Use of soluble recombinant TNF receptor to improve detection of TNF secretion in cultures of tumor infiltrating lymphocytes.

The accurate quantitation of picogram amounts of TNF is possible by ELISA and is useful in many areas of biomedical research, including studies of TNF release in vitro by stimulated lymphocytes and macrophages, and of serum levels in patients with cancer and sepsis. However, we show in this report that the detection of recombinant TNF standards by ELISA falls over time with incubation at 37 degrees C, and is further decreased when incubated with tumor infiltrating lymphocytes (TIL), making accurate quantitation difficult. We demonstrate that the soluble dimeric form of the TNF receptor can prevent this decrease, both in the presence and absence of TIL. In contrast, the soluble monomeric TNF receptor was much less effective in preventing this decrease. In addition, the dimeric but not the monomeric TNF receptor was found to inhibit bioactivity of TNF as measured by L929 cytotoxicity. The dimeric TNF receptor does not interfere with the detection of recombinant TNF standards by ELISA, and entirely stabilizes TNF levels incubated over 48 h at 37 degrees C in the presence and absence of TIL. This protection is specific, and the TNF receptor does not stabilize interferon-gamma. The dimeric form of the soluble TNF receptor has proven useful in detecting TNF released by TIL transduced with the TNF cDNA that are currently being used in studies of the gene therapy of cancer with TIL. The dimeric TNF receptor may also prove useful in the accurate quantitation of TNF released by stimulated lymphocytes and macrophages in vitro, and in the quantitation of serum TNF levels in patients.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Evaluation of RGS4 as a candidate gene for schizophrenia.

Several studies have suggested that the regulator of G-protein signaling 4 (RGS4) may be a positional and functional candidate gene for schizophrenia. Three single nucleotide polymorphisms (SNP) located at the promoter region (SNP4 and SNP7) and the intron 1 (SNP18) of RGS4 have been verified in different ethnic groups. Positive results have been reported in these SNPs with different numbers of SNP combinatory haplotypes. In this study, these three SNP markers were genotyped in 218 schizophrenia pedigrees of Taiwan (864 individuals) for association analysis. Among these three SNPs, neither SNP4, SNP7, SNP18 has shown significant association with schizophrenia in single locus association analysis, nor any compositions of the three SNP haplotypes has shown significantly associations with the DSM-IV diagnosed schizophrenia. Our results fail to support the RGS4 as a candidate gene for schizophrenia when evaluated from these three SNP markers.     

A founder mutation (R254X) of SLC22A5 (OCTN2) in Chinese primary carnitine deficiency patients

Mutations in the SLC22A5 gene, which encodes for the plasma membrane carnitine transporter OCTN2, cause primary carnitine deficiency (PCD). After our first report of OCTN2 mutations in Chinese, three more Chinese PCD patients were identified. The parents of these families were non-consanguineous and these families were unrelated. Two novel truncating mutations were found: R254X, a single-base mutation at cDNA position 981 (c.981C>T); and Y387X (c.1382T>G). Two probands, one each from Taiwan and Macau, were homozygous for R254X. The other proband from Taiwan carried both R254X and Y387X. Two additional heterozygote carriers of R254X were also identified among 250 control samples, while none was detected for Y387X. The population carrier rate for R254X would be about 1 in 125. Haplotypes of R254X alleles were examined and patients homozygous for R254X were also homozygous for the same haplotype of intragenic and microsatellites markers. Analysis of population frequencies of haplotypes revealed that the chance of 4 chromosomes having arisen as independent events was 0.016. We conclude that R254X is probably a founder mutation in Chinese. Other previously reported mutations found in the Japanese population were also screening in 250 control samples but no carrier was identified, indicating that they were either very rare or not present in Southern Chinese.     

Metformin use and the risks of herpes zoster and postherpetic neuralgia in patients with type 2 diabetes

Herpes zoster and postherpetic neuralgia cause substantial pain in patients. Persons with type 2 diabetes (T2D) are prone to zoster infection and postherpetic neuralgia due to compromised immunity. We conducted this study to evaluate the risks of herpes zoster and postherpetic neuralgia between metformin users and nonusers. Propensity score matching was utilized to select 47 472 pairs of metformin users and nonusers from Taiwan's National Health Insurance Research Database between January 1, 2000, and December 31, 2017. The Cox proportional hazards models were used for comparing the risks of herpes zoster and postherpetic neuralgia between metformin users and nonusers in patients with T2D. Compared with no-use of metformin, the adjusted hazard ratios (95% confidence interval) for metformin use in herpes zoster and postherpetic neuralgia were 0.70 (0.66, 0.75) and 0.510 (0.39, 0.68), respectively. A higher cumulative dose of metformin had further lower risks of herpes zoster and postherpetic neuralgia than metformin no-use. This nationwide cohort study demonstrated that metformin use was associated with a significantly lower risk of herpes zoster and postherpetic neuralgia than metformin no-use. Moreover, a higher cumulative dose of metformin was associated with further lower risks of these outcomes.     

Staging work-up and post-treatment surveillance of patients with melanoma

Strategies based on evidence are required to accurately stage and effectively follow patients with melanoma. The goal of staging is to define the extent of disease at the time of presentation to direct and assign prognosis. Patient surveillance is performed to assess treatment results and detect recurrences amenable to further treatment. Staging and surveillance require careful use of resources to be cost effective. This article addresses preoperative staging and post-treatment surveillance of patients with melanoma and outlines a method of follow-up based on available data.     

Systemic chemotherapy

The main use of systemic chemotherapy in metastatic melanoma remains palliative. Dacarbazine (dimethyl-1-triazeno imidazole-4-carboxamide [DTIC]) is the standard chemotherapy agent for advanced disease. The combination chemotherapy and biochemotherapy regimens have achieved higher response rates, but have not led to durable remission or improved survival. The field of systemic therapy remains in need of a more effective and less toxic treatment strategy.     

A comparison of the effects of different degrees of zona pellucida damage followed by cryopreservation on the postthaw development of mouse embryos

Purpose: The totally intact zona pellucida is not essential for the development of embryos. It is still unclear how much effect the degree of damages to the zona pellucida will have on the developmental potential of postthaw embryos after cryopreservation. We compared the developmental potential of cryopreserved mouse embryos after induction of two degrees of mechanical damage to the zonae pellucidae by micromanipulation. Methods: In experiment I, the development of 124 cryopreserved ICR mouse embryos to the blastocyst stage after zona pellucida penetration of two-cell embryos as in the procedures of subzonal sperm insertion (SUZI) was compared with the development of zona-intact cryopreserved embryos. In experiment II, the zonae pellucidae of 93 two-cell mouse embryos were dissected as in the procedures of partial zonal dissection (PZD), following which the embryos were frozen. This postthaw development was also compared with that of zona-intact two-cell cryopreserved embryos. All the embryos were thawed and cultured to the blastocyst stage. Additional controls were provided by culturing zonaintact and zona-penetrated or zona-dissected embryos without cryopreservation. Results: The development of unfrozen mouse embryos was not affected by either zona penetration (P=0.433) or zona dissection (P=0.659). The developmental potential of cryopreserved mouse embryos was significantly affected after zona dissection (blastocyst rate, 31% ZD vs 72%, control; P<0.001) but not after zona penetration (blastocyst rate, 59% ZP vs 64% control; P=0.441). Conclusions: The quality of cryopreserved embryos was affected by a large hole on the zona pellucida created by zona dissection but not by simple zona penetration.     

Impacts of early insulin treatment vs glimepiride in diabetic patients with background metformin therapy: A nationwide retrospective cohort study

Type 2 diabetes mellitus (T2DM) is a progressive disease. After metformin failure, the addition of insulin or sulfonylureas might increase the risk of hypoglycemia and cardiovascular (CV) morbidity. Here, the risk of all-cause mortality was compared between early insulin treatment and glimepiride use in T2DM patients with background metformin therapy.We conducted a 9-year retrospective cohort study from the population-based National Health Insurance Research Database in Taiwan. A total of 2054 patients with T2DM under insulin or glimepiride treatment were enrolled during 2004 to 2012. Overall event rates of all-cause mortality were compared between 1027 insulin users and 1027 matched glimepiride users.After the propensity score matching, the mortality rates were 72.5 and 4.42 per 1000 person-years for insulin users and glimepiride users. The adjusted hazard ratio of mortality was 14.47 (95% CI: 8.64–24.24; P value <.001) as insulin compared with glimepiride users. The insulin users had significantly higher risk of CV death (adjusted hazard ratio 7.95, 95% CI 1.65–38.3, P = .01) and noncardiovascular death (adjusted hazard ratio 14.9, 95% CI 8.4–26.3, P < .001).The nationwide study demonstrated that metformin plus insulin therapy was associated with higher risk of all-cause mortality.