Codon optimization and improved delivery/immunization regimen enhance the immune response against wild-type and drug-resistant HIV-1 reverse transcriptase, preserving its Th2-polarity.

DNA vaccines require a considerable enhancement of immunogenicity. Here, we optimized a prototype DNA vaccine against drug-resistant HIV-1 based on a weak Th2-immunogen, HIV-1 reverse transcriptase (RT). We designed expression-optimized genes encoding inactivated wild-type and drug-resistant RTs (RT-DNAs) and introduced them into mice by intradermal injections followed by electroporation. RT-DNAs were administered as single or double primes with or without cyclic-di-GMP, or as a prime followed by boost with RT-DNA mixed with a luciferase-encoding plasmid ("surrogate challenge"). Repeated primes improved cellular responses and broadened epitope specificity. Addition of cyclic-di-GMP induced a transient increase in IFN-γ production. The strongest anti-RT immune response was achieved in a prime-boost protocol with electroporation by short 100V pulses done using penetrating electrodes. The RT-specific response, dominated by CD4+ T-cells, targeted epitopes at aa 199-220 and aa 528-543. Drug-resistance mutations disrupted the epitope at aa 205-220, while the CTL epitope at aa 202-210 was not affected. Overall, multiparametric optimization of RT strengthened its Th2- performance. A rapid loss of RT/luciferase-expressing cells in the surrogate challenge experiment revealed a lytic potential of anti-RT response. Such lytic CD4+ response would be beneficial for an HIV vaccine due to its comparative insensitivity to immune escape.

Analysis of the Domains of Hepatitis C Virus Core and NS5A Proteins that Activate the Nrf2/ARE Cascade.

The hepatitis C virus (HCV) triggers a chronic disease that is often accompanied by a spectrum of liver pathologies and metabolic alterations. The oxidative stress that occurs in the infected cells is considered as one of the mechanisms of HCV pathogenesis. It is induced by the viral core and NS5A proteins. It is already known that both of these proteins activate the antioxidant defense system controlled by the Nrf2 transcription factor. Here, we show that this activation is mediated by domain 1 of the NS5A protein and two fragments of the core protein. In both cases, this activation is achieved through two mechanisms. One of them is mediated by reactive oxygen species (ROS) and protein kinase C, whereas the other is triggered through ROS-independent activation of casein kinase 2 and phosphoinositide 3-kinase. In the case of the HCV core, the ROS-dependent mechanism was assigned to the 37-191 a.a. fragment, while the ROS-independent mechanism was assigned to the 1-36 a.a. fragment. Such assignment of the mechanisms to different domains is the first evidence of their independence. In addition, our data revealed that intracellular localization of HCV proteins has no impact on the regulation of the antioxidant defense system.

Infant with unusually large choroid plexus papilloma undergoing emergency surgery. Case report with special emphasis on the surgical strategy.

Choroid plexus papillomas are one of the most common tumors of the nervous system in infants. The most frequent early symptoms, megalocephalia and vomiting, caused by elevated intracranial pressure often lead to a diagnosis only at a critical clinical stage. This study describes a case of a 3-month-old infant with a choroid plexus papilloma measuring 7 x 8 x 6 cm originating in the right lateral ventricle. The infant underwent emergency surgery in an acutely deteriorated state, i.e., acute herniation symptoms with fixed and dilated pupils. Despite of the size of the tumor, the proximity to eloquent cortex, and clinically deteriorated state, the infant recovered completely.

Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

The value of intraoperative angiography for surgical treatment of cerebral arteriovenous malformations in eloquent brain areas.

Intraoperative digital subtraction angiography (DSA) allows intraoperative assessment of outcome of cerebral arteriovenous malformations (AVM). This study reports on 21 patients with AVMs in eloquent areas of the brain extirpated between July 1995 to March 1998. Extirpation was always followed by intraoperative DSA. Intraoperative angiography disclosed an occult residual nidus in 4 cases (19%). Complete extirpation of the AVM was achieved in all cases. Following surgery the neurological condition improved in 15 cases (71%), remained unchanged in 5 (24%), and worsened in 1. There were no secondary postoperative haemorrhages, nor complications related to the angiography. These results indicate that intraoperative DSA should be considered in the course of surgical treatment of cerebral AVMs in eloquent areas of the brain.

Colorimeter for the intuitive manipulation of hue and saturation and its role in the study of perceptual distortion.

A simple optical method for mixing coloured light is described. The observer has intuitive and approximately independent control over hue and saturation at constant brightness. The method facilitates colour matching by unpracticed observers. It allows children with reading difficulties to select a colour that reduces perceptual distortion of text. The chromaticity coordinates of this colour vary from one observer to another but can be very specific. Complementary colours can exacerbate the distortions and induce pain. For the majority of children reporting beneficial perceptual effects, the u' coordinate is less than 0.25.

Electron microscopy after direct ultracentrifugation.

A direct ultracentrifugation technique was used in the preparation of skin lesion specimens for examination by electron microscopy. The concentration factor of centrifuged specimens was estimated to be in excess of 1,000-fold compared to conventional adsorption techniques. This resulted in an increase of over 300% in the detection rate of herpesviruses and poxviruses from skin lesion specimens.