PGL-III, a Rare Intermediate of Mycobacterium leprae Phenolic Glycolipid Biosynthesis, Is a Potent Mincle Ligand.

Although leprosy (Hansen's disease) is one of the oldest known diseases, the pathogenicity of Mycobacterium leprae (M. leprae) remains enigmatic. Indeed, the cell wall components responsible for the immune response against M. leprae are as yet largely unidentified. We reveal here phenolic glycolipid-III (PGL-III) as an M. leprae-specific ligand for the immune receptor Mincle. PGL-III is a scarcely present trisaccharide intermediate in the biosynthetic pathway to PGL-I, an abundant and characteristic M. leprae glycolipid. Using activity-based purification, we identified PGL-III as a Mincle ligand that is more potent than the well-known M. tuberculosis trehalose dimycolate. The cocrystal structure of Mincle and a synthetic PGL-III analogue revealed a unique recognition mode, implying that it can engage multiple Mincle molecules. In Mincle-deficient mice infected with M. leprae, increased bacterial burden with gross pathologies were observed. These results show that PGL-III is a noncanonical ligand recognized by Mincle, triggering protective immunity.

Developments in the Synthesis of Mycobacterial Phenolic Glycolipids.

The highly lipophilic outer barrier of mycobacteria, such as M. tuberculosis and M. leprae, is key to their virulence and intrinsic antibiotic resistance. Various components of this mycomembrane interact with the host immune system but many of these interactions remain ill-understood. This review covers several chemical syntheses of one of these components, mycobacterial phenolic glycolipids (PGLs), and outlines the interaction of these PGLs with the human immune system, as established using these well-defined pure compounds.

Synthetic Phenolic Glycolipids for Application in Diagnostic Tests for Leprosy.

Point-of-care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and prophylactic treatment strategies in leprosy control programs, thus positively contributing to clinical outcome and reducing transmission of this infectious disease. Levels of antibodies directed against the M. leprae-specific phenolic glycolipid I (PGL-I) closely correlate with an individual's bacterial load and a higher risk of developing leprosy. We describe herein the assembly of a set of PGL glycans carrying the characteristic phenol aglycon and featuring different methylation patterns. The PGL trisaccharides were applied to construct neoglycoproteins that were used to detect anti-PGL IgM antibodies in leprosy patients. ELISAs and quantitative lateral-flow assays based on up-converting nanoparticles (UCP-LFAs) showed that the generated PGL-I and PGL-II trisaccharide neoglycoconjugates can be applied for the detection of anti M. leprae IgM antibodies in POC tests.

Synthesis of the Staphylococcus aureus Strain M Capsular Polysaccharide Repeating Unit.

The synthesis of the Staphylococcus aureus strain M capsular polysaccharide repeating unit is reported. A postglycosylation oxidation strategy was utilized for the construction of the α-galactosaminuronic acid linkages, relying on a stereoselective 2-azido-4,6-O-di-tert-butylsilylidene galactopyranoside donor, for which the selectivity was assessed by model glycosylations. The α-fucosamine linkage was installed stereoselectively, using a reactive 2-azidofucosyl donor. An unexpected glycosidic bond cleavage during the TEMPO/PhI(OAc)2-mediated oxidation of a disaccharide intermediate was circumvented by a TEMPO/PhI(OAc)2-Pinnick oxidation protocol.

Couples with non-obstructive azoospermia are interested in future treatments with artificial gametes.

STUDY QUESTION: Would couples diagnosed with non-obstructive azoospermia (NOA) consider two future treatments with artificial gametes (AGs) as alternatives for testicular sperm extraction followed by ICSI (TESE-ICSI)? SUMMARY ANSWER: Most couples with NOA (89%) would opt for treatment with AGs before attempting TESE-ICSI and/or after failed TESE-ICSI. WHAT IS KNOWN ALREADY: Couples with NOA who undergo TESE-ICSI have a 25% chance of conceiving a child. Two future treatments that are being developed are 'ICSI with artificial sperm formed from somatic cells' (ICSI with AGs) and 'natural conception after autotransplantation of in vitro proliferated spermatogonial stem cells' (natural conception with AGs). It is unknown what treatment preferences patients have. STUDY DESIGN, SIZE, DURATION: A cross-sectional survey conducted in 2012-2013, addressing all 921 couples diagnosed with NOA and treated with TESE-ICSI in Dutch fertility clinics between 2007 and 2012. The coded questionnaires were sent by mail and followed up with two reminders. PARTICIPANTS/MATERIALS, SETTING, METHODS: We developed the questionnaire based on a literature review and previous qualitative interviews, and included treatment preference and the valuation of nine treatment characteristics. We assessed reliability of the questionnaires and calculated mean importance scores (MISs: 0-10) of each treatment characteristic. We assessed which patient and treatment characteristics were associated with a couple's hypothetical treatment preference using binominal regression. MAIN RESULTS AND THE ROLE OF CHANCE: The vast majority (89%) of the 494 responding couples (response rate: 54%) would potentially opt for AGs as a first and/or a last resort treatment option. More specifically, as a first treatment couples were likely (67%) to prefer natural conception with AGs over TESE-ICSI and less likely to prefer ICSI with AGs over TESE-ICSI (34%). After failed TESE-ICSI, the majority of couples (75%) would want to attempt ICSI with AGs as a last resort option. The most important characteristics of treatment were safety for children (MIS: 8.2), pregnancy rates (MIS: 7.7) and curing infertility (MIS: 6.8). Costs, burden, naturalness and technological sophistication were of about equal importance (MIS: 3.1-4.0). The majority of patients rated conception at home and moral acceptability as not important (MIS: 1.7 and 0.8, respectively), but the importance attributed to these variables did still affect patients' likeliness to opt for AGs. LIMITATIONS AND REASONS FOR CAUTION: Couples with NOA not opting for TESE-ICSI were not included and might have other perspectives. Couples' hypothetical choices for AGs might differ from their actual choices once data on the costs, safety and pregnancy rates become available from these new treatment options. WIDER IMPLICATIONS OF THE FINDINGS: The interest of couples with NOA in potential future treatments with AGs encourages further pre-clinical research. Priority setting for research and future decision-making on clinical application of AGs should take all characteristics important to patients into account. STUDY FUNDING/COMPETING INTERESTS: The authors report no financial or other conflict of interest relevant to the subject of this article.

Cytological evaluation of spermatogenesis: a novel and simple diagnostic method to assess spermatogenesis in non-obstructive azoospermia using testicular sperm extraction specimens.

Most of the non-obstructive azoospermia (NOA)-patients have only focal spermatogenesis which results in insufficient numbers of spermatozoa to reach the ejaculate. In ≈50% of these NOA-patients testicular sperm extraction (TESE) is successful and intracytoplasmic sperm injection (ICSI) is pursued. We studied whether (i) spermatogenesis can be evaluated by defining the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa in a testicular cell suspension, and (ii) these ratios are associated with the outcome of fertility treatment. A retrospective cohort study was conducted between June 2007 and August 2012. In this period, 441 consecutive ICSI-TESE cycles were performed in 212 couples. For each TESE biopsy, the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa were calculated. A control population of 32 vasectomized men was used to define cut-off values for complete spermatogenesis. Based on the pachytene to sperm ratio (P/Sp) and number of spermatozoa per 100 Sertoli cells (#Sp/100SC) groups were defined as complete spermatogenesis, hypospermatogenesis and partial maturation arrest (MA). Validation of the cytological diagnoses was performed by comparing the results of cytology to the histological evaluation of spermatogenesis in 40 cases. In 92.5%, a perfect match was observed and in the three remaining cases cytology corresponded well with the results of TESE. Couples with complete spermatogenesis have a higher ongoing pregnancy rate after the first treatment cycle compared to couples with hypospermatogenesis (34 vs. 16%; p = 0.02) and partial MA (34 vs. 19%; p = 0.11). In conclusion, pachytene spermatocytes, spermatozoa and Sertoli cells can be easily identified and counted in a cell suspension and their ratios can be successfully used to diagnose the level of spermatogenic impairment. This pilot study indicates that once successful spermatozoa retrieval is achieved, treatment outcome declines when spermatogenesis is impaired in NOA. The predictive value of cytological evaluation of spermatogenesis has to be established in a future prospective trial.

A novel cell-processing method 'AgarCytos' in conjunction with OCT3/4 and PLAP to detect intratubular germ cell neoplasia in non-obstructive azoospermia using remnants of testicular sperm extraction specimens.

STUDY QUESTION: Can we diagnose intratubular germ cell neoplasia (IGCN) using the immunohistochemical markers placental-like alkaline phosphatase (PLAP) and OCT3/4 using a novel cell-processing method 'AgarCytos', applied to the remnants of testicular sperm extraction (TESE) specimens and what is the prevalence of a testicular germ cell (pre)malignancy in men with a non-obstructive azoospermia (NOA) undergoing TESE for fertility treatment? SUMMARY ANSWER: IGCN can be successfully detected by immunohistochemical evaluation of AgarCytos, made of the remnants of TESE biopsies. The observed prevalence of a germ cell (pre)malignancy in this specific population was found to be 4.4%. WHAT IS KNOWN ALREADY: Infertile men are at higher risk for testicular cancer than the general population. IGCN can be detected by immunohistochemistry using PLAP and OCT3/4 in standard testicular biopsies and, with less accuracy, in semen. STUDY DESIGN, SIZE, DURATION: Between January 2011 and April 2012 a prospective cohort study was conducted at a Dutch tertiary care academic training hospital. All males with NOA (n = 182) undergoing a urological work-up followed by a diagnostic TESE for fertility treatment (n = 251) were included. PARTICIPANTS, SETTING, METHODS: After cryopreservation of sperm, if present, an AgarCyto was made of the remnants of the TESE biopsies. Sections were stained with haematoxylin-eosin for pathological examination as well as PLAP and OCT3/4 for immunohistochemistry to detect IGCN. MAIN RESULTS AND THE ROLE OF CHANCE: Eight men (4.4%) were diagnosed with a germ cell (pre)malignancy: six of them had seminoma, two without and four with concomitant IGCN, and two of them had IGCN only. Microscopic evaluation including immunohistochemical analysis of the AgarCytos diagnosed three (1.6%) more cases of a germ cell (pre)malignancy compared with scrotal ultrasound alone (one case of bilateral seminoma with concomitant IGCN and two cases of IGCN alone). No false-positive cytology results were found upon conventional histological evaluation. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study is lack of a simultaneously taken standard testicular biopsy, to compare the results of our novel diagnostic method with. Nevertheless, in all but one of our cases orchidectomy followed and the diagnosis was confirmed by histology. In the remaining case repeat TESE showed similar results. WIDER IMPLICATIONS OF THE FINDINGS: Simultaneous screening for IGCN is highly recommended to men with NOA undergoing TESE, because of the increased incidence of germ cell (pre)malignancies in this specific population. The principal advantage of our new method is that all available testicular tissue can be used for both sperm recovery and pathological evaluation, increasing the yield of spermatozoa as well as the chance to find (pre)malignant cells. In those cases where the disease is still in a premalignant stage, early diagnosis will allow for timely treatment and reduction of morbidity and mortality in this group of patients. STUDY FUNDING/COMPETING INTEREST(S): This study was (partially) funded by Merck Serono (the Netherlands). There are no conflicting interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

Release kinetics of intact and degraded troponin I and T after irreversible cell damage.

PURPOSE: We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage. METHODS: Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products. RESULTS: During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used. CONCLUSIONS: Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.

Release of cardiac troponin I from viable cardiomyocytes is mediated by integrin stimulation.

Elevated cardiac troponin-I (cTnI) levels have been demonstrated in serum of patients without acute coronary syndromes, potentially via a stretch-related process. We hypothesize that this cTnI release from viable cardiomyocytes is mediated by stimulation of stretch-responsive integrins. Cultured cardiomyocytes were treated with (1) Gly-Arg-Gly-Asp-Ser (GRGDS, n = 22) to stimulate integrins, (2) Ser-Asp-Gly-Arg-Gly (SDGRG, n = 8) that does not stimulate integrins, or (3) phosphate-buffered saline (control, n = 38). Cells and media were analyzed for intact cTnI, cTnI degradation products, and matrix metalloproteinase (MMP)-2. Cell viability was examined by assay of lactate dehydrogenase (LDH) activity and by nuclear staining with propidium iodide. GRGDS-induced integrin stimulation caused increased release of intact cTnI (9.6 +/- 3.0%) as compared to SDGRG-treated cardiomyocytes (4.5 +/- 0.8%, p < 0.001) and control (3.0 +/- 3.4%, p < 0.001). LDH release from GRGDS-treated cardiomyocytes (15.9 +/- 3.8%) equalled that from controls (15.2 +/- 2.3%, p = n.s.), indicating that the GRGDS-induced release of cTnI is not due to cell necrosis. This result was confirmed by nuclear staining with propidium iodide. Integrin stimulation increased the intracellular and extracellular MMP2 activity as compared to controls (both p < 0.05). However, despite the ability of active MMP2 to degrade cTnI in vitro, integrin stimulation in cardiomyocytes was not associated with cTnI degradation. The present study demonstrates that intact cTnI can be released from viable cardiomyocytes by stimulation of stretch-responsive integrins.

[Cystic teratoma of the ovary and cancer: structural and histochemical study]. / Teratoma quístico de ovário y cáncer: estudio estructural e histoquímico.

Three cases corresponding to surgical pieces which included cystic ovarian tumors were studied, with the purpose of characterizing their structural and cytochemical components. Techniques of Hematoxylin -eosin, P.A.S., P.A.S.-amylase, Cason's thrichromic and Ag (Senior-Munger) were employed, the last one to determine the presence of cytoplasmic secretory granules. In two the of cases, mature cystic teratomes were found, one of them with malignant development, typical of an adenocarcinoma. The last case corresponded to a monodermic teratoma--highly specialized struma of the ovary-carcinoid tumor. It is to be noted that, in this last case, the patient consulted for hypertensive crises.

Teratoma quístico de ovário y cáncer: estudio estructural e histoquímico. / [Cystic teratoma of the ovary and cancer: structural and histochemical study]

Three cases corresponding to surgical pieces which included cystic ovarian tumors were studied, with the purpose of characterizing their structural and cytochemical components. Techniques of Hematoxylin -eosin, P.A.S., P.A.S.-amylase, Casons thrichromic and Ag (Senior-Munger) were employed, the last one to determine the presence of cytoplasmic secretory granules. In two the of cases, mature cystic teratomes were found, one of them with malignant development, typical of an adenocarcinoma. The last case corresponded to a monodermic teratoma--highly specialized struma of the ovary-carcinoid tumor. It is to be noted that, in this last case, the patient consulted for hypertensive crises.