We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high-and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.Accumulating evidence indicates that persistent infection with high-risk human papillomaviruses (HPVs) is indeed a major causative factor in the development of cervical intraepithelial neoplasia and invasive cervical carcinoma (42,10,8,5,40,27,11,30). The HPV family includes over 100 genotypes, 30 to 40 of which are mucosotropic, and at least 15 types of the mucosotropic HPVs have been linked to cervical cancer (5,8,10,42). In addition, some of these types are also related to other cancers of the genital tract (21,22) and to cancers of other organs (14, 28). Light microscopic examination of a Papanicolaou (Pap)-stained smear is of primary importance for the detection of cervical cancer precursor lesions. It has been demonstrated that concomitant testing for DNA of the highrisk HPV types by the Pap test can clearly identify women at high risk for cervical cancer, particularly if persistent infection by high-risk HPV types is diagnosed (5,8,10,11,27,30,40). Furthermore, HPV genotyping is of critical importance for the investigation of the clinical behavior and the epidemiology of HPV infection, for population studies for HPV vaccination trials, and for monitoring of the efficacy of HPV vaccines. Several genotyping methods have been developed in order to identify high-risk HPV in liquid-based cytology (LBC) samples and tissue samples (1,12,34). The molecular techniques that have been applied for HPV DNA detect...

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1. In order to address the hypothesis that migrating fibroblasts have a different phenotype, human fetal lung fibroblasts (HFL-1) cells were evaluated in the Boyden blindwell chamber migration assay followed by immunoelectron microscopy. 2. HFL-1 cells were placed on nucleopore filters and incubated for 2 h using purified human plasma fibronectin (pFn) as a chemoattractant. Filters were then processed for immunoelectron microscopy using antibodies for alpha-smooth muscle (alpha-SM) actin as a marker for myofibroblasts, cellular fibronectin (cFn) and VLA-5. 3. Cells which had migrated to the bottom side of the filter were more likely to express alpha-SM actin, 29.1 +/- 3.4% of cells, compared with cells which did not migrate through the filter, 12.4 +/- 1.3% (P < 0.05). The total proportion of alpha-SM-actin-positive cells located on both sides of the filter showed no difference between those which had migrated toward pFn and controls (17.7 +/- 1.0% compared with 20.2 +/- 2.5%). 4. cFn-positive cells showed minimal differences compared with control cells, while perinuclear and endoplasmic reticulum staining of VLA-5 was observed only in the cells treated with pFn. 5. The results show that HFL-1 cells are heterogenous for alpha-SM actin expression. Short-term incubation with pFn did not change the proportion of alpha-SM-actin-positive HFL-1 cells. Cells which migrate, however, are enriched for alpha-SM actin expression. pFn-induced fibroblast chemotaxis can selectively recruit myofibroblasts with increased alpha-SM actin expression, a feature which may contribute to the altered population of cells at sites of fibrosis.

It is well known that corneal stroma cells (keratocytes) can transform to fibroblasts during the corneal wound healing. We have studied the expression of α-smooth muscle actin, vimentin and desmin in fibroblastic cells of the alkali-burneo and lacerated corneas in the rabbits by means of immunohistochemistry and electron microscopy.Methods. Rabbits were anesthetized, and central corneal alkali-burn and laceration were produced. The injured corneas healed for 1 day to 45 days, and 18 days embryonic rabbit corneas were immunostained with monoclonal antibodies against α-smooth muscle actin, vimentin and desmin. For transmission electron microscopy, two techniques were used for the staining of ultra-thin sections: 1) uranyl acetate and Reynold's lead citrate and 2) the tannic acid method of Kajikawa.Results. Antibody against α-smooth muscle actin reacted with fibroblastic cells in injured corneas (Fig 1) but not with stromal cells in normal and embryonic corneas. Antivimentin antibody strongly reacted with the fibroblastic cells in injured corneas (Fig 2) and stromal cells in normal and embryonic corneas.