6-O glucose linoleate supports in vitro human hair growth and lipid synthesis.

The hair follicle is a very active organ with a complex structure, which produces a hair fibre at a rate of 0.3 mm a day. Accordingly, the hair follicle is highly demanding in energy source, as the hair bulb matrix cells are endowed with one of the highest rates of proliferation in the human body. Moreover, recent data have shown the involvement of lipids in hair follicle function. As in vitro-grown hair follicle keeps producing a hair fibre that closely resembles the natural hair fibre, we decided to use this model to investigate the role of a new of glucose linoleate derivative (6-O-linoleyl-d-glucose: 6-O-GL) as a lipid precursor and energy provider. Our results demonstrated that 6-O-GL was (i) quite stable and surprisingly resistant to oxidative degradation, and (ii) readily taken up and metabolized by the hair follicle into various lipids, namely neutral lipids, ceramides and polar lipids. Moreover, it supported hair follicle growth and survival in a glucose- and linoleic-acid free medium. 6-O-GL thus appeared to be a bi-functional nutrient, ensuring both proper fibre quality and production by the hair follicle.

Protective effects of taurine on human hair follicle grown in vitro.

Taurine is a naturally occurring beta-amino acid produced by methionine and cysteine metabolism. It is involved in a variety of physiological functions, including immunomodulatory and antifibrotic. Taking advantage of the ability of human hair follicle grown in vitro to recapitulate most of the characteristic features of normal hair follicle in vivo, we studied (i) taurine uptake by isolated human hair follicles; (ii) its effects on hair growth and survival rate; and (iii) its protective potential against transforming growth factor (TGF)-beta1, an inhibitor of in vitro hair growth and a master switch of fibrotic program. We showed that taurine was taken up by the connective tissue sheath, proximal outer root sheath and hair bulb, promoted hair survival in vitro and prevented TGF-beta1-induced deleterious effects on hair follicle.

Electrophoretic study of the physico-chemical characteristics of Bence-Jones proteinuria and its association with kidney damage.

AIM: To identify a physico-chemical criterion, or set of criteria, explaining and possibly predicting the nephrotoxic behaviour of Bence-Jones proteins (BJP). METHODS: The electrophoretic mobility and isoelectric point (pI) of 92 BJP isolates were determined using various electrophoresis procedures on polyacrylamide gel. The proportions of monomers and dimers were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS PAGE) in 58 cases. PAGE data for 10 BJP isolates were used to construct Ferguson plots and titration curves. RESULTS: The distribution of electrophoretic mobility and pI values was bimodal and showed a positive correlation when the pI was above 6. The values of these two parameters in 22 patients with renal impairment were not significantly different from those in the patients without renal impairment, and the statistical analysis showed no predictive value for the onset of renal impairment. However, patients excreting the lambda light chain isotype had a 2.8-fold higher risk of developing renal impairment compared with the other patients. Studies of the charge variation of the protein with pH indicated three types of behaviour, suggesting that the charge of BJP is highly variable at physiological pH. CONCLUSION: It is important to study not only the positivity or negativity of the BJP charge at a given pH, but also its intensity. The study of the BJP titration curves in patients with renal impairment suggests that a low charge at physiological urinary pH could predict renal impairment.

Apolipoprotein E and multiple sclerosis: a biochemical and genetic investigation.

Apolipoprotein E (apo E) is postulated to be a major lipid carrier protein in the brain involved in brain development and repair. Multiple sclerosis (MS) is a major demyelinating disease characterized by destruction of myelin and marked alteration of myelin cholesterol and lipid metabolism. We have determined serum and cerebrospinal fluid (CSF) apo E concentrations using an original time-resolved immunofluorometric assay and calculated intrathecal apo E concentration. Apo E concentrations were determined in 13 control subjects and 129 neurological patients: 34 definite MS patients, 25 with Guillain-Barré syndrome (GBS), 32 with amyotrophic lateral sclerosis (ALS) and 38 with other neurological diseases. Seven clinical parameters (sex, age, age at MS onset, duration of the disease, course, clinical status and disability score) were considered in MS patients. Significant (P < 0.01) decrease in CSF apo E was observed in MS, linked to a decrease in intrathecal apo E. The decreased CSF apo E concentration in MS patients occur independent of the apo E genotype. Apo E is considered as a neurotrophic factor in the brain. Any decrease in intrathecal apo E synthesis would thus contribute to progression of neurological diseases, such as MS.

[Time-resolved fluorometry: principles and applications in clinical biology]. / Fluorimétrie en temps retardé: principe et applications en biologie clinique.

Time-resolved fluorometric assay is based on lanthanide fluorescence. This time-resolved fluorescence has a narrow-band emission line whose wavelength differs from that of emission-pulsed light and has a long decay-time. These characteristics make it possible to avoid background interference from sample constituents (protein, light-scattering particles, etc). Europium and its chelates are the most commonly used lanthanides. The europium-labelling of antigens or antibodies is followed by immunoassay. In the final step, fluorescence is measured, after enhancement, as counts per second. This assay has several advantages, including a wide working range, high sensitivity and good practicability. The method has widespread applications in the field of immunoassays in both clinical and research laboratories. The use of non-radioactive europium-labelled probes and the development of simultaneous multiple tests are possible future orientations.

Human hair shape is programmed from the bulb.

BACKGROUND: Few biological data on curly hair follicles have been reported in the literature. OBJECTIVES: To investigate the growth and morphology of curly hair follicles. METHODS: Follicles were dissected from scalp skin samples from African, Guyanese and caucasian volunteers and were observed macroscopically, in culture in William's E medium, and by immunohistochemistry. RESULTS: Macroscopic study of scalp biopsies obtained from African volunteers showed that the dermal implantation of follicles was curved with a retrocurvature at the level of the bulb, as opposed to a straight shape in caucasian follicles. The bulb itself was bent, in the shape of a golf club, while both the outer root sheath (ORS) and the connective tissue sheath were dissymmetrical along the follicle. In vitro growth of curly hair follicles was slightly slower than that of caucasian follicles but, more importantly, the curvature was maintained in the hair shaft produced in vitro. As shown by immunohistochemistry, the proliferative matrix compartment of curly hair follicles was asymmetrical, Ki-67-labelled cells being more numerous on the convex side and extending above the Auber line. On the convex part of the follicle, the ORS was thinner and the differentiation programmes of the inner root sheath and hair shaft were delayed. Furthermore, some ORS cells expressed alpha-smooth muscle actin protein on the concave side of the curvature, reflecting a mechanical stress. CONCLUSIONS: Hair curliness is programmed from the bulb and is linked to asymmetry in differentiation programmes.

Human hair greying is linked to a specific depletion of hair follicle melanocytes affecting both the bulb and the outer root sheath.

BACKGROUND: Although hair greying is a very common phenomenon characterized by loss of pigment in the hair shaft, the events that cause and control natural hair whitening with age in humans are still unclear. OBJECTIVES: To decipher the origin of natural hair whitening. METHODS: Human hair melanocytes were immunohistochemically characterized at different stages of whitening. RESULTS: Loss of hair shaft melanin was found to be associated with a decrease in both bulb melanin content and bulb melanocyte population. Although few melanocytes were present in the bulbs of grey hair, they still expressed tyrosinase and tyrosinase-related protein-1, synthesized and transferred melanins to cortical keratinocytes as seen by the presence of melanin granules. In white hair bulbs, no melanocytes could be detected either with pMel-17 or vimentin labelling. Pigmented hair follicles are known to contain inactive melanocytes in the outer root sheath (ORS), and grey and white hairs were also found to contain some of these quiescent melanocytes. However, their population was decreased compared with pigmented hair follicles, ranging from small to nil. This depletion of melanocytes in the different areas of white hairs was detected throughout the hair cycle, namely at telogen and early anagen stages. In contrast, the infundibulum and sebaceous gland of both pigmented and white hairs showed a similar distribution of melanocytes. Furthermore, other distinct cell populations located in the ORS, namely putative stem cells, Merkel cells and Langerhans cells were equivalently identified in pigmented and white hairs. CONCLUSIONS: Thus, hair greying appears to be a consequence of an overall and specific depletion of bulb and ORS melanocytes of human hair.

The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?

Up to now, the localization of stem cells in human anagen hair follicle relied on three complementary approaches; namely, detection of slow cycling cells, detection of high colony forming cells, and differential immunohistochemical staining. These techniques, however, gave conflicting results since stem cells were localized either as long label retaining cells in the so-called bulge area or as high colony forming cells in the lower third of the follicle. In the present study we investigated the expression of cytokeratin 19, a marker for putative stem cell-containing epithelial compartments, in order to characterize stem cell distribution in the human hair follicle throughout the hair cycle. We found that anagen human hair follicles contain two distinct reservoirs for stem cells located in the upper and lower thirds of the follicle. These two reservoirs fuse during the catagentelogen transition phase and individualize again in the newly forming anagen hair follicle.

Application of the time-resolved immunofluorometric assay to the study of C3 complement component glycation in vitro and in vivo.

A time-resolved immunofluorometric assay (TR-IFMA) was used for the measurement of glycated C3. The very high sensitivity of this technique allowed the direct measurement of glycated and non-glycated proteins (especially C3) in chromatography eluates. C3 glycation in vitro after incubation with 20 mmol/l glucose was always less than 3.5% by day 5. As determined with the TR-IFMA, the means +/- standard deviations of glycated C3 were 0.20% +/- 0.04 for non-diabetic subjects and 0.88% +/- 0.06 for insulin-dependent diabetic patients. The low percentages of glycated C3 in both our in vitro and in vivo studies show that this protein is subject to only moderate rates of glycation.

Thyroid hormone receptor beta1 is expressed in the human hair follicle.

To understand better the mechanisms by which thyroid hormone can exert its effects on the hair follicle, we looked for the expression of members of the thyroid hormone receptor (TR) family in human hair follicles. Immunoreactive TRs were detected in both dermal and epithelial compartments of the human pilosebaceous unit. Using reverse transcriptase-polymerase chain reaction, we established that TRbeta1 was the predominant form of TR expressed in the human hair follicle. In addition, we investigated the effects of 3,3', 5-triiodo-L-thyronine (T3) on the survival of human hair follicles in vitro, to understand the role of this thyroid hormone on hair follicle homeostasis. A physiological level of free T3 significantly enhanced human hair survival in vitro.

Time-resolved immunofluorometric assay of complement C3: application to cerebrospinal fluid.

Complement components have a role in various neurological disorders. Complement C3 can be measured by immunochemical methods, but only radioimmunoassays and electroimmunodiffusion assays (EIDs) are sufficiently sensitive to be applied to biological fluids in which the C3 concentration is low, especially cerebrospinal fluid (CSF). We report a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for C3 in CSF. The linearity (0.7-3650 micrograms/L) and intra- (CV < 4.8%) and inter-assay (CV < 10.9%) precision were satisfactory and the results agreed with those of EID. The assay is extremely sensitive (< 1 microgram/L) and its analytical range is large and well suited to clinical applications. This simple TR-IFMA is thus a nonisotopic alternative to radioimmunoassay for the quantification of complement C3 in CSF.

Decreased cervicovaginal production of both IgA1 and IgA2 subclasses in women with AIDS.

Paired sera and cervicovaginal secretions from 35 HIV-1-infected women representing different CDC stages of HIV infection were evaluated for total IgA, IgA1 and IgA2, for IgA, IgA1 and IgA2 to gp160, and for albumin. Age-matched healthy women (n = 45) served as controls. The secretion rates of total IgA, IgA1 and IgA2 were evaluated by calculating their relative coefficients of excretion by reference to albumin. In HIV-infected women, total IgA1 and IgA2 in sera and in cervicovaginal secretions increased proportionately as early as stages II + III and more markedly at stage IV. By contrast, the secretion rates of total IgA IgA1 and IgA2 were markedly reduced in AIDS women, the IgA2 secretion rate decreasing significantly as early as stages II + III. This apparent discrepancy was probably the result of increased transudation of serum-borne immunoglobulins into the vaginal cavity, since albumin levels in cervicovaginal secretions increased significantly according to the stages of disease. HIV-reactive IgA antibodies in serum, as in cervicovaginal secretions, were principally found within the IgA1 subclass. In women at stage IV, a high local production of IgA1 to gp160 occurred in spite of the impairment of cervicovaginal IgA synthesis, probably because of marked genital HIV replication at advanced stages.

A sensitive time-resolved immunofluorometric assay for the measurement of apolipoprotein B in cerebrospinal fluid. Application to multiple sclerosis and other neurological diseases.

Although low density lipoprotein receptors have been described on oligodendrocytes, apolipoprotein B was thought to be absent or present in only very small amounts in cerebrospinal fluid (CSF). Several immunoassays have been used for the measurement of apolipoprotein B in serum. However, the majority of methods cannot be used to measure small amounts of apolipoprotein B in CSF. In this study, we describe a highly sensitive time resolved immunofluorometric assay (TR-IFMA) using europium as label (detection limit: 0.3 microgram/l). The reliability of the TR-IFMA for the measurement of apolipoprotein B was first studied in serum. Serum and CSF apolipoprotein B concentrations were then determined in subjects free of neurological disorders and in patients with multiple sclerosis. Local intrathecal apolipoprotein B synthesis was calculated. Although the high sensitivity of the TR-IFMA allowed low amounts of apolipoprotein B in CSF to be detected (0.11 +/- 0.06; 0.12 +/- 0.06 mg/l in controls and multiple sclerosis patients, respectively), no apolipoprotein B could be detected in CSF by electroimmunodiffusion. As suggested by the blood/CSF apolipoprotein B ratio (about 6000), no apolipoprotein B synthesis was observed by both using apolipoprotein B index and formula. This indicates its probable serum origin. Moreover, there was no difference between controls and multiple sclerosis patients in CSF, serum, blood/CSF, index, and local intrathecal apoliprotein B synthesis. Finally, these results suggest that the role of apolipoprotein B in lipid transport in the central nervous system may be questionable.

Immunochemical characterization of monoclonal IgG containing mixed cryoglobulins.

Monoclonal IgG-containing type II cryoglobulins (CG) from 25 patients, most affected with autoimmune or inflammatory diseases, were characterized using a Western blotting procedure. Rheumatoid factors were measured by ELISA. Out of the 25 type II CG, 14 contained a single monoclonal IgG and 11 contained several distinct monoclonal Ig, as assessed by the electrophoretic mobility and isotypy of the narrow bands. Comparison of blots revealed by anti-IgG subclass antibodies in the patients' sera and CG showed a clear-cut enrichment of CG in both polyclonal and monoclonal IgG1 and IgG3. All monoclonal IgG in the CG belonged to the IgG1 or IgG3 subclasses. CG also contained more detectable RF of the IgM class than did the corresponding sera. Using conventional methods, the present CG either could not be typed or were all (immunoelectrophoresis) or often (immunofixation) classified as type III. The results challenge the classical classification of CG and show the necessity of the use of sensitive techniques for immunochemical characterization of mixed CG.

Expression of peroxisome proliferator activated receptors (PPARs) in human hair follicles and PPAR alpha involvement in hair growth.

Peroxisome proliferator-activated receptors (PPARs), which belong to the nuclear hormone receptor superfamily, have recently been described as potent key regulators of epidermal development. As 1,25-dihydroxyvitamin D3, retinoic acid and triiodothyronine are known to exert effects on skin and hair follicle growth through similar receptors, we decided to investigate both the expression pattern of the PPAR alpha, -delta and -gamma subtypes and their role in human hair follicles. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry, we established that PPAR alpha, -delta and -gamma were expressed in both dermal and epithelial human hair follicle cells. Additionally, we evaluated the dose effect of clofibrate, a PPAR alpha ligand, on the survival of human hair follicles in culture. A beneficial effect was observed within a narrow range of concentrations.

Frequency and relevance of IgM intrathecal synthesis in multiple sclerosis.

Using a new ELISA method we have measured the IgM concentration in the serum and the cerebrospinal fluid CSF from 110 neurological patients. Among there, 41 had multiple sclerosis (MS), 48 other inflammatory diseases (OID), including 30 AIDS, and 21 non-inflammatory neurological diseases (NID). A highly significant correlation was established between results with native IgM and the dithiothreitol reduced IgM. An intrathecal synthesis (ITS) of IgM was detected using the CSF IgM/CSF albumin ratio, the IgM index and a quantitative formula in 33 patients: nine MS, 23 OID (including 18 AIDS) and one NID. The frequency of IgM ITS was 22% in MS patients, 48% in the OID (60% in AIDS) and 5% in the NID groups. This ITS was not impaired by an increase in serum IgM concentration or by a blood-CSF barrier damage. These facts confirm that intrathecal immunity is not a "steady-state" related to the general immunity but a specific response restricted to the central nervous system. Conversely, CSF IgM increase and IgM ITS were closely related (p < 10(-6) ). In addition, IgM ITS and IgG ITS were found to be highly correlated in OID, especially in AIDS patients: such correlation was not observed in the MS group. No significant correlations were observed between IgM ITS and any of the clinical parameters in MS patients. These results suggest the probable specificity of IgM ITS in MS patients.

Characterization of cryoglobulins by immunoblotting.

We adapted an immunoblotting technique for the immunochemical characterization of cryoglobulins. We first compared the results from 157 samples with results obtained by immunofixation and immunoelectrophoresis and then with clinical observations in 125 cases. Full identification was possible in 98% of the cases by immunoblotting, in 54% by immunofixation, and in 28% by immunoelectrophoresis. Novel microheterogeneity aspects were observed by immunoblotting and immunofixation in 13% and 6% of the cases studied, respectively. Our results confirm the frequency of autoimmune and infectious diseases associated with mixed cryoglobulinemias, whereas a lymphoproliferative syndrome was observed only in cases of cryoglobulinemia with a monoclonal constituent or a microheterogeneity aspect, which was not always revealed by immunofixation or immunoelectrophoresis. The pathophysiological importance and classification of microheterogeneity aspects is not clear. These observations justify using a sensitive and specific method for identifying cryoglobulins, even when present at low concentrations, and call for long-term studies of these patients.