Trypanosoma cruzi cleaves galectin-3 N-terminal domain to suppress its innate microbicidal activity.

Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas' disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain - required for protein oligomerization - but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.

Is skin autofluorescence a marker of metabolic memory in pregnant women with diabetes?

AIM: To determine whether skin autofluorescence can help to detect those who have previously had abnormal glucose levels among women referred for diabetes during pregnancy. METHODS: Using an advanced glycation end product reader (AGE Reader(tm) (;) DiagnOptics BV, Groningen, the Netherlands), we measured forearm skin autofluorescence at 24-30 weeks of gestation in all women who were referred to our Nutrition Diabetology unit for diabetes during pregnancy. RESULTS: The study included 230 women (200 with gestational diabetes and 30 with pre-gestational diabetes, of whom 21 had Type 1 and nine had Type 2 diabetes) and a reference group of 22 normoglycaemic non-pregnant women. Skin autofluorescence was significantly higher in women with pre-gestational diabetes (1.97 ± 0.44 arbitary units) compared with gestational diabetes (1.77 ± 0.32 arbitary units; P = 0.003) and lower in the reference group (1.60 ± 0.32 arbitary units; P = 0.009 vs all pregnant women). Among women with gestational diabetes, 71 had a history of hyperglycaemia (i.e. gestational diabetes or macrosomia in a previous pregnancy or discovery of diabetes before 24th gestational week in the present pregnancy). These women had higher levels of skin autofluorescence (1.83 ± 0.35 arbitary units) than women with gestational diabetes without previous history of hyperglycaemia (1.73 ± 0.30 arbitary units; P = 0.04, non-significant, adjusted for age). Skin autofluorescence increased with the number of criteria present for previous hyperglycaemia (P for trend = 0.008) and was significantly associated with having two or three criteria for hyperglycaemia after adjusting for age (P = 0.02). CONCLUSIONS: Skin autofluorescence could reflect previous long-term hyperglycaemia in pregnant women, and could therefore be a marker of metabolic memory.

Interactions of human galectins with Trypanosoma cruzi: binding profile correlate with genetic clustering of lineages.

We report here the specific interaction between several members of the human galectin family with the three developmental stages of several genetic lineages of the protozoan parasite Trypanosoma cruzi. We provide data of specific and differential binding of human galectin (gal)-1, -3, -4, -7 and -8 to 14 strains of T. cruzi that belong to the six genetic lineages representing the genetic diversity of the parasite. It is shown that galectins preferentially bind forms present in the host, trypomastigotes and amastigotes, compared with the non-infective epimastigote present on the intestinal tract of the vector, reflecting the changes on glycosylation that occur during the metacyclogenesis and amastigogenesis process. Also, it is evidenced that galectin binding to the parasites promotes binding to the host cells and higher infection rates. In addition, evidence is provided indicating that the intracellular amastigotes may take over the cytosolic pool of some galectins when released to the extracellular medium. Finally, by applying unweighted pair group method analysis to the galectin-binding profile to either cell-derived trypomastigotes or amastigotes, we show that the differential-binding profile by the host galectins to the six lineages resembles the clustering based in genetic data. Therefore, the differential-binding profile for the six lineages could have implications in the immunopathology of Chagas' disease, affecting the complex network of immune responses on which galectins mediate, thus providing linkage clues to the notion that different lineages may be related to different clinical forms of the disease.

Purification of anti-thyroglobulin IgG from human serum.

The high prevalence of subjects in the general population and among patients with thyroid diseases who are positive for serum anti-thyroglobulin antibodies, together with the inconsistency of hybridoma techniques to obtain large amounts of anti-thyroglobulin antibodies, prompted us to prepare anti-thyroglobulin antibodies from human serum. An anti-thyroglobulin antibody positive serum was absorbed with CNBr-activated sepharose 4B conjugated to thyroglobulin and anti-thyroglobulin antibodies eluted by acid pH (3.0), basic pH (10.7) and detergent (SDS 3 g/l or 7 g/l). Preliminary experiments carried out with rabbit anti-human thyroglobulin allowed us to purify an Ig-fraction that retained its anti-thyroglobulin activity by immunoprecipitation and tanned red cell haemagglutination. When subjected to agarose gel electrophoresis and Silver stain, this fraction has the mobility of rabbit lg. Further experiments were carried out using Hashimoto's serum (tanned red cell haemagglutination 1:40620). pH 3.0 was found to give the best yield of stable antibodies. An IgG fraction was eluted at a concentration of 10+/-1.2 mg/l of serum. This fraction has the same electrophoretic mobility as human IgG and is close to pure human anti-thyroglobulin antibody. A dose-response curve was built up at a concentration range between 0.016 mg-2 mg of IgG anti-thyroglobulin antibody per litre. The slope of the curve parallels that of a dilution curve of the whole serum, suggesting that the purified antibodies are representative of the whole antibody population. In conclusion, we provide a method for preparing considerable amounts of highly purified anti-thyroglobulin antibodies from human serum for application in clinical medicine and basic research, and in particular which provides a standard for measuring anti-human thyroglobulin serum antibodies by weight rather than the presently used standard expressed in U/ml. Furthermore, this reagent could act as an ideal immuno-vector for the diagnosis and therapy of differentiated thyroid carcinoma.

Thyroglobulin determined in thyroid fine needle aspiration biopsies by radial immunodiffusion and electroimmunodiffusion.

Radial immunodiffusion and electroimmunodiffusion were used to measure thyroglobulin, the main component of thyroid colloid, in thyroid fine needle aspiration biopsies. A linear relationship was established between precipitation ring diameter and thyroglobulin concentration by radial immunodiffusion (0.5-3.0 g/l), and between "rocket" height and thyroglobulin concentration by electroimmunodiffusion (0.1-2.0 g/l). A nearly complete correlation was observed between the two methods (r = 0.97). In radial immunodiffusion the ring diameter is dependent on time of diffusion and on the antiserum concentration in the agar gel. In this study, the observation time was standardised at 48 h, and the rabbit anti-thyroglobulin serum concentration at 26 ml/l. The intrathyroidal concentration of thyroglobulin was determined by radial immunodiffusion and the thyroid find needle aspiration biopsy of 45 thyroid tumours with different cytological-laboratory- and clinical diagnoses. It was found that in colloid nodules or cysts thyroglobulin is markedly higher than in euthyroid nodular goitre (13.7 +/- 11.9 g/l vs. 1.35 +/- 0.8 g/l, p = 0.005). In conclusion radial immunodiffusion and electroimmunodiffusion are precise, easy to perform, low cost, non polluting methods, which do not require high sample dilution (in contrast, high sample dilution is necessary for measurement of thyroglobulin in thyroid fine needle aspiration biopsy by radial immunodiffusion). Measurement of thyroglobulin in thyroid fine needle aspiration biopsy provides a quantitative estimate of colloid, an important marker in the differential diagnosis of thyroid nodules.

Combined radiotherapy and CMF-B cytotoxic regimen in stage III-IV head and neck squamous cell carcinoma.

Twenty patients with stage III-IV squamous cell carcinoma of the head and neck were treated with the combination of a multidrug cytotoxic regimen CMF-B (cyclophosphamide, methotrexate, 5-fluorouracil, bleomycin) and radical radiotherapy. All the patients were evaluable: 7 (35%) achieved clinical complete response (CR), 10 (50%) partial response (PR) and 3 (15%) stable disease (SD). The median survival of the whole group is 14 months (range 3-26+); 21+ months for complete responders (range 4+-22+). Hematologic and gastrointestinal toxicity was moderate, 9 patients (45%) developed mucositis but only one needed total parenteral nutrition. It is concluded that the combination of CMF-B regimen and radiotherapy is effective and well tolerated in these patients and needs to be evaluated in further controlled studies.