Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

The Effect of Chronic Long-Term Intermittent Hypobaric Hypoxia on Bone Mineral Density in Rats: Role of Nitric Oxide

Intermittent hypoxia is the most common pattern of hypoxic exposure in humans. The effect of chronic long-term intermittent hypobaric hypoxia (CLTIHH) on bone metabolism is not investigated. We examined the effect of CLTIHH on bone metabolism and the role of nitric oxide (NO) in this process. The rats were divided into three groups in this study. The animals in groups I and II have been exposed to CLTIHH. The animals in group II were also treated with nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. To obtain CLTIHH, rats were placed in a hypobaric chamber (430 mm Hg; 5 h/day, 5 days/week, 5 weeks). The group III (control) rats breathed room air in the same environment. At the begining of the experiments, bone mineral density (BMD) of the animals were measured, and blood samples were collected from the tail vein. After the 5-week CLTIHH period, the same measurements were repeated. Parathyroid hormone, calcium, phosphate, bone alkaline phosphatase (b-ALP), NO, interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha levels were determined. The cytokines, NO levels, and BMD in CLTIHH-induced rats were higher compared with baseline and control values. The cytokines, b-ALP, and BMD increased while NO levels decreased in the group II compared with baseline values. BMD values of group II were lower than group I but higher than control group. Our results suggested that CLTIHH has positive effects on bone density. Intermittent hypoxia protocols may be developed for treatment and prevention of osteopenia and osteoporosis.     

Role of phytoestrogenic oils in alleviating osteoporosis associated with ovariectomy in rats

The objective of this study was to elucidate the effect of soybean oil (SbO) and sesame oil (SO) supplemented diets on bone biomarkers changes in OVX (ovariectomized) rats. The current data exhibited significant decrease in BMD (bone mineral density), accompanied with marked depletion in the level of Ca, P and Mg in both serum and bone of OVX rats. Also, serum estrogen, total protein, HDL-C (high density lipoprotein cholesterol), bone NO levels were decreased in OVX rats. However, a significant increase in the level of serum TL (total lipids), TC (total cholesterol), TG (triglycerides), LDL-C (low density lipoprotein cholesterol), VLDL-C (very low density lipoprotein cholesterol), urine minerals (Ca, P, Mg), as well as serum, bone and urine ALP (alkaline phosphatase) and ACP (acid phosphatase) activity were recorded in OVX rats. Further changes were also detected by the increased level of urine hydroxyproline, serum parathyroid hormone and osteocalcin, as well as urea and creatinine level in both serum and urine. On the other hand, when OVX rats were fed on SbO (soy bean oil) (15 % w/w) or SO (sesame oil) (10 % w/w) supplemented diets, the data recorded a significant improvement in all the above mentioned parameters. So, it can be concluded that consumption of SbO or SO supplemented diets might be considered as a functional food for retarding risks of osteoporosis associated with estrogen deficiency in OVX states.     

Short Term Survival after Admission for Heart Failure in Sweden: Applying Multilevel Analyses of Discriminatory Accuracy to Evaluate Institutional Performance

Background

Hospital performance is frequently evaluated by analyzing differences between hospital averages in some quality indicators. The results are often expressed as quality charts of hospital variance (e.g., league tables, funnel plots). However, those analyses seldom consider patients heterogeneity around averages, which is of fundamental relevance for a correct evaluation. Therefore, we apply an innovative methodology based on measures of components of variance and discriminatory accuracy to analyze 30-day mortality after hospital discharge with a diagnosis of Heart Failure (HF) in Sweden.

Methods

We analyzed 36,943 patients aged 45–80 treated in 565 wards at 71 hospitals during 2007–2009. We applied single and multilevel logistic regression analyses to calculate the odds ratios and the area under the receiver-operating characteristic (AUC). We evaluated general hospital and ward effects by quantifying the intra-class correlation coefficient (ICC) and the increment in the AUC obtained by adding random effects in a multilevel regression analysis (MLRA). Finally, the Odds Ratios (ORs) for specific ward and hospital characteristics were interpreted jointly with the proportional change in variance (PCV) and the proportion of ORs in the opposite direction (POOR).

Findings

Overall, the average 30-day mortality was 9%. Using only patient information on age and previous hospitalizations for different diseases we obtained an AUC = 0.727. This value was almost unchanged when adding sex, country of birth as well as hospitals and wards levels. Average mortality was higher in small wards and municipal hospitals but the POOR values were 15% and 16% respectively.

Conclusions

Swedish wards and hospitals in general performed homogeneously well, resulting in a low 30-day mortality rate after HF. In our study, knowledge on a patient’s previous hospitalizations was the best predictor of 30-day mortality, and this information did not improve by knowing the sex and country of birth of the patient or where the patient was treated.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

BAGY2 Retrotransposon Analyses in Barley Calli Cultures and Regenerated Plantlets

The stability of aging barley calli and regenerated plantlets from those calli was investigated by the BAGY2 retrotransposon-specific IRAP technique. Mature embryos of barley (Hordeum vulgare cv. Golden Promise) were cultured in Murashige and Skoog medium supplemented with 4 mg/L dicamba and maintained on the same medium for 45 and 90 days. Two IRAP-based primers were used, and the levels of variation of DNA isolated from 45- and 90-day-old calli and regenerated plantlets were found to be increased 0–21%, depending on the mature embryo material and the age of the callus. It has been observed that culture conditions cause genetic variations and evident BAGY2 retrotransposon alterations. Internal domains of BAGY2 were also analyzed by qPCR, and copy numbers were found to be increased. These findings are expected to contribute to understanding of how retrotransposons affect features like tissue culture (especially callus tissue) formation and genetic engineering studies.     

Molecular and immunological evaluation of the expression of cancer/testis gene products in human colorectal cancer

Tumor-specific gene products, such as cancer/testis (CT) antigens, constitute promising targets for the development of T cell vaccines. Whereas CT antigens are frequently expressed in melanoma, their expression in colorectal cancers (CRC) remains poorly characterized. Here, we have studied the expression of the CT antigens MAGE-A3, MAGE-A4, MAGE-A10, NY-ESO-1 and SSX2 in CRC because of the presence of well-described HLA-A2-restricted epitopes in their sequences. Our analyses of 41 primary CRC and 14 metastatic liver lesions confirmed the low frequency of expression of these CT antigens. No increased expression frequencies were observed in metastatic tumors compared to primary tumors. Histological analyses of CRC samples revealed heterogeneous expression of individual CT antigens. Finally, evidence of a naturally acquired CT antigen-specific CD8⁺ T cell response could be demonstrated. These results show that the expression of CT antigens in a subset of CRC patients induces readily detectable T cell responses.     

Investigation of asymptomatic visceral leishmaniasis cases using western blot in an endemic area in Turkey

In Turkey, Leishmania infantum is responsible for human visceral leishmaniasis (VL), which is seen mainly in the Aegean, Mediterranean, and Central Anatolia Regions. This study aimed to determine asymptomatic infections in an endemic area of VL in Turkey using the western blot technique. A total of 82 persons including children and adults were chosen randomly in Denizli province which is one of the endemic sites for VL. Serum samples were collected and screened using indirect immunofluorescent test (IFAT), enzyme-linked immunosorbent assay (ELISA) and western blot (WB). One year later, 35 of the 82 persons were sampled and screened serologically for the second time. Seven out of 82 samples were found to be positive by western blot analysis with the presence of 14 and/or 18 kDa bands. Two of these seven sera were also positive by IFAT, but only one of these two was positive by ELISA. Only one person showing seropositivity with all three tests had clinical symptoms and was diagnosed as VL with the presence of amastigotes in bone marrow aspirate. Because six people, including the one found to be seropositive in all two tests, had no clinical symptoms, they were accepted as asymptomatic carriers. The ratio of asymptomatic infection was calculated as 7.41% (6/81) in the region. In the second sampling, the western blot revealed antibodies against the same antigens in all seven subjects. Our findings showed that the presence of antibodies against 14 and 18 kDa antigens are important for the diagnosis of symptomatic and asymptomatic infections. Western blot was found to be effective in the detection of asymptomatic persons in the epidemiological studies in endemic areas.     

Ghrelin is present in teeth

Ghrelin belongs to the family of a gut-brain hormone that promotes food intake and controls energy balance. Recently, it has also been shown to regulate bone formation directly. Dental tissue shares several functional, developmental and anatomical similarities with bone, and in the present study we have investigated the presence of ghrelin in 44 human teeth using immunocytochemistry and radioimmunoassay. Both methods showed that the hormone is present in canines and molars, mainly in the odontoblasts but also in the pulp. Ghrelin could potentially play interesting physiological roles in teeth.