Receptor-Mediated Oral Delivery of a Bioencapsulated Green Fluorescent Protein Expressed in Transgenic Chloroplasts Into the Mouse Circulatory System

Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins

Central Nervous System Acting Drugs In Treatment Of Migraine Headache

Migraine is a primary headache disorder with an unknown pathophysiology. The growing evidence in recent years indicates migraine being a brain disorder, a sensory dysmodulation, and a system failure of normal sensory processing of the brainstem that involves the vascular tone and pain. At the moment, triptan family and NSAIDs are the first choice drugs for the treatment of acute migraine. There are several prophylactic drugs including the antiepileptic drugs (AEDs), betablockers, and Ca2+ channel blockers that are used for the treatment of migraine. Although many drugs including the triptans, NSAIDs, and others target the peripheral sites of activation, several novel drugs are being developed to target neural sites of action in the central nervous system (CNS). The first trigeminal synapses in the brain stem as well as the ascending and descending pathways and higher brain centers are involved in the transmission of pain and therefore be the main targets of several drugs some of which are in clinical trials. Central sensitization may also aggravate the headache and some drugs tend to alleviate pain by targeting neurotransmitters, receptors, or signalling molecules involved in this phenomenon. This article discusses the CNS acting novel drugs and those that are currently in use for the treatment of migraine. © 2012 Bentham Science Publishers

Pathophysiology of autism spectrum disorders: Revisiting gastrointestinal involvement and immune imbalance

Autism spectrum disorders (ASD) comprise a group of neurodevelopmental abnormalities that begin in early childhood and are characterized by impairment of social communication and behavioral problems including restricted interests and repetitive behaviors. Several genes have been implicated in the pathogenesis of ASD, most of them are involved in neuronal synaptogenesis. A number of environmental factors and associated conditions such as gastrointestinal (GI) abnormalities and immune imbalance have been linked to the pathophysiology of ASD. According to the March 2012 report released by United States Centers for Disease Control and Prevention, the prevalence of ASD has sharply increased during the recent years and one out of 88 children suffers now from ASD symptoms. Although there is a strong genetic base for the disease, several associated factors could have a direct link to the pathogenesis of ASD or act as modifiers of the genes thus aggravating the initial problem. Many children suffering from ASD have GI problems such as abdominal pain, chronic diarrhea, constipation, vomiting, gastroesophageal reflux, and intestinal infections. A number of studies focusing on the intestinal mucosa, its permeability, abnormal gut development, leaky gut, and other GI problem raised many questions but studies were somehow inconclusive and an expert panel of American Academy of Pediatrics has strongly recommended further investigation in these areas. GI tract has a direct connection with the immune system and an imbalanced immune response is usually seen in ASD children. Maternal infection or autoimmune diseases have been suspected. Activation of the immune system during early development may have deleterious effect on various organs including the nervous system. In this review we revisited briefly the GI and immune system abnormalities and neuropeptide imbalance and their role in the pathophysiology of ASD and discussed some future research directions

Receptor-Mediated Oral Delivery Of A Bioencapsulated Green Fluorescent Protein Expressed In Transgenic Chloroplasts Into The Mouse Circulatory System

Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins. © FASEB

Neuropeptides And Other Chemical Mediators, And The Role Of Antiinflammatory Drugs In Primary Headaches

Primary headaches including the migraine, cluster, and tension headaches are common neurological disorders which cause pain and disability to the patients. The pathomechanism of migraine is not very well understood however, current clinical findings indicate a possible primary brain disorder due to activation of the brain and brainstem as triggers for migraine. The headache phase of migraine may be due to activation of the peripheral nerves including the trigeminal nerve and others innervating the cranial blood vessels and release of vasoactive substances including the calcitonin generelated peptides (CGRP), possibly leading to vasodilation and brainstem activation. Several of our studies in an experimental model of pain using electrical stimulation of the trigeminal ganglion in rats focused on various neuropeptides release from the peripheral and central trigeminal nerve terminals, however, clinically only the CGRP in migraine and CGRP and vasoactive intestinal peptide (VIP) in cluster headache were found in patient\u27s blood. Although several drugs are used in the treatment of migraine, the non-steroid anti-inflammatory drugs (NSAIDs) and the triptan family of drugs are the first choice drugs recommended for the treatment of acute migraine headache. Although clinically very few studies detected other vasoactive/inflammatory molecules in the blood of migraine patients, sensitization of peripheral axons can involve many inflammatory mediators affecting the peripheral tissue substrates of pain. Moreover, central sensitization in the trigeminal nucleus can also contribute to additional pain responses. This article reviews neuropeptides and other molecules involved in primary headaches and major drugs proposed for their treatment in recent years. © 2010 Bentham Science Publishers Ltd

Major Neuroanatomical And Neurochemical Substrates Involved In Primary Headaches

Neuroanatomical structures involved in head pain are primarily the sensory distribution of four cranial nerves: the trigeminal-and to a lesser extent, facial, glossopharyngeal, and vagus-as well as the terminations of the upper three cervical nerves.In addition, various pain sensitive cranial structures including the scalp and its blood supply, the head and neck muscles, intracranial and meningeal arteries, and dura mater including the venous sinuses are the major anatomical substrates of various types of headaches. Although brain tumors, different types of hemorrhage, hypertension, and meningitis may present as a headache, the migraine, cluster, and tension headaches are the three major types of primary headaches. Current opinion suggests a primary central nervous system activation may initiate a migraine. Several triggering factors such as disturbances of brain oxygenation and metabolism, alterations in the serotonin levels, low levels of brain tissue magnesium, altered transport of ions across the cell membrane, abnormal mitochondrial energy metabolism, and genetic abnormalities including mutations of the P/Q type calcium channel gene, Na+/K+ pump ATP1A2, or sodium channel Nav1.1 mutations have been linked to the pathogenesis of migraines. Patients with mutations in the calcium channel gene are more sensitive to environmental factors, which results in a wave of cortical spreading depression in the patient after the attack is initiated.Moreover, several recent clinical and diagnostic studies indicate a dysfunction of the brainstem periaqueductal gray matter during migraine, or initiation of migraine by activation of the brainstem including the dorsal rostral and midline pons. Consistent with this, an active locus in the posterior hypothalamus has been implicated in cluster headache (CH). The headache phase involves the activation of the trigeminovascular system and possibly dilatation of the cranial blood vessels presumably mediated by the release of vasoactive substances and neuropeptides including the calcitonin gene-related peptide (CGRP). Increased serum CGRP levels were detected during migraine and CH. In addition, in CH, there is a release of parasympathetic peptide, vasoactive intestinal peptide. Currently, inhibiting the release of vasoactive substances and neuropeptides including the CGRP or nitric oxide, or blocking their receptors in the neuroanatomical substrates of head pain is a major focus in treatment of headaches. © 2010 by Nova Science Publishers, Inc. All rights reserved

Role Of Neuropeptides In Migraine: Where Do They Stand In The Latest Expert Recommendations In Migraine Treatment?

Many factors have been implicated in the pathogenesis of migraine headache, including activation of the trigeminovascular system, dysfunction of: cerebral blood vessels, circulating vasoactive substances, mitochondrial energy metabolism, brain oxygenation and metabolism, platelet disorder, alterations in serotonin levels, low levels of brain tissue magnesium, altered transport of ions across the cell membrane, and inheritance and dysfunction of the brainstem periaqueductal gray matter. The headache phase of migraine is associated with cerebral vasodilation and inflammation, presumably mediated by the release of vasoactive substances and neuropeptides including CGRP (calcitonin gene-related peptide). Increased serum CCRP levels have been detected during migraine and cluster headache. One strategy to treat migraine is to inhibit the release of neuropeptides or to block their receptors. This article briefly reviews some experimental and clinical investigations focused on neuropeptide involvement in migraine. © 2007 Wiley-Liss, Inc