Thus, therapeutic approaches aimed at inactivating PLK1 and/or reactivating PLK2-4 might be highly useful in the treatment of human liver cancer. (HEPATOLOGY 2010.) Polo-like kinase (PLKs) proteins play pivotal roles in cell cycle progression and response to DNA damage.1 Four members of this family of serine/threonine kinases were identified: PLK1, PLK2 (also known as SNK), PLK3 (also known as FNK or PRK), and PLK4 (or SAK).1 PLKs are characterized by a highly conserved N-terminal serine/threonine kinase domain and one or two polo boxes in the C-terminal region,

which are crucial for subcellular localization and binding of specific phosphopeptides.2 Expression of PLKs is tightly regulated during the cell cycle.1 PLK1 Crizotinib price is inhibited by numerous checkpoint genes, whereas PLK2-4 genes are activated by spindle checkpoints and DNA damage.1, 3 Despite the high sequence homology among the four members of the PLK family, their functions seem to diverge. PLK1 is involved mainly in the control of the G2/M phase, by promoting CDC25C phosphatase activity with subsequent activation of CyclinB1/CdK1 JAK inhibitor complex, and the degradation of early mitotic inhibitor-1 (EMI1), which inhibits the activated Anaphase-Promoting Complex/Cyclosome.1 PLK2 and PLK3 were identified as serum-inducible

growth responsive genes and are implicated in the stress-response.4 Analysis of PLK2 knockout mice indicated that PLK2 is implicated in embryonic development and cell cycle regulation, as confirmed by recent findings showing an involvement of PLK2 in promotion of S-phase entry and centriole duplication.5 Previously, levels of PLK3 have been described as either unchanged throughout the cell cycle6 or increased in mitosis.7 However, more recent evidence indicates that PLK3 expression peaks in G1 phase and is required for S phase entry through the regulation of cyclin E levels.8 Moreover, PLK3 is implicated in the regulation of Golgi apparatus fragmentation during cell cycle progression, and deregulated expression of PLK3 in vitro promotes cell cycle arrest and apoptosis, mainly due to microtubule disfunctions.9, 10 Like all other PLKs, PLK4 is

implicated in cell cycle regulation, because constitutive PLK4 expression leads to decreased cell growth and multinucleation in vitro.11, 12 Indeed, PLK4 is involved in the medchemexpress proper reproduction of centrosomes,13 and it is required for the APC-dependent destruction of cyclin B1, with the consequent exit from mitosis.11 Due to the critical role of PLKs in controlling cell cycle progression, their involvement in oncogenesis might be envisaged. An oncogenic role for PLK1 has been hypothesized, because its constitutive expression in NIH3T3 fibroblasts causes oncogenic foci formation and is tumorigenic in nude mice.14 Furthermore, PLK1 is overexpressed in a variety of human tumors,3 including human hepatocellular carcinoma (HCC).

RXRα is an important nuclear hormone receptor and acts as a heterodimer with other nuclear hormone receptors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR).14 RXR/PXR heterodimer is an Navitoclax chemical structure important regulator of CYP3A isoforms; however, the involvement of this complex in transcriptional regulation of CYP1A2 is not well established. CYP1A2 is mainly regulated by aryl hydrocarbon receptor; however, PXR-deficient mice and hepatocyte RXRα-deficient mice express lower hepatic messenger RNA (mRNA) levels of CYP1A2 and CYP3A11 compared to wildtype mice, particularly after APAP administration.15-17 Consequently,

these knockout mice are resistant to APAP-induced hepatotoxicity.15,

17 Thus, any changes in the expression of these nuclear hormone receptors in response to activation of antiviral pathways could potentially alter APAP-induced toxicity through modulation of NAPQI generation. Because viral infections can lead to significant induction of type I interferons (IFN), many groups have used IFN or IFN-inducing agents to study the impact of activation of antiviral responses on drug metabolism.18 One such agent is polyinosinic-polycytidylic acid (polyI:C), a viral double-stranded RNA (dsRNA) mimetic, which has been shown to impair drug metabolism.19 Although the effects of polyI:C on drug metabolism have been ascribed to its ability to induce IFN, Alpelisib mouse there has not been a conclusive study supporting this hypothesis. PolyI:C does induce other cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) that could affect activity or expression of CYPs. IFNs as well as TNF-α and IL-1 have all been shown to alter drug metabolism when administered in patients or in animal models.4, 20 Additionally, viral dsRNA and polyI:C are sensed by the

endosomal receptor, Toll-like receptor (TLR3), as well as recently discovered cytoplasmic receptors, such as RNA helicase retinoic acid-inducible gene-I (RIG-I).21 These receptors have cell-type and tissue-specific 上海皓元医药股份有限公司 roles in sensing polyI:C; however, it has not been characterized which receptors are involved in mediating the effects of polyI:C on hepatic drug metabolism.22 Here we used polyI:C and vesicular stomatitis virus (VSV), a dsRNA virus, to study how activation of antiviral responses can modulate APAP metabolism and hepatotoxicity. We provide a mechanism by which in vivo administration of polyI:C suppresses APAP-induced hepatotoxicity independent of IFN production or in the absence of TLR3 through transcriptional down-regulation of RXRα and PXR and their downstream CYPs.

RXRα is an important nuclear hormone receptor and acts as a heterodimer with other nuclear hormone receptors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR).14 RXR/PXR heterodimer is an click here important regulator of CYP3A isoforms; however, the involvement of this complex in transcriptional regulation of CYP1A2 is not well established. CYP1A2 is mainly regulated by aryl hydrocarbon receptor; however, PXR-deficient mice and hepatocyte RXRα-deficient mice express lower hepatic messenger RNA (mRNA) levels of CYP1A2 and CYP3A11 compared to wildtype mice, particularly after APAP administration.15-17 Consequently,

these knockout mice are resistant to APAP-induced hepatotoxicity.15,

17 Thus, any changes in the expression of these nuclear hormone receptors in response to activation of antiviral pathways could potentially alter APAP-induced toxicity through modulation of NAPQI generation. Because viral infections can lead to significant induction of type I interferons (IFN), many groups have used IFN or IFN-inducing agents to study the impact of activation of antiviral responses on drug metabolism.18 One such agent is polyinosinic-polycytidylic acid (polyI:C), a viral double-stranded RNA (dsRNA) mimetic, which has been shown to impair drug metabolism.19 Although the effects of polyI:C on drug metabolism have been ascribed to its ability to induce IFN, Cabozantinib mw there has not been a conclusive study supporting this hypothesis. PolyI:C does induce other cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) that could affect activity or expression of CYPs. IFNs as well as TNF-α and IL-1 have all been shown to alter drug metabolism when administered in patients or in animal models.4, 20 Additionally, viral dsRNA and polyI:C are sensed by the

endosomal receptor, Toll-like receptor (TLR3), as well as recently discovered cytoplasmic receptors, such as RNA helicase retinoic acid-inducible gene-I (RIG-I).21 These receptors have cell-type and tissue-specific medchemexpress roles in sensing polyI:C; however, it has not been characterized which receptors are involved in mediating the effects of polyI:C on hepatic drug metabolism.22 Here we used polyI:C and vesicular stomatitis virus (VSV), a dsRNA virus, to study how activation of antiviral responses can modulate APAP metabolism and hepatotoxicity. We provide a mechanism by which in vivo administration of polyI:C suppresses APAP-induced hepatotoxicity independent of IFN production or in the absence of TLR3 through transcriptional down-regulation of RXRα and PXR and their downstream CYPs.

High levels selleck screening library of functional HBc-specific T cells that display efficient antigen-restricted functions and are able to lyse HBV-infected hepatocytes could be elicited from both PBMCs and LILs of chronic HBV patients. Intrahepatic HBV-specific T cells are known to be in an exhaustion state.12 Despite this, specific T cells were strongly amplified from LILs, underlining the potency of the pDC-based strategy. Compared with current strategies developed to amplify HBV-specific T cells (peptides, mDCs), peptide-loaded pDCs induced greater numbers of specific T cells and faster immune responses.5, 16 HBeAg is known

to have an immunoregulatory function in promoting viral persistence through the modulation of the immune response to HBc antigen.29–31

Indeed, here HBeAg status was found to be a critical factor determining patients’ ability to elicit anti-HBV immune responses upon pDC stimulation. Two patients in our cohort switched their ability to respond to pDC stimulation within a 6-month interval. This switch was in line with modification of their HBeAg status. These observations highlight the major role of HBeAg in regulating specific T cell function. In accordance with our findings, mDCs pulsed with HBV-derived peptides elicited a stronger anti-HBV immunity in HBeAg-negative patients than in HBeAg-positive patients.32 In addition, HBeAg seroconversion has been shown to be associated with the restoration of pDC function in chronic HBV patients underlying IFN-α treatment.33 The fact that immunity to influenza antigen is also abrogated in RXDX-106 in vivo nonresponder

HBeAg-positive chronic HBV patients suggest that HBeAg not only modulates HBc antigen–specific responses but has wide-ranging effects on an individual’s ability to respond to specific immune stimulation. Our observations confirm that HBeAg is a critical factor determining the outcome of immunostimulation which should be taken into consideration when optimizing future approaches to HBV treatment. Moreover, our results demonstrate that other clinical parameters such as viral load, ALT levels, HBs antigen levels, or antiviral treatment are not related to the ability of chronic HBV patients to respond to the pDC stimulation. These observations therefore support 上海皓元 the hypothesis that treatment with nucleoside/nucleotide analogues is not associated with reinforced antiviral T cell responses. In addition to allowing the study of critical parameters of successful immune responses in the context of chronic HBV infection, the pDC cell line used as antigen-presenting cells is an interesting new tool to elicit HBV-specific T cells. It could also be used as a potential cell-based immunotherapeutic strategy in which its potent efficacy and simple design would be ideal. Virus-specific T cell responses are thought to be responsible not only for viral clearance but also for disease pathogenesis during HBV infection.

The 179 PSC patients without cytology or tissue biopsy evidence of cancer were analyzed for the number of deaths, liver transplants, active follow-up, presence of dominant strictures, results of cross-sectional imaging, cause of deaths, and reason for liver transplantation (Table 5). Four patients Autophagy inhibitor died of underlying infection causing septic shock and multiorgan failure. There was no confirmatory evidence of CCA at the time of autopsy in one patient. No autopsy was performed in the other patients. In patients with histological evidence of CCA, orthotopic liver transplantation was performed

in 29 patients, of whom 13 (45%) had evidence of CCA in the liver explants and 16 did not. The explant specimens typically showed extensive fibrosis and necrotic changes caused by intrabiliary radiation seed implantation, particularly around the hilar region, as expected with

the PSC/CCA liver transplant protocol.24 A total of 22 orthotopic liver transplants were performed in patients with positive FISH but no evidence of cancer on histology or cytology. In 13 of these patients, the main indication for the transplant was CCA.24 Residual tumor was found in none of these, but small tumors are often treated with the intense radiation of this protocol. We evaluated the performance of FISH tests in the diagnosis of CCA. The proportions of patients with and SP600125 order without CCA in patients with positive FISH tests are shown in Table 6. The sensitivity, specificity, positive

predictive value, and negative predicative values are depicted in Table 7. We also analyzed the clinical and laboratory characteristics of the patients with positive FISH polysomy who are actively being followed, without any evidence of CCA despite medchemexpress a median follow-up of 11 months (range, 7-34) with the patients who had a definitive diagnosis of CCA. The most important differences between these two groups showed that those who had CCA had significantly higher bilirubin and Mayo risk score, and most had dominant strictures, whereas none of those without CCA had dominant stricture (Table 8). FISH tests have been shown to play a potential role in the diagnosis of indeterminate biliary and pancreatic strictures. The results of our study seem to clarify some of the ambiguity associated with FISH tests in PSC and point to new directions in the evaluation of suspected CCA in PSC patients. As observed in our study and in agreement with other reports, FISH tests increase the sensitivity of cytology at the cost of a decrease in specificity. In agreement with Levy et al.19 regarding the questionable utility of trisomy 7 or 3 positive FISH results in PSC patients, our results indicated that FISH trisomy 7 or 3 has a very low sensitivity and limited specificity for CCA. Our results indicate that most patients with a positive trisomy 7 or 3 do not have CCA at the time of testing, and less than 20% manifest CCA on short-term (1-year) follow-up.

Heinisch, Berit A. Payer, Monika Ferlitsch Background: The hepatic vein pressure gradient (HVPG), an indirect measure of portal pressure, is a prognostic indicator for long term survival in cirrhosis. Bacterial/LPS/DNA translocation leads to activation of toll-like receptors (TLRs) resulting in the secretion

of inflammatory mediators into the circulation. Portal hypertension occurs in the presence of liver injury and inflammation even in the absence A-769662 cost of liver fibrosis in fulminant acute liver failure (Hepatology.10: 482; 1989), indicating that liver injury and inflammation can be sufficient and critical for the development of portal hypertension (with 50% of the patients having portal pressures > 12mmHg). Hypothesis: The rationale for screening inflammatory serum biomarkers of HVPG is based on the fact that portal hypertension is pathogenically related to liver injury and fibrosis, and that in turn these are associated with the activation of inflammatory pathways. Methods: This was a nested cohort study in the setting of Mitomycin C order a randomized clinical trial to assess the development of gastroesophageal varices (GEV) (N Engl J Med.353: 2254; 2005). Patients had

cirrhosis and portal hypertension but did not have GEV. A total of 90 patients that had baseline day-1 sera available were enrolled into the present study. The objective of this study was to determine whether novel inflammatory biomarkers could be used to develop a predictive paradigm for HVPG. Results: The correlations between HVPG and IL-1-beta (P= 0.0052); IL-1R-aipha (P= 0.0085); Fas-R (P= 0.0354) and serum VCAM-1(P= 0.0007) were highly significant. By using multivariate logistic regression analysis and selected parameters (TGF-beta; HSP70; at-risk alcohol use; and Child-Pugh B score) we can exclude HVPG egual or > 12 mmHg with 86 % accuracy (95% CI; 67.78 to 96.16 %) and the sensitivity was 87.01 % (95% CI; 69.68 to 96.34 %).

Therefore, the composite test could identify 86 % of compensated cirrhotic patients with HVPG below 12 mmHg and prevent unnecessary esophagogastroduodenoscopy with its associated morbidity and costs in these patients. As it is the case for estimating HVPG by MCE measuring liver stiffness (LS) with transient elastography (J Hepatol.56: 696; 2012), our diagnostic test was not efficient in predicting HVPG egual or >12 mmHg (PPV: 45.76 %; Specificity: 43.86 %). Conclusion: A blood test for HVPG could be performed virtually in all patients, including those unsuitable for LS measurements (e. g., patients with obesity, ascites, congestive heart failure and extrahepatic cholestasis) (Hepatology.51: 828; 2010). A simple test based on blood biomarkers would be very accessible worldwide. Disclosures: The following people have nothing to disclose: Mario Chojkier, Guadalupe Garcia-Tsao, Roberto J.

9–11 The reasons for this are not entirely clear, but it has been proposed that AFP could be a marker for hepatic progenitor cells or their subtypes.23,24 There is scant literature on low-AFP HCC patients, other than their prognosis being better than high HCC Carfilzomib cell line patients. To evaluate these patients, we interrogated our large HCC database, in which all patients were followed

from diagnosis until death. We found that 413 of our 1000 biopsy-proven, unresectable HCC patients had low AFP levels and these are the subject of this report. Low AFP levels were defined as <130 ng/mL, a commonly used cutoff for suspicion of HCC diagnosis in a patient with cirrhosis.25 We found differences between patients who had low and very low AFP levels (Table 1), according to survival. Even patients with a median AFP level of 40 ng/mL (range 55–215) were still only in the intermediate survival group of 560–800 days. Our previous whole-cohort analyses suggested to us that serum GGTP levels had important prognostic value.12,14 and had one of the highest hazard ratios of all the parameters that were investigated.14 We therefore examined the low AFP patients with respect to typical serum GGTP levels, and found a dichotomy by survival at GGTP levels

of 110 U/100 mL (Fig. 1). These two patient cohorts, Talazoparib supplier with typical GGTP levels above and below 110 U/mL could be further subdivided, based on prevalent tumor size (Figs 1,2). Patients with typical GGTP levels >110 only had large tumor sizes. By contrast, patients with lower typical GGTP levels had a range of tumor sizes, but could be subdivided, based on the presence or absence of PVT. Even patients in this grouping, who had low GGTP and smaller tumors and who had branch PVT, had longer survivals. The levels of GGTP in HCC patients with low AFP, thus appear to have useful prognostic significance. Others have also found a prognostic significance to high GGTP levels in serum or tumor of HCC patients.26–30 There may even be HCC-specific isoenzymes of GGTP.28,29 The biological significance of elevated GGTP for the growth of HCCs is not yet clear. It

is a membrane-bound enzyme that catalyzes the degradation of glutathione and other gamma-glutamyl compounds by hydrolysis of the gamma-glutamyl moiety or by its transfer to an acceptor. GGTP expression is highest in embryo livers medchemexpress and decreases rapidly to its lowest levels after birth, but then increases again in HCC development. The overexpression of GGTP in HCC may thus be related to the several possible mechanisms, including hypomethylation status of CCGG sites of GGT genes.29 Similar to AFP, this suggests that GGTP is a hepatic onco-developmental protein that is re-expressed in liver tumor development, as a consequence of methylation changes in its gene. The relationship between serum AFP and bilirubin levels that is shown in Figure 2a is unexpected. We had previously supposed that liver damage parameters (bilirubin) and tumor growth parameters (AFP) were independent.

Although staining for additional mesenchymal markers learn more would have strengthened the conclusions, the data are nonetheless compelling in demonstrating in the CCl4 model that hepatocytes and their derivatives do not express FSP1, type I collagen, or α-SMA and thus do not undergo EMT. Why do the authors of the two lineage tracing articles on hepatocyte EMT reach such different conclusions? Taura et al. propose that technical limitations associated with β-Gal staining yielded false-positive results in the Zeisberg

study. This hypothesis is supported by the observation that detection of β-Gal expression by immunostaining does not coincide with detection of β-Gal activity by X-gal.12 I would suggest that failure to rigorously

define EMT is another reason for the divergent findings. I-BET-762 supplier Zeisberg et al. define EMT through expression of the controversial and potentially nonspecific marker FSP1 but do not examine collagen synthesis, the feature ultimately most relevant to fibrosis. Taura et al., although focused on collagen synthesis as a primary marker of EMT, also demonstrate that hepatocytes in the fibrotic liver fail to express α-SMA, a finding of key importance given the many demonstrations (including in their study) that α-SMA–positive cells make up a large percentage of fibrogenic cells. Does the work of Taura et al. lay to rest the concept of hepatocyte EMT? The answer is a qualified yes. There are caveats, including the reality that neither the genetic background of the mice nor the injury model (CCl4) accurately model human disease. Regardless, this study effectively refutes the published MCE data that support hepatocyte EMT. Although it is still theoretically possible that hepatocyte EMT occurs in human disease,
s of evidence will be required for this to reemerge as a viable concept. Interestingly,

an exhaustive study has recently been published calling into question EMT in the kidney. Using two different epithelial cell–specific drivers, two different reporters, and two different models of renal fibrosis, Humphreys et al. find no evidence that epithelial cells of the kidney contribute to the myofibroblast population in vivo (or express FSP1).16 Like Taura and colleagues, this group suggests that nonspecific methods to detect the β-Gal reporter could have contributed to discordant findings in the literature. Thus, there is now convincing evidence that neither hepatocyte nor renal epithelial cell EMT occurs in fibrosis. Whether cholangiocyte EMT contributes to fibrosis in the liver is still an open question. Several groups, making use of both animal models and human tissue, have reported that cholangiocytes in fibrotic livers (from bile duct–ligated mice as well as humans with primary biliary cirrhosis, biliary atresia, and several other diseases) coexpress multiple epithelial and mesenchymal markers by immunostaining and are therefore likely to be undergoing EMT.

For ECC, neither HCV nor HBV status was a significant risk factor.53

A large, population-based, case-control study by Shaib et al. of Medicare-enrolled patients compared 625 cases of ICC with 90,834 controls. In a multivariate analysis, HCV was significantly associated with ICC. It was unclear whether patients with HCV also had a recorded diagnostic code for cirrhosis. However, nonspecific cirrhosis was strongly associated with ICC. The prevalence of HBV infection was similar in cases and controls.47 A similar population-based, case-control study by Welzel et al. of Medicare-enrolled patients examined risk factors for both ICC and ECC. There were 549 cases of ECC and 535 cases of ICC, compared with 102,782 controls. Significant risk factors for ICC included Adriamycin cost HCV and nonspecific cirrhosis. Regarding ECC, nonspecific cirrhosis was

also a risk factor, but HCV infection was not significant.28 A large cohort study of U.S. veterans by El-Serag et al. examined the association between HCV and both ICC and ECC in a cohort of 146,394 HCV-infected veterans and 572,293 uninfected controls. The risk for ICC in the HCV-infected cohort, though low at 4 per 100,000 person-years, was more than double that in the controls. The risk of ECC did not differ between the HCV-infected and uninfected veterans.54 The association of these risk factors with CC is not entirely clear, as studies have differing conclusions, and there is a paucity of population-based 上海皓元医药股份有限公司 or prospective cohort studies. In countries such as Korea and Thailand, where both HBV and CC are endemic, data show HBV, but not HCV, as a risk factor for ICC. On the other Belnacasan order hand, countries such as Japan and Western nations, including the United

States, where HCV is more prevalent, were more likely to show an association between HCV and ICC.27, 55 Diabetes and obesity have been examined as possible risk factors for CC. Most studies presented in this section were previously discussed in the section on viral hepatitis and cirrhosis (Table 6). The two SEER-Medicare studies showed a significant positive association between diabetes and CC.28, 47 Another large, population-based, case-control study from the United Kingdom by Grainge et al. found a significant association between diabetes and CC.56 Conversely, a population-based study by Welzel conducted in Denmark did not find a significant association between diabetes and ICC.48 Additionally, one hospital-based, case-control study showed a significant association between diabetes and ICC,27 whereas at least three others failed to show a signification association (Table 6).41, 51, 53 The data on diabetes as a risk factor for CC, especially ICC, are mostly indicative of a modest association, but are inconsistent. Data on obesity are limited (Table 6). Obesity was reported as a significant, but weak, risk factor for CC in two population-based, case-control studies. In the study by Grainge et al.

Of the 145 patients with SCC, 77 had carcinoma in situ (CIS), 31 had tumor invasion of the basement membrane that was confined to the lamina propria mucosae (m2), 24 had tumor invasion of the muscularis mucosae (m3) and 13 had tumor invasion of the upper submucosal layer (sm). The lesions in the 68 patients with tumor invasion of m2 or deeper (early invasive SCC) were

examined for the presence of a low-grade dysplasia component. For patients with multiple lesions, the lesion with the deepest invasion was examined as the main lesion (the lesion with the largest diameter being examined if the depths of invasion were the same). Characteristics of the patients are shown in Table 1. The depth of cancer invasion and tumor morphology were classified according to the criteria proposed by the Paris endoscopic selleck classification of Midostaurin datasheet superficial neoplastic lesions.11 The differences between clinicopathological factors in patients with m2 cancer, m3 cancer and sm cancer were insignificant. EMR was performed with a video-endoscope (Q-230, Q-240; Olympus, Tokyo, Japan). During the period from January

2002 to January 2006, 28 patients were confirmed to have small low-grade dysplasia of the esophagus (< 10 mm in the longest diameter) by endoscopic biopsy during endoscopic screening with iodine staining. The characteristics and natural courses of these 28 lesions were also studied. Characteristics of the patients are shown in Table 2. Morphological features of intraepithelial squamous neoplasia of the esophagus include both architectural and cytological abnormalities.8 The architectural

abnormality is characterized by disorganization of the epithelium and loss of normal cell polarity. Cytologically, the cells exhibit irregular and hyperchromatic nuclei, an increase in nuclear/cytoplasmic ratio and increased mitotic activity. Intraepithelial neoplasia in squamous epithelium of the esophagus is graded as low-grade dysplasia when both architectural and cytological abnormalities are confined to the lower half of the epithelium. The resected specimens were microscopically examined for the presence of a low-grade medchemexpress dysplasia component. If low-grade dysplasia components were observed (≥ 1 high-power field; HPF), the proportions of their areas to overall lesion size were calculated (quantified on longitudinal slices by cutting width). Subsequently, the degrees of architectural and cytological abnormalities of low-grade dysplasia components and those of tumor invasive fronts in the same lesions were studied. The degrees of abnormalities of the 28 small low-grade dysplasia lesions were also studied. They were classified and scored as mild (1 point), moderate (2 points) and severe (3 points). If various degrees of abnormalities were observed, those of the dominant part were recorded. Photomicrographs of the examined specimens are shown in Figures 1–4.