A case of ovarian small cell carcinoma of the pulmonary type that was observed as it developed.

INTRODUCTION: In the case reported here, the authors observed ovarian small cell carcinoma of the pulmonary type as it developed. CASE: The patient was a 48-year-old woman who underwent a hysterectomy for CIN3 in 2007. A year later, the woman underwent screening for ovarian cancer. A gradually growing ovarian mass was noted. This mass was found to be a mixed tumor. This mixed tumor grew to 36 mm in size, and six months later it had enlarged to 119 mm. After surgery, the tumor was pathologically diagnosed as an ovarian small cell carcinoma of the pulmonary type with a neuroendocrine nature that was positive for CD56 and synaptophysin. Postoperatively, the patient received six courses of combined therapy with irinotecan and cisplatin (CPT-P therapy), and the patient has survived disease- free for over two years. CONCLUSION: Findings suggested that ovarian small cell carcinoma of the pulmonary type is a type I ovarian malignancy that develops through an adenoma-carcinoma sequence.

Time-dependent gene expression and immunohistochemical analysis of the injured anterior cruciate ligament.

OBJECTIVES: This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation. METHODS: Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-ß1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3). RESULTS: Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-ß1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III. CONCLUSIONS: Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs' expressions.

Locally administered low-dose alendronate increases bone mineral density during distraction osteogenesis in a rabbit model.

We investigated the effect of locally administered bisphosphonate on distraction osteogenesis in a rabbit model and evaluated its systemic effect. An osteotomy on the right tibia followed by distraction for four weeks was performed on 47 immature rabbits. They were divided into seven equal groups, with each group receiving a different treatment regime. Saline and three types of dosage of alendronate (low, 0.75 microg/kg; mid, 7.5 microg/kg and high 75 microg/kg) were given by systemic injection in four groups, and saline and two dosages (low and mild) were delivered by local injection to the distraction gap in the remaining three groups. The injections were performed five times weekly during the period of distraction. After nine weeks the animals were killed and image analysis and mechanical testing were performed on the distracted right tibiae and the left tibiae which served as a control group. The local low-dose alendronate group showed a mean increase in bone mineral density of 124.3 mg/cm(3) over the local saline group (analysis of variance, p < 0.05) without any adverse effect on the left control tibiae. The findings indicate that the administration of local low-dose alendronate could be an effective pharmacological means of improving bone formation in distraction osteogenesis.

High IL-6 synthesis in cultured fibroblasts isolated from radicular cysts.

OBJECTIVE: Inflammatory cytokines have been reported to be related with inflammation and expansion of jaw cysts. In this study, to examine the relationship between radicular cysts and inflammatory cytokines, it was found that there was notable unique evidence on cytokine synthesis from fibroblasts isolated from radicular cysts. METHODS: The expression of such cytokines, namely, interleukin-1beta, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and granulocyte-macrophage colony-stimulating (GM-CSF) mRNA, in nine radicular cysts was examined and compared with that detected in six specimens of healthy gingival mucosa. Furthermore, separating all fibroblasts from their respective radicular cysts, healthy gingival mucosa, and healthy periodontal ligaments, these fibroblast groups were cultured without stimulators and a supernatant for each was obtained to analyse IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma by ELISA. RESULTS: Differences between radicular cysts and healthy gingival mucosa were not clearly shown by the expression of cytokine mRNA. Analysing inflammatory cytokine synthesis in fibroblast groups from these three kinds of tissues, surprisingly, the levels of IL-6 mRNA and protein were recognised to be higher in fibroblasts of radicular cysts than in those of control tissues by ELISA and a real-time RT-PCR. Significant differences in the cultured supernatants of these fibroblast groups were not recognised in the release of IL-1beta, IL-8, TNF-alpha, and IFN-gamma by ELISA. CONCLUSIONS: From these results, it was suggested that fibroblasts inducing IL-6 production might play important roles in the expansion of radicular cysts. It is considered that fibroblasts around radicular cysts may lead to high IL-6 synthesis over time in chronic inflammation.

Uni-axial cyclic stretch induces Cbfa1 expression in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin beta1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin beta1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.

Validity of simple mucosal biopsy criteria combined with endoscopy predicting patients with ulcerative colitis ultimately requiring surgery: a multicenter study.

BACKGROUND: Recent mucosal biopsy criteria combined with endoscopy effectively differentiate patients with ulcerative colitis ultimately requiring surgery (UC-S) from those receiving medication alone (UC-M). However, the criteria were inconvenient in practical use because of the need for complicated calculations, and the validity has not been verified in other institutes where the indications for surgery may differ. The aims of this multicenter study were to propose simple criteria in which calculation can be performed by mental arithmetic and to measure their validity. METHODS: Based on the above original criteria, we constructed simple criteria in which coefficients and constant were simplified to integral numbers. The criteria consisted of the diagnostic categories, highest-risk, high-risk, unpredictable, low-risk, and lowest-risk of surgery. The validity of these proposed criteria was evaluated in 121 patients with UC-S and 186 with UC-M from 11 institutes. RESULTS: The categories of high-risk and low-risk had sensitivities exceeding 86.0% and specificities exceeding 95.2%, and the validities were maintained at high levels in most individual institutes. There was little difference in validity between the proposed and original criteria when testing using the same patients. CONCLUSIONS: Despite simplified coefficients and constant, the proposed criteria reliably predicted the eventual clinical outcome of patients with ulcerative colitis and would be helpful in determining the necessity of surgery.

Regeneration of transformed verbena (Verbena x hybrida) by Agrobacterium tumefaciens.

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.

Biopsy pathology predicts patients with ulcerative colitis subsequently requiring surgery.

BACKGROUND: The clinical course of patients with ulcerative colitis (UC) is unpredictable, and 17%-38% ultimately require surgery. We hypothesized that mucosal histology may differ between patients requiring surgery and those receiving medication alone. The aim of this study was to elucidate comprehensive criteria consisting of specific histologic features enabling the prediction of failure to medical treatment. METHODS: We studied colorectal biopsy specimens from 67 patients ultimately requiring surgery (UC-S) and 90 receiving medication alone for more than 3 years (UC-M), and conducted multiple logistic regression analysis on 70 histologic features together with endoscopic disease extent and patient age. The analysis constructed an equation finding probability of UC-S (P(UC-S)). Based on a receiver-operating characteristic curve, we selected four cut-off values of P(UC-S), and determined criteria of five categories: highest-risk, higher-risk, unpredictable, lower-risk and lowest-risk of surgery. Sensitivity and specificity of criteria were evaluated in a 2 x 5 table. RESULTS: Statistically significant features predicting UC-S were deep ulceration (X1), frequent crypt abscesses (X2), focal and segmental mononuclear cell infiltration (X3 and X4), paucity of eosinophils (X5: eosinophil infiltration) and wide extent of the disease (X6). The regression equation was as follows: logitP(UC-S) = -16.26 + 3.20X1 + 4.83X2 + 11.65X3 + 5. 10X4 - 5.59X5 + 5.53X6. Higher-risk and lower-risk showed sensitivity exceeding 91.0% and specificity exceeding 98.5% in predicting the outcome. CONCLUSIONS: Our criteria incorporating specific histologic features and endoscopic disease extent reliably predict eventual clinical outcome, and are expected to prove useful in determining the necessity of surgery.

Malonyl-CoA:anthocyanin 5-O-glucoside-6"'-O-malonyltransferase from scarlet sage (Salvia splendens) flowers. Enzyme purification, gene cloning, expression, and characterization.

The orange to blue coloration of flowers in nature is, in most cases, provided by anthocyanins, a class of plant flavonoids, many of which are modified by malonyl group(s). However, the identity of the enzyme catalyzing the malonylation reaction remains to be established. Here, we describe for the first time the purification, characterization, and cDNA cloning of an anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (termed Ss5MaT1) was a monomeric 50-kDa protein catalyzing the regiospecific transfer of the malonyl group from malonyl-CoA to the 6"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Ss5MaT1 showed a k(cat) value of 7.8 s(-1) at 30 degrees C and pH 7.0 for the malonylation of bisdemalonylsalvianin (pelargonidin 3-(6"-O-caffeyl-beta-glucopyranoside)-5-beta-glucopyranoside) and K(m) values of 101 microm and 57 microm for bisdemalonylsalvianin and malonyl-CoA, respectively. p-Coumaric acid, which mimics an aromatic acyl group linked to the 3-glucosidic moiety of an anthocyanin substrate, was a competitive inhibitor with respect to the substrate. This strongly suggests that the presence of an aromatic acyl group at the 3-glucosidic moiety of anthocyanin is important for substrate recognition by the enzyme. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA encoding Ss5MaT1. Ss5MaT1 consisted of 462 amino acids and shared motifs that are commonly found among members of a versatile plant acyltransferase family, which was recently shown to include numerous homologs of unknown biochemical functions. Northern blot analysis revealed that the transcripts of Ss5MaT1 were detected in petals, sepals, bracts, and red stems, in accordance with the pigment accumulation patterns. Phylogenetic analysis suggests that the aliphatic and aromatic acylations of anthocyanins are generally catalyzed by subfamily members of the plant acyltransferase family.

Non-neoplastic glandular structures in a benign peripheral nerve sheath tumor.

It is a recognized fact that glandular structures sometimes occur in peripheral nerve sheath tumors (PNST). Reports indicate that epithelial potential could be expressed in malignant PNST, while the glands in most benign PNST could be trapped skin adnexa. We present a case of spindle cell tumor with glandular structures. The patient was a 55-year-old man who had a subcutaneous tumor excised. The spindle cell tumor had histological characteristics of neurofibroma. The glandular structures had a pattern of immunohistochemical staining that was similar to that of the secretory coils and excretory ducts of normal eccrine glands. Therefore, the glands were thought to be naturally existing eccrine glands. The glands were completely enclosed within the tumor and not connected to one another; a cluster formation was not observed. The upper portion of tumor included some glands of skin adnexa. The spindle cell tumor may have originated from the nerves distributed around the skin adnexa, and grown to the subcutaneous tissue. The glands may have been left behind rather than have been trapped in the tumor.

Graft incorporation within the tibial bone tunnel after anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft.

We described histologic changes in patellar tendon autografts that occur over time within the tibial tunnel in specimens harvested from patients undergoing revision anterior cruciate ligament reconstruction. Ten patients, averaging 21.2 years of age, were divided into two groups based on the time period between their original and revision surgery: early revision (less than 1 year, four patients) and late revision (more than 1 year, six patients). Among the early revision group, the tendon within the tunnel showed increased cellularity and random collagen bundles. A specimen from the shortest early revision case revealed a normal original bone-tendon junction, whereas others showed an obscured structure. Between the tendon and the tunnel wall, granulation tissue was seen and the bone-tendon junction was still immature. In the late revision group, the tendon appeared similar to normal ligament. The original bone-tendon junction was not seen, and the tendon continued completely to the tunnel wall with Sharpey-like fibers. Observations in the early revision group suggest that tendon remodeling and bone-tendon integration continue for at least several months after transplantation. The original bone-tendon junction appears to have shifted to the proximal patellar tendon-tunnel wall junction with time. These findings are in agreement with prior animal studies.

Specificity analysis and mechanism of aurone synthesis catalyzed by aureusidin synthase, a polyphenol oxidase homolog responsible for flower coloration.

Aureusidin synthase, which plays a key role in the yellow coloration of snapdragon flowers, is a homolog of plant polyphenol oxidase (PPO). The enzyme specifically acted on chalcones with a 4-monohydroxy or 3,4-dihydroxy B-ring to produce aurones, for whose production the oxidative cyclization of chalcones must be preceded by 3-oxygenation. However, it exhibited virtually no PPO activity toward non-chalcone phenolics. The enzyme was competitively inhibited by phenylthiourea, a specific PPO inhibitor. These results led us to propose a mechanism of aurone synthesis by aureusidin synthase on the basis of known PPO-catalyzed reactions and conclude that the enzyme is a chalcone-specific PPO specialized for aurone biosynthesis.

A murine osteosarcoma cell line with a potential to develop ossification upon transplantation.

An osteosarcoma cell line has been established from a soft tissue tumor that occurred spontaneously in a BALB / c mouse. This cell line showed ossification when transplanted into syngeneic mice. To examine the mechanism of bone formation, the expression of mRNAs for osteoblastic and chondroblastic markers and factors associated with ossification has been investigated. In culture, the cells exhibited a spindle shape in the growth phase, but had a polygonal shape in the stationary phase. Reverse transcription-polymerase chain reaction analysis showed that the cells expressed mRNAs for pro-alpha1(I) chain of type I collagen, alkaline phosphatase, osteopontin, osteocalcin, and core binding factor alpha1, suggesting differentiation into the stage of osteoblasts during the stationary phase. After transplantation, histological examination revealed small foci of pale blue material and basophilic networks that were scattered in the tumor tissues at one week. The former stained positive with alcian blue, suggesting a chondroid matrix. Pro-alpha1(II) chain of type II collagen mRNA was expressed at one week. A large part of tumors at two and three weeks consisted of basophilic networks, which stained positive via von Kossa's method, indicating a calcified woven bone. In situ hybridization analysis showed strong expression of osteopontin and osteocalcin mRNAs in tumor cells surrounding the bone matrix. Bone morphogenetic protein-6 and -7 mRNAs were detected in transplanted tumors, but not in cultured cells. These results suggest that the cell line has the properties of an osteoblastic lineage when cultured in vitro and has an ossifying ability through endochondral bone formation processes when transplanted in vivo.

Colonic intra-epithelial carcinoma occurring in a hyperplastic polyp via a serrated adenoma.

We present a case of intra-epithelial carcinoma occurring in a serrated adenoma of the colon. The pedunculated polyp, which measured 12 x 10 x 6 mm, was endoscopically removed from the ascending colon of a 78-year-old woman. Histologically, the polyp mainly consisted of serrated adenomatous glands, and had foci of intra-epithelial carcinoma at the top. Hyperplastic (metaplastic) areas were also present in both borders between the serrated adenomatous area and the surrounding normal mucosa. A sequential increase in the degree of dysplasia, and in the number of nuclei positively reactive for Ki-67 and p53 was evident from the hyperplastic areas toward the foci of carcinoma. The polyp described here may represent a carcinogenic potential of hyperplastic polyp via serrated adenoma.

Genes encoding the vacuolar Na+/H+ exchanger and flower coloration.

Vacuolar pH plays an important role in flower coloration: an increase in the vacuolar pH causes blueing of flower color. In the Japanese morning glory (Ipomoea nil or Pharbitis nil), a shift from reddish-purple buds to blue open flowers correlates with an increase in the vacuolar pH. We describe details of the characterization of a mutant that carries a recessive mutation in the Purple (Pr) gene encoding a vacuolar Na+/H+ exchanger termed InNHX1. The genome of I. nil carries one copy of the Pr (or InNHX1) gene and its pseudogene, and it showed functional complementation to the yeast nhx1 mutation. The mutant of I. nil, called purple (pr), showed a partial increase in the vacuolar pH during flower-opening and its reddish-purple buds change into purple open flowers. The vacuolar pH in the purple open flowers of the mutant was significantly lower than that in the blue open flowers. The InNHX1 gene is most abundantly expressed in the petals at around 12 h before flower-opening, accompanying the increase in the vacuolar pH for the blue flower coloration. No such massive expression was observed in the petunia flowers. Since the NHX1 genes that promote the transport of Na+ into the vacuoles have been regarded to be involved in salt tolerance by accumulating Na+ in the vacuoles, we can add a new biological role for blue flower coloration in the Japanese morning glory by the vacuolar alkalization.

Sesquiterpenes and monoterpenes from the bark of Inula macrophylla.

Eleven new sesquiterpenes (1--11) and two thymol derivatives (12, 13), along with 12 known sesquiterpenes and monoterpenes, were isolated from the bark of Inula macrophylla. Their structures were determined on the basis of spectral evidence (especially by HREIMS and 2D NMR) as well as chemical transformations. The structure of macrophyllic acid A (1) was confirmed by X-ray analysis, and the absolute configuration of 1 was determined on the basis of the appropriate chemical conversions and the application of a modified Mosher's method.

Enzymatic formation of aurones in the extracts of yellow snapdragon flowers.

The yellow coloration of snapdragon (Antirrhinum majus) flowers is mainly provided by the 6-glucosides of aureusidin and bracteatin. However, the biochemical mechanism of aurone biosynthesis is not well understood. In this study, we have identified aurone-biosynthesizing activity in the extracts of yellow snapdragon flowers. Incubation of 2',4',6',4-tetrahydroxychalcone (THC) with an enzyme preparation in the presence of H(2)O(2) caused the enzymatic formation of a single product, aureusidin, without the formation of a previously proposed 2-(alpha-hydroxybenzyl)coumaranone intermediate. The formation of aureusidin from THC was specifically observed with yellow flowers as well as aurone-accumulating flowers of other colors. The pH optimum for the enzymatic formation of aureusidin was around 5.4. Stoichiometric studies showed that one mole of aureusidin formation was accompanied by the consumption of one mole of oxygen with no detectable consumption of H(2)O(2), which may work as an enzyme activator. The oxidative formation of aureusidin from THC could be explained in terms of the action of a single enzyme, an internal monooxygenase catalyzing the 3-hydroxylation and oxidative cyclization of THC. Incubation of 2',4',6',3,4-pentahydroxychalcone (PHC) with an enzyme yielded both aureusidin and bracteatin at an approximate molar ratio of 6:1. In this case, H(2)O(2) was not required for enzyme activity but rather inhibited the reaction. The 4'-glucosides of THC and PHC could also act as substrates for the formation of the 6-glucosides of aurones. These results suggest that aureusidin can be produced from either THC or PHC, whereas bracteatin is not produced through the 5'-hydroxylation of aureusidin but arise solely from PHC.

Spatial distribution and histogenesis of colorectal Paneth cell metaplasia in idiopathic inflammatory bowel disease.

BACKGROUND AND AIM: Colorectal Paneth cell metaplasia (PCM) is known to be a sign of idiopathic inflammatory bowel disease (IBD), although its distribution and histogenesis are not fully understood. Objectives of this research were to investigate the spatial distribution of PCM in IBD and other forms of colitis (non-IBD), and to find stimuli causing PCM. METHODS: We studied multiple biopsy specimens from 181 patients with ulcerative colitis (UC), 159 with Crohn's disease (CD), 448 with non-IBD, and 78 normal controls. Paneth cell metaplasia frequency, at each colorectal site, was evaluated to find possible differences among diseases, phases of activity, and extents of disease. RESULTS: In non-IBD and controls, PCM was rarely (0-1.9%) seen at distal sites, but frequently (up to 48.7%) found at the ascending colon and cecum (P < 0.001). Paneth cell metaplasia frequency was significantly higher in IBD than in non-IBD patients and controls at distal sites (P < 0.001), but did not differ significantly between UC and CD, or among active, resolving, and quiescent phases. In UC, proctitis and left-sided colitis rarely displayed PCM at unaffected sites. Multiple logistic regression analysis revealed that PCM was positively associated with crypt distortion and mononuclear cell infiltration (P < 0.005), but negatively or not significantly associated with crypt atrophy, mucin depletion, acute inflammation, or phase of activity. CONCLUSIONS: Paneth cell metaplasia is a non-specific phenomenon in the proximal colon, but distal PCM, which occurs exclusively in affected mucosa, is a useful marker indicating IBD, even in the inactive phase. Regression analysis suggests that repair and regeneration may be the most potent stimuli causing PCM.

Observer variation of diagnoses based on simple biopsy criteria differentiating among Crohn's disease, ulcerative colitis, and other forms of colitis.

BACKGROUND AND AIM: Simple mucosal biopsy criteria proposed by authors reliably differentiate idiopathic inflammatory bowel disease (IBD) from other forms of colitis (non-IBD) and Crohn's disease involving the colon (CD) from ulcerative colitis (UC). The aim of this study is to investigate the reproducibility of these criteria. METHODS: Three established pathologists and two medical students blindly examined 20 sets of multiple biopsy slides from patients with CD, 20 from those with UC, and 20 from those with non-IBD. The students had been given instructions on histological definitions using another 15 sets previously. Each observer evaluated 10 histological items required in the criteria and determined categorical diagnoses such as definite IBD and probable UC. Interobserver agreement for the individual histological items was measured by using kappa analysis and Pearson's correlation, while it was measured for categorical diagnoses with the use of Spearman's rank correlation. RESULTS: All of the individual histological items expressed excellent or fair-to-good agreement among the five observers, although two items associated with the criteria for CD and UC had poor agreements among the students. With regard to categorical diagnoses based on the criteria for IBD and non-IBD, and those for CD and UC, coefficients for Spearman's rank correlation exceeded 0.92 and 0.86 among the pathologists, and 0.76 and 0.74 among the students, respectively; all of the coefficients were statistically significant (P < 0.05). CONCLUSIONS: The simple criteria were sufficiently reproducible and would help most pathologists to make an automated and objective diagnosis.