Assessment of reflectance confocal microscopy for non-invasive selection of optimal ovarian cortex fragments for autotransplantation.

RESEARCH QUESTION: Can reflectance confocal microscopy (RCM) be used to determine follicle density in human ovarian cortex fragments that are intended for fertility restoration? DESIGN: RCM was used on living cortex tissue fragments derived from five bovine ovaries and 13 human ovaries. All tissue fragments were cryopreserved and thawed before RCM analysis. Follicle numbers and distribution were determined by RCM and histology. Before and after RCM, general tissue viability and follicle integrity were assessed by a glucose uptake assay and neutral red staining, respectively. RESULTS: RCM can detect all stages of follicle development in living ovarian tissue to a maximum depth of 250 µm. In bovine tissue, all follicles were located within this 0-250 µm range. In human ovarian tissue, follicles were also present below the 250 µm RCM threshold, implying that only a percentage of the total number of follicles could be detected with RCM. The percentage of follicles detected by RCM appeared to be age dependent. The RCM procedure did not affect the glucose uptake by the tissue, whereas neutral red staining indicated a high level of follicle survival. CONCLUSION: In this proof of concept study, we have shown that RCM is a promising technique to determine the density of follicles ex vivo in living human ovarian cortex fragments, apparently without compromising the vitality of the tissue. Safety studies and further optimization of the RCM technique with a focus on increasing the penetration depth are required before clinical use of RCM.

The Decision-Making Process regarding Ovarian Tissue Cryopreservation in Girls with Turner Syndrome by Patients, Parents, and Healthcare Providers: A Mixed-Methods Study.

INTRODUCTION: Ovarian tissue cryopreservation (OTC) has proven to be effective in other patient groups, but the effectiveness in girls with Turner syndrome (TS) is still unclear. Guidelines for counselling about OTC in TS are lacking. The aim of this study was to gain insight into the experiences of patients, parents, and healthcare providers with the decision-making process regarding OTC in girls with TS. METHODS: Within a year after counselling, a survey was sent to 132 girls with TS and their parents. Furthermore, focus groups were conducted with (1) gynaecologists with subspeciality reproductive medicine, (2) paediatric endocrinologists, (3) parents of girls aged 2-12, and (4) parents of girls aged 13-18. Transcripts were analysed using a thematic analysis approach. RESULTS: The response rate of the survey was 45%. Of the survey respondents, 90% appreciated counselling regarding their future parenting options and considered it an addition to existing healthcare. Girls with TS and their parents indicated that the option of OTC raised hope for future genetic offspring and instantly made them feel that their only option was to seize this opportunity. Gynaecologists and paediatricians found it challenging to truly make families grasp a realistic perspective of OTC in girls with TS. DISCUSSION AND CONCLUSION: Offering young girls with TS the possibility of fertility preservation in an experimental setting raised high hopes and led to challenges for healthcare providers in ensuring a considered decision. The appropriate moment for counselling should be tailored to the individual and discussed with patient, parents, and paediatrician.

TurnerFertility trial: PROTOCOL for an observational cohort study to describe the efficacy of ovarian tissue cryopreservation for fertility preservation in females with Turner syndrome.

OBJECTIVE: To investigate the occurrence of live birth in women with Turner syndrome (TS) after ovarian tissue cryopreservation in childhood followed by auto transplantation in adulthood and to find reliable prognostic markers for estimating the ovarian reserve in girls with TS in the future. SETTING: An observational cohort study with long-term follow-up in a tertiary fertility clinic in the Netherlands. Patients recruitment between January 2018 and December 2021. PARTICIPANTS: 100 females aged 2 through 18 years with classical Turner (ie, 45,X0) or Turner variants (ie, 45,X mosaicism or structural anomalies). Girls with Y chromosomal content, minor X deletions with marginal impact on fertility, active HIV, hepatitis B or hepatitis C infection, and/or an absolute contra indication for surgery, anaesthesia or future pregnancy will be excluded. INTERVENTIONS: Ovarian cortical tissue will be harvested by performing a unilateral oophorectomy via laparoscopic approach. Ovarian cortex fragments will be prepared and cryopreserved. One fragment per patient will be used to determine follicular density by conventional histology, and to perform fluorescence in situ hybridisation analysis of ovarian cells. Routine chromosome analysis will be performed on both lymphocytes and buccal cells. A blood sample will be taken for hormonal analysis and all subjects will undergo a transabdominal ultrasound to determine the uterine and ovarian size. Patient characteristics, pregnancy rates and pregnancy outcomes will be collected from the patient's medical record. ETHICS AND DISSEMINATION: The study protocol has been approved by the Central Committee on Research Involving Human Subjects in November 2017 (CCMO NL57738.000.16). TRIAL REGISTRATION NUMBER: NCT03381300.

Clinical and microbiological features of dientamoebiasis in patients suspected of suffering from a parasitic gastrointestinal illness: a comparison of Dientamoeba fragilis and Giardia lamblia infections.

OBJECTIVES: To describe the clinical and microbiological features of Dientamoeba fragilis and Giardia lamblia infected patients, and to analyze the genetic variation of D. fragilis strains. METHODS: For a period of two years, all stool samples collected from patients suspected of having a parasitic gastrointestinal infection were examined according to our specific triple feces test (TFT) protocol. A retrospective case-control study was performed on D. fragilis and G. lamblia infected patients. Furthermore, PCR and genotyping by restriction fragment length polymorphism (RFLP) were performed upon the former. RESULTS: D. fragilis (6.3%) and G. lamblia (7.1%) were the most common pathogenic protozoa isolated out of 448 patients studied. Symptoms most frequently encountered with D. fragilis and G. lamblia infection were abdominal pain (69.2% and 72.4%, respectively) and diarrhea (61.5% and 79.3%, respectively). However, patients with D. fragilis infections suffered significantly less frequently from nausea and/or vomiting, anorexia and weight loss. After treatment, all D. fragilis and G. lamblia infected patients presenting a negative TFT follow-up also reported a complete resolution of their symptoms. Only genotype 1 could be detected in D. fragilis infected patients. CONCLUSIONS: D. fragilis and G. lamblia were the most frequently encountered parasites in our study population. Improved diagnostic tests are essential tools to study the prevalence and pathogenesis of D. fragilis.

The ocular humoral immune response in health and disease.

This review focuses on several aspects of humoral immunologic defence of the ocular surface and intraocular compartment. Secretory IgA (sIgA) is a major component of lacrimal fluid and contributes to the first line of defence against infection of the ocular surface. Recent findings show that part of the lacrimal gland (LG) IgA repertoire consists of so-called natural IgA antibodies. How B cells responsible for these natural IgA antibodies are distributed to effector mucosal sites like the LG is not known. Extensive data are now available on the regionalization of mucosal IgA responses in murine gut, involving peritoneal B cells, the prototypic natural antibody producing cells. By comparing elaborate experiments done in mice with the much less extensive data on the LG, it becomes clear that this gland is a unique, but poorly investigated effector organ of the mucosal immune system. In addition to the humoral immune response at the surface of the eye, the production of antibodies within the ocular compartment also has several fascinating features. The detection of pathogen-specific antibodies in intraocular fluid (IOF) of uveitis patients is accepted as a diagnostic tool, but the specificity of these intraocular antibodies was not investigated in much detail. Recent data however, demonstrate that even antibodies recognizing the same antigen in both serum and IOF still differ in the epitopes they recognize. This reveals that the intraocular compartment largely determines its own antibody profile in the defence against intra-ocular pathogens. Several models as to how an exclusive intra-ocular B cell repertoire may be generated are presented.

Drug resistance and genetic diversity of Plasmodium falciparum parasites from suriname.

Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine resistance transporter (pfcrt) gene (codon 76) and the pyrimethamine-sulfadoxine resistance markers in the dihydrofolate reductase (dhfr) gene (codons 16, 51, 59, 108, and 164) and dihydropteroate synthase (dhps) gene (codons 436, 437, 540, 581, and 613). Genetic variability was determined by sequence analysis of the polymorphic segments of the merozoite surface protein 2 (msp-2) and glutamate-rich protein (glurp) genes. Mutations in the pfcrt, dhps, and dhfr genes were found in all samples tested, suggesting that resistance to chloroquine and antifolate drugs is present at a high frequency. A low number of alleles was found for the msp-2 and glurp genes. This indicates limited genetic diversity and, based on geographic data, a genetically homogeneous P. falciparum population in Suriname.

ABCC6/MRP6 mutations: further insight into the molecular pathology of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a hereditary disease characterized by progressive dystrophic mineralization of the elastic fibres. PXE patients frequently present with skin lesions and visual acuity loss. Recently, we and others showed that PXE is caused by mutations in the ABCC6/MRP6 gene. However, the molecular pathology of PXE is complicated by yet unknown factors causing the variable clinical expression of the disease. In addition, the presence of ABCC6/MRP6 pseudogenes and multiple ABCC6/MRP6-associated deletions complicate interpretation of molecular genetic studies. In this study, we present the mutation spectrum of ABCC6/MRP6 in 59 PXE patients from the Netherlands. We detected 17 different mutations in 65 alleles. The majority of mutations occurred in the NBF1 (nucleotide binding fold) domain, in the eighth cytoplasmatic loop between the 15th and 16th transmembrane regions, and in NBF2 of the predicted ABCC6/MRP6 protein. The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. The second most frequent mutation, an intragenic deletion from exon 23 to exon 29 in ABCC6/MRP6, was detected in 11 (18.6%) of the patients. Our data include 11 novel ABCC6/MRP6 mutations, as well as additional segregation data relevant to the molecular pathology of PXE in a limited number of patients and families. The consequences of our data for the molecular pathology of PXE are discussed.

Protein disulfide isomerase of Toxoplasma gondii is targeted by mucosal IgA antibodies in humans.

Mass spectrometric analysis identified a 49 kDa antigen from Toxoplasma gondii as protein disulfide isomerase (PDI). This antigen is generally recognized by IgA in tears of healthy humans. We determined the complete open reading frame and expressed PDI recombinantly. Recombinant PDI was recognized by IgA in human tears known to contain antibodies specific for the 49 kDa antigen. High expression level and similarity to other protozoan PDIs suggest that T. gondii PDI might be a suitable target for recently described anti-protozoan drugs. PDI-specific antibodies clearly constitute part of the mucosal antibody repertoire possibly involved in defence against parasites.

Analysis of the frequent R1141X mutation in the ABCC6 gene in pseudoxanthoma elasticum.

PURPOSE: To characterize the ABCC6 R1141X nonsense mutation, which is implicated in more than 25% of a cohort of patients from The Netherlands with pseudoxanthoma elasticum (PXE). METHODS: A combination of single-strand conformational polymorphism (SSCP), PCR, sequencing, and Southern blot analysis was used to identify mutations in the ABCC6 gene in 62 patients. Haplotypes of 16 patients with the R1141X mutation were determined with eight polymorphic markers spanning the ABCC6 locus. The effect of the R1141X mutation on the expression of ABCC6 was studied in leukocytes and cultured dermal fibroblasts from affected skin in patients heterozygous or homozygous for the R1141X mutation. ABCC6 expression was analyzed by RT-PCR and immunocytochemistry with ABCC6-specific monoclonal antibodies. RESULTS: The ABCC6 R1141X mutation was found on 19 alleles in 16 patients with PXE and occurred in heterozygous, homozygous, or compound heterozygous form. All R1141X alleles were associated with a common haplotype, covering at least three intragenic ABCC6 markers. None of the patients or healthy control subjects had a similar ABCC6 haplotype. Furthermore, the results showed that the expression of the normal allele in R1141X heterozygotes was predominant, whereas no detectable, or very low, ABCC6 mRNA levels were found in R1141X homozygotes. Immunocytochemical staining of cultured dermal fibroblasts with ABCC6-specific monoclonal antibodies showed no evidence of the presence of a truncated protein in patients with PXE who were homozygous for R1141X. CONCLUSIONS: A specific founder effect for the R1141X mutation exists in Dutch patients with PXE. The R1141X mutation induces instability of the aberrant mRNA. Functional haploinsufficiency or loss of function of ABCC6 caused by mechanisms, such as nonsense-mediated decay (NMD), may be involved in the PXE phenotype.

Igm recognition of recombinant Toxoplasma gondii antigens by sera of acutely or latently infected humans.

Clinical non-relevant (CNR) IgM specific for Toxoplasma gondii is responsible for false-positive results in commercially available IgM assays. Using IgM immunoblotting, it is possible to distinguish between IgM in sera of acutely infected (AI) patients and CNR IgM. Especially the combination of staining of a 55 and 30 kD antigen in T.gondii lysate proved useful in this respect. The 55 kD antigen was identified as Rop1, while the 30 kD antigen was confirmed to be Sag1. However, the use of recombinant antigens instead of lysates for diagnostic assays would improve reproducibility. IgM recognized recombinant Rop1, but most CNR sera also had low anti-Rop1 titers. Although purified native Sag1 separated AI and CNR sera very well on immunoblot, IgM did not recognize recombinant Sag1 at all. Clearly, it is difficult to produce a recombinant Sag1 that can be recognized by IgM. Recombinant Rop1 might be suitable as one of the recombinant antigens in an IgM immunoblot assay, but has to be combined with at least one other immunogenic antigen.

Effects of the CDT6/ANGX gene on tumour growth in immune competent mice.

BACKGROUND: Cornea-derived transcript 6 (CDT6, also known as AngX) has been described to inhibit tumour growth in a human melanoma growing in nude mice. In another report, however, enhancement of tumour growth in comparable circumstances occurred. We therefore studied the effect of the same gene in different circumstances, using an immune competent mouse model. MATERIALS AND METHODS: In this report we describe the generation of a stably CDT6-expressing clone of the murine melanoma cell line B16-F10. These cells were implanted in female C57/B16 mice with empty vector transfected cells as control. RESULTS: We found no significant inhibition or stimulation of either lag-time or doubling-time of the tumours. In addition no effect of the CDT6 gene product was found on proliferation of endothelial cells in vitro. CONCLUSION: In contrast to an immune deficient mouse model, no anti-tumour effect could be detected with the CDT6 gene product in an immune competent mouse model.

Genotyping of Histomonas meleagridis isolates based on Internal Transcribed Spacer-1 sequences.

C-profiling is a novel genotyping method for protozoan pathogens, based on polymerase chain reaction and sequencing of AT-rich Internal Transcribed Spacer-1 sequences. It was applied to various Histomonas meleagridis isolates originating from outbreaks of histomoniasis in six Dutch turkey and chicken flocks. Three different H. meleagridis genotypes were identified. Type I and type II were associated with clinical disease. In two flocks, both having recovered from an outbreak of histomoniasis, a type III strain was found that was also morphologically slightly different from the type I and type II isolates. C-profiling is a promising technique to differentiate between H. meleagridis subtypes, making it useful for epidemiological studies.

Detection and identification of Enterocytozoon bieneusi and Encephalitozoon species in stool and urine specimens by PCR and differential hybridization.

Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. Enterocytozoon bieneusi and Encephalitozoon intestinalis are most commonly associated with chronic diarrhea. All Encephalitozoon species, including E. intestinalis, E. hellem, and E. cuniculi, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and Vittaforma corneae as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three Encephalitozoon species and V. corneae. An analytical sensitivity of 3.5 x 10(2) to 3.5 x 10(3) spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different Encephalitozoon species.

Carbohydrate moieties of microsporidian polar tube proteins are targeted by immunoglobulin G in immunocompetent individuals.

Microsporidia of the Encephalitozoon species are frequently found as opportunistic pathogens of immunocompromised patients, but very little is known about the prevalence and significance of Encephalitozoon infection in immunocompetent individuals. It was reported previously that 8% of Dutch blood donors and 5% of pregnant French women had an immunoglobulin G (IgG) immune response against specific organelles of Encephalitozoon intestinalis. These organelles, the so-called polar tube and anchoring disk, are used to penetrate membranes of host cells during infection. The unexpectedly high percentage of immunocompetent individuals with IgG against these organelles suggested that infection of humans with microsporidia might be more common than previously recognized. In the present study, we analyzed this anti-Encephalitozoon IgG response by using indirect immunofluorescence, Western blotting, two-dimensional gel electrophoresis, and chemical deglycosylation. Our results show that the antibody response is directed against the posttranslational carbohydrate modification of the major polar tube protein (polar tube protein 1) and carbohydrate moieties of proteins in the anchoring region of the polar tube of Encephalitozoon. In addition, the antibodies were found to decrease the infectivity of E. intestinalis in vitro. The significance and possible origin of these prevalent antibodies are discussed.

Use of rapid dipstick and latex agglutination tests and enzyme-linked immunosorbent assay for serodiagnosis of amebic liver abscess, amebic Colitis, and Entamoeba histolytica Cyst Passage.

A homemade enzyme-linked immunosorbent assay (ELISA) and a dipstick assay (Dipstick) for the detection of anti-Entamoeba histolytica antibodies in serum were developed and evaluated together with a commercially available latex agglutination test (LAT; Laboratoires Fumouze) for their use in serodiagnosis of amebiasis. The sensitivity of these assays was evaluated with sera from 27 patients with radiologically proven, cellulose acetate precipitation (CAP) test-positive amebic liver abscess, 7 patients with parasitologically and PCR-proven amebic colitis, and 11 patients with parasitologically and PCR-proven E. histolytica cyst passage. The sensitivities of the ELISA, Dipstick, and LAT were all 93.3% (42/45). With a combination of Dipstick and LAT, all abscess and colitis patients had at least one positive result. The specificity was assessed with 238 sera from patients with various parasitic, bacterial, viral, and fungal infectious diseases, sera containing autoimmune antibodies, and sera from healthy blood donors. The specificities of the ELISA, Dipstick, and LAT were 97.1%, 98.1%, and 99.5%, respectively. Of 61 sera from patients with PCR-proven E. dispar infection, 60 (98.4%) were negative in both Dipstick and LAT and 59 (96.7%) were negative in ELISA. Our data suggest that all three assays are sensitive serological tests. The rapid LAT and Dipstick provide fast results and can easily be applied in routine laboratories in order to facilitate the diagnosis of amebiasis.

Disruption of Abcc6 in the mouse: novel insight in the pathogenesis of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue, affecting mainly skin, eye and the cardiovascular system. PXE is characterized by dystrophic mineralization of elastic fibres. The condition is caused by loss of function mutations in ABCC6. We generated Abcc6 deficient mice (Abcc6-/-) by conventional gene targeting. As shown by light and electron microscopy Abcc6-/- mice spontaneously developed calcification of elastic fibres in blood vessel walls and in Bruch's membrane in the eye. No clear abnormalities were seen in the dermal extracellular matrix. Calcification of blood vessels was most prominent in small arteries in the cortex of the kidney, but in old mice, it occurred also in other organs and in the aorta and vena cava. Newly developed monoclonal antibodies against mouse Abcc6 localized the protein to the basolateral membranes of hepatocytes and the basal membrane in renal proximal tubules, but failed to show the protein at the pathogenic sites. Abcc6-/- mice developed a 25% reduction in plasma HDL cholesterol and an increase in plasma creatinine levels, which may be due to impaired kidney function. No changes in serum mineral balance were found. We conclude that the phenotype of the Abcc6-/- mouse shares calcification of elastic fibres with human PXE pathology, which makes this model a useful tool to further investigate the aetiology of PXE. Our data support the hypothesis that PXE is in fact a systemic disease.

Direct amplification and genotyping of Dientamoeba fragilis from human stool specimens.

Dientamoeba fragilis is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect D. fragilis DNA directly from human stool samples. The genetic diversity of D. fragilis from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that D. fragilis sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that D. fragilis shows remarkably little variation in its small-subunit rRNA gene.

Herpes simplex virus infection of the human eye induces a compartmentalized virus-specific B cell response.

Intraocular infection with herpes simplex virus (HSV) can cause uveitis, a potentially sight-threatening disease. The disease is characterized by an ocular infiltration of inflammatory cells such as macrophages and B and T cells. The characteristics of the local humoral and cellular immune responses elicited on intraocular HSV infection are poorly understood. The local HSV-specific antibody production, which are used routinely for confirmation of a clinical diagnosis of herpetic uveitis, has never been analyzed in detail. This study analyzed the humoral immune response against HSV type 1 (HSV-1) in paired samples of intraocular fluid and serum of patients with intraocular herpesvirus infection. In addition, the B cell epitope distribution on a single HSV-1 type-specific antigen, glycoprotein G, was compared for these paired samples. The results presented here indicate that the inflamed eyes of patients with HSV-induced uveitis display a compartmentalized B cell response directed toward the triggering virus.

Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans.

Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disulfide isomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were already detectable in tears of infants. To determine the epitope containing-regions on PDI, we generated truncated versions of recombinant PDI that differ by 8-10 amino acids in length. By IB, it was found that the sIgA epitopes were confined to conserved regions of PDI, including the functionally essential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of other species, which was confirmed by recognition of human PDI by IgA in tears. In contrast, anti-T. gondii PDI antibodies generated by immunization were not able to cross-react. Binding to the thioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constituting the same domain. By consecutive investigation of the function of the protein targeted by sIgA, the presence of antibody in relation to age and analysis of the epitope constituting regions on PDI we demonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore, analysis of antibody epitopes on an antigen is a useful method to distinguish conventional, affinity-matured antibodies from natural antibodies. The presence at early age and continuity of anti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encoded antibodies in human mucosa outside of the gut. Overall, the PDI-specific antibodies are clearly part of the natural antibody repertoire, suggesting an active role for these antibodies in the innate defense against pathogens.