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In der Vergangenheit traten in Pathologien häufig Grenzwertüberschreitungen für die inhalative Formaldehydbelastung auf. Aufgrund der krebserzeugenden Eigenschaft von Formaldehyd, der Festlegung eines neuen Formaldehydgrenzwerts und der Absenkung einiger Lösungsmittelgrenzwerte sollten die aktuellen Expositionen ermittelt und beurteilt werden. Mit Arbeitsplatzmessungen nach der Technischen Regel für Gefahrstoffe (TRGS) 402 ermittelten Messstellen der Unfallversicherungsträger und Bundesländer die inhalativen Gefahrstoffexpositionen während betrieblicher Routinetätigkeiten in 57 Pathologien in Deutschland, deren technische Schutzmaßnahmen subjektiv als ausreichend eingeschätzt wurden. Bei lang dauernden Tätigkeiten mit Formaldehydexposition überschritt der Acht-Stunden-Schichtmittelwert in bis zu 30 % der Fälle den zulässigen Arbeitsplatz- grenzwert (AGW) nach TRGS 900. Bei kurzzeitigen Tätigkeiten wurden die zulässigen Kurzzeitwertbedingungen in bis zu 60 % der Fälle nicht eingehalten, bei Arbeiten ohne Erfassung sogar in bis zu 70 %. Erhöhte Formaldehydkonzentrationen traten besonders bei unzureichend leistungsfähigen Absauganlagen und bei Formaldehyd emittierenden Tätigkeiten auf. Die Grenzwerte für Lösungsmittel wurden eingehalten.

The transmembrane pump halorhodopsin in halophilic archaea translocates chloride ions from the extracellular to the cytoplasmic side upon illumination. In the ground state a tightly bound chloride ion occupies the primary chloride-binding site (CBS I) close to the protonated Schiff base that links the retinal chromophore to the protein. The light-triggeredtrans–cisisomerization of retinal causes structural changes in the protein associated with movement of the chloride ion. In reverse, chemical depletion of CBS I inNatronomonas pharaonishalorhodopsin (NpHR) through deprotonation of the Schiff base results in conformational changes of the protein: a state thought to mimic late stages of the photocycle. Here, crystals ofHalobacterium salinarumhalorhodopsin (HsHR) were soaked at high pH to provoke deprotonation of the Schiff base and loss of chloride. The crystals changed colour from purple to yellow and the occupancy of CBS I was reduced from 1 to about 0.5. In contrast toNpHR, this chloride depletion did not cause substantial conformational changes in the protein. Nevertheless, two observations indicate that chloride depletion could eventually result in structural changes similar to those found inNpHR. Firstly, the partially chloride-depleted form ofHsHR has increased normalizedBfactors in the region of helix C that is close to CBS I and changes its conformation inNpHR. Secondly, prolonged soaking ofHsHR crystals at high pH resulted in loss of diffraction. In conclusion, the conformation of the chloride-free protein may not be compatible with this crystal form ofHsHR despite a packing arrangement that hardly restrains helices E and F that presumably move during ion transport.

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action. Copyright © 2019. Published by Elsevier Ltd.

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto-oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single-particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B-bound MET. We show that internalin B-bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B-bound receptors than for resting MET. Results of single-particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.

The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique., Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.

The cellular prion protein (PrPC) is a GPI-anchored cell-surface protein. A small subset of PrPC molecules, however, can be integrated into the ER-membrane via a transmembrane domain (TM), which also harbors the most highly conserved regions of PrPC, termed the hydrophobic core (HC). A mutation in HC is associated with prion disease resulting in an enhanced formation of a transmembrane form ((PrP)-Pr-ctm), which has thus been postulated to be a neurotoxic molecule besides PrPSc. To elucidate a possible physiological function of the transmembrane domain, we created a set of mutants carrying microdeletions of 2-8 aminoacids within HC between position 114 and 121. Here, we show that these mutations display reduced propensity for transmembrane topology. In addition, the mutants exhibited alterations in the formation of the Cl proteolytic fragment, which is generated by a-cleavage during normal PrPC metabolism, indicating that HC might function as recognition site for the protease(s) responsible for PrPC alpha-cleavage. Interestingly, the mutant G113V, corresponding to a hereditary form of prion disease in humans, displayed increased (PrP)-Pr-Ctm topology and decreased alpha-cleavage in our in vitro assay. In conclusion, HC represents an essential determinant for transmembrane PrP topology, whereas the high evolutionary conservation of this region is rather based upon preservation of PrPC alpha-cleavage, thus highlighting the biological importance of this cleavage. (C) 2010 Elsevier Inc. All rights reserved.

Hyaluronic acid was obtained from filtrates of heat-killed cultures of Streptococcus pyogenes group A, strain K56, by simple ethanol precipitation and treatment with an adsorbent. The hyaluronic acid is pure as judged from chemical and sedimentation analyses. Particles of streptococcal bacteriophage 12/12 were isolated from phage-lysed group A streptococci by polyethylene glycol precipitation and isopyenic centrifugation. Electron micrographs of negatively stained preparations showed a typical Bradley group B virus with a long, flexible, cross-striated tail and a knob- or star-like structure at the distal tip of the tail. The hyaluronic acid is depolymerized upon incubation with the phage 12/12 virions. After extensive digestion, a mixture of at least four oligosaccharides is formed, the two smallest of which are a tetra- and octasaccharide terminating in reducing N-acetyl-D-glucosamine. The tetrasaccharide shows an absorption maximum at 231.5 nm with a molar extinction coefficient epsilon = 4820 litres X mole-1 X cm-1, and it is therefore concluded that the bacteriophage-borne hyaluronidase catalyses a beta-elimination. Accordingly it is classified as a hyaluronate lyase (EC 4.2.99.1).

The Listeria monocytogenes surface protein InIB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InIB (InlB(321)) is sufficient for Met binding and activation. The complex between this Met-binding domain of InIB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB(321) consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB(321) in solution, highlighting the importance of Met Ig domains for InIB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB(321) binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB(321), These results call into question whether receptor dimerization is the basic underlying event in InlB(321)-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB(321) bind and activate the Met receptor. (C) 2008 Elsevier Ltd. All rights reserved.

PrP(C)-deficient mice expressing prion protein variants with large amino-proximal deletions (termed PrP(DeltaF)) suffer from neurodegeneration, which is rescued by full-length PrP(C). We now report that expression of PrP(DeltaCD), a PrP variant lacking 40 central residues (94-134), induces a rapidly progressive, lethal phenotype with extensive central and peripheral myelin degeneration. This phenotype was rescued dose-dependently by coexpression of full-length PrP(C) or PrP(C) lacking all octarepeats. Expression of a PrP(C) variant lacking eight residues (114-121) was innocuous in the presence or absence of full-length PrP(C), yet enhanced the toxicity of PrP(DeltaCD) and diminished that of PrP(DeltaF). Therefore, deletion of the entire central domain generates a strong recessive-negative mutant of PrP(C), whereas removal of residues 114-121 creates a partial agonist with context-dependent action. These findings suggest that myelin integrity is maintained by a constitutively active neurotrophic protein complex involving PrP(C), whose effector domain encompasses residues 94-134.

SummaryBackground 8-Methoxypsoralen–UVA (PUVA) and narrowband ultraviolet B (NB-UVB) are well-established treatments for chronic plaque-type psoriasis vulgaris. However, long-term risks of PUVA therapy include premature skin ageing and squamous cell carcinoma. Objectives To develop a device for targeted UV therapy of psoriatic plaques with protection of the healthy adjacent skin to reduce the risk for premature skin ageing and squamous cell carcinoma. Methods In total 28 patients with exacerbated psoriasis vulgaris were treated with the digital phototherapy device skintrek® [cream PUVA (n = 8), bath PUVA (n = 11) and UVB (n = 9)] or with conventional bath PUVA (n = 9) or NB-UVB (n = 4). Results The local Psoriasis Area and Severity Index (PASI) score was significantly reduced from a mean of 6·25 at baseline to 2·75 at the end of therapy in the skintrek cream PUVA group, from 6·4 to 3·0 in the skintrek bath PUVA group and from 5·5 to 2·0 in the skintrek UVB group. Treatment with skintrek cream PUVA reduced the mean local PASI by 54%, while skintrek bath PUVA did so by 51% and skintrek UVB by 63%. Targeted skintrek PUVA and skintrek UVB of inflamed psoriatic skin avoided skin pigmentation and were not inferior to conventional bath PUVA and NB-UVB therapy regimens. Conclusions Targeted UV therapy of psoriatic plaques with the digital phototherapy device skintrek is as effective as conventional cream and bath PUVA, as well as NB-UVB, while simultaneously sparing the healthy adjacent skin. It therefore potentially reduces the carcinogenic risk, reduces premature skin ageing and avoids tanning of healthy surrounding skin.

Background. Mesothelioma is a rare tumor, linked with occupational asbestos exposure. This association has been used to explain clustering of cases within families. Newer evidence, however, supports a possible genetic predisposition for this tumor. Case. Our patient's brother was diagnosed with advanced stage malignant peritoneal mesothelioma in August 1995 at age of 42, he underwent tumor-reductive surgery followed by chemotherapy. He underwent a repeat cytoreductive surgery in September 1996 and further chemotherapy. He died of disease in December 1996. Our patient underwent cytoreductive surgery in May 1999 at age of 49 for advanced stage malignant peritoneal mesothelioma with suboptimal debulking. She received multiple chemotherapy regimens, including most recently experimental targeted agents, for slow progressing disease. She is presently alive with clinical disease 6 years from diagnosis. Conclusion. This is the first report of two siblings of different gender with malignant peritoneal mesothelioma and only average environmental asbestos exposure. It is highly likely that the family described in this case report has some form of inherited susceptibility to malignancy cancer gene, HLA type, or tumor suppressor gene mutation.

Introduction: The temporal course of recovery of depressed patients' cognitive impairment is not fully understood. Methods: We used the California Verbal Learning Test (CVLT) to test declarative memory in 24 depressed patients before and after 35 days of antidepressive treatment as well as after long-term follow-up (> 12 months) in order to relate improvement of depression to recovery of cognitive impairment. Results: Patients with complete remission after 35 days had generally been less impaired at baseline. The disturbance of declarative memory in treatment responders as well as in non-responders did not change from baseline to end of treatment (day 35). However, our results revealed normal values in the CVLT sum score as well as in measures of short- and long-delay free-recall measures in both groups after long-term full remission. Discussion: We conclude that clinical response to antidepressive treatment precedes improvement of declarative memory. A low degree of impairment of declarative memory is associated with early complete remission of depression.

The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met. The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face. Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB. The mutants correspondingly display severely reduced binding to Met. To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required. We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion.

This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

Diffuse uterine leiomyomatosis is a rare condition distinguished from the common uterine leiomyomata by involvement of the entire myometrium by innumerable, ill-defined, often small and confluent, histologically benign smooth-muscle nodules. Fourteen cases have been previously described in the literature. We report a case of diffuse leiomyomatosis in a 39-year-old woman. Several microscopic foci of the process were microdissected for clonality analysis. All samples showed a non-random X-chromosome inactivation pattern, and thus were consistent with a monoclonal neoplastic population of cells. However, in different foci of tumor, different X chromosomes were inactivated, supporting the independent origin of neoplastic clones and rejecting the possibility of a single clonal origin of all tumor cells. The results of the molecular analysis suggest that diffuse uterine leiomyomatosis may be an exuberant example of diffuse and uniform involvement of the entire myometrium by multiple leiomyomata.

A tumor suppressor gene at 10q 23.3, designated PTEN, encoding a dual specificity phosphatase with lipid and protein phosphatase activity, has been shown to play an important role in the pathogenesis of a variety of human cancers. Germline mutations in PTEN cause Cowden syndrome (CS), which is characterized by multiple hamartomas and a high risk of breast and thyroid cancers. Frequent loss of heterozygosity at 10q is found in both early and advanced-stage sporadic melanomas; however, mutations or deletions in PTEN are detected mainly in melanoma cell lines. In this study, we examined PTEN expression in 34 unselected sporadic melanomas (4 primary melanomas, 30 metastases) using immunohistochemistry and correlated this with the results of structural studies of this gene. Immunostaining of 34 melanoma samples revealed no PTEN expression in 5 (15%) and low PTEN expression in 17 (50%), whereas the rest of the tumors (35%) had high levels of expression. Hemizygous deletion was found in 32% of the tumors but neither intragenic PTEN mutation nor biallelic deletion was found in any of the samples. Of the 5 melanomas showing no PTEN expression, 4 had no mutation or deletion of PTEN. Of the 13 tumors having weak PTEN immunoreactivity and informative loss of heterozygosity results, 6 had evidence of hemizygous allelic loss of PTEN while the remaining 7 had intact PTEN. These results strongly support PTEN as a major tumor suppressor on 10q involved in melanoma tumorigenesis and suggest an epigenetic mechanism of biallelic functional inactivation not previously observed in other cancers where PTEN might be involved.

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.

Objective.—Carcinosarcomas of urinary bladder are rare malignant neoplasms. Seventy-eight cases have been previously described. The histologic composition of these tumors is variable, but diagnosis requires the presence of both epithelial and mesenchymal malignant components. We report 4 additional cases, with an emphasis on unusual histologic features. Methods.—Histologic and immunohistochemical examinations were performed on bladder tumors from 4 patients. Clinicopathologic features of previously reported and current cases were reviewed and summarized. Results.—Four patients (3 men, 1 woman) age 54 to 77 years were found to have polypoid masses in the urinary bladder. In all cases, histologic examination showed biphasic neoplasms with distinct mesenchymal and epithelial components. The morphologic and immunohistochemical characteristics of the tumors varied. One of the cases was remarkable for the presence of liposarcoma, malignant peripheral nerve sheath tumor, and micropapillary urothelial carcinoma. Two of the patients died 2 years after diagnosis, which is consistent with the previously reported aggressive nature of urinary bladder carcinosarcomas. Conclusions.—Carcinosarcomas of the urinary bladder are rare, aggressive malignant neoplasms. To our knowledge, a liposarcomatous component has been reported in only 1 case previously, and components of micropapillary urothelial carcinoma and malignant peripheral nerve sheath tumor have not been reported previously in carcinosarcomas of the urinary bladder. Because of the aggressive biologic behavior of these tumors, they should be identified promptly and treated appropriately.

Differentiating between primary tumors of the liver and metastatic lesions can, at times, be difficult. Various histochemical and immunohistochemical methods have been used in an effort to better delineate between hepatocellular carcinoma (HCC), especially the microglandular variant, primary cholangiocarcinoma, and metastatic adenocarcinoma; these ancillary studies can yield less than satisfactory results. Recently, anti-MOC31, a monoclonal antibody directed against a cell surface glycoprotein, has been shown to be helpful in distinguishing between adenocarcinoma and mesothelioma. This study addresses whether this antibody might be helpful in distinguishing between HCC, primary cholangiocarcinoma, and metastatic adenocarcinoma in the liver. Formalin-fixed, paraffin-embedded tissue sections from 15 HCC (including 10 microglandular variants), 14 primary cholangiocarcinomas, and 33 metastatic adenocarcinomas (7 colon, 1 lung, 8 breast, 4 GE jct/gastric, 9 pancreas, 2 small intestine, 1 renal, 1 ovary) were immunostained with anti-MOC 31 (1:40, Dako) after protease digestion and biotin block using a modified ABC technique. Positive staining was limited to membrane based reactivity; controls stained appropriately. Immunoreactivity for MOC31 was observed in 14 of 14 cholangiocarcinomas and 33 of 33 metastatic tumors. Staining was diffuse, intense, and readily interpretable, with rare exceptions. All 15 cases of HCC were negative. We conclude that cholangiocarcinoma and metastatic adenocarcinoma from a variety of sites express MOC31; HCC is uniformly negative for this marker. Anti-MOC31 may prove useful in the evaluation of liver neoplasms where primary hepatocellular and adenocarcinoma enter the differential diagnosis; it is not useful in separating primary cholangiocarcinoma from metastatic adenocarcinoma.

The purpose of this study was to investigate the developmental potential of isolated rabbit blastomeres under various culture conditions to gain insight into their ability to form the two cell lineages of a viable blastocyst. Intact embryos at the 4-cell, 8-cell, 16-cell stages and blastomeres isolated from 4-, 8- and 16-cell rabbit embryos (1/4, 1/8 or 1/16 blastomeres respectively) were cultured in drops of one of three different media, each supplemented with either fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA). The effects of the extracellular matrix fibronectin (FN) on the development of isolated rabbit blastomeres were also investigated. Supplementation of the medium with FCS yielded a higher (P < 0.05) proportion of blastocysts than BSA or PVA, predominantly from 1/4 blastomeres. No major differences were found between the three basic culture media. In 1/4, 1/8 or 1/16 blastomeres, blastocyst formation rates were greater (P < 0.05) in groups cultured in matrix-free (54.5, 59.6 and 54.6% respectively) than in FN-coated groups (35.4, 46.0 and 26.1% respectively). Only in blastocysts derived from 1/4 blastomeres, were the numbers of inner cell mass (ICM) and total cells of blastocysts higher (P < 0.05) in FN-coated groups than in matrix-free groups (12.7 +/- 1.1 versus 8.5 +/- 0.7 ICM, 73.8 +/- 3. 7 versus 57.8 +/- 3.3 total cells). The percentage of blastocysts derived from single blastomeres with ICM cells decreased with increasing cell stage of the parent embryos in FN-coated (93.6, 78.3 and 44.0%, respectively) as well as matrix-free groups (96.2, 69.3 and 55.2%). In FN-coated groups, after 96 h (1/4) or 72 h (1/8 and 1/16) of culture, approximately 20-30% of blastomeres did not develop into normal blastocysts but formed sheets with 30-50 cells attached to the bottom of the dishes. These results indicate that the development of rabbit blastomeres shares important characteristics with those from mouse and domestic species and may thus aid in developing an efficient culture system for blastomeres, derived from human embryos.

The distal portion of chromosome 1p is one of the most commonly affected regions in human cancer. In this study of hereditary and sporadic colorectal cancer, a region of frequent deletion was identified at 32.2 centimorgans from 1ptel. Deletion breakpoints clustered in the vicinity of or inside the gene RIZ , which encodes a retinoblastoma protein-interacting zinc finger protein. Sequence analysis revealed frequent frameshift mutations of the RIZ gene. The mutations consisted of 1- or 2-bp deletions of a coding (A) 8 or (A) 9 tract and were confined to microsatellite-unstable colorectal tumors, being present in 9 of 24 (37.5%) primary tumors and in 6 of 11 (54.5%) cell lines; in 2 cell lines the mutation was homozygous/hemizygous. The mutations apparently were selected clonally in tumorigenesis, because similar poly(A) tracts in other genes were not affected. Two alternative products of the gene exist, RIZ1, which contains a PR ( P RDI-BF1- R IZ1) domain implicated in tumor suppressor function, and RIZ2, which is lacking this motif. Furthermore, the C-terminal region, which contains the poly(A) tracts, includes a PR-binding motif, possibly mediating interactions with other proteins or with RIZ itself (oligomerization). Four of eleven microsatellite-unstable colorectal cancer cell lines, three of which had frameshifts, showed reduced or absent mRNA expression of RIZ1. In a cell line that is homozygous/hemizygous for the typical frameshift mutation, immunoblotting showed truncated RIZ protein, whereas adenovirus-mediated RIZ1 expression caused G 2 /M arrest and apoptosis. We propose that RIZ is a target of the observed 1p alterations, with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.

BACKGROUND Evaluation of effusion specimens for the presence of adenocarcinoma often is complicated by the presence of reactive mesothelial cells that can mimic adenocarcinoma. Ancillary studies, in particular immunohistochemistry, can be helpful in making this distinction. MOC-31 is an antibody that recently was reported to be useful in distinguishing adenocarcinoma from mesothelioma in tissue specimens. In this study we examined the utility of this antibody in pleural effusions. METHODS Eighty-nine archival, formalin fixed, paraffin embedded cell blocks representing 59 adenocarcinomas, 12 other neoplasms (including 6 mesotheliomas), and 18 reactive effusions were retrieved. After protease digestion, recut slides were immunostained with the MOC-31 antibody utilizing a modified avidin-biotin complex technique. Only membrane-based reactivity was considered as positive. RESULTS In two adenocarcinomas there was insufficient material remaining in the cell block. Among the 57 remaining cases, reactivity was observed in 54 cases. Reactivity also was observed in one of six mesotheliomas and one small cell carcinoma. The remaining cases, including all 18 reactive effusions, were nonreactive. In distinguishing adenocarcinoma from reactive mesothelial cells, the presence of MOC-31 reactivity was found to be 95% sensitive and 100% specific with a positive predictive value of 100% and a negative predictive value of 95%. CONCLUSIONS MOC-31 is useful in differentiating between adenocarcinoma and reactive mesothelial cells in pleural effusion specimens. Cancer (Cancer Cytopathol) 1999;87:390–4. © 1999 American Cancer Society.

BACKGROUND Fine-needle aspiration biopsy (FNAB) is frequently used to diagnose mass lesions in the liver. Differentiating metastatic adenocarcinoma from primary hepatocellular carcinoma can be difficult. Despite a number of morphologic criteria, there remain occasional cases in which the cytologic features fail to resolve this differential reliably; in these cases ancillary studies may be useful. Recently, it has been reported that the antibody MOC-31 reliably separates metastatic adenocarcinoma from hepatocellular carcinoma. In this study we examine the utility of MOC-31 in liver FNAB material. METHODS Thirty-three archival, alcohol-fixed, paraffin-embedded cell blocks representing 17 cases of hepatocellular carcinomas and 16 cases of metastatic adenocarcinoma were retreived. After protease digestion, the sections were immunostained with the antibody MOC-31 (Dako, Carpinteria, CA) utilizing a modified avidin-biotin complex technique. Only membrane-based reactivity was considered positive. RESULTS In five cases there was insufficient diagnostic material remaining in the cell block for immunohistochemical staining. Among the remaining cases, MOC-31 reactivity was observed in 10 of 12 metastatic adenocarcinomas and 2 of 16 hepatocellular carcinomas. For metastatic adenocarcinoma the presence of MOC-31 reactivity yields a sensitivity of 83%, a specificity of 87%, a positive predictive value of 83%, and a negative predicitive value of 87%. CONCLUSIONS MOC-31 is useful in separating metastatic adenocarcinoma from hepatocellular carcinoma in FNAB cell block material. Cancer (Cancer Cytopathol) 1999;87:295–8. © 1999 American Cancer Society.

Objective: To determine the effect of routine second review of pathologic material that was sent to Ohio State University before initiation of therapy. Methods: All the gynecologic-oncologic histopathology review diagnoses made during a 1-year period were compared with original pathologic diagnoses. When there was a discrepant diagnosis with the second interpretation, the case was reviewed by at least two pathologists. Discrepancies were coded as no diagnostic disagreement, no diagnostic disagreement but pertinent information not included, diagnostic disagreement without clinical consequences, diagnostic disagreement with minor clinical significance, or diagnostic disagreement with major clinical significance. Proportions and confidence intervals were calculated. Results: Pathology reports from 295 referred patients were reviewed. Two hundred forty-five (83.1%) showed no discrepancy. Discrepancies were found in 50 cases (16.9%). There was significant information missing in four cases (1.4%), diagnostic disagreement with no clinical significance in 22 cases (7.5%), and diagnostic disagreement with minor clinical significance in 10 cases (3.4%). In 14 cases (4.7%, 95% confidence interval 2.28, 7.12) the changes in diagnoses had major therapeutic or prognostic implications that included changes from malignant or low malignant potential to benign (seven cases), malignant to low malignant potential (three cases), change in tumor type (two cases), and assessment of invasion (two cases). The cost of reviewing 295 specimens was approximately $39,235. The cost of identifying each major discrepancy was about $2802. Conclusion: Routine pathology review of gynecologic-oncologic cases before definite treatment revealed notable discrepancies in diagnoses. In 4.7% of cases, the change in diagnosis had a major effect on proper treatment planning or a significant prognostic implication.

Extraskeletal neoplasms with osteoclast-like giant cells are uncommon. These tumors are most frequently reported in the breast and pancreas, and are relatively rare in other sites. We report a case of primary gastric adenocarcinoma with an infiltrate of osteoclast-like giant cells. The patient is a 64-yr-old black woman who presented with epigastric pain and was found to have a mass in the gastric antrum. Histological examination showed a poorly differentiated adenocarcinoma with an infiltrate of osteoclast-like giant cells. The giant cells were present both in the primary gastric adenocarcinoma and in the lymph node metastases. Immunohistochemical stains demonstrated that the giant cells were of monocytic/histiocytic origin and probably represent a distinctive host response to the tumor. The patient is alive and well 12 months after resection. This is the second published report of gastric carcinoma with osteoclast-like giant cells. Based on this limited experience, gastric carcinoma with osteoclast-like giant cells may represent a distinct clinicopathological entity with a more favorable prognosis.

Rhabdoid tumors were originally described as a type of pediatric renal neoplasm that contains cells resembling rhabdomyoblasts but lacking muscle differentiation. Extrarenal rhabdoid tumors have since been reported in multiple anatomic sites in the pediatric and adult population. These tumors are characterized by an aggressive clinical course, resistance to treatment, and a rapidly fatal outcome. Eight cases of uterine neoplasms with rhabdoid differentiation have been previously reported. In the three cases where clinical follow-up was available, the patients died of disease within 3 to 17 months after the diagnosis was established. We report two cases of uterine malignant mixed Müllerian tumor (carcinosarcoma) with rhabdoid differentiation. The findings and clinical outcome confirm the aggressive nature of uterine tumors with rhabdoid differentiation. One of the patients died of disease 3 months after initial operative treatment while the other patient's tumor recurred in 1 month and she died within 10 weeks. The poor prognosis of these neoplasms makes their histopathologic recognition important.

Two cases of cervical melanosis, an uncommon process characterized by hyperpigmentation of the basal epithelium, with or without accompanying basal melanocytes, are reported. The etiology of cervical melanosis is uncertain but may represent a metaplastic change in response to trauma or chronic irritation. On gross examination, it is not possible to reliably separate the different causes of cervical pigmentation; consequently, any pigmented cervical lesion should be biopsied.

For many neoplasms, the status of regional lymph nodes is a critical prognostic factor with direct therapeutic implications. Despite the importance of lymph node status, there is no consensus on whether lymph nodes from node dissections should be submitted in their entirety. To study this issue, for a 4-month period, all lymph nodes from node dissections were submitted in their entirety. The larger nodes were submitted systematically to determine whether the additional tissue blocks contained metastases that were not identified in the initial tissue block. We also analyzed the cost of submitting, processing, and interpreting these extra blocks. During the study period, 149 patients underwent lymph node dissections, yielding a total of 2,915 lymph nodes. Submitting the larger nodes in their entirety resulted in processing 808 additional tissue blocks that demonstrated a total of 9 additional metastases in 7 patients. In 2 of these patients, the additional metastases changed the stage of the neoplasm and may have contributed to an alteration in therapy. By using median salary figures, the estimated cost for submitting, processing, and interpreting the additional blocks was $5,935.62. Whether the additional positive nodes justify this cost should be determined by individual pathology practices.

A constitutively dimeric truncated variant of internalin B (InlB321-CD), acting as stimulator of the receptor tyrosine kinase MET, was tested for dermal wound-healing potential. Due to a lack of the endogenous MET agonist HGF/SF in chronic wounds, HGF/SF substitution by an InlB321-CD-loaded hydrogel might be beneficial in chronic wound therapy. In this study, InlB321-CD in solution and incorporated in a hydrogel was tested for mitogenic effects on immortalized human dermal keratinocytes (HaCaT) with an MTT assay. Cell migration was investigated with a scratch assay on primary keratinocytes (PHK) and on HaCaT. For the latter, scratching needed to be mitomycin C-controlled. InlB321-CD effects on a model of human skin were analyzed histologically with respect to viability. InlB321-CD led to dose-dependent proliferative effects on HaCaT cells whereas the equimolar dose of monomeric InlB321 did not. Upon hydrogel incorporation of InlB321-CD its mitogenic activity for HaCaT cells was maintained thus confirming the hydrogel as a promising drug delivery system. Motogenic effects were shown on both HaCaT and PHK cells. InlB321-CD neither possesses cytotoxic effects on the viability of a human skin model nor alters its organotypic cell morphology.

Isothermal microcalorimetry was evaluated for the determination of the critical relative humidity (cRH) for three drug substances possessing different hygroscopic properties. Two extremely water-soluble substances (flupentixol,2HCl and Lu 25–109, water solubility over 1000 mg/ml) and a hydrophobic substance (sertindole, approximately 10 μ g/ml) were examined. The technique was evaluated with respect to sample size, duration of exposure time and temperature for the substance Lu 25–109, and further tested with the other compounds. The cRH values determined by isothermal microcalorimetry showed results similar to various weighing methods. This, together with the high sensitivity of the technique, which will enable a determination within hours, and the small sample amount needed, makes isothermal microcalorimetry a valuable screening technique for new drug candidates. The temperature effects on the cRH for Lu 25–109 showed a linear van't Hoff plot, but with a heat of interaction with water differing from the enthalpy of water condensation, however. By results obtained from solution calorimetry, this deviation was accounted for by the heat of formation of a saturated solution in the water layer surrounding the solid particles.

In this study we investigated effects of developmental stage at onset of individual in vitro culture on the progress of development in intact embryos and individual blastomeres derived from 4-cell and 8-cell porcine embryos (referred to as 1/4 and 1/8, respectively), the necessity of serum or BSA supplementation in culture media for embryos and blastomeres (in contrast to development in a defined medium), and the role of two concentrations of human leukemia inhibitory factor (hLIF) on development of blastomeres and embryos. More (p 0.05) lacked an ICM (blastocyst-like vesicles) than in defined medium without hLIF. It is concluded that 1) porcine intact embryos and isolated blastomeres can be cultured individually in defined medium up to the blastocyst stage from the 4-cell stage onwards; 2) more 1/4 isolated blastomeres develop to blastocysts with more total cells but with a lower ratio of ICM to total cells than blastocysts derived from 1/8 blastomeres; 3) the effects of hLIF are dependent on proteins present in the culture medium and on the embryonic stage; and 4) in defined medium, high concentrations of hLIF are inhibitory to blastocyst formation, but fewer blastocyst-like vesicles are formed. The defined culture system employed in this study allows examination of the effects of growth factors or cytokines in porcine early embryonic development.

The retinoblastoma (RB) gene was the first defined tumor suppressor gene. While originally described in retinoblastoma, more recently alterations in RB have been described in a number of other human neoplasms and there has been a suggestion that alteration of RB may play a significant role in the development of endometrial carcinoma. We examined RB protein expression by immunohistochemistry in a series of cases including normal endometrium, endometrial hyperplasia, and endometrial carcinoma. A relatively homogeneous pattern of staining was observed in proliferative endometrium, while weak or absent reactivity was noted in secretory endometrium. A heterogeneous pattern of reactivity was observed in 10/10 cases of hyperplasia, 66/70 cases of endometrial adenocarcinoma, and 7/7 cases of uterine carcinosarcoma. An altered pattern of reactivity was observed in the remaining 4/70 cases of adenocarcinoma. All of the cases with altered reactivity were high grade neoplasms. We conclude that alteration of RB protein expression is uncommon in endometrial adenocarcinoma and when it does occur, it may represent a late event in carcinogenesis.

Background Congenital hepatic fibrosis is an uncommon disease of undetermined etiology that may present clinically with portal hypertension or cholangitis. Histologically the liver is characterized by portal fibrosis and an increase in portal ductal structures. Case A 46-year-old male with a history of congenital hepatic fibrosis presented with abdominal pain and was found to have a 2 x 3-cm liver nodule. Fine needle aspiration biopsy showed hepatocytes and abundant bile duct epithelium. Conclusion This report expands the spectrum of nonneoplastic diseases that have been sampled by fine needle aspiration biopsy.

Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23.

Although cervical-vaginal telecytology is a promising tool, diagnostic accuracy has not been extensively evaluated. The authors examined the accuracy of five cytotechnologists who retrospectively reviewed 50 cervical-vaginal smears using the video monitor, and 2 months later, using the light microscope. Accuracy was expressed in terms of crude agreement with the original diagnosis and number of false positives (FPs) and false negatives (FNs). With a greater than one step difference as discrepant, the group crude agreement using the video monitor and the light microscope was 85.6% and 95.6%, respectively. The group number of FNs and FPs for the light microscope was 8 and 7, respectively, and for the video monitor was 34 and 7, respectively. There was a wide range of individual performance. We conclude that accuracy of telecytology is high, but less than that of light microscopy. The major reason for lower telecytologic accuracy was undercalling dysplasia.

This report presents the fine-needle aspiration biopsy (FNAB) findings of a multicystic renal tumor in a 52-yr-old woman. The aspirate smears contained clusters of cells with large, irregular nuclei and cytoplasmic vacuoles. Subsequent nephrectomy revealed a multicystic nephroma (MCN). Although most common in childhood, MCN should always be considered in the FNAB differential diagnosis of a multicystic renal mass in adult patients. Even in cases where the diagnosis of MCN is considered, it may be difficult to distinguish from cystic renal cell carcinoma on the basis of radiographic and FNAB findings.

The structural basis of ligand-induced dimerization of the receptor tyrosine kinase MET by its natural ligand hepatocyte growth factor/scatter factor (HGF/SF) is not well understood. However, interesting insight into the molecular mechanism of MET dimerization has emerged from crystal structures of MET in complex with a bacterial agonist, the invasion protein internalin B (InlB) from pathogenic Listeria monocytogenes. MET activation by InlB promotes uptake of bacteria into host cells. Structural and biophysical data suggest that InlB is monomeric on its own but dimerizes upon binding to the membrane-anchored MET receptor promoting the formation of a signaling active 2:2 complex. The dimerization interface is small and unusually located on the convex side of the curved InlB leucine-rich repeat (LRR) domain. As InlB does not dimerize in solution, the dimerization site could only be identified by studying packing contacts of InlB in various crystal forms and had to be proven by scrutinizing its biological relevance in cellular assays. InlB dimerization is thus an example of a low-affinity contact that appears irrelevant in solution but becomes physiologically significant in the context of 2-dimensional diffusion restricted to the membrane plane. The resulting 2:2 InlB: MET complex has an InlB dimer at its center with one MET molecule bound peripherally to each InlB. This model of ligand-mediated MET dimerization may serve as a blue-print to understand MET activation by NK1, a naturally occurring HGF/SF splice variant and MET agonist. Crystal structures of NK1 repeatedly show a NK1 dimer, in which residues implicated in MET-binding are located on the outside. Thus, MET dimerization by NK1 may also be ligand-mediated with a NK1 dimer at the center of the 2:2 complex with one MET molecule bound peripherally to each NK1. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases. (C) 2012 Elsevier B.V. All rights reserved.

This study investigated the appropriateness of different superovulatory protocols in various breeds of sheep for obtaining a maximum of zygotes suitable for microinjection. Animals were mated either once or two to three times to fertile rams. In Experiment 1, a 24 h interval between a two to three times mating and egg recovery resulted in 42.2% suitable zygotes whereas with single mating only 10.4% fertilized eggs were obtained. The extension of the interval to 40 h associated with a two to three times mating resulted in a recovery of 42.9% fertilized eggs but most (70%) of these were already at the two-cell stage. In Experiment 2, eCG resulted in similar superovulatory responses in Merino ewes as the more labour requiring FSH treatment (8.1 ± 4.5 versus 7.5 ± 4.1 corpora lutea (CL); 6.3 ± 3.0 versus 6.8 ± 4.0 oocytes/ zygotes; 39.4% versus 40.6% fertilization rate). In Experiment 3, following superovulation with pFSH (Folltropin®) the number of CL was not different among Merino, Finn, Crossbreds (Blackface X Finn) and Texel sheep (8.6 ± 5.2; 10.3 ± 4.5; 8.5 ± 3.8; 8.2 ± 2.8, respectively) as was the number of recovered oocytes/ zygotes (7.4 ± 5.6; 9.8 ± 4.3; 7.3 ± 3.8; 6.4 ± 2.9, respectively). However, the number of unfertilized ova was higher (P < 0.05) in Finn sheep as compared with Crossbreds and Texel sheep (5.0 ± 3.3 versus 2.2 ± 2.3 and 1.9 ± 2.6). Similarly, the fertilization rate was higher (P < 0.05) in Crossbreds and Texel sheep (64.4% and 65.5%) as compared with Finn and Merino sheep (38.3% and 42.5%). In Experiment 4, it was shown that in Merino sheep purified FSH supplemented with 68.6% LH resulted in lower (P < 0.05) superovulatory responses as compared with purified FSH supplemented with 133.1% LH or Folltropin (LH contamination 0.1%) (4.7 ± 3.3 versus 8.8 ± 3.8 and 8.6 ± 5.2 CL; 3.8 ± 2.5 versus 7.4 ± 3.6 and 7.4 ± 5.6 oocytes/zygotes, respectively). A three times repeated superovulatory treatment and oviductal flush per animal at monthly intervals did reduce (P < 0.05) the number of CL, but had no deleterious effect on zygote yields and the percentage of microinjectable zygotes. We conclude that (1) at least a two to three times mating is required to obtain acceptable fertilization rates; (2) the interval between mating and recovery should be 24–26 h in order to obtain zygotes; (3) eCG results in similar superovulatory responses as FSH; (4) Folltropin® is a suitable drug to induce superovulation in sheep; (5) the LH content of the FSH preparation plays a significant role in the superovulatory response of sheep; (6) superovulation and embryo recoveries can be repeated at least three times per animal without decrease in efficiency.

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P

Germ line mutations of the p53 gene have been described in the Li-Fraumeni Cancer Family Syndrome and occur in patients with multifocal gliomas, particularly those with a history of a metachronous cancer or a family history of cancer. p53 dysfunction is often associated with ovarian cancer. Patients with ovarian carcinoma frequently develop synchronous or metachronous cancers and may have a family history of this or related cancers. Thus, we hypothesized that germ line p53 mutations might be associated with a significant proportion of ovarian cancers. Germ line DNA isolated from peripheral leukocytes of 73 patients with ovarian carcinoma was screened for p53 sequence abnormalities utilizing single-strand conformation polymorphism analysis and direct PCR sequencing techniques. As many as 40% of this cohort of ovarian cancer patients from 67 families may represent familial phenotypes. Synchronous and metachronous cancers occurred in 19% of the cohort. Only two intron-based polymorphisms were found. Neither has been previously reported. One of these, in intron 6, occurred in three unrelated patients all of whom had a history of metachronous breast cancer. A polymorphism in intron 10 occurred in a patient with synchronous endometrial cancer. No classic germ line mutations of p53 were found.

The controversy of distinguishing keratoacanthomas (KAs) from well-differentiated squamous cell carcinomas (WDSCCs) is well established. A number of recent studies have attempted to separate these processes with the use of immunohistochemical stains. In corroboration, we have retrospectively reviewed three groups of patients with tissue biopsies with features of classical KA (n = 7), WDSCC (n = 8), and squamous cell carcinoma with KA-like features (SCC-KA) (n = 9). We compared their clinical and histological differences as well as their immunohistochemical differences using antibodies to proliferating cell nuclear antigen (PCNA), and wild- and mutant-type p53 protein. Classical KA showed a PCNA staining pattern located predominantly around the basal cell layers, in contrast to a relatively diffuse staining pattern seen in WDSCC. SCC-KA showed considerable overlap with these two types of staining patterns. The p53 staining showed basal, patchy, or diffuse patterns. These patterns were present in all three groups. Although both PCNA and p53 expression was more often present in SCC-KA, there were no statistical differences among the groups. In conclusion, the overlapping expression patterns of PCNA and p53 in keratocanthoma-like tumors support the hypothesis that these tumors represent a possible biologic spectrum. Because of the absence of significant statistical differences in the expression of these antigens, PCNA and p53 have not proved to be helpful in differentiating KA from KA-like squamous cell carcinoma.

Background. Resistance to chemotherapy is common in bulky hypoxic tumors such as epithelial ovarian cancer. Hyperbaric oxygen (HBO) oxygenates hypoxic tissues and promotes neovascularization. These unique properties of HBO may help overcome chemotherapy resistance by increasing both tumor perfusion and cellular sensitivity. This study was undertaken to determine if HBO increases the response of epithelial ovarian cancer to cisplatin chemotherapy. Methods. In Phase I,64 nu/nu mice were divided into four groups and subcutaneously inoculated with cells from the A2780 human epithelial ovarian cancer cell line. Group 1 served as controls. Group 2 received weekly intraperitoneal cisplatin (3.15 mg/kg). Group 3 was exposed to HBO (dives) at 2.4 atmospheres absolute pressure for 90 minutes, 7 days a week. Group 4 received both cisplatin and HBO. In Phase II, 72 mice were divided into two groups and similarly inoculated. Both groups received weekly intraperitoneal cisplatin (2.5 mg/kg). Group 1 was not exposed to HBO. Group 2 was exposed to HBO for 5 days a week. Results. Dramatic tumor neovascularization was found in tumors of mice exposed to HBO (P = 0.0001). There was significant (P = 0.014) tumor growth retardation in Phase I for mice receiving both cisplatin and HBO compared with those treated with cisplatin alone. This significance was noted after just two doses of cisplatin but subsequently lost due to reduced numbers of mice. In Phase II, neovascularization was detectable after 10 HBO treatments (2 weeks) and was maximal after 15 treatments (3 weeks). Conclusions. Hyperbaric oxygen increases vascularity in bulky tumors such as epithelial ovarian cancer. There appears to be a relationship between increased vascularity and enhanced response to chemotherapy that merits further investigation. Cancer 1995 ;75 :2313-22.

p53 is a nuclear phosphoprotein whose overexpression may portend a poor prognosis in a variety of neoplasms. In this immunohistochemical study we examined p53 overexpression in a variety of uterine smooth muscle tumors (34 leiomyosarcomas, 18 leiomyomas, and six smooth muscle tumors of uncertain malignant potential [STUMps]). p53 immunoreactivity was observed in none of 18 (0%) leiomyomas, one of six (17%) STUMPs, and 16 of 34 (47%) leiomyosarcomas. Reactivity was not observed in the surrounding nonneoplastic uterine smooth muscle. Strong p53 overexpression in the leiomyosarcomas was significantly associated with high grade morphology ( P = .013) and a high stage at the time of presentation ( P = .021). In 25 leiomyosarcoma patients with clinical follow-up, p53 overexpression was associated with shorter length of survival ( P = 0.024). However, this effect was not independent of tumor stage or grade. A regression analysis showed that tumor stage was the only independent predictor of length of survival. Our study size is small, and further studies are warranted to determine the significance and replicability of these findings.

ESD in the esophagus is an expert technique for ‘en bloc’ resection of mucosal cancers >2cm in size. I can provide a quasi-surgical specimen and significantly reduces the risk of local recurrence compared to piecemeal EMR. However, secondary stricture formation is the major drawback for resections >3cm and of more than 75% of the esophageal circumference. Multiple bouginage sessions or temporary stent placement may be required limiting the QoL. Experimental approches include transplantation of buccal or dermal cell grafts as well as systemic and local steroid applications. However, clinical experience is very limited so far. Since march 2011 be had been performing animal experiments concerning esophageal resection and re-transplantation of esophageal and gastric mucosal patches in pigs under an approved protocol (NLVL No: 33–42502–06/1151) with first preliminary but encouraging results when the following clinical presented. Case report: A 72 y male pat. with swallowing difficulty; tabacco use of 20 py until >15y ago. Prior rectal resection with sigma anus praeter for a T2 distal rectal cancer. EGD: Suspicion of early squamous cell cancer (Paris IIa; EUS UT1a, m, N0), >75% circum-ferential superficial tumor spread within the cervical esophagus and upper sphincter area (17–25cm aborally). Biopsy: SC HG-IEN. Discussion of the different treatment options in our interdisciplinary tumor board and with the patient: tubular endoscopic resection alone with 80–100% stricture probability, thermal ablation, radical surgery, primary chemoradiation or an experimental approach as compassionate use with primary ESD in the esophagus and secondary gastro-esophageal mucosal patch transplantation. On April 13, 2011 we performed an EGD under general anesthesia with tracheal intubation with first tubular ESD over 10cm from the lower hypopharynx through the UES from 17–27cm followed by a 9×4cm ESD in the gastric antrum. The healthy gastric specimen prelieved was cut longitudinally into 3 mucosal stripes which were attached to the denuded esophageal muscular layer by means of hemoclips. The specimen were gently pressed against the wall by a non-covered self-expanding metal stent to allow luminal and vascular nutrition of the specimen. The sphincter area of 1.5cm length had to be spared in order to not compress the lower larynx with the stent and to avoid unsupportable pain for the patient. After a control the following day the patient could be extubated. The esophageal specimen showed a non-invasive low horny early squamous cell cancer (pT1a G2 L-, V-) and and curative resection (R0; invasion depth of lamina propria max. 150 microns). Stent removal was performed at day 20 pp and was cumbersome due to local mucosal hyperplasia. However, multiple islets of gastric mucosa had successfully grown at the esophageal resection site. The patient was discharged on day 24 and regulary seen as outpatient. We observed a stepwise circular spread out of the reddish layer over an area of 5–6cm within the next 6 month and successful prevention of stenosis. Biopsy proved antral HP-negative mucosa. Unfortunately a 1–1.5cm non-transplant covered segment in the sphincter area showed a repetive tendency to stricture formation serving quasi as ‘natural’ control and requiring multiple ambulatory bouginage treatments but allowing a (semi-) solid diet without restrictions for most food products (DG1). Conclusions: The so far unique case of a gastro-esophageal endoscopic mucosal transplant with one year follow-up after wide-spread ESD in the esophagus for an early squamous cell cancer opens a new perspective for systematic research in this field.

Somatotropin and FSH act synergystically on insulin-like growth factor-I (IGF-I) synthesis in ovarian follicles; IGF-I regulates several granulosa cell specific functions and may thereby be beneficial in bovine superovulation. In a series of 3 experiments we investigated the effects of recombinant bovine somatotropin (rBST) on several parameters of the superovulatory response in dairy cows. A total of 81 Holstein Friesian crossbred dairy cows received either 640 mg rBST or the vehicle (controls) on Day 4 or 13 of the superovulation schedule. Superovulation was induced with 2500 IU PMSG on Day 9. The cows were artificially inseminated on Day 13. In Experiment 1, on Days 4, 8, 11, 13 and 17 4 to 5 animals each were slaughtered to obtain follicular fluid, endometrium and plasma. The rBST application increased IGF-I contents in plasma and follicular fluid on Days 8, 11 and 13 (P0.05) in the treated cows when compared with that of the controls. Plasma and follicular IGF-I contents were correlated closely (rBST: r=0.90, n=10; control: r=0.94, n=9). The number of antral follicles increased following rBST treatment, and on the day of artificial insemination (AI) twice as many follicles4 mm were counted in the rBST treated animals than in the control group. In Experiment 2, the flushing of 38 donors on Day 7 after AI resulted in more transferable embryos in the rBST group than in the control group (4.2+/-1.0 vs 2.5+/-0.7; P0.05). In contrast, in Experiment 3 involving 21 animals when rBST was administered at the time of AI the superovulation response was not altered. It is concluded that rBST increases follicular and plasma IGF-I contents and thereby has profound effects on follicular and early embryonic development.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: To determine whether an extended sector biopsy of the prostate will increase the detection of prostate cancer, without causing an increase in morbidity. Materials and Methods: A total of 74 men with a mean age of 62.3 years (46-98 years) who either had an elevated PSA or an abnormal digital rectal exam underwent a transrectal ultrasound guided needle biopsy. Beginning on 7/1/98, an extended sector biopsy technique was performed on 74 patients by one urologist (RRB). Each transrectal ultrasound guided needle biopsy included 12 total cores (normal sextant biopsy, 2 in each peripheral zone, and 2 in the transition zone). We retrospectively reviewed the biopsy results for the location of cancer. PSA data and morbidity of the procedures were reviewed. Results: Of 74 total patients, 40 (54.1%) were positive for adenocarcinoma of the prostate. There were 10 positive results detected only in the additional zones. If one looks at the total number of cancers detected (40), then 10/40 (25%) of the cancers detected were found in the additional regions only or in 13.5% of all patients biopsied. Of the 10 patients with sector only prostate cancer, 8 were detected in the peripheral zone, 1 in the transition zone and 1 in both zones. All 10 patients had a Gleason pattern score 3+3=6 or 4+3=7. There were no atypical or PIN cores found in the sector zones only. PSA ranged from 1.2-142 (median 6.0 ng/ml). The median PSA was 6.2 ng/ml in all patients found to have cancer, and 6.0 ng/ml in the cancers detected only in the additional zones. There was 1 (1.4%) complication of urinary retention and fever. Conclusion: Our study suggests that an extensive sector biopsy may increase the detection of prostate cancer by 13.5% over a routine sextant biopsy, without demonstrable serious morbidity.