Immunogenicity of DNA Vaccine against H5N1 Containing Extended Kappa B Site: In Vivo Study in Mice and Chickens

Influenza is one of the most important illnesses in the modern world, causing great public health losses each year due to the lack of medication and broadly protective, long-lasting vaccines. The development of highly immunogenic and safe vaccines is currently one of the major problems encountered in efficient influenza prevention. DNA vaccines represent a novel and powerful alternative to the conventional vaccine approaches. To improve the efficacy of the DNA vaccine against influenza H5N1, we inserted three repeated kappa B (κB) motifs, separated by a 5-bp nucleotide spacer, upstream of the cytomegalovirus promoter and downstream of the SV40 late polyadenylation signal. The κB motif is a specific DNA element (10pb-long) recognized by one of the most important transcription factors NFκB. NFκB is present in almost all animal cell types and upon cell stimulation under a variety of pathogenic conditions. NFκB is released from IκB and translocates to the nucleus and binds to κB sites, thereby leading to enhanced transcription and expression of downstream genes. We tested the variants of DNA vaccine with κB sites flanking the antigen expression cassette and without such sites in two animal models: chickens (broilers and layers) and mice (BALB/c). In chickens, the variant with κB sites stimulated stronger humoral response against the target antigen. In mice, the differences in humoral response were less apparent. Instead, it was possible to spot several gene expression differences in the spleens isolated from mice immunized with both variants. The results of our study indicate that modification of the sequence outside of the sequence encoding the antigen might enhance the immune response to the target but understanding the mechanisms responsible for this process requires further analysis

PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects

<p>Abstract</p> <p>Background</p> <p>The clinical treatment of various types of pain relies upon the use of opioid analgesics. However most of them produce, in addition to the analgesic effect, several side effects such as the development of dependence and addiction as well as sedation, dysphoria, and constipation. One solution to these problems are chimeric compounds in which the opioid pharmacophore is hybridized with another type of compound to incease antinociceptive effects. Neurotensin-induced antinociception is not mediated through the opioid system. Therefore, hybridizing neurotensin with opioid elements may result in a potent synergistic antinociceptor.</p> <p>Results</p> <p>Using the known structure-activity relationships of neurotensin we have synthesized a new chimeric opioid-neurotensin compound PK20 which is characterized by a very strong antinociceptive potency. The observation that the opioid antagonist naltrexone did not completely reverse the antinociceptive effect, indicates the partial involvement of the nonopioid component in PK20 in the produced analgesia.</p> <p>Conclusions</p> <p>The opioid-neurotensin hybrid analogue PK20, in which opioid and neurotensin pharmacophores overlap partially, expresses high antinociceptive tail-flick effects after central as well as peripheral applications.</p

In vivo antinociception of potent mu opioid agonist tetrapeptide analogues and comparison with a compact opioid agonist - neurokinin 1 receptor antagonist chimera

<p>Abstract</p> <p>Background</p> <p>An important limiting factor in the development of centrally acting pharmaceuticals is the blood-brain barrier (BBB). Transport of therapeutic peptides through this highly protective physiological barrier remains a challenge for peptide drug delivery into the central nervous system (CNS). Because the most common strategy to treat moderate to severe pain consists of the activation of opioid receptors in the brain, the development of active opioid peptide analogues as potential analgesics requires compounds with a high resistance to enzymatic degradation and an ability to cross the BBB.</p> <p>Results</p> <p>Herein we report that tetrapeptide analogues of the type H-Dmt¹-Xxx²-Yyy³-Gly⁴-NH₂are transported into the brain after intravenous and subcutaneous administration and are able to activate the μ- and δ opioid receptors more efficiently and over longer periods of time than morphine. Using the hot water tail flick test as the animal model for antinociception, a comparison in potency is presented between a side chain conformationally constrained analogue containing the benzazepine ring (BVD03, Yyy³: Aba), and a "ring opened" analogue (BVD02, Yyy³: Phe). The results show that in addition to the increased lipophilicity through amide bond N-methylation, the conformational constraint introduced at the level of the Phe³side chain causes a prolonged antinociception. Further replacement of NMe-D-Ala²by D-Arg²in the tetrapeptide sequence led to an improved potency as demonstrated by a higher and maintained antinociception for AN81 (Xxx²: D-Arg) vs. BVD03 (Xxx²: NMe-D-Ala). A daily injection of the studied opioid ligands over a time period of 5 days did however result in a substantial decrease in antinociception on the fifth day of the experiment. The compact opioid agonist - NK1 antagonist hybrid SBCHM01 could not circumvent opioid induced tolerance.</p> <p>Conclusions</p> <p>We demonstrated that the introduction of a conformational constraint has an important impact on opioid receptor activation and subsequent antinociception in vivo. Further amino acid substitution allowed to identify AN81 as an opioid ligand able to access the CNS and induce antinociception at very low doses (0.1 mg/kg) over a time period up to 7 hours. However, tolerance became apparent after repetitive i.v. administration of the investigated tetrapeptides. This side effect was also observed with the dual opioid agonist-NK1 receptor antagonist SBCHM01.</p

Affinity of fentanyl and its derivatives for the sigma(1)-receptor

Fentanyl and its 11 commercially available derivatives were investigated as to their affinity for the sigma(1) receptor. The parent compound is a rather poor binder (IC50 = 4973 nM), but its close derivatives (benzylfentanyl or p-fluorofentanyl) have submicromolar affinities. Modelling provides a structural basis for the observed trends in activity

Ocena wysokości przestrzeni międzykręgowej na podstawie punktów anatomicznych w celu wykonania nakłucia lędźwiowego zawodzi w ponad 30% przypadków

BACKGROUND: The anatomical landmark which is used to identify the correct level for lumbar puncture is the line connecting both iliac crests. This crosses the vertebra column at the level of the L4–L5 intervertebral space or L4 vertebra. It can be difficult to determine in a group of orthopaedic patients due to chronic orthopaedic disorders, chronic pain, overweight, or difficulties with positioning for lumbar puncture. The objective of this study was to determine if identification of intervertebral space by a physical exam differs from that of an ultrasound assessment.METHODS: Adult patients scheduled for lower limb surgery under spinal block were enrolled in this study. The intervertebral space suitable for lumbar puncture was determined by physical exam by an anaesthetist in the sitting or lateral position. This was followed by a lumbar ultrasound. Primarily, a transducer was placed in paramedian sagittal view followed by transverse interlaminar view to confirm the identification of the interlaminar spaces. The ‘counting-up’ approach starting with the L5–S1 space was applied.RESULTS: One hundred and twenty two patients (122) were included in this study. Lumbar intervertebral spaces were identified by ultrasound in all cases. There was concordance of intervertebral space identification (between clinical and ultrasound examination) in 78 cases (64%). Mean deviation of inacuracy was one intervertebral space with no statistical difference among cephalad and caudal direction. There were no statistically significant differences found in terms of demographic data (sex, age, height, weight, or BMI), positioning for lumbar puncture, or intervertebral space chosen for the puncture between the concordant and the nonconcordant identification groups. The only statistically significant difference found was the difference in the years of experience of the anaesthetist performing the clinical assessment and puncture.CONCLUSIONS: The concordance rate between clinical examination and using assessment of intervertebral space identification for lumbar puncture is 64% among patients undergoing lower limb surgery. No special parameters were found which could make an anaesthetist aware that a patient is at greater risk of inadequate intervertebral space level assessment. Spinal ultrasound can reduce the incidence of inappropriate lumbar puncture level in orthopaedic patients.  BACKGROUND: The anatomical landmark which is used to identify the correct level for lumbar puncture is the line connecting both iliac crests. This crosses the vertebra column at the level of the L4–L5 intervertebral space or L4 vertebra. It can be difficult to determine in a group of orthopaedic patients due to chronic orthopaedic disorders, chronic pain, overweight, or difficulties with positioning for lumbar puncture. The objective of this study was to determine if identification of intervertebral space by a physical exam differs from that of an ultrasound assessment.METHODS: Adult patients scheduled for lower limb surgery under spinal block were enrolled in this study. The intervertebral space suitable for lumbar puncture was determined by physical exam by an anaesthetist in the sitting or lateral position. This was followed by a lumbar ultrasound. Primarily, a transducer was placed in paramedian sagittal view followed by transverse interlaminar view to confirm the identification of the interlaminar spaces. The ‘counting-up’ approach starting with the L5–S1 space was applied.RESULTS: One hundred and twenty two patients (122) were included in this study. Lumbar intervertebral spaces were identified by ultrasound in all cases. There was concordance of intervertebral space identification (between clinical and ultrasound examination) in 78 cases (64%). Mean deviation of inacuracy was one intervertebral space with no statistical difference among cephalad and caudal direction. There were no statistically significant differences found in terms of demographic data (sex, age, height, weight, or BMI), positioning for lumbar puncture, or intervertebral space chosen for the puncture between the concordant and the nonconcordant identification groups. The only statistically significant difference found was the difference in the years of experience of the anaesthetist performing the clinical assessment and puncture.CONCLUSIONS: The concordance rate between clinical examination and using assessment of intervertebral space identification for lumbar puncture is 64% among patients undergoing lower limb surgery. No special parameters were found which could make an anaesthetist aware that a patient is at greater risk of inadequate intervertebral space level assessment. Spinal ultrasound can reduce the incidence of inappropriate lumbar puncture level in orthopaedic patients.

The substantial impact of ultrasound-guided regional anaesthesia on the clinical practice of peripheral nerve blocks

BACKGROUND: Ultrasound-guided (US-guided) regional anaesthesia has gained worldwide popularity in recent years owing to the benefits the method offers to patients. The 1st Department of Anaesthesiology and Intensive Care of Warsaw Medical University was one of the first centres in Poland to employ US-guided peripheral nerve blocks (PNB) on a routine basis. The technique was incorporated into the institution’s clinical practice from 2007. The purpose of this study was to retrospectively assess changes in the clinical practice of US-guided versus non US-guided PNBs over six years of experience with the technique.METHODS: Retrospective analysis assessing the prevalence of PNB methods, local anaesthetic (LA) injection techniques (i.e. single injection vs. multiple), LA volumes used, success rates and the incidence of complications.RESULTS: This study included 4,066 PNBs performed between January 2006 and June 2012. The results showed systematic growth in the prevalence of US-guided blocks in the total number of PNBs, from 8.6% in 2007 up to 53.3% in 2012. The mean LA volume used in PNB was significantly lower in US-guided blocks compared to traditional PNB techniques (respectively, 21.83 mL vs. 31.41 mL, P &lt; 0.05) without a decrease in the success rate (respectively, 76% vs. 74%, P &gt; 0.05). A shift in the prevailing block technique from single injection to multiple injections was observed, regardless of the nerve location technique employed (from 29% up to 84% of PNBs performed using multiple injection technique).CONCLUSIONS: The use of ultrasound optimizes the technique of peripheral blocks and the amount of local anaesthetic used. Ultrasonography does not affect the safety of peripheral blocks.BACKGROUND: Ultrasound-guided (US-guided) regional anaesthesia has gained worldwide popularity in recent years owing to the benefits the method offers to patients. The 1st Department of Anaesthesiology and Intensive Care of Warsaw Medical University was one of the first centres in Poland to employ US-guided peripheral nerve blocks (PNB) on a routine basis. The technique was incorporated into the institution’s clinical practice from 2007. The purpose of this study was to retrospectively assess changes in the clinical practice of US-guided versus non US-guided PNBs over six years of experience with the technique.METHODS: Retrospective analysis assessing the prevalence of PNB methods, local anaesthetic (LA) injection techniques (i.e. single injection vs. multiple), LA volumes used, success rates and the incidence of complications.RESULTS: This study included 4,066 PNBs performed between January 2006 and June 2012. The results showed systematic growth in the prevalence of US-guided blocks in the total number of PNBs, from 8.6% in 2007 up to 53.3% in 2012. The mean LA volume used in PNB was significantly lower in US-guided blocks compared to traditional PNB techniques (respectively, 21.83 mL vs. 31.41 mL, P &lt; 0.05) without a decrease in the success rate (respectively, 76% vs. 74%, P &gt; 0.05). A shift in the prevailing block technique from single injection to multiple injections was observed, regardless of the nerve location technique employed (from 29% up to 84% of PNBs performed using multiple injection technique).CONCLUSIONS: The use of ultrasound optimizes the technique of peripheral blocks and the amount of local anaesthetic used. Ultrasonography does not affect the safety of peripheral blocks

Chirality and Rigidity in Triazole-Modified Peptidomimetics Interacting with Neuropilin-1

The interaction of Neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has been shown to promote angiogenesis under physiological and pathological conditions. Angiogenesis around tumors is a major factor allowing for their growth and spread. Disrupting NRP-1/VEGF complex formation is thus a promising pathway for the development of new anticancer pharmaceuticals. A large body of work has been produced in the last two decades detailing the development of inhibitors of NRP-1/VEGF complex formation. Among those were peptide A7R and its smaller derivatives KXXR and K(Har)XXR. It has been previously reported that replacement of the XX backbone with triazole residues has a positive effect on the proteolytic stability of inhibitors. It has also been reported that a higher dihedral angle range restriction of the XX backbone has a positive effect on the activity of inhibitors. In this work, we have designed new triazole derivatives of K(Har)XXR inhibitors with substitution allowing for higher range restriction of the XX backbone. The obtained peptidomimetics have greater activity than their less restricted counterparts. One of the newly obtained structures has greater affinity than the reference peptide A7R

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response

Can keratin scaffolds be used for creating three-dimensional cell cultures?

Three-dimensional (3D) cell cultures were created with the use of fur keratin associated proteins (F-KAPs) as scaffolds. The procedure of preparation F-KAP involves combinations of chemical activation and enzymatic digestion. The best result in porosity and heterogeneity of F-KAP surface was received during pepsin digestion. The F-KAP had a stable structure, no changes were observed after heat treatment, shaking and washing. The 0.15-0.5 mm fraction had positive effect for formation of 3D scaffolds and cell culturing. Living rat mesenchymal cells on the F-KAP with no abnormal morphology were observed by SEM during 32 days of cell culturing