Monocyte Trafficking,Engraftment, and Delivery of Nanoparticles and an Exogenous Gene into the Acutely Inflamed Brain Tissue – Evaluations on Monocyte-Based Delivery System for the Central Nervous System

The ability of monocytes and monocyte-derived macrophages (MDM) to travel towards chemotactic gradient, traverse tissue barriers, and accumulate precisely at diseased sites makes them attractive candidates as drug carriers and therapeutic gene delivery vehicles targeting the brain, where treatments are often hampered by the blockade of the blood brain barrier (BBB). This study was designed to fully establish an optimized cell-based delivery system using monocytes and MDM, by evaluating their homing efficiency, engraftment potential, as well as carriage and delivery ability to transport nano-scaled particles and exogenous genes into the brain, following the non-invasive intravenous (IV) cell adoptive transfer in an acute neuroinflammation mouse model induced by intracranial injection of Escherichia coli lipopolysaccharides. We demonstrated that freshly isolated monocytes had superior inflamed-brain homing ability over MDM cultured in the presence of macrophage colony stimulating factor. In addition, brain trafficking of IV infused monocytes was positively correlated with the number of adoptive transferred cells, and could be further enhanced by transient disruption of the BBB with IV administration of Mannitol, Bradykinin or Serotonin right before cell infusion. A small portion of transmigrated cells was detected to differentiate into IBA-1 positive cells with microglia morphology in the brain. Finally, with the use of superparamagnetic iron oxide nanoparticles SHP30, the ability of nanoscale agent-carriage monocytes to enter the inflamed brain region was validated. In addition, lentiviral vector DHIV-101 was used to introduce green fluorescent protein (GFP) gene into monocytes, and the exogenous GFP gene was detected in the brain at 48 hours following IV infusion of the transduced monocytes. All together, our study has set up the optimized conditions for the more-in-depth tests and development of monocyte-mediated delivery, and our data supported the notion to use monocytes as a non-invasive cell-based delivery system for the brain.     

Prepubertal treatment with estrogen or testosterone precipitates the loss of regular estrous cyclicity and normal gonadotropin secretion in adult female rats

To examine the effects of prepubertal steroid environment on subsequent estrous cyclicity and gonadotropin secretion, Silastic implants containing 25, 50 or 100% 17 beta-estradiol (E2;n=34), 50% diethylstilbestrol (DES; n=16) or 50% testosterone (T; n=17) were placed into female rats at 12 days of age and removed on the day of vaginal opening (18-24 days of age). At 80 days of age, the percentages of regularly cycling females in the E2-(three groups combined), DES- and T-implanted groups were 59%, 0% and 59%, respectively. By 110 days of age, the percentages were reduced to 24%, 0% and 0%, and at 140 days of age 6%, 0% and 0%, respectively. Many of these females displayed irregular estrous cycles followed by a persistent estrous (PE) state. By contrast, 89% of the control females (blank implants or no implant) maintained regular cycles up to 140 days of age. At 150 days of age, an i.p. injection of gonadotropin-releasing hormone (GnRH; 100 ng/100 g BW) markedly increased serum luteinizing hormone (LH), but not follicle-stimulating hormone (FSH), in intact PE females treated prepubertally with E2 implants. After the test with GnRH, PE rats were ovariectomized (OVX). Thirty days after OVX, similar GnRH administration significantly increased serum levels of both LH and FSH, but these responses were significantly (P less than 0.01) reduced when compared with those in OVX controls. Progesterone administration to estradiol benzoate-primed, acutely (3 days) OVX, or long-term (43 days) OVX-PE females did not increase LH or FSH release. These results indicate that exposure to exogenous estrogen or T prior to puberty precipitates the decline in estrous cyclicity associated with the loss of gonadotropin surge response, presumably due to an alteration in hypothalamic GnRH release.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

Myc-Induced Liver Tumors in Transgenic Zebrafish Can Regress in tp53 Null Mutation

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53^-/- mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53^-/- mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53.     

Large Cohort Screening of G6PD Deficiency and the Mutational Spectrum in the Dongguan District in Southern China

Background

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymatic disorder of the erythrocytes that affects 400 million people worldwide. We developed a PCR-reverse dot blot (RDB) assay to screen twenty genotypes of seventeen Chinese G6PD mutations and investigate the spectrum of G6PD deficiency mutations in Dongguan District, Guangdong Province, in southern China.

Method

The PCR-RDB assay consists of multiplex PCR amplification of seven fragments in the G6PD target sequence of wild-type and mutant genomic DNA samples followed by hybridization to a test strip containing allele-specific oligonucleotide probes. A total of 16,464 individuals were analyzed by a combination of phenotypic screening and genotypic detection using the PCR-RDB assay and DNA sequence analysis.

Results

The PCR-RDB assay had a detection rate of 98.1%, which was validated by direct sequencing in a blind study with 100% concordance. The G6PD deficiency incidence rate in Dongguan District is 4.08%. Thirty-two genotypes from 469 individuals were found. The two most common variants were c.1376G>T and c.1388G>A, followed by c.95A>G, c.871G>A, c.392G>T, and c.1024 C>T. In addition, two rare mutations (c.703C>A and c.406C>T) were detected by DNA sequencing analysis. In our study, 65 cases harbored the C1311T/IVS polymorphism and 67 cases were homozygote.

Conclusion

The PCR-RDB assay we established is a reliable and effective method for screening G6PD mutations in the Chinese population. Data on the spectrum of mutations in the Dongguan District is beneficial to the clinical diagnosis and prevention of G6PD deficiency.     

Gender specific effect of LIPC C‐514T polymorphism on obesity and relationship with plasma lipid levels in Chinese children

Hepatic lipase (LIPC) is a key rate‐limiting enzyme in lipoprotein catabolism pathways involved in the development of obesity. The C‐514T polymorphism in the promoter region is associated with decreased LIPC activity. We performed a case‐controlled study (850 obese children and 2119 controls) and evaluated the association between LIPC C‐514T polymorphism, obesity and plasma lipid profile in Chinese children and adolescents. Additionally, we conducted a meta‐analysis of all results from published studies as well as our own data. A significant association between the polymorphism and obesity is observed in boys (P = 0.042), but not in girls. And we observed a significant relationship of the polymorphism with total cholesterol (TC) and high density lipoprotein cholesterol (HDL‐C) independent of obesity in boys. The T allele carriers have higher levels of low density lipoprotein cholesterol (LDL‐C) in obese boys, and triglyceride (TG), TC and LDL‐C in non‐obese girls (all P < 0.05). In the meta‐analysis, under dominant model the T allele increased body mass index (BMI) level in boys, while it decreased BMI in girls, and increased the levels of TC both in the overall and subgroups, TG and HDL‐C in the overall and boys, and LDL‐C in the overall (all P < 0.05). Our results suggest that the T allele might carry an increased risk of obesity in Chinese boys. The meta‐analysis suggests that T allele acts as a risk allele for higher BMI levels in male childhood, while it is a protective allele in female childhood. And the polymorphism is associated with the levels of plasma lipids, which may be modulated by obesity and gender.     

Ornamentation of achene silica walls and its contribution to the systematics of <Emphasis Type="Italic">Eleocharis</Emphasis> (Cyperaceae)

Micromorphology of the achene surface of 26 Brazilian species of Eleocharis was studied by scanning electron microscopy in order to evaluate its usefulness in the taxonomy of the genus. The results point out two patterns of cell organization according to silica structures of achenes. The first corresponds to a group of species (group A) that have small to medium cells arranged vertically. The second is found in those species (group B) with medium to large cells arranged horizontally. These data were useful in separating species of Eleocharis subgenus Scirpidium and E. subgenus Limnochloa (group B) from E. subgenus Eleocharis (group A). However, group A shows considerable variation in silica wall arrangement. Eleocharis squamigera, previously considered as part of E. subgenus Eleocharis, shows features rather similar to those of Scirpidium, confirming recent phylogenies. The subgenus Limnochloa was clearly distinguished from others by achenes with large cells (over 55 μm width), presence of crenate or repand anticlinal walls, and some orifices near the wall in some species. The silica wall ornamentation seems to be a useful morphological tool for studying relationships between subgenera and distinguishes Limnochloa from the other subgenera.