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.

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.

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.

Multiple forms with glucose 6-phosphate dehydrogenase activity in Musca domestica L. as revealed by electrophoresis on cellulose acetate gel.

Single newly emerged males of Musca domestica, WHO strain, usually show five electrophoretic bands of glucose 6-phosphate dehydrogenase (G6PD) activity. Of these five molecular forms, designated with Roman numerals in order from the origin, we have considered the first three: these have been characterized with respect to their substrate and coenzyme specificity and to their sensitivity to some sulfhydryl inhibitors. The data show band III to be G6P specific, nicotinamide adenine dinucleotide phosphate dependent and to be a type I enzyme according to Kamada and Hori's classification. Bands I and II, on the other hand, show wide substrate specificity and low sensitivity to the sulfhydryl inhibitors assayed. In addition, in the absence of an exogenous substrate and in the presence of nicotinamide adenine dinucleotide as a coenzyme, fairly weak bands, which can be ascribed to the so called "nothing dehydrogenase" effect, are seen in the position I and II. Nevertheless, the data reported do not allow a clear definition of the enzymatic type corresponding to bands I and II of G6PD activity.

The in vitro conversion of a specific molecular form of glucose 6-phosphate dehydrogenase from Musca domestica L.

A nicotinamide adenine dinucleotide phosphate dependent glucose 6-phosphate dehydrogenase (G6PD), belonging to type I of Kamada and Hori's classification, is present on the zymograms of newly emerged males of Musca domestica. It is capable of undergoing tryptic degradation and being thus transformed into a different active enzymatic form, with some of its catalytic properties unchanged, but with different electrophoretic mobility. We show in this paper that this specific G6PD form of gut origin in M. domestica is not a tissue-specific enzyme, but rather a product of hydrolytic degradation by gut proteinases which act during the process of homogenization. Besides, the G6PD of type I in the housefly is shown to be sensitive to the "storage effect" and to protection by mercaptoethanol, contrary to its hydrolytic gut form which is not sensitive to these processes. In this connection, we discuss the possible reasons for these differences in behavior.

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.

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.

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.

Angiopolarity of cell carriers: directional angiogenesis in resorbable liver cell transplantation devices.

The purpose of this study was to obtain directional angiogenesis of small blood vessels and capillaries to an implant made from a resorbable polymer for hepatocyte transplantation. It was intended to mimic the native acinar structure of the liver in order to facilitate replication of the cells and organ growth. The implant device structure was designed for injection to minimize surgical trauma. Hollow microspheres with an open porous wall structure and one large central opening were made from poly(d,l-lactic-co-glycolic acid) (85:15 lactic:glycolic). This polymeric scaffold was seeded with hepatocytes and implanted into the abdominal wall muscle of syngeneic Fisher rats. Specimens explanted up to 56 days p.o. showed hepatocyte survival and the development of a directional blood supply. This phenomenon is coined "angiopolarity". The study should help in addressing the issue as to whether avascular cell implants with post-transplantation organ growth should be attempted. Processing options in applying heat to the polymer solution allow manufacturing of larger microspheres with different diameters of central openings. This would allow the use of the scaffold for other cell transplantations than hepatocytes.

Future directions in biomaterials.

Biomaterials have made a great impact on medicine. However, numerous challenges remain. This paper discusses three representative areas involving important medical problems. First, drug delivery systems; major considerations include drug-polymer interactions, drug transformation, diffusion properties of drugs and, if degradation occurs, of polymer degradation products through polymer matrices developing a more complete understanding of matrix degradation in the case of erodible polymers and developing new engineered polymers designed for specific purposes such as vaccination or pulsatile release. Second, cell-polymer interactions, including the fate of inert polymers, the use of polymers as templates for tissue regeneration and the study of polymers which aid cell transplantation. Third, orthopaedic biomaterials, including basic research in the behaviour of chondrocytes, osteocytes and connective tissue-free interfaces and applied research involving computer-aided design of biomaterials and the creation of orthopaedic biomaterials.

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.

Corneal epithelial wound healing on bilayer composite hydrogels.

We demonstrate that a bilayer composite hydrogel composed of corneal stroma crosslinked to poly(ethylene oxide) provides a substrate suitable for wound healing behavior of corneal epithelial cells and for formation and maintenance of a stable multilayered epithelium. Potential diffusion-limitation of nutrients or regulatory molecules across the hydrogel was investigated experimentally with a new in vitro ocular assay, using epithelial cell migration as an index of molecular diffusion limitations. Corneal epithelial cells explanted on the composite hydrogel in vitro exhibited morphology similar to those in vivo, and migrated effectively over the stromal surface. Importantly, our system yielded multilayered epithelium like that found in normal corneal tissue under conditions that closely simulate the in vivo physiologic arrangement. In addition, our results indicate that molecules of substantially greater molecular weight than glucose appear to control the cell migration rate. Thus, engineering design of this composite hydrogel system may allow it to be useful in corneal wound healing applications.

The mesentery as a laminated vascular bed for hepatocyte transplantation.

The small bowel mesentery provides a unique structure of a large vascularized surface area to support hepatocyte transplantation. Cell-seeded polymeric matrices can be juxtaposed in a relatively atraumatic manner between leaves of mesentery such that adequate exchange of nutrients and diffusion of gases can proceed in the interim while neovascularization occurs. Hepatocytes obtained from (RHA) Wistar rats by collagenase perfusion were seeded onto non-woven filamentous sheets of polyglycolic acid 1 x 3 cm in size and 2 mm thickness to a density of 500,000 cells/cm2. Twenty-six recipient Gunn rats (UDP-glucuronyl transferase deficient) underwent laparotomy. Hepatocyte-ladened polymer sheets were placed between leaves of mesentery. Eight sheets were placed per animal and the leaves were approximated, creating a functional implant 1 x 3 x 2 cm. Biopsies between 5-99 days after implantation revealed neovascularization, moderate inflammatory reaction and the presence of viable hepatocytes in 96% (25/26). Immunoperoxidase studies using anti-albumin antibody substantiated hepatocyte specific function in implants. HPLC profiles of bile from Gunn rats transplanted with hepatocytes from congeneic (RHA) rats demonstrated the presence of bilirubin conjugates. There were no conjugation fractions seen in control gunn rats without hepatocyte transplantation. Although total serum bilirubin did not significantly decrease, conjugated bilirubin was identified in 46% (12/26) animals after transplantation with congeneic hepatocytes. We conclude that the mesentery of the small bowel provides a large vascularized surface for cell transplantation. Large numbers of metabolically active hepatocytes can engraft, vascularize, and show function. The mesentery may be a potential bed for clinical hepatocyte transplantation.

Are calcitonins analgesic and/or hyperalgesic?

The acetylcholine writhing test and the hot plate test were used in mice to evaluate peripheral and central analgesia after porcine, salmon and human calcitonin administrations. ICV and IV injection of calcitonins caused a peripheral analgesia that persisted for at least 2 hr. SC injection of calcitonins did not produce peripheral analgesia. However, when administered by IP route, an increasing peripheral analgesia was observed. Although it had a slow onset, it was as powerful as after ICV or IV administration. Employing the hot plate test to determine the entity of a central analgesic response, the IP administration of calcitonin surprisingly revealed a hyperalgesic effect. It started soon after the injection, reaching its maximum after about 2 hr. At this point the hyperalgesic effects were fully antagonized by EDTA.2 Na ICV, or by Ca++ICV. Moreover only the latter produced a strong and long acting analgesia. Applying a central or peripheral test, calcium seems to play different roles on the algesia variations induced by calcitonins.

Antinociceptive effect of some carboxypeptidase A inhibitors in comparison with D-phenylalanine.

It had previously been shown that D-phenylalanine and hydrocinnamic acid, two in vitro inhibitors of carboxypeptidase A, possess an analgesic action when injected i.p. in mice. We have studied the in vivo effects of indole-3-acetic acid, another carboxypeptidase A inhibitor, and of the following analogs of D-phenylalanine substituted in position 4: D-tyrosine, p-fluoro-D-phenylalanine and trifluoroacetyl-p-fluoro-D-phenylalanine. Whereas indole-3-acetic acid caused a higher and shorter analgesia in comparison with D-phenylalanine, p-fluoro-D-phenylalanine and its N-trifluoroacetyl derivative yielded both a greater and a much longer lasting analgesic effect. Since the latter compound showed only slight inhibitory activity on carboxypeptidase A in vitro, we suggest that inhibition of this enzyme and analgesia might not be directly correlated.

Influence of glycine on morphine-induced antinociception in mice.

The effects of glycine on morphine-induced antinociception were investigated in mice, using a cutaneous thermal test (hot-plate), a visceral chemical test (acetylcholine writhing test), and a locomotor activity test. When glycine (200 mg/kg p.o.) and morphine (5 mg/kg s.c.) were given together during the first 30 min, glycine first antagonized the morphine-induced antinociception then this was followed by a synergistic effect. The two-phase influence of glycine on morphine-induced antinociception may be due to the interaction of glycine with different receptors.

Analgesic properties of N-terminal substituted phenylalanyl-alanine.

Phenylalanylalanine, an in vitro inhibitor of enkephalinase, and some of its N alpha-derivatives are shown to possess an analgesic action when injected i.p. and i.c.v. into mice in the presence or absence of Leu5-enkephalin. In the second case a synergistic response is observed. The intensity of the analgesic response depends markedly on the nature of the N-terminal substituent which affects the hydrophobic character of the resulting dipeptide, its subsequent transport and probably its rate of biotransformation by cleaving enzymes.

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.