Sudhanshu P. Raikwar

Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration

Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson’s disease (PD), Alzheimer’s disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1–42 (Aβ1–42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can also enter into the peripheral system through defective BBB, recruit immune cells into the brain, and exacerbate neuroinflammation. We suggest that mast cell-associated inflammatory mediators from systemic inflammation and brain could augment neuroinflammation and neurodegeneration in the brain. This review article addresses the role of some atypical inflammatory mediators that are associated with mast cell inflammation and their activation of glial cells to induce neurodegeneration.

Human iPS cell-derived insulin producing cells form vascularized organoids under the kidney capsules of diabetic mice.

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.

Insulin producing cells derived from embryonic stem cells: Are we there yet?†

Derivation of insulin producing cells (IPCs) from embryonic stem (ES) cells provides a potentially innovative form of treatment for type 1 diabetes. Here, we discuss the current state of the art, unique challenges, and future directions on generating IPCs. J. Cell. Physiol. 218: 256–263, 2009. Published 2008 Wiley‐Liss, Inc.

Transcriptional targeting modalities in breast cancer gene therapy using adenovirus vectors controlled by α-lactalbumin promoter

The breast-specific antigen α-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human α-lactalbumin promoter, we investigated the activity of a 762-bp human α-lactalbumin promoter. α-Lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer–restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of α-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single α-lactalbumin promoter. Both breast cancer–restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support α-lactalbumin promoter. AdE1aALAE1b showed better breast cancer–restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with α-lactalbumin promoter controlling both E1a and E1b gene expression is superior to α-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that α-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that α-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer. [Mol Cancer Ther 2005;4(12):1850–9]

Characterization of Intestinal and Pancreatic Dysfunction in VPAC1-Null Mutant Mouse

Objectives: These studies examined the effect of homozygous deletion of vasoactive intestinal peptide receptor type 1 (VPAC1) on development and function of intestines and pancreas. Methods: Genetically engineered VPAC1-null mutant mice were monitored for growth, development, and glucose homeostasis. Expression of VPAC1 was examined during embryonic development using VPAC1 promoter-driven &bgr;-galactosidase transgenic mice. Results: Homozygous deletion of VPAC1 resulted in fetal, neonatal, and postweaning death owing to failure to thrive, intestinal obstruction, and hypoglycemia. Histological findings demonstrated disorganized hyperproliferation of intestinal epithelial cells with mucus deposition and bowel wall thickening. The pancreas demonstrated small dysmorphic islets of Langerhans containing &agr;, &bgr;, and &dgr; cells. Expression of a VPAC1 promoter-driven transgene was observed in E12.5 and E14.5 intestinal epithelial and pancreatic endocrine cells. Vasoactive intestinal peptide receptor type 1-null mutant animals had lower baseline blood glucose levels compared to both heterozygous and wild-type littermates. Vasoactive intestinal peptide receptor type 1-deficient mice responded to oral glucose challenge with normal rise in blood glucose followed by rapid hypoglycemia and failure to restore baseline glucose levels. Insulin challenge resulted in profound hypoglycemia and inadequate glucose homeostasis in VPAC1-null mutant animals. Conclusions: These observations support a role for VPAC1 during embryonic and neonatal development of intestines and endocrine pancreas.

Combination therapy of androgen-independent prostate cancer using a prostate restricted replicative adenovirus and a replication-defective adenovirus encoding human endostatin-angiostatin fusion gene

Although prostate-restricted replicative adenovirus has exhibited significant antitumor efficacy in preclinical studies, it is necessary to develop more potent adenoviruses for prostate cancer gene therapy. We evaluated the synergistic killing effect of prostate-restricted replicative adenovirus and AdEndoAngio, a replication-defective adenovirus expressing the endostatin-angiostatin fusion protein (EndoAngio). When coadministered with AdEndoAngio, prostate-restricted replicative adenovirus significantly elevated EndoAngio expression, suggesting that AdEndoAngio coreplicates with prostate-restricted replicative adenovirus. Conditioned medium from prostate cancer cells infected by prostate-restricted replicative adenovirus plus AdEndoAngio inhibited the growth, tubular network formation, and migration of human umbilical vein endothelial cells better than conditioned medium from prostate cancer cells infected by AdEndoAngio alone. Furthermore, in vivo animal studies showed that the coadministration of prostate-restricted replicative adenovirus plus AdEndoAngio resulted in the complete regression of seven out of eight treated androgen-independent CWR22rv tumors, with a tumor nodule maintaining a small size for 14 weeks. The residual single tumor exhibited extreme pathologic features together with more endostatin-reactive antibody-labeled tumor cells and fewer CD31-reactive antibody-labeled capillaries than the AdEndoAngio-treated tumors. These results show that combination therapy using prostate-restricted replicative adenovirus together with antiangiogenic therapy has more potent antitumor effects and advantages than single prostate-restricted replicative adenovirus and deserves more extensive investigation. [Mol Cancer Ther 2006;5(3):676–84]

An improved total synthesis of PET HSV-tk gene expression imaging agent 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG)

Abstract An improved total synthesis of [18F]FHPG starting from 1,3‐dibenzyloxy‐2‐propanol and guanine has been developed. [18F]FHPG was prepared by nucleophilic substitution of the appropriate precursor with [18F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified Silica Sep‐Pak solid‐phase extraction (SPE) method in 10–15% radiochemical yield, and 70 min synthesis time from end of bombardment (EOB).

An improved total synthesis of PET HSV-tk gene reporter probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG)

Abstract An improved total synthesis of [18F]FHBG starting from triethyl‐1,1,2‐ethanetricarboxylate and 2‐amino‐6‐chloropurine is reported. [18F]FHBG was prepared by nucleophilic substitution of the appropriate precursor with [18F]KF/Kryptofix 2.2.2 followed by a quick deprotection reaction and purification with a simplified Silica Sep‐Pak solid‐phase extraction (SPE) method in 20–25% radiochemical yield.

Cross-Talk between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

Parkinson’s disease (PD) is a progressive movement disorder characterized by neuroinflammation and dopaminergic neurodegeneration in the brain. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces the release of inflammatory mediators from glial cells and neurons. Glia maturation factor (GMF), a brain proinflammatory protein, MPP+, and mast cell-derived inflammatory mediators induce neurodegeneration which eventually leads to PD. However, the precise mechanisms underlying interaction between glial cells, neurons and mast cells in PD still remain elusive. In the present study, mouse bone marrow-derived mast cells (BMMCs) and mouse fetal brain-derived mixed glia/neurons, astrocytes and neurons were incubated with MPP+, GMF and mast cell-derived inflammatory mediators mouse mast cell protease-6 (MMCP-6), MMCP-7 or tryptase/brain-specific serine protease-4 (tryptase/BSSP-4). Inflammatory mediators released from these cells in the culture medium were quantitated by enzyme-linked immunosorbent assay. Neurodegeneration was quantified by measuring total neurite outgrowth following microtubule-associated protein-2 immunocytochemistry. MPP+-induced significant neurodegeneration with reduced total neurite outgrowth. MPP+induced the release of tryptase/BSSP-4 from the mouse mast cells, and tryptase/BSSP-4 induced chemokine (C-C motif) ligand 2 (CCL2) release from astrocytes and glia/neurons. Overall our results suggest that MPP+, GMF, MMCP-6 or MMCP-7 stimulate glia/neurons, astrocytes or neurons to release CCL2 and matrix metalloproteinase-3. Additionally, CD40L expression is increased in BMMCs after incubation with MPP+ in a co-culture system consisting of BMMCs and glia/neurons. We propose that mast cell interaction with glial cells and neurons during neuroinflammation can be explored as a new therapeutic target for PD.

Real-Time Non-invasive Imaging of ES Cell-Derived Insulin Producing Cells

ES cells are a potential source for insulin producing cells (IPCs). However, two major handicaps are establishing reliable differentiation protocols and the lack of imaging techniques that allow monitoring of these cells post-transplantation. Here, we describe a new approach for monitoring the in vitro differentiation and real-time, non-invasive imaging of ES cell-derived IPCs in vivo. ES cells were molecularly engineered so that the rat insulin promoter (RIP) driven luciferase (Luc) expression was specifically turned on and up regulated following their differentiation into IPCs. The rationale underlying this approach is that the transcriptional activation of RIP leads to Luc expression in IPCs providing an extremely sensitive reporter for monitoring the earliest differentiation events in real-time both in vitro and in vivo.