Influenza A Virus: Cellular Entry.

The frequent emergence of pathogenic viruses with pandemic potential has posed a significant threat to human health and economy, despite enormous advances in our understanding of infection mechanisms and devising countermeasures through developing various prophylactic and therapeutic strategies. The recent coronavirus disease (COVID-19) pandemic has re-emphasised the importance of rigorous research on virus infection mechanisms and highlighted the need for our preparedness for potential pandemics. Although viruses cannot self-replicate, they tap into host cell factors and processes for their entry, propagation and dissemination. Upon entering the host cells, viruses ingeniously utilise the innate biological functions of the host cell to replicate themselves and maintain their existence in the hosts. Influenza A virus (IAV), which has a negative-sense, single-stranded RNA as its genome, is no exception. IAVs are enveloped viruses with a lipid bilayer derived from the host cell membrane and have a surface covered with the spike glycoprotein haemagglutinin (HA) and neuraminidase (NA). Viral genome is surrounded by an M1 shell, forming a "capsid" in the virus particle. IAV particles use HA to recognise sialic acids on the cell surface of lung epithelial cells for their attachment. After attachment to the cell surface, IAV particles are endocytosed and sorted into the early endosomes. Subsequently, as the early endosomes mature into late endosomes, the endosomal lumen becomes acidified, and the low pH of the late endosomes induces conformational reaggangements in the HA to initiate fusion between the endosomal and viral membranes. Upon fusion, the viral capsid disintegrates and the viral ribonucleoprotein (vRNP) complexes containing the viral genome are released into the cytosol. The process of viral capsid disintegration is called "uncoating". After successful uncoating, the vRNPs are imported into the nucleus by importin α/ß (IMP α/ß), where viral replication and transcription take place and the new vRNPs are assembled. Recently, we have biochemically elucidated the molecular mechanisms of the processes of viral capsid uncoating subsequent viral genome dissociation. In this chapter, we present the molecular details of the viral uncoating process.

A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.

Diagnostic significance of apical membranous and cytoplasmic dot-like CD26 expression in encapsulated follicular variant of papillary thyroid carcinoma: a useful marker for capsular invasion.

Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and invasive encapsulated follicular variant of papillary thyroid carcinoma (EFV-PTC) are indistinguishable preoperatively. CD26 expression in follicular tumor-uncertain malignant potential (FT-UMP) is reported to be clearly higher than in that without capsular invasion. To verify the diagnostic significance of CD26 immunostaining in EFV-PTC, we examined the expression pattern of CD26 in non-invasive EFV-PTC (NIFTP) and invasive EFV-PTC. We performed immunohistochemical analysis using CD26 antibody for 37 NIFTPs and 54 EFV-PTCs (34 minimally invasive EFV-PTCs and 20 widely invasive EFV-PTCs). Most NIFTP samples showed an apical membranous pattern or a cytoplasmic diffuse pattern of expression. Invasive EFV-PTCs more frequently showed a cytoplasmic dot-like pattern, and the labeling indices of tumor cells with cytoplasmic dot-like patterns were significantly higher than those in NIFTPs. The sizes of dots seen in NIFTPs (mean: 1.12 µm) were significantly smaller than in invasive EFV-PTCs (1.33 µm), minimally invasive EFV-PTC (1.27 µm), and widely invasive EFV-PTC (1.38 µm). We, therefore, conclude that cytoplasmic diffuse and/or cytoplasmic dot-like CD26 expression, particularly the larger CD26-positive dots, could be useful markers for capsular invasion in EFV-PTC. CD26 immunostaining, using cell blocks or cytological specimens, may preoperatively distinguish between NIFTP and invasive EFV-PTC.

Structural insights into HDAC6 tubulin deacetylation and its selective inhibition.

We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.

RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway.

Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

Human iPSC-derived CD4+ Treg-like cells engineered with chimeric antigen receptors control GvHD in a xenograft model.

CD4+ T cells induced from human iPSCs (iCD4+ T cells) offer a therapeutic opportunity for overcoming immune pathologies arising from hematopoietic stem cell transplantation. However, most iCD4+ T cells are conventional helper T cells, which secrete inflammatory cytokines. We induced high-level expression of FOXP3, a master transcription factor of regulatory T cells, in iCD4+ T cells. Human iPSC-derived, FOXP3-induced CD4+ T (iCD4+ Treg-like) cells did not secrete inflammatory cytokines upon activation. Moreover, they showed demethylation of the Treg-specific demethylation region, suggesting successful conversion to immunosuppressive iCD4+ Treg-like cells. We further assessed these iCD4+ Treg-like cells for CAR-mediated immunosuppressive ability. HLA-A2 CAR-transduced iCD4+ Treg-like cells inhibited CD8+ cytotoxic T cell (CTL) division in a mixed lymphocyte reaction assay with A2+ allogeneic CTLs and suppressed xenogeneic graft-versus-host disease (GVHD) in NSG mice treated with A2+ human PBMCs. In most cases, these cells suppressed the xenogeneic GvHD progression as much as natural CD25+CD127- Tregs did.

Notch2 signaling is required for proper mast cell distribution and mucosal immunity in the intestine.

Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.

HLA-C matching status does not affect rituximab-mediated antibody-dependent cellular cytotoxicity by allogeneic natural killer Cells.

Risk of leukemia relapse after T cell-depleted hematopoietic stem cell transplantation is lower in the "HLA-C mismatched" recipient-donor combinations. This might be attributable to increased natural killing by allogeneic NK cells carrying a KIR that does not bind to HLA-C on target cells (HLA-C-uncoupled KIR). Considering a new strategy of allogeneic NK cell transfer with rituximab to treat B-cell lymphomas, however, it is unknown whether the HLA-C matching status also affects rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). To address this issue, we investigated the levels of ADCC by purified NK cells carrying an HLA-C-uncoupled KIR, where the NK cell donors had either matched or mismatched HLA-C combination with target cells. Purified NK cells carrying an HLA-C-uncoupled KIR consistently showed enhanced ADCC against target cells when NK cell donors had an HLA-C-mismatch. When NK cell donors did not have an HLA-C mismatch, it was inconsistent whether HLA-C-uncoupled KIR caused ADCC enhancement. When the levels of ADCC by whole NK cells were compared, there were substantial differences among the donors regardless of the HLA-C matching status. Subjects with HLA-C mismatch may not have an advantage when cytoimmunotherapy using allogeneic NK cells is considered in combination with rituximab.

Diagnostic utility of notch-1 immunocytochemistry in endometrial cytology.

OBJECTIVE: It was the aim of this study to evaluate the diagnostic utility of Notch-1 immunocytochemistry in distinguishing endometrial glandular and stromal breakdown (EGBD) from endometrial adenocarcinoma in endometrial cytology. STUDY DESIGN: Samples of normal endometrium, EGBD and endometrial adenocarcinoma were subjected to immunocytochemical staining for Notch-1, and we examined the labeling index (LI) of Notch-1 (the ratio of intranuclear Notch-1-positive cells to total cells). We compared (1) the Notch-1 LI in normal endometrium, (2) the Notch-1 LI between normal endometrium and endometrial adenocarcinoma, and (3) the Notch-1 LI in normal endometrium, EGBD and endometrial adenocarcinoma. RESULTS: In analysis item 1, the LI of Notch-1 was 32.9 ± 8.4, 19.4 ± 8.2 and 12.5 ± 7.5% in proliferative endometrium, secretory endometrium and atrophic endometrium, respectively. In analysis item 2, the LI of Notch-1 in endometrial adenocarcinoma was 45.2 ± 7.4%, which was significantly higher than that in normal endometrium. In analysis item 3, the LI of Notch-1 in EGBD was 31.3 ± 8.3%, which was significantly lower than that in endometrial adenocarcinoma. CONCLUSION: In conclusion, Notch-1 immunocytochemistry is a useful method for distinguishing between EGBD and endometrial carcinoma in endometrial cytology.

Disrupting the HDAC6-ubiquitin interaction impairs infection by influenza and Zika virus and cellular stress pathways.

The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.

Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease.

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy.

Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.

Aberrant DNA polymerase alpha is excluded from the nucleus by defective import and degradation in the nucleus.

DNA polymerase alpha is essential for the onset of eukaryotic DNA replication. Its correct folding and assembly within the nuclear replication pre-initiation complex is crucial for normal cell cycle progression and genome maintenance. Due to a single point mutation in the largest DNA polymerase alpha subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase alpha activity and S phase arrest at restrictive temperature. In this study, we show that an aberrant form of endogenous p180 in tsFT20 cells (p180(tsFT20)) is strictly localized in the cytoplasm while its wild-type counterpart enters the nucleus. Time-lapse fluorescence microscopy with enhanced green fluorescent protein-tagged or photoactivatable green fluorescent protein-tagged p180(tsFT20) variants and inhibitor analysis revealed that the exclusion of aberrant p180(tsFT20) from the nucleus is due to two distinct mechanisms: first, the inability of newly synthesized (cytoplasmic) p180(tsFT20) to enter the nucleus and second, proteasome-dependent degradation of nuclear-localized protein. The nuclear import defect seems to result from an impaired association of aberrant de novo synthesized p180(tsFT20) with the second subunit of DNA polymerase alpha, p68. In accordance, we show that RNA interference of p68 results in a decrease of the overall p180 protein level and in a specific increase of cytoplasmic localized p180 in NIH3T3 cells. Taken together, our data suggest two mechanisms that prevent the nuclear expression of aberrant DNA polymerase alpha.

Expression of immunoreactivity and genetic mutation in eosinophilic and ciliated metaplastic changes of endometrial glandular and stromal breakdown: cytodiagnostic implications.

Various metaplastic changes may be present in endometrium, in which also cellular atypias may often be observed. Particularly, eosinophilic and ciliated changes (ECCs) occur in both nonneoplastic and neoplastic endometrium. This may cause confusion in the cytodiagnosis. This study was enterprised to investigate the possible help of immunocytochemical and cytogenetic study in the diagnostic and biologic assessment of ECC cells. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the material consists of 40 cases of cytologic smears examined by direct sampling of the endometrial cavity comprising 30 cases of ECC in endometrial glandular and stromal breakdown (EGBD) and 10 cases of well-differentiated adenocarcinoma (G1). After cytodiagnosis, immunostaining for p53 protein, Ki-67, and cyclin A was performed on multiple wet-fixed slides from each single case to evaluate the immunoreactivity, intensity of nuclear staining, and nuclear labeling index (N-LI). The intensity of nuclear staining was scored as negative (0), weak (1), moderate (2), or strong (3), and the N-LI was scored as less than 10% (0), from 10% to 25% (1), from 26% to 50% (2), or more than 50% (3), and the final score was calculated by adding both partial scores. A statistical significance test was performed by using Mann-Whitney U test, and the result was judged as significant when the P value was less than .05. For genetic mutation analysis of p53, the material comprised 6 cases of EGBD in which a high score was measured with immunocytochemistry for p53 protein, and the presence of ECC was confirmed on the hematoxylin and eosin. The ECC cells on paraffin-embedded specimens were captured using laser capture microdissection technology. Mutations in p53 gene (exons 5-8) were examined using DNA sequencing analysis. In immunocytochemistry for p53 protein, Ki-67, and cyclin A, the proportions of immunoreactive cells for p53 were statistically higher in ECC compared with those of G1 (P < .05). The proportions of the immunoreactive cells for Ki-67 and cyclin A were statistically higher in G1 compared with those of ECC (P < .05). (2) In genetic mutation analysis of p53, DNA sequencing of p53 in 6 cases revealed no mutations. The percentage of immunoreactive cells for p53 protein were higher in ECC than in G1, but the mutation point was not confirmed in genetic mutation analysis. The differential expression of these biologic parameters in ECC cells could be considered of possible relevance to the cytopathologic diagnosis in the future.

Influenza virus uses transportin 1 for vRNP debundling during cell entry.

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.

Utility of thin-layer preparations in endometrial cytology: immunocytochemical expression of PTEN, beta-catenin and p53 for benign endometrial lesions.

This article focuses on the characteristic features of morphology and molecular biology of PTEN, beta-catenin, and p53 immunocytochemistry in normal endometrium (proliferative, secretory, and atrophic) and endometrial glandular and stromal breakdown (EGBD) using thin-layer specimens. During a 6-month period, 120 endometrial samples were collected directly using the Uterobrush and a thin-layer specimen was prepared. Immunocytochemical expression of PTEN, beta-catenin, and p53 were investigated using 30 cases each of proliferative endometrium (PE), secretory endometrium (SE), atrophic endometrium (AE), and EGBD.PTEN expression of normal endometrial glandular epithelial cells changes with the hormonal status; PE produce very high expression, SE creates attenuation or disappearance of PTEN expression and AE diminished more in comparison with SE. PTEN expression of EGBD showed a tendency towards attenuation compared with PE, but showed high expression in comparison with SE and AE. As for the immunoreactivity of beta-catenin, in all phases (PE, SE, AE, and EGBD), it was observed in the cytoplasm of glandular epithelial cells, but not nuclei, and showed strong membranous staining. As for the p53 immunoreactivity, p53 positivity was not observed in the glandular epithelial cells in all phases (PE, SE, AE, and EGBD) with the exception of some metaplastic cells. The presence of p53 immunoreactivity of a weak, low ratio in metaplastic cells was unexpected. In the current study, the expression manner of PTEN, beta-catenin, and p53 immunocytochemistry was observed in the normal endometrium (PE, SE, and AE) and EGBD.

Purification of Unanchored Polyubiquitin Chains from Influenza Virions.

Influenza A virus (IAV) is an enveloped virus with a segmented single-stranded negative-strand RNA genome. In general, the role of virally encapsidated host cell proteins in the viral life cycle is unclear. The virion contains abundant ubiquitin molecules some of which have been identified as unanchored polyubiquitin chains. These ubiquitin chains have been postulated to play a role in recruiting histone deacetylase 6 (HDAC6) to the cytosolic surface of late endosomes (LEs), promoting IAV uncoating via aggresome processing-a cellular machinery that disposes of protein waste. HDAC6, a class II HDAC, is unusual because it resides mostly in the cytosol instead of the nucleus. It is a unique protein consisting of two catalytic domains (CDs) and a zinc-finger ubiquitin-binding domain (ZnF-UBP) close to its C-terminus. This ZnF-UBP recognizes the unconjugated ubiquitin C-terminus (di-Gly motif) with very high affinity. Biochemical analyses showed that free di-Gly motifs are present in the form of unanchored ubiquitin inside IAV virions. These motifs are exposed following low pH-triggered viral fusion at the LEs and attract HDAC6 transiently to the cytosolic surface of vesicles. The binding of the two components promotes viral uncoating via HDAC6 interaction with cellular motor proteins dynein and myosin II and the viral M1 capsid. The cellular mechanism involved is related to aggresome processing, a pathway that promotes degradation of misfolded protein aggregates. K63-linked ubiquitin chains are thought to be the trigger for aggresome processing, though it is still not clear whether such types of chains are prevalent within the IAV capsid. Here, we present methods using purified ZnF-UBP domain of HDAC6 to immunoprecipitate viral unanchored ubiquitin chains, which can then be used for further biochemical analyses of ubiquitin chain linkage.

Diabetes mellitus reduces the therapeutic effectiveness of interferon-alpha2b plus ribavirin therapy in patients with chronic hepatitis C.

AIM: Patients with chronic hepatitis C (CHC) often have diabetes mellitus (DM). However, it is unknown whether DM affects patient response to interferon (IFN) plus ribavirin therapy. Therefore, the aim of this study was to examine the influence of DM on the outcome of IFN-alpha2b plus ribavirin therapy. METHODS: In a cohort of 110 patients with CHC, the outcome of 6 months of IFN-alpha2b plus ribavirin therapy was evaluated by comparing the patients with and without DM. RESULTS: There were 46 sustained-responders; 64 patients did not become sustained responders. Higher age (P = 0.015), lower platelet counts (P = 0.036), hepatitis C virus (HCV) serotype 1 (P = 0.001), advanced liver fibrosis (P = 0.004), and the presence of DM (P = 0.007) were significantly associated with not becoming a sustained-responder. Seventeen CHC (15%) patients had DM. Sex ratio, age, body mass index, alanine aminotransferase levels, HCV-RNA titer, and HCV serotypes did not significantly differ between the patients with and without DM, while fasting plasma glucose, hemoglobin A1c and liver histological staging were significantly different. On multiple logistic regression analysis, HCV serotype 1 (odds ratio 8.743, 95% confidence interval 2.215-34.517; P = 0.002) and the presence of DM (odds ratio 8.657, 95% confidence interval 1.462-51.276; P = 0.014) were independently associated with not becoming a sustained-responder. CONCLUSIONS: The findings indicate that DM reduces the response to IFN-alpha2b plus ribavirin therapy in CHC patients.

Endometrial glandular and stromal breakdown, part 2: cytomorphology of papillary metaplastic changes.

Careful cytomorphologic evaluation of abnormal endometrial lesions has made accurate and reproducible microscopic assessment possible. Histopathology of patients with dysfunctional uterine bleeding due to an anovulatory cycle usually contain endometrial glandular and stromal breakdown (EGBD) and papillary metaplasia on the endometrial surface epithelium, if an appropriate sample has been collected. We often recognized abnormal cell clumps in the cytological smears with EGBD cases. They were composed of metaplastic cells, and some irregular small projection figures were observed from the margins of the cell clumps. We describe the quantitation of metaplastic clumps with irregular protrusions (MCIP) in endometrial endocyte samples. The current study presents the cytomorphological characteristics of the metaplastic changes recognized in EGBD cases. During a 7-yr period, 144 cases for which histopathological diagnoses were obtained following endometrial curettage, after collecting a direct endometrial sample using the endocyte. The material comprised 49 cases of normal proliferative endometrium (NPE) (patients aged 28-51, average 39.9), 32 cases of EGBD (patients aged 30-67, average 49.6), and 63 cases of endometrial hyperplasia without atypia (EH) (patients aged 35-65, average 47.7). The following points were investigated: (1) the occurrence of metaplastic cells; (2) the occurrence and the frequency of MCIP; and (3) the occurrence of MCIP that contains condensed stromal clusters. Metaplastic cells were seen in 93.8% of the EGBD cases. Cytomorphologic pattern identified with MCIP was 90.6%, and its frequency showed 16.1%. The occurrence of MCIP that contain condensed stromal clusters (93.1%) showed a strong association in comparison with other lesions, such as NPE and EH. Our data appear to indicate that the appearance of MCIP with condensed stromal clusters originated from the papillary metaplasia, which occurred on the endometrial surface epithelium. The cytologic observation of those cells may be a useful indicator for providing the nature of EGBD endometrium.

Endometrial glandular and stromal breakdown, part 1: cytomorphological appearance.

Endometrial carcinoma is the most common invasive neoplasm of the female reproductive tract. Early detection and accurate diagnosis of these lesions and its precursor by endometrial cytology is now accepted in Japan and regarded as an effective primary method of evaluating endometrial pathology (atypical hyperplasia or carcinoma). Careful cytomorphologic evaluation of the abnormal endometrial lesions has made possible an accurate and reproducible microscopic assessment. The current study was conducted to determine the significance of endometrial cytology on disordered endometrium associated with anovulation when compared with endometrial hyperplasia. From January 1998 through April 2004, 144 cases on which histopathological diagnoses were obtained by endometrial curettage after taken direct endometrial sample by Endocyte. The materials comprise 49 cases of normal proliferative endometrium, and 63 cases of endometrial hyperplasia without atypia were prepared as control cases. The cytomorphology was examined involving so-called endometrial glandular and stromal breakdown (EGBD). EGBD cases evidenced significant numbers of stromal cells condensed and formed compact nests with hyperchromatic nuclei and little or no cytoplasm. They were often associated with fragmented clusters of endometrial glands with condensed cluster of stromal cells. Both the fragmented cluster of endometrial glands and condensed cluster of stromal cells are a characteristic cytologic feature of EGBD endometrium on the cyto-architectural diagnosis. The combination of these cellular patterns is highly specific to this abnormal pathological condition in EGBD endometrium. To improve the accuracy of the cytodiagnosis, it is important that the cytology of the EGBD endometrium should be diagnosed negative; as a result, we can achieve successful endometrial cytology with cyto-architectural criteria for the endometrial pathology.