Sensor-controlled scalp cooling to prevent chemotherapy-induced alopecia in female cancer patients.

BACKGROUND: Scalp cooling has been used since the 1970s to prevent chemotherapy-induced alopecia, one of the most common and psychologically troubling side effects of chemotherapy. Currently available scalp cooling systems demonstrate varying results in terms of effectiveness and tolerability. METHODS: For the present prospective study, 55 women receiving neoadjuvant, adjuvant, or palliative chemotherapy were enrolled. The aim was to assess the effectiveness of a sensor-controlled scalp cooling system (DigniCap: Sysmex Europe GmbH, Norderstedt, Germany) to prevent chemotherapy-induced alopecia in breast or gynecologic cancer patients receiving 1 of 7 regimens. Clinical assessments, satisfaction questionnaires, and alopecia evaluations [World Health Organization (who) grading for toxicity] were completed at baseline, at each cycle, and at completion of chemotherapy. RESULTS: Of the 55 patients, 78% underwent scalp cooling until completion of chemotherapy. In multivariate analysis, younger women and those receiving paclitaxel weekly or paclitaxel-carboplatin experienced less alopecia. The compound successful outcome ("no head covering" plus "who grade 0/1") was observed in all patients 50 years of age and younger receiving 4 cycles of docetaxel-cyclophosphamide or 6 cycles of paclitaxel-carboplatin. Conversely, alopecia was experienced by all women receiving triplet polychemotherapy (6 cycles of docetaxel-doxorubicin-cyclophosphamide). For women receiving sequential polychemotherapy regimens (3 cycles of fluorouracil-epirubicin-cyclophosphamide followed by 3 cycles of docetaxel or 4 cycles of doxorubicin-cyclophosphamide followed by 4 cycles of docetaxel), the subgroup 50 years of age and younger experienced a 43% success rate compared with a 10% rate for the subgroup pf older women receiving the same regimens. CONCLUSIONS: The ability of scalp cooling to prevent chemotherapy-induced alopecia varies with the chemotherapy regimen and the age of the patient. Use of a compound endpoint with subjective and objective measures provides insightful and practical information when counselling patients.

Sequential exposure to fibroblast growth factors (FGF) 2, 9 and 18 enhances hMSC chondrogenic differentiation.

OBJECTIVE: To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. DESIGN: Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. Quantitative real time polymerase chain reaction (qRT-PCR) and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. RESULTS: FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with transforming growth factor-beta (TGF-ß), FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤ d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. CONCLUSIONS: The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-ß, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented extracellular matrix (ECM) production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

Course of cognitive functioning during stroke rehabilitation.

The aim of the study was to determine the course of cognitive functioning within the subacute phase (< 4 months) after stroke during rehabilitation. Stroke patients admitted to a rehabilitation centre were submitted to a neuropsychological examination on admission (1 month post-stroke) and upon discharge (4 months post-stroke). Cognitive domains studied were attention, executive functioning, memory, and visual attention. Forty-two patients (mean age = 57.1 years; SD = 7.7) participated. At admission more than half of the patients showed deficits in attention and memory. Patients improved significantly on these domains; the largest improvement was seen in the domain of visual attention, while executive functioning did not improve significantly. A differential course of cognitive functioning was found in the subacute phase after stroke. The prognosis of visual attention is the most prominent.

[Life-threatening macroglossia following cleft palate palatoplasty]. / Lebensbedrohliche Makroglossie nach Gaumenspaltenverschluss.

The case of a 13-month-old child who developed a life-threatening macroglossia with airway obstruction following palatoplasty for a cleft palate is reported. As direct laryngoscopy was not feasible a laryngeal mask (LM) was inserted to secure the airway. Under fiber optic guidance an endotracheal tube was then introduced via the LM. In this article the incidence, pathophysiology, clinical dynamics, options for emergency anesthesia management and organizational implications of this rare but typical complication in the field of oral and craniomaxillofacial surgery are reported.

Suppression of third-order intermodulation in a klystron by third-order injection.

The first observations and measurements are reported on suppression of the third-order intermodulation (IM3) product arising from nonlinear mixing of two drive frequencies in a klystron, by externally injecting a wave at the IM3 product frequency. Optimum amplitude and phase of the injected wave for maximum suppression are examined. Results indicate that suppression of the IM3 product by as much as 30 dB can be achieved. Experimental results compare favorably with predictions of a 1D simulation code that takes into account all kinematical and dynamical effects including charge overtaking and space charge forces.

Maximal oxygen uptake during field running does not exceed that measured during treadmill exercise.

Modern ergometric equipment enables the simulation of laboratory maximal oxygen uptake (.VO(2max)) testing in the field. Therefore, it was investigated whether the improved event specificity on the track might lead to higher .VO(2max) measurements in running. Identical protocols were used on the treadmill and on the track (speed was indicated by a computer-driven flashing light system). Ambulatory measurements of gas exchange were carried out throughout both tests, which were executed in randomized order. There were no significant differences ( P=0.71) in .VO(2max) between treadmill [4.65 (0.51) ml.min(-1)] and field tests [4.63 (0.55) ml.min(-1)]. However, the test duration differed significantly ( P<0.001) by approximately 5%: treadmill 691 (39) s; field test 727 (42) s. With the exception of maximum heart rate (HR(max); significantly higher in the field with P=0.02) all criteria for the degree of effort were similar between the two tests. However, the difference in HR(max) at less than 2 beats.min(-1), was practically negligible. Submaximal measurements of oxygen uptake and minute ventilation were significantly higher on the treadmill ( P<0.001 for both parameters). In summary, field tests with incremental running protocols do not result in higher .VO(2max) measurements compared to laboratory treadmill exercise. A better running economy on the track results in higher maximal velocities and longer exercise durations being sustained. The determination of .VO(2max) is not a reasonable application for ambulatory gas exchange measurements because laboratory values are not surpassed.

N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues as vanilloid receptor ligands.

The vanilloid receptor represents a promising target for drug development. Building on our previous strategies which have generated potent agonists for VR1, we now describe a series of novel N-(3-acyloxy-2-benzylpropyl)-N'-dihydroxytetrahydrobenzazepine and tetrahydroisoquinoline thiourea analogues, several of which are potent VR1 antagonists. We report here the rationale for the design, the synthesis, and the in vitro characterization of activity in assays for [(3)H]resiniferatoxin binding and (45)Ca influx using heterologously expressed rat VR1.

Thapsigargin suppresses phorbol ester-dependent human involucrin promoter activity by suppressing CCAAT-enhancer-binding protein alpha (C/EBPalpha) DNA binding.

Human involucrin (hINV) is a keratinocyte differentiation marker expressed in the suprabasal epidermal layers. In cultured keratinocytes hINV mRNA levels are increased 10-fold by a 24-h treatment with 50 ng/ml PMA, an agent that promotes keratinocyte differentiation. Previous studies show that thapsigargin (TGN), an agent that depletes intracellular calcium stores, inhibits keratinocyte differentiation. In the present study we show that TGN inhibits the PMA-dependent, differentiation-associated, increase in hINV mRNA levels and hINV promoter activity. Inhibition is half-maximal at 10 nM and maximal at 100 nM TGN. Neither basal hINV promoter activity nor glyceraldehyde-3-phosphate dehydrogenase mRNA levels are inhibited. Mutation of a functionally important CAATT-enhancer-binding protein (C/EBP) site within the hINV promoter proximal regulatory region eliminates the regulation, suggesting that TGN may effect C/EBP-dependent promoter activation. Consistent with this hypothesis, TGN inhibits C/EBPalpha-dependent promoter activation via a mechanism that involves inhibition of C/EBPalpha binding to DNA without changing C/EBPalpha protein levels. These results suggest that TGN interferes with hINV expression by interfering with C/EBP transcription-factor function.

Vaccination against canine distemper virus infection in infant ferrets with and without maternal antibody protection, using recombinant attenuated poxvirus vaccines.

Canine distemper virus (CDV) infection of ferrets is clinically and immunologically similar to measles, making this a useful model for the human disease. The model was used to determine if parenteral or mucosal immunization of infant ferrets at 3 and 6 weeks of age with attenuated vaccinia virus (NYVAC) or canarypox virus (ALVAC) vaccine strains expressing the CDV hemagglutinin (H) and fusion (F) protein genes (NYVAC-HF and ALVAC-HF) would induce serum neutralizing antibody and protect against challenge infection at 12 weeks of age. Ferrets without maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 5) or ALVAC-HF (n = 4) developed significant neutralizing titers (log(10) inverse mean titer +/- standard deviation of 2.30 +/- 0.12 and 2.20 +/- 0.34, respectively) by the day of challenge, and all survived with no clinical or virologic evidence of infection. Ferrets without maternal antibody that were vaccinated intranasally (i.n.) developed lower neutralizing titers, with NYVAC-HF producing higher titers at challenge (1.11 +/- 0.57 versus 0.40 +/- 0.37, P = 0.02) and a better survival rate (6/7 versus 0/5, P = 0.008) than ALVAC-HF. Ferrets with maternal antibody that were vaccinated parenterally with NYVAC-HF (n = 7) and ALVAC-HF (n = 7) developed significantly higher antibody titers (1.64 +/- 0. 54 and 1.28 +/- 0.40, respectively) than did ferrets immunized with an attenuated CDV vaccine (0.46 +/- 0.59; n = 7) or the recombinant vectors expressing rabies glycoprotein (RG) (0.19 +/- 0.32; n = 8, P = 7 x 10(-6)). The NYVAC vaccine also protected against weight loss, and both the NYVAC and attenuated CDV vaccines protected against the development of some clinical signs of infection, although survival in each of the three vaccine groups was low (one of seven) and not significantly different from the RG controls (none of eight). Combined i.n.-parenteral immunization of ferrets with maternal antibody using NYVAC-HF (n = 9) produced higher titers (1.63 +/- 0. 25) than did i.n. immunization with NYVAC-HF (0.88 +/- 0.36; n = 9) and ALVAC-HF (0.61 +/- 0.43; n = 9, P = 3 x 10(-7)), and survival was also significantly better in the i.n.-parenteral group (3 of 9) than in the other HF-vaccinated animals (none of 18) or in controls immunized with RG (none of 5) (P = 0.0374). Multiple routes were not tested with the ALVAC vaccine. The results suggest that infant ferrets are less responsive to i.n. vaccination than are older ferrets and raises questions about the appropriateness of this route of immunization in infant ferrets or infants of other species.

CCAAT/enhancer-binding proteins. A role in regulation of human involucrin promoter response to phorbol ester.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of keratinocyte differentiation and of involucrin gene expression. In the present study we show that a CCAAT/enhancer-binding protein (C/EBP) site in the proximal regulatory region is required for the phorbol ester response. Mutation of the C/EBP site results in the loss of basal and TPA-responsive activity. Gel mobility supershift analysis shows that C/EBPalpha binding to this site is increased by TPA treatment. Moreover, cotransfection of the human involucrin reporter plasmid with C/EBPalpha increases promoter activity to an extent comparable with TPA treatment. Mutation of the C/EBP-binding site eliminates these responses. Transfection experiments using GADD153 to create C/EBP-null conditions confirm that C/EBP factors are absolutely required for promoter activity and TPA responsiveness. C/EBPbeta and C/EBPdelta inhibit both TPA- and C/EBPalpha-dependent promoter activation, indicating functional differences among C/EBP family members. These results suggest that C/EBP transcription factor activity is necessary for basal promoter activity and TPA response of the involucrin gene.

Mucosal vaccination with recombinant poxvirus vaccines protects ferrets against symptomatic CDV infection.

Canine distemper virus (CDV) infection of ferrets causes a disease characterized by fever, erythema, conjunctivitis and leukocytopenia, similar clinically to measles except for the fatal neurologic sequelae of CDV. We vaccinated juvenile ferrets twice at 4-week intervals by the intranasal or intraduodenal route with attenuated vaccinia (NYVAC) or canarypox virus (ALVAC) constructs containing the CDV hemagglutinin and fusion genes. Controls were vaccinated with the same vectors expressing rabies glycoprotein. Animals were challenged intranasally 4 weeks after the second vaccination with virulent CDV. Body weights, white blood cell (WBC) counts and temperatures were monitored and ferrets were observed daily for clinical signs of infection. WBCs were assayed for the presence of viral RNA by RT-PCR. Intranasally vaccinated animals survived challenge with no virologic or clinical evidence of infection. Vaccination by the intraduodenal route did not provide complete protection. All control animals developed typical distemper. Ferrets can be effectively protected against distemper by mucosal vaccination with poxvirus vaccines.

Regulation of human involucrin promoter activity by a protein kinase C, Ras, MEKK1, MEK3, p38/RK, AP1 signal transduction pathway.

Involucrin is a marker of keratinocyte terminal differentiation. Our previous studies show that involucrin mRNA levels are increased by the keratinocyte differentiating agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Welter, J. F., Crish, J. F., Agarwal, C., and Eckert, R. L. (1995) J. Biol. Chem. 270, 12614-12622). We now study the signaling cascade responsible for this regulation. Protein kinase C and tyrosine kinase inhibitors inhibit both the TPA-dependent mRNA increase and the TPA-dependent increase in hINV promoter activity. The relevant response element is located within the promoter proximal regulatory region and includes an AP1 site, AP1-1. Co-transfection of the hINV promoter with dominant negative forms of Ras, MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun inhibit the TPA-dependent increase. Wild type MEKK1 enhances promoter activity and the activity can be inhibited by dominant negative MEKK1, MEK1, MEK7, MEK3, p38/RK, and c-Jun. In contrast, wild type Raf-1, ERK1, ERK2, MEK4, or JNK1 produced no change in activity and the dominant negative forms of these kinases failed to suppress TPA-dependent transcription. Treatment with an S6 kinase (S6K) inhibitor, or transfection with constitutively active S6K produced relatively minor changes in promoter activity, ruling out a regulatory role for S6K. These results suggest that activation of involucrin transcription involves a pathway that includes protein kinase C, Ras, MEKK1, MEK3, and p38/RK. Additional pathways that transfer MEKK1 activation via MEK1 and MEK7 also may function, but the downstream targets of these kinases need to be identified. AP1 transcription factors appear to be the ultimate target of this regulation.

Characterization of human involucrin promoter distal regulatory region transcriptional activator elements-a role for Sp1 and AP1 binding sites.

Human involucrin (hINV) is an important precursor of the keratinocyte cornified envelope that is specifically expressed in the suprabasal layers of stratifying epithelia. Previous truncation and mutagenesis experiments have shown that an activator protein 1 (Ap1) site, AP1-5, located 2100bp upstream of the transcription start site, is required for optimal promoter activity. These previous studies suggest that AP1-5 is part of a distal regulatory region spanning nucleotides -2473 to -2088. In the present report, we study the distal regulatory region (DRR), which surrounds AP1-5. Our studies show that this region contains weak and strong activator elements spanning nucleotides -2473/-2216 and -2140/-2088, respectively. The strong activator element contains AP1-5 and an adjacent specificity protein 1 (Sp1) site. The AP1-5 site is absolutely required for DRR activity, as its mutation reduces transcription to basal levels. Mutagenesis studies of the AP1-5 and Sp1 sites in the presence or absence of the weak activator element indicate that the Sp1 site and the weak activator element synergistically activate the AP1-5 site-dependent transcription. The cooperation between the Sp1 and AP1-5 sites is also observed in the context of the full-length promoter. Gel mobility shift and supershift studies show that Sp1, but not Sp2, Sp3 or Sp4 binds to the Sp1 site. When the Sp1 site is mutated or the distance between the AP1-5 and Sp1 site is increased, the binding of AP1 factors to AP1-5 is markedly reduced. Surprisingly, gel shift studies suggest that activation does not require the formation of a stable AP1/Sp1/DNA ternary complex. These studies suggest that the AP1-5 site is absolutely required for transcriptional activation, that the weak activator element and Sp1 sites serve to enhance this activation, and that the Sp1 site is required for optimal AP1 factor binding at the AP1-5 site.

[Diagnostic value of somatostatin receptor scintigraphy with indium-111 pentetreotide in small-cell bronchial carcinoma]. / Diagnostische Wertigkeit der Somatostatinrezeptorszintigraphie mit Indium-111 Pentetreotid beim Kleinzelligen Bronchialkarzinom.

Small-cell lung cancer (SCLC) cells may express somatostatin receptors [14]. Receptor-positive tissue can be visualised in vivo by scintigraphy with radiolabelled somatostatin analogues. In a prospective study we examined 18 patients with histologically proven SCLC for the diagnostic value of somatostatin receptor scintigraphy using indium-111 pentetreotide. Planar whole body scanning was performed 4 and 24 hours after administration. Additional SPECT imaging of the thorax and the abdomen was done at 24 hours. The results were compared with conventional staging procedures: ultrasound, x-ray, computed tomography and bone scintigraphy. In all 18 patients the primary tumour was correctly identified. Out of 13 patients with mediastinal lymphoma formation 10 patients showed positive SRS. In 2 more patients SRS showed mediastinal uptake while CT scanning was negative. The detection of distant metastases in patients with extensive disease was true positive in 8 cases (OSS, HEP, BRA), false negative in 4 cases (PLE, ADR, HEP), corresponding to a sensitivity of 67%. In 2 patients cerebral metastases were no longer detectable by SRS after previous local irradiation. Even though the method is limited in respect of revealing distant metastases in the upper abdominal area due to physiological uptake in liver, spleen and kidneys, differentiation between limited disease (LD) and extensive disease (ED) was possible in all cases. We conclude that [111In]pentetreotide scintigraphy is a suitable method for the detection of SCLC primary tumours and a substantial tool for differentiation between LD and ED if combined with ultrasonography of the upper abdomen.

The epidermis: genes on - genes off.

The epidermal keratinocyte stem cell is distinguished by a relatively undifferentiated phenotype and an ability to proliferate. As part of a carefully orchestrated process, the offspring of these stem cells lose the ability to proliferate and begin a process of morphologic and biochemical transformation that results in their conversion into corneocytes. This process requires the coordinated expression of a host of cellular genes. The mechanisms responsible for regulation of these genes is an area of intense interest. In keratinocytes, as in other cell types, the expression of most genes is regulated at the transcriptional level by a class of proteins called transcription factors. Transcription factors are nuclear proteins that regulate transcription by mediating the final steps in the relay of information from the cell surface to the nucleus and the gene. These factors bind to specific DNA sequence elements located within the target gene. In this brief review we summarize evidence implicating activator protein 1 (AP1), AP2, Sp1, POU domain, CCAAT enhancer binding protein, and several other transcription factors as regulators of expression of keratinocyte genes.

S100A11, S100A10, annexin I, desmosomal proteins, small proline-rich proteins, plasminogen activator inhibitor-2, and involucrin are components of the cornified envelope of cultured human epidermal keratinocytes.

The cornified envelope (CE) is an insoluble sheath of epsilon-(gamma-glutamyl)lysine cross-linked protein, which is deposited beneath the plasma membrane during keratinocyte terminal differentiation. We have probed the structure of the CE by proteolytic cleavage of purified CE fragments isolated from CEs formed spontaneously in cell culture. CNBr digestion, followed by trypsin and then proteinase K treatment released 25%, 42%, and 18%, respectively, of the CE protein. Purification and sequencing of released peptides has identified two novel CE precursors, S100A11 (S100C, calgizzarin) and S100A10 (calpactin light chain). We also sequenced peptides derived from annexin I and plasminogen activator inhibitor 2, two putative envelope precursors, as well as portions of the well established CE precursor proteins SPR1A, SPR1B, and involucrin. Many desmosomal components were identified (desmoglein 3, desmocolin A/B, desmoplakin I, plakoglobin, and plakophilin), indicating that desmosomes become cross-linked into the CE. Fragments derived from envoplakin, the recently sequenced 210-kDa membranous CE precursor protein, which also appears to be a desmosomal component, were also identified. Analysis of the pattern of peptide release following the sequential digestion indicates that S100A11 is anchored to the envelope via Gln102 and/or Lys103 at the carboxyl terminus and at Lys3, Lys23, and/or Gln22 in the amino terminus. A similar type of analysis indicates that small proline-rich proteins 1A and 1B (SPR1A and SPR1B) become cross-linked at the amino terminus (residues 1-23) and the carboxyl terminus (residues 86-89). No loricrin, cystatin A, or elafin peptides were detected.

Canine distemper virus (CDV) infection of ferrets as a model for testing Morbillivirus vaccine strategies: NYVAC- and ALVAC-based CDV recombinants protect against symptomatic infection.

Canine distemper virus (CDV) infection of ferrets causes an acute systemic disease involving multiple organ systems, including the respiratory tract, lymphoid system, and central nervous system (CNS). We have tested candidate CDV vaccines incorporating the fusion (F) and hemagglutinin (HA) proteins in the highly attenuated NYVAC strain of vaccinia virus and in the ALVAC strain of canarypox virus, which does not productively replicate in mammalian hosts. Juvenile ferrets were vaccinated twice with these constructs, or with an attenuated live-virus vaccine, while controls received saline or the NYVAC and ALVAC vectors expressing rabies virus glycoprotein. Control animals did not develop neutralizing antibody and succumbed to distemper after developing fever, weight loss, leukocytopenia, decreased activity, conjunctivitis, an erythematous rash typical of distemper, CNS signs, and viremia in peripheral blood mononuclear cells (as measured by reverse transcription-PCR). All three CDV vaccines elicited neutralizing titers of at least 1:96. All vaccinated ferrets survived, and none developed viremia. Both recombinant vaccines also protected against the development of symptomatic distemper. However, ferrets receiving the live-virus vaccine lost weight, became lymphocytopenic, and developed the erythematous rash typical of CDV. These data show that ferrets are an excellent model for evaluating the ability of CDV vaccines to protect against symptomatic infection. Because the pathogenesis and clinical course of CDV infection of ferrets is quite similar to that of other Morbillivirus infections, including measles, this model will be useful in testing new candidate Morbillivirus vaccines.

Epidermal keratinocytes - genes and their regulation.

In recent years, the human epidermal keratinocyte has been extensively studied. These studies have shown that epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), retinoids, phorbol ester, vitamin D and other agents regulate keratinocyte proliferation, differentiation, apoptosis and gene expression. We review progress in understanding the mechanisms that regulate keratinocyte structural gene expression. In most cases little is known regarding the factors that regulate gene expression in response to a physiological agent. However, the available results suggest a role for a variety of transcription factors, including STAT factors, NFkappaB, octamer site (OCT) binding proteins and activator protein 1 (AP1) factors in regulating expression of these genes. Among these transcriptional regulators, AP1 appears to play a central role. We review the current literature regarding the regulation of involucrin, loricin, transglutaminase type 1 and cytokeratin gene expression. This survey indicates that the AP1 family of transcriptional regulators is implicated in the regulation of nearly all of these genes. We also discuss recent studies which describe the distribution of the AP1 factors, c-jun, junB, junD, Fra-1,Fra-2, c-fos and fosB, in epidermis.