Invasive pulmonary aspergillosis with candidiasis: usefulness of molecular and ultrastructural morphological analysis on FFPE tissue for invasive fungal infections.

Invasive pulmonary aspergillosis (IPA) is one of the most frequent forms of invasive fungal infections (IFI); however, it is often difficult to identify the pathogenic fungal species and to select appropriate treatments for patients with IFI including IPA. Here, we describe the detailed pathophysiology of an autopsy case of severe respiratory failure due to IPA with candidiasis. The patient developed severe respiratory failure after influenza infection and died, and the autopsy revealed a mixed disease of IPA with candidiasis. In this study, in addition to the routine pathological examination, we further examined formalin-fixed paraffin-embedded (FFPE) tissues by scanning electron microscopy (SEM) and partial genomic DNA sequencing. Although optical microscopy alone was insufficient to identify the pathogenic organisms, SEM clearly depicted the characteristic morphology of Aspergillus sp. and Candida sp. as closely overlapping in a nested fashion, providing evidence of mixed infection of both fungal species in a focal site. The technique using FFPE tissue in combination with ultrastructural observation by SEM, elemental analysis by SEM-EDX, and DNA sequencing is promising for analyzing the pathophysiology of IFI.

Occludin induces microvillus formation via phosphorylation of ezrin in a mouse hepatic cell line.

Apical and basolateral cell membranes are separated by tight junctions (TJs). Microvilli are limited to the apical cell membrane. TJs and microvilli are the landmarks for epithelial cell polarity. However, the direct relationship between TJ proteins (TJPs) and the components of microvilli remains unclear. In this study, we investigated whether occludin, which is considered to be a functional TJP, is involved in microvillus formation. In occludin knockout mouse hepatic cells (OcKO cells), the microvillus density was less than that in wild-type (WT) cells and the length of microvilli was short. Immunoreactivity of ezrin was decreased in OcKO cells compared with that in WT cells. Although there was no change in the expression level of ezrin, phosphorylation of ezrin was decreased in OcKO cells. The microvillus density and the length of microvilli were increased in OcKO cells by transfection of full-length mouse occludin and COOH-terminal domains of occludin. These results suggested that occludin induced microvillus formation via phosphorylation of ezrin and that the COOH-terminal domain of occludin, which is localized in non-TJ areas, might be able to induce microvilli formation. Our results provide new insights into the function of occludin.

[Clinical significance of chronologic immature platelet fraction analysis in TAFRO syndrome].

TAFRO syndrome is characterized by thrombocytopenia with unknown etiology. The assessment of immature platelet fraction (IPF) is useful for differential diagnoses that include thrombocytopenia. However, the significance of IPF in cases of TAFRO syndrome remains to be reported. We present a case of TAFRO syndrome wherein the patient demonstrated a marked increase in IPF without thrombocytopenia, which offers vital information concerning TAFRO diagnosis and the serial measurements of IPF during treatment. A 65-year-old man presenting with fever was admitted to our hospital. He exhibited mild splenomegaly and lymphadenopathy, as well as rapidly worsening renal failure and fluid retention. These indications prompted the initiation of corticosteroid therapy. A normal platelet count and aberrantly high IPF implied abnormal thrombopoiesis, and subsequent bone-marrow findings suggested TAFRO syndrome. The platelet counts started to decrease following the corticosteroid therapy, but the treatment refractoriness prompted the urgent administration of rituximab. Thereafter, the platelet count nadir remained for approximately one month, whereas the decreasing IPF trend preceded platelet recovery. In the present case, a high pre-treatment IPF was demonstrated before the emergence of thrombocytopenia, and a decreasing trend of IPF was observed before platelet recovery during treatment. Therefore, serial IPF measurements could be useful for the early diagnosis and prognostication of TAFRO syndrome.

Aberrant expression of claudin-4 and -7 in hepatocytes in the cirrhotic human liver.

The liver comprises hepatocytes and non-parenchymal cells such as bile duct epithelial cells. Claudin-4 and -7 are not expressed in hepatocytes under physiological conditions. It was reported that claudin-7 increased in human pulmonary fibroses. We therefore investigated claudin-4 and -7 expressions in human cirrhotic livers, in which hepatocyte proliferation is severely delayed. We examined liver tissues from 50 patients with liver tumors. The expression of claudin-4 and -7 in hepatocytes significantly increased with the grade of fibrosis, not with inflammatory activity, in the liver tissues of chronic hepatitis. The number of claudin-4- and -7-positive cells observed was greater than that of alpha-fetoprotein-positive hepatic progenitor cells. In primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 was not induced by treatment with proinflammatory cytokines. In immunohistochemical analysis of liver tissues of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice and primary cultures of mouse hepatocytes, the expression of claudin-4 and -7 increased with proliferation of progenitor cells. However, the claudin-4- and -7-positive cells were not always progenitor cells. Thus, claudin-4 and -7 were observed in hepatocytes of severely damaged mouse and human livers. These findings suggest that claudin-4- and -7-positive hepatocytes may exist during the process of differentiation from progenitor cells into mature hepatocytes.

Claudins-4 and -7 might be valuable markers to distinguish hepatocellular carcinoma from cholangiocarcinoma.

The claudin family members are the functional components of tight junctions. Expression and localization of claudins vary among organs and tumor types. In this study, we examined expression and localization of tight junction proteins (TJP) in human liver tumors, to estimate their usefulness as differential diagnostic markers. The materials used for immunohistochemical analysis were 47 liver tumor specimens including 29 cases of hepatocellular carcinoma (HCC), 15 cases of cholangiocarcinoma (CC), 3 cases of combined HCC and CC (CHC), and 3 cases of cholangiolocellular carcinoma (CoCC). Samples were examined using semiquantitative and statistical analysis of immunoreactivity. In HCC, claudin-1, occludin, tricellulin, and JAM-A were expressed on the cell membrane as well as in hepatocytes. In CC, claudins-1, -4, and -7, tricellulin, and JAM-A were expressed on the cell membrane and occludin was predominantly expressed in the apicalmost areas of the cell membrane. Significant differences in the immunohistochemical scores of claudin-4 and claudin-7 were observed when comparing HCC and CC. CHC was positive for all of the TJPs examined in this study. The expression pattern of CoCC was found to be similar to that of CC. There were differences in the distribution of intensity scores of claudins-4 and -7 and occludin between CoCC and HCC. In addition, CHC was positive for Glypican-3 and CK-19. CoCC was positive for only CK-19. The results suggest that claudins-4 and -7 might be valuable markers for distinguishing HCC and CC and that CoCC might arise from hepatic ductal cells.

Analysis of the expression and localization of tight junction transmembrane proteins, claudin-1, -4, -7, occludin and JAM-A, in human cervical adenocarcinoma.

OBJECTIVE: Tight junction proteins have recently been reported to be useful for distinguishing between neoplastic and non-neoplastic tissues. In this study, we evaluated the expression and localization of tight junction transmembrane proteins in human cervical adenocarcinoma and adenocarcinoma in situ (AIS), and we determined whether their expression patterns could distinguish cervical adenocarcinoma from non-neoplastic cervical glands. METHODS: Fifty-five patients with cervical adenocarcinoma or AIS were included in this study. Surgical specimens were immunohistochemically stained for claudin (CLDN) -1, -4, -7, occludin, and JAM-A. RESULTS: Significantly higher expression levels of CLDNs and JAM-A were found in cervical AIS and adenocarcinoma than in non-neoplastic glands. In cervical AIS and adenocarcinoma, localization of CLDN1 and JAM-A was extended throughout the whole cell membranes, whereas they were predominantly expressed at the most apical cell-cell junction in non-neoplastic glands. ROC curve analysis revealed that immunoreactivities of CLDN-1 or JAM-A successfully distinguished neoplasms from non-neoplastic cervical glands with high specificity (CLDN-1, 79.1%; JAM-A, 79.1%) and high sensitivity (CLDN-1, 84.1%; JAM-A, 95.5%). CONCLUSIONS: As expected, there were immunohistochemical differences between cervical adenocarcinoma and non-neoplastic cervical glands by using antibodies against tight junction transmembrane proteins. These results suggest that CLDN-1 and JAM-A are potential biomarkers for cervical adenocarcinoma.