Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-step in vitro culture

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5-7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.     

Associations of recurrent miscarriages with chromosomal abnormalities,thrombophilia allelic polymorphisms and/or consanguinity in Saudi Arabia

Background

Recurrent pregnancy loss (RPL) or recurrent spontaneous abortion is an obstetric complication that affects couples at reproductive age. Previous reports documented a clear relationship between parents with chromosomal abnormalities and both recurrent miscarriages and infertility. However, limited data is available from the Arabian Peninsula which is known by higher rates of consanguineous marriages. The main goal of this study was to determine the prevalence of chromosomal abnormalities and thrombophilic polymorphisms, and to correlate them with RPL and consanguinity in Saudi Arabia.

Methods

Cytogenetic analysis of 171 consent patients with RPL was performed by the standard method of 72-h lymphocyte culture and GTG banding. Allelic polymorphisms of three thrombophilic genes (Factor V Leiden, Prothrombin A20210G, MTHFR C677T) were performed using PCR-RFLP (restriction fragment length polymorphism) and gel electrophoresis.

Results

Data analysis revealed that 7.6 % of patients were carrier of numerical or structural chromosomal abnormalities. A high rate of translocations (46 %) was associated to increased incidence of RPL. A significant correlation between consanguineous RPL patients and chromosomal abnormalities (P?<?0.05) was found. Both Factor V Leiden and Prothrombin A20210G allelic polymorphisms were significantly associated with a higher prevalence of RPL.

Conclusions

This study demonstrated a strong association between RPL and the prevalence of chromosomal abnormalities and inherited thrombophilia. Given the high rate of consanguineous marriages in the Saudi population, these results underline the importance of systematic cytogenetic investigation and genetic counseling preferably at the premarital stage or at least during early pregnancy phase through preimplantation genetic diagnosis (PGD).

     

A case–control evaluation of 143 single nucleotide polymorphisms for breast cancer risk stratification with classical factors and mammographic density

Panels of single nucleotide polymorphisms (SNPs) stratify risk for breast cancer in women from the general population, but studies are needed assess their use in a fully comprehensive model including classical risk factors, mammographic density and more than 100 SNPs associated with breast cancer. A case–control study was designed (1,668 controls, 405 cases) in women aged 47–73 years attending routine screening in Manchester UK, and enrolled in a wider study to assess methods for risk assessment. Risk from classical questionnaire risk factors was assessed using the Tyrer–Cuzick model; mean percentage visual mammographic density was scored by two independent readers. DNA extracted from saliva was genotyped at selected SNPs using the OncoArray. A predefined polygenic risk score based on 143 SNPs was calculated (SNP143). The odds ratio (OR, and 95% confidence interval, CI) per interquartile range (IQ-OR) of SNP143 was estimated unadjusted and adjusted for Tyrer–Cuzick and breast density. Secondary analysis assessed risk by oestrogen receptor (ER) status. The primary polygenic risk score was well calibrated (O/E OR 1.10, 95% CI 0.86–1.34) and accuracy was retained after adjustment for Tyrer–Cuzick risk and mammographic density (IQ-OR unadjusted 2.12, 95% CI% 1.75–2.42; adjusted 2.06, 95% CI 1.75–2.42). SNP143 was a risk factor for ER+ and ER− breast cancer (adjusted IQ-OR, ER+ 2.11, 95% CI 1.78–2.51; ER− 1.81, 95% CI 1.16–2.84). In conclusion, polygenic risk scores based on a large number of SNPs improve risk stratification in combination with classical risk factors and mammographic density, and SNP143 was similarly predictive for ER-positive and ER-negative disease.     

Expression of HMP/AN2, a melanoma associated antigen,in murine cerebral gliomas: potential for radioimmunotargeting

Human melanoma proteoglycan (HMP), a melanoma-associated antigen, is expressed in both human melanomas and gliomas. We used HMP-specific monoclonal antibody (mAb) VT68.2 to investigate whether murine GL261 cerebral gliomas express the HMP homologue AN2 and to determine whether AN2 could be targeted for selective delivery of radiation in vivo. HMP-specific mAb VT68.2 stained murine GL261 glioma cells grown in culture and intracerebrally in syngeneic C57BL/6 mice. Positron emission tomography with radiolabeled mAb VT68.2 showed high-contrast, positive images of gliomas against a negative background. At 96 h after injection, glioma uptake of radiolabeled mAb VT68.2 was 10× greater than that of the isotype control mAb and 20× greater than that detected in normal cerebral tissue. Our results show murine GL261 cerebral gliomas express AN2 and HMP-specific mAb VT68.2 accumulates selectively and specifically at high concentration and is retained within murine cerebral gliomas. Thus, HMP is a potential target for antibody-mediated selective delivery of radiation to cerebral gliomas in vivo.     

Regulatory T cells and cytokines in malignant pleural effusions secondary to mesothelioma and carcinoma

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immunosuppressive factors of the tumor and host, particularly certain cytokines and CD4(+) CD25(+) regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4(+) CD25(+) T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4(+) CD25(+) T cells. Activated CD8(+) T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4(+) CD25(+) pleural effusion T cells and low percentages of CD8(+) CD25(+) pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immunosuppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.     

Combined atorvastatin and coenzyme Q10 improve the left ventricular function in isoproterenol-induced heart failure in rat

The effect of atorvastatin on cardiac remodeling, function, and homodynamic parameters in isoproterenol-induced heart failure was evaluated in the present study. A subcutaneous injection of isoproterenol (5mg/kg/day) for 10 days was used for the induction of heart failure. Isoproterenol administration produced intensive myocardial necrosis and fibrosis with a significant decrease in the arterial pressure indices, heart rate, contractility (LVdP/dt(max)) and relaxation (LVdP/dt(min)), but an increase in the left ventricular end-diastolic pressure. Rats were randomly assigned to control, treatment with only atorvastatin, and treatment with atorvastatin plus coenzyme Q10. Histopathological analysis showed a marked attenuation of myocyte necrosis and interstitial fibrosis in all atorvastatin treated groups (P<0.001). A low dose of atorvastatin (5mg/kg/day) significantly improved the left ventricular systolic pressure, contractility and relaxation (P<0.01). On the contrary, a high dose of atorvastatin (20mg/kg/day) worsened the isoproterenol-induced left ventricular dysfunction by a further reduction of LVdP/dt(max) from +2780 ± 94 to +1588 ± 248 (mmHg/s; P<0.01) and LVdP/dt(min) from -2007 ± 190 to -2939 ± 291 (mmHg/s; P<0.05). Co-administration of coenzyme Q10 with atorvastatin reversed the hemodynamic depression and the left ventricular dysfunction to a high level (P<0.001). There was a lower level of LVEDPs in the atorvastatin+coenzyme Q10 treated groups (3 ± 1 and 4 ± 1.4 versus 8 ± 3.5 and 14 ± 3.6 mmHg, respectively), thereby suggesting improvement in the myocardial stiffness by the combined coenzyme Q10 and atorvastatin treatment. The atorvastatin therapy attenuated myocardial necrosis and fibrosis in isoproterenol-induced heart failure. However, a high dose of the drug considerably worsened the left ventricular dysfunction and hemodynamic depression, which was reversed by coenzyme Q10 co-administration.     

Urrets-Zavalia syndrome following penetrating keratoplasty for keratoconus

Background

Urrets-Zavalia syndrome (UZS) consists of a fixed dilated pupil associated with iris atrophy. It is a poorly understood complication following penetrating keratoplasty (PKP) for keratoconus (KC). In this work, we aim to establish the incidence, visual outcomes, and an understanding of UZS.

Methods

This was a retrospective single-center study in a tertiary eye service in the United Kingdom of consecutive patients with UZS following PKP for KC in a 10-year period. Post-operative complications, including raised intraocular pressure (IOP), were recorded. UZS patients and age-matched control patients who had undergone PKP for KC without developing UZS attended a comprehensive clinical assessment. Anterior segment indocyanine green (ICG) angiography assessed iris perfusion.

Results

The incidence of UZS was 16.2 %. There was no difference in LogMAR VA or Pelli–Robson contrast sensitivity between groups. There was higher first-day post-operative IOP in UZS (p?=?0.02). UZS patients had increased pupil size (p?=?0.09) with reduced response to pilocarpine 2 % (p?<?0.001). ICG angiography revealed delayed/reduced iris vasculature filling in UZS compared with normal filling patterns of controls.

Conclusions

Elevated post-operative IOP within 24 h was a significant factor in the development of UZS. Visual function in UZS patients was unaffected. UZS patients developed longstanding mydriasis with reduced reactivity to topical pilocarpine 2 %. ICG angiography confirmed iris vessel ischemia; supporting the theory that iris ischemia resulting from occlusion of iris root vessels due to elevated IOP causes UZS. We advocate rigorous intraoperative management of ocular viscoelastic devices and aggressive postoperative IOP control in patients undergoing PKP for KC.     

Limiting youth access to tobacco: comparing the long-term health impacts of increasing cigarette excise taxes and raising the legal smoking age to 21 in the United States

Although many states in the US have raised cigarette excise taxes in recent years, the size of these increases have been fairly modest (resulting in a 15% increase in the per pack purchase price), and their impact on adult smoking prevalence is likely insufficient to meet Healthy People 2010 objectives. This paper presents the results of a 75-year dynamic simulation model comparing the long-term health benefits to society of various levels of tax increase to a viable alternative: limiting youth access to cigarettes by raising the legal purchase age to 21. If youth smoking initiation is delayed as assumed in the model, increasing the smoking age would have a minimal immediate effect on adult smoking prevalence and population health, but would affect a large drop in youth smoking prevalence from 22% to under 9% for the 15-17-year-old age group in 7 years (by 2010)-better than the result of raising taxes to increase the purchase price of cigarettes by 100%. Reducing youth initiation by enforcing a higher smoking age would reduce adult smoking prevalence in the long-term (75 years in the future) to 13.6% (comparable to a 40% tax-induced price increase), and would produce a cumulative gain of 109 million QALYs (comparable to a 20% price increase). If the political climate continues to favor only moderate cigarette excise tax increases, raising the smoking age should be considered to reduce the health burden of smoking on society. The health benefits of large tax increases, however, would be greater and would accrue faster than raising the minimum legal purchase age for cigarettes.