Re-evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis.

Excessive amounts of melanin pigments may hamper histopathological assessments of melanocytic lesions by obscuring cellular morphology and hindering antibody-antigen interactions. To determine the optimal melanin-bleaching conditions for histopathological examination, heavily pigmented melanomas were treated with warm hydrogen peroxide (H2O2) diluted with various diluents (1% disodium hydrogen phosphate 12H2O (Na2 HPO4); phosphate buffer 0.05 M, pH 7.4 (PB); and PBS 0.05 M, pH 7.4) at varying temperatures (50°C, 55°C, and 60°C) and for varying incubation times (0.5, 1, 2, and 3 h). The effect of the sequential order of antigen retrieval and bleaching on preserving tissue morphology was then evaluated. Additionally, the effect of melanin bleaching using warm diluted H2O2 on the antigenicity of melanoma-related markers (HMB-45, MART-1, and S-100) and other markers used for histopathology was examined in amelanotic melanomas and tonsil tissue. Optimal and complete bleaching was achieved using warm 3% H2O2 in PB treatment at 55°C for 2 h following antigen retrieval with microwaving or digestion with trypsin. Under these conditions, the tissue morphology and antigenicity of various immunohistochemical markers were also well preserved. Bleaching with warm 3% H2O2 PB is a fast and efficient method of bleaching melanin pigments and performing immunohistochemical examination in heavily melanin-pigmented lesions.

Altered expression of CDX-2, PDX-1 and mucin core proteins in "Ulcer-associated cell lineage (UACL)" in Crohn's disease.

The ulcer-associated cell lineage (UACL) induced at the site of ileac chronic ulceration in Crohn's disease has been reported to show histological differentiation resembling gastric pyloric mucosa. To clarify the significance of homeobox gene-encoded transcription factors in the formation of the UACL in Crohn's disease, we investigated the immunohistochemical expression of gastrointestinal mucins (MUC5AC for gastric surface mucous cells; MUC6 for gastric gland mucous cells, and MUC2 for intestinal goblet cells) and homeobox gene-encoded transcription factors (CDX-2 for intestinal mucosa and PDX-1 for pyloric mucosa) in the UACL. The analysis was undertaken on ileal mucosa obtained from ileal resections performed in 19 patients with active Crohn's disease of the small bowel. The UACL was observed in nine patients. In the UACL, expression of mucous cells with a foveolar-structure showed immunoreactivity to MUC5AC, and the mucous cells with a glandular structure showed immunoreactivity to MUC6, and the expression of MUC2 was decreased. In addition, we detected the decreased expression of CDX-2 along with the increased expression of PDX-1 in the UACL. The UACL showed histological differentiation simulating gastric pylori mucosa. The down-regulation of CDX-2 and the up-regulation of PDX-1 could be an important mechanism in the induction of the UACL.

In vivo bactericidal activities of Japanese rice-fluid against H. pylori in a Mongolian gerbil model.

PURPOSE: The antibiotic effect of rice-fluid on Helicobacter pylori infection was investigated using a Mongolian gerbil model. METHODS: Gerbils were divided into four groups: H. pylori -infected, rice-fluid-treated animals (group A); H. pylori -infected, untreated animals (group B); uninfected, rice-fluid-treated animals (group C); and uninfected, untreated animals (group D). Group A and B animals were killed 14 weeks after H. pylori infection and group C and D animals were killed at the same age. The stomachs were examined for histology, 5'-bromo-2'-deoxyuridine (BrdU) labeling, and the bacterial burden. Serum anti-H. pylori antibody titers were also tested. RESULTS: The positive incidence of H. pylori -culture was 25 and 84 % in groups A and B, respectively (p<0.01). Both the degree of inflammation and the BrdU labeling index in group A were significantly lower than those in group B. CONCLUSIONS: Rice-fluid showed an antibiotic effect on H. pylori and an anti-inflammatory effect on the H. pylori -associated gastritis.

In vitro bactericidal activities of Japanese rice-fluid against Helicobacter pylori strains.

BACKGROUND: Helicobacter pylori has now been widely recognized as a causative agent of gastroduodenal diseases. The development of safer anti- H. pylori compounds is desirable due to the antibiotic-resistant strains emerged to date. METHODS: We successfully developed the compounds of Rice-fluid derived from unpolished, polished, and usually cooked Japanese rice, and investigated their in vitro antibacterial activities by means of the Time-Kill-Curve methods against various species of bacteria including H. pylori strains. RESULTS: All of the compounds revealed keen bactericidal activities against H. pylori, followed by Streptococcus pneumoniae and Campylobacter jejuni strains, but failed to affect the viability of other bacterial species investigated including staphylococci, enterococci, Pseudomonas aeruginosa, and other gram-negative rods belonging to the family Enterobacteraceae. The bactericidal activities were demonstrated to be time- and concentration-dependent. CONCLUSIONS: The compounds of Rice-fluid are considered to be potentially new and safe therapeutic regimens against H. pylori infections. The mechanism of their bactericidal activities against H. pylori strains remains to be elucidated.

Three-dimensional analysis of alveolar structure in usual interstitial pneumonia.

The etiology of usual interstitial pneumonia (UIP), a progressive lung disease, remains unclear. We examined alveolar structure in UIP three-dimensionally. Lung biopsy specimens from five patients with idiopathic pulmonary fibrosis were used. Sections 150-microm thick were stained with elastica solution for elastic fibers, with alpha-smooth muscle actin antibody for myofibroblasts, with anti-Thomsen-Friedenreich antibody for type-II pneumocytes and with anti-CD34 antibody for blood vessels. We examined them three-dimensionally using a laser confocal microscope or light microscope. In the fibrotic lesions, the thick elastic fibers forming the alveolar framework were not particularly dense considering the reduction in alveolar volume. Near the fibrotic lesions, some of the thin elastic fibers in the alveolar wall were slightly sinuous and ended with rounded tips. Type-II pneumocytes had proliferated and were distributed uniformly over the alveolar surface. Smooth muscle actin filaments were detected only around the alveolar orifice. These findings show that in UIP destruction of the elastic fiber framework of the alveoli may lead to irreversible focal alveolar collapse after damage to the alveolar epithelial cells, and proliferation of type-II pneumocytes may be involved with this elastolysis.

Distribution of cathepsin D granules in normal and pathologic conditions in human prostate.

Although cathepsin D has been implicated in prostate cancer invasion and metastasis, the distribution of this enzyme in normal human prostate is unknown. We investigated immunohistochemically the distribution of cathepsin D granules in normal prostatic tissues with or without androgen ablation. We also examined the cancer tissues after androgen ablation and the hyperplastic tissues. Changes in the distribution pattern of the larger cathepsin D-positive granules (apoptotic bodies) were observed in the normal prostate as well as in the normal tissue of the anti-androgen-treated cancer specimens. While the apoptotic bodies were denser in the proximal duct of the normal adult prostate, they were more abundant in the normal peripheral acini of the anti-androgen-treated cases. There were few apoptotic bodies in the adenoma tissues, but many in the hormonally treated cancer tissues. These results showed that the distribution pattern and density of cathepsin D granules well reflected the status of the human prostatic cells in relation to age, hormonal environment and hyperplastic or neoplastic change.

Aberrant expression of TFF1, TFF2, and PDX1 and their diagnostic value in lobular endocervical glandular hyperplasia.

Lobular endocervical glandular hyperplasia (LEGH) is a distinct benign glandular lesion expressing gastric gland mucous cell-type mucin (N-acetylglucosaminα1 → 4galactose → R [GlcNAcα1 → 4Gal → R]). To investigate histogenesis and diagnostic markers of LEGH, we examined the immunohistochemical expression profile of gastric surface mucous cell (MUC5AC and TFF1), gastric gland mucous cell (MUC6, TFF2, and GlcNAcα1 → 4Gal → R), gastric pyloric epithelial cell (PDX1), and endocervical cell (keratan sulfate) markers in normal endocervix samples and benign glandular lesions (nabothian cysts, tunnel clusters, and LEGHs). MUC5AC and MUC6 were expressed in normal endocervical mucosa and benign glandular lesions. TFF1, TFF2, GlcNAcα1 → 4Gal → R, and PDX1 were expressed only in LEGH. Keratan sulfate was expressed in normal endocervical mucosa and benign glandular lesions. In LEGH, gastric surface mucous cell and gastric gland mucous cell differentiation were demonstrated, and transdifferentiation from endocervical mucosa into gastric pyloric mucosa was suggested. In addition to GlcNAcα1 → 4Gal → R, TFF1, TFF2, and PDX1 are additional useful markers for LEGH.

Trefoil factor 2 in gland mucous cell mucin in the mucous gel covering normal or damaged gastric mucosa using the Mongolian gerbil model.

OBJECTIVE: Trefoil factor 2 (TFF2) is localized in gastric gland mucous cells. The purpose of the study was to determine whether TFF2 and gastric mucin are localized in mucous cells and in the surface mucous gel layer (SMGL) of the normal gastric mucosa or in the mucoid cap adherent to gastric mucosal lesions in Mongolian gerbils. MATERIAL AND METHODS: Gastric mucosal lesions were induced in Mongolian gerbils using oral administration of Helicobacter pylori (H. pylori), subcutaneous administration of indomethacin, or oral administration of 30% ethanol. Tissue samples were fixed in Carnoy's solution for preservation of the SMGL, dehydrated, and embedded in paraffin. Histochemical staining for gastric mucins and immunostaining for TFF2 were performed. RESULTS: It was found that surface mucous cell mucin and gland mucous cell mucin were segregated in the SMGL covering the normal gastric mucosa, and the mucin of the mucoid cap covering the mucosal lesions was primarily gland mucous cell mucin. There was a co-localization of TFF2 in gland mucous cell mucin in gland mucous cells, the SMGL, and the mucoid cap. CONCLUSIONS: The co-localization of TFF2 in gland mucous cells and in the adherent mucus suggests a physical interaction between TFF2 and gland mucous cell mucin, and the participation of TFF2 trapped in the adherent mucus functions in mucosal defense, healing, and repair.

Helicobacter pylori infection increases cell kinetics in human gastric epithelial cells without adhering to proliferating cells.

We investigated the effect of H. pylori infection on cell proliferation of gastric mucosa using immunostaining for H. pylori or Ki67. H. pylori cells attached to surface mucous cells covering luminal surface and the upper part of gastric foveolae, and up-regulated the proliferative activity of gastric epithelial cells without adhering to the proliferating epithelial cells.

Anatomical and histological studies of so-called Müllerian duct cyst.

BACKGROUND: We examined so-called Müllerian duct cysts both histologically and immunohistochemically with anatomical observation to investigate the etiology of the 'Müllerian duct cyst'. METHODS: Five cystic lesions located in the prostatic midline were obtained from surgical specimens. A communication between the cystic lesion and the urethra via the utricular orifice was looked for and the specimens were subjected to histological and immunohistochemical testing. RESULTS: A communication between the cyst and the urethra was confirmed in four cases, but not in one case. Histological and immunohistochemical examinations of the epithelium lining indicated that its characteristics were identical to those of the prostatic utricle in all cases. CONCLUSIONS: The so-called Müllerian duct cyst exhibits features comparable to those previously described in the prostatic utricle. There is no evidence that these cystic lesions originate from the Müllerian duct remnant, at least in the epithelial lining. We suggest that they should be termed a prostatic utricular cyst or cystic dilation of the prostatic utricle, depending on whether an outlet to the urethra is absent or present, respectively.

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas.

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.

Large intestinal type-urachal adenocarcinoma with focal expression of prostatic specific antigen.

We present a case of colonic-type adenocarcinoma, which might arise from an urachal remnant through a villous adenoma. The cancer tissue in the present case showed focal immunoreaction to prostate-specific antigen (PSA). This is the first report of urachal adenocarcinoma expressing PSA.

Cell kinetic study of the endometrium by nonisotopic in situ hybridization for histone H3 messenger RNA and immunohistochemistry for Ki-67 and for estrogen and progesterone receptors.

We investigated the cell kinetics of the endometrium in hysterectomy specimens taken for leiomyoma from 22 women with regular ovulatory menstrual cycles. Formalin-fixed, paraffin-embedded tissue sections were examined for proliferating activity using histone H3 messenger RNA in situ hybridization (H3 mRNA-ISH) and immunostaining for the Ki-67 antigen. The relationship of the proliferative activity of endometrial cells to the immunohistochemical expression of the estrogen receptor (ER) and the progesterone receptor (PR) was also examined. During the menstrual cycle, H3 mRNA expression was observed in both the epithelial cells and the stromal cells of the endometrium. In the functional layer, the labeling indices for H3 mRNA (H3 mRNA-LIs) in the epithelial cells peaked in the late proliferative phase, decreased sharply in the early secretory phase, and remained unchanged thereafter. On the other hand, H3 mRNA-LIs of stromal cells displayed two peaks: one in the midproliferative phase and the other in the late secretory phase, the former peak being the greater. In the basal layer, epithelial cells and stromal cells showed low H3 mRNA-LIs and no significant variation throughout the menstrual cycle. The H3 mRNA-LIs correlated well with the Ki-67-LIs and were lower than the corresponding Ki-67-LIs. The regression coefficient (H3 mRNA-LIs against the Ki-67-LIs) was 0.33 for epithelial cells and 0.49 for stromal cells, suggesting that the cell cycle time was longer for epithelial cells than for stromal cells. The proliferative activity of endometrial cells showed close relationships with the expressions of ER and PR in the endometrium. When used in combination with other proliferative markers in paraffin-embedded tissue sections, H3 mRNA-ISH could open broader perspectives on the cell kinetics of the endometrium.

ERCP features in 27 patients with autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis has been proposed as a new clinical entity. However, the ERCP features of this entity have not been well described. METHODS: Clinical and radiographic features in 27 patients with a diagnosis of autoimmune pancreatitis were evaluated. RESULTS: All 27 patients were at first suspected to have pancreatic cancer. The patients were predominantly elderly men and presented with jaundice or mild symptoms and pancreatic enlargement, but no attack of acute pancreatitis. A cholestatic biochemical profile and serum IgG elevation were usually present. A diffusely or segmentally irregular and narrow main pancreatic duct and a positive response to corticosteroid therapy were characteristic. During observation without treatment, serial pancreatography in 2 patients demonstrated progression of diffuse, irregular narrowing of the main pancreatic duct over periods, respectively, of 2 and 6 months. Both the irregular narrowing of the main pancreatic duct and distal bile duct strictures improved in various degrees in response to corticosteroid therapy. CONCLUSIONS: Segmental or diffuse irregular narrowing of the main pancreatic duct are ERCP features of autoimmune pancreatitis.

Effects of angiotensin-converting enzyme inhibition on changes in left ventricular myocardial creatine kinase system after myocardial infarction: their relation to ventricular remodeling and function.

We assessed the effects of angiotensin-converting enzyme (ACE) inhibition on changes in the myocardial intracellular creatine kinase (CK) system in relation to left ventricular (LV) remodeling and function in heart failure after myocardial infarction (MI) in rats. We compared the findings at 4 weeks after MI to those at 12 weeks after MI. LV weight and chamber size were significantly increased and percent fractional shortening (%FS) was decreased in untreated MI rats compared with normal control animals both at 4 and 12 weeks after MI. Animals with MI and treated with the ACE inhibitor temocapril showed significantly reduced LV weight and chamber size and increased %FS compared with untreated MI rats at 12 weeks after MI, but not at 4 weeks after MI. At 4 weeks after MI, no significant changes were found in the total creatine and relative distribution of each CK isoenzyme in either the temocapril-treated or untreated animals with MI compared with the normal controls. In contrast, at 12 weeks after MI, untreated MI rats showed significant reductions in the total creatine and mitochondrial and MM-CK fractions and increases in the MB- and BB-CK fractions compared with the controls. The alterations in the mitochondrial and MB-CK fractions were significantly attenuated after 12 weeks of ACE inhibition. Thus, LV myocardial energy metabolism is progressively impaired and its alteration is not related to the magnitude of geometric changes and LV dysfunction after MI. Most of the beneficial effects of ACE inhibition were observed at 12 weeks after MI. Our results may provide an insight into the therapeutic strategy of ACE inhibition in chronic heart failure after MI.

Loss of calponin h1 in renal angiomyolipoma correlates with aggressive clinical behavior.

OBJECTIVES: To investigate whether any immunohistochemical differences exist between two types of renal angiomyolipomas (AMLs). Renal AMLs are generally considered to be benign in nature. However, a few of these tumors have been reported to involve other organs. METHODS: Tissue specimens from 3 cases of clinically aggressive and 9 cases of clinically indolent renal AMLs were examined histologically and immunohistochemically using antibodies against calponin h1 and alpha-smooth muscle actin. Immunostaining was evaluated semiqualitatively as no staining to strong staining. RESULTS: The tumor cells in the myomatous component of the aggressive type did not show any reaction to the antibody to calponin h1 protein, but they showed strong immunoreactions with the antibody to the alpha-smooth muscle actin antigen. All cases of the nonaggressive AMLs demonstrated strong immunoreactions with both antibodies used. CONCLUSIONS: Our initial results in a small series of cases suggest a potential molecular difference between the aggressive and nonaggressive type of AMLs. Loss of calponin h1 from the tumor cells of the aggressive type might be related to their pathologically invasive features, and this aggressive type might be categorized into an intermediate type between the benign and malignant types.

Are neuroendocrine cells responsible for the development of benign prostatic hyperplasia?

OBJECTIVES: To examine the possible relationship between the distribution of neuroendocrine (NE) cells and the development of benign prostatic hyperplasia (BPH) in the human prostate, we performed an NE cells-distribution analysis and made morphological observations of acinous structures in different-aged prostates. METHODS: Forty-three human prostates obtained from surgical and autopsy cases aged from 2 months to 86 years were examined immunohistochemically using Chromogranin A and analyzed with special reference to the development of BPH. RESULTS: NE cells were distributed predominantly in the verumontanum and main prostatic ducts and were fewer in number in the terminal acini. As BPH development progressed, the NE cells were greatly diminished in number or completely lost from most adenoma nodules. CONCLUSIONS: The NE cells of the prostate may be distributed and transported from the periurethral region near the verumontanum to the terminal acini during the development of the acinar structures. The distribution pattern is relatively consistent among prostates of all ages. However, NE cells do not appear in acquired tissue within BPH nodules as the nodules develop. Thus, the distribution of NE cells does not seem to be related to the development of BPH.