Formulation,characterization of Quercus infectoria (Olivier) emulsions,and in vitro,in vivo evaluation as cosmeceutical formulation

Background

Pharmacological properties of Quercus infectoria Olivier (galls) have been determined to be astringent, antidiabetic, antipyretic, anti-tremor, local anesthetic, and anti-parkinsonism. The galls of Quercus infectoria have been used for millennia in traditional oriental medicine in Asian nations to treat inflammatory illnesses.

Aims

The study's objective was to create a Quercus infectoria Olivier gall extract in stable water in oil (w/o) emulsion and to check its effects on the mechanical properties of skin and antiaging effects.

Method

The galls were macerated in absolute methanol. Quercus infectoria Olivier gall extract's antioxidant property was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. Stearic acid, cetyl alcohol, KOH, glycerin, and distilled water were used to create the emulsion. The test (with extract) and control (without extract) emulsions were made, respectively, using the same process. Stability tests (color, liquefaction, microscopy, phase separation, and pH) are performed in in vitro, lasted 72 days at four distinct storage temperatures that is 8°C, 25°C, 40°C, and 40°C + 75% RH for both the control and test formulations. By using spectrophotometry, the (SPF) sun protection factors of the two formulations were calculated at various concentrations. Extract from Quercus infectoria underwent phytochemical investigation as well.

Results

The results showed that Quercus infectoria Olivier has antioxidant and (SPF) sun protection properties, reduce sebum, increases elasticity and stable emulsion containing 04% Quercus infectoria gall extract which might be used as topical antiaging formulation.     

In vitro antioxidant activity and in vivo efficacy of topical formulations containing vitamin C and its derivatives studied by non-invasive methods

Background/purpose: Vitamins C and its derivatives, mainly due to their antioxidant properties, are being used in cosmetic products to protect and to reduce the signs of ageing. However, there are no studies comparing the effects of vitamin C [ascorbic acid (AA)] and its derivatives, magnesium ascorbyl phosphate (MAP) and ascorbyl tetra‐isopalmitate (ATIP), when vehiculated in topical formulations, mainly using objective measurements, which are an important tool in clinical efficacy studies. Thus, the objective of this study was to determine the in vitro antioxidant activity of AA and its derivatives, MAP and ATIP, as well as their in vivo efficacy on human skin, when vehiculated in topical formulations. Methods: The study of antioxidant activity in vitro was performed with an aqueous and a lipid system. The in vivo methodology consisted of the application of these formulations on human volunteers' forearm skin and the analysis of the skin conditions after 4‐week period daily applications in terms of transepidermal water loss (TEWL), stratum corneum moisture content and viscoelasticity using a Tewameter^®, Corneometer^® and Cutometer^®, respectively. Results: In vitro experiments demonstrated that in an aqueous system, AA had the best antioxidant potential, and MAP was more effective than ATIP, whereas in the lipid system ATIP was more effective than MAP. In in vivo studies, all formulations enhanced stratum corneum moisture content after a 4‐week period daily applications when compared with baseline values; however, only the formulation containing AA caused alterations in TEWL values. The formulations containing MAP caused alterations in the viscoelastic‐to‐elastic ratio, which suggested its action in the deeper layers of the skin. Conclusion: AA and its derivates presented an in vitro antioxidant activity but AA had the best antioxidant effect. In in vivo efficacy studies, only the formulation containing AA caused alterations in TEWL values and the formulation containing MAP caused alterations in the viscoelastic‐to‐elastic ratio. This way, vitamin C derivatives did not present the same effects of AA on human skin; however, MAP showed other significant effect‐improving skin hydration, which is very important for the normal cutaneous metabolism and also to prevent skin alterations and early ageing.     

Influence of wetness on the ultraviolet protection factor (UPF) of textiles: in vitro and in vivo measurements

BACKGROUND/PURPOSE: Clothing is an important product for sunburn protection and skin cancer prevention. The moisture content of a fabric, which can increase during its wearing, may decrease the fabric's capability of protecting the skin from solar UV radiation, that is, lower its UPF (ultraviolet protection factor). Due to limited data about the effect of fabric wetness on UPF, this study was undertaken to investigate the following: (a) the effect of saturating a variety of fabrics with tap water and with salt water on fabric UPF and (b) whether wetted-fabric UPF values reflect only the fact that the fabric is wet during testing or the fact that the skin is hydrated and the fabric is wet. METHODS: For objective a, 69 summer fabrics were spectrophotometrically (in vitro) assessed when "dry" and when saturated with tap and salt water. In vitro UPFs, percent UVA transmission and percent UVB transmission values were calculated from the transmission data. For objective b, 100% cotton and 100% polyester fabrics were tested in vivo to determine in vivo UPF values. The minimal erythema dose (MED) was determined for each of the 12 subjects on unprotected "dry" skin and on "hydrated" unprotected skin. MEDprotected was determined when the subject's skin was covered with "dry" and with saturated fabric. In vivo UPFs were calculated using this data. Student's paired t-tests were used to determine the effect of wetting. RESULTS: With one exception, in vitro UPF values were the same when the fabrics were saturated with tap water and when they were saturated with salt water. However, saturating the fabrics with water had different effects on the UPF, UVA transmission, and UVB transmission values. For linen, viscose and polyester fabrics, UPF significantly increased. For the cotton fabrics and the polyester + TiO2 fabrics, UPF significantly decreased. For the modal + TiO2 fabrics and the polyester crepe + TiO2 fabrics, UPF significantly increased. From the in vivo testing, the MED of the "hydrated unprotected" skin was not different than the MED of "dry unprotected skin." Values obtained from subtracting dry-fabric in vivo UPF values from dry-fabric in vitro values and subtracting wet-fabric in vivo UPF values from wet-fabric in vitro values are not different. CONCLUSION: Fabrics do not need to be tested when saturated with tap and with salt water. Testing fabrics wet and dry should be done, as the effect of saturating fabric on UPF value varies. Fortunately, UPF values for wetted fabrics reveal only the effect of increased moisture content in the fabric and have nothing to do with wetting of the skin by the fabric.     

Augmentation of sebaceous lipogenesis by an ethanol extract of Grifola frondosa (Maitake mushroom) in hamsters in vivo and in vitro

Abstract:  Grifola frondosa (Maitake mushroom) is an edible and medicinal mushroom with versatile effects such as antitumor and immunomodulating actions. Here, we demonstrated that an ethanol extract of G. frondosa fruiting body (Maitake extract) augmented intracellular lipid droplet formation and the production of triacylglycerols (TG), a major component of sebum, along with the activation of diacylglycerol acyltransferase, a rate-limiting enzyme of TG synthesis in cultured hamster sebocytes. The topical treatment of Maitake extract on the skin of hamster auricles augmented sebum accumulation in sebaceous glands and ducts. However, in comparison with the Maitake extract, another ethanol extract prepared from Agaricus blazei Murill showed less activity in sebaceous lipogenesis in hamsters in vivo and in vitro . These results provide novel evidence that Maitake extract augments sebaceous lipogenesis in hamsters in vivo and in vitro . Thus, Maitake extract is likely to be a unique agent leading to the remission of dry skin.     

In vivo quantitative analysis of the effect of hydration (immersion and Vaseline treatment) in skin layers using high‐resolution MRI and magnetisation transfer contrast*

Background/aims: Many claims are made as to the efficacy of topical preparations in moisturising the skin, yet most of these claims cannot be substantiated by scientific study for the skin layers beneath the stratum corneum, and yield no information on the remainder of the epidermis and dermis. This argues for an in vivo quantitative method for measuring the effect of water loading extended to various layers of the skin. Methods: Detailed high‐resolution in vivo MRI studies of hydration and dehydration of finger pad skin layers were conducted on one normal subject using two moisturisation methods (topical white soft paraffin (Vaseline) and water immersion). The dehydration study was carried out immediately following removal from prolonged skin moisturisation. Inter‐individual variability for skin hydration (group study) was studied in seven healthy volunteers at 0 and 7 h hydration with Vaseline. Location dependence in skin hydration was investigated on the same subject by looking into the hydration of forearm and finger pad skin. System stability and measurement reproducibility was verified through a detailed phantom study. Results: Images of normal and hydrated human skin were obtained in vivo at voxel dimensions of 50 μm×150 μm×1000 μm. The effect of hydration and dehydration as a function of exposure to moisturiser (i.e. water and Vaseline) on the image signal intensity, observed T1, and interaction of free and bound water in specific tissues were identified and correlated with existing physiological knowledge. Swelling of stratum corneum due to hydration was expressed as an in vivo model of tissue hydration. Conclusion: Results of the dehydration study showed that the changes due to the previous hydration of the skin are reversible for all skin layers. For both moisturisation methods (i.e. Vaseline and skin bathing), the effects of hydration and dehydration on the skin were similar. The trends of the MRI parameters for finger pad and arm skin were similar. The group study showed low inter‐subject variability of hydration on stratum corneum and epidermis.     

Reduction of pain and side effects in the treatment of solar lentigines with pneumatic skin flattening (PSF)

Background: Energy densities utilized in the treatment of pigmented lesions such as solar lentigines with intense pulsed light systems are often limited by pain and post‐treatment erythema and edema. The sensation of pain associated with the treatment is immediate and acute. Application of topical anesthesia is time‐consuming, with only very moderate pain relief. Objective: (a) To test pain reduction as well as the reduction of post‐treatment erythema and edema when using pneumatic skin flattening (PSF). This new technology utilizes an evacuation chamber to generate skin compression and activates tactile neural receptors in the skin. The result is an afferent inhibition of pain transmission in the dorsal horn (the ‘gate theory’). (b) To test the efficacy of PSF. Methods: Twenty patients were treated for solar lentigines. The patients were treated by three different IPLs. The evaluation of acute pain and post‐treatment erythema and edema was performed on all 20 patients: one to three sites per patient treated with PSF and the same number of control sites without PSF. Identical energies and IPL were applied to both sites on each patient. The pain evaluation was performed on a 10‐level scale modified McGill Pain Questionnaire. The clinical response to treatment was also evaluated. Results: All 20 patients completed the study and preferred the PSF treatment side over the non‐PSF side. Substantial pain reduction was observed in 19/20 patients (95%). The average reduction of pain was by two levels, from very painful to very mild pain. Erythema reductions were observed on 14/18 (77%) patients and edema reduction on 8/9 (88%) patients. Treatment efficacy on PSF sites was identical to that of non‐PSF sites. Conclusion: The pneumatic skin flattening (PSF) technology considerably reduces pain, erythema and edema in the treatment of solar lentigines by IPLs. Treatment efficacy is preserved. The enhanced safety of PSF enables the increase of energy density and the acceleration of results.     

Ex vivo analysis of desmoglein 1-responsive T-helper (Th) 1 and Th2 cells in patients with pemphigus foliaceus and healthy individuals

Pemphigus foliaceus (PF) is a severe autoimmune bullous disorder, characterized by autoantibodies (autoAb) against desmoglein 1 (Dsg1). As T cells may be critical in the pathology of PF, the aim of the present study was to identify and characterize autoaggressive T-helper cells reactive to Dsg1 in PF patients and healthy individuals. Eight patients with the clinical diagnosis of PF and six HLA class II-matched healthy individuals were examined. By magnetic cell-sorting (MACS) cytokine-secretion assay, Dsg1-responsive T-helper (Th) 1 and Th2 cells were isolated and cloned by limiting dilution. The generated T-cell clones (TCC) were characterized regarding proliferative response, TCR Vbeta-chain usage, and cytokine profile upon in vitro stimulation with Dsg1. Both Dsg1-reactive Th1 and Th2 cells were detected in PF patients and controls at similar frequencies. A total of 15 Th1 and Th2 clones were isolated from patients and 27 TCC from healthy controls. Analysis of TCR Vbeta-chain usage of autoreactive T cells from both groups revealed no predominance of a specific Vbeta chain. Noteworthy, the isolated TCC showed a polarized Th1- or Th2-like phenotype upon in vitro culture and stable expression of Th1 or Th2 cytokines during long-term in vitro culture. In summary, our data demonstrate that T-cell autoreactivity against Dsg1 is not restricted to patients with PF. Moreover, both Th1 and Th2 cells were present in patients and healthy donors, suggesting that the loss of B-cell tolerance against Dsg1 in PF is not exclusively determined by the presence of autoaggressive T cells.     

Significant improvement in crow's feet after treatment with Jet-M and a mixed solution of copper–GHK,oligo-hyaluronic acid,rhodiolar extract,tranexamic acid,and β-glucan (GHR formulation)

Jet-M (Tav-Tech Ltd., Israel) is an instrument for skin resurfacing. When it sprays microdroplets of solution or shoots air on the skin, exfoliation and stretching of superficial layers can occur. Thus, it will increase percutaneous absorption of vitamins and other cosmetic agents. A cosmetic preparation containing copper–glycyl-L-histidyl-L-lysine, oligo-hyaluronic acid, rhodiolar extract, tranexamic acid, and β-glucan was used with Jet-M in one patient.

Anesthesia was not administered and there was no pain during the treatment. A male aged 59 years was treated once a week for 12 weeks. In the clinical photographs, wrinkles around the treated eye were greatly decreased. Skin biopsies were taken from treated and untreated areas. Hematoxylin and eosin and Masson's trichrome staining showed increased collagen production in the upper dermis. On the other hand, collagen IV production was slightly increased. Fibrillin-1 and procollagen type 1 were greatly increased and tropoelastin was also increased. There was no adverse effect during and after treatment.     

Effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on migration of epidermal keratinocytes in vitro and wound healing in vivo

Tissue inhibitors of metalloproteinases (TIMP), common inhibitors of matrix proteinases, have cell-promoting activity. We studied the effects of recombinant human tissue inhibitor of metalloproteinases-2 (rh-TIMP-2) on the migration of normal human epidermal keratinocytes (NHEK). An in vitro migration assay revealed that rh-TIMP-2 enhanced random migration (up to 170%, p<0.05) in a dose-dependent manner. When we applied rh-TIMP-2 solution (20 microg/20 microl/wound) daily to full-thickness wounds made with an 8-mm punch on the backs of healthy (n=8), aged (n=9), and diabetic (n=15) rodents, we observed faster wound closure (p<0.05) than in vehicle-treated controls. Accelerated wound closure was dose-dependent (0-20 microg/wound) in diabetic mice (n=6), and the optimal concentration was 10-20 microg of rh-TIMP-2/wound. Histological examinations performed on days 0, 5, 10, 15, and 20 in diabetic mice revealed faster migration of epidermal keratinocytes from wound edges. These results suggest that rh-TIMP-2 plays an important role in wound healing.     

In vitro and in vivo evidence of tyrosinase inhibitory activity of a synthesized (Z)‐5‐(3‐hydroxy‐4‐methoxybenzylidene)‐2‐thioxothiazolidin‐4‐one (5‐HMT)

Tyrosinase is a key enzyme that catalyses the initial rate‐limiting steps of melanin synthesis. Due to its critical role in melanogenesis, various attempts were made to find potent tyrosinase inhibitors although many were not safe and effective in vivo. We evaluated tyrosinase inhibitory activity of six compounds. Among them, (Z)‐5‐(3‐hydroxy‐4‐methoxybenzylidene)‐2‐thioxothiazolidin‐4‐one (5‐HMT) had the greatest inhibitory effect and potency as the IC₅₀ value of 5‐HMT was lower than that of kojic acid, widely‐known tyrosinase inhibitor. Based on in silico docking simulation, 5‐HMT had a greater binding affinity than kojic acid with a different binding conformation in the tyrosinase catalytic site. Furthermore, its skin depigmentation effect was confirmed in vivo as 5‐HMT topical treatment significantly reduced UVB‐induced melanogenesis in HRM2 hairless mice. In conclusion, our study demonstrated that 5‐HMT has a greater binding affinity and inhibitory effect on tyrosinase and may be a potential candidate for a therapeutic agent for preventing melanogenesis.     

Differential expression and in vivo secretion of the antimicrobial peptides psoriasin (S100A7), RNase 7, human beta‐defensin‐2 and ‐3 in healthy human skin

Antimicrobial peptides (AMP) are key players in the skin's defense system. Previous observations suggest a site‐ and age‐dependent expression of individual AMP. We investigated the expression and secretion patterns of four important AMP in a representative collective of healthy human skin samples. Levels of psoriasin, RNase 7 and hBD‐3 expression – assessed by immunohistochemistry – varied between different body localisations. Older individuals expressed hBD‐2 more frequently. No gender‐related expression was observed. The in vivo secretion of psoriasin, measured in skin washing fluids using ELISA, was related to body localisation and age, whereas RNase 7 secretion showed no significant differences regarding these variables. HBD‐2 and ‐3 secretion could not be detected. Our findings suggest the usage of control samples matching localisation and approximate age (in the case of hBD‐2) for comparative immunohistochemical analysis. To avoid bias through great interindividual differences, sufficient large collectives should be used for in vivo secretion analyses.     

Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.     

Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis

BACKGROUND: Toll-like receptors (TLRs) are part of the innate immune system involved in the response to microbial pathogens. TLR2 recognizes various ligands expressed by Gram-positive bacteria, while TLR3, TLR4 and TLR5 are specific for double-stranded RNA, Gram-negative lipopolysaccharides and bacterial flagellin, respectively. OBJECTIVES: To determine, firstly, whether epidermal keratinocytes of normal skin express TLRs and, secondly, whether modulation of TLR expression occurs in psoriasis, an inflammatory skin disease associated with certain microorganisms such as streptococci, staphylococci and yeasts. METHODS: Eight samples of normal, and 15 samples of lesional and nonlesional psoriatic skin were stained with polyclonal antibodies specific for TLR1-5 using an avidin-biotin-peroxidase technique. RESULTS: Epidermal keratinocytes in normal skin constitutively expressed TLR1, TLR2 and TLR5, while TLR3 and TLR4 were, in most cases, barely detectable. Cytoplasmic TLR1 and TLR2 were expressed throughout the epidermis, with higher staining of the latter on basal keratinocytes, while TLR5 expression was concentrated in the basal layer. In contrast, in lesional epidermis from patients with psoriasis, TLR2 was more highly expressed on the keratinocytes of the upper epidermis than on the basal layer, while TLR5 was downregulated in basal keratinocytes compared with corresponding nonlesional psoriatic epidermis. In addition, nuclear TLR1 staining was observed in the upper layers of both nonlesional and lesional psoriatic epidermis, but not in that of normal skin. CONCLUSIONS: These findings suggest that TLRs expressed by epidermal keratinocytes constitute part of the innate immune system of the skin. The relevance of altered keratinocyte TLR expression in psoriasis remains to be determined.     

In vivo microautoradiography of [3H]1,24(OH)2D3 (tacalcitol) following topical application to normal rats and in vitro metabolism in human keratinocytes

This study was conducted to investigate the mechanism of topical absorption of [ ³ H]1,24(OH) ₂ D ₃ (1,24-dihydroxyvitamin D ₃ ; tacalcitol) by applying an ointment containing 4 μg/g [ ³ H]1,24(OH) ₂ D ₃ to the skin of rats using an occlusion method. Microautoradiography of the skin at the application site 1 h after topical treatment showed a high concentration of radiolabel in the stratum corneum, the epidermis and around the hair follicles. Radiolabel was also seen in the epidermis and hair follicle areas 8 h and 24 h after application. The radiolabel was distributed to a minor extent to the subcutaneous fat layer. Microautoradiography showed two routes of purcutaneous absorption of 1,24(OH) ₂ D ₃ : through the stratum corneum and epidermis into the microvessels, and through hair follicle areas into the bloodstream. After topical application of an ointment containing 4 μg/g or 40 μg/g [ ³ H]1,24(OH) ₂ D ₃ to the shaved neck skin of rats, the absorption rate, estimated by excretion in the urine and faeces, was about 30% of the total applied radioactivity. The main excretion route after topical application was in the faeces. Furthermore, 1,24(OH) ₂ D ₃ added to human adult keratinocytes was not metabolized into other compounds, and only the unchanged compound was detected. These findings strongly suggest that 1,24(OH) ₂ D ₃ distributed into the epidermis acts on epidermal keratinocytes. Topical application of 1,24(OH) ₂ D ₃ appears to be a possible approach to the treatment of psoriasis and other skin diseases through its action on the 1,25(OH) ₂ D ₃ receptor, which reportedly plays a very important role in the regulation of proliferation and differentiation of keratinocytes. Received: 18 April 1995     

Influence of cultured dermal fibroblasts on human melanoma cell proliferation,matrix metalloproteinase-2 (MMP-2) expression and invasion in vitro

The dermis is the main site of melanoma invasion. Matrix metalloproteinases (MMPs), especially MMP-2, produced by melanoma or surrounding stromal cells, are essential for the destruction of dermal extracellular matrix. Here, we examined how dermal fibroblasts influenced proliferation, MMP-2 secretion and invasion of human melanoma cell lines in vitro. Human melanoma cell lines M3 Da and M1Dor were cocultured with dermal fibroblasts under non-contact and contact conditions in order to assess both soluble and insoluble factors, respectively. Zymographic analysis showed that the levels of MMP-2 and TIMP-2 in melanoma cells were not altered in non-contact cocultures when compared with those in individual cultures. However, in contact cocultures, the expression of MMP-2 in membrane extracts was enhanced. Under our coculture conditions, dermal fibroblasts failed to upregulate melanoma cell invasion through a three-dimensional type I collagen matrix. Since stromal and cancer cell contacts have been shown to occur after disruption of the extracellular matrix, we hypothesized that fibroblasts may influence melanoma cell invasion after the beginning of tumor progression through the dermis.     

Application of the Food and Drug Administration (FDA) bioequivalent guidance of topical dermatological corticosteroid in yellow-skinned Japanese population: validation study using a chromameter

The American Food and Drug Administration (FDA) bioequivalent guidance of topical dermatological corticosteroids in 1995 (the Guidance) requires measurement of the skin blanching response with a chromameter for evaluation of cutaneously applied corticosteroid formulations. The Japanese government decided to apply the same guidelines in 2003, despite there having been no reported trial for the yellow-skinned races. The purpose of this study was to obtain basic data of corticosteroid-induced skin blanching response measured with a chromameter on yellow-skinned races. Four studies were performed according to the Japanese version of the Guidance for Industry using a chromameter on the forearms of healthy Japanese volunteers. This involved: (i) a validation study of proper duration of treatment exposure (dose duration); (ii) a comparison study of two dermatological corticosteroid products that represented different potency classes; (iii) inspection of reproducibility using right and left forearms; and (iv) study of seasonal difference. We showed that: (i) the same medication can give different values of ED(50) (the dose duration required to achieve 50% of the fitted areas under the effect curves [AUEC](max) value) under different dose durations; (ii) ED(50) do not always represent the potency of the corticosteroid; (iii) the results of AUEC at maximum duration were similar, but AUEC at an approximate ED(50) duration time varied widely; and (iv) the results of AUEC were different according to season. In conclusion the dose duration relationships, determination of the AUEC(max), and the ED(50) could be obtained on yellow-skinned races using the FDA method. However, negligible differences were found in our study regarding dose duration, reproducibility and seasonal change.     

T-cell subsets and NCI (nuclear contour index) of their skin infiltrates in mycosis fungoides (MF)--a comparison of pre-and post-treatments using ACNU [1-(4-amino-2 methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl-3-nitrosourea)] and interferon alpha and the effectiveness of interferon alpha on Sézary cells in vitro

ACNU solution was applied to plaque lesions once daily for 4 weeks, while 3 million IU/day of Interferon α was injected locally into tumor lesions for 4 weeks. Clinically, ACNU and Interferon α were very effective. To determine the cytological effectiveness of both treatments, T-cell subsets and the NCI of their skin infiltrates in mycosis fungoides (MF) were compared pre- and post-treatment. Using either ACNU and Interferon α, in pre-treatment skin tissue, Leu3a positive cells were dominant, whereas in post-treatment skin tissue, Leu2a positive cells were dominant. As a result, the helper/suppressor T-cell (H/S) ratio in plaque lesion (3.1) was remarkably decreased (0.44) by ACNU treatment and the H/S ratio in tumor lesions was also reduced from 9.3 to 2.6 after injection of Interferon α. Moreover, the NCI of T-cells in plaque lesions decreased from 7.57 ± 1.71 to 4.30 ± 0.75 following treatment with ACNU, and that in tumor lesions was also reduced from 8.62 ± 1.89 to 4.35 ± 1.17 with Interferon α treatment. These immunohistos-chemical staining patterns and immunoelectron microscopic features in post-treatment periods are similar to those of control skin tissue which is not malignant. It was of interest that the antiproliferative effect of Interferon α on Hut 78 cells (Sézary cells) in vitro was demonstrated. Therefore, Interferon α might have direct cytotoxic effects on MF cells similar to that with Sézary cells. These results indicate that both treatments, ACNU and Interferon α, are effective not only clinically but also cytologically. The data also indicate that the NCI of T-cell and T-cell subsets might be used as an indicator of a treatment's effectiveness against MF.