Hanan A. Assaf

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type‐specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.

Expression pattern of p75 neurotrophin receptor protein in human scalp skin and hair follicles: Hair cycle-dependent expression

BACKGROUND The p75 neurotrophin receptor (p75NTR) is a death factor (apoptosis-promoting protein) that belongs to the tumor necrosis factor receptor superfamily of membrane proteins. In the murine hair follicle (HF) model, p75NTR plays a critical role during HF morphogenesis, functioning as a receptor that negatively controls HF development. p75NTR signaling is involved in the control of keratinocyte apoptosis during catagen. To date, knowledge about the expression pattern of p75NTR protein in human scalp skin and HFs is limited. In this investigation we hypothesized that p75NTR protein is expressed in human scalp skin and its expression in HFs fluctuates with the transitions from anagen --> catagen --> telogen stages. METHODS To test this hypothesis, the immunoreactivity of p75NTR protein was examined in human scalp skin by immunofluorescent and immunoalkaline phosphatase methods. A total of 50 normal-appearing human scalp skin biopsy specimens were examined (healthy women age 53-57 years). In each case, 50 HFs were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). RESULTS We found variations in p75NTR protein expression with HF cycling. p75NTR expression was negligible in early, mid, and mature anagen and weak during late anagen. p75NTR expression was moderate during anagen-catagen transition. It was strong in both catagen and telogen HF. Also, p75NTR protein expression was strong in the stratum corneum (epidermis), dermal fibroblasts, blood vessels, nerve endings, adipocytes, and both sebaceous and sweat glands. LIMITATIONS Our knowledge about other proteins (prosurvival and pro-apoptotic molecules) interacting with p75 is incomplete. CONCLUSIONS Our investigation reports, for the first time, the expression patterns of p75NTR in human scalp skin and HFs. p75NTR protein expression exhibited significant hair cycle-dependent fluctuation, suggesting a possible role in human HF biology.

Expression patterns of the glial cell line–derived neurotrophic factor, neurturin, their cognate receptors GFRα-1, GFRα-2, and a common signal transduction element c-Ret in the human skin hair follicles

BACKGROUND Glial cell line-derived neurotrophic factor (GDNF) and a related family member, neurturin (NTN), and their cognate receptors (GFRalpha-1 and GFRalpha-2, for GDNF and NTN, respectively) are distal members of the transforming growth factor-beta superfamily. They are involved in the control of murine hair follicle (HF) cycling. This study tests the hypothesis that GDNF and NTN, and their cognate receptors, are expressed in the human HF and their expression varies in the different stages of the HF cycle. METHODS The expression pattern of these proteins was examined in human HF by immunofluorescence, immunoalkalinephosphatase staining methods, and reverse transcription-polymerase chain reaction (GDNF). The functional effects (GDNF and NTN) were examined in organ culture of the microdissected HFs. RESULTS GDNF, NTN, GFRalpha-1, GFRalpha-2, and c-Ret proteins were weakly expressed in catagen and telogen HFs. In contrast, they were strongly expressed in the epithelial and mesenchymal compartments of the anagen HF. GDNF gene was transcribed, both in the human scalp skin and in the isolated anagen HFs (reverse transcription-polymerase chain reaction). In HF organ culture, GDNF (but not NTN) increased the number of the proliferating HF keratinocytes (Ki 67 + cells). GDNF partially protected HFs from transforming growth factor-beta2-induced premature catagen transition. LIMITATIONS None. CONCLUSIONS GDNF, NTN, GFRalpha-1, GFRalpha-2, and c-Ret proteins are differentially expressed in the different stages of HF cycle. GFRalpha-mediated signaling involves c-Ret and may play a role in human HF biology.

Expression of nerve growth factor and its high-affinity receptor, tyrosine kinase A proteins, in the human scalp skin

Background:  Nerve growth factor (NGF) and its high‐affinity receptor, tyrosine kinase A (TrkA), are members of the neurotrophin family. NGF–TrkA are involved in murine hair morphogenesis and cycling. To date, their expression in human hair follicle (HF) is unknown. In this investigation, we hypothesize that NGF–TrkA proteins are expressed in the human scalp skin. Moreover, NGF–TrkA expression in HF changes with the transitions from anagen→catagen→telogen stages.

Expression of bone morphogenetic protein-7 in human scalp skin and hair follicles

SIR, Previous studies have indicated that bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-b superfamily, control cell proliferation and differentiation in the skin. The BMP family consists of more than 20 secreted proteins, including BMP-2 ⁄4, BMP-3, BMP-7, BMP-9, BMP-10 and BMP-15. They exert their biological effects through binding to transmembrane serine ⁄ threonine kinase BMP receptors leading to phosphorylation and translocation of intracellular signalling molecules into the nucleus. Hair follicle (HF) cycling transitions include phases of relative quiescence (telogen), rapid growth (anagen) and apoptosis-driven regression (catagen). These phases and epidermal homeostasis are controlled by BMPs such as BMP-4. To date, BMP-7 protein expression in normal scalp skin is still unknown. In this investigation we hypothesize that BMP-7 protein is expressed in human scalp skin and that BMP-7 protein expression in the HF changes with the transitions from anagen to catagen to telogen stages. To test our hypothesis BMP-7 protein expression was examined in human scalp skin. We examined 50 normal human scalp skin (frontal and temporal regions) specimens, obtained from 50 women (age range 53–57 years) undergoing cosmetic surgery, using immunohistological methods. Samples were maintained in Williams E medium at 4 C. Skin specimens were frozen in liquid nitrogen and stored at )80 C. Samples were embedded and processed for longitudinal cryosections (8 lm). Sections were dried, fixed in acetone and stored at )20 C. Cryosections of skin were immunostained using the immunofluorescent tyramide signal amplification (TSA) labelling technique (PerkinElmer Life Science, Boston, MA, U.S.A.). Cryosections were washed in Tris-acid-Tween buffer (TNT, pH 7Æ5), followed by washing in 3% hydrogen peroxide. Sections were incubated overnight at 4 C with primary antibodies (polyclonal rabbit antihuman BMP-7 IgG antibody; 1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). Sections were washed in TNT and incubated with tetramethylrhodamine isothiocyanate (TRITC)-conjugated F(ab)2 fragments of goat antirabbit IgG secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.) diluted in Tris-acidblocking buffer (TNB, pH 7Æ2) for 30 min at 37 C. Sections were incubated with streptavidin–horseradish peroxidase (1 : 50 in TNB) for 30 min at 37 C. TRITC–tyramide amplification reagent was administered for 30 min at 37 C, followed by counterstaining with 4¢,6¢-diamidino-2phenylindole and mounting in levamisole. All sections were batched stained. The TSA signals were visualized under a fluorescence microscope. The positive control consisted of glial cells and neurones of the mouse cerebellar cortex. Additional sections, run in parallel but with omission of the primary antibodies, served as the negative controls. Semiquantification of BMP-7 protein expression was done following protocols used by other groups. When the immunohistochemical staining results were examined, the staining intensity of BMP-7 protein was statistically significantly higher (P < 0Æ001) in anagen HF than in catagen or telogen HF (Table 1). In human scalp skin, BMP-7 protein was expressed prominently in the epidermis, papillary dermis and extrafollicular structures including sebaceous glands, sweat glands, arrector pili muscles and blood vessels (Fig. 1). Within the HF compartment, BMP-7 immunoreactivity (IR) was prominent in the outer root sheath, inner root sheath, dermal papilla, precorteocytes and corteocytes of the hair shaft and connective tissue sheath (CTS) (Fig. 2). In catagen and telogen HF, BMP-7 IR was weak in all compartments except the CTS (Fig. 2).

Age‐associated decrease of CD1d protein production in normal human skin

Background  CD1d belongs to a family of antigen‐presenting molecules structurally related to the classical major histocompatibility complex class I proteins.

Human scalp skin and hair follicles express neurotrophin-3 and its high-affinity receptor tyrosine kinase C, and show hair cycle-dependent alterations in expression.

Background  Neurotrophin (NT)‐3 and its high‐affinity receptor tyrosine kinase C (Trk C) are essential for nervous system development. These members of the NT family are also involved in murine hair morphogenesis and cycling. However, their role in human hair follicle (HF) biology remains to be elucidated.

Analysis of the expression pattern of glial cell line-derived neurotrophic factor, neurturin, their cognate receptors GFRα-1 and GFRα-2, and a common signal transduction element c-Ret in the human scalp skin

Background:  Glial cell line‐derived neurotrophic factor (GDNF) and a related family member, neurturin (NTN), as well as their cognate receptors (GDNF receptors, GFRα‐1 and GFRα‐2, respectively) are involved in nervous system development and murine hair cycle control. To date, their expression in human scalp skin is still unknown.

Survivin as a Novel Biomarker in the Pathogenesis of Acne Vulgaris and Its Correlation to Insulin-Like Growth Factor-I

Survivin, a member of the inhibitor of apoptosis protein family, has an important role in cell cycle regulation. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone with wide range of biologic effects including stimulation of lipogenesis in sebaceous glands. Their overexpression in some fibrotic disorders suggests a possible implication of both IGF-I and survivin in the pathogenesis of acne and/or acne scars. The current study aimed to assess and correlate serum levels of IGF-I and survivin in patients with active acne vulgaris and postinflammatory acne scars and to evaluate their lesional expressions in comparison to healthy controls. Serum IGF-I and survivin were estimated using commercially available ELISA kits and their tissues expressions were investigated using Western blotting. Our findings suggest that IGF-I and survivin could play potential roles in the pathogenesis of active acne vulgaris and more importantly in postinflammatory acne scars with significant positive correlation coefficient between serum levels of IGF-I and survivin which support IGF-I-/PI3K-/AKT-mediated downregulation of nuclear expression of FoxO transcription factors resulting in enhanced survivin expression.

Expression of Ras homologous B protein in the human scalp skin and hair follicles: hair follicle cycle stages-associated changes

Background: RhoB belongs to the Ras homologous (Rho) subfamily which consists of low molecular weight mass GTP‐binding proteins. Rho proteins are regulatory molecules that mediate changes in cell shape, contractility, motility and gene expression.