Ultrasound as predictor of histologic subtypes linked to recurrence in basal cell carcinoma of the skin.

BACKGROUND: Basal cell carcinoma (BCC) recurrences, especially in the facial region, represent a complex cosmetic problem. To date the possibility of predicting recurrence is supported solely by the histologic subtype. OBJECTIVE: To evaluate the relationship between BCC histologic subtypes linked to high and low risk of recurrence and the presence of hyperechoic spots on sonography. METHODS: Retrospective analysis of the pre-surgical ultrasound examinations of primary BCC tumours with visualization and counting of intra-tumoural hyperechoic spots. The data were then correlated with the corresponding histologic subtype. RESULTS: Thirty one patients with histologically proven BCC were included in the study. Hyperechoic spots were detected in all cases and there was a positive, statistically significant association between hyperechoic spots count and high recurrence risk histologic subtypes. Higher hyperechoic spots count was found in the recurrence-prone micronodular, sclerosing variant and morpheiform BCC subtypes. Low risk and high risk of recurrence showed a significant difference on the mean hyperechoic spots count of 5.5 (range: 3-25) and 8 (4-81). A cut-off point ≥7 hyperechoic spots presented a sensitivity of 79% and specificity of 53% for predicting the high risk of recurrence subtypes. CONCLUSION: The presence and count of hyperechoic spots within BCC lesions may help predicting the high risk of recurrence histologic subtypes.

Sonography in pathologies of scalp and hair.

Disorders of the scalp often result in severe cosmetic interference with quality of life, creating the need for optimal medical surveillance. We tested the latest generation of ultrasound machines in patients with scalp pathology and prepared a cross-sectional library encompassing a wide assortment of conditions. Normative data on the sonographic anatomy of scalp and human hair, and important methodological considerations, are also included.

Ultrasound detection and identification of cosmetic fillers in the skin.

BACKGROUND: While the incidence of cosmetic filler injections is rising world-wide, neither exact details of the procedure nor the agent used are always reported or remembered by the patients. Thus, although complications are reportedly rare, availability of a precise diagnostic tool to detect cutaneous filler deposits could help clarify the association between the procedure and the underlying pathology. OBJECTIVES: The aim of this study was to evaluate cutaneous sonography in the detection and identification of cosmetic fillers deposits and, describe dermatological abnormalities found associated with the presence of those agents. METHODS: We used ultrasound in a porcine skin model to determine the sonographic characteristics of commonly available filler agents, and subsequently applied the analysis to detect and identify cosmetic fillers among patients referred for skin disorders. RESULTS: Fillers are recognizable on ultrasound and generate different patterns of echogenicity and posterior acoustic artefacts. Cosmetic fillers were identified in 118 dermatological patients; most commonly hyaluronic acid among degradable agents and silicone oil among non-degradable. Fillers deposits were loosely scattered throughout the subcutaneous tissue, with occasional infiltration of local muscles and loco-regional lymph nodes. Accompanying dermatopathies were represented by highly localized inflammatory processes unresponsive to conventional treatment, morphea-like reactions, necrosis of fatty tissue and epidermal cysts; in the case of non-degradable agents, the associated dermatopathies were transient, resolving upon disappearance of the filler. CONCLUSIONS: Cosmetic filler agents may be detected and identified during routine ultrasound of dermatological lesions; the latter appear to be pathologically related to the cosmetic procedure.

Melatonin maintains mitochondrial membrane potential and attenuates activation of initiator (casp-9) and effector caspases (casp-3/casp-7) and PARP in UVR-exposed HaCaT keratinocytes.

Melatonin is a recognized antioxidant with high potential as a protective agent in many conditions related to oxidative stress such as neurodegenerative diseases, ischemia/reperfusion syndromes, sepsis and aging. These processes may be favorably affected by melatonin through its radical scavenging properties and/or antiapoptotic activity. Also, there is increasing evidence that these effects of melatonin could be relevant in keratinocytes, the main cell population of the skin where it would contribute to protection against damage induced by ultraviolet radiation (UVR). We therefore investigated the kinetics of UVR-induced apoptosis in cultured keratinocytes characterizing the morphological and mitochondrial changes, the caspases-dependent apoptotic pathways and involvement of poly(ADP-ribose) polymerase (PARP) activation as well as the protective effects of melatonin. When irradiated with UVB radiation (50 mJ/cm(2)), melatonin treated, cultured keratinocytes were more confluent, showed less cell blebbing, more uniform shape and less nuclear condensation as compared to irradiated, nonmelatonin-treated controls. Preincubation with melatonin also led to normalization of the decreased UVR-induced mitochondrial membrane potential. These melatonin effects were followed by suppression of the activation of mitochondrial pathway-related initiator caspase 9 (casp-9), but not of death receptor-dependent casp-8 between 24 and 48 hr after UVR exposure. Melatonin down-regulated effector caspases (casp-3/casp-7) at 24-48 hr post-UV irradiation and reduced PARP activation at 24 hr. Thus, melatonin is particularly active in UV-irradiated keratinocytes maintaining the mitochondrial membrane potential, inhibiting the consecutive activation of the intrinsic apoptotic pathway and reducing PARP activation. In conclusion, these data provide detailed evidence for specific antiapoptotic mechanisms of melatonin in UVR-induced damage of human keratinocytes.

Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines.

Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.

CRH functions as a growth factor/cytokine in the skin.

We tested the effect of CRH and related peptides in a large panel of human skin cells for growth factor/cytokine activities. In skin cells CRH action is mediated by CRH-R1, a subject to posttranslational modification with expression of alternatively spliced isoforms. Activation of CRH-R1 induced generation of both cAMP and IP3 in the majority of epidermal and dermal cells (except for normal keratinocytes and one melanoma line), indicating cell type-dependent coupling to signal transduction pathways. Phenotypic effects on cell proliferation were however dependent on both cell type and nutrition conditions. Specifically, CRH stimulated dermal fibroblasts proliferation, by increasing transition from G1/0 to the S phase, while in keratinocytes CRH inhibited cell proliferation. In normal and immortalized melanocytes CRH effect showed dichotomy and thus, it inhibited melanocyte proliferation in serum-containing medium CRH through G2 arrest, while serum free media led instead to CRH enhanced DNA synthesis (through increased transition from G1/G0 to S phase and decreased subG1 signal, indicating DNA degradation). CRH also induced inhibition of early and late apoptosis in the same cells, demonstrated by analysis with the annexin V stains. Thus, CRH acts on epidermal melanocytes as a survival factor under the stress of starvation (anti-apoptotic) as well as inhibitor of growth factors induced cell proliferation. In conclusion, CRH and related peptides can couple CRH-R1 to any of diverse signal transduction pathways; they also regulate cell viability and proliferation in cell type and growth condition-dependent manners.

Melatonin increases survival of HaCaT keratinocytes by suppressing UV-induced apoptosis.

Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P < 0.01) and 10(-3) m (P < 0.001). Additional postirradiation treatment with melatonin showed no increase in the pre-UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.

Corticotropin-releasing hormone triggers differentiation in HaCaT keratinocytes.

BACKGROUND: Corticotropin-releasing hormone (CRH) is proposed to be involved in the regulation of the proliferative capacity of keratinocytes, based on its significant actions in the skin. These are mediated by CRH-R1alpha and represented by adenylate cyclase activation, Ca2+ influx, inhibition of cell proliferation and modifications in intracellular signal transduction by NF-kappaB. OBJECTIVES: To define CRH action in the cell cycle we investigated its effects on the differentiation programme using the HaCaT keratinocytes model. METHODS: HaCaT keratinocytes were incubated with CRH in Dulbecco's modified Eagles's medium (containing 1.8 mmol L(-1) calcium) or EpiLife (containing 0.06 mmol L(-1) calcium) medium. Cell proliferation was assessed with the MTT assay. Flow cytometry was used for the measurement of DNA content, cell size and granularity and the expression of cytokeratin 14, cytokeratin 1 and involucrin. The electrophoretic mobility shift assay was used to determine DNA binding activity by AP-1 transcription factor. Expression of cytokeratin 1 was also assessed with immunofluorescence microscopy. RESULTS: CRH did produce inhibition of proliferation, which was dose-dependent; the shape of the inhibition curve was determined by the media calcium concentration. CRH action was pinpointed at inhibition of the G0/1 to the S phase transition of the cell cycle. CRH also increased AP-1 binding activity, cell granularity, cytokeratin 1 and involucrin expression, and inhibited cytokeratin 14 expression. CONCLUSIONS: These results are consistent with CRH induction of the keratinocyte differentiation programme. Thus, the overall CRH cutaneous actions connote protective functions for the epidermis, that appear to include the triggering or acceleration of the differentiation programme.

Self-regulated endocrine systems in the skin.

The skin is the main physical barrier between the environment and internal homeostasis; functionally it is highly complex and expresses endocrine activities with self-regulatory properties. This skin neuroendocrine system comprises locally produced neuro-endocrine mediators that interact with corresponding specific receptors through para or autocrine mechanisms. While there are also systemic effects of cutaneously produced hormones exemplified by vitamin D3 and PTHrP, the most important function of this system would be the modulation of responses to noxious agents. Solar radiation, the most significant environmental stressor is already known to significantly affect cutaneous endocrine activities. Ultimately, the skin neuroendocrine system would act to preserve cutaneous structural and functional integrity to maintain systemic homeostasis.

Microassay of phosphate provides a general method for measuring the activity of phosphatases using physiological, nonchromogenic substrates such as lysophosphatidic acid.

Since measurement of lysophosphatidate phosphatase activity is important in studies of tumorigenesis, we attempted to develop a simpler alternative to the more complex methods currently available. Measuring the phosphate released would permit use of the same method for a variety of phosphatases with physiological substrates, many of which are nonchromogenic. The Malachite green method of K. Itaya and M. Ui (1966, Clin. Chim. Acta 14, 361) has adequate sensitivity for quantitating phosphatase activity in biological samples. In samples with high endogenous phosphate concentrations pretreatment with 50 mg Dowex 1 x 10 (100-200 mesh, OH- form) usually permitted reliable determination of phosphatase activity. For 34 consecutive runs the mean relative difference [(phosphorus activity--vitamer activity)/phosphorus activity] obtained from the simultaneous measurement of both the phosphate released and the corresponding organic product (pyridoxal and pyridoxine) was -0.03 +/- 0.09. The within run and between run coefficients of variation (three runs of four to five replicates) were 0.05 and 0.04, respectively. Pyridoxine 5'-phosphate hydrolase activity (pH 10) in cultured skin cells (normal and cancerous) ranged from 2 to 12 nmol phosphorus/min. mg protein. Lysophosphatidate phosphatase activity (pH 7.4) ranged from 3 to 14 nmol phosphorus/min. mg protein. The current approach permits the measurement of phosphatase activity with a single method using a variety of substrates and incubation conditions.

Steroidogenesis in the human skin: 21-hydroxylation in cultured keratinocytes.

We have evaluated the metabolism of radiolabeled progesterone (P) by the microsomal fraction isolated from HaCaT keratinocytes. P was widely metabolized to different compounds that included DOC (5-7% conversion) thus demonstrated 21-hydroxylase (21-OHase) activity, a key step in adrenal synthesis of gluco- and mineralocorticoids. However, RT-PCR amplification for the CYPc21 transcript of the corresponding gene showed no evidence for gene expression in HaCaT cells suggesting that the 21-OHase enzyme present in keratinocytes is different from that described in adrenal gland. Further characterization showed that whereas estradiol stimulated markedly P metabolism by HaCaT microsomes, with generation of new unidentified compounds, Lineweaver-Burk analysis of keratinocyte 21-OHase activity showed that the K(m) and V(max) were unaffected by estrogen. The apparent K(m) was 0.6 microM without estradiol and 0.7 microM with estradiol, while the respective V(max) values were 60 and 76 nmol/l/min. To conclude, we found extensive metabolism of P in human keratinocytes, we also provide the first demonstration of 21-OHase activity in this cell system and further showed that it is coded by a gene different from the adrenal CYPc21.

Malignant melanoma.

CONTEXT: The rapidly developing fields of melanoma research are revolutionizing the current concepts on melanoma etiology and pathogenesis and are introducing newer diagnostic techniques and potential therapeutic approaches. OBJECTIVES: To present the most current concepts on the etiology and pathogenesis of melanoma and to introduce the recent diagnostic techniques and the potential therapeutic approaches. METHODS: Data sources were reports on melanoma published in the English language literature and observations made using specimens available at Harvard University, Johns Hopkins Medical Center, Albany Medical College, Loyola University Medical Center, and University of Tennessee Health Science Center. RESULTS: Studies on melanoma containing chromosomal or genetic evaluation were selected for further analysis. Current clinical and pathologic categories with the reported genetic abnormalities were related to the latest information on pigment biology. The data extracted were used to develop a conceptual framework on the pathogenesis of melanoma; the generated model was then evaluated and used to suggest potential therapeutic approaches. CONCLUSIONS: (1) Melanoma is not genetically homogeneous, and the existing differences between the pathologic categories, particularly in areas such as type of growth phase (radial vs vertical growth), total vertical dimension, ulceration of primary tumor, and metastatic process, have profound prognostic and therapeutic implications. (2) Chromosomal aberrations and gene mutations are found in sporadic and familial melanomas; among the most important are those affecting the 9p21, which contains the p16 locus, a site known to be critical for normal progression of the cell cycle. Aberrant p16 expression is associated with more aggressive behavior. (3) Melanoma cells possess a remarkable repertoire of biosynthetic capacities represented by the production of hormones, growth factors, and their receptors that may sustain and accelerate tumor development and progression. For example, expression of the tumoral products alpha-melanocyte-stimulating hormone and adrenocorticotropic hormone is regulated in vitro by ultraviolet light, a known carcinogen. (4) Melanomas differ from other tumors in their intrinsic capability to express melanogenic enzymes with the corresponding structural proteins to actually synthesize melanin. Melanogenesis-related proteins are rapidly entering the clinical arena, being used not only as diagnostic markers, but also as potential targets for melanoma therapy.

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.

Studies in mammalian skin have shown expression of the genes for corticotropin-releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH-Rs). These CRH-Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)-mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypothalamic-pituitary-adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/POMC system, the CRH-Rs may be a central element.

Celiac disease-associated autoimmune endocrinopathies.

Celiac disease (CD) is an autoimmune disorder induced by gluten intake in genetically susceptible individuals. It is characterized by the presence of serum antibodies to endomysium, reticulin, gliadin, and tissue transglutaminase. The incidence of CD in various autoimmune disorders is increased 10- to 30-fold in comparison to the general population, although in many cases CD is clinically asymptomatic or silent. The identification of such cases with CD is important since it may help in the control of type I diabetes or endocrine functions in general, as well as in the prevention of long-term complications of CD, such as lymphoma. It is believed that CD may predispose an individual to other autoimmune disorders such as type I diabetes, autoimmune thyroid, and other endocrine diseases and that gluten may be a possible trigger. The onset of type I diabetes at an early age in patients with CD, compared to non-CD, and the prevention or delay in onset of diabetes by gluten-free diet in genetically predisposed individuals substantiates this antigen trigger hypothesis. Early identification of CD patients in highly susceptible population may result in the treatment of subclinical CD and improved control of associated disorders.

Adrenal insufficiency in high-risk surgical ICU patients.

STUDY OBJECTIVES: To examine the incidence and response to treatment of adrenal insufficiency (AI) in high-risk postoperative patients. DESIGN: Prospective observational case series. SETTING: Large urban tertiary-care surgical ICU (SICU). PARTICIPANTS: Adults > 55 years of age who required vasopressor therapy after adequate volume resuscitation in the immediate postoperative period. INTERVENTIONS: Each patient underwent a cosyntropin (ACTH) stimulation test; at the discretion of the clinical team, some patients were empirically given hydrocortisone (100 mg IV q8h for three doses) before serum cortisol values became available. MEASUREMENTS: Adrenal dysfunction (AD), defined as serum cortisol < 20 microg/dL at all time points, with Delta cortisol (60 min post-ACTH minus baseline) of < or = 9 microg/dL; functional hypoadrenalism (FH), defined as serum cortisol < 30 microg/dL at all time points or Delta cortisol (60 min post-ACTH minus baseline) < or = 9 microg/dL; and AI, as the presence of either AD or FH. RESULTS: One hundred four patients were enrolled with a mean age (SD) of 65.2 +/- 16.9 years. AI (AD plus FH) was found in 34 of 104 patients (32.7%): AD was found in 9 patients (8.7%), FH in 25 patients (24%), and normal adrenal function in 70 patients (67.3%). The absolute eosinophil count was significantly higher in the combined AD and FH groups compared with the group with normal adrenal function (p < 0.05). Forty-six of 104 patients (44.2%) received hydrocortisone; 29 (63%) could be weaned from treatment with vasopressors within 24 h. This beneficial effect of hydrocortisone reached statistical significance in the FH group when compared with untreated patients (p < 0.031); a similar trend was seen in the AD group (p = 0.083). Mortality was also lower in the hydrocortisone-treated AI patients (5 of 23 [21%] vs 5 of 11 [45%] in those not receiving hydrocortisone; p < 0.01). CONCLUSION: There is a high incidence of AI among SICU patients > 55 years of age with postoperative hypotension requiring vasopressors. There is also a significant association between hydrocortisone replacement therapy, resolution of vasopressor requirements, and improved survival.

Pleiotropic effects of corticotropin releasing hormone on normal human skin keratinocytes.

The hypothalamic-pituitary-adrenal (HPA) axis is the major stress response system. Several components of the HPA axis, such as corticotropin-releasing hormone (CRH) and POMC peptides and their receptors are also present in the skin. In earlier studies, we showed that CRH inhibits cellular proliferation of immortalized human keratinocytes. We now examine further the functional activity of the HPA axis in the skin, by characterizing the actions of CRH on normal foreskin keratinocytes. The CRH receptor was detected as CRH-R1 antigen at 47 kDa in the cultured keratinocytes by Western blotting, and immunohistochemistry demonstrated its presence in the epidermal and follicular keratinocytes. CRH is also biologically active in cultured keratinocytes, where it inhibits proliferation and enhances the interferon-gamma-stimulated expression of the hCAM and ICAM-1 adhesion molecules and of the HLA-DR antigen. These effects were concentration-dependent, with maximal activity at CRH 10(-7) M. Thus, in the keratinocyte, the most important cellular component of the epidermis, CRH appears to induce a shift in energy metabolism away from proliferation activity, and toward the enhancement of immunoactivity. Therefore, similar to its central actions, cutaneous CRH may also he involved in the stress response, but at a highly localized level.

Role of molecular biology in diagnostic pathology of melanoma.

Cutaneous melanoma is the most rapidly increasing malignancy in the white European population; its clinical significance is enhanced because it can affect younger individuals (1-3). The high mortality rate among melanoma patients, second to lung cancer, is related to melanoma's resistance to therapy once the metastastic process has started (4-6). The tumor derives from epidermal melanocytes, either activated or genetically altered; thus, important precursors include activated melanocytes present within solar lentigo or forming prema- lignant lesions such as lentigo maligna (7-10). Melanoma can also arise from relatively benign or atypical nevomelanocyte lesions (7-10). Benign lesions that can nevertheless result in melanoma include congenital melanocytic nevus, nevus of Ota, nevus of Ito, and cellular blue nevus. The atypical lesions with the same possible outcome are represented by acquired dysplastic melanocytic nevus, melanocytic dysplasia on the acral or mucosal surface, spindle cell and/or atypical epithelioid melanocytic nevus (Spitz nevus), and dysplastic and/or congenital nevus spilus (7-10).

Human skin expresses growth hormone but not the prolactin gene.

Using sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) methods, we showed the expression of mRNA for growth hormone (GH) but not prolactin (PRL) in whole human skin (normal and basal cell carcinoma (BCC)). These RNAs for PRL and GH were below detectability in human epidermal keratinocytes and in human and hamster malignant melanocytes. This is in agreement with previous studies showing GH gene expression in dermal fibroblasts. GH peptide was not detected (by immunocytochemistry) in human skin specimens (normal and pathologic) in either dermal or epidermal compartments. The mRNA coding for the GH mediator insulin-like growth factor-1 (IGF-1) was detectable in whole skin and in malignant melanocytes. Therefore, in the present investigation of hormonal mediators of the cutaneous (epidermal) response to environmental stress, we have excluded the direct participation of PRL and GH in that reaction. Thus the analogy previously noted between the systemic (central) and skin responses to stress, as represented by cutaneous expression of hypothalamic-pituitary-adrenal axis components, does not extend to other pituitary hormones also involved in that response such as PRL and GH.

Liquid chromatography-mass spectrometry detection of corticotropin-releasing hormone and proopiomelanocortin-derived peptides in human skin.

We have previously shown expression of CRH and POMC genes and peptides in the human skin. To ascertain the identity of those peptides, we used methods of peptide extraction and purification combined with the highly specific technique of liquid chromatography-mass spectrometry. Testing extracts of human skin, we identified endogenous peptides with masses and retention times corresponding to CRH, ACTH 1-39, ACTH 1-13, and alpha-MSH standards. Thus, conclusive evidence is provided for the presence of CRH and the POMC-derived ACTH 1-39, ACTH 1-13, and alpha-MSH peptides in human skin. Direct identification of these peptides is consistent with translation of the corresponding genes, and it also suggests intermediate pituitary lobe-like POMC peptide processing.