Clinicopathological and prognostic relevance of Rap1-GAP expression in melanocytic tumors

Rap1-GAP protein has been identified as an inactivator of Rap1 activity, a putative endogenous antagonist of Ras proteins. The Rap1-GA1 locus maps to 1p36.1–35, the region which may harbor a gene for familial melanoma. In the present immunohistochemical study we analyzed the clinicopathological and prognostic relevance of Rap1-GAP expression in 60 benign and 103 malignant melanocytic tumors. Cytoplasmic immunoreactivity was detected in the cells of 27/60 nevi (45%) and 59/103 melanomas (57%). In the latter group the frequency of Rap1-GAP expression increased ( P < 0.05) with the thickness of primary tumors and was highest in metastatic lesions. Rap1-GAP protein was detected in 15/19 subsequently recurring primary melanomas (79%) but only in 32/67 tumors (47%) of patients who remained free of disease ( P < 0.05) for at least 6 years. Five out of six recurring thin melanomas (< 2 mm) were found to be immunoreactive. Although being no indicator for malignant transformation of melanocytic lesions, Rap1-GAP overexpression may represent a useful marker for identifying thin high-risk melanomas. Cytoplasmic expression of Rap1-GAP has also been observed in the cells of skin appendages and in keratinocytes, particularly in suprabasal layers of the epidermis. Therefore, Rap1-GAP is likely to be associated with cellular growth and/or differentiation. However, the present study did not provide evidence that this gene, despite its chromosomal localization, represents an early melanoma gene. Received: 16 September 1996     

Blockade of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) down-regulates induction of contact sensitivity by haptenated epidermal cells

The hapten, trinitrobenzene sulphonic acid, induced weak B7-1 (CD80) and moderate B7-2 (CD86) expression on Langerhans cells and mRNA expression of both molecules in organ-cultured murine skin. The intradermal injection of hapten-treated epidermal cells induced hapten-specific contact sensitivity in synergic mice. Cells of the keratinocyte cell line, Pam 212, or epidermal cells treated with a mixture of anti-Ia/thy 1·2/γδ antibody plus complement, did not show any sensitizing ability. When hapten-treated epidermal cells were injected into mice after incubation with anti-B7-2 (CD86) or B7-1 (CD80) antibody the resultant contact sensitivity reaction was decreased to less than 50% of the control reaction, a reduction which was similar to that seen with the anti-ICAM-1 and anti-LFA-1 antibody-induced inhibition of contact sensitivity. Anti-B7-1 (CD80) or anti-B7-2 (CD86) antibody also inhibited hapten-specific lymphocyte proliferation or the allogenic mixed lymphocyte and epidermal cell reaction in vitro, although the inhibitory effect of anti-B7-1 antibody was not as significant as that of anti-B7-2 antibody. These results indicate that costimulatory signals induced by a hapten on epidermal Langerhans cells play an important role in the induction of hapten-specific contact sensitivity in mice.     

Failure of periodic ultraviolet radiation treatments to prevent sensitization to nitrogen mustard: a case report

We have reported that prior ultraviolet radiation (UVR) exposure significantly delayed development of contact sensitivity to nitrogen mustard (Halprin et al. 1981). We felt that this effect was due to disruption of functional Langerhans cells in skin by UVR and suggested that periodic UVR treatments might prevent sensitization to the mustard. We now report on a patient with mycosis fungoides whose epidermal Langerhans cell count was monitored with the ATP-ase stain in order to determine when such ‘booster’ UVR therapy was to be given. Our attempts to interfere with Langerhans cell function in this manner failed to prevent delayed contact sensitivity to nitrogen mustard and may have been partly responsible for the development of contact urticaria to nitrogen mustard after 28 days of use. Whether the reaction was a delayed, cell-mediated reaction, or an antibody mediated reaction is not clear, but the use of UVR did fail to prevent contact sensitivity to the nitrogen mustard in our patient.     

Comparison of patch test contact sensitivity to acetone and aqueous extracts of Parthenium hysterophorus in patients with airborne contact dermatitis

Our aim was to compare the degree of patch test positivity to acetone and aqueous extracts of Parthenium hysterophorus in patients with airborne contact dermatitis. We performed patch testing with the Indian standard series (which includes aqueous extracts of parthenium, xanthium and chrysanthemum), and with 1 : 100 and 1 : 200 dilutions of an acetone extract of parthenium in 72 patients with airborne contact dermatitis. All patients showed contact sensitivity to the 1 : 100 dilution and 67 patients had positive allergic reactions to the 1 : 200 dilution of the acetone extract, whereas only 45 patients showed a positive reaction to the aqueous extract of P. hysterophorus. Our results confirm that parthenium allergens are more soluble in acetone than in water, and that the acetone extract is significantly better in detecting contact sensitivity to parthenium in patients with suspected plant dermatitis. Hence, the acetone extract is recommended for routine patch testing.     

Aluminium allergy in patients hyposensitized with aluminium-precipitated antigen extracts

During hyposensitization therapy with aluminium-precipitated antigen solutions, a small % of patients develop persistent subcutaneous nodules at the injection site; the existence of delayed sensitivity to aluminium has been implicated in the pathogenesis of these nodules. We studied the prevalence of aluminium sensitivity (using patch, prick and intradermal tests) and common contact allergens (TRUE Test™) in 20 healthy subjects, and in 40 patients treated with aluminium-containing extracts, 20 of whom had persistent subcutaneous nodules that remained for more than 2 months, the other half having no nodular reactions or nodules that remained for less than 2 months. Aluminium sensitivity was found only in those patients of the treated group who had persistent nodular reactions, 4 cases of positivity to an aluminium chloride patch test being found. All 4 cases were women, nodules remained for more than 6 months, and intracutaneous tests were negative. 3 of them also had contact sensitivity to nickel. In 2 cases, nodules were removed for histological and histochemical examination, showing non-specific inflammatory granulomas, and aluminium crystals being found in only 1 case. It is concluded that delayed sensitivity to aluminium appears to be implicated in the pathogenesis of persistent nodular reactions, but sensitivity to aluminium was not found in patients treated with aluminium-precipitated extracts without persistent nodular reactions.     

Possible functional impairment of Langerhans' cells in vitiliginous skin. Reduced ability to elicit dinitrochlorobenzene contact sensitivity reaction and decreased stimulatory effect in the allogeneic mixed skin cell lymphocyte culture reaction

We used patch testing to compare the ability to elicit contact sensitivity to dinitrochlorobenzene (DNCB) of uninvolved with vitiliginous skin of 31 patients with vitiligo. The induction of DNCB contact sensitivity was possible in the vitiliginous skin in the same way as in normal skin. The DNCB contact sensitivity reactions, however, were generally diminished in vitiliginous skin, although the number of cases showing similar DNCB contact reactivity between normal and vitiliginous skin increased when the sensitization procedure was performed in vitiliginous skin instead of normal skin. On the other hand, delayed skin reactions to intradermally injected Candida albicans antigen were not suppressed in vitiliginous skin. The number of Langerhans' cells was not decreased in vitiliginous skin as compared with that of normal skin. The epidermal cells derived from vitiliginous skin, however, tended to show a lower stimulatory effect in the allogeneic mixed skin cell lymphocyte culture reaction than those from normal skin. These results suggest a possibility of functional impairment of Langerhans' cells in vitiliginous skin.     

Suppression of mouse contact hypersensitivity after treatment with antibodies to leukocyte function-associated antigen-1 and intercellular adhesion molecule-1

We investigated the effects of the antibodies against the adhesion molecules leucocyte function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on mouse allergic contact hypersensitivity. Mice were injected intraperitoneally with both antibodies before sensitization by an epicutaneous application of dinitrofluorobenzene (DNFB). Simultaneous administration of the antibodies induced suppression of ear swelling in the effector phase of contact hypersensitivity. To show the effect of antibodies in vitro, lymph node cells (LNC) of mice treated with antibodies or with phosphate-buffered saline (PBS) were cultured in the presence of dinitrobenzene sulphonic sodium salts (DNBS), and the production of interleukin-2, interleukin-4 and interleukin-10 in the culture supernatants was measured using an enzyme-linked immunosorbent assay (ELISA). It was found that the production of interleukin-2 in the cells of mice treated with the antibodies was significantly lower than in the cells of PBS-treated mice. On the other hand, no difference was noted in the production of interleukin-4 or interleukin-10. Our results indicate that in vivo simultaneous administration of antibodies to cell adhesion molecules before hapten sensitization induces the suppression of contact hypersensitivity in mice and that the suppression may be due to the inhibition of the production of interleukin-2. Received: 6 December 1995     

Suboptimal non-inflammatory concentrations of haptens may elicit a contact sensitivity reaction when used as a mix

Contact dermatitis is a cutaneous inflammatory reaction mediated by hapten-specific T cells. We used a murine model to investigate the contact sensitivity reaction elicited by different concentrations (optimal and suboptimal) of the haptens DNFB and oxazolone applied singly or in combination. The simultaneous application of DNFB and oxazolone at optimal concentrations (0.2% and 0.4% respectively) did not significantly increase the ear swelling response induced by each of the allergens when applied singly. No contact sensitivity response was observed when the haptens were tested individually at subthreshold concentrations (0.05% and 0.1% respectively). However mixing the 2 molecules at the same concentrations gave rise to a clinical contact sensitivity reaction. The simultaneous application of the haptens at a 2× higher, but still suboptimal concentrations (0.1% and 0.2% respectively), elicited an inflammatory response that was significantly greater than the responses elicited by either of the haptens when applied separately. These results suggest that a "false-positive" reaction to a mix may reveal a genuine sensitization to the constituents.     

Peroxynitrite production, DNA breakage, and poly(ADP-ribose) polymerase activation in a mouse model of oxazolone-induced contact hypersensitivity.

Peroxynitrite-induced poly(ADP-ribose) polymerase activation has been implicated in the pathogenesis of various inflammatory conditions. Here we have investigated whether peroxynitrite and poly(ADP-ribose) polymerase may play a role in the pathophysiology of the elicitation phase of contact hypersensitivity. We have detected nitrotyrosine, DNA breakage, and poly(ADP-ribose) polymerase activation in the epidermis of mice in an oxazolone-induced contact hypersensitivity model. As tyrosine nitration is mostly mediated by peroxynitrite, a nitric-oxide-derived cytotoxic oxidant capable of causing DNA breakage, we have applied peroxynitrite directly on mouse skin and showed poly(ADP-ribose) polymerase activation in keratinocytes and in some scattered dermal cells. We have also investigated the cellular effects of peroxynitrite in HaCaT cells, a human keratinocyte cell line. We found that peroxynitrite inhibited cell proliferation and at higher concentrations also caused cytotoxicity. Peroxynitrite activates poly(ADP-ribose) polymerase in HaCaT cells and poly(ADP-ribose) polymerase activation contributes to peroxynitrite-induced cytotoxicity, as indicated by the cytoprotective effect of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. The cytoprotective effect of 3-aminobenzamide cannot be attributed to inhibition of apoptosis, as apoptotic parameters (caspase activation and DNA fragmentation) were not reduced in the presence of 3-aminobenzamide in peroxynitrite-treated cells. Moreover, poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide dose-dependently reduced interferon-induced intercellular adhesion molecule 1 expression as well as interleukin-1beta-induced interleukin-8 expression. Our results indicate that peroxynitrite and poly(ADP-ribose) polymerase regulate keratinocyte function and death in contact hypersensitivity.     

Effects of in vivo administration of anti-Ia antibodies on contact sensitivity

Our previous studies of the effects of the in vivo administration of anti-la (anti-class II major histocompatibility antigens) antibodies in mice demonstrated that, although the antibodies bind to epidermal Langerhans' cells, they do not affect their antigen-presenting capacity in vitro. In this study we investigated the effects of the in vivo administration of these antibodies on the induction and expression of contact sensitivity. We found that the antibodies inhibit the induction of contact sensitivity significantly and affect the elicitation phase to a much lesser extent. The inhibition is short-lived and probably not attributable to the induction of suppressor cells.     

Heme oxygenase-1 inhibits T cell-dependent skin inflammation and differentiation and function of antigen-presenting cells

Heme oxygenase-1 (HO-1) is increased in psoriatic skin. We asked for the impact of physiological and pharmacological HO-1 induction on skin immunity and the mechanisms involved in HO-1-induced immunomodulation. We found cutaneous HO-1 expression upregulated comparable with suppressors of cytokine signalling (SOCS)1 and SOCS3 in psoriasis and atopic eczema and temporarily increased in murine ovalbumin-induced late phase reaction (LPR) and 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). Cutaneous inflammation was enhanced by HO-1 inhibition and was abrogated by treatment with the HO-1 inducer cobaltic protoporphyrin (CoPP) both when applied around sensitization or before challenge. HO-1 inhibition specifically prevented the anti-inflammatory CoPP effect. CoPP inhibited T cell proliferation in splenocytes of treated mice and in human mixed leukocyte reaction and lymphocyte transformation test. CoPP induced HO-1 in antigen-presenting cells and depressed monocytic accessory molecule expression and the differentiation and maturation of monocyte-derived dendritic cells (MDDC). It decreased tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 production while increasing IL-10 secretion. The antigen-presenting capacity was diminished in CoPP-treated and HO-1-transduced MDDC. We demonstrate for the first time the physiological role of HO-1 in the limitation of skin inflammation and implement pharmacological HO-1 induction as a therapeutic approach for T cell-dependent inflammatory dermatoses. Suppression of antigen-presenting cells may represent a main anti-inflammatory mechanism of HO-1.     

Induction of inflammatory cytokines in murine keratinocytes upon in vivo stimulation with contact sensitizers and tolerizing analogues

In order to elucidate the role of keratinocytes (KCs) in the induction of contact sensitivity, we applied various contact sensitizers [2,4-dinitrofluorobenzene (DNFB), urushiol, 3-n-pentadecylcatechol (PDC), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone)] and tolerizing compounds [2,4-dinitrothiocyanobenzene (DNTB), 5-methyl-3-n-pentadecyl-catechol (5-Me-PDC)] onto the earskin of non-sensitized Balb/c mice. In addition, we applied croton oil as a non-sensitizing, but stimulatory agent. Cytokine production was demonstrated by Northern blot hybridization of the total cellular RNA extracted from epidermal cells depleted by Langerhans cells and Thy 1+ dendritic cells using radiolabeled DNA probes encoding for the murine cytokines IL-1 alpha, -2, -3, -4, TNF alpha, IFN tau, GM-CSF and G-CSF. From all cytokines tested, TNF alpha and IL-1 alpha were markedly increased upon in vivo stimulation with contact sensitizers and also after application of croton oil. Both light and electron microscopic immunostaining with a polyclonal and monoclonal antibody demonstrated the presence of TNF alpha in the epidermis. This staining was most pronounced in KCs of the suprabasal epidermis upon application of contact sensitizers or croton oil, but not with tolerizing analogues. Using a functional assay significantly more TNF alpha was found in the supernatants of KCs treated in vitro with DNFB or LPS than with DNTB. GM-CSF was found in untreated epidermis as well as in stimulated cells. The results suggest that the sensitizing properties of contact sensitizers may partly be dependent on their ability to induce proinflammatory mediators. The induction and release of TNF alpha and IL-1 alpha in KCs by contact sensitizers may play an important role in the early response to immunogenic or inflammatory signals in vivo, whereby tolerance induction seems to be less dependent on these cytokines.     

Modulation of suppressor mechanisms in allergic contact dermatitis: 5. Evidence that inhibition of suppressor T lymphocytes by Corynebacterium parvum is mediated by interferon

Using a contact hypersensitivity model in BALB/c mice we have previously been able to show that Corynebacterium parvum or C. parvum serum (C.p.s.) of mice treated 24 hr before with C. parvum inhibited the suppressor T-lymphocyte (Ts-cell) response induced by epicutaneous antigen overload with 2,4-dinitrofluorobenzene (DNFB) or by i.v. injection of 2,4-dinitrobenzene sulfonic acid (DNBSO3) without inhibition of the T effector cell response (TDH-cell). In the present investigation we further analyzed the factor responsible for Ts-cell inhibition. Treatment of the C.p.s. with sheep-antimouse interferon (IFN) globulin neutralized its Ts-cell inhibitory effect. Intravenous (i.v.) injection of a crude mouse fibroblast IFN (340 U per mouse) 2 hr after i.v. application of a dose of DNBSO3 inducing tolerance had a similar Ts-cell inhibitory effect as observed with C.p.s. Injection of an electrophoretically pure alpha- and beta-IFN preparation (1000 U per mouse) increased contact sensitivity in mice sensitized with an antigen overload and inhibited the induction of Ts-cells by DNBSO3 i.v. This result is highly suggestive that the Ts-cell inhibitory factor in serum of C. parvum-treated mice is IFN and it shows that Ts-cells as compared to TDH-cells are susceptible to the inhibitory effect of highly purified IFN. This finding suggests that IFN may be an important immunoregulatory factor of delayed hypersensitivity not only in contact allergy but also in bacterial and viral defense.     

ATPase positive epidermal Langerhans cells: inhibition of ATPase by ammonium bituminosulfonate (Ichthyol) and pix lithanthracis]

Both, ammonium bituminosulfonate (Ichthyol) and Pix lithanthracis reduce the number of ATPase-positive epidermal Langerhans cells (ELC) in the epidermis of BALB/c mice: vaselinum flavum alone vs vaselinum flavum +5% Ichthyol: P less than 0.01; vaselinum flavum vs vaselinum flavum +5% Pix lithanthracis: P less than 0.001. In contrast to this, the number of Ia-positive cells was not changed under identical conditions. These results allow the conclusion that Ichthyol and Pix lianthracis are able to inhibit the enzyme ATPase on the surface of ELC. We infer that inhibition of ELC ATPase may be important in the regulation of ELC function (inhibition of cutaneous contact hypersensitivity).     

Contact dermatitis II. Clinical aspects and diagnosis

Contact dermatitis (CD) is an altered state of skin reactivity induced by exposure to an external agent. "Eczema" and "dermatitis" are often used synonymously to denote a polymorphic pattern of inflammation of the skin characterized, at least in its acute phase, by erythema, vesiculation and pruritus. Substances that induce CD after single or multiple exposures may be irritant or allergic in nature. The clinical presentation may vary depending on the identity of the triggering agent and the reactivity of the subject, but in all cases the lesions are primarily confined to the site of contact. According to the mechanism of elicitation, the following types of contact reactions may be distinguished: (1) allergic contact dermatitis (ACD); (2) irritant contact dermatitis (ICD); (3) phototoxic and photoallergic contact dermatitis, and (4) immediate type contact reactions. The present review will focus on allergic contact dermatitis. ACD is the clinical presentation of contact sensitivity in humans. The pathophysiology of the contact sensitivity reaction has been reviewed in a preceding issue of this journal [1].     

The loss of contact sensitization in man

Little is yet known about the duration of contact sensitivity, but frequent exposure of a target to allergen seems to reduce skin reactivity. The aim of this study was to study the persistence of a specific contact sensitivity in 66 patients with alopecia areata, previously sensitized to DNCB (31 patients) and SADBE (35 patients) between 1978-1985. Patch tests were performed with 0.020 ml of different concentrations of DNCB or SADBE in acetone (0.05%, 0.10%, 0.20%, 1%). The results were read in a standardized manner. Of 66 patients, 47 (71%) had positive reactions and 19 (29%) negative. 8 of the 19 negative patients had been treated with DNCB, 11 with SADBE. Approximately 1/3 of the patients previously sensitized had lost their original sensitivity, and this did not seem to be time-dependent. This phenomenon seemed to be clinically correlated because the majority of the patients were from the "low responders" group. We think that acquired unresponsiveness to topical antigen in man is a possible phenomenon, but that it occurs more rarely than in mice and guinea pigs.     

Topical application of artesunate on guinea pig allergic contact dermatitis

Artesunate is derived from the Chinese medicinal herb qinghao. Many animal studies suggest that systemic artesunate has immunoregulatory activity. We investigated the effect of topical artesunate on contact sensitivity in guinea pigs sensitized with DNCB. Female hairless guinea pigs were used and the contact reaction graded by visual (5 point) score and erythema measured with a color analyzer. Topical artesunate inhibited the elicitation reaction of contact hypersensitivity when given at the 1st and 12th h after challenge ( p < 0.05), but showed no effect when given from day 3 to day I before challenge ( p > 0.05). Artesunate had no effect on toxic (irritant) contact dermatitis from 20% croton oil ( p > 0.05). The results indicate that topical application of artesunate may offer a novel treatment of allergic contact dermatitis and other cutaneous immune-mediated disorders.     

Alpha-melanocyte stimulating hormone modulates contact hypersensitivity responsiveness in C57/BL6 mice

The neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) can act as an antagonist to interleukin 1 (IL-1) bioactivities such as inhibition of fever production, thymocyte proliferation, and inhibition of release of acute phase inflammatory molecules from the liver. In this report we have found that epicutaneous application of alpha-MSH suppresses both the sensitization and elicitation limbs of the cutaneous immune response (CIR) to potent contact sensitizers like dinitrofluorobenzene (DNFB) or oxazalone (OX) in mice. Further, the loss of contact hypersensitivity due to applications of alpha-MSH could be reconstituted by either intradermal or intravenous injections of epidermal thymocyte activating factor (ETAF)/interleukin-1. Topical application of alpha-MSH did not cause an alteration in Ia+ dendritic cells (i.e., Langerhans cells) but did produce a significant reduction in the expression of Thy1.2 marker on the Thyl+ dendritic epidermal cells (Thy1+DEC). It has no effects on the phenotypic expression of asialo GM-1 on these same cells. These observations suggest that alpha-MSH, a peptide classically isolated from the pituitary but found in many other tissues and cells of the body, may represent an additional biologic modifier than can modulate suppression of the contact hypersensitivity responses to various haptens. However, the mechanisms by which alpha-MSH or potentially other peptides found in the skin produce these suppressive effects have not been elucidated.     

Junctional adhesion molecules (JAM)-B and -C contribute to leukocyte extravasation to the skin and mediate cutaneous inflammation

Leukocyte extravasation is a finely tuned process, in which transmigration is the final step. Transmigration depends on molecules located at borders of endothelial cells; e.g., junctional adhesion molecules (JAM-A, -B and -C). In vivo blockade of JAM-A lead to decreased migration of monocytes into the skin. In contrast, the role of JAM-B and -C in development of cutaneous inflammation is unknown. We therefore elicited an allergic contact dermatitis in mice using 2,4-dinitro-1-fluorobenzene. RT-PCR and immunofluorescent staining of healthy skin revealed a constitutive JAM-B (66.4%+/-6.7% of all vessels) and -C expression (88.6+/-13.2%), which remained constant after induction of contact dermatitis. Functional studies, in which either JAM-B or -C neutralizing antibodies were injected into sensitized mice prior to allergen challenge showed a concentration-dependent reduction of the contact dermatitis. Decreased ear swelling was accompanied by reduction of leukocyte infiltration as analyzed by hematoxylin and eosin (H&E) histology and enzyme activity. Combined antibody treatment at doses of 1.25 mg per kg bodyweight lead to additive inhibition of allergic contact dermatitis, indicating that JAM-B and -C may have distinct functions. In conclusion, interactions with JAM-B and -C are essential for development of cutaneous inflammation.     

Akt blockade downregulates collagen and upregulates MMP1 in human dermal fibroblasts

Acutely transforming retrovirus AKT8 in rodent T-cell lymphoma (Akt) is a serine/threonine kinase that plays important roles in survival, cell-cycle progression, and cell proliferation, and has recently been implicated in collagen regulation. The aim of this study was to determine the role of Akt in collagen deposition by normal dermal fibroblasts, and to determine the sensitivity of cultured systemic sclerosis (SSc) fibroblasts to Akt inhibition. We show that blockade of Akt using pharmacological inhibitors, small interfering RNA (siRNA), and a dominant-negative Akt mutant led to inhibition of the basal type I collagen production. Furthermore, inhibition of Akt upregulated basal matrix metalloproteinase 1 (MMP1) production and reversed the inhibitory effect of transforming growth factor-beta (TGF-beta) on MMP1 gene expression. In addition, SSc fibroblasts were more sensitive to Akt inhibition, with respect to collagen and MMP1 production. These findings suggest that in human dermal fibroblasts, Akt has dual profibrotic effects, increasing collagen synthesis and decreasing its degradation via downregulation of MMP1. Akt could directly contribute to elevated collagen in SSc fibroblasts and it may represent an attractive target for therapy of SSc fibrosis.