Luminal and cancer cells in the breast show more rapid telomere shortening than myoepithelial cells and fibroblasts

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to clarify telomere length variations within and between individuals using our tissue quantitative fluorescence in situ hybridization method. We obtained 3 novel findings. (1) In corresponding normal tissues, telomere length decreased in the order myoepithelial cells > normal-appearing fibroblasts > luminal epithelial cells, and telomere lengths were characteristic in these 3 cell types within each individual. (2) As expected, cancer cells had significantly shorter telomeres than myoepithelial cells (P < .0001) and normal-appearing fibroblasts (P = .0161), but there was no significant difference in telomere length between luminal cells and cancer cells (P = .6270). (3) Fibroblasts adjacent to cancer had longer telomeres than normal-appearing fibroblasts distant from cancer (P < .0001). This study, which represents the first reported assessment of telomere length variations in the 5 cell types comprising breast tissue within and between individuals, revealed that normal luminal epithelial cells and cancer cells had the shortest telomeres. Our new findings indicate that telomeres of background luminal cells are as short as those of cancer cells. Tissue quantitative fluorescence in situ hybridization, applicable to analysis of individual cells in tissue sections, is considered to be a powerful technique with considerable promise for studies in oncology.     

Risky sexual behavior among patients on long-term antiretroviral therapy: a prospective cohort study in urban and rural Uganda

Background

While the effects of initiation of antiretroviral treatment (ART) on risky sexual behavior have been extensively studied, less is known about the long-term changes in risky sexual behavior over time in resource-poor settings.

Methods

We conducted a secondary longitudinal analysis of one rural and one urban cohort of patients who initiated ART in Uganda between April 2004 and July 2007 followed up-to 2016. Data on sexual behavior were collected every 6 months for 3.5 years in individuals on ART?≥?4 years (baseline) when a behavioral questionnaire was introduced. Risky sexual behavior was defined as sexual intercourse with?≥?2 partners or inconsistent or no condom use in previous 6 months. We report characteristics overall, and by cohort. We used multivariable generalized estimating equations logistic regression to assess the effects of time on ART on risky sexual behavior.

Results

Of 1012 participants, 402 (39.8%) were urban and 610 (60.2%) were rural residents. Mean age was 42.8 years (SD 8.5). Mean duration of follow-up was 51.3 months (SD 15.3), but longer for urban than rural participants (64.5 vs 36.4 months). Risky sexual behavior declined from 33.1% at baseline to 9.6% after 3.5 years of follow-up in the rural cohort (p?≤?0.01 for the test of trend) and was unchanged from 9.7% at baseline to 9.9% after 3.5 years in the urban cohort (p?=?0.51). Receiving care at a rural clinic (aOR 4.99, 95% CI 3.64–6.84); male gender (aOR 1.66, 95% CI 1.26–2.19) and being younger (aOR 5.60, 95% CI 3.80–8.25 for 18–34 years and aOR 2.34, 95% CI 1.74–3.14 for 35–44 years) were associated with increased odds of risky sexual behavior. Not being married (aOR 0.25; 95% CI 0.19–0.34), and longer time on ART (aOR 0.71 95% CI 0.67–0.76) were associated with reduced odds of risky sex.

Conclusions

We observed a decline in risky sexual behavior in rural people on long-term (≥?4 years) ART. Rural, male and young individuals had higher odds of self-reported risky sexual behavior. ART programs should continue to emphasize risk reduction practices, especially among people receiving care in rural health facilities, males, younger individuals and those who are married.

     

GPT2 mutations cause developmental encephalopathy with microcephaly and features of complicated hereditary spastic paraplegia

Various genetic defects can cause intellectual and developmental disabilities (IDDs). Often IDD is a symptom of a more complex neurodevelopmental or neurodegenerative syndrome. Identifying syndromic patterns is substantive for diagnostics and for understanding the pathomechanism of a disease. Recessive glutamate pyruvate transaminase (GPT2) mutations have recently been associated with IDD in 4 families. Here, we report a novel recessive GPT2 stop mutation p.Gln24* causing a complex IDD phenotype in a homozygous state in 5 patients from 2 consanguineous Arab families. By compiling clinical information of these individuals and previously described GPT2 patients a recognizable neurodevelopmental and potentially neurodegenerative phenotype can be assigned consisting of intellectual disability, pyramidal tract affection with spastic paraplegia, microcephaly and frequently epilepsy. Because of the consistent presence of pyramidal tract affection in GPT2 patients, we further suggest that GPT2 mutations should be considered in cases with complex hereditary spastic paraplegia.     

Real-time PCR assay compared to nested PCR and antigenemia assays for detecting cytomegalovirus reactivation in adult T-cell leukemia-lymphoma patients

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of >/=50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.     

Influence of resveratrol supplementation on IVF–embryo transfer cycle outcomes

Research questionDoes resveratrol, a polyphenolic compound, affect IVF–embryo transfer outcomes?DesignThis single-centre, cross-sectional retrospective study was designed to compare the outcomes of embryo transfer cycles in women receiving resveratrol supplementation (200 mg/day) continuously (RES group) with a control group (non-RES group). Of 8686 embryo transfer cycles, 1409 cycles with poor prognostic factors were excluded, including cycles in women aged ≥43 years and those with poor-quality embryos. The RES group (204 cycles, 102 women) was compared with the non-RES group (7073 cycles, 2958 women).ResultsAfter matching patients by age at the time of oocyte retrieval, grade and developmental stage of embryos, number of embryos transferred, and fresh or vitrified-warmed embryo transfer, multivariate logistic regression analysis showed that resveratrol supplementation is strongly associated with a decrease in clinical pregnancy rate [odds ratio (OR) 0.539, 95% confidence interval (CI) 0.341–0.853] and an increased risk of miscarriage (OR 2.602, 95% CI 1.070–6.325).ConclusionsResveratrol supplementation during embryo transfer cycles appears to be detrimental for pregnancy outcomes. An analysis of the supplementation protocol and randomized controlled studies are needed.     

Combination Treatment of Preoperative Embryo Cryopreservation and Endoscopic Surgery (Surgery-ART Hybrid Therapy) in Infertile Women with Diminished Ovarian Reserve and Uterine Myomas or Ovarian Endometriomas

Study ObjectiveTo analyze the clinical outcomes of and predictive factors for the therapeutic effect of combination treatment of preoperative embryo cryopreservation and endoscopic surgery (surgery-assisted reproductive technology [ART] hybrid therapy) in infertile women with diminished ovarian reserve (DOR) with uterine fibroids and/or ovarian endometriomas.DesignRetrospective cohort study.SettingData from all patients who underwent surgery-ART hybrid therapy at Juntendo University Hospital and Sugiyama Clinic between 2014 and 2016 were analyzed retrospectively. We compared women who experienced live birth (success group) and implantation failure or miscarriage (failure group) after surgery-ART hybrid therapy and evaluate the predictive factors for live birth.PatientsA total of 39 infertile women underwent surgery-ART hybrid therapy with 86 embryo transfer cycles.InterventionsAll women underwent ART treatment for embryo cryopreservation preoperatively, reproductive surgery, and warmed embryo transfer after the postoperative contraceptive interval (surgery-ART hybrid therapy) for women with DOR (anti-Müllerian hormone <1.0 ng/mL) and/or advanced reproductive age (>40 years) with uterine myomas and/or ovarian endometriomas who required surgery.ResultsAmong 39 women underwent surgery-ART hybrid therapy, 1 woman acquired no embryo after oocyte retrieval trials and abandoned efforts to conceive, 14 experienced childbirth (success group) and 24 (63.2%) experienced implantation failure or miscarriage (failure group) after surgery-ART hybrid therapy. The median patient age was 40 years (interquartile range [IQR], 38–41 years) in the success group and 41.5 years (IQR, 41–42 years) in the failure group (p = .032). The respective serum anti-Müllerian hormone levels were 2.5 ng/mL (range, 0.1–8.6 ng/mL) and 1.3 ng/mL (range, 0.1–4.2 ng/mL) (p = .396), and the respective numbers of preoperative frozen were 5.0 (range, 4.0–6.0) and 2.0 (range, 1.0–3.0) (p < .001). There were no significant differences in surgical findings of myomas and endometriosis between the 2 groups. Compared with the 24 women who experienced hybrid therapy failure, the 14 who underwent successful surgery-ART hybrid therapy were significantly younger and had a greater number of cryopreserved embryos.ConclusionSuccessful surgery-ART hybrid therapy requires a sufficient preoperative age-specific number of frozen embryos, establishment of ART treatment with stable pregnancy outcomes and skillful reproductive surgery, and a strong desire of the patient and doctor for pregnancy.     

Outcome of implantable cardioverter defibrillator therapy for congenital heart disease

Background: The use of implantable cardioverter defibrillator (ICD) therapy for congenital heart disease (CHD) has been increasing, but few studies have reported on the efficacy of ICD therapy in Japanese CHD patients. Method: Twelve CHD patients (median age, 35 years) with first ICD implantation were examined. Median follow‐up duration was 2.9 years. Demographic information, implant electrical parameters, appropriate and inappropriate discharge data and complications were recorded for all implants from 2003 to 2010. Results: Implant indication was primary prevention in two patients and secondary prevention in 10. Overall four patients received one or more discharges; three patients (25%) with secondary prevention received nine appropriate discharges. Inappropriate discharge attributed to sinus tachycardia occurred in two patients (16.7%). Only one patient experienced the late complication of skin erosion at the generator implantation site. Conclusions: Patients with CHD experienced significant rates of appropriate discharges and lower complications. But given that the indications of ICD implantation were mostly for secondary prevention, the ratio of appropriate shocks might be lower than in previous studies. In the primary prevention patients, the benefit of ICD was not clear because no appropriate discharges were seen during follow up. Although ICD implantation for CHD is beneficial for preventing sudden cardiac death, careful decision making and a large, long‐term prospective study is required for the determination of the efficacy of ICD therapy in Japanese patients with CHD.     

Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells

Since dendritic cells (DC) play pivotal roles in both innate and adaptive immunity, DC can be a good target for immuno-gene therapy. However, the optimal generation method for gene-modified DC has not yet been well exploited. CD34+ cells from cord blood (CB), bone marrow (BM), or peripheral blood (PB) were expanded in a medium containing stem cell factor (SCF), flt 3 ligand (Flt3L) and thrombopoietin (TPO) with or without HESS-5, a murine BM stromal cell line, for 2 weeks (the first expansion step), then differentiated to DC in a medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF), flt 3 ligand (Flt3L), stem cell factor (SCF), tumor necrosis factor-alpha (TNF-alpha), IL-4, and lipopolysaccharide (LPS) for 9 days (the second differentiation step). DC progenitors were transduced with human immunodeficiency virus (HIV) vectors at different time points during the second step. Use of HESS-5 during the first step resulted in more DC generation than without it (cell expansion: CB, 10,461 vs. 354-fold; BM, 962 vs. 225-fold; peripheral blood mononuclear cell (PBMC), 8,506 vs. 240-fold; %DC: CB, 83.4% vs. 76.9%; BM, 83.6 vs. 69.8%; PBMC, 85.9 vs. 60.5%). Gene transduction to the in vitro expanded DC progenitors at day 3 during the second step, resulted in better final yield of the gene-modified DC than that to those at day 0 or day 6 (as much as 44% of DC expressed green fluorescence protein (GFP) as a transgene) and the transduction efficiency correlated with endocytic ability and percent of S phase. DC transduced with an HIV vector encoding a melanoma antigen, MART-1, were adequately recognized by specific anti-MART-1 CTL. The two-step culture method with HESS-5 is useful for rapid expansion of DC progenitors and subsequent lentiviral gene transduction to DC.     

Granule types and their morphological changes in terminal cluster and acinar cells in the late pre- and early postnatal rat sublingual gland

The developmental characteristics of serous cells appearing in the rat sublingual gland from the late prenatal to the early postnatal period were investigated in this study. Particular attention was paid to the morphological changes observed in the secretory granules at the histochemical and ultrastructural level. On prenatal day 18, granules with homogeneous high electron density (Type I granules), and mottled granules (Type II granules) with heterogeneous electron density appeared in the narrow luminar cytoplasm of cells constituting the terminal clusters. On prenatal day 19, these granules decreased in number and were replaced by bipartite granules (Type III granules) composed of a highly electron-dense core and a more electron-lucent rim. Pronase treatment almost completely digested the Type I and II granules and the electron-dense core of the Type III granules, although some of the Type I and II granules in serous demilunes at a later stage were insufficiently digested. On prenatal day 19.5, homogeneous granules of low electron density (Type IV granules) appeared in the terminal clusters and acini, and increased in number daily, making up 92.8% of the total granules on postnatal day 28. The granule morphology on electron microscopy, Alcian blue, and periodic acid-Schiff staining strongly suggested that Type I and II granules were serous granules, Type IV granules were mucous granules, and Type III granules were transforming-type granules. None of the secretory cells showed chromatin condensation, which is a characteristic of apoptosis. These findings suggest that the developing rat sublingual gland from the late prenatal to early postnatal period has numerous serous granules in the terminal clusters and acini, and that the majority of granules are replaced by mucous granules through transforming-type granules. In addition, because apoptotic figures of secretory cells could not be detected, it appears that most of the serous cells in the developing rat sublingual gland might have changed to mucous cells.     

Murine strain differences in airway inflammation induced by diesel exhaust particles and house dust mite allergen

BACKGROUND: Differences in allergic airway inflammation induced by ovalbumin (OVA) + diesel exhaust particles (DEP) in various murine strains have already been reported. However, there is no report that different murine strains respond differently towards house dust mites or DEP, which are known to aggravate allergic asthma. METHODS: The Dermatophagoides farinae allergens Der f (1 microg) or Der f (1 microg) + DEP (50 microg) were administered intratracheally to two different mouse strains (CBA/JN and C57BL/6N). Histological changes in the lung tissues, asthma-relevant cytokines in the lungs, and allergen-specific immunoglobulins in plasma were investigated. RESULTS: Der f treatment led to the proliferation of goblet cells, production of mucus plugs, and the recruitment of eosinophils and lymphocytes to the airways of the mice. The manifestation of the airway inflammation in the C57BL/6N mouse was much greater than in the CBA/JN mouse. The protein levels of interleukin (IL)-4 and IL-5, regulated on activation, normal T cell expressed, and presumably secreted (RANTES), and eotaxin in the lung tissue of C57BL/6N mice were higher than those in CBA/JN mice by a factor of 1.26 (IL-4), 5.26 (IL-5), 2.07 (RANTES) and 3.27 (eotaxin). DEP aggravated the manifestations of the eosinophilic inflammation in CBA/JN mice through goblet cell proliferation. However, the exact effect of DEP could not be evaluated in C57BL/6N because of its severe enhancement of the inflammation. DEP enhanced the local expression of IL-5, RANTES, and eotaxin in the CBA/JN mouse, and consequently triggered an increased IgG1 production in both strains. Allergen-specific IgE antibodies were lower than 1 titer in both mice. CONCLUSION: The murine strain differences in the pathogenesis of allergic airway disease caused by mite allergen might be related to the local expression of the cytokines we screened. The aggravating effect of DEP may be mediated by an increase in the local expression of IL-5, RANTES, eotaxin, and the production of an antigen specific to IgG1.