The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth-modulatory effects of agents in this assay, one critical read-out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organcultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.Key words: classification -eflornithine -gp100 -hair follicle cycle -melanin -Philpott assay Please cite this paper as: Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.

SummaryBackgroundBullous pemphigoid is a blistering skin disorder with increased mortality. We tested whether a strategy of starting treatment with doxycycline gives acceptable short-term blister control while conferring long-term safety advantages over starting treatment with oral corticosteroids.MethodsWe did a pragmatic, multicentre, parallel-group randomised controlled trial of adults with bullous pemphigoid (three or more blisters at two or more sites and linear basement membrane IgG or C3). Participants were randomly assigned to doxycycline (200 mg per day) or prednisolone (0·5 mg/kg per day) using random permuted blocks of randomly varying size, and stratified by baseline severity (3–9, 10–30, and >30 blisters for mild, moderate, and severe disease, respectively). Localised adjuvant potent topical corticosteroids (<30 g per week) were permitted during weeks 1–3. The non-inferiority primary effectiveness outcome was the proportion of participants with three or fewer blisters at 6 weeks. We assumed that doxycycline would be 25% less effective than corticosteroids with a 37% acceptable margin of non-inferiority. The primary safety outcome was the proportion with severe, life-threatening, or fatal (grade 3–5) treatment-related adverse events by 52 weeks. Analysis (modified intention to treat [mITT] for the superiority safety analysis and mITT and per protocol for non-inferiority effectiveness analysis) used a regression model adjusting for baseline disease severity, age, and Karnofsky score, with missing data imputed. The trial is registered at ISRCTN, number ISRCTN13704604.FindingsBetween March 1, 2009, and Oct 31, 2013, 132 patients were randomly assigned to doxycycline and 121 to prednisolone from 54 UK and seven German dermatology centres. Mean age was 77·7 years (SD 9·7) and 173 (68%) of 253 patients had moderate-to-severe baseline disease. For those starting doxycycline, 83 (74%) of 112 patients had three or fewer blisters at 6 weeks compared with 92 (91%) of 101 patients on prednisolone, an adjusted difference of 18·6% (90% CI 11·1–26·1) favouring prednisolone (upper limit of 90% CI, 26·1%, within the predefined 37% margin). Related severe, life-threatening, and fatal events at 52 weeks were 18% (22 of 121) for those starting doxycycline and 36% (41 of 113) for prednisolone (mITT), an adjusted difference of 19·0% (95% CI 7·9–30·1), p=0·001.InterpretationStarting patients on doxycycline is non-inferior to standard treatment with oral prednisolone for short-term blister control in bullous pemphigoid and significantly safer in the long-term.FundingNIHR Health Technology Assessment Programme.

Context: Both insufficient and excess levels of thyroid hormones (T 3 and T 4 ) can result in altered hair/skin structure and function (e.g. effluvium). However, it is still unclear whether T 3 and T 4 exert any direct effects on human hair follicles (HFs), and if so, how exactly human HFs respond to T 3 /T 4 stimulation.Objective: Our objective was to asses the impact of T 3 /T 4 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 T 3 /T 4 . Results:Studying microdissected, organ-cultured normal human scalp HFs, we show here that T 4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T 3 and T 4 . T 4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-␤2, 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 T 4 to T 3 . Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T 3 and T 4 (CK6 is enhanced, CK14 down-regulated). Both T 3 and T 4 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 T 3 and/or T 4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation. (J Clin Endocrinol Metab 93: 4381-4388, 2008) C linically, it has long been observed that patients with thyroid dysfunction may show prominent hair abnormalities (1-4) and several in vivo studies have demonstrated (partially conflicting) hair growth-modulatory effects of thyroid hormone (TH) in sheep, rats, and mice (5-8). In humans, hypothyroidism can be associated with telogen effluvium, along with the presentation of dry, brittle, and dull hair shafts (2-4). Confusingly, hyperthyroid states can also lead to effluvium, together with thinned hair shaft diameter and brittle, greasy hair (1, 9 -11), despite an apparently increased hair matrix proliferation (3). Hair shafts of patients with hyperthyroidism also show substantially reduced tensile strength (10). Early graying has been claimed to be related to autoimmune thyroid disease, hypothyroidism, and hyperthyroidism (11, 12), whereas darkening of

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.