Triple-negative breast carcinomas of low malignant potential: review on diagnostic criteria and differential diagnoses.

Triple-negative breast carcinomas constitute a wide spectrum of lesions, mostly being highly aggressive. Nevertheless, some special histologic subtypes can have low malignant potential. The purpose of the present paper is to review diagnostic criteria and prognostic parameters of breast neoplasms of special histotypes. Specifically, adenoid cystic carcinoma, adenomyoepithelioma, acinic cell carcinoma, mucoepidermoid carcinoma, tall cell carcinoma with reverse polarity, and secretory carcinoma will be discussed. For each tumour, definition and morphological and molecular features, together with prognostic parameters, will be presented. Paradigmatic cases will be illustrated.

Oil Red O Is a Useful Tool to Assess Donor Liver Steatosis on Frozen Sections During Transplantation.

Oil Red O is a useful tool to assess donor liver steatosis on frozen sections during transplantation. Steatosis is a frequent finding in liver evaluation during transplantation, accounting for 9% to 26% of biopsied donor liver. The degree of macrovesicular steatosis is classified as mild, moderate, and severe; the latter is considered an absolute contraindication to liver transplantation because it is associated with poor allograft outcome. Because of the scarcity of organs, there is a debate whether livers with less severe macrovesicular steatosis are still suitable for transplant. Consequently, tools or methods that allow a more accurate intraoperative assessment of steatosis on frozen sections are mandatory. The aim of this study is to improve intraoperative evaluation of steatosis during transplantation using Oil Red O stain on liver biopsies. METHODS: Twenty consecutive liver biopsies of donors were collected during transplantation procedures from September 2017 to February 2018 at the Institute of Pathology of the University and Hospital Trust of Verona, Italy. Each liver biopsy was cut at a different thickness (3, 5, and 8 µm) and stained with both Oil Red O and conventional hematoxylin and eosin for intraoperative consultation. The degree (percentage of hepatocytes involved) of fatty changes was recorded. The results obtained during the intraoperative consultation were finally compared with the formalin-fixed and paraffin-embedded permanent section. RESULTS: Assessment of steatosis on hematoxylin and eosin frozen sections was reported as mild in 17 cases (85%), moderate in 2 cases (10%) and severe in 1 case (5%). Oil Red O frozen sections reported the following results: mild steatosis in 16 cases (80%), moderate in 2 cases (10%), and severe in 2 cases (10%). The percentage of liver steatosis obtained with Oil Red O was consistent in all cases with that of the permanent sections. The staining procedure for Oil Red O required approximately 18 minutes. CONCLUSIONS: Oil Red O special stain is a fast and inexpensive tool to improve the assessment of steatosis on frozen biopsies during liver transplantation.

Fast Chromotrope Aniline Blue Special Stain Is a Useful Tool to Assess Fibrosis on Liver Biopsy During Transplantation.

BACKGROUND: Assessment of potential liver allograft donors with frozen sections has clinical relevant consequences for the transplant recipient. Several clinical risk factors have been identified that increase the risk of transplantation failure and it is critical for the pathologist to become familiar with the histologic criteria for donor liver suitability. In this setting an accurate and reliable assessment of fibrosis is crucial. We sought to report the value of the rapid chromotrope aniline blue stain (CAB) in a transplantation clinical work-flow for scoring liver fibrosis. MATERIALS AND METHODS: Twenty consecutive intraoperative donor liver biopsy specimens were evaluated by a pathologist at the Transplant Pathology Board Room, AOUI Verona, during 24-hour on-call service. The stage of fibrosis was evaluated according to Ishak score ranging from 0 to 6 (absent to cirrhosis) using hematoxylin and eosin stain (H&E) plus rapid CAB special stain. After a 3-week washout period, only the slides stained with H&E were re-assessed for fibrosis stage by the same pathologist blinded to donor patient data. RESULTS: Combination H&E-CAB staging fibrosis score was higher in 20%, lower in 10%, and the same in 70% of biopsy specimens as determined using only H&E stain alone. Rapid CAB stain takes 20 minutes longer than H&E stain alone. CONCLUSIONS: CAB staining may be performed on frozen tissue from liver biopsy during a transplantation process without a significant delay in diagnosis. Combination H&E-CAB staining improves sensibility of interpretation of fibrosis.

Ependymoma with diffuse signet-ring features: report of a case and review of the literature.

Signet-ring cell ependymoma is a rare variant of ependymoma with only seven cases described in literature. Biological behavior and prognosis of this entity are not well-known until now. We present a case of a 49-year-old female with a history of headache and gait instability. Magnetic resonance imaging showed an upper cervical tumor with cystic component and mural nodule. The patient underwent surgery. Microscopically some cells displayed an eccentric nucleus compressed to the periphery by vacuolated cytoplasm. Perivascular pseudorosettes and ependymal rosettes were seen only focally. The cells were positive for glial fibrillary acidic protein and epithelial membrane antigen. The diagnosis was ependymoma with diffuse signet-ring features, grade II according to the World Health Organization. It may be difficult to diagnose this unusual variant of ependymoma especially on small biopsies or frozen sections. A complete examination of the specimen is recommended with immunohistochemical confirmation to rule out potential morphologic mimics, such as metastatic adenocarcinomas and gliomas in the differential diagnosis.

Anti-inflammatory effect of mud-bath applications on adjuvant arthritis in rats.

OBJECTIVE: The real effects of mud-bath applications on the inflammatory process are still not clarified. We studied these effects on rat adjuvant-induced arthritis. METHODS: Arthritis was induced in 30 rats by subplantar injection of Freund's complete adjuvant (FCA) into the right hind paw. Ten days after FCA injection, the rats were randomized in 3 groups of 10 each: the first one was submitted to a cycle of mud-bath applications, the second one was treated with indomethacin, the third one received only saline per os (control group). The paw volume, measured by plethysmometry, and the serum levels of TNFalpha and IL-1beta were considered as evaluation parameters. RESULTS: FCA injection caused a progressive enhancement of paw volume and a rapid increase of TNFalpha and IL-1beta serum levels. After the randomization, mud-bath applications reduced inflammation and at the end of the treatment the paw volume and the TNFa and IL-1beta serum levels were significantly tapered in comparison to the controls (p < 0.01). CONCLUSION: The results of the study suggest an anti-inflammatory effect of mud-bath applications on adjuvant arthritis in rats. These results could explain the beneficial effects of thermal treatments observed in some inflammatory rheumatic diseases.

Norbormide: a calcium entry blocker with selective vasoconstrictor activity in rat peripheral arteries.

Norbormide is a unique vasoactive substance endowed with species- and tissue-specific, endothelium independent, vasoconstrictor activity that is restricted to the peripheral arteries of rat. In rat aorta and in all tested arteries of other species norbormide exhibits vasorelaxant property presumably due to the blockade of calcium channels. A calcium entry blocker effect of norbormide has also been described in isolated, perfused guinea pig hearts. In these preparations norbormide produced coronary vasodilator, as well as negative inotropic and dromotropic effects. In single ventricular myocytes of guinea pigs norbormide reduces L-type calcium current. The mechanism underlying the selective vasoconstrictor effect of norbormide is unknown. In rat caudal artery, a vessel contracted by norbormide, the drug activates phospholipase C (PLC) signal cascade which is the biochemical pathway involved in the contractile effect triggered by most receptor-activating vasoactive agents. Therefore, norbormide-induced contraction of rat peripheral vessels is likely to be due to the activation of a PLC-coupled receptor abundantly or selectively expressed in vascular smooth muscle cells. The identification of this putative receptor could facilitate the development of tissue-selective pharmacological agents.

Signaling mechanisms for the selective vasoconstrictor effect of norbormide on the rat small arteries.

Norbormide (NRB) is a selective vasoconstrictor agent of the rat small vessels. The mechanisms underlying the selective vasoconstrictor effect of NRB are unknown. To investigate whether phospholipase C (PLC) signaling pathway plays a role in NRB-induced vasoconstriction, we performed experiments in NRB-contracted tissues, namely, rat caudal arteries (RCA) and smooth muscle cells derived from rat mesenteric arteries (MVSMCs). An NRB-insensitive vessel, namely rat aorta (RA), served as a control tissue. In RCA and RA we measured either isometric tension or formation of inositol phosphates (IPs), the latter taken as an index of PLC activation. In MVSMCs, we measured intracellular free calcium concentration ([Ca2+]cyt). In the presence of external Ca2+, NRB (2-50 microM) stimulated IPs formation in RCA but not in RA, and increased [Ca2+]cyt in MVSMCs. In the absence of external Ca2+, NRB (50 microM) increased IPs formation in RCA but was unable to increase [Ca2+]cyt in MVSMCs. In RCA, in the presence of external Ca2+, NRB-induced contraction was inhibited by calphostin C (0.2-1 microM), an inhibitor of protein kinase C (PKC), and by SK&F 96365 (30 microM), an inhibitor of the store-operated calcium channels, but was poorly affected by verapamil, an L-type calcium channel blocker. However, verapamil was much more effective when external Ca2+ was substituted by Sr2+. These results suggest that NRB elicits its tissue and species-selective vasoconstrictor effect by stimulating PLC-PKC pathway and increasing Ca2+ influx through both verapamil-sensitive and -insensitive calcium channels. Ca2+ release from sarcoplasmic reticulum seems not involved in NRB vasoconstriction.

Synthesis of flavone-2'-carboxylic acid analogues as potential antitumor agents.

Some flavone-2'-carboxylic acid analogues are described. Direct in vitro toxicity of the synthesized compounds was evaluated towards four tumoral cell lines, and the ability of these compounds to stimulate mouse peritoneal macrophages in culture to become tumoricidal (indirect toxicity) was also studied. Direct cytotoxic activity was very low for all derivatives. However, almost all compounds showed a remarkable increase of indirect cytotoxicity. In particular, compound 3i, which has an F atom in the 7 position of the flavone ring, was able to increase significantly the macrophage's lytic properties, and has been selected for further investigations.

Synthesis, cytotoxicity and SAR of simple geiparvarin analogues.

Some simple geiparvarin analogues, in which the coumarin moiety has been replaced with an X-substituted benzene ring, are described. The compounds were tested on LoVo cells (human colon carcinoma cell line) and some of them show a cytotoxicity comparable with that of the prototype. A QSAR analysis was also attempted, but it did not provide satisfactory results, mainly because of the limited range of variation of the biological activity.

Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes.

1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol. 2. [3H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [3H]-5-HT uptake with apparent Kis of 0.27 +/- 0.04 and 0.76 +/- 0.04 microM, respectively. Morphine essentially failed to inhibit [3H]-5-HT uptake (Ki 0.50 +/- 0.30 M). 3. Methadone, morphine and tramadol were active in the hot plate test with ED50s of 3.5, 4.3 and 31 mg kg-1, respectively. At the highest tested dose (80 mg kg-1) tramadol produced only 77 +/- 5.3% of the maximal possible effect. 4. When [3H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg-1, s.c., equal to the ED50 in the hot plate test) and methadone (35 mg kg-1, s.c., equal to the ED90 in the hot plate test) decreased uptake. 5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg-1 and 15 to 120 mg kg-1, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg-1, s.c.) or tramadol (31 mg kg-1, s.c.) was administered. [3H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%. 6. Rats chronically-treated with methadone showed a significant increase in [3H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [3H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals. 7. These results further support the hypothesis that [3H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [3H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

Modulation of pharmacological profile of diphenylethane (lefetamine-type) derivatives.

With the aim to split the pharmacological properties of lefetamine (CAS 14148-99-3), some structural modifications of this compound have been studied. The basic group of lefetamine has been shifted from the alkyl chain to the vicinal phenyl ring and the N-substitution has been changed. The dimethylaminomethyl derivatives and chiefly the o-morpholinometyhl exhibited a strong anti-visceral chemical antinociception activity stripped of thermal antinociception properties and physical dependence liability. Furthermore, through the introduction of a diethylaminomethyl group in the lefetamine structure some derivatives were selected exhibiting besided a significant increase in the anti-visceral chemical antinociception activity, remarkable local anesthetic properties.

Some geiparvarin bioisosteres and homologues: synthesis and biological evaluation against human colon carcinoma cells (LoVo).

Some geiparvarin bioisosteres and homologues are described. The compounds were tested on LoVo cells (human colon carcinoma cell line). The EC50 values of the synthesized compounds ranged from 35.04 microM (compound 3) to 11.88 microM (compound 4), while the EC50 value of geiparvarin 1 was 13.30 microM.

Calcium-antagonist effects of norbormide on isolated perfused heart and cardiac myocytes of guinea-pig: a comparison with verapamil.

1. Cardiac effects on norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea-pigs. 2. In ventricular myocytes, norbormide 50 microM inhibited the peak calcium current (ICa) by 49.6 +/- 3.9% without altering the shape of the current-voltage relationship; verapamil 1 microM inhibited ICa by 83.2 +/- 3.3%. Neither norbormide nor verapamil affected ICa at the first beat after a 3 min quiescence period; during repeated depolarizations, both drugs cumulatively blocked ICa (use-dependence), with time constants of 23.0 +/- 7.0 s for norbormide and 91.3 +/- 8.4 s for verapamil. 3. In constant-flow perfused hearts electrically driven at 2.5 Hz or 3.3 Hz, both norbormide and verapamil concentration-dependently decreased ventricular contractility (dP/dtmax), atrio-ventricular (AV) conduction velocity and coronary pressure. Intraventricular conduction velocity was slightly decreased by norbormide but not by verapamil. At an equivalent change in AV conduction, norbormide depressed heart contractility less than verapamil. The effects of norbormide on AV conduction, intraventricular conduction, and contractility were frequency-dependent. Furthermore, the curves correlating the mechanical and electrical effects of norbormide at the two frequencies used were apparently coincident, while those of verapamil were clearly separated. 4. In spontaneously beating right atria, norbormide and verapamil decreased the frequency of sinus node (SA) in a concentration-dependent way. At an equivalent effect on the AV conduction, norbormide exerted a greater effect on sinus frequency than verapamil. 5. These results indicate that in guinea-pig heart norbormide has the pharmacological profile of a Ca-antagonist with strong electrophysiological properties. In comparison with verapamil, norbormide is more selective on SA and AV node tissues and exerts a weaker negative inotropic effect on ventricles. In principle, this pattern of effects may be an advantage in treating supraventricular tachyarrhythmias in patients with heart failure. The effect of norbormide on intraventricular conduction may represent an additional antiarrhythmic mechanism.

Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase.

We have tethered epidermal growth factor (EGF) to a solid substrate in a manner permitting the factor to retain its biological activity as assessed by both mitogenic and morphological assays. Mouse EGF was covalently coupled to aminosilane-modified glass via star poly(ethylene oxide) (PEO), which allows the ligand to retain significant mobility and active conformation. Tethered EGF was as effective as soluble EGF in eliciting DNA synthesis and cell rounding responses of primary rat hepatocytes under different surface conditions. In contrast, physically adsorbed EGF at comparable surface concentrations showed no activity. Presentation of growth factors in this manner may help to expedite their clinical use by permitting greater control of temporal and spatial availability in the extracellular environment.

Motility behavior of hepatocytes on extracellular matrix substrata during aggregation.

Aggregation of hepatocytes in culture is an important phenomenon to control in tissue engineering applications. Aggregation generally enhances maintenance of differentiated functions but inhibits cell growth. At present there exists insufficient information for rational design of substrata that control aggregation. Indeed, the cellular mechanism(s) underlying the aggregation process is poorly understood, although cell motility is generally considered to be an essential phenomenon. In this article we provide the first study investigating the relationship between hepatocyte aggregation and motility behavior on various extracellular matrix substrata, including Matrigel, laminin, and fibronectin. We find that the extent of aggregation depends on the concentration of the extracellular matrix proteins, as well as on the type. Furthermore, we find that the extent of aggregation appears to be independent of classical single-cell locomotion. In fact, under conditions giving rise to substantial aggregation, the fraction of cells exhibiting classical locomotion is essentially negligible. Instead, aggregation appears to involve intracellular contacts accomplished via a different form of cell motility: active cell membrane extensions followed by adhesive cell-cell interactions. An implication of these findings is that aggregation may be largely governed by relative strengths of cell-cell versus cell-substratum interactions. These observations could be helpful for improved design of cell transplantation devices and cell culture substrata. (c) 1996 by John Wiley & Sons, Inc.

In vitro cell response to differences in poly-L-lactide crystallinity.

Many different processing techniques are currently being used to produce tissue regeneration devices from polyesters in the polylactide/polyglycolide family. While it is generally well recognized that processing techniques influence bulk mechanical and degradation properties of these materials, the effects on surface properties are relatively less well studied. We thus investigated the effects of processing conditions that are known to change bulk properties, but not composition, on the surface properties of poly-L-lactide (PLLA). Specifically, we investigated the role of bulk crystallinity of PLLA substrates on several physiochemical aspects of the surface and on the attachment, morphology, and differentiated function of cultured primary hepatocytes and growth of 3T3 fibroblasts. We fabricated smooth, clear PLLA films of 13-37% crystallinity. Glancing angle X-ray diffraction indicated that low crystallinity films lacked order in the first 50 A of the surface while relatively high crystallinity films had detectable order in this range. In other aspects, the surfaces of all PLLA substrates appeared identical with XPS, SEM, and advancing contact angle analysis, but contact angle hysteresis was slightly greater for more crystalline films. Although the physicochemical properties of the surfaces appeared almost identical, we observed differences in cell behavior on less crystalline versus more crystalline films. Hepatocytes formed spheroids on all PLLA substrates, but spheroid formation was faster (24-48 H) on crystalline substrates. quantitative image analysis was used to assess the average cell area as a function of time in culture, and our data confirm previous reports that retention of differentiated function is inversely related to cell spreading where function was assessed by P-450 enzyme activity. In addition, the growth rate of 3T3 fibroblasts was lower on crystalline substrates than on amorphous substrates. An important conclusion from this work is that processing techniques that lead to seemingly inconsequential changes in bulk and surface properties of these polymers may influence biological response.

Synthesis and antitumor activity of some analogues of flavone acetic acid.

Some coumarin-, flavonol- and flavanon-acetic acids are described. The cytotoxicity of the synthesized compounds was determined on a human colon carcinoma cell line (LoVo) through evaluation of neutral red uptake, performed by the Riddel method. All tested derivatives were able to induce a statistically significant reduction of lysosomal neutral red uptake at 5 x 10(-5) M concentration. Some compounds were more active than the reference compound flavon-8-acetic acid.

Hepatocyte culture on carbohydrate-modified star polyethylene oxide hydrogels.

We describe the synthesis and in vitro biological characterization of a new class of carbohydrate-modified hydrogels based on radiation-cross-linked star polyethylene oxide (PEO). Hydrogels were synthesized from either of two types of PEO star molecules in order to vary the terminal hydroxyl content of the gels while keeping other gel properties such as molecular weight between cross-links and water content constant. The resulting gels were covalently modified with monosaccharide ligands and the behaviour of primary rat hepatocytes on the modified gels was evaluated under culture conditions. Hepatocytes exhibited a sugar-specific adhesion to the modified gels, adhering to gels bearing galactose but not glucose. Cell spreading was observed on both types of galactose-modified PEO star gels; moreover, the gels supported long-term (6 d) culture and differentiated function of primary hepatocytes. Further, on comparing the cell spreading behaviour observed on the PEO star gels with that reported previously for galactose-modified polyacrylamide, we find that our gels elicit spreading at ligand concentrations lower by an order of magnitude. A simple mechanistic analysis indicates that this enhanced ability of PEO star gels to support spreading of primary hepatocytes on low concentrations of immobilized galactose derives from freedom of the immobilized ligands to come within sufficiently close proximity to mimic a high-affinity branched oligosaccharide.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1. The effects of norbormide on the contractility of endothelium-deprived rat, guinea-pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2. In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5-50 microM) induced a concentration-dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10-800 microM) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3. In resting rat and guinea-pig aortae, guinea-pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10-100 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) was ineffective in phenylephrine-contracted guinea-pig and rat aorta. 4. In A7r5 cells, a cell line from rat aorta, norbormide prevented high K(+)- but not 5-hydroxytryptamine-induced intracellular calcium transients. 5. These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.