myocardial infarction: 17.2%; stable coronary artery disease: 14.3%; patients without coronary and carotid artery atherosclerosis: 25%). Furthermore, no significant differences in plasma HHV-6 load were observed amongst the three groups of patients. These findings indicate that coronary instability is not associated specifically with active HHV-6 or HHV-8 infection. However, an unusually high rate of active HHV-6 infection was documented among patients without atherosclerosis admitted to hospital with cardiac disease. "

Aim Immunophenotype peripheral blood T cells from renal transplant recipients (RTR) using cellular markers of regulatory T cells (Tregs) and flow cytometry, including Foxp3, and correlate these findings with clinical parameters. Methods Expression of phenotypic markers of Tregs was assessed by flow cytometric analysis of peripheral blood lymphocytes (PBL) from (i) RTR (n = 95); (ii) patients with end-stage renal failure (ESRF) awaiting transplantation (n = 17); and (iii) normal healthy controls (n = 18). Results The percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) cell population did not significantly alter at different time points post-transplant. However, the percentage of CD4(+) CD25(+) Foxp3(+) cells within the CD4(+) population was significantly lower in RTR compared with patients with ESRF. In contrast, RTR and ESRF had a similar percentage of CD4(+) CD25(+) cells expressing Foxp3. Multivariate analysis of PBL and clinical parameters demonstrated (i) a positive linear relationship between the percentage CD4(+) CD25(+) cells expressing Foxp3 and estimated glomerular filtration rate and (ii) a higher percentage of CD4(+) CD25(+) cells in the CD4(+) cell population in patients with malignancy (the majority were skin cancers). Malignancy also correlated strongly with time post-transplant and age of the RTR. Conclusion Immune monitoring of the PBL phenotype in RTR using CD4, CD25 and Foxp3 may stratify RTR and predict graft outcome and function, and risk of complications from immunosuppression. Longitudinal and functional studies of Tregs are essential to extend the findings of the present study.

Glycosaminoglycans are natural heteropolysaccharides that are present in every mammalian tissue. They are composed of repeating disaccharide units that consist of either sulfated or non-sulfated monosaccharides. Their molecular size and the sulfation type vary depending on the tissue, and their state either as part of proteoglycan or as free chains. In this regard, glycosaminoglycans play important roles in physiological and pathological conditions. During recent years, cell biology studies have revealed that glycosaminoglycans are among the key macromolecules that affect cell properties and functions, acting directly on cell receptors or via interactions with growth factors. The accumulated knowledge regarding the altered structure of glycosaminoglycans in several diseases indicates their importance as biomarkers for disease diagnosis and progression, as well as pharmacological targets. This review summarizes how the fine structural characteristics of glycosaminoglycans, and enzymes involved in their biosynthesis and degradation, are involved in cell signaling, cell function and cancer progression. Prospects for glycosaminoglycan-based therapeutic targeting in cancer are also discussed.

In this work, we have applied Density Functional Theory calculations to investigate the electronic and spacial effects of different phosphorus ligands on the selectivity of the olefin (propene and styrene) insertion reaction into the RhH bond of the complexes HRh(PR3)(CO)2(olefin), where the modified ligand PR3, is a phosphine (R = H, F, Et, Ph) or phosphite (R = OEt, OPh). M06/SBKJC/cc-pVDZ calculations revealed that the olefin coordination and insertion reaction are dominated by the electronic effects of the phosphorus ligands. A very good correlation between the Tolman electronic factor, χ, with the backdonated charges from the metallic center to the olefin and also with the interaction energy of the olefin with the four-coordinated HRh(CO)2(PR3) catalyst was found. Using the propene as the substrate and for all the phosphorus ligands investigated, the insertion always proceeds through the reaction path leading to the linear metal-alkyl intermediate. However, when styrene is used, the branched metal-alkyl intermediate is always favored. The structural results obtained for the transition states do not support the existence of a π-allilic intermediates. The regioselectivity obtained for the insertion reaction of styrene results from thermodynamic aspects of the reaction in which the branched metal-alkyl intermediate is always favored by ∼5 kcal/mol. The M06/SBKJC/cc-pVDZ results are in good agreement with the experimental findings. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Diswall M, Gustafsson A, Holgersson J, Sandrin MS, Breimer ME. Antigen-binding specificity of anti-αGal reagents determined by solid-phase glycolipid-binding assays. A complete lack of αGal glycolipid reactivity in α1,3GalT-KO pig small intestine. Xenotransplantation 2011; 18: 28–39. © 2011 John Wiley & Sons A/S. Abstract: Background: αGal-specific lectins, monoclonal and polyclonal antibodies (Abs) are widely used in xenotransplantation research. Immunological assays such as immunohistochemistry, flow cytometry, Western blot and thin layer chromatography are often the only applicable characterization procedures when limited amount of tissue is available and biochemical characterization is impossible. Hence, detailed knowledge of the Ab/lectin carbohydrate-binding specificity is essential. Methods: The binding specificity of human blood group AB serum, three different affinity-purified human polyclonal anti-Gal Ab batches, and two anti-Gal mAb clones (TH5 and 15.101) as well as Griffonia simplicifolia isolectin B4 and Marasmius oreades agglutinin were examined for reactivity with glycolipid fractions isolated from human and pig (wild-type and α1,3GalT-KO) tissues using thin layer chromatogram and microtiter well binding assays. Results: All anti-Gal-specific reagents reacted with the pentaglycosylceramide Galα1,3nLc4, and several 6–12 sugar compounds in wild-type pig kidneys. However, their staining intensity with different αGal antigens varied considerably. Some, but not all, anti-Gal reagents cross-reacted with a pure iGb3 glycolipid reference compound. No reactivity with glycolipids isolated from α1,3GalT-KO pig small intestine or human tissues was found, confirming the specificity of the anti-Gal reagents in those tissues for α1,3Gal-epitopes produced by the α1,3GalT (GGTA1). Conclusions: Different anti-Gal reagents vary in their carbohydrate epitope specificity. Mono-/polyclonal Abs and lectins have different carbohydrate epitope fine specificity toward pig glycolipids as well as purified Galα1,3nLc4, and iGb3. Despite the difference in αGal specificity, all reagents were completely non-reactive with glycolipids isolated from α1,3GalT-KO pig small intestine.

Nanotube-based sensors are high-sensitivity devices that can probe a number of nano-sized impurities through doping-induced variations in their conducdvity. One key question for designing efficient sensors is to establish which impurity/ nanotube combination will generate maximum sensitivity. Standard ways for selecting a good nanotube/impurity combination consists of an ad hoc approach that involves sifting through an immense universe of possibilities. This trial-and-error approach, despite being time-consuming and computationally demanding, does not necessarily result in sensitivity optimization. A mathematically transparent theoretical formalism was adopted to introduce an inverse way of thinking about the nanotube/impurity combination by establishing a set of characteristics these components must have to generate high sensitivity devices. In this way guidelines were generated that can narrow the search for the ideal components of nanotube-based sensors. We illustrate this method with a few examples of nanotubes doped with a different single-atom impurities.

Carbohydrate structures influence many aspects of cell biology. Manipulating the glycosyltransferase enzymes, that sequentially add carbohydrate moieties to proteins and lipids as they pass through the Golgi and secretory pathway, can alter these carbohydrate epitopes. We previously demonstrated that the eight amino acid cytoplasmic tail of α1,2fucosyltransferase (FT) contained a sequence for Golgi localisation. In this study, we examined the localisation of the closely related secretor type α1,2fucosyltransferase (Sec) which has a smaller, yet apparently unrelated, five amino acid cytoplasmic tail. In contrast to the Golgi localisation of FT, Sec displayed atypical cytoplasmic vesicular-like staining. However, replacing just the five amino acid tail of Sec with FT was sufficient to relocalise the enzyme to a perinuclear region with Golgi-like staining. The biological significance of this relocalisation was this chimaeric enzyme was more effective than FT at competing for N-Acetyl-lactosamine and thus was superior in reducing expression of the Galα(1,3)Gal xenoepitope. Copyright © 2009 John Wiley & Sons, Ltd.

BACKGROUND: Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically-modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti-pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8(+) and gammadelta T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co-stimulation of T cells. METHODS: Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell-cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D-Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. RESULTS AND CONCLUSIONS: In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.

Indirect exchange coupling plays a central role in determining the magnetic order in systems composed of adsorbed magnetic moments on a metallic host. For low "dimensional metallic structures, such as nanotubes, this interaction is predicted to decay slowly. Ab-initio calculations have nevertheless been unable to reproduce this prediction. We show that for Armchair and Zigzag Nanotubes the rate of decay is very sensitive to the location of the impurities within the nanotube host, the range being much shorter when the impurities are located in the') centre of a nanotube hexagon. This explains the diffi- i culty in probing the long range character of the indirect' exchange coupling from standard ab-initio calculations. In so doing, we confirm the general long-ranged character of the coupling but provide a condition for observing shorter range decay.

Background: Porcine dendritic cells (DC) are likely to be pivotal cells in the initiation of stimulatory and potential tolerogenic responses to xenoantigens, however, there are limited studies characterizing these antigen presenting cells. Methods: Porcine PBMC (CD172a+) were cultured with GM-CSF and IL-4 and phenotype and functional capabilities assessed. Lipopolysaccharide (LPS), IL-10, and IL-3 were added to the GM-CSF/IL-4 DC cultures to determine phenotypic and functional changes. Quantitative real-time polymerase chain reaction (PCR) for key cytokines was performed and the modified porcine DC were further assessed by primary mixed lymphocyte reaction to determine the effect of LPS, IL-10, and IL-3 on stimulatory capability. Results: Porcine PBMC (CD172+) cultured with GM-CSF and IL-4 produced cells with DC morphology, which were major histocompatability complex (MHC) class II+, CD14−/lo, and CD1alo. Addition of IL-10 or IL-3 to GM-CSF/IL-4 DC cultures produced cells with lower levels of MHC class II and higher levels of antigen uptake consistent with less mature DC. Quantitative real-time PCR of DC showed the addition of IL-10 induced an increase in IL-10 mRNA, no detectable IL-12, and reduced IL-6 mRNA. The addition of IL-3 to DC cultures decreased IL-12, IL-6 and tumor necrosis factor (TNF), with no change in IL-10 mRNA. GM-CSF/IL-4 DC induced strong human lymphocyte proliferation, compared with significantly reduced stimulatory capacity induced by IL-10 and IL-3 treated DC cultures. Conclusions: The profound effect on differential DC cytokine profile and reduced human anti-pig responses has important therapeutic implications in xenotransplantation. The mechanism of altered regulation warrants further investigation.

The isolectin B4 (IB4) stains a subset of small and medium-sized dorsal root ganglion (DRG) neurons by binding to terminal α-galactose on glycoproteins and glycolipids. The enzymes α(1,3)galactosyltransferase (1,3GT) and isoglobotriaosylceramide synthase (iGb3S) synthesize the galactose-α(1,3)-galactose group, which is the most common carbohydrate containing terminal α-galactose. 1,3GT preferentially glycosylates proteins whereas iGb3S glycosylates lipids. We generated antibodies against rat 1,3GT and iGb3S that were used for immunohistochemical staining of DRG cells. Virtually all neurons that bound IB4 expressed both enzymes, suggesting that IB4 binds to both glycoproteins and glycolipids in IB4-positive neurons. 1,3GT immunoreactivity was observed in small and medium-sized neurons and satellite cells. iGb3S immunoreactivity was observed in neurons of varying sizes. Many neurons that expressed these enzymes did not bind IB4. Additionally, the majority of neurons that expressed substance P expressed both enzymes but did not bind IB4. Ultrastructual studies revealed that 1,3GT was predominantly associated with the Golgi apparatus, whereas iGb3S was found near the Golgi apparatus and in large, clear vesicles throughout the soma. These data suggest that, although expression of 1,3GT and/or iGb3S appears to be necessary for IB4 binding, expression of these enzymes is not sufficient to impart IB4 binding. J. Comp. Neurol. 501:70–82, 2007. © 2007 Wiley-Liss, Inc.

The current success rate of transplant surgery and immunosuppression has led to a demand for organs that has outstripped the supply. This has required investigation of alternate strategies. Therefore, allotransplantation across the ABO blood group barrier has commenced, and pig-to-human xenotransplantation is under consideration. The first immunological barrier to both these types of transplantation is the prevention of the antibody-mediated rejection. This rejection is a result of natural preformed antibodies circulating in the serum of the recipient binding to either ABO (for allo) or alpha-galactose (alpha-Gal) (for xeno) antigens expressed on the donor tissue. These antibodies recognise antigens that are, in both cases, carbohydrate molecules with the characteristic feature that the nonreducing terminal carbohydrate is either a Gal or N-acetlygalactosamine residue in an alpha1,3 linkage. These epitopes are synthesised by closely related members of a single family of glycosyltransferases. This review discusses the carbohydrate antigens, the enzymes involved in their synthesis and the consequences of natural antibodies binding these antigens.

Background Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Galalpha(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind alphaGal or other potential xenoepitopes, such as N-acetyllactosamine (NAcLac), created by the deletion of alpha1,3galactosyltransferase (GT) in animals. Methods and results Initial analysis suggested the human C-type lectin NKRP1A bound to a pool of glycoconjugates, the majority of which contained the terminal Galalpha(1,3)Gal epitope. This was confirmed by high level binding of cells expressing NKRP1A to mouse laminin, which contains a large number of N-linked oligosaccharides with the Galalpha(1,3)Gal structure. The consequence of removing the terminal alphaGal was then investigated. Elevated NAcLac levels were observed on thymocytes from GT-/- mice. Exposing NAcLac on laminin, by alpha-galactosidase treatment, resulted in a significant increase in NKRP1A binding. Conclusions NKRPIA binds to the alphaGal epitope. Moreover, exposing NAcLac by removal of alphaGal resulted in an increase in binding. This may be relevant in the later phases of xenotransplant rejection if GT-/- pigs, like GT-/- mice, display increased NAcLac expression.

Mulley WR, Wee JL, Christiansen D, Milland J, Ierino FL, Sandrin MS. Lentiviral expression of CTLA4Ig inhibits primed xenogeneic lymphocyte proliferation and cytokine responses. Xenotransplantation 2006; 13: 248–252. © Blackwell Munksgaard, 2006 Abstract: Background: Co-stimulatory blockade is known to inhibit lymphocyte responses and to prolong allograft and xenograft survival. The present study examines the effect of transgenic expression of cytotoxic T lymphocyte-associated molecule-4 immunoglobulin (CTLA4Ig) by a porcine endothelial cell line (PIEC) transduced by a lentiviral vector, on primed xenogeneic T-cell proliferative and cytokine responses. Methods: Splenocytes from mice primed with PIEC were used as responder cells in a secondary proliferative assay. CTLA4Ig transduced and wild-type PIEC were used as stimulator cells. Responder cells were assayed for proliferation and cytokine production. Results: Proliferation was profoundly inhibited by CTLA4Ig transduced cells compared with control cells. Cytokine analysis by enzyme linked immunospot demonstrated that production of interferon-γ, IL4 (interleukin 4) and IL10 was inhibited by CTLA4Ig transduced cells compared with control cells. Conclusion: CTLA4Ig inhibited primed indirect xenogeneic T-cell proliferative and cytokine responses in vitro. Expression of immunomodulatory molecules by xenogeneic tissues has potential therapeutic applications for future xenotransplantation.

The human indoleamine 2,3-dioxygenase (HuIDO) baculoviral construct, for expression of HuIDO protein with a hexa-histidine and FLAG (DYKDDDDK) tag, was produced using the BacPAK Baculovirus Expression System. HuIDO baculovirus was used to infect Sf21 insect cells to produce functionally active protein in large amounts. Conditions for protein purification by metal affinity chromatography were determined and optimized. Addition of haemin ensured optimal activity of the purified heme-containing oxygenase. The soluble purified protein was used to immunize a chicken to produce large quantities of polyclonal IgY against HuIDO. The anti-HuIDO IgY antibody specifically detected HuIDO produced by a range of cell types including transfectants and native HuIDO expression induced in IFN-gamma-stimulated cells. The antibody detected HuIDO in cell lysates by western blotting and in the cytoplasm of cells by microscopy. The antibody was unable to block the function of the enzyme, indicating that this antibody binds outside the active site of HuIDO.

Natural killer (NK) cells represent the first line of defence against viral infections but, in the case of hepatitis B virus (HBV), may also be involved in liver injury. We here compared NK-cell activity of 11 patients with acute HBV infection, either HIV-positive or HIV-negative, with that of 11 healthy subjects. One of the HIV-positive patients, characterized by a severe immunodeficiency, died 3 weeks after hospitalization for HBV-related fulminant hepatitis (FH). He displayed a remarkable NK-cell cytotoxicity against both cell lines and autologous dendritic cells, whereas the NK-cell activity of the remaining patients was significantly reduced as compared with healthy individuals. Our findings suggest that NK-cell-mediated cytotoxicity could contribute to the development of HBV-related acute liver failure in HIV-positive patients with severe immunodeficiency. An immunopathological model of FH in immunocompromised patients was proposed.

We have previously reported that islets present in cultured fetal pig pancreas (FPP) are resistant to destruction by Galalpha(1,3)Gal antibodies and compliment, but are susceptible to the 'secondary' antibody response which occurs on transplanting pig islet tissue to Galo/o murine recipients. In a model of antibody-mediated graft rejection, we tested the resistance of porcine islets to antibody. Using FPP from human CD46 transgenic pigs, we now report that the complement regulator, CD46, affords protection from antibody-mediated rejection when mouse anti-pig serum (MAPS) was administered to scid mice bearing PFF grafts from either CD46 transgenic or normal pigs. Indeed, whereas normal pig islets were destroyed by an intraperitoneal (i.p.) injection of 0.1 to 0.2 ml of MAPS antibody, destruction of CD46-expressing transgenic islets required 0.5 ml, i.e. up to five times the amount. In contrast, there was no prolongation of the survival of CD46 transgenic mouse skin or heart major histocompatibility complex-compatible or -incompatible allografts--rejected by predominantly cellular immune mechanisms, as opposed to xenograft rejection. Although complement regulators have been examined for their protective role in hyperacute rejection of vascularized xenografts, it is clear that they also have protective effects in the later, antibody-mediated responses, but are unlikely to effect the inflammatory response in cell-mediated rejection.

Xenotransplantation (XTx) provides a potential solution to the shortage of human organs and tissues, and has several advantages over other possible solutions to this problem. However, a number of scientific and ethical barriers exist, and need to be addressed in order to advance the field of XTx in a manner that optimizes its potential to benefit society and minimizes its risk. Some of the most pressing ethical issues are discussed, and the position of the Ethics Committee of the International Xenotransplantation Association is presented.

Although removal of the Galα(1,3)Gal antigen from pigs would prevent hyperacute graft rejection, the technique of homologous recombination to knock out the α 1,3 galactosyltransferase gene is not available for pigs, and an alternative strategy is presented. As both α 1,3 galactosyltransferase and α 1,2 fucosyltransferase use the same substrate (N-acetyl lacto-samine), competition between the transferases in vitro and in vivo was examined. The data show that there is indeed a hierarchy of these gly-cosyltransferases competing for the same substrate, and that α 1,2 fuco-syltransferase takes precedence over α 1,3 galactosyltransferase: a) COS cells simultaneously transfected with cDNA clones encoding α, 2 fuco-syltransferase and α 1,3 galactosyltransferase show preferential expression of the H substance (synthesised by α 1,2fucosyltransferase) rather than Galα(1,3)Gal (synthesised by α 1,3galactosyltransferase), even though α 1,3galactosyltransferase mRNA and functional enzyme was present, b) In a pig kidney cell line that expressed both the Galα(1,3)Gal and H, the increased expression of H induced by the transfection and stable expression of α 1,2fucosyltransferase resulted in decreased expression of Galα(1,3)Gal. c) Coexpression of α 1,2fucosyltransferase and α 1,3galactosyltransferase in either COS cells or the pig cell line resulted in decreased human antibody binding and complement-mediated cell lysis, d) Transgenic mice, ubiquitously expressing α 1,2fucosyltransferase show a major decrease in Galα-(1,3)Gal expression and a decrease in natural human antibody binding. These findings have important implications for xenotransplantation in that α,2fucosyltransferase transgenic pigs could be a source of donors for xenotransplantation to humans.

Strategies are now being designed to overcome the hyperacute rejection of vascularized pig xenografts by human natural anti-Galα(1,3)Gal antibodies. We now demonstrate that a synthetic octapeptide, Gal pep 1 (DAHWESWL), isolated from a peptide epitope library using the α-galactosyl specific lectin IB4, “mimics” the carbohydrate epitope Galα(1,3)Gal. In vitro studies demonstrated that the binding of the IB4 lectin or human natural antibodies to pig cells could be specifically blocked by Gal pep 1, in several different serological assays: a) the hemagglutnation of pig erythrocytes; b) cytofluorographic analysis of pig lymphocytes and endothelial cells; c) cytotoxicity of human serum against pig endothelial cells; d) an ELISA assay. The relative affinity of the peptide for the IB4 lectin was similar to that of α-galactosyl sugars, although it was bound at a lower affinity by human antibodies. The implications of these observations to xenotransplantation are that peptides could theoretically be used to block hyperacute rejection.

Transplantation of tissues that are vascularized by ingrowth of host vessels, as opposed to primarily vascularized grafts, may be one way of avoiding hyperacute rejection (HAR). Pretransplant treatment of the graft to eliminate from it highly immunogenic cells may also improve graft survival. We examined organ cultured fetal pig pancreas in prediabetic NOD/Lt female mice treated with peritransplant anti-CD4 MAb (GK 1.5) and in separate experiments also studied the expression of Gal(α 1–3)Gal, an epitope that may be a major target of a human anti-pig response. Immunocytochemistry to detect insulin, glucagon, and somatostatin and Gal(α 1–3)Gal showed that differentiated (i.e., hormone containing) endocrine cells were Gal(α 1–3)Gal negative but hormone-negative ductal cells showed strong Gal(α1–3)Gal staining on their luminal border and interstitial cells were also positive particularly early after transplantation. Rejection occurred in all animals, but its rate was markedly altered by transient immunosuppression with the depleting anti-CD4 MAb. Cell infiltration was first detected in controls 2 days posttransplant, was pronounced by 4 days, and grafts showed advanced rejection by 7 days whether or not they had been “immunomodified” by 2 days exposure to 90% O2. In contrast, peritransplant immunosuppression with GK 1.5 improved graft survival but rejection was advanced by 28 days. The control graft sites had many eosinophils, macrophages and mast cells by 4 days, but little infiltration was seen until after 14 days in the immunosuppressed mice and was predominantly by mononuclear cells. Use of “immunomodified” grafts was of no benefit. Thus, graft immunomodification per se, that often allows islet allografts to survive in rodents, had no effect on xenograft survival in this model. A major potential target antigen for natural Ab in humans, Gal(α 1–3)Gal, is not present on differentiated graft endocrine cells.

In pig-to-human transplantation, one of the major obstacles is the expression of Galα(1,3)Galactose by the endothelium of vascularized human tissues and the presence in all humans of IgG and IgM antibodies to this epitope: xenotransplantation would be followed by hyperacute rejection (HAR). However, the findings in endothelial cells of all vessels and the parenchyma of tissues such as kidney and liver do not extend to islet cells. Histological studies demonstrate that the adult pig islet (apart from endothelial cells) does not express Galα(1,3)Gal, nor do fresh neonatal or fetal pig islet cells; after tissue culture (under a variety of conditions) large amounts of Galα(1,3)Gal are expressed by the pig islet cells. However, double staining studies show that Gal+ cells do not secrete insulin, glucagon, or somatostatin and these cells may be spared from potential antigen antibody-mediated rejection after transplantation. The relevance of Gal expression in islet cells is discussed–short term studies–preferably in pig to Old World Monkey transplants could provide the answer to the relevance of Galα(1,3)Gal expression in islet cells. Thus far it appears that some islets can be destroyed by antibody; others are spared and it is important to determine the nature of the sparing mechanism.

Previous histological studies of porcine tissues using the Galα(l–3)Gal specific lectin, IB4-biotin, and an immunoperoxidase technique demonstrated the widespread distribution of Galα(l–3)Gal in pigs—particularly in the endothelium of all vessels and in the parenchyma of liver and kidney. An exception was the endocrine (islets) and exocrine tissue of the pig pancreas—adult islets did not express Galα(l–3)Gal and apart from ducts, and the exocrine pancreas was also nonreactive. Because of the potential use of islet xenotransplantation for the treatment of diabetes, a more extensive study was undertaken of fetal pancreas and of cultured islet cell clusters. Like the adult pancreas, islets from fetuses at term or earlier, essentially did not express Galα(l–3)Gal; however, during culture, under a variety of conditions, they became strongly reactive with IB4 and expressed large amounts of Galα(l–3)Gal. Staining for pancreatic hormones (insulin, glucagon, and somatostatin) demonstrated that true islets were being examined, although double staining for insulin and Galα(l,3)Gal indicated that cells secreting insulin were not Galα(l–3)Gal positive. The origin of the Galα(l–3)Gal+ cells is not apparent, nor is whether they are precursors of hormone secreting cells. It was of interest that, after transplantation to nude mice and allowing for maturation, the islets resembled the adult phenotype and were Galα(l–3)Gal negative. The implications of the findings are not clear but indicate that some cells in the cultured fetal islet (other than insulin secreting cells) could be the target of antibody mediated destruction.