In vivo gene transfer: focus on the kidney

Renal gene transfer techniques are being developed as a novelexperimental approach to understand the pathogenesis of renaldisease and to potentially develop new therapeutic tools. Wereview the currently available technology to introduce foreigngenetic material into renal tissue, i.e., retroviral, adenoviral,and liposomal transfer systems with their respective advantagesand caveats. Today, the transfer efficiency of these methodsappears to be sufficiently high to study the effects of transducedgenes on renal function and morphology in rat kidney. This willallow (i) the elucidation of the function of genes on the courseof renal disease in experimental animal models and (ii) themodulation of local expression of endogenous genes which presumptivelycontribute to renal pathology in these models. One strategyto accomplish this aim is the use of recombinant DNA technologyto design antisense DNA constructs or oligonucleotides, whichinterfere with the renal expression of target genes. We willalso discuss some of the shortcomings of the currently usedtechniques with respect to potential therapeutic use of genetransfer systems and gene modulation.     

"Reverse" DMA hybridization method for the rapid identification of subgingival microorganisms

A "reverse" hybridization method is described, in which whole chromosomal DNA was extracted from 10-20 colonies of "unknown" strains in pure culture and labelled with digoxigenin by a random primer technique. DNA probes were prepared from a total of 23 strains and hybridized with targets containing 100 ng purified, denatured DNA from 38 reference strains fixed to nitrocellulose. 21/23 digoxigenin-labelled DNA probes successfully detected all members of the homologous species present on filters. Probes to Fusobacterium nucleatum strains 364 and MG detected 3/4 and 1/4 members of this species, respectively; 13/23 probes were 100% specific, but cross reactions between 10 probes and DNA targets from closely related, heterologous species occurred in 15/834 possible instances. False-positive reactions that occurred between closely related species were, however, easily distinguished and did not prevent the accurate identification of probe strains. Digoxigenin-labelled probes were capable of detecting 100 pg of homologous DNA. The reverse hybridization procedure allows identification or grouping of a large number of isolates within 3 days and provides a more economical means of characterizing subgingival isolates than predominant cultivable techniques and conventional phenotypic testing. This method could be adapted for the direct identification of microorganisms in subgingival plaque samples.     

Restriction endonuclease analysis of varicella-zoster vaccine virus and wild-type DNAs

The DNA from several clinical isolates of varicella-zoster virus (VZV) were compared with the DNA from the vaccine strain VZV using three restriction endonucleases: BamHI, BgII, and HpaI. When electrophoresed through an agarose gel, the vaccine DNA digestion pattern was significantly different from the digestion patterns of the wild-type DNAs. Variations in the digestion pattern of the separate clinical isolates were also observed.     

HLA-B44 subtyping in a Spanish population: further evidence of Caucasian population diversity

HLA-B44 is the most frequent HLA-B allele in Caucasian populations. Several B44 subtypes, B*4402-B*4406, have been identified in individuals with this ethnic origin. Mismatches among B44 subtypes have been described as major targets for allogeneic responses in bone marrow transplantation. We have developed a PCR-SSO method, based on a B12- specific DNA amplification of exon 2 through exon 3 and subsequent non radioactive hybridization with eight probes, which allow us to discriminate all B12 homozygous combinations. We applied this method to determine the frequency of B44 subtypes in a Spanish population, as well as their HLA-A.-C.-DRB1,-DRB3/DRB4/DRB5.-DQA1 and -DQB1 associated haplotypes. A total of 141 healthy unrelated Spanish individuals and 31 B44-bearing haplotypes were investigated. Four B44 alleles were identified, B*4402 (33%), B*4403 (66%), B*4404 (0.7%), and B*4405 (0.7%). Haplotype analysis showed a clear differentiated distribution pattern for the two major B44 subtypes. B*4402 is associated with Cw5 (11/13) and A2 antigens (10/13). In contrast, B*4403 is mainly found together with DRB1*0701 (14/16). An inverted B*4402/B*4403 frequency in comparison with other European and North American Caucasian populations, revealed the existence of an extended haplotype diversity between populations of the same ethnic origin. Apart from anthropological studies, high resolution typing for HLA class I antigens presenting molecular polymorphism will be of great relevance in unrelated bone marrow transplantation.     

鼻咽癌旁上皮细胞核DNA含量分析

应用显微分光光度计测定15例鼻咽癌19处癌旁病变的上皮细胞核DNA含量并与浸润癌相比较。中、重度异型增生上皮细胞核DI及超过2.5c细胞的百分数处于单纯增生+轻度异型增生与浸润癌之间,3组DI及超过2.5c细胞的百分数差异显著。中、重度异型增生以非整倍体为主,其细胞核DNA含量组方图相似于浸润癌。从细胞核DNA含量角度来看,中、重度异型增生是重要的癌前病变。     

Assessment of class-specific antibodies against denatured DNA in patients with systemic lupus erythematosus

In this retrospective study 103 serum samples from 16 females with systemic lupus erythematosus (SLE), obtained during a mean follow-up time of 2 years, were investigated for the presence of anti-denatured [single-stranded (ss)] DNA antibodies of the IgG, IgM, and IgA classes. The anti-ssDNA antibodies were determined by an enzyme-linked immunosorbent assay (ELISA), and the results were expressed in three ways: as units derived from a single serum dilution and as two parameters,E andA, calculated from the dose-response curve,E being an estimate of the effective amount of antibodies andA a function of the reaction constant between the antigen and the antibody. The simultaneous occurrence of anti-ssDNA antibodies of all three immunoglobulin classes was seen most often in the patients with the shortest duration of the disease. Clinically active disease was found to correlate with high reaction constants of the IgA anti-ssDNA antibodies. There was also an association between the IgA anti-ssDNA antibody levels and the presence of nephritis. Great fluctuations in the amounts of effective antibodies of the IgG class were seen in seven patients, in six of whom changes in the disease activity also were seen. Changes in the disease activity were unaccompanied by fluctuations in the IgG anti-ssDNA levels in four patients; two of these patients were positive for antibodies against extractable nuclear antigens. We conclude that it is of value to express the results of the anti-ssDNA ELISA as a function of the dose-response curve when monitoring patients with SLE and that immunoglobulin class-specific determinations of anti-ssDNA antibodies may provide information about the disease activity in many patients with SLE.     

Chromophobe renal cell carcinoma

Chromophobe renal cell carcinoma (RCC) is a recently established subtype of RCC, which has rarely been reported in Japan. In this communication, the authors report two Japanese cases of chromophobe RCC together with the immunohistochemical findings. The tumors were composed of sheets and cribriform glands formed by tumor cells with cloudy and reticular cytoplasm. Ultrastructurally, the cytoplasm was filled with numerous microvesicles. The tumor cells were positive for cytokeratin, epithelial membrane antigen, and Tamm-Horsfall protein. Occasionally, LeuM1-positive cells were also noted. Vimentin was negative, unlike the usual RCC. Reactivity for peanut agglutinin was more frequent than that to Lotus tetragonolobus agglutinin. The results of this study suggest that the tumor cellq possessed phenotypes similar to the distal nephron rather than to the proximal tubular cells.     

Establishment and Characterization of Cell Lines from Human Adenovirus Type 12-induced Murine Tumors Producing Endogenous Virus Particles

Two cell lines designated IC KMS and D KMS were established from human adenovirus type 12 induced tumors of C3Hf/OK mouse. The cell lines retained the characteristics of the original tumor i.e., production of numerous C type and intracisternal A-type particles, integration of Adl2 El region DNA and amplification of the myc gene family. Chromosomal analysis revealed chromosome aberrations in both IC KMS and D KMS cells. The modal chromosome number of IC KMS cells was 54 and that of D-KMS cells was 48. Metacentric chromosomes and mini-chromosomes were found. Trisomy of chromosome 3, 7 and 12 was seen frequently in D KMS cells. Although DNA aneuploidy was revealed by flow cytometry, the DNA indices of these cells showed no relation to the copy number of integrated Adl2 DNA. These cells have been propagated by serial culture during the past 17 months. Production of endogenous virus particles is a unique characteristic of IC KMS and D KMS cells. These cell lines would be useful materials for examining the contribution of Adl2 carcinogenesis to activation of endogenous virus particles, and also the correlation between Adl2 carcinogenesis and cancer related genes. Acta Pathol Jpn 42: 242-248, 1992.