[Indications for p16/Ki-67 in cervical cytology]. / p16-/Ki-67 in der Zervix-Zytologie: Indikationen.

BACKGROUND: The p16/Ki-67 immunocytochemistry has been used for more than five years in cervical cytology to detect transforming HPV infections. MATERIAL AND METHODS: Based on findings in the study presented here and data in the literature, practically relevant indications for this test are to be determined. The clinical course of 1109 patients with varying primary cytology results and simultaneous immunocytochemistry is analyzed. Short-term observations considering currently present lesions and long-term follow-up evaluation aimed at the prognostic evaluation are studied separately and compared with the literature. RESULTS: The p16/Ki-67 immunocytochemistry effectiveness is described by the relative risk (RR) for the positive endpoint CIN2+. For short-term observations (n = 409) a RR of 3.79 (CI 95% 2.15 to 6.67) and for long-term follow-up (n = 700) after an average 34.7 months a RR of 8.72 (CI 95% 5.77 to 13.17) was found. The highest RR of 6.32 (CI 95% 3.71 to 10.76) was determined for the group IIID1/LSIL, followed by 3.98 (95% CI 1.45 to 10.91) for the group III-p (ASC-H). DISCUSSION: Regardless of the study designs and significant differences of the resulting statistics in the literature, there is consensus concerning the significantly higher specificity and positive prediction of the p16/Ki-67 immunocytochemistry compared to cytology or HPV DNA test results. Therefore, p16/Ki-67 immunocytochemistry is useful in cases of persistent group IIID1/LSIL and equivocal cytological findings (group III-p/ASC-H). Especially in the former group, the frequency of colposcopic examinations can be reduced. In this respect, adding p16/Ki-67 immunochemistry likely improves patient management. However, an indication for treatment solely based upon a positive immunocytochemical finding is unjustified.

Comparison of cost- and energy-efficient signal modulations for next generation passive optical networks.

Extensive numerical investigations are undertaken to analyze and compare, for the first time, the performance, techno-economy, and power consumption of three-level electrical Duobinary, optical Duobinary, and PAM-4 modulation formats as candidates for high-speed next-generation PONs supporting downstream 40 Gb/s per wavelength signal transmission over standard SMFs in C-band. Optimization of transceiver bandwidths are undertaken to show the feasibility of utilizing low-cost and band-limited components to support next-generation PON transmissions. The effect of electro-absorption modulator chirp is examined for electrical Duobinary and PAM-4. Electrical Duobinary and optical Duobinary are power-efficient schemes for smaller transmission distances of 10 km SMFs and optical Duobinary offers the best receiver sensitivity albeit with a relatively high transceiver cost. PAM-4 shows the best power budget and cost-efficiency for larger distances of around 20 km, although it consumes more power. Electrical Duobinary shows the best trade-off between performance, cost and power dissipation.

Nyquist-shaped dispersion-precompensated subcarrier modulation with direct detection for spectrally-efficient WDM transmission.

The use of single-sideband subcarrier modulation (SCM) with Nyquist (N) pulse shaping for cost-effective spectrally-efficient wavelength division multiplexed transmission with direct detection is described. Transmission of digitally pre-compensated 7 × 11 GHz-spaced QPSK SCM channels at 14 Gb/s per channel is experimentally demonstrated over distances of up to 800 km of uncompensated standard single-mode fiber (SSMF) (13440 ps/nm chromatic dispersion).

Antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian medicinal plant Eremophila neglecta.

AIMS: To determine the antibacterial spectrum and cytotoxic activities of serrulatane compounds from the Australian plant Eremophila neglecta. METHODS AND RESULTS: Antimicrobial activities of serrulatane compounds 8,19-dihydroxyserrulat-14-ene (1) and 8-hydroxyserrulat-14-en-19-oic acid (2) were tested against Gram-negative and Gram-positive bacteria including human and veterinary pathogens and some multidrug-resistant isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the compounds were determined by broth microdilution assay. Both compounds exhibited antibacterial activity against all Gram-positive test strains. They showed antimycobacterial activity against isolates of Mycobacterium fortuitum and Mycobacterium chelonae. Of the five Gram-negative bacteria tested, only Moraxella catarrhalis showed susceptibility to the compounds. Cytotoxic activities were tested in the Vero cell line. Compound 1 showed more activity than 2 in both antibacterial and cytotoxicity assays with cytotoxicity at concentrations similar to the MBC. CONCLUSIONS: Serrulatane compounds showed significant activity against medically important bacteria, with 1 exhibiting stronger antibacterial activity. However, they also displayed toxicity to mammalian cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Serrulatanes are of interest as novel antibacterial compounds for use in biomedical applications; this study reports data obtained with a range of bacterial strains and mammalian cells, essential for assessing the capabilities and limitations of potential applicability of these compounds.

Molecular-level removal of proteinaceous contamination from model surfaces and biomedical device materials by air plasma treatment.

Established methods for cleaning and sterilising biomedical devices may achieve removal of bioburden only at the macroscopic level while leaving behind molecular levels of contamination (mainly proteinaceous). This is of particular concern if the residue might contain prions. We investigated at the molecular level the removal of model and real-life proteinaceous contamination from model and practical surfaces by air plasma (ionised air) treatment. The surface-sensitive technique of X-ray photoelectron spectroscopy (XPS) was used to assess the removal of proteinaceous contamination, with the nitrogen (N1s) photoelectron signal as its marker. Model proteinaceous contamination (bovine serum albumin) adsorbed on to a model surface (silicon wafer) and the residual proteinaceous contamination resulting from incubating surgical stainless steel (a practical biomaterial) in whole human blood exhibited strong N1s signals [16.8 and 18.5 atomic percent (at.%), respectively] after thorough washing. After 5min air plasma treatment, XPS detected no nitrogen on the sample surfaces, indicating complete removal of proteinaceous contamination, down to the estimated XPS detection limit 10ng/cm(2). Applying the same plasma treatment, the 7.7at.% nitrogen observed on a clinically cleaned dental bur was reduced to a level reflective of new, as-received burs. Contact angle measurements and atomic force microscopy also indicated complete molecular-level removal of the proteinaceous contamination upon air plasma treatment. This study demonstrates the effectiveness of air plasma treatment for removing proteinaceous contamination from both model and practical surfaces and offers a method for ensuring that no molecular residual contamination such as prions is transferred upon re-use of surgical and dental instruments.

Surface-MALDI mass spectrometry in biomaterials research.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used for over a decade for the determination of purity and accurate molecular masses of macromolecular analytes, such as proteins, in solution. In the last few years the technique has been adapted to become a new surface analysis method with unique capabilities that complement established biomaterial surface analysis methods such as XPS and ToF-SSIMS. These new MALDI variant methods, which we shall collectively summarize as Surface-MALDI-MS, are capable of desorbing adsorbed macromolecules from biomaterial surfaces and detecting their molecular ions with high mass resolution and at levels much below monolayer coverage. Thus, Surface-MALDI-MS offers unique means of addressing biomaterial surface analysis needs, such as identification of the proteins and lipids that adsorb from multicomponent biological solutions in vitro and in vivo, the study of interactions between biomaterial surfaces and biomolecules, and identification of surface-enriched additives and contaminants. Surface-MALDI-MS is rapid, experimentally convenient, overcomes limitations in mass resolution and sensitivity of established biochemical techniques such as SDS-PAGE, and can in some circumstances be used for the quantitative analysis of adsorbed protein amounts. At this early stage of development, however, limitations exist: in some cases proteins are not detectable, which appears to be related to tight surface binding. This review summarizes ways in which Surface-MALDI-MS methods have been applied to the study of a range of issues in biomaterials surfaces research.

The control of Staphylococcus epidermidis biofilm formation and in vivo infection rates by covalently bound furanones.

In order to overcome the continuing infection rate associated with biomaterials, the use of covalently bound furanones as an antibiofilm coating for biomaterials has been investigated. Furanones have previously been shown to inhibit growth of Gram-positive and Gram-negative bacteria. The aim of these studies were to covalently bind furanones to polymers and to test their efficacy for inhibiting biofilm formation of Staphylococcus epidermidis and in vivo infection rate. Two methods of covalent attachment of furanones were used. The first, a co-polymerisation with a styrene polymer, and second, a plasma-1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) reaction to produce furanone-coated catheters. Biofilm formation by S. epidermidis in vitro was inhibited by 89% for polystryene-furanone disks and by 78% by furanone-coated catheters (p<0.01). In an in vivo sheep model we found furanones were effective at controlling infection for up to 65 days. Furanones have potential to be used as a coating for biomaterials to control infection caused by S. epidermidis.

XPS and surface-MALDI-MS characterisation of worn HEMA-based contact lenses.

XPS and MALDI-MS were used to analyse initial adsorption events in the fouling of HEMA-based contact lenses. All of the lenses tested accumulated tear film deposits within 10 min of wear. XPS indicated the presence of mainly proteinaceous deposits, with indications of some contributions by mucins or lipids on some lenses and the nature of the deposit being influenced by the lens chemistry. MALDI-MS detected the presence of surface-adsorbed species with molecular weights < 15 kDa. While lysozyme could be identified by comparison of MALDI-MS signals with known protein mass and assignments are suggested for some other signals, several other species, with MWs less than that of lysozyme, could not be identified as no ocular proteins with corresponding MWs had been reported in previous biochemical tear film analyses. These species, and others, were also detected in MALDI-MS analysis of reflex tear film, suggesting that the adsorbed unidentified species were not simply products of surface-induced dissociation of adsorbing higher-MW proteins. This short-term wear study detected rapid interface conversion and demonstrated the utility and surface sensitivity of XPS and MALDI-MS in characterising contact lens deposits at the initial stages when sub-monolayer adsorbed amounts are present on lenses.

Primary lymph node plasmacytomas (plasmacytic lymphomas).

To determine whether primary lymph node plasmacytoma (PLNP) is a distinct entity among other types of plasma cell neoplasia, we analyzed a large series of PLNPs from 2 large lymphoma registries to compare histologic, immunophenotypic, and clinical features of PLNPs, nonnodal extramedullary plasmacytomas, and multiple myeloma. Twenty-five PLNPs (clinical data on 15 cases) were compared with 10 non-lymph node plasmacytomas and 51 cases of multiple myeloma; 36 cases of reactive plasmacytoses were used as controls. The histologic features of PLNP and other extramedullary plasmacytomas were similar. The histologic features of PLNPs were more immature than those of reactive plasmacytoses and less immature than in multiple myeloma. The immunophenotype of PLNPs significantly differed from that of reactive plasmacytoses, other extramedullary plasmacytomas, and multiple myeloma. PLNPs did not progress to multiple myeloma, unlike other extramedullary plasmacytomas, even though survival in PLNPs and other extramedullary plasmacytomas was similar. Our findings suggest that PLNPs may be distinct from other plasma cell dyscrasias.

[Cytopathology--current trends]. / Zytopathologie--zukünftige Trends.

Modern imaging techniques help to detect minute tumours, even in remote locations of the body. This increases the demand to sample cellular material by minimal invasive procedures, such as fine needle aspiration (FNA). For optimal treatment, tumour cells should be characterized for prognostically relevant features. Primary and metastatic neoplasms, and individual disseminated cells of solid tumours are targets for FNA and exfoliative cytology. Recognition and typing of tumour cells is aided by immunochemistry and PCR based molecular studies. Especially immunostaining requires laboratory procedures adapted to cytology samples. Screening for precancerous lesions, highly successful in cervical cancer prevention, extends into areas such as bladder and lung cancer in selected patient groups. Cell image analysis, especially for DNA ploidy, and chromosomal FISH of interphase cells can have predictive value enhancing the information derived from the purely morphologic assessment. Prognostic tumour cell markers are studied using immunochemistry and PCR on cytological material. Cytology has to foster translational research and test the results from molecular cytogenetics, including comparative genomic hybridisation (CGH) and cDNA microarray techniques on dissociated solid tumour cells. Newly identified genetic abnormalities can be evaluated for their functional relevance and may correlate with distinct structural alterations of the tumour cells. Proper training, dedication to diagnostic cytologic work and molecular studies, and sufficient staffing will enable cytology to get the most for the patient out of limited cell material. This approach helps to distinguish genetically defined tumour subtypes by recognizing properties of the cell gestalt.

Significantly different bcl-2 expression profiles in gastric and non-gastric primary extranodal high-grade B-cell lymphomas.

Fifty-five cases of primary extranodal high-grade B-cell non-Hodgkin's lymphoma were investigated for bcl-2 and p53 protein expression as well as for t(14;18) translocations and p53 mutations. Phenotypic and genotypic profiles were compared between tumours of gastric (27 cases) and non-gastric (28 cases) origin. bcl-2 protein expression was significantly lower in gastric (11/27) than in non-gastric (28/28) lymphomas (p<0.0001), while nuclear p53 protein expression did not differ significantly between these two groups. In the stomach, there were no significant differences in either bcl-2 or p53 expression profiles between high-grade lymphomas with (n=14) and without (n=13) evidence of a low-grade component of MALT type. However, secondary high-grade lymphomas showed a significant down-regulation of bcl-2 protein (p<0.0001) and, conversely, an up-regulation of p53 protein (p<0.0001) as compared with their low-grade tumour components. In extranodal high-grade B-cell lymphomas, bcl-2 protein expression was not associated with t(14;18) translocation. Only one gastric lymphoma had a p53 point mutation with potential alteration of the amino acid sequence. These findings indicate that primary gastric high-grade B-cell lymphomas are immunohistologically distinct from primary extranodal high-grade B-cell lymphomas of an origin other than in the stomach.

Detection of c-kit mutation Asp 816 to Val in microdissected bone marrow infiltrates in a case of systemic mastocytosis associated with chronic myelomonocytic leukaemia.

BACKGROUND/AIMS: The occurrence of myeloid leukaemia in patients with systemic mastocytosis is a well recognised phenomenon. However, the pathophysiological basis of such a coevolution has not been clarified. Recent data have shown that the c-kit mutation Asp 816 to Val is detectable in neoplastic mast cells in most patients with systemic mastocytosis, including those who have associated haematological disorders. The aim of this study was to study clonal disease evolution by analysing bone marrow cells from a patient with systemic mastocytosis and associated chronic myelomonocytic leukaemia (CMML) for the presence of this mutation. METHODS: The DNA of microdissected bone marrow cells from a patient with systemic mastocytosis and associated CMML was analysed for the presence of the c-kit mutation Asp 816 to Val by means of HinfI digestion and direct sequencing of semi-nested polymerase chain reaction (PCR) products. RESULTS: The two neoplasms could easily be identified and discriminated in paraffin wax embedded bone marrow sections by tryptase and chloroacetate esterase staining. A total number of 10 tryptase positive systemic mastocytosis infiltrates and 10 tryptase negative CMML infiltrates were removed by microdissection. As assessed by HinfI digestion and direct sequencing of semi-nested PCR products, the c-kit mutation Asp 816 to Val was detected in five of seven systemic mastocytosis infiltrates and four of six CMML infiltrates. By contrast, no c-kit mutation Asp 816 to Val was found in bone marrow infiltrates in patients with CMML without associated systemic mastocytosis (n = 20). CONCLUSION: These data support a monoclonal evolution of systemic mastocytosis and concurrent CMML in the patient studied.

Peptoid-containing collagen mimetics with cell binding activity.

Collagen mimetic peptides containing the peptoid residue Nleu (Goodman Bhumralkar, Jefferson, Kwak, Locardi. Biopolymers 1998;47:127-142) were tested for interactions with epithelial cells and fibroblasts. Molecules containing the sequence Gly-Pro-Nleu with a minimum of nine repeats showed cell binding activity. The activity of these molecules appeared to be conformationally sensitive, with the triple-helical form being preferred. When immobilized on a surface, the (Gly-Pro-Nleu)(10)-Gly-Pro-NH(2) sequence stimulated the attachment and growth of corneal epithelial cells and fibroblasts and the migration of epithelial tissue. The peptide sequence KDGEA inhibited cell attachment to the (Gly-Pro-Nleu)(10)-Gly-Pro-NH(2) sequence, suggesting that cell binding to this collagen mimetic involves the alpha2beta1 heterodimer integrin receptor. Interestingly, peptides containing the sequence (GlyNleu-Pro-)(10)-NH(2) did not have cell binding activity. The discovery that triple-helical peptides containing the Gly-Pro-Nleu sequences interact with cells opens up new opportunities in the design of collagen mimetic biomaterials.

Epithelialization of a synthetic polymer in the feline cornea: a preliminary study.

PURPOSE: This study examined the potential of a synthetic polymer to support stable epithelial growth when implanted in the feline cornea. METHODS: A perfluoropolyether-based polymer was cast into lenticules that were coated with collagen I and implanted in four feline corneas. Epithelial growth onto the lenticules was monitored clinically for 6 weeks, after which time the animals were killed, and three corneas were evaluated histologically. Immunohistochemistry was used to identify proteins associated with the formation of a basement membrane (laminin) and adhesion complexes (bullous pemphigoid antigen and collagen VII). Electron microscopy was used to examine the tissue-polymer interface for evidence of the assembly of these adhesive structures. RESULTS: Postoperative epithelial growth began on days 2 to 3, and lenticules were fully epithelialized by days 5 to 9. Lenticules were clinically well tolerated and histology showed epithelium consisting of multiple layers adherent to the lenticule's surface. Laminin, bullous pemphigoid antigen and collagen VII were identified at the tissue-polymer interface using immunohistochemistry. Ultrastructural examination showed evidence of assembly of these proteins into a recognizable basement membrane and hemidesmosomal plaques. CONCLUSIONS: A perfluoropolyether-based polymer coated with collagen I was implanted in the feline cornea and supported epithelial growth that showed signs of persistent adhesion, both clinically and histologically. This polymer shows potential for ophthalmic applications that require sustained epithelialization.

Elimination of stick-slip of elastomeric sutures by radiofrequency glow discharge deposited coatings.

Fine elastomeric sutures intended for cardiovascular surgery can exhibit "stick-slip" behavior as they are pulled through tissue; the resulting oscillatory force can damage delicate tissue or cause sutures to snap. To eliminate this undesirable effect, sutures were surface-modified using a radiofrequency glow discharge in a vapor of either hexamethyldisiloxane or hexamethyldisilazane, to produce a thin polymeric coating on the suture. The same coatings were also deposited onto aluminized tape to facilitate their characterization by measurement of air/water contact angles and by X-ray photoelectron spectroscopy. Coatings from both monomers were found to be very hydrophobic. The hexamethyldisiloxane glow discharge coatings underwent negligible oxidation when stored in air, and thus remained stable over a shelf-life period akin to what may be required of sutures. The hexamethyldisilazane glow discharge coatings, in contrast, incorporated substantial amounts of oxygen over a 3-month period. The coatings did not measurably alter the tensile properties of the sutures. The frictional properties of coated sutures were assessed by measuring the dynamic friction between the suture and ovine myocardium. Both coatings were effective in removing the inherent stick-slip behavior of polybutester sutures in this model. The coatings remained intact after several passes and proved to be robust and efficacious under various strain regimes.

Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer.

The relative effects of porosity and surface chemistry on the migration of epithelial tissue over the surface of a polymer were determined in vitro. These studies compared nonporous polymers with those having 0.1-microm diameter track-etched pores and were conducted on polycarbonate and polyester. Epithelial tissue migration over the polymer surface was stimulated by the presence of these pores. The surface chemistries of the polymers were modified by deposition of various polymer films using radio frequency gas deposition, giving a range of surfaces that varied in air:water sessile contact angle (SCA) of between 26 and 100 degrees. Tissue migration on the nonporous surfaces was affected by the surface chemistry, being generally linear as a function of the SCA and higher on hydrophilic than on hydrophobic surfaces but reduced if the hydrophilic surface had a mobile chemistry. The effects of the 0.1-microm diameter pores and the surface hydrophilicity were additive with the maximal level of epithelial tissue migration occurring on a porous, hydrophilic polymer surface.

Lymphomas in patients with connective tissue disease. Comparison of p53 protein expression and latent EBV infection in patients immunosuppressed and not immunosuppressed with methotrexate.

Cell cycle dysregulation as measured by p53 protein expression and latent Epstein-Barr (EBV) infection are important in the pathogenesis of lymphoma, particularly in immunosuppressed patients. Although latent EBV commonly is detected in lymphomas arising in patients with connective tissue disease who are immunosuppressed with methotrexate, p53 protein expression has not been reported. We compared the immunohistologic expression of p53 protein and the incidence of latent EBV infection in lymphomas arising in patients with connective tissue disease treated and not treated with methotrexate. Increased p53 staining was detected in 10 of 11 lymphomas arising in patients after methotrexate therapy vs 5 of 11 in patients not treated with methotrexate. Latent EBV was detected in 7 of 13 lymphomas arising in patients after methotrexate therapy vs 2 of 11 in patients not treated with methotrexate. Concordant p53 expression and latent EBV were detected in 5 of 7 lymphomas arising after treatment with methotrexate, including 1 that regressed after methotrexate therapy was withdrawn. These findings suggest that cell cycle dysregulation and EBV-related transformation are important in the pathogenesis of lymphomas arising in patients with connective tissue disease who are immunosuppressed with methotrexate.