Major histocompatibility complex binding peptides: a target for therapeutic development.

Peptides that bind with high affinity to major histocompatibility complex molecules could represent useful tools in treating class II-associated autoimmune diseases such as rheumatoid arthritis, type 1 diabetes and multiple sclerosis. Although the concept has been validated in experiments with both purified receptor systems in vitro and cellular systems in vivo, many challenging problems need to be resolved before efficacious therapeutic agents are obtained.     

NMEGylation: a novel modification to enhance the bioavailability of therapeutic peptides

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.     

Postnatal development of the cholecystokinin-gastrin family of peptides in the brain and gut of rat

The average levels of CCK-8- and CCK-like peptides present in aqueous and acidic extracts of the brain of the newborn rat were, respectively, 5.3 and 2.9 pmol/g wet weight. These low levels increased 20- and 10-fold, respectively, so as to attain, at 25–30 days post partum, values comparable to those found in adult brain. There was also a rapid deposition of CCK-gastrin-like peptides in the whole gut between day 20 post coitum and day 10 post partum, when maximal concentrations were attained. Later, less CCK-gastrin peptides than other gut components were accumulated during the progressive weaning of the young rats, so that the average concentrations of CCK-gastrin-like peptides were, at day 30, similar to those observed in adult gut.     

Microglia in development: linking brain wiring to brain environment

Microglia are enigmatic non-neuronal cells that infiltrate and take up residence in the brain during development and are thought to perform a surveillance function. An established literature has documented how microglia are activated by pathogenic stimuli and how they contribute to and resolve injuries to the brain. However, much less work has been aimed at understanding their function in the uninjured brain. A series of recent in vivo imaging studies shows that microglia in their resting state are highly motile and actively survey their neuronal surroundings. Furthermore, new data suggest that microglia in their resting state are able to phagocytose unwanted synapses and in this way contribute to synaptic pruning and maturation during development. Coupled with their exquisite sensitivity to pathogenic stimuli, these data suggest that microglia form a link that couples changes in brain environment to changes in brain wiring. Here we discuss this hypothesis and propose a model for the role of microglia during development in sculpting brain connectivity.     

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

Summary The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.     

Gastrointestinal peptides in the brain.

Both immunoreactive intact cholecystokinin (CCK33) and its COOH-terminal octapeptide (CCK8) are detected in brain and gut extracts of monkey, dog, and pig using an antiserum with equivalent sensitivities for detecting CCK8 in the free form or when incorporated in the intact molecule. The failure to detect intact cholecystokinin in extracts from monkey or dog by using an antiserum developed by immunization with porcine CCK33 is due to marked species differences in the NH2-terminal portion of the molecule. Immunohistochemical staining reveals the presence of CCK peptides in rabbit cerebral cortical tissue neurons. Subcellular fractionation of rat cerebral cortical tissue demonstrates that CCK immunoreactivity is concentrated in the pellet identified by electron microscopy to contain a high proportion of synaptic vesicles. A converting enzyme that differs from trypsin has been partially purified from canine and porcine cerebral cortical extracts. It converts porcine CCK to smaller immunoreactive forms, but fails to convert big gastrin to heptadecapeptide gastrin. This enzyme differs from trypsin not only in substrate specificity but also in several physicochemical properties. Cerebral cortical extracts from hyperphagic ob/ob mice have strikingly lower contents of CCK than those from their lean littermates and other normal mice. These studies taken together are consistent with a role for CCK as a neurotransmitter involved in the overall regulation of appetite.     

Studies on the early floral development inCleomoideae (Capparaceae) with emphasis on the androecial development

Floral ontogeny ofCleome spinosa, Cleome violacea andPolanisia dodecandra subsp.trachysperma was studied in the context of the question whether the fascicled androecium ofReseda andCapparis (with fused fascicles) or the 2 + 4-pattern of theBrassicaceae is primitive in theCapparales. InPolanisia dodecandra, the 9–18 stamens show unidirectional initiation from the adaxial side toward the abaxial side of the flower. InCleome violacea, the six stamens also are formed in an unidirectional order, but development starts abaxially and a zigzag-like pattern is superimposed. InCleome spinosa, two stamen primordia in transversal (lateral) position are followed by four stamens which arise on a somewhat higher level in two pairs in front of the median sepals. It is assumed that the evolutionary steps in the androecial development proceed fromReseda viaCapparis andPolanisia/Cleome toBrassicaceae. This interpretation is supported byrbcL-studies (Chase & al. 1993,Rodman & al. 1993).Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

Permeability of the blood-brain barrier to peptides: An approach to the development of therapeutically useful analogs

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics. This shows how the principles elucidated for peptide passage across the BBB can be used to develop therapeutic peptides and how those peptides can be further tested in complementary in vivo and in vitro systems.     

Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes

To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.     

Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure

In order to elucidate how brain natriuretic peptides (NPs) are affected by experimentally induced heart failure, we have measured the immunoreactive (IR) levels of the NP in extracts from 10 regions of ovine brain, including pituitary, and clarified their molecular forms using high performance liquid chromatography (HPLC). Using species-specific radioimmunoassay (RIA), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were all detected in extracts taken from control animals and sheep that had undergone rapid ventricular pacing for 7 days to induce heart failure. CNP was the most abundant NP as assessed by specific RIA, and the pituitary contained the highest IR levels for all three NP. Compared with control animals, the pituitary content of BNP in animals with heart failure was reduced by 40% (control, 0.26±0.02 pmol/g wet weight versus heart failure 0.16±0.01; P<0.01, n=7). No other significant changes were observed. The molecular forms of ANP and CNP in whole brain extracts as assessed by HPLC were proANP and CNP22, CNP53 and proCNP, respectively. BNP in pituitary extracts was assessed to be primarily proBNP with a minor component of mature BNP26.     

Effects of intracranial self-stimulation on brain opioid peptides

The neurochemical system(s) underlying brain stimulation reward (ICSS) has been investigated for many years. The catecholamine hypothesis is currently most accepted with predominant emphasis on the role of dopamine. The present report examines the role of three opioid peptides--Methionine and Leucine Enkephalin (ME and LE) and beta-Endorphin (beta-E) in this behavior. Peptide levels from pituitary, hypothalamus and whole brain were determined by independent RIAs and analyzed according to treatment: low, moderate and high ICSS responders, sham controls, animals receiving nonspecific stimulation, and naloxone--with and without ICSS. Not only did naloxone reduce ICSS from high responders by 74%, it also was able to reduce peptide levels--most notably for ME and beta E in most regions. Additionally, the effects of ICSS on endorphin levels was found to be related to the rate category of responding. Since endorphins are known to interact with dopamine systems, it is therefore considered likely that the endogenous opioid peptides play an important role in ICSS either directly or indirectly via their influence on catecholamine systems.     

Cannabinoids and brain injury: therapeutic implications

Mounting in vitro and in vivo data suggest that the endocannabinoids anandamide and 2-arachidonoyl glycerol, as well as some plant and synthetic cannabinoids, have neuroprotective effects following brain injury. Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission and reduce the production of tumour necrosis factor-alpha and reactive oxygen intermediates, which are factors in causing neuronal damage. The formation of the endocannabinoids anandamide and 2-arachidonoyl glycerol is strongly enhanced after brain injury, and there is evidence that these compounds reduce the secondary damage incurred. Some plant and synthetic cannabinoids, which do not bind to the cannabinoid receptors, have also been shown to be neuroprotective, possibly through their direct effect on the excitatory glutamate system and/or as antioxidants.     

Antifungal peptides: Origin, activity, and therapeutic potential

Antifungal peptides have been identified in a wide range of life forms which include plants, mammals, and microorganisms. Their structures are as varied as their antifungal properties. Semisynthetic and fully synthetic analogs have been developed from a few of these natural peptides that are superior to the parent compound. A few of these peptides hold promise in combating fungal infections and have entered clinical trials.     

The neurobiology of gliomas: from cell biology to the development of therapeutic approaches

Gliomas are the most common type of primary brain tumour and are often fast growing with a poor prognosis for the patient. Their complex cellular composition, diffuse invasiveness and capacity to escape therapies has challenged researchers for decades and hampered progress towards an effective treatment. Recent molecular characterization of tumour cells combined with new insights into cellular diversification that occurs during development, and the modelling of these processes in transgenic animals have enabled a more detailed understanding of the events that underlie gliomagenesis. Combining this enhanced understanding of the relationship between neural stem cell biology and the cell lineage relationships of tumour cells with model systems offers new opportunities to develop specific and effective therapies.     

Technical aspects of telepathology with emphasis on future development.

Pathology undergoes presently changes due to new developments in diagnostic opportunities and cost saving efforts in health care. Out of the wide field of telepathology the paper selects three prototype applications: telepathology in teleeducation, expert advice for preselected details of a slide and finally telepathology for remote diagnosis. The most challenging field for remote diagnosis is the application in the frozen section scenario. The paper starts with the mental experiment to map conventional procedures to counterparts in telepathology. Technical opportunities and economical restrictions of telepathology equipment are discussed with respect to the components: electronic camera, display devices, haptic sensors and displays, available telecommunication channels and telepathology software. As an example and for illustration of the state of the art for an advanced telemicroscopy system able to perform remote frozen section diagnosis, the HISTKOM equipment is presented in more details. The section concerning future developments regards the aspects of the acceptance by tentative users, legal aspects, costs and affordability of equipment, the market for equipment components and the adequate telecommunication services. Further is regarded the mutual influence of properties of existing systems and application experiences gained with them on the next generation of equipment and application software. Conclusions and references close the paper.