Expression of CD44 in effusions of patients diagnosed with serous ovarian carcinoma – diagnostic and prognostic implications

CD44 is a family of cell adhesion molecules involved in a variety of cellular functions. The present study analysed the expression of two CD44 isoforms in serous effusions of patients diagnosed with ovarian carcinoma and corresponding primary and metastatic lesions. Fifty-eight effusions, 23 primary ovarian tumours, and 44 metastatic lesions were studied for protein expression of CD44s and v3-10 using immunohistochemistry. Results were correlated with clinical parameters. CD44v3-10 was seen in carcinoma cells in the majority of cases at all sites. Malignant effusions showed an up-regulation of CD44s compared to both primary tumours and metastatic solid lesions. Mesothelial cells frequently expressed CD44s, but were rarely immunoreactive for v3-10. CD44s immunoreactivity in cancer cells in effusions was significantly more often observed in patients with FIGO stage 3 than in stage 4 patients (P = 0.045). Staining results did not correlate with age, effusion site, metastatic site, tumour grade or residual tumour mass after initial surgery. Likewise, comparison of overall and disease-free survival with expression of the CD44 isoforms studied did not reveal any statistically significant associations. The up-regulation in CD44 levels in effusions, primarily in stage 3 disease, suggests that adhesion of ovarian carcinoma cells to mesothelium may be regulated at the level of CD44s expression, and provides further evidence of phenotypic alteration in the transition from primary tumour cell clones to effusions. The similar expression profile of CD44 in carcinoma cells in peritoneal and pleural effusions supports our previous observations and the hypothesis that carcinoma cells in peritoneal effusions are truly metastatic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of two European strains of Ehrlichia phagocytophila of equine origin

We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.     

Survival and immunogenicity of dissociated allogeneic fetal neural dopamine-rich grafts when implanted into the brains of adult mice

Summary The survival of grafts of dissociated allogeneic fetal neural dopamine (DA) rich tissue in the striatum has been studied after transplantation between inbred strains of mice differing at defined immunogenetical loci between donor and recipient. Six to 7 weeks and 15 weeks after grafting, surviving grafted DA neurons were found in the brains of all the recipients, albeit with a large variation in numbers, located either within the striatum or within the adjacent lateral ventricle. The mean number of surviving DA neurons did not differ between the syngeneic controls and the histoincompatible donor-host combinations, and there was no difference in survival between grafts that differed at single or multiple major histocompatibility complex (MHC) loci, and those that differed at multiple non-MHC loci. The amount of inflammatory cells in the graft area did not differ between the groups, and none of the animals showed massive infiltration of inflammatory cells. The in situ immunogenicity of the grafted neural tissue after intracerebral implantation was monitored by means of Simonsen's alloimmunization test, at 6–7 weeks after transplantation, which provides a sensitive measure primarily of the cellular immunological response. Most, but not all, graft recipients showed immunization with a Spleen Index (S.I.) close to that seen in recipients of an orthotopical skin graft of the same histoincompatibility combination. In contrast to the prolonged survival of the intracerebral neural transplants, none of the skin grafts survived longer than 3 weeks, thus demonstrating the immunologically privileged status of the brain. We conclude that intracerebrally grafted allogeneic neural tissue is capable of provoking a cellular immune response. Despite host immunization, however, the dissociated fetal neural allografts survived for at least 15 weeks without any overt signs of rejection, regardless of the donor-host combination used.     

Eccentric and concentric torque-velocity characteristics of the quadriceps femoris in man

Summary The primary purpose of this investigation was to study the eccentric and concentric torque-velocity characteristics of the quadriceps femoris in man using a recently developed combined isometric, concentric and eccentric controlled velocity dynamometer (the SPARK System). A secondary purpose was to compare the method error associated with maximal voluntary concentric and eccentric torque output over a range of testing velocities. 21 males (21–32 years) performed on two separate days maximal voluntary isometric, concentric and eccentric contractions of the quadriceps femoris at 4 isokinetic lever arm velocities of 0° · s^–1 (isometric), 30° · s^–1 120° · s^–1 and 270° · s^–1. Eccentric peak torque and angle-specific torques (measured every 10° from 30° to 70°) did not significantly change from 0° · s^–1 to 270° · s^–1 (p>0.05) (with the exception of angle-specific 40° torque, which significantly increased;p<0.05). The mean method error was significantly higher for the eccentric tests (10.6%±1.6%) than for the concentric tests (8.1%±1.7%) (p<0.05). The mean method error decreased slightly with increasing concentric velocity (p>0.05), and increased slightly with increasing eccentric velocity (p>0.05). A tension restricting neural mechanism, if active during maximal eccentric contractions, could possibly account for the large difference seen between the present eccentric torque-velocity results and the classic results obtained from isolated animal muscle.     

Comparative dosimetry of diode and diamond detectors in electron beams for intraoperative radiation therapy

The aim of the present study is to examine the validity of using silicon semiconductor detectors in degraded electron beams with a broad energy spectrum and a wide angular distribution. A comparison is made with diamond detector measurements, which is the dosimeter considered to give the best results provided that dose rate effects are corrected for. Two-dimensional relative absorbed dose distributions in electron beams (6-20 MeV) for intraoperative radiation therapy (IORT) are measured in a water phantom. To quantify deviations between the detectors, a dose comparison tool that simultaneously examines the dose difference and distance to agreement (DTA) is used to evaluate the results in low- and high-dose gradient regions, respectively. Uncertainties of the experimental measurement setup (+/- 1% and +/- 0.5 mm) are taken into account by calculating a composite distribution that fails this dose-difference and DTA acceptance limit. Thus, the resulting area of disagreement should be related to differences in detector performance. The dose distributions obtained with the diode are generally in very good agreement with diamond detector measurements. The buildup region and the dose falloff region show good agreement with increasing electron energy, while the region outside the radiation field close to the water surface shows an increased difference with energy. The small discrepancies in the composite distributions are due to several factors: (a) variation of the silicon-to-water collision stopping-power ratio with electron energy, (b) a more pronounced directional dependence for diodes than for diamonds, and (c) variation of the electron fluence perturbation correction factor with depth. For all investigated treatment cones and energies, the deviation is within dose-difference and DTA acceptance criteria of +/- 3% and +/- 1 mm, respectively. Therefore, p-type silicon diodes are well suited, in the sense that they give results in close agreement with diamond detectors, for practical measurements of relative absorbed dose distributions in degraded electron beams used for IORT.     

Streptococcal protein G: A sensitive tool for detection of antibodies to human immunodeficiency virus proteins in Western blot analysis

Protein G is a cell wall protein of group C and G streptococci which binds human IgG antibodies of all four subclasses with high affinity. This property of the molecule was utilized to develop a sensitive Western blot assay to detect antibodies against HIV proteins in patient sera.     

Significant expression of IGFBP2 in breast cancer compared with benign lesions

BACKGROUND/AIM: Insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) play a role in the normal development of breast tissue, and possibly in breast cancer aetiology. IGFBP2, one of six members of the IGFBP superfamily, acts as regulator of the IGFs and has pleiotropic effects in normal and neoplastic tissues. Because IGFs have mitogenic effects on mammary epithelia, this study investigated IGFBP2 expression in mammary tissues of different benign and malignant entities. METHODS: Immunohistochemistry was used to study correlations between the presence and intensity of IGFBP2 staining and tumour type and grade, in addition to steroid hormone receptor status, in 120 breast specimens. Expression was measured by quantitative colour video image analysis and semiquantitative evaluation, and the measurements correlated well (r = 0.92; p<0.05). RESULTS: Both methods found no significant expression of IGFBP2 in normal glandular cells and hyperplasia (group I). Atypical hyperplasia showed a slightly increased cytoplasmic expression of IGFBP2, and carcinoma in situ showed a distinctive, membrane associated and cytoplasmic expression (group II). Infiltrating carcinomas strongly expressed cytoplasmic IGFBP2 (group III). There were significant differences between group I and II, and between group II and III. There were no significant differences between invasive lobular and invasive ductal carcinoma, or between grades I, II, and III within these entities. There was no significant correlation between IGFBP2 immunostaining and oestrogen or progesterone receptor positivity within the malignant group. CONCLUSIONS: IGFBP2 mitogenic signals of autocrine/paracrine regulatory mechanisms may be responsible for the growth of breast carcinomas and IGFBP2 may be an independent indicator of malignancy.     

Functional reactivation of the deafferented hippocampus by embryonic septal grafts as assessed by measurements of local glucose utilization

Summary Transection of the septo-hippocampal connections through fimbria-fornix damage in the rat results in profound hippocampal cholinergic deafferentation, and, when applied bilaterally, leads to severe and long-lasting impairments in learning and memory. Previous studies have shown that intrahippocampal septal grafts can reestablish a new cholinergic innervation in the inititally denervated hippocampal formation and at least partly compensate for the lesion-induced learning impairments in fimbria-fornix lesioned rats. The purpose of the present study was to determine the magnitude of lesion-induced alterations in cerebral function as reflected in local glucose use measured by (¹⁴C)-2-deoxyglucose (2-DG) autoradiography, and the degree to which this index of functional activity could be normalized following reinnervation from transplants of fetal cerebral tissue from the primordial septal region. Six months after unilateral fimbriafornix transection the rate of glucose utilization was reduced markedly throughout the ipsilateral hippocampus when compared to the intact contralateral side, while in the neocortex only the cingulate cortex showed long-lasting reductions in glucose use. Rats that received a transplant of fetal septal-diagonal band tissue at the time of fimbria-fornix transection, and were sacrificed 6 months later, displayed significantly greater glucose utilization in the ipsilateral hippocampus and cingulate cortex than was measured in these areas in rats with lesion alone. The recovery in glucose use was paralleled by a significant increase in acetylcholinesterase (AChE) staining in several areas of the ipsilateral hippocampal formation and cingulate cortex. This index of graft-induced cholinergic reinnervation was, moreover, significantly correlated with the rate of glucose use. Thus, in the fimbria-fornix transected animals the magnitude of glucose depression correlated with the extent of reduction in AChE staining, and in the grafted animals the degree of normalization of glucose use was correlated with the graft-induced increase in AChE-staining density. These results thus indicate that the 2-DG autoradiographic technique can provide a unique opportunity to map both altered functional activity in localized areas of the brain following specific lesions and the extent to which transplant-derived reinnervation of the host may induce a return to normal functional levels in the target site.ETP and Royal Society (London) visiting fellow     

Increase in vascular permeability induced by leukotriene b4 and the role of polymorphonuclear leukocytes

Leukotriene B₄ (LTB₄), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB₄ on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB₄ (150 nM-5 M) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later larger venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB₄ used. Subsequently, a dose-dependent increase in vascular permeability was seen at postcapillary and larger venules, with a maximum 10–20 min after application; the maximum occurred later the higher the dose of LTB₄. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB₄, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB₄-mediated permeability increase. LTB₄ may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.     

Characterisation of recombinant human IgE-Fc fragments expressed in baculovirus-infected insect cells

IgE mediates its effector functions through the Fc region and it has been demonstrated that structures in the Cvarepsilon3-domain are crucial for FcvarepsilonR-binding. In order to further study structures of importance for the function of IgE, such as the carbohydrates, fragments with unmodified amino acid sequence were blunt-end cloned and expressed in baculovirus-infected Sf9 cells. Two fragments of human IgE, one encompassing the entire Fc-region (rCvarepsilon2-4) and a smaller one comprising the second and third domain (rCvarepsilon2-3), were produced and characterised with respect to epitope expression, glycosylation and FcvarepsilonR-binding. N-terminal analysis showed the expected VCSRDF-sequence of the Cvarepsilon2-domain, confirming correct cleavage of the secretion signal. Immunoblotting and gel permeation chromatography demonstrated that rCvarepsilon2-4 mainly formed a dimer, whereas rCvarepsilon2-3 also existed as monomers and oligomers. Endoglycosidase-treatment revealed that both fragments were N-glycosylated. In inhibition ELISA, rCvarepsilon2-4 and myeloma protein IgE(DES) reacted in a near equimolar way with monoclonal antibodies against the Cvarepsilon2-, Cvarepsilon3- and Cvarepsilon4-domains, whereas rCvarepsilon2-3 only reacted with anti-Cvarepsilon2 mAbs. Moreover, in FACS analysis rCvarepsilon2-4 interacted with two cell-lines constitutively expressing FcvarepsilonRI or FcvarepsilonRII, whereas rCvarepsilon2-3 lacked reactivity. A substantial reduction in the ability of rCvarepsilon2-4, following endoglycosidase treatment, to react with recombinant alpha-chain of the high affinity receptor for IgE in sandwich ELISA, indicated a role of N-linked oligosaccharides in stabilising receptor binding structures. Taken together, our results show that rCvarepsilon2-4, but not rCvarepsilon2-3, will be useful in studies of structure-function relationships of IgE, including the role of N-glycosylation, since it demonstrated appropriate epitope expression, conformation and ability to bind Fcvarepsilon-receptors.