L-Asparaginase delivered by Salmonella typhimurium suppresses solid tumors

Bacteria can be engineered to deliver anticancer proteins to tumors via a controlled expression system that maximizes the concentration of the therapeutic agent in the tumor. L-asparaginase (L-ASNase), which primarily converts asparagine to aspartate, is an anticancer protein used to treat acute lymphoblastic leukemia. In this study, Salmonellae were engineered to express L-ASNase selectively within tumor tissues using the inducible araBAD promoter system of Escherichia coli. Antitumor efficacy of the engineered bacteria was demonstrated in vivo in solid malignancies. This result demonstrates the merit of bacteria as cancer drug delivery vehicles to administer cancer-starving proteins such as L-ASNase to be effective selectively within the microenvironment of cancer tissue

Microvasculature remodeling in the mouse lower gut during inflammaging

Inflammaging is defined as low-grade, chronic, systemic inflammation in aging, in the absence of overt infection. Age-associated deterioration of gastrointestinal function could be ascribed to the inflammaging, although evidence is yet to emerge. Here we show that microvessels in aging mouse intestine were progressively deprived of supportive structures, microvessel-associated pericytes and adherens junction protein vascular endothelial (VE)-cadherin, and became leaky. This alteration was ascribed to up-regulation of angiopoetin-2 in microvascular endothelial cells. Up-regulation of the angiopoietin-2 was by TNF-α, originated from M2-like residential CD206 + macrophages, proportion of which increases as animal ages. It was concluded that antigenic burdens encountered in intestine throughout life create the condition of chronic stage of inflammation, which accumulates M2-like macrophages expressing TNF-α. The TNF-α induces vascular leakage to facilitate recruitment of immune cells into intestine under the chronic inflammatory setting. © Author(s) 2017.1

Label-Free White Blood Cell Classification Using Refractive Index Tomography and Deep Learning

Objective and Impact Statement. We propose a rapid and accurate blood cell identification method exploiting deep learning and label-free refractive index (RI) tomography. Our computational approach that fully utilizes tomographic information of bone marrow (BM) white blood cell (WBC) enables us to not only classify the blood cells with deep learning but also quantitatively study their morphological and biochemical properties for hematology research. Introduction. Conventional methods for examining blood cells, such as blood smear analysis by medical professionals and fluorescence-activated cell sorting, require significant time, costs, and domain knowledge that could affect test results. While label-free imaging techniques that use a specimen’s intrinsic contrast (e.g., multiphoton and Raman microscopy) have been used to characterize blood cells, their imaging procedures and instrumentations are relatively time-consuming and complex. Methods. The RI tomograms of the BM WBCs are acquired via Mach-Zehnder interferometer-based tomographic microscope and classified by a 3D convolutional neural network. We test our deep learning classifier for the four types of bone marrow WBC collected from healthy donors (n=10): monocyte, myelocyte, B lymphocyte, and T lymphocyte. The quantitative parameters of WBC are directly obtained from the tomograms. Results. Our results show >99% accuracy for the binary classification of myeloids and lymphoids and >96% accuracy for the four-type classification of B and T lymphocytes, monocyte, and myelocytes. The feature learning capability of our approach is visualized via an unsupervised dimension reduction technique. Conclusion. We envision that the proposed cell classification framework can be easily integrated into existing blood cell investigation workflows, providing cost-effective and rapid diagnosis for hematologic malignancy

Transcriptional Regulation of the Outer Membrane Protein A in Acinetobacter baumannii

Acinetobacter baumannii is known for its virulence in severely ill, hospitalized patients and for exhibiting multidrug resistance. A. baumannii infection treatment poses a serious problem in clinical environments. The outer membrane protein A (OmpA) of the Acinetobacter genus is involved in bacterial virulence. Regulatory factors of OmpA in the post-transcriptional stage have been previously identified. However, the regulatory factors that act before the transcriptional stage remain unclear. We investigated the A1S_0316 gene that encodes a putative transcription factor for OmpA expression in A. baumannii. A1S_0316 was purified and examined using size-exclusion chromatography, which revealed that it forms an oligomer. The binding affinity of A1S_0316 to the OmpA promoter region was also examined. We compared the binding affinity to the OmpA promotor region between A1S_0316 and the AbH-NS protein. A1S_0316 showed higher binding affinity to the OmpA promotor region than did H-NS. We examined the regulatory effect of these proteins on OmpA expression in A. baumannii using real-time qPCR and various in vitro tools. Our results indicated that A1S_0316 acts as an anti-repressor on the promotor region of the OmpA gene by inhibiting the binding of the AbH-NS protein. This study was the first demonstration of the transcriptional regulation of OmpA expression

Immunohistochemical study on the distribution of phosphorylated extracellular signal-regulated kinase (ERK) in the central nervous system of SOD1G93A transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of phosphorylated extracellular signal-regulated kinases (pERK) in the central nervous system of SOD1(G93A) transgenic mice. In symptomatic transgenic mice, pERK-immunoreactive astrocytes were detected in the spinal cord, brainstem, central gray and cerebellar nuclei. In contrast to symptomatic mice, no pERK-immunoreactive astrocytes were observed in any brain region of wtSOD1 and presymptomatic mice, and the number and intensity of stained neurons were not different at the age of 8 weeks and 13 weeks. Interestingly, region-specific alterations in pERK immunoreactivity were observed in the hippocampal region and cerebellum. These results provide the first evidence that pERK-immunoreactive astrocytes were found in the CNS of SOD1(G93A) transgenic mice after clinical symptoms, showing a possible consequence of the pathological process of ALS. This study has also demonstrated that pERK increases in the hippocampus and cerebellum, suggesting a role of pERK in an abnormality of cognitive and/or motor function in ALS, respectively. However, the mechanisms underlying the increased immunoreactivity for pERK and the functional implications require elucidation

A novel balanced-lethal host-vector system based on glmS.

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number

Immunohistochemical localization of insulin-like growth factor binding protein 2 in the central nervous system of SOD1(G93A) transgenic mice

In the present study, we performed immunohistochemical studies to investigate the changes of insulin-like growth factor binding protein 2 (IGFBP2) in the central nervous system of SOD1(G93A) mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS). Decreased immunoreactivity for IGFBP2 was observed in the cerebral cortex, hippocampus and brainstem of SOD1(G93A) transgenic mice. In the cerebral cortex, the number of IGFBP2-positive cells was decreased in the somatomotor area, somatosensory area, auditory area, visual area, entorhinal area, piriform area and prefrontal area. In the hippocampal formation, IGFBP2 immunoreactivity was significantly decreased in the CA1-3 areas and the dentate gyrus. In the brainstem, few IGFBP2-immunoreactive cells were observed in the medullary and pontine reticular formation, vestibular nucleus, trigeminal motor nucleus, facial nucleus, hypoglossal nucleus and raphe nucleus. In the spinal cord, IGFBP2 immunoreactivity was not significantly decreased in SOD1(G93A) transgenic mice. This study showing decreased IGFBP2 in different brain regions of SOD1(G93A) transgenic mice may provide clues for understanding differential susceptibility of neural structures in ALS.Kang DW, 2008, ANN ANAT, V190, P502, DOI 10.1016/j.aanat.2008.08.001Chesik D, 2007, CYTOKINE GROWTH F R, V18, P267, DOI 10.1016/j.cytogfr.2007.04.001GOODALL EF, 2006, EXPERT REV MOL MED, V8, P1, DOI 10.1017/S1462399406010854WILCZAK N, 2005, ENDOCRIN DEV, V9, P160Pirttila T, 2004, ACTA NEUROL SCAND, V109, P337, DOI 10.1111/j.1600-0404.2004.00223.xEkestern E, 2004, NEURODEGENER DIS, V1, P88, DOI 10.1159/000080049Chung YH, 2003, BRAIN RES, V994, P253, DOI 10.1016/j.brainres.2003.09.047Kaspar BK, 2003, SCIENCE, V301, P839Wilczak N, 2003, LANCET, V361, P1007Chowen JA, 2002, J NEUROENDOCRINOL, V14, P163, DOI 10.1046/j.0007-1331.2001.00758.xCleveland DW, 2001, NAT REV NEUROSCI, V2, P806Rowland LP, 2001, NEW ENGL J MED, V344, P1688Wang JM, 2000, BRAIN RES, V859, P381Torres-Aleman I, 1998, NEUROLOGY, V50, P772CLEMMONS DR, 1997, CYTOKINE GROWTH F R, V8, P45DErcole AJ, 1996, MOL NEUROBIOL, V13, P227Dore S, 1996, MOL BRAIN RES, V41, P128CollettSolberg PF, 1996, ENDOCRIN METAB CLIN, V25, P591JONES JI, 1995, ENDOCR REV, V16, P3LOGAN A, 1994, ENDOCRINOLOGY, V135, P2255GURNEY ME, 1994, SCIENCE, V264, P1772KERKHOFF H, 1994, ACTA NEUROPATHOL, V87, P411

Age-related changes in glycogen synthase kinase 3beta (GSK3beta) immunoreactivity in the central nervous system of rats

Although glycogen synthase kinase 3beta (GSK3beta) is emerging as a prominent drug target in the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and stroke, very little is known about age-related changes in GSK3beta expression and GSK3beta phosphorylation. Therefore, we examined age-related changes in immunoreactivities for GSK3beta and phosphorylated GSK3beta (pGSK3beta) in the central nervous system. In aged rats, there were significant increases in GSK3beta immunoreactivity in the cell bodies and processes of pyramidal cells in most cortical regions. GSK3beta immunoreactivity was also significantly increased in the pyramidal layer of CA1-3 regions, and the granule cell layer of dentate gyrus. Age-related increases were prominent in lateral septal nuclei, compared to the medial septal nuclei. Interestingly, both GSK3beta and pGSK3beta was increased in the prefrontal cortex, while GSK3beta and pGSK3beta was differentially localized in the cerebellar cortex. The first demonstration of age-related alterations in immunoreactivities for GSK3beta and pGSK3beta in the basal forebrain area and cholinergic projection targets may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases including AD

Anti-tumoral effect of the mitochondrial target domain of Noxa delivered by an engineered Salmonella typhimurium.

Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD) as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP) derived from a voltage-gated potassium channel (Kv2.1). The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD , a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma

Maintenance of <i>pLux</i> in <i>E. coli</i> proliferating in tumor tissue.

<p>(A) GlmS⁻ mutant bacteria (CKS1001) and parental wild type <i>E. coli</i> (CH1436) carrying <i>^E.cGlmS⁺ pLux</i> were injected into CT26 tumor-bearing mice through the tail vein (1×10⁸ CFU). Bioluminescent signals from <i>pLux</i> were monitored at the indicated times using an <i>in vivo</i> imaging system. (B) The photon intensity of the tumor region was plotted as a function of time for GlmS⁻ mutant and wild-type <i>E. coli</i>. The region of interest (ROI) was selected manually over the tumor region and the area was kept constant. Photon intensity was recorded as the maximum intensity (photons s⁻¹ cm⁻² sr⁻¹) within the ROI. Data represent the means and SEM of three independent experiments. (C) Tumor tissues were sampled on the indicated days. The total number of bacteria and the number of bacteria carrying <i>^E.cGlmS⁺ pLux</i> was assessed by CFU determination.</p