Comparison of Wet Fractionation Methods for Processing Broccoli Agricultural Wastes and Evaluation of the Nutri-Chemical Values of Obtained Products.

The main objective of this study was to increase the economic value of broccoli green agro-waste using three wet fractionation methods in the shadow of green biorefinery and the circular economy. Product candidates were obtained directly by using a mechanical press, and indirectly by using microwave coagulation or via lactic acid fermentation of green juice. The leaf protein concentrates (LPC) fractions displayed significantly higher dry matter content and crude protein content (34-39 m/m% on average) than the green juice fraction (27.4 m/m% on average), without considerable changes in the amino acids composition ratio. UHPLC-ESI-ORBITRAP-MS/MS analysis showed that kaemferol and quercetin are the most abundant flavonols, forming complexes with glycosides and hydroxycinnamic acids in green juice. Lacto-ermentation induced a considerable increase in the quantity of quercetin (48.75 µg·g-1 dry weight) and kaempferol aglycons (895.26 µg·g-1 dry weight) of LPC. In contrast, chlorogenic acid isomers and sulforaphane disappeared from LPC after lactic acid fermentation, while microwave treatment did not cause significant differences. These results confirm that both microwave treatment and lacto-fermentation coagulate and concentrate most of the soluble proteins. Also, these two processes affect the amount of valuable phytochemicals differently, so it should be considered when setting the goals.

Thyrotropin-releasing hormone selectively stimulates human hair follicle pigmentation.

In amphibians, thyrotropin-releasing hormone (TRH) stimulates skin melanophores by inducing secretion of α-melanocyte-stimulating hormone in the pituitary gland. However, it is unknown whether this tripeptide neurohormone exerts any direct effects on pigment cells, namely, on human melanocytes, under physiological conditions. Therefore, we have investigated whether TRH stimulates pigment production in organ-cultured human hair follicles (HFs), the epithelium of which expresses both TRH and its receptor, and/or in full-thickness human skin in situ. TRH stimulated melanin synthesis, tyrosinase transcription and activity, melanosome formation, melanocyte dendricity, gp100 immunoreactivity, and microphthalmia-associated transcription factor expression in human HFs in a pituitary gland-independent manner. TRH also stimulated proliferation, gp100 expression, tyrosinase activity, and dendricity of isolated human HF melanocytes. However, intraepidermal melanogenesis was unaffected. As TRH upregulated the intrafollicular production of "pituitary" neurohormones (proopiomelanocortin transcription and ACTH immunoreactivity) and as agouti-signaling protein counteracted TRH-induced HF pigmentation, these pigmentary TRH effects may be mediated in part by locally generated melanocortins and/or by MC-1 signaling. Our study introduces TRH as a novel, potent, selective, and evolutionarily highly conserved neuroendocrine factor controlling human pigmentation in situ. This physiologically relevant and melanocyte sub-population-specific neuroendocrine control of human pigmentation deserves clinical exploration, e.g., for preventing or reversing hair graying.

Thyroid-stimulating hormone, a novel, locally produced modulator of human epidermal functions, is regulated by thyrotropin-releasing hormone and thyroid hormones.

Several elements of the hypothalamic-pituitary-thyroid axis (HPT) reportedly are transcribed by human skin cell populations, and human hair follicles express functional receptors for TSH. Therefore, we asked whether the epidermis of normal human skin is yet another extrathyroidal target of TSH and whether epidermis even produces TSH. If so, we wanted to clarify whether intraepidermal TSH expression is regulated by TRH and/or thyroid hormones and whether TSH alters selected functions of normal human epidermis in situ. TSH and TSH receptor (TSH-R) expression were analyzed in the epidermis of normal human scalp skin by immunohistochemistry and PCR. In addition, full-thickness scalp skin was organ cultured and treated with TSH, TRH, or thyroid hormones, and the effect of TSH treatment on the expression of selected genes was measured by quantitative PCR and/or quantitative immunohistochemistry. Here we show that normal human epidermis expresses TSH at the mRNA and protein levels in situ and transcribes TSH-R. It also contains thyrostimulin transcripts. Intraepidermal TSH immunoreactivity is up-regulated by TRH and down-regulated by thyroid hormones. Although TSH-R immunoreactivity in situ could not be documented within the epidermis, but in the immediately adjacent dermis, TSH treatment of organ-cultured human skin strongly up-regulated epidermal expression of involucrin, loricrin, and keratins 5 and 14. Thus, normal human epidermis in situ is both an extrapituitary source and (possibly an indirect) target of TSH signaling, which regulates defined epidermal parameters. Intraepidermal TSH expression appears to be regulated by the classical endocrine controls that determine the systemic HPT axis.

Corticotropin-releasing hormone stimulates the in situ generation of mast cells from precursors in the human hair follicle mesenchyme.

Hair follicles (HFs) maintain a peripheral, functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis, whose most proximal element is corticotropin-releasing hormone (CRH). The mast cell (MC)-rich connective-tissue sheath (CTS) of mouse vibrissa HFs harbors MC precursors. Differentiation of these MC precursors into mature MCs can be induced by stem cell factor (SCF). We have investigated whether the MC progenitors of normal human scalp HF CTS respond to stimulation with CRH. Microdissected anagen HFs and full-thickness scalp skin were treated with CRH (10(-7) M). CRH treatment induced the degranulation of CTS MCs, in addition to increasing the number of CTS MCs in full-thickness skin and HF organ cultures in situ. In the latter, cells with characteristic MC features emigrated from the CTS. CRH-receptor protein expression in the CTS was colocalized with Kit expression on some CTS MCs in situ. CRH treatment upregulated SCF mRNA and protein expression within the HF epithelium. In skin organ culture, CRH-induced degranulation of CTS MCs was abolished by anti-SCF antibody. We demonstrate that human skin is an extramedullary reservoir for MC precursors, and we have identified a regulatory loop between CRH and SCF signaling. This highlights a previously unpublished finding about neuroendocrine control of human MC biology.

Thyrotropin powers human mitochondria.

Here we demonstrate that the neuropeptide hormone thyrotropin (TSH), which controls thyroid hormone production, exerts a major nonclassical function in mitochondrial biology. Based on transcriptional, ultrastructural, immunohistochemical, and biochemical evidence, TSH up-regulates mitochondrial biogenesis and consequently activity in organ-cultured normal human epidermis in situ. Mitochondrial activity was assessed by measuring 2 key components of the respiratory chain. The abundance of mitochondria was assessed employing 2 independent morphological techniques: counting their numbers in human epidermis by high-magnification light microscopy of skin sections immunostained for mitochondria-selective cytochrome-c-oxidase subunit 1 (MTCO1) and transmission electron microscopy (TEM). Treatment with 10 mU/ml of TSH for 6 d strongly up-regulates the number of light-microscopically visualized, MTCO1-demarcated mitochondria. On the ultrastructural level, TEM confirms that TSH indeed stimulates mitochondrial proliferation and biogenesis in the perinuclear region of human skin epidermal keratinocytes. On the transcriptional level, TSH up-regulates MTCO1 mRNA (quantitative RT-PCR) and significantly enhances complex I and IV (cytochrome-c-oxidase) activity. This study pioneers the concept that mitochondrial energy metabolism and biogenesis in a normal, prototypic human epithelial tissue underlies potent neuroendocrine controls and introduces human skin organ culture as a clinically relevant tool for further exploring this novel research frontier in the control of mitochondrial biology.

A simple assay for the study of human hair follicle damage induced by ionizing irradiation.

Due to its rapidly proliferating matrix keratinocytes, the hair follicle is highly sensitive to ionizing irradiation (IR)-induced skin damage and thus an instructive and clinically relevant model organ for investigating the effects of IR on rapidly dividing epithelial-mesenchymal interaction systems. Here, we have assessed the impact of IR on organ-cultured human scalp hair follicles. We show that IR significantly inhibits the proliferation and induces apoptosis of hair follicle matrix keratinocytes, disrupts normal hair follicle pigmentation, and upregulates a number of quantitative toxicity and viability markers (oxidative stress indicators, DNA oxidative damage, LDH release). This introduces human hair follicle organ culture as an excellent novel research tool for radiobiology and invites exploitation as a preclinical assay system for testing candidate radioprotectants.

Thyrotropin releasing hormone (TRH): a new player in human hair-growth control.

Thyrotropin-releasing hormone (TRH) is the most proximal component of the hypothalamic-pituitary-thyroid axis that regulates thyroid hormone synthesis. Since transcripts for members of this axis were detected in cultured normal human skin cells and since human hair follicles (HFs) respond to stimulation with thyrotropin, we now have studied whether human HF functions are also modulated by TRH. Here we report that the epithelium of normal human scalp HFs expresses not only TRH receptors (TRH-R) but also TRH itself at the gene and protein level. Stimulation of microdissected, organ-cultured HFs with TRH promotes hair-shaft elongation, prolongs the hair cycle growth phase (anagen), and antagonizes its termination by TGF-beta2. It also increases proliferation and inhibits apoptosis of hair matrix keratinocytes. These TRH effects may be mediated in part by reducing the ATM/Atr-dependent phosphorylation of p53. By microarray analysis, several differentially up- or down-regulated TRH-target genes were detected (e.g., selected keratins). Thus, human scalp HFs are both a source and a target of TRH, which operates as a potent hair-growth stimulator. Human HFs provide an excellent discovery tool for identifying and dissecting nonclassical functions of TRH and TRH-mediated signaling in situ, which emerge as novel players in human epithelial biology.

Does erythropoietin modulate human hair follicle melanocyte activities in situ?

Erythropoietin (EPO) is now appreciated for not only drive erythopoiesis, but also to exert additional functions. Since we had previously shown that human hair follicles (HFs) are both an extra-renal source and an extra-medullary target of EPO, we have now studied whether one such function is the regulation of HF pigmentation. Human anagen VI HFs were treated with EPO (100 IU/ml) in serum-free organ culture. Unexpectedly, we noticed greatly divergent pigmentary effects of EPO, since both up- and down-regulation of HF melanin content and tyrosinase activity in situ was seen in HF derived from different individuals. These divergent effects could not be attributed to differences in skin regions, the total HF melanocyte number or specific traits of individual HF donors. Our pilot study provides first evidence suggesting that EPO may modulate normal human melanocyte functions under physiologically relevant conditions in situ.

Leptin and the skin: a new frontier.

Here, we examine the currently available information which supports that the adipokine, leptin, is a major player in the biology and pathology of mammalian skin and its appendages. Specifically, the potent metabolic effects of leptin and its mimetics may be utilized to improve, preserve and restore skin regeneration and hair cycle progression, and may halt or even partially reverse some aspects of skin ageing. Since leptin can enhance mitochondrial activity and biogenesis, this may contribute to the wound healing-promoting and hair growth-modulatory effects of leptin. Leptin dependent intracellular signalling by the Janus kinase 2 dependent signal transducer and activator of transcription 3, adenosine monophosphate kinase, and peroxisome proliferator-activated receptor (PPAR) gamma coactivator/PPAR converges to mediate mitochondrial metabolic activation and enhanced cell proliferation which may orchestrate the potent developmental, trophic and protective effects of leptin. Since leptin and leptin mimetics have already been clinically tested, investigative dermatology is well-advised to place greater emphasis on the systematic exploration of the cutaneous dimensions and dermatological potential of this pleiotropic hormone.

Erythropoietin and the skin: a role for epidermal oxygen sensing?

Erythropoietin (EPO), long appreciated as the chief endocrine regulator of red blood cell formation, is now recognized to exert many additional functions outside the bone marrow. Thus, the quest is on to define the full range of EPO functions in the physiology and pathology of non-hematopoietic tissues. Two recent studies in man and mice have highlighted the importance of the mammalian skin as one peripheral tissue with a previously unsuspected role in EPO biology; both, as a target and as a source of EPO. In addition, the skin has been proposed to function as an oxygen sensor. The present hypothesis essay critically reviews the currently available evidence for this and provides a unifying theoretical scenario for intracutaneous EPO functions and for a potential role of the skin in the control of EPO production. Mainly, we propose that the skin itself directly contributes to the up-regulation of EPO plasma levels in response to hypoxia.

A human folliculoid microsphere assay for exploring epithelial- mesenchymal interactions in the human hair follicle.

The search for more effective drugs for the management of common hair growth disorders remains a top priority, both for clinical dermatology and industry. In this pilot study, we report a pragmatic organotypic assay for basic and applied hair research. The patented technique produces microdroplets, which generate human folliculoid microspheres (HFMs), consisting of human dermal papilla fibroblasts and outer root sheath keratinocytes within an extracellular matrix that simulates elements of the hair follicle mesenchyme. Studying a number of different markers (for example, proliferation, apoptosis, cytokeratin-6, versican), we show that these HFMs, cultured under well-defined conditions, retain several essential epithelial-mesenchymal interactions characteristic for human scalp hair follicle. Selected, recognized hair growth-modulatory agents modulate these parameters in a manner that suggests that HFMs allow the standardized preclinical assessment of test agents on relevant human hair growth markers under substantially simplified in vitro conditions that approximate the in vivo situation. Furthermore, we show by immunohistochemistry, reverse transcriptase-PCR, and DNA microarray techniques that HFMs also offer a useful discovery tool for the identification of target genes and their products for candidate hair drugs. HFM thus represent an instructive modern experimental and screening tool for basic and applied hair research in the human system.

Human female hair follicles are a direct, nonclassical target for thyroid-stimulating hormone.

Pituitary thyroid-stimulating hormone (TSH) regulates thyroid hormone synthesis via receptors (TSH-R) expressed on thyroid epithelial cells. As the hair follicle (HF) is uniquely hormone-sensitive and, hypothyroidism with its associated, increased TSH serum levels clinically can lead to hair loss, we asked whether human HFs are a direct target for TSH. Here, we report that normal human scalp skin and microdissected human HFs express TSH-R mRNA. TSH-R-like immunoreactivity is limited to the mesenchymal skin compartments in situ. TSH may alter HF mesenchymal functions, as it upregulates alpha-smooth muscle actin expression in HF fibroblasts. TSH-R stimulation by its natural ligand in organ culture changes the expression of several genes of human scalp HFs (for example keratin K5), upregulates the transcription of classical TSH target genes and enhances cAMP production. Although the functional role of TSH in human HF biology awaits further dissection, these findings document that intracutaneous TSH-Rs are fully functional in situ and that HFs of female individuals are direct targets for nonclassical, extrathyroidal TSH bioregulation. This suggests that organ-cultured scalp HFs provide an instructive and physiologically relevant human model for exploring nonclassical functions of TSH, in and beyond the skin.

Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair.

Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound.

Thyroid hormones directly alter human hair follicle functions: anagen prolongation and stimulation of both hair matrix keratinocyte proliferation and hair pigmentation.

CONTEXT: Both insufficient and excess levels of thyroid hormones (T3 and T4) can result in altered hair/skin structure and function (e.g. effluvium). However, it is still unclear whether T3 and T4 exert any direct effects on human hair follicles (HFs), and if so, how exactly human HFs respond to T3/T4 stimulation. OBJECTIVE: Our objective was to asses the impact of T3/T4 on human HF in vitro. METHODS: Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. HFs from euthyroid females between 40 and 69 yr (average, 56 yr) were cultured and treated with T3/T4. RESULTS: Studying microdissected, organ-cultured normal human scalp HFs, we show here that T4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T3 and T4. T4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-beta2, the key anagen-inhibitory growth factor. Because we show here that human HFs transcribe deiodinase genes (D2 and D3), they may be capable of converting T4 to T3. Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T3 and T4 (CK6 is enhanced, CK14 down-regulated). Both T3 and T4 also significantly stimulate intrafollicular melanin synthesis. CONCLUSIONS: Thus, we present the first evidence that human HFs are direct targets of thyroid hormones and demonstrate that T3 and/or T4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation.

T-oligos as differential modulators of human scalp hair growth and pigmentation: a new "time lapse system" for studying human skin and hair follicle biology in vitro?

Small DNA oligonucleotides homologous to the 3' overhang of human telomeres, called T-oligos, stimulate pigmentation in human epidermal melanocytes in vitro and in vivo. They induce UV-mimetic effects in the absence of DNA-damage, however, it is unknown how T-oligos affect human hair follicle keratinocyte and melanocyte functions in situ. Here, we present the first evidence that these oligonucleotides are powerful modulators of pigmentation and growth of microdissected, organ-cultured human scalp hair follicles. Hair follicles were incubated with T-oligo or vehicle control and were then assessed for changes in hair shaft length, follicle morphology, pigmentation, proliferation and apoptosis. After only 48 h, T-oligos induced a fourfold increase in pigmentation of human anagen VI hair bulbs, while hair matrix keratinocyte proliferation was reduced by 65%, without apparent changes in hair bulb cell apoptosis. This corresponded well with a significant inhibition of hair shaft elongation, which was not accompanied by premature catagen induction in anagen VI hair follicles. These diametrically opposed effects of T-oligos on human hair follicle melanocytes (stimulation of melanogenesis) versus human hair bulb keratinocytes (inhibition of proliferation) in situ illustrate that human hair follicle organ culture offers an excellent tool for T-oligo research. They suggest that T-oligos deserve to be further explored for the management of clinical hair growth and pigmentation disorders, and raise the possibility that this model may offer a unique "time lapse system" for studying skin and hair follicle biology and DNA repair strategies under physiologically relevant conditions.

Probing the effects of stress mediators on the human hair follicle: substance P holds central position.

Stress alters murine hair growth, depending on substance P-mediated neurogenic inflammation and nerve growth factor (NGF), a key modulator of hair growth termination (catagen induction). Whether this is of any relevance in human hair follicles (HFs) is completely unclear. Therefore, we have investigated the effects of substance P, the central cutaneous prototypic stress-associated neuropeptide, on normal, growing human scalp HFs in organ culture. We show that these prominently expressed substance P receptor (NK1) at the gene and protein level. Organ-cultured HFs responded to substance P by premature catagen development, down-regulation of NK1, and up-regulation of neutral endopeptidase (degrades substance P). This was accompanied by mast cell degranulation in the HF connective tissue sheath, indicating neurogenic inflammation. Substance P down-regulated immunoreactivity for the growth-promoting NGF receptor (TrkA), whereas it up-regulated NGF and its apoptosis- and catagen-promoting receptor (p75NTR). In addition, MHC class I and beta2-microglobulin immunoreactivity were up-regulated and detected ectopically, indicating collapse of the HF immune privilege. In conclusion, we present a simplistic, but instructive, organ culture assay to demonstrate sensitivity of the human HF to key skin stress mediators. The data obtained therewith allow one to sketch the first evidence-based biological explanation for how stress may trigger or aggravate telogen effluvium and alopecia areata.

Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.

Chemotherapy-induced alopecia represents one of the major unresolved problems of clinical oncology. The underlying molecular pathogenesis in humans is virtually unknown because of the lack of adequate research models. Therefore, we have explored whether microdissected, organ-cultured, human scalp hair follicles (HFs) in anagen VI can be exploited for dissecting and manipulating the impact of chemotherapy on human HFs. Here, we show that these organ-cultured HFs respond to a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC), in a manner that resembles chemotherapy-induced HF dystrophy as it occurs in vivo: namely, 4-HC induced melanin clumping and melanin incontinence, down-regulated keratinocyte proliferation, massively up-regulated apoptosis of hair matrix keratinocytes, prematurely induced catagen, and up-regulated p53. In addition, 4-HC induced DNA oxidation and the mitochondrial DNA common deletion. The organ culture system facilitated the identification of new molecular targets for chemotherapy-induced HF damage by microarray technology (eg, interleukin-8, fibroblast growth factor-18, and glypican 6). It was also used to explore candidate chemotherapy protectants, for which we used the cytoprotective cytokine keratinocyte growth factor as exemplary pilot agent. Thus, this novel system serves as a powerful yet pragmatic tool for dissecting and manipulating the impact of chemotherapy on the human HF.