Perlecan controls neurogenesis in the developing telencephalon

Background  

Perlecan is a proteoglycan expressed in the basal lamina of the neuroepithelium during development. Perlecan absence does not impair basal lamina assembly, although in the 55% of the mutants early disruptions of this lamina conducts to exencephaly, impairing brain development. The rest of perlecan-null brains complete its prenatal development, maintain basal lamina continuity interrupted by some isolated ectopias, and are microcephalic. Microcephaly consists of thinner cerebral walls and underdeveloped ganglionic eminences. We have studied the mechanisms that generate brain atrophy in telencephalic areas where basal lamina is intact.     

Tumorigenic Potential of Olfactory Bulb-Derived Human Adult Neural Stem Cells Associates with Activation of TERT and NOTCH1

Background

Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation.

Methodology/Principal Findings

Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with mainteinance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity.

Conclusions/Significance

Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.     

CADM1 Is a Key Receptor Mediating Human Mast Cell Adhesion to Human Lung Fibroblasts and Airway Smooth Muscle Cells

Background

Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.

Objective

In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.

Methods

CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.

Results

Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.

Conclusion and Clinical Relevance

Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.     

The mycobacterial DNA-binding protein 1 (MDP1) from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG influences various growth characteristics

Background  

Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene.     

Inhibition of Human MCF-7 Breast Cancer Cells and HT-29 Colon Cancer Cells by Rice-Produced Recombinant Human Insulin-Like Growth Binding Protein-3 (rhIGFBP-3)

Background

Insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I) to form a complex (IGF-I/IGFBP-3) that can treat growth hormone insensitivity syndrome (GHIS) and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells.

Methodology/Principal Findings

We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3) in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL) were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively) and HT-29 colon cancer cells (65.14 ±3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively) when compared with wild type rice.

Conclusion/Significance

These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.     

Conflicting phylogenetic signals in the SlX1/Y1 gene in Silene

Background  

Increasing evidence from DNA sequence data has revealed that phylogenies based on different genes may drastically differ from each other. This may be due to either inter- or intralineage processes, or to methodological or stochastic errors. Here we investigate a spectacular case where two parts of the same gene (SlX1/Y1) show conflicting phylogenies within Silene (Caryophyllaceae). SlX1 and SlY1 are sex-linked genes on the sex chromosomes of dioecious members of Silene sect. Elisanthe.     

Enhancing engineered vascular networks in vitro and in vivo: The effects of IGF1 on vascular development and durability

Objectives

Creation of functional, durable vasculature remains an important goal within the field of regenerative medicine. Engineered biological vasculature has the potential to restore or improve human tissue function. We hypothesized that the pleotropic effects of insulin‐like growth factor 1 (IGF1) would enhance the engineering of capillary‐like vasculature.

Materials and methods

The impact of IGF1 upon vasculogenesis was examined in in vitro cultures for a period of up to 40 days and as subcutaneous implants within immunodeficient mice. Co‐cultures of human umbilical vein endothelial cells and human bone marrow‐derived mesenchymal stem cells in collagen‐fibronectin hydrogels were supplemented with either recombinant IGF1 protein or genetically engineered cells to provide sustained IGF1. Morphometric analysis was performed on the vascular networks that formed in four concentrations of IGF1.

Results

IGF1 supplementation significantly enhanced de novo vasculogenesis both in vitro and in vivo. Effects were long‐term as they lasted the duration of the study period, and included network density, vessel length, and diameter. Bifurcation density was not affected. However, the highest concentrations of IGF1 tested were either ineffective or even deleterious. Sustained IGF1 delivery was required in vivo as the inclusion of recombinant IGF1 protein had minimal impact.

Conclusion

IGF1 supplementation can be used to produce neovasculature with significantly enhanced network density and durability. Its use is a promising methodology for engineering de novo vasculature to support regeneration of functional tissue.     

Human TGFalpha-derived peptide TGFalphaL3 fused with superantigen for immunotherapy of EGFR-expressing tumours

Background  

Monoclonal antibodies have been employed as targeting molecules of superantigen for the preclinical treatment of a variety of tumours. However, other targeting molecules, such as tumour-related ligands or peptides, are less exploited. Here, we tested other targeting molecules by genetically fusing the third loop of transforming growth factor alpha (TGFalphaL3) to mutant staphylococcal enterotoxin A (SEA_D227A).     

Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

Background

Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.

Methodology/Principal Findings

The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.

Conclusions/Significance

The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.     

Recombinant Human Endostatin Endostar Suppresses Angiogenesis and Lymphangiogenesis of Malignant Pleural Effusion in Mice

Background

Malignant pleural effusion (MPE) is a common complication of lung cancer. One widely used treatment for MPE is Endostar, a recombined humanized endostatin based treatment. However, the mechanism of this treatment is still unclear. The aim of this study was to investigate the effects of Endostar in mice with MPE.

Methods and Materials

Lewis lung carcinoma (LLC) cell line expressing enhanced green fluorescent protein (EGFP) was injected into pleural cavity to establish MPE mice model. Mice were randomly divided into four groups. High dose of Endostar (30 mg/kg), low dose of Endostar (8 mg/kg), normal saline, or Bevacizumab (5 mg/kg) was respectively injected into pleural cavity three times with 3-day interval in each group. Transverse computed tomography (CT) was performed to observe pleural fluid formation 14 days after LLC cells injection. Mice were anesthetized and sacrificed 3 days after final administration. The volume of pleural effusion n was measured using 1 ml syringe. Micro blood vessel density (MVD), Lymphatic micro vessel density (LMVD), the expression level of vascular endothelial growth factor A (VEGF-A) and VEGF-C were observed by immunohistochemistry (IHC) staining.

Results

The volume of pleural effusion as well as the number of pleural tumor foci, MVD and the expression of VEGF-A were significantly reduced in high dose of Endostar treat group. More importantly, LMVD and the expression of VEGF-C were markedly lower in treat group than those in the other three control groups.

Conclusion

Our work demonstrated that Endostar played an efficient anti-cancer role in MPE through its suppressive effect on angiogenesis and lymphangiogenesis, which provided a certain theoretical basis for the effectiveness of Endostar on the MPE treatment.     

Health-Related Quality of Life of Young Adults Treated with Recombinant Human Growth Hormone during Childhood

Background

Since recombinant human growth hormone (rhGH) became available in 1985, the spectrum of indications has broadened and the number of treated patients increased. However, long-term health-related quality of life (HRQoL) after childhood rhGH treatment has rarely been documented. We assessed HRQoL and its determinants in young adults treated with rhGH during childhood.

Methodology/Principal Findings

For this study, we retrospectively identified former rhGH patients in 11 centers of paediatric endocrinology, including university hospitals and private practices. We sent a questionnaire to all patients treated with rhGH for any diagnosis, who were older than 18 years, and who resided in Switzerland at time of the survey. Three hundred participants (58% of 514 eligible) returned the questionnaire. Mean age was 23 years; 56% were women; 43% had isolated growth hormone deficiency, or idiopathic short stature; 43% had associated diseases or syndromes, and 14% had growth hormone deficiency after childhood cancer. Swiss siblings of childhood cancer survivors and the German norm population served as comparison groups. HRQoL was assessed using the Short Form-36. We found that the Physical Component Summary of healthy patients with isolated growth hormone deficiency or idiopathic short stature resembled that of the control group (53.8 vs. 54.9). Patients with associated diseases or syndromes scored slightly lower (52.5), and former cancer patients scored lowest (42.6). The Mental Component Summary was similar for all groups. Lower Physical Component Summary was associated with lower educational level (coeff. -1.9). Final height was not associated with HRQoL.

Conclusions/Significance

In conclusion, HRQoL after treatment with rhGH in childhood depended mainly on the underlying indication for rhGH treatment. Patients with isolated growth hormone deficiency/idiopathic short stature or patients with associated diseases or syndromes had HRQoL comparable to peers. Patients with growth hormone deficiency after childhood cancer were at high risk for lower HRQoL. This reflects the general impaired health of this vulnerable group, which needs long-term follow-up.     

Generation,Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

Background

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.

Methodology/Principal Findings

We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.

Conclusions/Significance

Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.     

BYPASS1: synthesis of the mobile root-derived signal requires active root growth and arrests early leaf development

Background  

The Arabidopsis bypass1 (bps1) mutant root produces a biologically active mobile compound that induces shoot growth arrest. However it is unknown whether the root retains the capacity to synthesize the mobile compound, or if only shoots of young seedlings are sensitive. It is also unknown how this compound induces arrest of shoot growth. This study investigated both of these questions using genetic, inhibitor, reporter gene, and morphological approaches.     

Regulation of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> body size and male tail development by the novel gene <Emphasis Type="Italic">lon-8</Emphasis>

Background  

In C. elegans and other nematode species, body size is determined by the composition of the extracellular cuticle as well as by the nuclear DNA content of the underlying hypodermis. Mutants that are defective in these processes can exhibit either a short or a long body size phenotype. Several mutations that give a long body size (Lon) phenotype have been characterized and found to be regulated by the DBL-1/TGF-β pathway, that controls post-embryonic growth and male tail development.     

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

Background  

Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs).     

Mechanism of G1 arrest in the <Emphasis Type="Italic">Drosophila</Emphasis>eye imaginal disc

Background  

Most differentiating cells are arrested in G1-phase of the cell cycle and this proliferative quiescence appears important to allow differentiation programmes to be executed. An example occurs in the Drosophila eye imaginal disc, where all cells are synchronized and arrested in G1 phase prior to making a fate choice either to initiate the first round of photoreceptor differentiation or to re-enter one terminal mitosis.     

Mitochondrial Function in Human Neuroblastoma Cells Is Up-Regulated and Protected by NQO1, a Plasma Membrane Redox Enzyme

Background

Recent findings suggest that NADH-dependent enzymes of the plasma membrane redox system (PMRS) play roles in the maintenance of cell bioenergetics and oxidative state. Neurons and tumor cells exhibit differential vulnerability to oxidative and metabolic stress, with important implications for the development of therapeutic interventions that promote either cell survival (neurons) or death (cancer cells).

Methods and Findings

Here we used human neuroblastoma cells with low or high levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS modulates mitochondrial functions and cell survival. Cells with elevated NQO1 levels exhibited higher levels of oxygen consumption and ATP production, and lower production of reactive oxygen species. Cells overexpressing NQO1 were more resistant to being damaged by the mitochondrial toxins rotenone and antimycin A, and exhibited less oxidative/nitrative damage and less apoptotic cell death. Cells with basal levels of NQO1 resulted in increased oxidative damage to proteins and cellular vulnerability to mitochondrial toxins. Thus, mitochondrial functions are enhanced and oxidative stress is reduced as a result of elevated PMRS activity, enabling cells to maintain redox homeostasis under conditions of metabolic and energetic stress.

Conclusion

These findings suggest that NQO1 is a potential target for the development of therapeutic agents for either preventing neuronal degeneration or promoting the death of neural tumor cells.     

Genomic and proteomic profiling II: Comparative assessment of gene expression profiles in leiomyomas, keloids, and surgically-induced scars

Background  

Leiomyoma have often been compared to keloids because of their fibrotic characteristic and higher rate of occurrence among African Americans as compared to other ethnic groups. To evaluate such a correlation at molecular level this study comparatively analyzed leiomyomas with keloids, surgical scars and peritoneal adhesions to identify genes that are either commonly and/or individually distinguish these fibrotic disorders despite differences in the nature of their development and growth.     

IL-1 does not reverse the anti-proliferative effect of aspirin in breast cancer cells

Objectives

Interleukin- 1 (IL-1) is a multifunctional proinflammatory cytokine. There have been studies suggesting a role in affecting growth and invasiveness of malignant breast cells by either blocking or stimulating growth of cultured MCF-7 breast cancer cells. This effect may be mediated by induction of COX-2. Aspirin is an inhibitor of COX-2 and has been implicated, with other non-steroidal anti-inflammatory drugs (NSAIDS) in prevention and treatment of breast cancer. In this study the in vitro effects of IL-1 and aspirin on growth of MCF-7 human breast cancer cells was examined.

Methods

MCF-7 cells were treated with various concentrations of IL-1 and aspirin alone and in combination. Cell growth was assessed by cell number measurement.

Results

Aspirin significantly decreased growth rate in a dose-dependant manner, alone and as a combined treatment with IL-1 with a maximum reduction in growth rate at 300 mg/ml (P < 0.05). Treatment with IL-1 alone showed no significant effect on growth rate of MCF-7 cells (P > 0.05).

Conclusion

This study confirms that aspirin suppresses the proliferation rate of MCF-7 cells both as a single agent and in combination with IL-1. It also suggests that IL-1 alone does not stimulate or inhibit growth of MCF-7 cells.