A major determinant in the virulence of Salmonella and Shigella spp. is the ability of these organisms to invade epithelial cells of the gastrointestinal mucosa and multiply intracellularly. The invasion of cell culture monolayers is a convenient experimental system to evaluate eucaryotic cell penetration and is correlated with the potential of a strain to cause human disease. We have developed an agarose-L agar overlay technique which allows for the convenient quantitation of the number of infected tissue culture cells in a monolayer. Bacterial strains were introduced onto antibiotic-free HleLa cell monolayers. Infected monolayers were washed, and noninternalized bacteria were counterselected with kanamycin (50 ,ug/ml). The number of infected HeLa cells present was determined by overlaying the monolayer with distilled water-agarose (0.5 to 1.5%) followed by an equal volume of 2X L agar. Bacterial colonies formed over infected cells in 24 h at 37°C, and wells were counted with a dissecting microscope under x 2 power. Bacterial colonies were not observed with noninvasive variants of Shigella spp. To obtain countable wells (20 to 200 CFU) the multiplicity of infection or invasion times were adjusted. With a 90-min invasion time, the invasive potential of a strain was reflected by the multiplicity of infection needed to produce countable wells. The quantitation of bacterium-invaded cells by using standard bacteriological methods is a convenient and rapid method to evaluate the invasive potential of bacterial strains. Additionally, parameters essential for the invasive process can easily be investigated.

The expression pattern of transforming growth factor-beta 1 (TGF-beta 1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5%, 7,12-dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF-beta 1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF-beta 1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF-beta 1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF-beta 1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF-beta 1 was not detected in these tumors. The expression of TGF-beta 1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF-beta 1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF-beta 1 has functions other than its growth-repressing activity.

The presence of an activating mutation in the Ha-ras gene in hamster cheek pouch tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) complete carcinogenesis was investigated. The normal sequence of a fragment of genomic DNA encompassing codon 61 of the Ha-ras gene was amplified by the polymerase chain reaction using primers designed for a highly conserved region of the mouse Ha-ras-1 gene. The sequence of the amplified fragment was determined by a direct sequencing technique and exhibited 83.3% and 87.5% homology with the corresponding human and mouse sequences, respectively. At the amino acid level, the sequence was identical among the three species. Paraffin sections of 11 squamous cell carcinomas of the cheek pouch were used to detect mutated Ha-ras alleles. DNA sequencing of the tumors showed that six of 11 tumors presented an A----T transversion in the second position of codon 61, resulting in an amino acid change from glycine to leucine. As has been demonstrated in other systems, we have shown a specific mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch, further supporting the role of this oncogene in chemical carcinogenesis.

We have previously shown that the precursors of cutaneous papillomas in SENCAR mice initiated with 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate are focal hyperplastic lesions that we refer to as squamous cell hyperplastic foci (SCHF). Ha-ras gene codon 61 mutations were frequently found in SCHF, providing evidence that these lesions represent clones of initiated cells. We report here the pathogenesis of multiple hair follicle involvement in more advanced SCHF and describe the role of the hair follicle in papilloma histogenesis. Detailed histological evaluation of 83 SCHF and 25 early papillomas revealed a morphological continuum from the least developed SCHF, involving only one hair follicle, to advanced SCHF and early papillomas, which involved more than 10 hair follicles. These results provide evidence of the recruitment of additional hair follicles as SCHF progress. In advanced SCHF and early papillomas the bulk of the epithelial component in all cases consisted of several markedly hyperplastic adjacent hair follicles, whereas the involved interfollicular epidermis (IFE) was generally less hyperplastic. All of the hair follicles involved in SCHF appeared to have been preexisting, based on their pattern of spacing, that they were consistently normal appearing below the level of the sebaceous glands, and that they were in the same phase of the hair cycle as surrounding, uninvolved hair follicles. Also, no evidence of follicular neogenesis was observed in serially sectioned SCHF, and coalescence of smaller lesions was rare. To investigate whether the involvement of multiple hair follicles in SCHF was due to expansion of initiated cells into existing hair follicles or, possibly, to a paracrine mechanism, we analyzed different levels of three serially sectioned SCHF and one early papilloma for Ha-ras mutations. These analyses revealed cells with Ha-ras gene codon 61 mutations at multiple levels that involved different hair follicles. Overall, our results provide evidence that as initiated cells clonally expand, they spread across the IFE and populate the upper permanent portions of existing hair follicles. The abnormal proliferation of the infundibula of the hair follicles involved in SCHF appears to give rise to most of the epithelial component of papillomas.

A 2.15‐kb Pst I fragment of the 140‐megadalton (MDa) plasmid of Shigella flexneri which encodes Congo red (CR) dye binding activity was isolated by recombinant techniques. The recombinant plasmid was shown to encode the structural gene for CR binding but failed to restore the ability of noninvasive strains to penetrate HeLa cell monolayers. A partial restriction map was constructed and polypeptides specified by the 140‐MDa insert DNA identified in a minicell system. The putative polypeptide responsible for CR binding activity was identified by its thermoregulated expression in minicells. The Mr of the putative CR binding protein was 24000.