Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation.

Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cyclic adenosine monophosphate (cAMP)-dependent signaling pathway and downstream mediators. To investigate protein-protein interaction partners of FSHR at resting state and upon FSH stimulation, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. Interestingly, while only WT FSHR interacted with FSH, insulin-like growth factor 1 receptor (IGF1R), for example, interacted with both WT and A189V FSHR upon FSH stimulation. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR.

The CD34+ Cell Dose Matters in Hematopoietic Stem Cell Transplantation with Peripheral Blood Stem Cells from Sibling Donors.

The effect of CD34+ cell dose in allogeneic hematopoietic stem cell transplantation (HSCT) on overall survival (OS) and incidence of acute and chronic graft-versus-host disease (GvHD) has not been established and few studies have been performed. Our single center analysis included 189 patients with hematological malignancies who received peripheral blood stem cell (PBSC) grafts from sibling donors. Myeloablative conditioning was used in 88 cases and 101 received reduced intensity conditioning. The median CD34+ cell dose was 5.6 × 106/kg (0.6-17.0). In the multivariate analysis, a CD34 cell dose of 6-7 × 106/kg was associated with better OS and lower transplant-related mortality (TRM), while a dose of <5 × 106/kg led to increased relapse and reduced chronic GVHD (cGVHD). A high CD34 cell-dose (>6.5 × 106/kg) correlated with less acute GVHD (aGVHD) II-IV. We conclude that the CD34 cell dose has an impact on the outcome of HSCT from sibling donor PBSCs.

Transcriptional profiling reveals monocyte-related macrophages phenotypically resembling DC in human intestine.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

Diet adherence and gluten exposure in coeliac disease and self-reported non-coeliac gluten sensitivity.

BACKGROUND/OBJECTIVES: Adherence to gluten-free diet in self reported non-coeliac gluten sensitive subjects is scarcely researched. Objectives of the study were to compare dietary adherence in coeliac disease (CD) subjects and in non-coeliac gluten sensitive (NCGS) subjects, and to estimate gluten exposure based on weighed food records and analysis of gluten content in selected food items. SUBJECTS/METHODS: Twenty-three subjects with biopsy verified CD on a gluten-free diet and 34 HLA-DQ2+ NCGS subjects on a self-instituted gluten-free diet were enrolled. The latter group was under investigation of CD. Dietary adherence was assessed by frequency questionnaire and structured forms supplied by weighed food records. For the analyses of food samples, the sandwich R5-ELISA, Ridascreen® Gliadin competitive method was used. RESULTS: There was no difference in dietary adherence between CD and NCGS subjects (83% vs 68%, p = 0.21). NCGS subjects were mainly self-educated in gluten-free diet compared to CD subjects (91% and 39%, respectively, p < 0.001). In non-adherent subjects, there was no difference in gluten exposure between CD and NCGS (10 vs 138 mg/day, p = 0.83). There was no difference in BMR-factor between CD and NCGS subjects, or between adherent and non-adherent subjects. CONCLUSIONS: Both CD and NCGS subjects were largely adherent, and adherence did not differ between the groups. Gluten exposure varied greatly, and some CD and NCGS subjects reached gluten intake above 500 mg/day, which might have considerable health effects on the individual, especially in case of coeliac disease.

Responsive population dynamics and wide seeding into the duodenal lamina propria of transglutaminase-2-specific plasma cells in celiac disease.

A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells in the lamina propria as well as antibodies in the subepithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of two untreated patients, both for TG2-specific and non-TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen-specific antibodies should be applicable to other antibody-mediated diseases.

Pancreas transplantation with enteroanastomosis to native duodenum poses technical challenges--but offers improved endoscopic access for scheduled biopsies and therapeutic interventions.

To facilitate endoscopic access for rejection surveillance and stenting of the pancreas, we have abandoned the duodenojejunostomy (DJ) in favor of duodenoduodenostomy (DD) in pancreas transplantation (PTx). From September 2012 to September 2013 we performed 40 PTx with DD; 20 solitary-PTx (S-PTx) and 20 simultaneous pancreas and kidney transplantation (SPK). We compared the outcomes with results from 40 PTx-DJ (10 S-PTx and 30 SPK) from the preceding era. The DD-enteroanastomoses were performed successfully. Endoscopic pancreas biopsies (endoscopic ultrasound examination [EUS]) yielded representative material in half of the cases. One exocrine fistula was treated by endoscopic stenting. PTxs-DD were associated with a higher rate of thrombosis compared to PTx-DJ (23% vs. 5%) and reoperations (48% vs. 30%), as well as inferior graft survival (80% vs. 88%). Time on waiting list, HLA A + B mismatches and reoperations were associated with graft loss. Only recipient age remained an independent predictor of patient death in multivariate analysis. PTx-DD showed a higher rate of thrombosis and inferior results, but facilitated a protocol biopsy program by EUS that was feasible and safe. Given that technical difficulties can be solved, the improved endoscopic access might confer long-term benefits, yet this remains to be proven.

Amalgam tattoo mimicking mucosal melanoma: a diagnostic dilemma revisited.

Mucosal melanoma of the oral cavity is a rare but highly aggressive neoplasm. However, the clinicians need to be aware of the other and more frequent etiologies of intraoral pigmentation, such as amalgam tattoos. As amalgam has been extensively used for dental restorations and can cause pigmentations in the oral mucosa, this is a differential diagnosis not to be forgotten. We describe the characteristics of these two phenomena and present a case vignette illustrating the differential diagnostic issues. Other causes of intraoral pigmentation are summarized.

Androgen receptor genotypes predict response to endocrine treatment in breast cancer patients.

BACKGROUND: The androgen receptor (AR) is frequently expressed in breast cancers. The AR genotype may affect disease-free survival and response to endocrine therapy. METHODS: In all, 634 women undergoing breast cancer surgery between 2002 and 2008 were followed until 30 June 2010. Six haplotype-tagging single-nucleotide polymorphisms in the AR, and the resulting AR diplotypes, were examined in relation to breast cancer patient characteristics, tumour characteristics, disease-free survival, and response to endocrine treatment. RESULTS: Five common AR diplotypes were found. Seventeen rare variants were combined into a composite group. The resulting six AR diplotype groups were clustered into two subgroups, groups A (n=128) and B (n=499), with three diplotypes in each. Patients in group B had larger total breast volume (P=0.024), higher body mass index (BMI) (P=0.050), more axillary lymph node involvement (P(trend)=0.020), and higher histological grade (P(trend)=0.031). There were 59 breast cancer events in the 569 patients with invasive cancers and no preoperative treatment. Patients in group B also had shorter disease-free survival (P=0.037) than patients in group A. Among patients in group B with oestrogen receptor α positive tumours, tamoxifen (TAM) treatment was associated with longer disease-free survival (P=0.008), while treatment with aromatase inhibitors (AIs) was not (P=0.94). Response to endocrine treatment could not be predicted based on BMI, suggesting that the effect of AR diplotypes went beyond that of a higher BMI. CONCLUSION: A marker for a group of patients who responded to TAM, but not to AIs, was identified. If this finding is confirmed, AR genotyping may provide useful information for selection of endocrine treatment of breast cancer patients.

Density of CD163+ CD11c+ dendritic cells increases and CD103+ dendritic cells decreases in the coeliac lesion.

Coeliac disease is a chronic inflammation of the intestinal mucosa controlled by gluten-specific T cells restricted by disease-associated HLA-DQ molecules. We have previously reported that mucosal CD11c(+) dendritic cells (DCs) are responsible for activation of gluten-reactive T cells within the coeliac lesion. In mice, intestinal CD11c(+) DCs comprise several functionally distinct subsets. Here, we report that HLA-DQ(+) antigen-presenting cells (APCs) in normal human duodenal mucosa can be divided into four subsets with striking similarities to those described in mice: CD163(+) CD11c(-) macrophages (74%), and CD11c(+) cells expressing either CD163 (7%), CD103 (11%) or CD1c (13%). CD103(+) and CD1c(+) DCs belonged to partly overlapping populations, whereas CD163(+) CD11c(+) APCs appeared to be a distinct population. In the coeliac lesion, we found increased density of CD163(+) CD11c(+) APCs, whereas the density of CD103(+) and CD1c(+) DCs was decreased, suggesting that distinct subpopulations of APCs in coeliac disease may exert different functions in the pathogenesis.

ESHRE PGD Consortium/Embryology Special Interest Group--best practice guidelines for polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS).

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:35-48.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHRE's recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos.

Fatty acid-spermine conjugates as DNA carriers for nonviral in vivo gene delivery.

The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules-fatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C(16) and C(18) hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.

Association between polymorphisms in the prostate-specific antigen (PSA) promoter and release of PSA.

Variations in serum prostate-specific antigen (PSA) have been ascribed to A/G nucleotide polymorphisms located at -158 bp (rs266882) and -4643 bp (rs925013), relative to the transcription start site within the promoter of the PSA gene. PSA is also an androgen receptor target (AR) gene and polymorphisms in AR gene are known to affect AR function. Our objective was to compare the impact of these A/G polymorphisms separately or in combination with AR CAG micro satellite on regulation of PSA secretion into seminal plasma and blood in young men. Leukocyte DNA was extracted from 291 conscripts and genotyping performed with the Sequenom Mass Array System. PSA was measured with an immunofluorometric assay. Linear regression analysis was used to test the association of polymorphism frequencies with serum and seminal plasma levels of PSA. PSA gene polymorphisms at -158 bp or -4643 bp did not alone influence total PSA (tPSA) levels in seminal plasma or in blood. Homozygotes for the A-allele at -158 bp in combination with CAG > 22 had significantly higher serum levels of tPSA than subjects carrying the G-allele (p = 0.01). In conclusion, the PSA gene polymorphisms did not importantly influence the levels of tPSA in seminal plasma or in blood. tPSA in serum was influenced by interactions between PSA promoter variants and AR CAG polymorphism.

Diagnosis and treatment of celiac disease.

The understanding of the pathogenesis of celiac disease has made huge advances in recent years. The disease is caused by an inappropriate immune response to dietary gluten proteins. This immune response is controlled by CD4(+) T cells in the lamina propria that recognize gluten peptides in the context of disease predisposing HLA-DQ2 and HLA-DQ8 molecules.(1, 2) These T cells are specific for proline- and glutamine-rich gluten peptides that are resistant to proteolysis and that have been become deamidated by the enzyme transglutaminase 2 (TG2). Strikingly, celiac disease patients produce antibodies to this same enzyme when exposed to dietary gluten. Here we discuss how the new insight in the pathogenesis has lead to development of new diagnostics and nourished research into novel treatments.

Androgen receptor gene GGN repeat length and reproductive characteristics in young Swedish men.

BACKGROUND: The human androgen receptor (AR) gene contains two polymorphisms of CAG and GGN repeats respectively. The GGN repeat function is still largely unknown and to date there are no in vivo data on this segment with respect to the general population. METHODS: We investigated the impact of CAG and GGN repeats on male reproductive function, one by one and in interaction with each other, in 220 adolescent men from the general Swedish population. Physical examination and semen analysis, including accessory sex gland markers and measurement of reproductive hormone levels, were performed. Lifestyle-associated factors, including maternal smoking during pregnancy, were recorded. GGN and CAG repeat lengths were determined by sequencing. RESULTS: GGN<23 was associated with lower semen volume when compared to GGN=23 (mean difference -0.6 ml, P=0.02) and GGN>23 (mean difference -0.9 ml, P=0.002). Men with GGN<23, exposed to maternal smoking during pregnancy, had higher body mass index compared to men with other GGN lengths, no matter whether their mother had smoked or not during pregnancy (mean difference 4.8 kg/m2, P<0.001). CONCLUSIONS: Short GGN repeats seem to be associated with decreased semen volume, possibly due to suboptimal AR activity. Body composition may be influenced by the combination of fetal exposure to maternal smoking and certain AR genotypes.

The effects of atorvastatin on gluten-induced intestinal T cell responses in coeliac disease.

Various experimental models suggest that the cholesterol-lowering drugs statins may also modulate immune responses. Cellular level studies on human disorders are needed, however, to provide a rational basis for clinical testing of statins as immune therapy. Coeliac disease, a chronic small intestinal inflammation driven by HLA-DQ2 restricted mucosal T cells that are specific for ingested wheat gluten peptides, is in many ways ideal for this purpose. In addition, there is a need for alternative treatment to the gluten-free diet in this disorder. Here we have assessed the effects of atorvastatin on gluten-reactive T cells, dendritic cells and the coeliac mucosa by in vitro culture of biopsies. Atorvastatin inhibited gluten-induced proliferation and specific cytokine production of human intestinal gluten-reactive T cell clones and lines. Dendritic cells exposed to atorvastatin displayed a reduced expression of the costimulatory molecule CD83 upon maturation with lipopolysaccharide. Incubation of intestinal biopsy specimens with atorvastatin in vitro, however, did not influence gluten-induced cytokine release. In conclusion, atorvastatin has specific effects on isolated gluten-reactive T cells and dendritic cells, but does not shut down the gluten-induced production of proinflammatory cytokines in intestinal biopsies.

Impact of therapy and androgen receptor polymorphism on sperm concentration in men treated for testicular germ cell cancer: a longitudinal study.

BACKGROUND: Testicular cancer (TC) patients have a high survival rate, and the question of post-therapy recovery of sperm production and its dependence on genetic predisposition is of major interest. METHODS: Ejaculates were obtained from 112 TC patients at one or more of the following time points: post-orchidectomy, or 6, 12, 24, 36 and 60 months post-therapy. The lengths of the androgen receptor (AR) function modulating CAG and GGN repeats in leukocyte DNA were also analysed. RESULTS: No significant decrease in sperm concentration was seen in men who received 1-2 cycles of adjuvant chemotherapy (ACT). Radiotherapy (RT) or more than two cycles of chemotherapy (HCT) caused an initial decline in sperm concentration, which returned to pre-treatment levels 2-5 years after therapy. In the HCT group, sperm concentration 12-24 months post-treatment (T(12-24)) was inversely correlated with CAG length (rho = -0.72, P = 0.03). The type of treatment, but not the concentration at T(0), was an independent predictor of sperm concentration at T(6) (P < 0.0005) and T(12-24) (P = 0.004). CONCLUSION: ACT did not induce a significant decline in sperm concentration. After HCT and RT, a significant reduction of sperm concentration was observed, recovering to pre-treatment levels 2-5 years post-treatment. In HCT-treated patients, the AR CAG length influenced the recovery of spermatogenesis.

Adding functional entities to plasmids.

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.