Antibodies to hair follicles in alopecia areata

Although alopecia areata is suspected to be an autoimmune disease, no direct evidence of an altered immune response to components of the hair follicle has been reported. We studied whether antibodies to normal human anagen scalp hair follicles are present in individuals with alopecia areata. Thirty-nine alopecia areata sera and 27 control sera were tested by Western immunoblotting for antibodies to 6 M urea-extractable proteins of normal anagen scalp hair follicles. At serum diluted 1:80, all alopecia areata subjects (100%), but only 44% of control individuals, had antibodies directed to one or more antigens of approximately 57, 52, 50, 47, or 44 kD. The incidence of antibodies to individual hair follicle antigens in alopecia areata was up to seven times more frequent than in control sera and their level up to 13 times greater and was statistically significant for all five antigens. Tissue specificity analysis indicated that these antigens were selectively expressed in hair follicles. These findings indicate that individuals with alopecia areata have abnormal antibodies directed to hair follicle antigens, and support the hypothesis that alopecia areata is an autoimmune disease.     

Unmyelinated innervation of sinus hair follicles in rats

Unmyelinated nerve fibres comprise approximately one third of the innervation of rodent sinus hair follicles but their function is unknown. They may play a role as high-threshold sensory fibres, or may be autonomic efferents controlling the vascular sinus. In the present experiments capsaicin and surgical sympathectomy were used to establish whether these unmyelinated fibres are afferent fibres or autonomic efferents. The deep vibrissal nerves of mystacial follicles (C1 and C4) and a non-mystacial follicle (the postero-orbital, PO) were assessed in normal adult animals (n = 6) and compared with those treated with neonatal capsaicin (n = 6) or bilateral superior cervical ganglionectomy (n = 7). In capsaicin-treated animals, counts of fibres in the deep vibrissal nerves from all follicles showed normal numbers of myelinated axons, but approximately 80% reduction in unmyelinated fibres (normal mean +/- SD: C1 94 +/- 10, C4 89 +/- 9, PO 85 +/- 6; after neonatal capsaicin: C1 17 +/- 8, C4 16 +/- 6, PO 18 +/- 6; n = 6, P < 0.001 for all follicles). After sympathectomy there was no significant reduction in myelinated or unmyelinated fibre numbers. Labelling of PO follicles with WGA-HRP showed minimal numbers of labelled cells (0-10) within the superior cervical ganglion, also suggesting minimal sympathetic innervation. This sparse sympathetic supply to the follicle was further demonstrated by a lack of tyrosine hydroxylase reactivity within the follicle complex; tissues outside the dermal capsule showed reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ageing-associated large-scale deletions of mitochondrial DNA in human hair follicles

Hair follicles plucked from the bi-temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing-associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi-quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05% +/- 0.01%, 0.00%, 0.55% +/- 0.05%, 0.52% +/- 0.24%, 0.65% +/- 0.17%, 1.33 +/- 0.25%, and 1.89% +/- 0.81% for the subjects in the age groups of 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D-loop region of mtDNA by PCR with back-to-back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large-scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non-invasively obtained from the human, we suggest that the aged-dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.     

Different populations of melanocytes are present in hair follicles and epidermis

Melanocytes in human skin reside both in the epidermis and in the matrix and outer root sheath of anagen hair follicles. Comparative study of melanocytes in these different locations has been difficult as hair follicle melanocytes could not be cultured . In this study we used a recently described method of growing hair follicle melanocytes to characterize and compare hair follicle and epidermal melanocytes in the scalp of the same individual. Three morphologically and antigenically distinct types of melanocytes were observed in primary culture. These included (1) moderately pigmented and polydendritic melanocytes derived from epidermis; (2) small, bipolar, amelanotic melanocytes; and (3) large, intensely pigmented melanocytes; the latter two were derived from hair follicles. The three sub-populations of cells all reacted with melanocyte-specific monoclonal antibody. Epidermal and amelanotic hair follicle melanocytes proliferated well in culture, whereas the intensely pigmented hair follicle melanocytes did not. Amelanotic hair follicle melanocytes differed from epidermal melanocytes in being less differentiated, and they expressed less mature melanosome antigens. In addition, hair follicle melanocytes expressed some antigens associated with alopecia areata, but not antigens associated with vitiligo, whereas the reverse was true for epidermal melanocytes. Thus antigenically different populations of melanocytes are present in epidermis and hair follicle. This could account for the preferential destruction of hair follicle melanocytes in alopecia areata and of epidermal melanocytes in vitiligo.     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Using stereological methods, two cerebral cortical areas from AIDS brains were investigated. Neuronal density, profile area of neurons, and perikaryon volume fraction were measured and compared to age-matched control brains. In the fronto-orbital cortex (area 11) of AIDS brains, a significant loss of neurons was seen. The perikaryon volume fraction was likewise decreased. The size of neurons did not differ between control and AIDS brains. In patients with clinical signs of progressive dementia and in brains with human immunodeficiency virus (HIV)-specific neuropathology (HIV-leukoencephalopathy and/or HIV-encephalitis) as compared to patients lacking these features, a small decrease in neuronal density was noted but this difference did not reach the level of statistical significance (P = 0.16). In the superior parietal lobule (area 7) of AIDS brains, no loss of nerve cells was noted. AIDS patients with progressive dementia and brains with HIV-specific neuropathology showed no difference in neuronal densities as compared to those without such features. We conclude that the fronto-orbital cortex, in contrast to the parietal cortex, is mainly damaged in AIDS brains. Neuronal loss was not significantly correlated with development of dementing symptoms and of HIV-specific neuropathology.     

Fiber growth initiation in hair follicles of goats treated with melatonin

Amphotericin B (AmB) is the drug of choice for most systemic fungal infections, but doses are frequently reduced because of nephrotoxicity. We investigated the role of thromboxane as a mediator for this nephrotoxicity. Vehicle or amphotericin (0.60 mg/kg) was infused into the left renal artery in four groups of rats, and renal plasma flow (RPF) and glomerular filtration rate (GFR) were measured. Group 1 received vehicle for 90 min. Group 2 received vehicle followed by a 30 minute AmB infusion which caused a significant and reversible fall in the RPF and GFR. Group 3 received vehicle followed by AmB infusion, but were infused with a bolus of ibuprofen (20 mg/kg) 45 minutes before AmB. This group exhibited an insignificant attenuation in the fall in RPF and GFR. Group 4 received vehicle followed by AmB, but were infused with a bolus and continuous infusion of the thromboxane receptor antagonist SQ29,548. This group demonstrated an attenuation in the fall in RPF and a significant decrease in GFR compared to AmB control rats. In addition, the rat glomeruli were incubated with AmB (4 ug/ml). Supernatant levels of thromboxane B2 were significantly elevated in the presence of AmB vs buffer alone. We conclude that the reduction in RPF and GFR observed with AmB infusion in the rat is partially mediated by release of thromboxane.     

Measurement of apoptotic fragments in growing hair follicles following gamma-ray irradiation in mice

Feeding ticks are generally spatially distributed in clusters on vertebrate hosts. To test the effect of clustering on transmission of a tick-borne pathogen, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner-infected Ixodes ricinus L. nymphs and uninfected I. ricinus larvae were allowed to feed together in retaining chambers on uninfected AKR/N mice. Engorged infective nymphs dropped off at days 5, 6, and 7, and the 1st infected larvae that fed in the chambers together with the infected nymphs dropped off at day 5. In contrast, ear biopsies and xenodiagnostic larvae placed on the head remained negative during that period. These results suggest that a cofeeding transmission occurred between B. burgdorferi-infected ticks and noninfected ones in the absence of a disseminated infection. Further investigations are being undertaken to determine whether the mechanism responsible for this cofeeding transmission is similar to that described previously with virus-infected ticks.     

Endocrine activity of induced persistent follicles in sheep

This experiment was designed to examine gonadotropin requirements for the induction and maintenance of persistent ovarian follicles in sheep. At the time of prostaglandin (PG) treatment on the tenth day of an induced estrous cycle, 8 ewes (with one ovary autotransplanted to the neck) received an injection of a GnRH antagonist ([Ac-d-Nal1, d-4-C-1-Phe2, d-Trp3, d-Arg6, d-Ala10] GnRH.HOAc; 50 microg/kg s.c.), and continuous hourly injections of exogenous ovine LH (equivalent to 1.25 microg NIH-oLH-S26) began simultaneously with this first antagonist injection (time zero). Antagonist was given three times at 3-day intervals. On Day 6, LH injections were stopped in 4 ewes (group 2) but continued in 4 other ewes (group 1) until the end of the 10-day experiment. Ovarian vein blood was sampled daily every 15 min for a 2-h period around two injections of exogenous LH (this sampling included group 2 after Day 6). Additional jugular and ovarian vein blood samples were collected every 8 h throughout the experiment. Daily ultrasound examination revealed the presence of at least one large follicle (range 4- to 7.5-mm diameter) from Day 3 to Day 10 in all ewes, but no new growing follicles (> 2 mm) were detected for at least 6 days. After Day 2, secretion of estradiol was positively correlated with that of inhibin (r = 0.83, p < 0.001), whereas FSH concentrations were inversely related to inhibin (r = -0.71, p < 0.001) and estradiol (r = -0.81, p < 0.001). In the absence of an LH surge, estradiol and androstenedione secretion (range 5-20 ng steroid/min) was maintained from Day 1 to Day 8 in group 1; but in group 2, secretion decreased abruptly when the LH injections stopped. Thus, continued low-amplitude, high-frequency LH pulses were required to maintain estradiol secretion when concentrations of FSH were < 0.5 ng/ml. However, estradiol and androstenedione secretion decreased (and FSH concentrations increased) between Days 8 and 10 in the ewes that received continued LH injections (group 1), showing that atresia in estrogenic follicles was not due to a lack of gonadotropin availability but to changes within the follicle. For the first 3 days after administration of PG, androstenedione secretion was greater than that of estradiol (p < 0.05), but from Day 4 to 6 the secretion rates were similar (p < 0.1), suggesting that aromatase may be limiting in the first 3 days whereas provision of androstenedione precursors was altered as the follicle persisted. In group 2 on Days 7 and 8 when hourly LH injections had stopped, neither androstenedione nor estradiol secretion increased after one test injection of LH; in contrast, androstenedione but not estradiol secretion increased after a second LH test injection 1 h later, suggesting that secretion of androstenedione is controlled by repeated exposure to LH. In conclusion, persistent estrogenic follicles were produced in the follicular phase in sheep by treatment with a combination of GnRH antagonist and hourly pulses of LH. Secretion of estradiol was dependent on continued hourly LH pulses of approximately 1 ng/ml and the follicles remained estrogenic for 8 days, after which time the ability to secrete estradiol and androstenedione declined even with continued LH injections.     

Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells

Neurofilaments, the neuron-specific intermediate filaments, are composed of three immunochemically distinct subunits: NF-L, NF-M and NF-H that can be either phosphorylated or unphosphorylated. In mammals, the distribution of these subunits has been described in vestibular ganglion neurons, but there are no reports on the presence of neurofilaments in vestibular hair cells. We investigated, by immunocytochemistry, neurofilaments in vestibular hair cells from rat and guinea-pig using antibodies against the three subunits and to dephosphorylated NF-H (clone SMI 32, recognizes also NF-M on immunoblots), on Vibratome sections of the vestibular end-organs and on isolated hair cells. Various immunostaining protocols were used, as appropriate for the method of observation: laser scanning confocal microscopy (immunofluorescence) and transmission electron microscopy (immunoperoxidase, pre-embedding technique). In rat and guinea-pig cristae and utricles, neurofilament immunoreactivity was observed in axons inside and below the sensory epithelia. In guinea-pig, in addition to this staining, intensely immunoreactive annular structures were found in the basal regions of hair cells. These rings were detected with anti-NF-L, -NF-M and -dephosphorylated NF-H/M antibodies, but not with anti-phosphorylation-independent NF-H. Ring-containing hair cells were present in all regions of the sensory epithelia but were more abundant in the peripheral areas. All levels of observation (Vibratome and thin sections, and isolated hair cells) showed that only the guinea-pig type I hair cells contained a neurofilament ring. High-resolution observations showed that the ring was located below the nucleus, often close to smooth endoplasmic reticulum and the cell membrane.     

An improved assay for hexokinase activity in human tissue homogenates

An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement, at 340 nm, of NADPH formed according to the reaction scheme: [formula: see text] In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase, EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9; glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.     

A potassium current in guinea-pig outer hair cells activated by ion channel blocker DCDPC

Fenamate compounds have been reported to inhibit ion channels in a number of tissues, including a non-selective cation channel in the mammalian outer hair cell (OHC). We have further investigated the effects of 3'-5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) on OHC currents using the whole-cell configuration of the patch clamp technique. Extracellular application of 10 microM DCDPC rapidly and reversibly activated an inward current at hyperpolarized potentials. The DCDPC-activated current appeared in the shorter OHCs from the basal turns of the cochlea. The reversal potential of the inward current was dependent on the external K+ ion concentration. An outwardly rectifying K+ current, found predominantly in OHCs from apical turns, was reversibly inhibited by DCDPC. These results suggest that DCDPC has a significant effect on OHC physiology at all tonotopic locations along the basilar membrane and so may have implications for cochlear function during fenamate intake.     

Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer's disease

OBJECTIVE: To investigate the levels of glutathione transferase (GST), a protective enzyme against aldehydes, and especially 4-hydroxynonenal (HNE) in the brain and ventricular CSF of autopsied AD and normal control subjects. BACKGROUND: Studies have implicated increased levels of oxidative stress in the brain in the pathogenesis of AD. Decreased levels of polyunsaturated fatty acids and increased levels of markers of lipid peroxidation have been reported in the brain in AD, particularly in areas severely affected in the disease. HNE, one marker of lipid peroxidation, is neurotoxic in neuronal culture and in vivo and is elevated in AD brain and CSF. METHODS: We measured levels of GST activity and protein in multiple brain regions and ventricular CSF in short-postmortem-interval AD patients and age-matched prospectively evaluated control subjects. RESULTS: A decrease in GST activity in all brain areas was observed in AD compared with controls with significant decreases in the amygdala, hippocampus and parahippocampal gyrus, inferior parietal lobule, and nucleus basalis of Meynert. Levels of GST protein also were depleted in most brain regions in AD. A significant decrease in GST activity and protein levels was also found in ventricular CSF in AD. CONCLUSION: Reduced levels of GST, a protective mechanism against HNE, may have a role in the pathogenesis of neuron degeneration in AD.     

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol

A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of apparent equilibrium between all three forms (active tetramer, inactive monomer, inactive tetramer) was reached. When tryptase was extensively cross-linked with several heterobifunctional photoactivatable reagents, this modified enzyme exhibited a long-term stability in low-ionic-strength buffer and at elevated temperature, unlike that of the native enzyme. Its is suggested that cross-linking prevents the spontaneous inactivation and dissociation of tryptase by 'freezing' the normal association state of the enzyme and supports the hypothesis that the dissociation of native tetrameric tryptase into inactive monomer is the primary event for the entire process of spontaneous inactivation of this enzyme.     

Activin from secondary follicles causes small preantral follicles to remain dormant at the resting stage

The sequences controlling the expression of the Rhodobacter capsulatus recA and uvrA genes belonging to the SOS DNA repair system have been identified by PCR mutagenesis. Data obtained demonstrated that the GTTCN7GTAC and GAACN7GAAC motifs present upstream of the recA gene and the GTTCN7GTTC motif found upstream of the uvrA gene are required for their respective DNA damage-mediated induction. Alignment of recA promoters of R. capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis with the uvrA promoters of R. capsulatus and R. sphaeroides has identified the consensus sequence GTTCVYVYTWTGTTC as the SOS operator site of the Rhodospirillaceae family.     

Changing patterns of gap junctional intercellular communication and connexin distribution in mouse epidermis and hair follicles during embryonic development

This paper is concerned with whether transport accident risk tends to peak at particular times, in relation to both time of day and time on task, and with the underlying causes of such peaks. Macro-analyses confirmed the presence of a clear circadian (ca 24 hour) rhythm in road accident risk with a major peak at ca 03:00 but suggested that this rhythm could not be entirely accounted for in terms of drivers falling asleep at the wheel. Sleep propensity clearly shows a pronounced circadian rhythm and performance efficiency in wakeful subjects shows a similar trend implying that the 03:00 road accident peak may simply reflect lowered performance capabilities. However, there are 'residual' peaks in accidents at certain times of day that are difficult to account for in terms of circadian rhythmicity. It is suggested that these may reflect a time on task effect which shows a pronounced, but transient, 2-4 hour peak in risk. Only when individuals had been on duty for 12 hours or more did the risk exceed that found during the 2-4 hour peak. While an explanation for this transient peak is offered, the underlying reason for it is, as yet, uncertain and clearly warrants investigation in view of its practical implications. It is concluded that there are 'black times' when accidents are far more likely and that there is a strong need to investigate possible countermeasures.