Trophoblast organoids as a model for maternal-fetal interactions during human placentation.

The placenta is the extraembryonic organ that supports the fetus during intrauterine life. Although placental dysfunction results in major disorders of pregnancy with immediate and lifelong consequences for the mother and child, our knowledge of the human placenta is limited owing to a lack of functional experimental models1. After implantation, the trophectoderm of the blastocyst rapidly proliferates and generates the trophoblast, the unique cell type of the placenta. In vivo, proliferative villous cytotrophoblast cells differentiate into two main sub-populations: syncytiotrophoblast, the multinucleated epithelium of the villi responsible for nutrient exchange and hormone production, and extravillous trophoblast cells, which anchor the placenta to the maternal decidua and transform the maternal spiral arteries2. Here we describe the generation of long-term, genetically stable organoid cultures of trophoblast that can differentiate into both syncytiotrophoblast and extravillous trophoblast. We used human leukocyte antigen (HLA) typing to confirm that the organoids were derived from the fetus, and verified their identities against four trophoblast-specific criteria3. The cultures organize into villous-like structures, and we detected the secretion of placental-specific peptides and hormones, including human chorionic gonadotropin (hCG), growth differentiation factor 15 (GDF15) and pregnancy-specific glycoprotein (PSG) by mass spectrometry. The organoids also differentiate into HLA-G+ extravillous trophoblast cells, which vigorously invade in three-dimensional cultures. Analysis of the methylome reveals that the organoids closely resemble normal first trimester placentas. This organoid model will be transformative for studying human placental development and for investigating trophoblast interactions with the local and systemic maternal environment.

Should be a locally advanced colon cancer still considered a contraindication to laparoscopic resection?

BACKGROUND: The role of elective laparoscopic resection (LR) for the treatment of locally advanced colon cancer (LACC) is unclear. Most studies have retrospectively investigated the outcomes of LR for pT4 cancers, while clinical T4 (cT4) cancers are excluded in the large randomized controlled trials comparing LR and open resection (OR). The aim of this study was to investigate the outcomes in patients undergoing elective LR for LACC. METHODS: A prospective single-institution database including consecutive patients undergoing elective LR for clinical LACC (high-risk T3 or T4 N0-2) between March 1996 and March 2017 was retrospectively reviewed. A multivariate analysis was performed to identify predictors of conversion to OR and risk factors for adverse oncologic outcomes. RESULTS: A total of 300 patients undergoing LR for LACC were included. A multi-visceral resection was needed in 17 (5.7%) patients. A total of 63 (21%) LRs were converted to OR, mainly due to suspected adjacent organ invasion (82.5%) or obesity (9.5%). Overall postoperative Clavien-Dindo 3-4 complication rate was 4.7%, with no significant differences between completed and converted LRs. Final pathology showed 18 (6%) pT2, 215 (71.7%) pT3, 54 (18%) pT4a, and 13 (4.3%) pT4b cancers. A R0 resection was achieved in 98.3% of patients. On multivariate analysis, tumor size ≥ 7 cm and tumor site (splenic flexure) were the independent risk factors for conversion to OR. A pT4 colon cancer and LNR of 0.25 or greater, but not conversion to OR, were independently associated with both poorer OS and DFS. CONCLUSION(S): Clinical LACC should not be considered a contraindication to LR itself. Bulky tumors ≥ 7 cm and splenic flexure cancers are at higher risk of conversion to OR; however, there is no increased postoperative morbidity or adverse oncologic outcomes in converted patients.

Analysis of Early and Long-Term Oncologic Outcomes After Converted Laparoscopic Resection Compared to Primary Open Surgery for Rectal Cancer.

BACKGROUND: Laparoscopic rectal resection (LRR) for cancer is a challenging procedure, with conversion to open surgery being reported in up to 30% of cases. Since only a few studies with short follow-up have compared converted LRR and open RR (ORR), it is unclear if conversion to open surgery should be prevented by preferring an open approach in those patients with preoperatively known risk factors for conversion. The aim of this study was to compare early postoperative outcomes and long-term survival after completed LRR, converted LRR or ORR for non-metastatic rectal cancer. METHODS: A prospective database of consecutive curative LRRs and ORRs for rectal cancer was reviewed. Patients undergoing LRR who required conversion (CONV group) were compared with those who had primary open rectal surgery (OPEN group) and completed LRR (LAP group). A multivariate analysis was performed to identify predictors of poor survival. RESULTS: A total of 537 patients were included in the study: 272 in the LAP group, 49 in the CONV group and 216 in the OPEN group. There were no significant differences in perioperative morbidity, mortality and length of hospital stay between the three groups. Five-year overall survival and disease-free survival rates did not significantly differ between LAP, CONV and OPEN patients: 83.9 versus 77.8 versus 81% (P = 0.398) and 74.5 versus 62.9 versus 72.7% (P = 0.145), respectively. Similar 5-year OS and DFS rates were observed between patients who had converted LRR for locally advanced tumor or for non-tumor-related reasons: 81.2 versus 80.8% (P = 0.839) and 62.5 versus 63.7% (P = 0.970), respectively. Poor grade of tumor differentiation, lymphovascular invasion and a lymph node ratio of 0.25 or greater, but not conversion, were independently associated with poorer survival. CONCLUSION: Conversion to open surgery does not impair short-term outcomes and does not jeopardize 5-year survival in patients with rectal cancer when compared to primary open surgery.

Regulatory T Cell Responses in Participants with Type 1 Diabetes after a Single Dose of Interleukin-2: A Non-Randomised, Open Label, Adaptive Dose-Finding Trial.

BACKGROUND: Interleukin-2 (IL-2) has an essential role in the expansion and function of CD4+ regulatory T cells (Tregs). Tregs reduce tissue damage by limiting the immune response following infection and regulate autoreactive CD4+ effector T cells (Teffs) to prevent autoimmune diseases, such as type 1 diabetes (T1D). Genetic susceptibility to T1D causes alterations in the IL-2 pathway, a finding that supports Tregs as a cellular therapeutic target. Aldesleukin (Proleukin; recombinant human IL-2), which is administered at high doses to activate the immune system in cancer immunotherapy, is now being repositioned to treat inflammatory and autoimmune disorders at lower doses by targeting Tregs. METHODS AND FINDINGS: To define the aldesleukin dose response for Tregs and to find doses that increase Tregs physiologically for treatment of T1D, a statistical and systematic approach was taken by analysing the pharmacokinetics and pharmacodynamics of single doses of subcutaneous aldesleukin in the Adaptive Study of IL-2 Dose on Regulatory T Cells in Type 1 Diabetes (DILT1D), a single centre, non-randomised, open label, adaptive dose-finding trial with 40 adult participants with recently diagnosed T1D. The primary endpoint was the maximum percentage increase in Tregs (defined as CD3+CD4+CD25highCD127low) from the baseline frequency in each participant measured over the 7 d following treatment. There was an initial learning phase with five pairs of participants, each pair receiving one of five pre-assigned single doses from 0.04 × 106 to 1.5 × 106 IU/m2, in order to model the dose-response curve. Results from each participant were then incorporated into interim statistical modelling to target the two doses most likely to induce 10% and 20% increases in Treg frequencies. Primary analysis of the evaluable population (n = 39) found that the optimal doses of aldesleukin to induce 10% and 20% increases in Tregs were 0.101 × 106 IU/m2 (standard error [SE] = 0.078, 95% CI = -0.052, 0.254) and 0.497 × 106 IU/m2 (SE = 0.092, 95% CI = 0.316, 0.678), respectively. On analysis of secondary outcomes, using a highly sensitive IL-2 assay, the observed plasma concentrations of the drug at 90 min exceeded the hypothetical Treg-specific therapeutic window determined in vitro (0.015-0.24 IU/ml), even at the lowest doses (0.040 × 106 and 0.045 × 106 IU/m2) administered. A rapid decrease in Treg frequency in the circulation was observed at 90 min and at day 1, which was dose dependent (mean decrease 11.6%, SE = 2.3%, range 10.0%-48.2%, n = 37), rebounding at day 2 and increasing to frequencies above baseline over 7 d. Teffs, natural killer cells, and eosinophils also responded, with their frequencies rapidly and dose-dependently decreased in the blood, then returning to, or exceeding, pretreatment levels. Furthermore, there was a dose-dependent down modulation of one of the two signalling subunits of the IL-2 receptor, the ß chain (CD122) (mean decrease = 58.0%, SE = 2.8%, range 9.8%-85.5%, n = 33), on Tregs and a reduction in their sensitivity to aldesleukin at 90 min and day 1 and 2 post-treatment. Due to blood volume requirements as well as ethical and practical considerations, the study was limited to adults and to analysis of peripheral blood only. CONCLUSIONS: The DILT1D trial results, most notably the early altered trafficking and desensitisation of Tregs induced by a single ultra-low dose of aldesleukin that resolves within 2-3 d, inform the design of the next trial to determine a repeat dosing regimen aimed at establishing a steady-state Treg frequency increase of 20%-50%, with the eventual goal of preventing T1D. TRIAL REGISTRATION: ISRCTN Registry ISRCTN27852285; ClinicalTrials.gov NCT01827735.

Investigation of soluble and transmembrane CTLA-4 isoforms in serum and microvesicles.

Expression of the CTLA-4 gene is absolutely required for immune homeostasis, but aspects of its molecular nature remain undefined. In particular, the characterization of the soluble CTLA-4 (sCTLA-4) protein isoform generated by an alternatively spliced mRNA of CTLA4 lacking transmembrane-encoding exon 3 has been hindered by the difficulty in distinguishing it from the transmembrane isoform of CTLA-4, Tm-CTLA-4. In the current study, sCTLA-4 has been analyzed using novel mAbs and polyclonal Abs specific for its unique C-terminal amino acid sequence. We demonstrate that the sCTLA-4 protein is secreted at low levels following the activation of primary human CD4(+) T cells and is increased only rarely in the serum of autoimmune patients. Unexpectedly, during our studies aimed to define the kinetics of sCTLA-4 produced by activated human CD4(+) T cells, we discovered that Tm-CTLA-4 is associated with microvesicles produced by the activated cells. The functional roles of sCTLA-4 and microvesicle-associated Tm-CTLA-4 warrant further investigation, especially as they relate to the multiple mechanisms of action described for the more commonly studied cell-associated Tm-CTLA-4.

Large-scale analysis of viral nucleic acid spectrum in temporal lobe epilepsy biopsies.

OBJECTIVE: Chronic inflammatory processes are important promotors of temporal lobe epilepsy (TLE) development. Based on human herpesvirus 6 (HHV-6) DNA detection in brain tissue from patients with TLE, an association of persistent viral infection with TLE has been discussed. Individual studies reported increased HHV-6 DNA in patients with clinical signs of previous inflammatory brain reaction, that is, febrile seizures or meningoencephalitis. However, detection rates vary considerably between different studies. Here we performed a large-scale analysis of viral DNA/RNA spectrum in high-quality TLE biopsies. In addition to all Herpesviridae, we addressed potentially relevant neurotropic RNA viruses. METHODS: DNA and RNA were extracted from 346 fresh-frozen tissue samples removed by epilepsy surgery. Real-time polymerase chain reaction (PCR) and nested PCR were performed for Herpesviridae and RNA viruses, respectively. Clinical data were analyzed for earlier signs of inflammatory brain reactions. Fresh-frozen hippocampal tissue samples from patients without chronic central nervous system (CNS) disease served as controls (n = 62). Seven previous PCR studies with overall 178 TLE patients were additionally analyzed regarding a correlation of clinical parameters and HHV-6 detection. RESULTS: PCR revealed HHV-6B DNA in 34 specimens (9.8%) from TLE patients. HHV-6B DNA was also present in eight control samples (12.9%; p > 0.05), but showed a lower virus concentration (p < 0.001). Other herpesviruses and RNA viruses were virtually absent. In patients with clinical signs of previous brain inflammation, HHV-6B DNA was observed in 15.0%, whereas only 6.3% of the samples from patients without febrile seizures or meningoencephalitis were positive for HHV-6B DNA (p < 0.05). A meta-analysis of the eight HHV-6 PCR studies revealed similar results. SIGNIFICANCE: This biopsy-based study shows no differences in frequency of HHV-6B DNA detection between TLE patients and controls. These results do not support the hypothesis of a persistent HHV-6B infection as a major pathogenetic factor in TLE. However, the higher virus load in TLE patients and the increased detection rate of HHV-6B DNA in patients with previous inflammatory brain reactions require further investigations.

Postthymic expansion in human CD4 naive T cells defined by expression of functional high-affinity IL-2 receptors.

As the thymus involutes with age, the maintenance of peripheral naive T cells in humans becomes strongly dependent on peripheral cell division. However, mechanisms that orchestrate homeostatic division remain unclear. In this study we present evidence that the frequency of naive CD4 T cells that express CD25 (IL-2 receptor α-chain) increases with age on subsets of both CD31(+) and CD31(-) naive CD4 T cells. Analyses of TCR excision circles from sorted subsets indicate that CD25(+) naive CD4 T cells have undergone more rounds of homeostatic proliferation than their CD25(-) counterparts in both the CD31(+) and CD31(-) subsets, indicating that CD25 is a marker of naive CD4 T cells that have preferentially responded to survival signals from self-Ags or cytokines. CD25 expression on CD25(-) naive CD4 T cells can be induced by IL-7 in vitro in the absence of TCR activation. Although CD25(+) naive T cells respond to lower concentrations of IL-2 as compared with their CD25(-) counterparts, IL-2 responsiveness is further increased in CD31(-) naive T cells by their expression of the signaling IL-2 receptor ß-chain CD122, forming with common γ-chain functional high-affinity IL-2 receptors. CD25 plays a role during activation: CD25(+) naive T cells stimulated in an APC-dependent manner were shown to produce increased levels of IL-2 as compared with their CD25(-) counterparts. This study establishes CD25(+) naive CD4 T cells, which are further delineated by CD31 expression, as a major functionally distinct immune cell subset in humans that warrants further characterization in health and disease.

Autosomal recessive Stickler syndrome due to a loss of function mutation in the COL9A3 gene.

Stickler syndrome (STL) is a clinically variable and genetically heterogeneous syndrome characterized by ophthalmic, articular, orofacial, and auditory manifestations. STL has been described with both autosomal dominant and recessive inheritance. The dominant form is caused by mutations of COL2A1 (STL 1, OMIM 108300), COL11A1 (STL 2, OMIM 604841), and COL11A2 (STL 3, OMIM 184840) genes, while recessive forms have been associated with mutations of COL9A1 (OMIM 120210) and COL9A2 (OMIM 120260) genes. Type IX collagen is a heterotrimeric molecule formed by three genetically distinct chains: α1, α2, and α3 encoded by the COL9A1, COL9A2, and COL9A3 genes. Up to this time, only heterozygous mutations of COL9A3 gene have been reported in human and related to: (1) multiple epiphyseal dysplasia type 3, (2) susceptibility to an intervertebral disc disease, and (3) hearing loss. Here, we describe the first autosomal recessive Stickler family due to loss of function mutations (c.1176_1198del, p.Gln393Cysfs*25) of COL9A3 gene. These findings extend further the role of collagen genes family in the disease pathogenesis.

Human uterine natural killer cells regulate differentiation of extravillous trophoblast early in pregnancy.

In humans, balanced invasion of trophoblast cells into the uterine mucosa, the decidua, is critical for successful pregnancy. Evidence suggests that this process is regulated by uterine natural killer (uNK) cells, but how they influence reproductive outcomes is unclear. Here, we used our trophoblast organoids and primary tissue samples to determine how uNK cells affect placentation. By locating potential interaction axes between trophoblast and uNK cells using single-cell transcriptomics and in vitro modeling of these interactions in organoids, we identify a uNK cell-derived cytokine signal that promotes trophoblast differentiation at the late stage of the invasive pathway. Moreover, it affects transcriptional programs involved in regulating blood flow, nutrients, and inflammatory and adaptive immune responses, as well as gene signatures associated with disorders of pregnancy such as pre-eclampsia. Our findings suggest mechanisms on how optimal immunological interactions between uNK cells and trophoblast enhance reproductive success.

How Do Uterine Natural Killer and Innate Lymphoid Cells Contribute to Successful Pregnancy?

Innate lymphoid cells (ILCs) are the most abundant immune cells in the uterine mucosa both before and during pregnancy. Circumstantial evidence suggests they play important roles in regulating placental development but exactly how they contribute to the successful outcome of pregnancy is still unclear. Uterine ILCs (uILCs) include subsets of tissue-resident natural killer (NK) cells and ILCs, and until recently the phenotype and functions of uILCs were poorly defined. Determining the specific roles of each subset is intrinsically challenging because of the rapidly changing nature of the tissue both during the menstrual cycle and pregnancy. Single-cell RNA sequencing (scRNAseq) and high dimensional flow and mass cytometry approaches have recently been used to analyse uILC populations in the uterus in both humans and mice. This detailed characterisation has significantly changed our understanding of the heterogeneity within the uILC compartment. It will also enable key clinical questions to be addressed including whether specific uILC subsets are altered in infertility, miscarriage and pregnancy disorders such as foetal growth restriction and pre-eclampsia. Here, we summarise recent advances in our understanding of the phenotypic and functional diversity of uILCs in non-pregnant endometrium and first trimester decidua, and review how these cells may contribute to successful placental development.

Non-anatomical colonic resections: splenic flexure and transverse colectomy. Central vascular ligation is crucial for survival.

BACKGROUND: Cancers of the transverse colon (TC) and of the splenic flexure (SF) are rather uncommon and their prognosis has been reported significantly poorer as compared to right- and left-sided colon cancers. Several studies tried to investigate which is the optimal surgery for oncological and survival outcomes reporting extended colectomies as more effective than limited resections. METHODS: All consecutive patients with diagnosis of colorectal cancer submitted to surgical resection with anastomosis between January 2005 and December 2016 at the Division of Surgical Oncology and Digestive Surgery, Department of Oncology, at the University of Turin, were included. Based on tumor location, patients were enrolled in arm A (cancer of the ascending colon, right (hepatic) flexure, left and sigmoid colon) or arm B (cancer of the transverse colon and of the left (splenic) flexure). RESULTS: Out of 1433 patients registered, 500 patients fulfilled all inclusion and exclusion criteria and were allocated in arm A (N.=425) or in arm B (N.=75). Central vascular ligation and the achievement of at least 5 cm of healthy margin were always performed in all procedures of both arms. Patients' population of the two arms was homogeneous as concerns demographic characteristics and stage of the disease. In arm A, resections were performed more frequently by mini-invasive approach as compared to arm B. Operative time was statistically longer in arm B. Postoperative complications rate was extremely low and comparable in both arms (13.4 and 8.0 in arms A and B, respectively). No postoperative mortality was observed. Overall 5-year survival rates were similar in arm A and B (82.3% and 73.05%, respectively; P=0.29). Arm B patients were treated either with more limited resections (resection of the TC and of the SF) or with extended colectomies (right- and left-sided, standard or enlarged, hemicolectomies), both associated with central vascular ligation. Despite a shorter length of surgical specimen after limited resections, postoperative complications, lymph node yield, and survival were absolutely comparable in both types of surgical treatments. CONCLUSIONS: Limited resections for TC and SF cancers can be performed with the same clinical, oncological and survival outcomes as compared to more extended colectomies if an adequate size of the specimen for the achievement of at least 5 cm of health margin is associated to Central Vascular Ligation.

Neoadjuvant chemotherapy for gastric cancer. Is it a must or a fake?

AIM: To investigate the neoadjuvant chemotherapy (NAC) effect on the survival of patients with proper stomach cancer submitted to D2 gastrectomy. METHODS: We proceeded to a review of the literature with PubMed, Embase, ASCO and ESMO meeting abstracts as well as computerized use of the Cochrane Library for randomized controlled trials (RCTs) comparing NAC followed by surgery (NAC + S) with surgery alone (SA) for gastric cancer (GC). The primary outcome was the overall survival rate. Secondary outcomes were the site of the primary tumor, extension of node dissection according to Japanese Gastric Cancer Association (JGCA) performed in both arms, disease-specific (DSS) and disease-free survival (DFS) rates, clinical and pathological response rates and resectability rates after perioperative treatment. RESULTS: We identified a total of 16 randomized controlled trials comparing NAC + S (n = 1089) with SA (n = 973) published in the period from January 1993 - March 2017. Only 6 of these studies were well-designed, structured trials in which the type of lymph node (LN) dissection performed or at least suggested in the trial protocol was reported. Two out of three of the RCTs with D2 lymphadenectomy performed in almost all cases failed to show survival benefit in the NAC arm. In the third RCT, the survival rate was not even reported, and the primary end points were the clinical outcomes of surgery with and without NAC. In the remaining three RCTs, D2 lymph node dissection was performed in less than 50% of cases or only recommended in the "Study Treatment" protocol without any description in the results of the procedure really perfomed. In one of the two studies, the benefit of NAC was evident only for esophagogastric junction (EGJ) cancers. In the second study, there was no overall survival benefit of NAC. In the last trial, which documented a survival benefit for the NAC arm, the chemotherapy effect was mostly evident for EGJ cancer, and more than one-fourth of patients did not have a proper stomach cancer. Additionally, several patients did not receive resectional surgery. Furthermore, the survival rates of international reference centers that provide adequate surgery for homogeneous stomach cancer patients' populations are even higher than the survival rates reported after NAC followed by incomplete surgery. CONCLUSION: NAC for GC has been rapidly introduced in international western guidelines without an evidence-based medicine-related demonstration of its efficacy for a homogeneous population of patients with only stomach tumors submitted to adequate surgery following JGCA guidelines with extended (D2) LN dissection. Additional larger sample-size multicentre RCTs comparing the newer NAC regimens including molecular therapies followed by adequate extended surgery with surgery alone are needed.

Large-scale analysis of herpesviridae in epilepsy-patients with signs of autoimmune encephalitis.

PURPOSE: Epilepsy is one of the most common primary brain disorders. Nonparaneoplastic autoimmune encephalitis is increasingly recognized as an important cause of adult onset epilepsy. However, only in rare cases an initiating factor of the syndrome can be identified. Autoantibody detection after central nervous herpesvirus infection indicates a postviral etiology in a subgroup of patients. In order to analyze a possible underrecognition of postinfectious autoimmunity we performed a large-scale analysis of herpesvirus DNA in cerebrospinal fluid samples from patients with clinical signs of autoimmune encephalitis. METHODS: Real time PCR for HSV 1/2, CMV, EBV, VZV, HHV-6A, HHV-6B, HHV-7 and HHV-8 was performed in cerebrospinal fluid samples from 113 patients with epilepsy and suspected autoimmune encephalitis. Indirect immunofluorescence analysis was used for autoantibody analysis. RESULTS: Antineuronal autoantibodies could be identified in 48 patients with definite autoimmune encephalitis. No autoantibodies were detected in 65 additional patients with probable or possible autoimmune encephalitis. Real-time PCR analysis revealed in three autoantibody-negative patients positive results for HSV, but no evidence for further virus DNA. CONCLUSION: The findings argue against longstanding herpesvirus infection of the CNS as frequent trigger for autoimmunity. However, appearance of autoantibodies after a short period of active virus infection cannot be excluded.

A second locus mapping to 2q35-36 for familial pseudohyperkalaemia.

Familial pseudohyperkalaemia (FP) is a symptomless, dominantly inherited red cell trait, which shows a 'passive leak' of K+ cations into the plasma upon storage of blood at room temperature (or below). There are no haematological abnormalities. The loss of K+ is due to a change in the temperature dependence of the leak. The Scottish case initially described, FP Edinburgh, maps to 16q23-qter. Here we studied a large kindred of Flemish descent with FP, termed FP Lille, which was phenotypically identical to the Edinburgh FP. In FP Lille, however, the responsible locus mapped to 2q35-36, with a Lod score of 8.46 for marker D2S1338. We infer that FP Edinburgh and FP Lille, although they are phenocopies of one another, stem from two distinct loci, FP1 (16q23-qter) and FP2 (2q35-36), respectively. This duality hints at the possibility that the protein mediating the leak might be a heterodimer. No mutation was found in three plausibly candidate genes: the KCNE4 gene, the TUBA1 gene and a predicted gene located in genomic contig NT_005403.

Genetics of food preferences: a first view from silk road populations.

Food preferences are the main factor driving food intake and choice. There are good reasons to suspect some genetic influence on food acceptance, not least because genetic factors are implicated in a number of factors that are likely to be related to food choice. In addition, some food dislikes show themselves early in life, before there is any evidence for aversive experiences. Although taste has been widely studied in regards of pure tastes such as bitter or sweet perception, the relationship between taste-related genes and food preferences has seldom been explored. In this work we investigated relationship of 37 taste-related genes with food preferences. The study was carried out during a scientific expedition through Caucasus and Central Asia (Silk Road) analyzing more than 400 samples from 5 different countries. A food preference questionnaire was administered to each participant and a DNA sample was obtained. Other information, such as age, sex, life style and anthropometrical measures, were also collected. We found significant associations with variants of: (1) TAS1R2 [Correction added after initial online publication on 27 Aug 2012. TAS1R3 was changed to TAS1R2.] gene and liking of Vodka (P= 1.6 × 10(-3)), white wine (P= 4.0 × 10(-4)) and lamb meat (P= 1.6 × 10(-3)); (2) PCLB2 gene and preference for Hot Tea (P= 8.0 × 10(-4)); (3) TPRV1 gene and beet liking (P= 3.8 × 10(-5)); and (4) ITPR3 gene and liking of both lamb meat (5.8 × 10(-4)) and sheep cheese (8.9×10(-4)). These findings give a new insight on a better understanding, of genetic factors influencing food preferences which is critical to the development of effective dietary interventions, especially for people that may be genetically not predisposed for liking specific nutrients.

Multiplexed immunophenotyping of human antigen-presenting cells in whole blood by polychromatic flow cytometry.

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (APCs; e.g., dendritic cells, monocytes and B lymphocytes) in minimally manipulated whole blood samples. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, in order to show the quantification of surface expression levels of molecules involved in chemotaxis (e.g., CX(3)CR1 and CCR2), adhesion (e.g., CD11b and CD62L), antigen presentation (e.g., CD83, CD86 and CD209) and immune regulation (e.g., CD101) on circulating APCs. Each immunostaining reaction requires as little as 50-100 microl of peripheral whole blood and no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h.

Fluorescence intensity normalisation: correcting for time effects in large-scale flow cytometric analysis.

A next step to interpret the findings generated by genome-wide association studies is to associate molecular quantitative traits with disease-associated alleles. To this end, researchers are linking disease risk alleles with gene expression quantitative trait loci (eQTL). However, gene expression at the mRNA level is only an intermediate trait and flow cytometry analysis can provide more downstream and biologically valuable protein level information in multiple cell subsets simultaneously using freshly obtained samples. Because the throughput of flow cytometry is currently limited, experiments may need to span over several weeks or months to obtain a sufficient sample size to demonstrate genetic association. Therefore, normalisation methods are needed to control for technical variability and compare flow cytometry data over an extended period of time. We show how the use of normalising fluorospheres improves the repeatability of a cell surface CD25-APC mean fluorescence intensity phenotype on CD4(+) memory T cells. We investigate two types of normalising beads: broad spectrum and spectrum matched. Lastly, we propose two alternative normalisation procedures that are usable in the absence of normalising beads.

Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy.

OBJECTIVE: Variation in the bitter-taste receptor gene, TAS2R38 confers the ability to taste 6-n-propylthiouracil (PROP). The objective of this study was to relate TAS2R38 haplotypes and PROP-tasting phenotypes to adiposity in a genetically isolated population. We hypothesized that the nontaster phenotype would be associated with higher BMI and waist circumference (WC) in females, and that dietary restraint would mediate this relationship. METHODS AND PROCEDURES: Participants were 540 healthy inhabitants of the genetically isolated village of Carlantino in southern Italy who were 15-89 years of age at the time of the study. Haplotype analyses were performed and PROP tasting was assessed using a filter paper method. Height, weight, and WC were measured and restrained eating was assessed using a brief questionnaire. RESULTS: Nontaster females had higher BMI and WC than females who were phenotypic tasters, and this relationship was specific to females with low dietary restraint. Regression analysis showed that BMI declined by 1.7 units across taster groups in females when the model included the PROP by restraint interaction. PROP phenotype was not significantly associated with WC in the regression models. Polymorphisms in TAS2R38 were not associated with BMI or WC in females. Neither TAS2R38 haplotype nor PROP phenotype was strongly related to BMI or WC in males. DISCUSSION: These data support previous findings of a relation between the nontaster phenotype and higher BMI in females that is modified by dietary restraint. Assessment of PROP phenotypes might provide unique information about adiposity that is not captured by haplotype analysis alone.

Commonality in the genetic control of Type 1 diabetes in humans and NOD mice: variants of genes in the IL-2 pathway are associated with autoimmune diabetes in both species.

Variants within the IL-2 (interleukin 2) and CD25 genes are associated with T1DM (Type 1 diabetes mellitus) in mice and humans respectively. Both gene products are essential for optimal immune tolerance and a partial failure to tolerize is linked to the autoimmune responses to insulin and other beta-cell proteins that precede T1DM onset. Gene variants that contribute to common disease susceptibility often alter gene expression only modestly. Small expression changes can be technically challenging to measure robustly, especially since biological variation usually contributes negatively to this goal. The present review focuses on allele-specific expression assays that can be used to quantify genotype-determined expression differences such as those observed for IL-2, where the susceptibility allele is transcribed 2-fold less than the resistance allele.