Spirulina diet to lactating mothers protects the antioxidant system and reduces inflammation in post-natal brain after systemic inflammation.

OBJECTIVES: This study concerns: (1) the long-term effects of peripheral lipopolysaccharide (LPS) in neonatal rats on inflammation and antioxidant parameters in brain and (2) the effects of a Spirulina-enriched diet given to lactating mothers on protective and inflammatory parameters in brains of suckling pups subjected to peripheral inflammation. METHODS: Five-day old rat pups were treated with LPS (i.p. 2 mg/kg). After 3, 7, 30, and 65 days, mRNA, miRNA, and protein levels of pro-inflammatory cytokines and the Nuclear factor E2-related factor 2 (Nrf2)-system were examined. In a sub-group, a Spirulina-enriched diet was given to the mothers 24 hours before the pups were treated with LPS, then the effects on antioxidant and inflammatory parameters were evaluated. RESULTS: The main findings were: (1) interleukin 1 beta (IL-1ß) was upregulated in cortex 3, 7, and 30 days after LPS treatment, (2) Nrf2 and the catalytic subunit of γ-glutamylcysteinyl ligase were decreased in cortex 7 days after LPS in parallel with increased levels of phosphorylated p38 and decreased levels of histone H3 acetylation, and (3) a Spirulina-enriched diet to lactating mothers normalized both the increased IL-1ß expression and the decreased antioxidant parameters after LPS. The protective effects of Spirulina were correlated with decreased levels of phosphorylated p38 and high levels of the antioxidant miRNA-146a. DISCUSSION: A Spirulina diet given to lactating mothers can protect against neuroinflammation and decreased antioxidant defence in brain of suckling pups subjected to peripheral inflammation, possibly via decreased activation of p38 and high levels of the antioxidant miRNA-146a.

Sustained Effects of Neonatal Systemic Lipopolysaccharide on IL-1ß and Nrf2 in Adult Rat Substantia Nigra Are Partly Normalized by a Spirulina-Enriched Diet.

BACKGROUND/AIM: Neonatal infection can sensitize the adult substantia nigra (SN) to secondary insults, causing a decrease in antioxidant capacity which may lead to Parkinson's disease in adults. We studied the prolonged effect of systemic infection by (i.p.) administration of lipopolysaccharide (LPS) on interleukin (IL)-1ß, the antioxidant regulator nuclear factor-erythroid 2-related factor 2 (Nrf2), and the peroxisome proliferator-activated receptor γ coactivator (PGC)-1α in rat SN. METHOD AND RESULTS: Five-day-old rat pups were treated with LPS (i.p. 2 mg/kg). After 65 days, the mRNA level of IL-1ß was significantly increased, in parallel with a decrease in that of the rate-limiting enzyme in glutathione synthesis, the γ-glutamylcysteine ligase catalytic subunit (γGCLc), Nrf2, and brain-derived neurotrophic factor (BDNF). Protein levels of γGCLc and Nrf2 were decreased while IL-1ß protein was significantly increased. These LPS-induced long-term changes correlated with a decrease in phosphorylated (active) AKT (pAKT) and phosphorylated (inactive) GSK-3ß (pGSK-3ß). In another set of experiments, a 0.1% Spirulina-containing diet was given to lactating mothers 24 h before the LPS treatment of the pups. The Spirulina-supplemented diet decreased IL-1ß protein expression in SN and elevated the mRNA level of γGCLc, Nrf2 protein, PGC-1α protein, and pAKT. CONCLUSION: Early-life infection can negatively affect Nrf2, pAKT, and pGSK-3ß for a long time in SN. A diet enriched with antioxidant and anti-inflammatory phytochemicals can partly restore some, but not all, of the effects on the antioxidant defense, possibly via normalizing effects on pAKT.

Understanding the Specific Implications of Amino Acids in the Antibody Development.

As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody's architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies.

Time-dependent effects of systemic lipopolysaccharide injection on regulators of antioxidant defence Nrf2 and PGC-1α in the neonatal rat brain.

BACKGROUND/AIMS: Both excitotoxicity and neuroinflammation are associated with oxidative stress. One transcription factor, nuclear factor E2-related factor 2 (Nrf2), and one transcription cofactor, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), increase the endogenous antioxidant defence and can thus modulate neuronal cell death. Here, we investigated the temporal effects (after 24 and 72 h) of systemic (i.p.) administration of lipopolysaccharide (LPS) on the cerebral Nrf2 and PGC-1α systems. METHODS AND RESULTS: Seven-day-old rat pups were injected with LPS (0.3 mg/kg). After 24 h, the protein levels of γ-glutamylcysteine ligase modulatory subunit, γ-glutamylcysteine ligase catalytic subunit, Nrf2, PGC-1α and manganese superoxide dismutase (MnSOD) were increased in parallel with decreased levels of Keap1. These effects were correlated with an increased level of phosphorylated Akt and elevated acetylation of histone 4. In contrast, 72 h following LPS, a decrease in the components of the Nrf2 system in parallel with an increase in Keap1 was observed. The down-regulation after 72 h correlated with phosphorylation of p38 mitogen-activated protein kinase, while there were no changes in PGC-1α and MnSOD protein levels or the acetylation/methylation pattern of histones. CONCLUSION: Systemic LPS in neonatal rats induced time-dependent changes in brain Nrf2 and PGC-1α that correlated well with the protective effect observed after 24 h (pre-conditioning) and the deleterious effects observed after 72 h (sensitizing) of systemic LPS reported earlier. Collectively, the results point towards Nrf2 and PGC-1α as a possible mechanism behind these effects.

Significance of antibody numbering systems in the development of antibody engineering.

Immunotherapy has become increasingly popular in recent years for treating a variety of diseases including inflammatory, neurological, oncological, and auto-immune disorders. The significant interest in antibody development is due to the high binding affinity and specificity of an antibody against a specific antigen. Recent advances in antibody engineering have provided a different view on how to engineer antibodies in silico for therapeutic and diagnostic applications. In order to improve the clinical utility of therapeutic antibodies, it is of paramount importance to understand the various molecular properties which impact antigen targeting and its potency. In antibody engineering, antibody numbering (AbN) systems play an important role to identify the complementarity determining regions (CDRs) and the framework regions (FR). Hence, it is crucial to accurately define and understand the CDR, FR and the crucial residues of heavy and light chains that aid in the binding of the antibody to the antigenic site. Detailed understanding of amino acids positions are useful for modifying the binding affinity, specificity, physicochemical features, and half-life of an antibody. In this review, we have summarized the different antibody numbering systems that are widely used in antibody engineering and highlighted their significance. Here, we have systematically explored and mentioned the various tools and servers that harness different AbN systems.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels.

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.

NRF2-regulation in brain health and disease: implication of cerebral inflammation.

The nuclear factor erythroid 2 related factor 2 (NRF2) is a key regulator of endogenous inducible defense systems in the body. Under physiological conditions NRF2 is mainly located in the cytoplasm. However, in response to oxidative stress, NRF2 translocates to the nucleus and binds to specific DNA sites termed "anti-oxidant response elements" or "electrophile response elements" to initiate transcription of cytoprotective genes. Acute oxidative stress to the brain, such as stroke and traumatic brain injury is increased in animals that are deficient in NRF2. Insufficient NRF2 activation in humans has been linked to chronic diseases such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. New findings have also linked activation of the NRF2 system to anti-inflammatory effects via interactions with NF-κB. Here we review literature on cellular mechanisms of NRF2 regulation, how to maintain and restore NRF2 function and the relationship between NRF2 regulation and brain damage. We bring forward the hypothesis that inflammation via prolonged activation of key kinases (p38 and GSK-3ß) and activation of histone deacetylases gives rise to dysregulation of the NRF2 system in the brain, which contributes to oxidative stress and injury.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers.

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

Angiotensinergic and noradrenergic neurons in the rat and human heart.

Although the physiological and pharmacological evidences suggest a role for angiotensin II (Ang II) with the mammalian heart, the source and precise location of Ang II are unknown. To visualize and quantitate Ang II in atria, ventricular walls and interventricular septum of the rat and human heart and to explore the feasibility of local Ang II production and function, we investigated by different methods the expression of proteins involved in the generation and function of Ang II. We found mRNA of angiotensinogen (Ang-N), of angiotensin converting enzyme, of the angiotensin type receptors AT(1A) and AT2 (AT(1B) not detected) as well as of cathepsin D in any part of the hearts. No renin mRNA was traceable. Ang-N mRNA was visualized by in situ hybridization in atrial ganglial neurons. Ang II and dopamine-ß-hydroxylase (DßH) were either colocalized inside the same neuronal cell or the neurons were specialized for Ang II or DßH. Within these neurons, the vesicular acetylcholine transporter (VAChT) was neither colocalized with Ang II nor DßH, but VAChT-staining was found with synapses en passant encircle these neuronal cells. The fibers containing Ang II exhibited with blood vessels and with cardiomyocytes supposedly angiotensinergic synapses en passant. In rat heart, right atrial median Ang II concentration appeared higher than septal and ventricular Ang II. The distinct colocalization of neuronal Ang II with DßH in the heart may indicate that Ang II participates together with norepinephrine in the regulation of cardiac functions: produced as a cardiac neurotransmitter Ang II may have inotropic, chronotropic or dromotropic effects in atria and ventricles and contributes to blood pressure regulation.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia.

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.