Differences In Staining Patterns Research Articles

The distribution of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the lungs of congenital diaphragmatic hernia patients and age‐matched controls

In congenital diaphragmatic hernia (CDH), the pathogenesis of abnormal pulmonary morphology is still incompletely understood. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are known to play an important role in the turnover of the extracellular matrix (ECM) during development and in remodelling of tissue. The aim of this study was to investigate differences in the expression of MMPs and TIMPs between CDH lungs and controls, against the background of the abnormal pulmonary vasculature in CDH. We studied 12 lungs of term CDH patients who died < 24 h after birth and 11 normal age-matched control lungs, by immunohistochemistry with antibodies against human MMP-1, -2, -9, TIMP-1 and -2. There was a clear increase in the number of MMP-1-reactive capillaries and fibroblasts in CDH lungs compared with controls. In contrast, TIMP-2 reactivity in these structures was decreased in CDH lungs. The arterial endothelium and medial smooth muscle expressed MMP-2, -9 and TIMP-2 in both CDH and control lungs. In small arteries (< 100 microm in diameter), the positive surface area of MMP-2, -9 and TIMP-2 was significantly larger in CDH lungs than in controls. There was no difference in the distribution and expression of TIMP-1 between CDH lungs and normal controls. The differences in staining pattern of MMPs and TIMPs between normal and CDH lungs suggest that these enzymes might play a role in the abnormal remodelling of the interstitium and the pulmonary arteries in CDH lungs. This could contribute to our understanding of the abnormal lung morphology and the occurrence of pulmonary hypertension, which forms one of the major obstacles to the successful treatment of these patients.

Dynamic Changes to the Inositol 1,4,5-Trisphosphate and Ryanodine Receptors during Maturation of Bovine Oocytes

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RyR) have been identified as two ligand-gated calcium channels which play a critical role in mediating calcium release in many different types of cells and tissues. The physiological significance of the two receptors in regulation of intracellular calcium during meiotic maturation and fertilization in the bovine oocyte was evaluated. Metabolic labeling of bovine oocytes by Met-Cys 35S during early and late maturation was followed by immunoprecipitation of both RyR and IP3R using specific antibodies against these two receptors. Results indicate that IP3R is translated throughout the maturation period; in contrast, RyR is only translated during the late maturation period of bovine oocytes. In addition, the experiments reported here investigate the temporal and spatial relationships between these calcium channels and the endoplasmic reticulum (ER) and cortical granules (CG). Immunocytochemistry, fluorescence staining and confocal microscopy were applied at four oocyte developmental stages: the germinal vesicleintact (GV-intact), metaphase I (MI) and metaphase II (MII) stages of maturation and the fertilized egg at 6 h post insemination (hpi). Although oocytes demonstrated some differences in staining patterns and localization, both receptor types showed apparent dynamic changes during meiotic maturation and dramatic decreases in signals after insemination. These results indicate the changes in the number and distribution of IP3R and RyR may account for the increased intracellular calcium responsiveness at fertilization. The IP3R appears to associate with the ER at the sub-vitelline membrane cortex in bovine oocytes. In addition, RyR appears to associate with the CG. In conclusion, although these two receptors may have different functional roles in regulation of calcium release during meiotic maturation and fertilization, it appears that both IP3R and RyR contribute to the significant increase of intracellular calcium during fertilization and activation in the bovine oocyte.

Analyzing Patterns of Staining in Immunohistochemical Studies: Application to a Study of Prostate Cancer Recurrence

Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis.

Aberrant DNA methylation patterns in cultured mouse embryos

Abstract Mouse early embryos undergo gernome-wide demethylation and remethylation events during pre-implantation development. Abnormal methylation reprogramming is thought to be associated with development arrest. Using immunofluorescence staining with an antibody against 5-methylcytosine (MeC), we examined the genome methylation patterns of mouse embryos cultured in vitro. The results did not show the difference in staining patterns between development-blocked two-cell embryos that cultured in vitro and the two-cell embryos that were freshly collected from the donor mice. But in vitro-arrested morulae displayed a strong positive staining when compared to the morulae freshly collected from the donor mice. At the blastocyst stage, although most embryos showed the expected methylation patterns, with highly stained inner cell mass (ICM) and weekly stained trophectoderm (TE), a proportion of embryos were dimly stained in both ICM and TE. These results indicated that the methylation profile of the embryos coul...

Identification of the receptor for advanced glycation end products in synovial tissue of patients with rheumatoid arthritis.

Generation of advanced glycation end products (AGE) is an inevitable process in vivo and can be accelerated under pathologic conditions such as oxidative stress, e.g. in rheumatoid arthritis (RA). This process is mediated by the AGE-specific receptor (RAGE). In this study we analysed the presence of RAGE in RA and osteoarthritic (OA) synovial tissue using immunohistology. Frozen synovial tissue samples from 11 RA patients and 12 OA patients were treated with goat anti-RAGE immunoglobulin G (IgG) and rabbit antigoat IgG. Immunostaining was visualised with streptavidin horse radish peroxidase (chromogen amino-ethyl-carbazole). Cell differentiation was performed with antibodies against CD68, CD45RO, and CD20. In 9/11 RA and 8/12 OA synovial specimens, RAGE was detected in synovial lining, sublining, and stroma. In RA, many T cells (CD45RO(+)) and some macrophages (CD68(+)) showed positive immunostaining for RAGE, whereas B cells were mostly negative. We found no difference in staining patterns between the RA and OA samples. We detected RAGE in RA and OA synovial tissue. The presence of RAGE on macrophages, T cells, and some B cells suggests its role in the pathogenesis of inflammatory joint disease.

Melan A (A103) is expressed in adrenocortical neoplasms but not in renal cell and hepatocellular carcinomas.

Most adrenocortical neoplasms, renal cell carcinomas, and hepatocellular carcinomas are diagnosed by a combination of clinical and morphologic features. However, occasionally this histologic differential diagnosis requires additional ancillary tests, such as immunohistochemistry. The authors investigated the potential value of A103 in the differential diagnosis of these tumors. Thirty-two adrenocortical neoplasms, 86 renal cell carcinomas, and 57 hepatocellular carcinomas were evaluated by immunohistochemistry using a monoclonal antibody A103 and a standard ABC method. The adrenocortical neoplasms were 21 adenomas and 11 carcinomas. Thirty-one of the 32 adrenocortical neoplasms showed strong and diffuse granular cytoplasmic staining for Melan A. No nuclear reaction was observed. There were no differences in staining patterns between adrenocortical adenomas and carcinomas. With the exception of one clear cell renal cell carcinoma, all non-adrenocortical neoplasms were negative. The authors conclude that A103 is a useful addition to the immunohistochemical panel in the differential diagnosis of adrenocortical neoplasms from both renal cell and hepatocellular carcinomas. This marker, however, does not separate benign from malignant adrenocortical neoplasms.

Periurethral connective tissue status of postmenopausal women with genital prolapse with and without stress incontinence.

Connective tissue consists mainly of collagen and structural glycoproteins and is considered an important factor of the supportive structures of the genitourinary region. Few data have been published as yet with respect to the role of connective tissue in pelvic floor relaxation and genuine stress incontinence (GSI). We compared periurethral connective tissue structures between continent and incontinent postmenopausal women with genital prolapse. Twenty-nine women underwent pelvic floor reconstructive surgery (anterior colporrhaphy: n = 29, sacrospinous fixation: n = 12), with biopsies obtained from the periurethral region. Fifteen patients and 14 women were incontinent and continent, respectively. Tissue samples were investigated for localization and distribution of collagen (types I, III, IV, V, VI) and glycoproteins (fibronectin, laminin, vitronectin) using immunofluorescent microscopic techniques. Collagen types I, III, IV, V, and VI were found in all tissue samples. Compared with the continent women, the tissue samples of the incontinent women showed a significantly weaker immunohistochemical staining of type I, III, and VI collagen. No difference in staining patterns of collagen type IV and V, fibronectin, and laminin was observed between the two groups. Intact vitronectin was found in the periurethral tissues of all the continent women, whereas this glycoprotein was either absent or fragmented in the incontinent women. Our results indicate an altered metabolism of connective tissue in the periurethral region with a significant decrease of collagen and vitronectin expression in postmenopausal women with pelvic floor relaxation with and without GSI.

The WFS1 gene, responsible for low frequency sensorineural hearing loss and Wolfram syndrome, is expressed in a variety of inner ear cells.

Heterozygous mutations in the WFS1 gene are responsible for autosomal dominant low frequency hearing loss at the DFNA6/14 locus, while homozygous or compound heterozygous mutations underlie Wolfram syndrome. In this study we examine expression of wolframin, the WFS1-gene product, in mouse inner ear at different developmental stages using immunohistochemistry and in situ hybridization. Both techniques showed compatible results and indicated a clear expression in different cell types of the inner ear. Although there were observable developmental differences, no differences in staining pattern or gradients of expression were observed between the basal and apical parts of the cochlea. Double immunostaining with an endoplasmic reticulum marker confirmed that wolframin localizes to this organelle. A remarkable similarity was observed between cells expressing wolframin and the presence of canalicular reticulum, a specialized form of endoplasmic reticulum. The canalicular reticulum is believed to be involved in the transcellular movements of ions, an important process in the physiology of the inner ear. Although there is nothing currently known about the function of wolframin, our results suggest that it may play a role in inner ear ion homeostasis as maintained by the canalicular reticulum.

Chromogranin A in the human gastrointestinal tract: an immunocytochemical study with region-specific antibodies.

We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411-424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 17-38 and 176-195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.

Expression of endoglin in human melanocytic lesions.

Angiogenesis plays an important role in progression of various tumours including malignant melanoma. It is possible that the immunostaining of angiogenic markers could differentiate benign and malignant melanocytic lesions or that the immunostaining pattern with angiogenic markers could vary with tumour thickness and thus be a prognostic marker. We were interested to see whether there was any correlation between endoglin (CD 105; EDG) expression with tumour thickness in primary cutaneous malignant melanomas (MM), any correlation between EDG expression and clinical outcome in patients with primary cutaneous MMs and any difference in staining pattern between cutaneous MMs and Spitz naevi which could be of diagnostic value. Tissue sections from 14 primary cutaneous MM lesions with a Breslow thickness of > 2 mm, 10 primary cutaneous MM lesions with a Breslow thickness of 1-2 mm, and six Spitz and 10 compound naevi were stained for EDG. EDG expression was compared with survival in patients with primary cutaneous MMs. Overall, EDG staining was positive in 96% of MMs and 94% of benign melanocytic naevi. Very strong (++++) and strong (+++) EDG staining was found in 58% of MMs and 56% of benign melanocytic lesions. EDG expression did not vary significantly with the thickness of the lesion in primary cutaneous melanoma. Survival of melanoma patients did not correlate with the degree of EDG expression. Therefore, expression of this marker alone is not sufficient to differentiate benign and malignant melanocytic lesions.

H-TYPE 1 CARBOHYDRATE ANTIGEN EXPRESSION BY OVINE ENDOMETRIAL CELLS

Our objective was to determine whether H-Type 1 carbohydrate antigen is expressed by ovine endometrial epithelial cells. Endometrium was obtained from sheep on days (D) 1, 5, 11, 13, and 15 of the estrous cycle or pregnancy and D17 and D19 of pregnancy. Immunofluorescence microscopy of frozen tissue sections revealed intense staining on the apical surface of glandular uterine epithelial (GE) cells from D11 to D17 of pregnancy. Light punctate staining of luminal uterine epithelial (LE) cells was present from D15 to D19 of pregnancy, with isolated areas of intense staining observed only on D15 of pregnancy. There were no noticeable differences in staining patterns on equivalent d of the estrous cycle. Immortalized sheep LE and GE cells were used to determine whether estradiol (E), progesterone (P), or E + P, with or without interferon tau (IFNtau), regulates H-Type 1 antigen expression in vitro. Intermittent punctate surface staining was observed on both cell lines independent of steroid treatment. Treatment with P or IFNtau increased H-Type 1 antigen expression (P < 0.01) and resulted in large aggregates of punctate staining. Domain-specific biotinylation and Western blotting of cell lysates from LE and GE cells were used to identify proteins carrying the H-Type 1 antigen. For both cell types, major immunoreactive apical membrane proteins were detected at 31, 33, 42, 55, 60, and 70 kDa. Therefore, the H-type 1 antigen is expressed mainly on GE cells during pregnancy recognition in utero and up-regulated by P and IFNtau on LE and GE cells in vitro.

Skeletal Muscle Fibers Express Major Histocompatibility Complex Class II Antigens Independently of Inflammatory Infiltrates in Inflammatory Myopathies

The aim of our study was to address the question of whether muscle fibers express major histocompatibility complex (MHC) class II in inflammatory myopathies. For this purpose we performed a systematic study of MHC class II antigen expression on muscle fiber membranes in muscle tissue from polymyositis and dermatomyositis patients in various stages of disease activity. Thirty-two patients with classical clinical signs of myositis were divided into subgroups depending on duration of clinical signs of myositis and presence or absence of inflammatory infiltrates in muscle tissue. Immunohistochemistry as well as double-immunofluorescence stainings were used to identify the presence of MHC class II in muscle tissue. MHC class I was included for comparison. Quantification of positive staining was performed using an image analysis system in addition to evaluation by manual microscopic scoring and laser confocal microscopy. It was demonstrated that a significant proportion of skeletal muscle fibers in inflammatory myopathies express MHC class II as well as MHC class I and that MHC antigen expression is independent of the inflammatory cell infiltration. Furthermore, there were no differences in staining pattern between polymyositis and dermatomyositis patients. Our results indicate that MHC class II and MHC class I molecules may be involved in initiating and maintaining the pathological condition in myositis rather than only being a consequence of a preceding local inflammation.

Value of topoisomerase II alpha, MIB-1, p53, E-cadherin, retinoblastoma gene protein product, and HER-2/neu immunohistochemical expression for the prediction of biologic behavior in adrenocortical neoplasms.

Prediction of biologic behavior in adrenocortical neoplasms is difficult because of the lack of availability of reliable clinical, biochemical, and pathologic prognostic markers. Reliable objective markers predictive of clinical outcome in adrenocortical neoplasms are needed to assign optimal treatment of potentially malignant tumors. In the current article, the authors evaluated a set of molecular markers (topoisomerase II alpha (Topo II alpha), MIB-1, p53, human epithelial cadherin (E-cadherin), retinoblastoma gene protein product, and HER-2/neu) and correlated their expression with histologic diagnosis and clinical outcome. Paraffin-embedded, formalin-fixed tissue blocks from 30 cases of adrenocortical neoplasms (15 benign and 15 malignant) were obtained from the surgical pathology archives at the University of Utah Health Sciences Center (Salt Lake City, UT) and the Medical College of Wisconsin (Milwaukee, WI). Age, gender, recurrence, tumor size and weight, hemorrhage, necrosis, pleomorphism, mitotic count, capsular and lymphovascular invasion, hyaline globules, intranuclear inclusions, and immunohistochemical expression of Topo II alpha, p53, MIB-1, E-cadherin, retinoblastoma gene protein product, and HER-2/neu were studied. Clinical data were obtained from the clinical charts, or communication with the treating physician, or both. Adrenocortical neoplasms with hemorrhage, necrosis, large size (>5 cm), weight more than 100 g, nuclear pleomorphism, lymphovascular invasion, and brisk mitotic rate (more than 5 per 30 high-power fields) were more likely to behave in a malignant fashion (P approximately 0.001-0.009). The difference in proliferation indices in benign and malignant neoplasms was statistically significant (P < 0.001). The difference in p53 staining in benign and malignant neoplasms also was statistically significant (P < 0.001). Higher p53 labeling index (>20%) was present in 73% (11/15) of malignant lesions but was found in only 1 of 15 (6.6%) benign lesions. The difference in retinoblastoma staining between benign and malignant neoplasms was statistically significant (P = 0.004). There was no significant difference in staining pattern of E-cadherin expression between benign and malignant lesions. HER-2/neu overexpression was not observed in any of the benign or malignant adrenocortical neoplasms.

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum, hippocampus and spinal cord using monoclonal antibodies with different epitope specificities.

The HNK-1 carbohydrate, an unusual 3'-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated alpha2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.

Expression patterns of biologic markers in small breast cancers and preneoplastic breast lesions

When does proliferating breast epithelium turn malignant? Single parameter analyses have not answered this question. We have tried to answer this through an analysis of immunohistochemical staining patterns in the following morphologically defined breast lesions: atypical ductal hyperplasia (ADH, 23 cases), papilloma (12 cases), ductal cancer in situ (DCIS, 28 cases), and mammographically detected small primary cancers (34 cases). The seven antibodies used were c-neu, bcl-2, p53, p21, CD44, MIB 1, and FAS. Staining patterns were compared within groups and between groups of lesions. Interesting differences in staining patterns were seen between invasive ductal cancer and invasive lobular cancer: invasive lobular cancer was less p53-positive and more CD44-positive than invasive ductal cancer. We found no common pattern in the different proliferating epithelia to show when they become malignant.

Expression of transforming growth factor-alpha, epidermal growth factor, and epidermal growth factor receptor in follicles of human ovarian tissue before and after cryopreservation

Objective: To study the expression of transforming growth factor-alpha (TGF-α), epidermal growth factor (EGF), and EGF receptor in follicles of human ovarian tissue. Design: A retrospective, controlled comparative study. Setting: In vitro fertilization laboratory of a university hospital. Patient(s): Fifteen women with regular menstrual cycles who underwent laparoscopy and the biopsy of ovarian tissue. Intervention(s): Paraffin sections were prepared from ovarian tissues, followed by immunohistochemical staining of TGF-α, EGF, and EGF receptor. Main Outcome Measure(s): Immunostaining for TGF-α, EGF, and EGF receptor in follicles of fresh and frozen ovarian tissues. Result(s): Immunoreactivities for TGF-α and EGF receptor were observed simultaneously in the oocytes of primordial, primary, preantral, and antral follicles. Strong staining for TGF-α and EGF receptor was present in thecal cells. The TGF-α and EGF receptor was also expressed in some granulosa cells of primary to antral follicles. The EGF only stained weakly in the oocytes of primordial and primary follicles and in thecal cells. There was no difference in staining patterns for TGF-α, EGF, and EGF receptor between fresh and frozen ovarian tissues. Conclusion(s): The TGF-α and EGF receptor was expressed in primordial to antral follicles, indicating a role of TGF-α in regulating follicular development through binding to the EGF receptor. Freeze-thawing did not substantially alter immunoreactivites for TGF-α, EGF, and EGF receptor in frozen ovarian tissue.

Expression of Indian Hedgehog, Parathyroid Hormone-Related Protein, and Their Receptors in the Postnatal Growth Plate of the Rat: Evidence for a Locally Acting Growth Restraining Feedback Loop After Birth

A locally acting growth restraining feedback loop has been identified in the murine embryonic growth plate in which the level of parathyroid hormone-related peptide (PTHrP) expression regulates the pace of chondrocyte differentiation. To date, it is largely unknown whether this feedback loop also regulates the pace of chondrocyte differentiation in the growth plate after birth. We therefore characterized the spatio-temporal expression of Indian hedgehog (IHH), PTHrP, and their receptors in the postnatal growth plate from female and male rats of 1, 4, 7, and 12 weeks of age. These stages are representative for early life and puberty in rats. Using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) on growth plate tissue, IHH and components of its receptor complex, patched (PTC) and smoothened (SMO), PTHrP and the type I PTH/PTHrP receptor messenger RNA (mRNA) were shown at all ages studied irrespective of gender. Using in situ hybridization, IHH, PTHrP, and PTH/PTHrP receptor mRNA were detected in prehypertrophic and hypertrophic chondrocytes in both sexes during development. In addition, especially in the younger age groups, faint expression of PTH/PTHrP receptor mRNA also was shown in stem cells and proliferative chondrocytes. Immunohistochemistry confirmed the observations made with in situ hybridization, by showing the presence of IHH, PTC, PTHrP, and PTH/PTHrP receptor protein in prehypertrophic and hypertrophic chondrocytes. In addition, staining for hedgehog, PTC, and PTHrP also was observed in growth plate stem cells. No differences in staining patterns were observed between the sexes. Furthermore, no mRNA or protein expression of the mentioned factors was detected in the perichondrium. Our data suggest that in contrast to the proposed feedback loop in the early embryonic growth plate, which requires the presence of the perichondrium, a feedback loop in the postnatal growth plate can be confined to the growth plate itself. In fact, two loops might exist: (1) a loop confined to the transition zone and early hypertrophic chondrocytes, which might in part be autocrine and (2) a loop involving the growth plate stem cells.

Dissociation of intra- and extracellular domains of desmosomal cadherins and E-cadherin in Hailey-Hailey disease and Darier's disease.

In order to clarify the pathomechanism of acantholysis in Hailey-Hailey disease (HHD) and Darier's disease (DD), the distribution of desmosomal and adherens junction-associated proteins was studied in the skin of patients with HHD (n = 4) and DD (n = 3). Domain-specific antibodies were used to determine the cellular localization of the desmosomal transmembrane glycoproteins (desmogleins 1 and 3 and desmocollin), desmosomal plaque proteins (desmoplakin, plakophilin and plakoglobin) and adherens junction-associated proteins (E-cadherin, alpha-catenin, beta-catenin and actin). A significant difference in staining patterns between intra- and extracellular domains of desmosomal cadherins and E-cadherin was demonstrated in acantholytic cells in both HHD and DD, but not in those in pemphigus vulgaris and pemphigus foliaceus samples used as controls. In acantholytic cells in HHD and DD, antibodies against attachment plaque proteins and intracellular epitopes of desmosomal cadherins exhibited diffuse cytoplasmic staining, whereas markedly reduced staining was observed with antibodies against extracellular epitopes of the desmogleins. Similarly, membrane staining of an intracellular epitope of E-cadherin was preserved, while immunoreactivity of an extracellular epitope of E-cadherin was destroyed. While the DD gene has been identified as ATP2A2, the gene for HHD has not been clarified. The dissociation of intra- and extracellular domains of desmosomal cadherin and E-cadherin is characteristic of the acantholytic cells in HHD and DD, and not of pemphigus. This common phenomenon in HHD and DD might be closely related to the pathophysiological mechanisms in both conditions.

The distribution of choline acetyltransferase- and acetylcholinesterase-like immunoreactivity in the palmar skin of patients with palmoplantar pustulosis.

The distribution of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in involved skin in patients with palmoplantar pustulosis (PPP) and in normal palmar skin in healthy non-smokers and smokers has been studied by immunohistochemistry, especially in relation to the sweat gland apparatus. The sweat gland and its duct showed ChAT- and AChE-like immunoreactivity (LI) of varying intensity in all three groups and with stronger reactivity than in the epidermis. ChAT-LI was present in the coil and in the duct except in the corneal layer. Smokers and patients with PPP displayed significantly fewer ChAT+ acrosyringia than non-smokers. In the patients with PPP, the granulocytes in the pustules and in the papillary dermis displayed ChAT-LI. Western blot analysis of granulocytes from peripheral blood from healthy donors confirmed the presence of ChAT-like proteins in large amounts in neutrophils and small amounts in eosinophils. AChE-LI of varying intensity was found in all parts of the sweat gland apparatus in all three groups. The strongest AChE-LI in the acrosyringia was seen in the lowest part of the stratum corneum, where the PPP pustules are located. No significant differences in staining pattern or intensity were found between the coils, nerve fibres surrounding the coils or ducts. The number of mast cells in the papillary dermis was about four times larger in the patients with PPP than in the control subjects. AChE-LI was observed in about 25% of the mast cells in non-smoking control subjects and in patients with PPP, but only in 10% of those in the smoking control subjects. Our findings indicate that the (non-neuronal) cholinergic system may be involved in cutaneous inflammatory processes.

Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated.

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.