Sustained gene expression in transplanted skin fibroblasts in rats

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Absorption of sunscreens across human skin: an evaluation of commercial products for children and adults

Aims Topical sunscreens are routinely applied to the skin by a large percentage of the population. This study assessed the extent of absorption of a number of common chemical sunscreen agents into and through human skin following application of commercially available products. Methods Sunscreen products were applied to excised human epidermis in Franz diffusion cells with the amount penetrating into and across the epidermis assessed by h.p.l.c. for 8 h following application. Results All sunscreen agents investigated penetrated into the skin (0.25 g m(-2) or 14% of applied dose), but only benzophenone-3 passed through the skin in significant amounts (0.08 g m(-2) or 10% of the applied dose). With one exception, suncreen agents in corresponding products marketed for adults and children had similar skin penetration profiles. Conclusions Whilst limited absorption across the skin was observed for the majority of the sunscreens tested, benzophenone-3 demonstrated sufficiently high penetration to warrant further investigation of its continued application.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis, To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes, Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes, Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Kinetics of propranolol uptake in muscle, skin, and fat of the isolated perfused rat hindlimb

The tissue distribution kinetics of a highly bound solute, propranolol, was investigated in a heterogeneous organ, the isolated perfused limb, using the impulse-response technique and destructive sampling. The propranolol concentration in muscle, skin, and fat as well as in outflow perfusate was measured up to 30 min after injection. The resulting data were analysed assuming (1) vascular, muscle, skin and fat compartments as well mixed (compartmental model) and (2) using a distributed-in-space model which accounts for the noninstantaneous intravascular mixing and tissue distribution processes but consists only of a vascular and extravascular phase (two-phase model). The compartmental model adequately described propranolol concentration-time data in the three tissue compartments and the outflow concentration-time curve (except of the early mixing phase). In contrast, the two-phase model better described the outflow concentration-time curve but is limited in accounting only for the distribution kinetics in the dominant tissue, the muscle. The two-phase model well described the time course of propranolol concentration in muscle tissue, with parameter estimates similar to those obtained with the compartmental model. The results suggest, first that the uptake kinetics of propranolol into skin and fat cannot be analysed on the basis of outflow data alone and, second that the assumption of well-mixed compartments is a valid approximation from a practical point of view las, e.g., in physiological based pharmacokinetic modelling). The steady-state distribution volumes of skin and fat were only 16 and 4%, respectively, of that of muscle tissue (16.7 ml), with higher partition coefficient in fat (6.36) than in skin (2.64) and muscle (2.79. (C) 2000 Elsevier Science B.V. All rights reserved.

Probability of axillary node involvement in patients with tubular carcinoma of the breast

The aim of this study was to investigate the frequency of axillary metastasis in women with tubular carcinoma (TC) of the breast. Women who underwent axillary dissection for TC in the Western Sydney area (1984-1995) were identified retrospectively through a search of computerized records. A centralized pathology review was performed and tumours were classified as pure tubular (22) or mixed tubular (nine), on the basis of the invasive component containing 90 per cent or more, or 75-90 per cent tubule formation respectively. A Medline search of the literature was undertaken to compile a collective series (20 studies with a total of 680 patients) to address the frequency of nodal involvement in TC. A quantitative meta-analysis was used to combine the results of these studies. The overall frequency of nodal metastasis was five of 31 (16 per cent); one of 22 pure tubular and four of nine mixed tumours (P = 0.019). None of the tumours with a diameter of 10 mm or less (n = 16) had nodal metastasis compared with five of 15 larger tumours (P = 0.018). The meta-analysis of 680 women showed an overall frequency of nodal metastasis in TC of 13.8 (95 per cent confidence interval 9.3-18.3) per cent. The frequency of nodal involvement was 6.6 (1.7-11.4) per cent in pure TC (n = 244) and 25.0 (12.5-37.6) per cent in mixed TC (n = 149). A case may be made for observing the clinically negative axilla in women with a small TC (10 mm or less in diameter).

Treatment implications of a positive sentinel lymph node biopsy for patients with early-stage breast carcinoma

BACKGROUND. Sentinel lymph node (SLN) mapping and biopsy is emerging as an alternative to axillary lymph node dissection (ALND) in determining the lymph node status of patients with early-stage breast carcinoma. The hypothesis of the technique is that the SLN is the first lymph node in the regional lymphatic basin that drains the primary tumor. Non-SLN (NSLN) metastasis in the axilla is unlikely if the axillary SLN shows no tumor involvement, and, thus, further axillary interference may be avoided. However, the optimal treatment of the axilla in which an SLN metastasis is found requires ongoing evaluation. The objectives of this study were to evaluate the predictors for NSLN metastasis in the presence of a tumor-involved axillary SLN and to examine the treatment implications for patients with early-stage breast carcinoma. METHODS. Between June 1998 and May 2000, 167 patients participated in the pilot study of SLN mapping and biopsy at Westmead Hospital. SLNs were identified successfully and biopsied in 140 axillae. All study patients also underwent ALND. The incidence of NSLN metastasis in the 51 patients with a SLN metastasis was correlated with clinical and pathologic characteristics. RESULTS. Of 51 patients with a positive SLN, 24 patients (47%) had NSLN metastases. The primary tumor size was the only significant predictor for NSLN involvement. NSLN metastasis occurred in 25% of patients (95% confidence interval [95%CI], 10-47%) with a primary tumor size less than or equal to 20 mm and in 67% of patients (95%CI, 46-83%) with a primary tumor size > 20 mm (P = 0.005). The size of the SLN metastasis was not associated significantly with NSLN involvement. Three of 7 patients (43%) with an SLN micrometastasis (< 1 mm) had NSLN involvement compared with 38 of 44 patients (48%) with an SLN macrometastasis (greater than or equal to 1 mm). CONCLUSIONS. The current study did not identify a subgroup of SLN positive patients in whom the incidence of NSLN involvement was low enough to warrant no further axillary interference. At present, a full axillary dissection should be performed in patients with a positive SLN. (C) 2001 American Cancer Society.