Július Rajčáni

Biodistribution and pharmacokinetics of 125I‐labeled monoclonal antibody M75 specific for carbonic anhydrase IX, an intrinsic marker of hypoxia, in nude mice xenografted with human colorectal carcinoma

Carbonic anhydrase IX (CA IX) is frequently expressed in human carcinomas and absent from the corresponding normal tissues. Strong induction by tumor hypoxia predisposes CA IX to serve as a target for cancer diagnostics and therapy. Here we evaluated targeting properties and pharmacokinetics of CA IX‐specific monoclonal antibody (MAb) M75. Binding parameters of 125I‐labeled M75, including equilibrium dissociation constant, hypoxia‐related binding to various cell lines and internalization, were analyzed in vitro. Biodistribution of 125I‐M75 in nude mice bearing HT‐29 human colorectal carcinoma xenografts with hypoxic pattern of CA IX expression was studied by measurements of radioactivity in dissected tissues and macroautoradiography of tissue sections. Pharmacokinetics of intravenously administered 125I‐M75 was described using a 2‐compartment model. Blood clearance showed a distribution phase t1/2(α) = 3.4 hr and an elimination phase t1/2(β) = 55.3 hr postinjection. Despite predominant CA IX localization in less accessible perinecrotic regions, 125I‐M75 exhibited specific accumulation in xenograft, with a mean uptake of 15.3 ± 3.6% of injected dose per gram of tumor tissue at 48 hr postadministration. Specificity of M75 localization was confirmed by low tumor uptake of control antibody. Altogether, our data demonstrate that M75 MAb is a promising tool for selective immunotargeting of hypoxic human tumors that express CA IX.

Peculiarities of Herpes Simplex Virus (HSV) Transcription: An overview

The herpes simplex virus (HSV) has a 152 kbp dsDNA encoding probably 84 proteins. The approximate number of ORFs is 94, from which seven are doubled. The most probable number of single copy ORFs is 84 after omitting the two latency associated transcripts (LAT)/ORFs and the putative UL27.5 ORF. The high gene number creates a “crowded” genome with several overlapping transcripts. The unique long (UL) segment has at least 10 interposed ORFs, the existence of which was not obvious at first sequence analysis, while the unique short (US) segment has two such genes. The surplus of ORFs causes complex transcription patterns: (1) Transcripts with common initiation signals but different termination; (2) Transcripts with different initiation sites but co-terminal ends; (3) “Nested” transcripts differing at both, the initiation as well as termination signals, having partially collinear sequences. At least three or possibly four ORF (gene) pairs (UL9.5/UL10; UL27/UL27.5; UL43/UL43.5; ICP34.5/ORF P and O) occupy both DNA strands at complementary positions rising anti-sense transcripts expressed by an antagonistic mechanism of mutual exclusion. The anti-sense mRNA mechanism might also operate when either LAT or ICP0 ORFs are expressed during latency assuring the absence of lytic virus replication. In contrast, during productive replication the cascade regulation of gene expression predominates, based on stepwise activation of immediate early (IE), early (E), early late (EL) and late (L) promoters. The promoters of different expression kinetic classes (α, β, γ−1 and γ−2) are equipped with different number of cellular transcription factor binding and/or enhancer motifs. Surprisingly, only a few HSV mRNAs are being spliced (ICP0, UL15, US1, US12/ICP47). As reviewed here, the transcription pattern of the great majority of overlapping ORFs within the HSV-1 was quite convincingly elucidated, with exception of the putative UL27.5 gene. The UL27.5 transcript was not identified yet. Since the existence of the UL27.5 gene was based on indirect rather than direct evidence, it needs final confirmation.

Multiorgan distribution of human influenza A virus strains observed in a mouse model.

Multiorgan spread and pathogenesis of influenza infection with three human influenza A viruses was studied in mice. Mouse-adapted viruses A/Dunedin/4/73(H3N2), A/Mississippi/1/85(H3N2), and A/PR/8/34(H1N1) differed considerably in virulence (p.f.u./LD50): 79,000 p.f.u. for Dunedin, 5,000 p.f.u. for Mississippi, and 65 p.f.u. for PR/8, which qualified Dunedin as low virulent, Mississippi as intermediate, and PR/8 as highly virulent. All three viruses were detected in lungs, heart, and thymus by cultivation and RT-PCR. Moreover, vRNA of all viruses was found in liver and spleen, of Dunedin and PR/8 also in kidneys and that of Dunedin and Mississippi in blood. Only vRNA of Dunedin was demonstrated in brain. Lung damage accompanied by histopathological changes and thymus reduction were most extensive after infection with the highly virulent virus PR/8. We assume that the ability to spread to multiple organs may be a more common property of influenza viruses in mammalian hosts than previously believed.

Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection

During the last decade, new data accumulated describing the early events during herpes simplex virus 1 (HSV-1) replication occurring before capsid formation and virion envelopment. The HSV virion carries its own specific transcription initiation factor (α-TIF), which functions together with other components of the cellular transcriptase complex to mediate virus-specific immediate early (IE) transcription. The virus-coded IE proteins are the transactivator and regulatory elements modulating early transcription and subsequent translation of nonstructural virus-coded proteins needed mainly for viral DNA synthesis and for the supply of corresponding nucleoside components. They also cooperate at the late transcription and translation of the viron (capsid, tegument and envelope) proteins. In addition, the transactivator, IE proteins down-regulate their own transcription, while others facilitate viral mRNA processing or interfere with the presentation of newly synthesized virus antigens. Establishment of latency is closely related to the transcription of a separate category of transcripts, termed latency-associated (LAT). Formation of LATs occurs mainly in nondividing neurons which are metabolically less active and express lower levels of cellular transcription factors (nonpermissive cells). Expression of the stable non-spliced (2 kb), and especially of stable spliced (1.5 and 1.45 kb) LATs is a prerequisite for HSV reactivation. Different HSV genomes (from various HSV strains) do not undergo IE transcription at the same rate. Restricted IE transcription and the absence of viral DNA synthesis favors LAT formation and persistence of the silenced genome. Uneven levels of LAT expression and differences in the metabolic state of carrier neurons infleunce the reactivation competence. Under artificial or natural activation conditions, sufficient amounts of IE transactivator proteins and proteins promoting nucleoside metabolism are synthesized even in the absence of the viral α-TIF facilitating reactivation.

Transcription at Early Stages of Herpes Simplex Virus 1 Infection and during Reactivation

Objective: The kinetics of immediate early (IE) and early (E) herpes simplex virus 1 (HSV-1) mRNA transcription was followed in explanted trigeminal ganglia from rabbits with established latency. Methods: The expression of IE and E mRNAs was first assessed in infected Vero cells by RT-PCR and then in explanted trigeminal ganglia by nested RT-PCR. Results: In infected Vero cells, IE mRNAs [for infected cell protein (ICP) 0, ICP4 and ICP27] were first detected 1–2 h post-inoculation (p.i.), peaking at 3 h p.i. The transcription of E mRNAs [for thymidine kinase (TK), RR1 and UL9], which were first detected from 3 h p.i., peaked between 5 and 10 h p.i. In explanted ganglia, the ICP0, ICP4 and ICP27 mRNAs were first detected after 4 h in culture. This was followed by the appearance of TK mRNA at 8 h and then by the UL9 mRNA, detected from 12 h post-explantation. A further E mRNA (RR1), as well as the late gC mRNA, were first observed after 24 h in culture. Moreover, ICP4 mRNA could be found in non-cultured ganglia. Conclusions: During reactivation of latent HSV-1 in explanted ganglia, the onset of ICP0 and ICP27 transcription at 4 h in culture was followed by TK transcription (at 8 h). Thus, in the rabbit reactivation model, ICP0 gene transcription rather than ICP4 transcription represents the relevant indicator of latency reactivation.

Activation of latent herpesvirus hominis in explants of rabbit trigeminal ganglia: the influence of immune serum.

SummaryMore than fifty albino rabbits were inoculated into the right scarified cornea with 107 PFU of the Kupka strain of human herpes virus type 1 (HHV-1). At intervals ranging from 4–280 days post infection (p.i.), both gasserian ganglia, both trigeminal nerve trunks and pieces from brain stem and from both corneas were explanted. Activation of the latent HHV-1 was found mainly in the homolateral ganglion tissue, but also in explants originating from the opposite ganglia. Within 24–72 hours, prior to the release of virus into the medium, one infectious unit of HHV was recovered from 104–105 cells of the ganglion explant. In addition, a few neurons and satellite cells revealed the presence of virus-specific antigens when the explants were examined by immunofluorescence in serial sections. If the ganglia were explanted in the presence of immune serum, the virus recovery rate was at least twice lower as compared to the virus activation in explants kept in the absence of immune serum.

Developments in herpes simplex virus vaccines: Old problems and new challenges

Vaccination has remained the best method for preventing virus spread. The herpes simplex virus (HSV) candidate vaccines tested till now were mostly purified subunit vaccines and/or recombinant envelope glycoproteins (such as gB and gD). In many experiments performed in mice, guinea pigs and rabbits, clear-cut protection against acute virus challenge was demonstrated along with the reduction of the extent of latency, when established in the immunized host. The immunotherapeutic effect of herpes vaccines seems less convincing. However, introduction of new adjuvants, which shift the cytokine production of helper T-cells toward stimulation of cytotoxic T-cells (TH1 type cytokine response), reveals a promising development. Mathematical analysis proved that overall prophylactic vaccination of seronegative women, even when eliciting 40–60 % antibody response only, would reduce the frequency of genital herpes within the vaccinated population. Even when partially effective, immunotherapeutic vaccination might represent a suitable alternative of chronic chemotherapy in recurrent labial and genital herpes.

Herpes Simplex Virus 1 (HSV-1) Strain HSZP Glycoprotein B Gene: Comparison of Mutations among Strains Differing in Virulence

The nonpathogenic HSZP strain of HSV-1 induces large polykaryocytes due to a syn3 mutation (His for Arg at residue 858) in the C-terminal endodomain of glycoprotein B (gB) (40). We determined the nucleotide (nt) sequence of the UL27 gene specifying the gB polypeptide of HSZP (gBHSZP) and found 3 mutations in its ectodomain at aminoacids (aa) 59, 79 and 108. The ANGpath virus, which also has a syn3 mutation in the C-terminal endodomain of gB (Val for Ala at residue 855) is pathogenic for adult mice (39), but can be made nonpathogenic by replacing the gBANGpath gene by the corresponding gBKOS sequence (21). The gBANGpath had three ectodomain mutations (at aa 62, 77 and 285), while gBKOS had at least four ectomain mutations (aa 59, 79, 313, and 553). Two mutations (aa 59 and 79) in the latter, located in the variable antigenic site IV/D1 were common for gBKOS and gBHSZP. These together with the gBANGpath mutations at aa 62 and 77 create a cluster of 4 mutations in diverse region of the N-terminal part of gB (between aa 59-79), in which the gBs of pathogenic ANGpath and 17 viruses differ from the gBs of nonpathogenic HSZP and KOS viruses. The lower pathogenicity of KOS as related to gBKOS, is furthermore associated with the change of Ser to Thr at aa 313 (locus III/D2). The possibility is discussed that mutations in both above mentioned antigenic loci could result in higher immunogenicity of the corresponding antigenic epitopes, which, in turn, would contribute to the decreased virulence of HSZP and KOS viruses.

GLYCOPROTEIN K OF HERPES SIMPLEX VIRUS : A TRANSMEMBRANE PROTEIN ENCODED BY THE UL53 GENE WHICH REGULATES MEMBRANE FUSION

Glycoprotein K (gK) encoded by the UL53 gene is the ninth out of eleven HSV glycoproteins (gps). The precursor gK (pgK) is a transmembrane protein with four hydrophobic domains, which consists of 338 amino acids. The UL53 gene has two initiation codons: the upper overlaps with the UL52 ORF, while the lower is located 55 codons downstream and specifies a truncated precursor of the gK polypeptide. The UL53 gene and the upstream located UL52 gene have a common polyadenylation signal downstream from the UL53 stop codon so that the UL53 mRNA is completely nested within the UL52 transcript. The syn1 mutations in several KOSsyn mutants and in the MPsyn virus, which had been fine mapped to DNA coordinates 0.735–0.740, were later on located to the UL53 gene, especially to its portion which specifies the first 120 amino acids (aa) from the N-terminus (most frequently residue 40) and to a less precisely defined locus between aa 301–310 (close to the C-terminus). Point mutations in the N-terminal ectodomain of gK, which are related to syn formation, impair the putative ability of this region to down-regulate membrane fusion. The two N-glycosylated mannose core oligosaccharides are attached to the Asn residues of the gK polypeptide at positions 48 and 58, respectively. In infected cells, gK is localized mainly in the nuclear and endoplasmic reticulum (ER) membranes. It is not clear, whether gK becomes incorporated into the envelope of mature HSV particles. Studies with the insertion/deletion gK mutants showed the importance of gK for capsid envelopment, for the transportation and egress or virions from infected cells. It seems that gK has an essential role in virion egress, even though this glycoprotein acts in accord with gH and with another membrane protein encoded by the UL20 gene.

Relation between expression pattern of p53 and survivin in cutaneous basal cell carcinomas

Summary Background The tumor suppressor gene p53 is a key regulator of cell division and/or apoptosis. Survivin is a multifunctional member of the inhibitor of apoptosis family. Survivin and p53 represent diametrically opposed signals that influence the apoptotic pathway. Material/Methods To determine the role of p53 and survivin in basal cell carcinoma (BCC), we evaluated the expression pattern of both proteins with regard to the percentage of positively immunostained tumor cells, the intensity of staining, and subcellular localization among 31 subjects with BCC. Results Overexpression of p53 protein was found in 28 of 31 cases (90.3%), whereas survivin accumulation was seen in 27 (87.1%). For p53, moderate and/or strong immunoreactivity was seen in 20 of 28 cases (71.4%), and 26 of 28 cases (92.9%) showed more than 25% reactive tumor cells. Nuclear p53 staining was detected in 23 of 28 cases (82.1%), whereas combined nuclear and cytoplasmic localization was found in only 5 of 28 cases (17.9%). Survivin revealed mild intensity of immuno-reaction in 22 of 27 cases (71%), and 25 of 27 cases (92.6%) showed less than 25% labeled tumor cells. Combined nuclear and cytoplasmic survivin localization was present in 26 of 27 cases (96.3%). Statistically significant differences were detected in the assessed expression parameters between those proteins. Conclusions Our results suggest that overexpression of wild type p53 protein may suppress the expression of survivin and its antiapoptotic activity in BCC cells.