Biological evaluation of multivalent lewis X-MGL-1 interactions.

Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin-carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le(X) ) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal Le(X) , were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying Le(X) structures, compared to monovalent Le(X) trisaccharide. Multivalent presentation of Le(X) on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le(X) -OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate-lectin interactions can be exploited to modulate immune responses.

Defined presentation of carbohydrates on a duplex DNA scaffold.

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors.

Three German fibrinogen Aalpha-chain amyloidosis patients with the p.Glu526Val mutation.

Plasma protein fibrinogen variants cause fibrinogen A alpha-chain (AFib) amyloidosis, which presents with hypertension, proteinuria, and azotemia. Six AFib mutations have been reported thus far. We identified three patients who presented with marked proteinuria and serum creatinine elevations. Their kidney biopsies revealed destruction of the glomerular architecture by amyloid deposits with typical, apple-green birefringence in polarized light after Congo red staining. We found immunoreactivity against fibrinogen, which is typical for this type of amyloidosis. We sequenced the FGA exon 5 and demonstrated heterozygosity for the p.Glu526Val mutation in all three cases. This amino acid substitution is the most common fibrinogen A alpha-chain variant causing AFib amyloidosis. The mutation has been reported in individuals of European and American descent but not yet in German patients. AFib amyloidosis should therefore be considered an important differential diagnosis in German patients with renal amyloidosis. In the cases described here, the use of antibodies directed against fibrinogen, followed by direct gene sequencing, revealed the underlying cause.

Evidence of insulin resistance in adult uncomplicated malaria: result of a two-year prospective study.

The study aimed at investigating the effects of adult uncomplicated malaria on insulin resistance. Fasting levels of blood glucose (FBG), glycosylated hemoglobin (HbA1c), and serum insulin were measured in 100 diabetics and 100 age-matched controls before and during Plasmodium falciparum malaria. Insulin resistance and beta cell function were computed by homeostatic models assessment of insulin resistance (HOMAIR) and beta cell function (HOMAB) formulae, respectively. Body mass index (BMI) was computed. At baseline, diabetics had significantly (P < 0.05) higher levels of BMI, FBG, HbA1c, and HOMAIR but lower level of HOMAB than controls. Baseline insulin levels were comparable (P > 0.05) between the two study groups. During malaria, diabetics maintained significantly (P < 0.05) higher levels of BMI, FBG, and HbA1c but lower levels of insulin and HOMAB than controls. Malaria-induced HOMAIR levels were comparable (P > 0.05) between the two study groups but higher than baseline levels. Apart from BMI and HOMAB, mean levels of all the remaining parameters increased in malaria-infected controls. In malaria-infected diabetics, significant (P < 0.05) increase was only observed for insulin and HOMAIR but not the other measured parameters. Uncomplicated malaria increased insulin resistance in diabetics and controls independent of BMI. This finding may have implications for the evolution of T2DM in malaria-endemic regions.

Role of the C-type lectin receptors MCL and DCIR in experimental colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. Though its exact etiology is still unclear, it is proposed that an imbalance in the intestinal homeostasis leads to a disturbed interaction between commensal microbiota and the mucosal immune system. Previous studies have shown that both innate and adaptive immunity are involved in an overwhelming colon inflammation, and thus contribute to the pathogenesis of IBD. In innate immunity, several pattern recognition receptors such as Toll-like receptors, NOD-like receptors or C-type lectin receptors (CLRs) are involved in IBD pathogenesis. Myeloid CLRs are mainly expressed by antigen-presenting cells and bind to glycan structures present on self or foreign antigens. The Macrophage-restricted C-type lectin (MCL) and the Dendritic cell immunoreceptor (DCIR) are two poorly characterized members of the CLR family. In this study, we investigated the role of MCL and DCIR in the pathogenesis of murine colitis. Both CLRs bound to intestinal microbiota to a different extent. They modulated the production of pro-inflammatory cytokines by antigen-presenting cells upon stimulation with heat-killed microbiota and impacted subsequent T cell responses. To analyze whether MCL and DCIR contribute to the pathogenesis of IBD, the dextran sulfate sodium (DSS) murine colitis model was employed. MCL-/- as well as DCIR-/- mice exhibited only a slightly increased severity of disease compared to wild-type mice indicating a limited role for MCL and DCIR in the regulation of intestinal immunity.

A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

The C-Type Lectin Receptor SIGNR3 Binds to Fungi Present in Commensal Microbiota and Influences Immune Regulation in Experimental Colitis.

Inflammatory bowel disease is a condition of acute and chronic inflammation of the gut. An important factor contributing to pathogenesis is a dysregulated mucosal immunity against commensal bacteria and fungi. Host pattern-recognition receptors (PRRs) sense commensals in the gut and are involved in maintaining the balance between controlled responses to pathogens and overwhelming innate immune activation. C-type lectin receptors (CLRs) are PRRs recognizing glycan structures on pathogens and self-antigens. Here we examined the role of the murine CLR specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 (SIGNR3) in the recognition of commensals and its involvement in intestinal immunity. SIGNR3 is the closest murine homolog of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) receptor recognizing similar carbohydrate ligands such as terminal fucose or high-mannose glycans. We discovered that SIGNR3 recognizes fungi present in the commensal microbiota. To analyze whether this interaction impacts the intestinal immunity against microbiota, the dextran sulfate sodium-induced colitis model was employed. SIGNR3(-/-) mice exhibited an increased weight loss associated with more severe colitis symptoms compared to wild-type control mice. The increased inflammation in SIGNR3(-/-) mice was accompanied by a higher level of TNF-α in colon. Our findings demonstrate for the first time that SIGNR3 recognizes intestinal fungi and has an immune regulatory role in colitis.

Amyloid in endomyocardial biopsies.

The prognosis of cardiac amyloidosis depends on the nature and origin of the amyloid protein deposited. However, little is known about the prevalence and origin of amyloid in heart muscle biopsies. We therefore examined retrospectively the distribution and origin of amyloid in a consecutive series of endomyocardial biopsies. Endomyocardial biopsies with verified presence of amyloid from 101 patients were included. Amyloid was classified immunohistochemically in each of them. Our collective comprised 63 men and 38 women, with a mean age of 66 years (range 37-85 years). Cardiac amyloidosis was the most common of the AL (54 patients) or ATTR type (42 patients). In five individuals, amyloid remained unclassified. AL amyloidosis was subdivided into ALlambda (45 patients) and ALkappa amyloid (nine patients). AA amyloid was not found in any individual. The amount of amyloid was higher in AL than in ATTR amyloidosis. Genomic DNA was extracted and examined by DNA sequencing in 19 patients with ATTR amyloidosis. Five (26%) individuals carried TTR mutations (p.Val20Ile, p.Val30Met (twice), p.Asp39Val and p.Glu54Asp) and were classified as suffering from hereditary ATTR amyloidosis. Amyloid in endomyocardial biopsies is most commonly of immunoglobulin light chain origin, followed by non-hereditary and hereditary-type ATTR amyloid.

Localized insulin-derived amyloidosis in patients with diabetes mellitus: a case report.

Three patients with insulin dependent type 1 diabetes mellitus and one with insulin dependent type 2 diabetes mellitus developed localized amyloid tumors at their general insulin injection sites. All 4 patients (two women and two men) were treated with modern recombinant human insulin or insulin analogues. In addition, 1 patient had used both recombinant and animal insulin. The amyloid tumors were resected and examined histologically using Congo red staining and immunohistochemistry. Insulin was found to be the major component of the amyloid tumors in all four patients. These 4 cases were diagnosed recently within a relatively short period of time, which leads to the conjecture that local insulin-derived amyloid tumors remain principally a differential diagnosis of skin tumors in insulin-dependent diabetic patients.

Prevalence and origin of amyloid in kidney biopsies.

We aimed to reassess renal amyloidosis in kidney biopsies with a focus on possibly misclassified or unclassified cases and changes in the prevalence of different amyloid types. Two hundred thirty-three kidney biopsies obtained from 231 patients diagnosed with amyloid during the period from 1990 to 2007 years were included in this retrospective study. Amyloid was identified by Congo red staining and polarization microscopy. Immunohistochemical classification was made with antibodies directed against AA amyloid, apolipoprotein A1, fibrinogen, lysozyme, lambda-light chain, kappa-light chain, beta2-microglobulin, transthyretin, and amyloid P-component. Amyloid was present in each biopsy as vascular, tubulo-interstitial and/or glomerular deposits. Immunoglobulin light chain-derived (AL) amyloidosis was most prevalent and diagnosed in 123 (53.2%) patients. It was categorized into AL amyloid of lambda-light chain (ALlambda) [105 (85.4%) patients] and kappa-light chain origin (ALkappa) [10 (8.1%)]. The amyloid deposits of 8 (6.5%) patients were not clearly distinguishable into ALlambda amyloid or ALkappa amyloid and categorized as AL amyloid, not otherwise specified. Reactive systemic amyloid A (AA) amyloidosis was the second most common type and was found in 93 patients (40.3%). Overall 7 patients were found to suffer from fibrinogen A alpha-chain-[amyloid of fibrinogen (AFib); 4 (1.7%) patients], transthyretin-[amyloid of transthyretin (ATTR); 2 (0.9%)], or apolipoprotein A1-derived (AApoAI) amyloidosis [1 (0.4%)]. In 8 patients (3.4%) the amyloid deposits remained unclassifiable. After additional immunostaining and further clinical information the diagnoses of 12 patients (5.1%) were modified (2 ALlambda amyloid, 4 ALkappa amyloid, 1 amyloid unclassified, 3 mixed-type amyloidosis, AA+ATTR, ALlambda+ATTR, and ALkappa+ATTR, 1 AFib, and 1 AApoAI). Although the histologic and immunohistochemical reevaluation confirmed the classifications in 221 (95.7%) patients. Renal amyloidosis is most commonly of ALlambda-origin, followed by AA amyloidosis. AFib amyloidosis was found to be the most prevalent type of hereditary renal amyloidosis, illustrating the necessity of a thorough classification of the amyloid proteins.

Prevalence of germline mutations in the TTR gene in a consecutive series of surgical pathology specimens with ATTR amyloid.

Transthyretin-derived amyloidosis (ATTR) amyloidosis is the third most prevalent amyloid type in surgical pathology and may occur as a hereditary disease with germline mutations in the TTR gene or as senile systemic amyloidosis (SSA) without mutations. Distinction between hereditary ATTR amyloidosis and SSA is of central importance, as the former necessitates genetic counseling and can be treated by liver transplantation. However, little is known about the prevalence of hereditary ATTR amyloidosis in surgical pathology specimens. We have examined the distribution of hereditary ATTR amyloidosis and SSA in a consecutive series of surgical pathology specimens with histologically and immunohistochemically confirmed ATTR amyloid. Thirty-three consecutive patients were retrieved from the Amyloid Registry of the Charité University Hospital. Genomic DNA was extracted from formalin-fixed and paraffin-embedded tissue or patient blood and examined by DNA sequencing. ATTR amyloid was found in the gastrointestinal tract, endomyocardium, peripheral nerve, carpal tunnel ligament, synovia, breast, and testicle. Amyloid fibrils were present as interstitial and vascular deposits, as evidenced by Congo red staining. TTR gene mutations were detected in 12 of 30 patients, with p.Val30Met being the most prevalent (5 patients). Furthermore, 2 novel mutations (p.Asp39Val and p.Glu54Asp) were found. In patients carrying a mutation, ATTR amyloid was found in the gastrointestinal tract, myocardium, nerve, and testicles. To conclude, the hereditary form of ATTR amyloid seems to be more common in elderly patients than previously thought. It is, therefore, important to genetically test every patient when diagnosing ATTR amyloidosis.

Hereditary apolipoprotein AI-associated amyloidosis in surgical pathology specimens: identification of three novel mutations in the APOA1 gene.

Apolipoprotein AI-derived (AApoAI) amyloidosis may present either as a non-hereditary form with wild-type protein deposits in atherosclerotic plaques or as a hereditary form due to germline mutations in the APOA1 gene. Currently, more than 50 apoAI variants are known, and 13 are associated with amyloidosis. We describe six patients with AApoAI amyloidosis due to APOA1 germline mutations that affect the larynx, small intestine, large intestine, heart, liver, kidney, uterus, ovary, or pelvic lymph nodes. In each patient, the amyloid showed a characteristic apple green birefringence when viewed under polarized light after Congo red staining and was immunoreactive with antibodies against apoAI. Sequence analyses revealed one known (p.Leu75Pro) and three novel APOA1 mutations that included gene variations leading to two different frameshifts (p.Asn74fs and p.Ala154fs) and one amino acid exchange (p.Leu170Pro). These three novel mutations extend our knowledge about both the location of the mutations and the organ distribution in hereditary AApoAI amyloidosis. Thirteen of the now sixteen amyloidogenic mutations are localized in two hot-spot regions that span residues 50 to 93 and 170 to 178. The organ distribution and clinical presentation of AApoAI amyloidosis seems to depend on the position of the mutation. Patients with alterations in codons 1 to 75 mostly develop hepatic and renal amyloidosis, while carriers of mutations in residues 173 to 178 mainly suffer from cardiac, laryngeal, and cutaneous amyloidosis.

Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli.

Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl(2) reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.