Faculty Bibliography

STUDY OBJECTIVE/OBJECTIVE:To clarify the relationship of hypogastric nerves (HNs) with several pelvic anatomic landmarks and to assess any anatomic differences between the 2 sides of the pelvis, both in cadaveric and in vivo dissections. DESIGN/METHODS:Prospective observational study. SETTING/METHODS:An anatomic theater for cadaveric dissections and a university hospital for in vivo laparoscopy. PATIENTS/METHODS:Five nulliparous female cadavers underwent laparotomic dissection; 10 nulliparous patients underwent laparoscopic surgery for rectosigmoid endometriosis without posterolateral parametrial infiltration. INTERVENTIONS/METHODS:Measurements of the closest distance between HNs and ureters, the midsagittal plane, the midcervical plane, and uterosacral ligaments on both hemipelvises. A comparison of anatomic data of the 2 hemipelvises was conducted. MEASUREMENTS AND MAIN RESULTS/RESULTS:The right and left HNs were identified in all specimens, both on cadavers and in vivo dissections. A wide anatomic variability was reported. Regarding the differences between the 2 hemipelvises, we found that the right HN was significantly (p <.001) farther to the ureter (mean = 14.5 mm; range, 10-25 mm) than the left one (mean = 8.6 mm; range, 7-12 mm). The HN was closer to the midsagittal plane on the right side (mean = 14.6 mm; range, 12-17 mm) than on the left side (mean = 21.6 mm; range, 19-25 mm). The midcervical plane was found 2.7 mm (range, 2-4 mm) to the left of the midsagittal one. The right HN was found to be nonsignificantly closer to the midcervical plane and the uterosacral ligament on the right side than on the left side (p >.05). CONCLUSIONS:Despite a wide anatomic variability of position and appearance, the HNs are reproducibly identifiable using an "interfascial" technique and considering the ureters and uterosacral ligaments as anatomic landmarks.

Hair follicle morphogenesis and cycling were examined in transgenic mice that overexpress the bone morphogenetic protein (BMP) inhibitor Noggin under the control of the neuron-specific enolase promoter. The Noggin transgene was misexpressed in the proximal portion of the hair follicle, primarily the matrix cells, apart from the usual expression in neurons. Transgene expression appeared only after induction of both the primary (tylotrich) and secondary (nontylotrich) pelage hair follicles had already occurred, thus allowing examination of the role of BMP signaling in follicles that had been induced normally in the presence of BMPs. The overexpression of Noggin in these animals resulted in a dramatic loss of hair postnatally. There was an apparently normal, but shortened period of postnatal hair follicle morphogenesis, followed by premature initiation of hair follicle cycling via entry into the first catagen transformation. This resulted in a complete loss of hair shafts from the nontylotrich hair follicles in these mice while the tylotrich hair follicles were normal. The onset of anagen of the first postnatal hair follicle cycle was also accelerated in the transgenic mice. Our results show that BMP signaling is specifically required for proper proliferation and differentiation during late morphogenesis of nontylotrich hair follicles and that inhibition of this signaling pathway may be one of the triggers for the onset of catagen when the follicles are in anagen and the onset of anagen when the follicles are in telogen. Ectopic sebocyte differentiation was another hallmark of the phenotype of these transgenic mice suggesting that BMP signaling may be an important determinant of lineage selection by common progenitor cells in the skin. BMPs likely promote a hair follicle-type differentiation pathway of keratinocytes while suppressing the sebaceous differentiation pathway of skin epithelium

Filaggrin and trichohyalin are keratin intermediate filament-associated proteins, and are primarily expressed in the granular cells of the epidermis and in the inner root sheath cells of the hair follicles, respectively. These two proteins are, however, occasionally co-expressed in some tissues. To gain more insights into the mechanisms for expression and processing of (pro)filaggrin and trichohyalin during various pathways of epithelial differentiation, we compared their localization by double immunostaining techniques in normal and psoriatic epidermis, tongue filiform papillae and cultured human epidermal keratinocytes. In normal foreskin, trichohyalin immunoreactive cells were observed only occasionally and, when present, they always co-expressed filaggrin. Trichohyalin expression occurred, however, in filaggrin-negative cells as well as in filaggrin-positive cells in the psoriatic epidermis, tongue papillae and cultured keratinocytes. Filaggrin and trichohyalin were induced independently at different times following calcium shift in cultured keratinocytes. Immunoelectron microscopy demonstrated the distinct intracellular distribution of filaggrin and trichohyalin. Some filaggrin granules were observed in the cells where trichohyalin was diffusely distributed. Likewise some trichohyalin granules were found in the cells with diffuse filaggrin labelling. These results suggest the existence of distinct expression/processing mechanisms of filaggrin and trichohyalin in differentiating keratinocytes

BACKGROUND. The antitrichohyalin antibody AE 15 is effective for identifying the cell lineage that undergoes the pathway of inner root sheath-type differentiation. Unfortunately, the AE 15 does not react with trichohyalin in tissue that is formalin-fixed and embedded in paraffin according to routine procedures. METHODS. We attempted to retrieve the trichohyalin antigenicity in formalin-fixed, paraffin-embedded biopsy specimens that included normal skin as well as skin tumors such as trichofolliculoma and pilotricoma. RESULTS. We found that the use of a metal solution in combination with microwave oven heating improves the trichohyalin immunoreactivity substantially. Further, trichohyalin was found to be expressed not only in the secondary hair structure in trichofolliculoma but also in a certain cell lineage that differentiates to squamoid cells in pilomatricoma. CONCLUSIONS. Our findings established that surgical specimens processed under routine procedures can be successfully investigated with AE 15 using the microwave irradiation method. Studies of epidermal diseases expressing trichohyalin should provide valuable insights into our understanding the functional significance of trichohyalin during abnormal keratinization

BACKGROUND: Recently, in the filiform papillae epithelium of mouse dorsal tongue, we showed the presence of hybrid granules in which filaggrin and trichohyalin were both present, but physically segregated. Further, trichohyalin was also detected in scattered granular cells of a number of hyperplastic skin diseases. METHODS: The epidermis infected with molluscum contagiosum virus (MCV) was studied by conventional electron microscopy in conjunction with light and electron-microscopic immunohistochemistry, using both antifilaggrin and antitrichohyalin antibodies as probes. RESULTS: We found that the granular cells of MCV-infected epidermis contained both filaggrin and trichohyalin. Subsequent electron-microscopic examination showed that the granular cells contained morphologically heterogeneous granules that appeared to be composed of discrete areas of distinct electron densities. Double-labeling, using antibodies to filaggrin and trichohyalin, clearly indicated that filaggrin and trichohyalin were both present in the hybrid granules and that the electron-dense regions contained trichohyalin while the more electron-lucent regions contained filaggrin. CONCLUSIONS: The expression of trichohyalin was a common feature observed in the epidermis from a heterogenous group of hyperplastic conditions, including MCV infection. This finding has led us to speculate that trichohyalin may be specifically or preferentially involved in interacting with the hyperproliferation-related keratin pair (K6/K16), whereas the function of filaggrin is more closely linked to the skin-type keratin pair (K1/K10) that are normal keratins found in the differentiated epidermis

Epidermolytic hyperkeratosis (EH) is a genetic disorder of keratins associated with epidermal differentiation. Affected individuals carry gene mutations for conserved sequences of keratins K1 or K10. The structural alterations of tonofilaments in EH seem to be a direct consequence of the keratin gene mutations. EH epidermis, however, shows many other unexplained abnormalities including acanthosis, hypergranulosis, and hyperkeratosis. To further elucidate the pathogenetic mechanism of EH, we studied distribution patterns of other keratinization-associated molecules including involucrin, small proline-rich protein (SPRR) 1, loricrin and trichohyalin in the skin of four patients by light and electron microscopic immunohistochemistry in conjunction with conventional transmission electron microscopy. The middle to upper epidermal cells showed moderate to strong immunoreactivities to involucrin, SPRR1 and loricrin antibodies. Both intracellular staining and cell peripheral staining was seen for involucrin and SPRR1 antibodies. Loricrin labelling was prematurely associated with the plasma membrane of granular cells, possibly relating to abnormal keratin filament aggregation and cellular vacuolization. Some loricrin labelling was localized on the keratin aggregates, suggesting intermolecular associations between keratin and loricrin. Trichohyalin, hardly detectable in normal epidermis, was present in some granular and cornified cells in EH in association with keratin filaments, suggesting that it may function as an intermediate filament-associated protein. While cornified cell envelopes were intensely labelled only with loricrin antibodies in normal skin, they were immunoreactive to involucrin, SPRR1 and loricrin antibodies in EH. Sequential change in electron density of the cornified cell envelopes, a constant feature in normal skin, was often absent in EH. These results suggest an altered assembly process of cornified cell envelopes in EH

Trichohyalin is a protein of relatively high molecular weight (approximately 200 kDa), associated with intermediate filaments, that was for many years thought to be expressed only in the inner root sheath and medulla of the hair follicle. We show here, however, that this protein is expressed in association with (pro)filaggrin in the granular layer of many non-follicular, keratinizing, stratified epithelia which also express keratins K6/K16, including those of the filiform papillae of dorsal tongue epithelia. In this epithelium, which elaborates morphologically heterogeneous keratohyalin granules in its upper cell layers, trichohyalin forms hybrid granules with filaggrin, the major intermediate filament associated protein found in keratohyalin granules, which is normally expressed in advanced epidermal differentiation. These two intermediate filament-associated proteins remain physically segregated in the hybrid granules, but they share the same fate, as they both become dispersed in transitional cells, and are undetectable in cornified cells. Trichohyalin was also detected in nail matrix epithelia, the epithelium of Hassal's corpuscles of the thymus, and newborn foreskin epidermis. It is essentially absent from normal trunk and scalp epidermis, but is expressed in a few scattered cells of the granular layer that are also filaggrin-positive. In addition, trichohyalin is expressed in the epidermis in a number of hyperplastic skin diseases. These findings demonstrate that trichohyalin is not peculiar to a small number of hair follicle cells, but is expressed in a number of normal and pathological epithelia where it is uniquely associated with filaggrin. In addition, since all these trichohyalin-expressing keratinocytes also synthesize keratins K6 and K16 (the markers for an 'alternative' pathway of keratinocyte differentiation), this raises the possibility that the trichohyalin protein is specifically (or preferentially) involved in aggregating intermediate filaments containing the K6/K16 keratins

Lamellar granules are specialized lipid-rich organelles present in epidermal granular cells. They fuse with the apical cell surface and discharge their contents into the intercellular space forming lamellar sheets. It was previously shown by electron microscopy that lamellar granules in biopsies of infants affected with harlequin ichthyosis are either absent or abnormal and no intercellular lamellae could be detected. A monoclonal antibody (AE17) directed against a protein component of lamellar granules was used for immunoblotting and immunohistochemical studies as an indication of both the presence and function of lamellar granules. Epidermal extracts from all harlequin and normal specimens tested showed an immunoreactive protein of 25-28 kD. Immunohistochemical staining of normal skin using AE17 showed apical cytoplasmic staining in the granular layer and intercellular staining between the granular and stratum corneum cells. Harlequin samples showed variable degrees of staining ranging from little to heavy apical cytoplasmic staining of granular cells. No intercellular staining was detected. The immunohistochemical staining pattern correlated with the electron microscopic localization of abnormal vesicles and the absence of intercellular lamellae in the affected samples. We conclude that the vesicles represent lamellar granules that contain the AE17 antigen but are structurally abnormal and defective in their ability to discharge both their lipid and protein contents into the intercellular space. We suggest that this defect in the lamellar granules represents the underlying basis for stratum corneum cell retention and subsequent accumulation of scale in harlequin ichthyosis

Filaggrin and trichohyalin are the two major intermediate filament associated proteins which interact with keratin filaments in the skin. These two proteins initially accumulate in cytoplasmic granules called keratohyalin or trichohyalin granules which provide prominent morphological hallmarks of differentiation in the epidermis and the inner root sheath of hair follicles, respectively. The contents of each of these granules are modified and subsequently released into the cytoplasm of the fully mature cells where they function in the role of aggregating keratin filament bundles. We are beginning to identify several important aspects relative to the unique biological functions of both filaggrin and trichohyalin during the late stages of keratinocyte differentiation. This overview summarizes recent work on these proteins and will also highlight the existence of novel cytoplasmic granules, keratohyalin-trichohyalin hybrid granules, in dorsal tongue epithelia

'Trichohyalin' is a 220-kD protein found in trichohyalin granules that are present as major differentiation products in the medulla and inner root sheath cells of human hair follicles. It was unclear whether this protein served as an intermediate filament precursor in the inner root sheath or as an intermediate-filament-associated (matrix) protein. We have produced a panel of monoclonal antibodies (AE15-17) to this protein and used them to trace its fate during inner root sheath differentiation. These studies have allowed us to define three immunologically distinct forms of this trichohyalin protein. They are 1) the AE15-positive form, which is found throughout all trichohyalin granules; 2) the AE16-positive form, which is localized as discrete punctae on the surface of trichohyalin granules; and 3) the AE17-positive, intermediate-filament-bound form, which associates with the inner root sheath filaments with a regular, 400-nm periodicity. From these results we suggest that the 220-kD trichohyalin protein is an intermediate-filament-associated protein that may play a role in the lateral aggregation, precise alignment, and stabilization of inner root sheath filament bundles