Organ Perfusion for Uterus Transplantation in Non-Human Primates With Assumed Procurement of a Uterus From a Brain-Dead Donor.

BACKGROUND: Clinical studies of uterus transplantation have been performed to treat uterine factor infertility. Because the uterus is a pelvic visceral organ, the method of perfusion for the procurement of vital organs from a brain-dead donor should be modified for removal of the uterus. Herein, we report the results of a preliminary study in cynomolgus monkeys of a new perfusion method for uterus transplantation with assumed procurement of a uterus from a brain-dead donor. METHODS: Cynomolgus monkeys were used; thoracolaparotomy was performed on the donor. A perfusion catheter was then placed into the unilateral femoral artery and/or external iliac artery. Cross-clamping was performed for the aorta under the diaphragm and the inferior vena cava was divided in the pleural space. The perfusion solution was then administered via the catheter to perfuse all organs in the abdominal cavity, including those in the pelvic cavity. After the perfusion, gross observation and histopathological examination of abdominal organs were conducted. RESULTS: Gross findings showed that all abdominal organs turned white in all specimens, indicating favorable perfusion of the uterus and all other organs in the abdomen. Pathological findings showed that almost no hemocytes were observed in the vessels of each organ. CONCLUSIONS: With perfusion via the femoral artery and/or external iliac artery, all organs in the abdominal cavity, including the uterus, could be perfused. It was suggested that this technique could be useful for uterus transplantation assuming the procurement of a uterus from a brain-dead donor.

Recurrent herpes simplex virus infection in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy associated with L29P and IVS9-1G>C compound heterozygous autoimmune regulator gene mutations.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) has characteristic clinical features with organ-specific autoimmune polyendocrine diseases and candidiasis, caused by the mutations of autoimmune regulator (AIRE) gene. Although almost all patients are complicated with mucocutaneous candidiasis, no apparent susceptibility to other infections has yet been reported. We herein report that a patient with APECED suffered from recurrent herpes simplex virus type 1 (HSV-1) infection after severe primary herpetic stomatitis, associated with sequential HSV-1 isolates of the same genomic profile, consistent with endogeneous recurrence. Thus, not only candidiasis but also HSV infection should receive more attention in patients with APECED, with treatment being administered accordingly.

Cleavage in and around the DR1 element of the A sequence of herpes simplex virus type 1 relevant to the excision of DNA fragments with length corresponding to one and two units of the A sequence.

The A sequence of herpes simplex virus type 1 (HSV-1) is a region bracketed by two direct repeats named DR1. Concatemeric HSV-1 DNA, the product of DNA replication, is cleaved at a specific site on the second DR1 distal from the S component (authentic cleavage) to yield unit-length linear HSV-1 DNA prior to or during packaging of HSV-1 DNA. The presence of two DNA bands, of 0.25 kb (shorter band) and 0.5 kb (longer band), the lengths of which correspond to one and two units of the A sequence, was identified using acrylamide gel electrophoresis of HSV-1 DNA preparations extracted by the method of Hirt. Twelve DNA fragments from each band were molecularly cloned, and nucleotide sequences were determined. Both termini of eight (67%) DNA clones from the shorter band corresponded to the specific cleavage site on DR1. Five (41%) DNA clones from the longer band had a terminus corresponding to the specific cleavage site on DR1 on one side, but not on the opposite side. Thirteen (54%) of 24 termini of 12 analyzed DNA clones from the longer band were in and around DR1. Thus, cleavage events of DR1 can be classified into three categories: (i) authentic cleavage; (ii) site-specific cleavage on the third DR1 distal from the S component (secondary site-specific cleavage), which is related to the generation of the shorter DNA band in combination with authentic cleavage; and (iii) less-specific cleavage events in and around other DR1 elements which relate to the generation of the longer DNA band.

Molecular epidemiology of herpes simplex virus type 1 genital infection in association with clinical manifestations.

The antigenic types of herpes simplex virus (HSV), HSV-1 and HSV-2 are considered to be the etiology of genital herpes. Symptoms of primary HSV-1 and HSV-2 genital infections are similar, however, recurrence of the infection is less frequent after the HSV-1-related genital infection. We determined genotypes of 79 HSV-1 strains isolated from genital lesions in women (43 from primary and 36 from recurrent infections), by analyzing restriction fragment length polymorphism of the HSV-1 strains. Each proportion of genotypes of F1, F12, and F41 in strains isolated from recurrent genital lesions was higher than the corresponding proportion in strains from primary genital lesions. Genotypes of HSV-1 strains isolated two or more times from recurrent genital lesions of each of three subjects were genotype F1, thereby supporting the hypothesis that the F1 genotype is closely associated with recurrence. While the possibility of a genotype preference at the site of infection was not ruled out, genotypes of more than half the number of HSV-1 strains from genital lesions were the same as those from non-genital (mainly oral-facial) lesions analyzed in our foregoing studies, thus indicating that most HSV-1 genotypes are apparently shared by genital and non-genital lesions.

[Study of alpha-herpesvirus: present state and future developments].

The herpesvirus is a ubiquitous infectious agent present in a variety of vertebrates including humans. The virus has a duplex DNA molecule of 120-240 kbp within an icosahedral capsid surrounded by an envelope. Latency is a central feature of herpesvirus biology. Studies of herpesviruses relate to 1) diagnosis, prevention, and the treatment of diseases associated with herpesvirus infections, 2) elucidation of biological mechanisms of mammalian cells using herpesviruses, 3) construction of vaccine and vector systems for gene therapy, and 4) establishment of putative associations of herpesviruses with host organisms during evolution (suggesting the host-linked evolution of herpesvirus).

[Structure and classification of alpha-herpesvirus].

Virion of herpesviruses (100 to 200 nm in diameter) consists of a core containing a linear double-stranded DNA, an icosahedral capsid, an amorphous layer designated as the tegument which surrounds the capsid, and a protein-containing lipid membrane designated as the envelope. Terminal and internal reiterated sequences are present on herpesvirus genomes, giving a diverse range of DNA structures. The family Herpesviridae is divided into three subfamily of Alpah-, Beta- and Gammaherpesvirinae. Herpesviruses seem to have diversified from a common ancestor, in a manner mediating co-speciation of herpesviruses with host species through species-specific latent infection.

Restriction fragment length polymorphism analysis using random chromosomal gene probes for epidemiological analysis of Campylobacter jejuni infections.

We have evaluated the ability of a new genotyping method for Campylobacter jejuni based on restriction fragment length polymorphisms using random chromosomal gene probes. DNAs from C. jejuni strains digested with each of three restriction enzymes, HhaI, HaeIII, and HpaII, were analyzed by Southern hybridization using each of two unrelated cosmid clones, P14 and P15 (respectively containing 30- and 35-kb genomic DNA fragments of C. jejuni strain OH4384). The method reported provides a stable and discriminating means for identifying C. jejuni strains and should be useful for epidemiological analyses.

Mechanism and application of genetic recombination in herpesviruses.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that latently infects sensory ganglia and encodes over 80 genes in a 152 kbp DNA genome. This well characterised virus provides a model for analysing genetic recombination in herpesviruses, a fundamental biological process by which new combinations of genetic materials are generated. The frequency of homologous recombination was estimated to be 0.0048-0.007 (0.48%-0.7%)/kb of the HSV-1 genome, determined using physical markers. The double-strand break repair model, the current model of homologous recombination, adequately explains L-S inversion of herpesvirus genomes and the recombinogenicity of the a sequence. Several herpesvirus genomes, including HSV-1 consist of a unique sequence bracketed by a pair of inverted repeat sequences. This arrangement is attributed to illegitimate recombination between molecules arranged in an inverse orientation. Junctions of unique and repeated sequences that correspond to the crossover site of illegitimate recombination are recombinogenic. Recombination is important for virus evolution, construction of mutated virus, gene therapy and vaccination in which the potential for recombination between engineered input virus and wild type virus has to be considered.

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.

Evolution of herpes simplex virus type 1 under herpesviral evolutionary processes.

Herpesviruses, the genomes of which are double-stranded DNA of 120 kilobase pairs or more, infect a wide range of vertebrates from mammals to fish. Herpes simplex virus type 1 (HSV-1), a representative of family Herpesviridae, is a ubiquitous human pathogen. HSV-1 relates to common mucocutaneous diseases, while HSV-1 infection can mean a serious outcome, e.g. blindness and insult to the central nervous system. Evolution of herpesviruses includes DNA rearrangements, often generating tandemly or invertedly repeated sequences. Studies of HSV-1 DNA dynamics substantiated these processes of DNA recombination involved in the evolution of herpesvirus. Herpesviruses seem to have diversified from a common ancestor, in a manner mediating co-speciation of herpesviruses with host species through species-specific latent infections. Thus, the notion of host-linked evolution of herpesviruses is given support. Relationships between HSV-1 genotypes and human ethnic groups can be traced by analyses of DNA polymorphisms of HSV-1 strains present in populations of various countries. A close association of an HSV-1 genotype with a particular historical human population seems probable. Such being the case, the host-linked mode is likely to be linked to diversification of HSV-1 in human populations.

Populations of two eastern countries of Japan and Korea and with a related history share a predominant genotype of herpes simplex virus type 1.

Herpes simplex virus type 1 (HSV-1), a common human pathogen of non-epidemic nature is linked closely to the individual by latent infection. HSV-1 genotypes usually differ with race. Based on a "dual structure model" for population history of the Japanese, modern Japanese populations are assumed to have derived from two major migration events. The Jomon people arrived in Japan > 10,000 years ago and the Yayoi people began migrating to Japan from the Korean peninsula approximately 2,300 years ago. The presence of two predominant genotypes of F1 and F35 was noted in HSV-1 strains isolated in Japan. As the F1 genotype also predominated in Korea, peoples of Japan and Korea share the F1 genotype. Regional differences in the frequency of F1 and F35 genotypes within Japan seem to relate to differences in the dispersion of descendants of the Yayoi and Jomon peoples. Our hypothesis is that the F35 genotype relates to the Jomon people, earlier residents in japan, while the F1 genotype relates to the Yayoi people who migrated much later from the Korean peninsula.

Inhibition of generation of authentic genomic termini of herpes simplex virus type 1 DNA in temperature-sensitive mutant BHK-21 cells with a mutated CCG1/TAF(II)250 gene.

A temperature-sensitive (ts) mutant from the BHK-21 hamster cell line, tsBN462, has a defect in progression of the G1 phase at the nonpermissive temperature of 39.5 degrees C. The ts mutation in tsBN462 is located in the CCG1 gene, encoding the general transcription factor TAF(II)250. In tsBN462 at 39.5 degrees C, infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and generation of authentic genomic termini of HSV-1 was inhibited. HSV-1 concatemers containing L components in two possible orientations were produced in tsBN462 at 39.5 degrees C; hence, the generation of authentic genomic termini seemed to be dispensable for inversion of the L component. As production of mRNAs of HSV-1 genes of three kinetic classes in the tsBN462 at 39.5 degrees C was comparable to findings under permissive conditions, the sequential and regulated manner in which HSV-1 gene expression is processed is likely to be maintained in the nonpermissive condition.

Replication of herpes simplex virus type 1 DNA is inhibited in a temperature-sensitive mutant of BHK-21 cells lacking RCC1 (regulator of chromosome condensation) and virus DNA remains linear.

tsBN2, a temperature-sensitive (ts) growth mutant of the hamster cell line BHK-21, has a point mutation in the RCC1 (regulator of chromosome condensation) gene, and prematurely enters mitosis at 39.5 degrees C, a nonpermissive temperature. In this mutant at 39.5 degrees C infectious progeny of herpes simplex virus type 1 (HSV-1) was not produced and replication of HSV-1 DNA was inhibited. HSV-1 DNA from virus particles is normally circularized upon infection, and circularized HSV-1 DNA molecules can serve as template for DNA replication. In tsBN2 at 39.5 degrees C, HSV-1 DNA appeared to remain linear after infection, suggesting the obstruction of HSV-1 DNA circularization, which could account for failure of HSV-1 DNA replication. In transient replication assays performed in tsBN2 at 39.5 degrees C, through superinfection with HSV-1 helper virus, there was no evidence of replication of circular DNA of the hybrid plasmid containing the HSV-1 replication origin. Production of mRNAs of HSV-1 early genes required for HSV-1 DNA replication was decreased in tsBN2 at 39.5 degrees C. Therefore, RCC1 was assumed to be involved in the formation of an HSV-1 DNA configuration suitable for replication (that is circularization) and the supply of proteins required for replication of the circularized HSV-1 DNAs.

Comparison of the association with eczema herpeticum in the two predominant genotypes of herpes simplex virus type 1.

Eczema herpeticum, sometimes called Kaposi's varicelliform eruption, is usually caused by a disseminated herpes simplex virus infection in a patient whose underlying skin disease in atopic dermatitis. Herpes simplex virus type 1 (HSV-1), a widespread infectious agent in human populations, is the etiologic agent of eczema herpeticum. Analyses of restriction fragment length polymorphism (RFLP) of HSV-1 strains isolated in Japan, using restriction endonucleases, revealed the presence of two predominant genotypes of F1 and F35. The number of HSV-1 strains of F1 genotype was over twice that of the F35 genotype, and the nucleotide change between F1 and F35 was estimated to be 1.5%. The question of whether the genomic difference between two predominant genotypes could influence clinical manifestations remained to be addressed. On the basis of RFLP, we determined genotypes of HSV-1 strains isolated from the patients in Japan, including those with eczema herpeticum. Two of four HSV-1 strains of F35 genotype were from patients with eczema herpeticum, whereas none of 12 HSV-1 strains of F1 genotype was from those with eczema herpeticum. Thus, the F35 genotype seemed to be associated more frequently with eczema herpeticum than the F1 genotype.

Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress.

The ferritin-encoding gene (cft) of Campylobacter jejuni was cloned and sequenced. The nucleotide sequence of cft had a 501 bp open reading frame for a protein with 167 amino acids and a predicted molecular mass of 19 180 Da, and showed a high similarity to that of Helicobacter pylori and Escherichia coli ferritin genes. To determine the biological function of ferritin in C. jejuni, a ferritin-deficient mutant was constructed. The growth of ferritin-deficient strain SNA 1 was clearly inhibited under iron deprivation. The ferritin-deficient mutant was more sensitive to killing by H2O2 and paraquat than the isogenic parent strain. These findings demonstrate that ferritin in C. jejuni makes a significant contribution to both iron storage and protection from intracellular iron overload, and resulting iron-mediated oxidative stress.