Grating coupled SPR microarray analysis of proteins and cells in blood from mice with breast cancer.

Biomarker discovery for early disease diagnosis is highly important. Of late, much effort has been made to analyze complex biological fluids in an effort to develop new markers specific for different cancer types. Recent advancements in label-free technologies such as surface plasmon resonance (SPR)-based biosensors have shown promise as a diagnostic tool since there is no need for labeling or separation of cells. Furthermore, SPR can provide rapid, real-time detection of antigens from biological samples since SPR is highly sensitive to changes in surface-associated molecular and cellular interactions. Herein, we report a lab-on-a-chip microarray biosensor that utilizes grating-coupled surface plasmon resonance (GCSPR) and grating-coupled surface plasmon coupled fluorescence (GCSPCF) imaging to detect circulating tumor cells (CTCs) from a mouse model (FVB-MMTV-PyVT). GCSPR and GCSPCF analysis was accomplished by spotting antibodies to surface cell markers, cytokines and stress proteins on a nanofabricated GCSPR microchip and screening blood samples from FVB control mice or FVB-MMTV-PyVT mice with developing mammary carcinomas. A transgenic MMTV-PyVT mouse derived cancer cell line was also analyzed. The analyses indicated that CD24, CD44, CD326, CD133 and CD49b were expressed in both cell lines and in blood from MMTV-PyVT mice. Furthermore, cytokines such as IL-6, IL-10 and TNF-α, along with heat shock proteins HSP60, HSP27, HSc70(HSP73), HSP90 total, HSP70/HSc70, HSP90, HSP70, HSP90 alpha, phosphotyrosine and HSF-1 were overexpressed in MMTV-PyVT mice.

[Denervation of the patella: influence on mid-term results after total knee arthroplasty]. / Retropatellare Denervierung : Einfluss auf die mittelfristigen Ergebnisse von Knietotalendoprothesenimplantationen.

BACKGROUND: The purpose of the study was to determine possible differences in the mid-term results of total knee arthroplasty in patients treated with and without denervation of the patella. PATIENTS AND METHODS: This study included 80 total knee replacements in 71 patients who were treated with total knee replacement, either with (n = 40) or without (n = 40) simultaneous denervation of the patella out of a total population with 122 knee replacements in 100 patients. Comparability of both groups was achieved by applying matching criteria. All patients were reviewed by isokinetic tests, physical and radiological examination. The mean follow-up time was 2.2 years. RESULTS: The mean hospital for special surgery (HSS) score revealed no statistically significant differences between both groups (with denervation 77.9 ± 11.1 and without denervation 77.8 ± 11.0, p = 0.976). The isokinetic torque measurements with low angle velocity (60°/s) indicated slightly higher values during extension (60.2 ± 32.2 Nm versus 55.8 ± 25.2 Nm, p = 0.497) and flexion (52.4 ± 28.3 Nm versus 46.1 ± 22.3 Nm, p = 0.272) movements of the affected knee joint. However, the differences did not reach statistical significance. At high angle velocity (180°/s) no differences could be found between both groups. No cases of postoperative necrosis of the patella were observed. Anterior knee pain after denervation was reported in 6 cases (15 %) compared to 10 cases (25 %) in patients who were treated without denervation (p = 0.402). CONCLUSION: No statistically significant differences could be found between patients with and without denervation of the patella for total knee arthroplasty.

Bioactive impact of manuka honey and bone char incorporated into gelatin and chitosan cryogels in a rat calvarial fracture model.

Bone tissue engineered scaffolds are designed to mimic the natural environment for regeneration when typical healing is inhibited. Autografts are the current gold standard for treatment but are limited by available bone and supplementary surgical sites that broaden complications and comorbidities. Cryogels are an ideal scaffold in bone regeneration due to their mechanical integrity and marcoporous structure that elicits angiogenesis and subsequently new bone tissue formation. To aid in bioactivity and osteoinductivity, manuka honey (MH) and bone char (BC) were added to gelatin and chitosan cryogels (CG). Manuka honey has powerful antimicrobial properties to aid against graft infection, and bone char is composed of 90% hydroxyapatite, a well-studied bioactive material. These additives are natural, abundant, easy to use, and cost effective. CG cryogels incorporated with either BC or MH, and plain CG cryogels were implanted into rat calvarial fracture models for cortical bone regeneration analysis. We found indication of bioactivity with both bone char and manuka honey through the presence of woven bone structure in histology stains and micro computed tomography (microCT) data. Overall, plain CG cryogels supported greater bone regeneration capabilities than the BC or MH incorporated cryogels due to a lack of advanced organized tissue formation and collagen deposition after 8 weeks of implantation; however, future work should explore varying additive concentrations and delivery methods to further assess additive potential.

Effect of brace design on patients with ACL-ruptures.

Different designs of functional knee braces for ACL-injury rehabilitation exist. In addition to the mechanical stabilization provided by rigid shell braces, sleeve braces also address proprioceptive mechanisms, but little is known if this leads to benefits for ACL-deficient subjects. Therefore the aim of this study was to investigate the effect of 2 different functional brace designs (shell and sleeve brace) on functional achievements in ACL-deficient patients. 28 subjects with ACL-ruptured knees performed tests for knee joint laxity, joint position sense, static and dynamic balance and isometric and dynamic lower limb extension strength in non-braced, sleeve braced and shell braced condition. The results showed a significant decrease in knee joint laxity for sleeve (33%; p<0.001) and rigid shell bracing (14%, p=0.039). The sleeve brace revealed a significant increase in dynamic balance after perturbation (20%; p=0.024) and a significant increase in dynamic lower limb peak rate of force development (17%; p=0.015) compared to the non-braced condition. The effects might be caused by the flexible area of support and the incorporated mechanisms to address proprioceptive aspects. Braces might not be needed in simple daily life tasks, but could provide beneficial support in more dynamic settings when patients return to sporting activities after an ACL-injury.

Development and validation of a method for simultaneous analysis of hair underivatized amino acids and damage biomarkers, using liquid chromatography-tandem mass spectrometry.

The forensic and medical fields are seeing growing interest in the amino acid and damage biomarker composition of hair, in order to identify adulteration of drug hair testing and for diagnostic purposes. Therefore, there is an increased demand for quick and accurate analytical methods. This study presents the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of hair amino acids and four damage biomarkers, which also implements an isotopic dilution strategy to improve recovery and precision of the acid hydrolysis-sensitive analytes. The applied strategy enabled a recovery of the hydrolysis-sensitive amino acids between 83 and 120% (vs. 33-77%, without isotopic dilution) for two different protein standards, and a precision with a relative standard deviation (RSD) between 1.3 and 7.5% (vs. 9.0-29.4%, without isotopic dilution). All 21 analytes could be measured without interferences by matrix and sample components, thus demonstrating satisfactory selectivity of the method. For spiked samples of hair hydrolyzate, recovery was between 88 and 120%, whereas precision and intermediate precision were below 10.1%. The high sensitivity of the method made it possible to reduce sample preparation to a 10000-fold dilution of the raw hydrolyzate. The wide linear range displayed by the method allowed the simultaneous quantification of minor (0.3 µmol/g of hair) and major (up to 1000 µmol/g of hair) components of the biological fiber. This method was successfully applied to the analysis of real hair samples submitted to six different treatments. Statistical data analysis by means of t-test and principal component analysis (PCA) showed a clear discrimination of the treated from the untreated hair samples and of the different treatments. Since these hair treatments can interfere with hair drug testing, the method possesses the ability of identifying hair samples with potential for attempted drug test evasion. In addition, lanthionine emerged as a new biomarker for heat damaged hair.

Studies on rabbit lymphocytes in vitro. IX. The suppression of antiallotype-induced blast transformation in lymphocyte cultures from allotypically suppressed donors.

The number of lymphocytes transformed in vitro by antiallotype sera in cultures obtained from allotypically suppressed rabbits is significantly less than that induced in cultures from normal rabbits. There is a compensatory increase in the amount of transformation induced by antiallotype sera directed toward the unsuppressed allotype. Thus the control of allotypic expression is suppressed rabbits appears to be the same for lymphocytes and for plasma cells.

Studies on rabbit lymphocytes in vitro. VI. The induction of blast transformation with sheep antisera to rabbit IgA and IgM.

Sheep antisera specific for the three major immunoglobulin groups of the rabbit, i.e. IgG (gamma-chain), IgA (alpha-chain), and IgM (micro-chain), are each able to induce blast transformation of the peripheral lymphocytes of the rabbit when added to in vitro cultures. The per cent of lymphocytes transformed with each antiserum indicate that one-fourth of the peripheral lymphocytes carry or have the capacity to synthesize molecules of all three of the major immunoglobulin groups, and that the remaining three-fourths carry or have the capacity to synthesize only two (IgG and IgM). The data do not permit direct conclusions concerning the ability of a single cell to produce molecules belonging to more than one immunoglobulin group at the same time, or the ability of a given cell to make a transition from the synthesis of molecules of one immunoglobulin group to those of another group.

Studies on rabbit lymphocytes in vitro. V. The induction of blast transformation with sheep antisera to rabbit IgG subunits.

The addition of sheep antisera to rabbit IgG or to rabbit IgG subunits (L chain, H chain, Fab piece, Fc piece) to in vitro cultures of rabbit peripheral lymphocytes may induce up to 80-90% of the small lymphocytes to transform into immature "blast" cells. As many as 1 x 10(7) molecules of antibody may be required to stimulate each cell. It is concluded that each peripheral lymphocyte carries the antigenic specificities of the entire IgG molecule and thus could carry, and may produce specific antibody molecules. Such a conclusion supports the theory that the small lymphocyte may be the site of specific recognition of antigens in the primary response.

Studies on rabbit lymphocytes in vitro. 8. The relationship between heterozygosity and homozygosity of lymphocyte donor and per cent blast transformation induced by antiallotype sera.

Rabbit antisera to immunoglobulin allotype Ab4 will stimulate a maximum of 77% (mean +/- SEM = 39.78 +/- 4.8) blast transformation of Ab4Ab4 homozygous lymphocytes in vitro and a maximum of 39% (mean +/- SEM = 19.77 +/- 1.6) blast transformation of heterozygous Ab4Ab5 lymphocytes. Similarly anti-Ab5 sera will induce a maximum of 82% (mean +/- SEM = 50.83 +/- 7.0) blast transformation of Ab5Ab5 homozygous lymphocytes in vitro and a, maximum of 42% (mean +/- SEM = 16.1 +/- 1.6) blast transformation of heterozygous Ab4Ab5 lymphocytes. Thus, the lymphocytes of an allotypically heterozygous rabbit appear to be selected to produce or express only one of the two genetically supplied allotypic determinants controlled by the "b" locus. A similar conclusion has been made in regard to allotypic expression by immunoglobulin-producing plasma cells. Previous data indicate that each lymphocyte carries or expresses more than one immunoglobulin group specificity (IgG, IgA, IgM) while each plasma cell carries only one. One possible interpretation of these data is that, while the production of immunoglobulin groups and allotypic specificities are under similar control in both the lymphocyte and the plasma cell, the lymphocyte produces immunoglobulin that remains in or attached to itself and the plasma cell produces immunoglobulins that are secreted rapidly.

In vitro studies of the rabbit immune system. V. Suppressor T cells activated by concanavalin A block the proliferation, not the induction of antierythrocyte plaque-forming cells.

The late B-cell proliferative phase of the in vitro antibody response by rabbit spleen cells is highly susceptible to suppression by activated T cells. The in vitro antisheep erythrocyte plaque-forming cell (PFC) response by spleen cells from normal or primed rabbits can be suppressed by adding concanavalin A (Con A), Con A-prestimulated peripheral blood or spleen lymphocytes, or supernates from Con A-prestimulated peripheral blood lymphocytes. The suppression is not mediated by a direct interaction of Con A with responding cells as shown by the effectiveness of prestimulated cells. Primed spleen cultures remain sensitive to Con A suppression as late as 72 h after initiation, and the addition of Con A after 24-72 h rapidly stops the increase in the number of PFC. T cells are required for Con A addition to be effective but the suppression can be induced at a time when T-helper cells are no longer necessary. Further, the suppressive effect of Con A addition is abrogated by specific antisera to rabbit T cells. We propose that Con A activates suppressor T cells which then exert their effects on proliferating PFC or their immediate precursor B cells. The early inductive or recruitment phase of the response is probably not blocked by suppressor cells. Also, there is an apparent relationship between the number of proliferating B cells and the number of suppressor cells required. Finally, the difficulties in inducing a stimulatory effect by Con A and the prolonged period that Con A addition is suppressive suggests that the rabbit has relatively more and/or longer-lived suppressor cells than the mouse and may be a particularly useful species for studying suppressive phenomena and their mechanisms.

Immunoglobulin M allotypes of the rabbit: identification of a second specificity.

Rabbit antiserums, originally prepared to react specifically with rabbit immunoglobulin allotypes Ab5 and Ab6, also react with some normal rabbit serums that clearly do not contain Ab5 or Ab6 allotypes. This reaction is due to antigenic specificity present on rabbit immunoglobulin M(IgM) and not on rabbit immunoglobulin G. Serums that contain this IgM allotype do not react with an antiserum that reacts with the known rabbit IgM allotype, Ms1. This specificity may therefore be identified as a second rabbit IgM allotype, Ms2.

Rabbit blood lymphocytes may be T cells with surface immunoglobulins.

Rabbit peripheral blood lymphocytes and thymus cells do not respond to lipopolysaccharide mitogen in vitro, whereas spleen cells do. Soluble concanavalin A consistently stimulates 80 to 90 percent of rabbit peripheral blood lymphocytes, and the morphologic changes associated with such transformation may be observed within 18 hours after stimulation. Approximately 80 percent of rabbit peripheral blood lymphocytes have demonstrable immunoglobulin markers. These and other observations suggest that most rabbit peripheral blood lymphocytes are T cells with surface immunoglobulins.

Physical mapping of distinct 7q22 deletions in uterine leiomyoma and analysis of a recently annotated 7q22 candidate gene.

Uterine leiomyoma (UL) is a benign, smooth muscle tumor of the uterus affecting a significant proportion of women of reproductive age. Deletions involving chromosome 7q22 are common in UL and vary in length. Previously reported 7q22 deletion intervals were physically mapped using information from the recently completed human genome sequence. Four distinct deletion intervals, which included a microdeletion reported by our laboratory, were identified. This microdeletion contains two known genes, ORC5L and LHFPL3. The single deleted marker in the microdeletion was mapped within the LHFPL3 locus. The ORC5L gene has been studied in UL. Conversely, LHFPL3 has been annotated only recently, and has therefore not been studied in UL. The predicted LHFPL3 protein sequence contained a polyalanine domain, and a signature sequence for the PMP22 Claudin protein family. Members of this family are transmembrane proteins with roles in differentiation, proliferation, and extracellular matrix formation, and have been implicated in other tumors. Differences in LHFPL3 expression were observed in both human and Eker rat UL. Our results provide evidence for four distinct 7q22 deletion intervals, each with multiple candidate genes, including the recently identified LHFPL3 gene.

Yolk sac carcinoma grown from rat egg cylinders.

Eight-day rat egg cylinders transplanted in toto under the kidney capsules of adult histocompatible recipients gave rise to small nodules composed of parietal yolk sac cells surrounded by Reichert's membrane-like material. These yolk sac cells could grow and give rise to malignant, transplantable tumors identical to mouse yolk sac carcinomas derived from teratocarcinoma. The sera of rats with yolk sac tumors contained elevated concentrations of alpha1 fetoprotein.

Tissue sites of alpha fetoprotein synthesis by the rat during pregnancy and hepatoma growth.

The tissue sites of alpha1 fetoprotein (AFT) synthesis by the rat during gestation and hepatoma growth were determined by specific incorporation of a radiolabeled amino acid precursor into AFP by tissue cultures in vitro. During gestation, AFP were produced by the yolk sac, the fetal liver, and in small amounts by the fetal gastrointestinal tract; there was no synthesis by maternal rat tissues. During growth of a transplantable hepatoma, only the hepatoma tissue synthesized AFP; the nontumor tissue of the host contained AFP but did not produce it.

PIP: Alpha fetoprotein (AFP) synthesis by adult rats during gestation and hepatoma growth was determined in vitro with specific precipitations of radiolabeled AFP antisera after incubation of Spinner cultures of various rat tissues in arginine-free culture medium containing radiolabeled arginine. In general, AFP was synthesized by fetal liver, yolk sac, small intestine, and transplantable (tumor) tissue; none of the normal adult tissues, including testis or ovary, produced AFP. AFP synthesis (measured over 22 hours) was confined to the fetal liver (367 ng), yolk sac (1,368 ng), and to a small extent, the gastrointestinal tract during 19-day gestation. None of the maternal tissues produced AFP. When measured during growth of a transplantable hepatoma, AFP was synthesized only by the hepatoma tissue, though the nontumor tissue of the host contained AFP, due to release of AFP from the cultured tissue as it degenerated in vitro, but did not produce it (noninvolved tissues of hepatoma-bearing rats did not incorporate labeled arginine into AFP in vitro). Identifying fetal organs responsible for AFP synthesis explains observed AFP concentration changes in the postpartum period in rats, since elevated AFP in the mother is caused by AFP produced by the fetus which crosses the placenta or yolk sac to maternal circulation. Elevations above normal (.06 mcg/ml) adult rat concentrations occur in 3 circumstances in the nonpregnant rat: 1) development of AFP-producing tumors; 2) proliferation by normal liver cells; and 3) exposure to chemical carcinogens.

Expression of fibronectin and laminin in the rat liver after partial hepatectomy, during carcinogenesis, and in transplantable hepatocellular carcinomas.

The distribution in the F344 rat liver of two extracellular matrix and basement membrane components, fibronectin and laminin, was studied by immunofluorescence. Fibronectin was found diffusely in normal liver lining the sinusoids and in connective tissue surrounding blood vessels and bile ducts; laminin was present predominantly in the basement membranes of blood vessels and bile ducts and was only inconsistently seen lining the sinusoids. After partial hepatectomy (PH), there was a transient decrease of fibronectin in the central and midzone sinusoidal hepatic areas. This decrease was most marked on day 3 after the PH. Carcinogens caused marked changes in the distribution of fibronectin. Large extracellular deposits of fibronectin were seen in areas of oval cell proliferation in livers of rats treated with N-2-fluorenylacetamide (2-FAA) while being fed a choline-deficient diet. In contrast, the nodules that developed in these livers were almost completely devoid of fibronectin staining. Neoplastic nodules produced in rats by cyclic feedings of 2-FAA r by injections of diethylnitrosamine also contained little or no fibronectin. Laminin staining did not change markedly during these treatments, but increased staining was seen associated with the newly formed ductlike structures and oval cells in liver of rats treated with carcinogens. Transplantable hepatomas varied in their fibronectin staining from fibronectin-negative hepatomas to ones with fibronectin staining within or around every tumor cell. Laminin was only found around the vascular structures within the tumors. The presence or absence of fibronectin in hepatomas did not show an obvious correlation to growth rate or metastatic potential of the tumors studied.

Immunohistology of malignant rabbit fibroma virus--a comparative study with rabbit myxoma virus.

Malignant rabbit fibroma virus (MV) causes a syndrome that consists of disseminated malignant tumors and immunosuppression complicated by severe Pasteurella multocida infection and death. Tissues from rabbits given MV and rabbit myxoma virus were examined by direct immunofluorescence with the use of antibody against virus antigens. Primary and metastatic tumors caused by MV and rabbit myxoma virus were composed of soft tissue cells containing virus antigens. Skin appendages and epidermis overlying the respective tumors showed scant MV but abundant myxoma virus antigen. Both viruses were present systemically in the reticuloendothelial system. Epithelial cells from the liver, kidney, and lung of myxoma virus-infected rabbits contained virus, whereas in MV tumor-bearing rabbits, these cells were uninvolved. However, nasal mucosal and conjunctival epithelia, the locations of Pasteurella infection, showed squamous metaplasia and contained large amounts of MV and myxoma antigens. By analogy to other respiratory tract pathogens, these epithelial changes were probably etiologically significant for development of pasteurellosis in rabbits bearing virus-induced tumors. Thus by immunopathologic as well as clinical examination, MV produces a syndrome distinct from that seen with rabbit myxoma virus. MV induced severe immunosuppression despite T-lymphocyte hyperplasia in the lymphoid tissues observed. The combination of a systemic virus infection, epithelial alterations that impaired clearance mechanisms, and immunologic dysfunction is likely to contribute to the inability of rabbits given MV to survive their gram-negative infection.

An immunohistologic study of Shope fibroma virus in rabbits: tumor rejection by cellular reaction in adults and progressive systemic reticuloendothelial infection in neonates.

The development of lesions in adult and neonatal New Zealand White rabbits following intradermal inoculation of Shope fibroma virus was studied by immunofluorescence for viral antigens. T-cells, and immunoglobulin. In adults a self-limiting local fibroxanthosarcomatous tumor was rejected within 10-12 days in association with a dense infiltration of T-cells. In neonates expanding skin lesions were associated with systemic presence of virus in the reticuloendothelial system. In surviving infected neonates, granulomas formed at the site of infection after 3 weeks. These reactions may have limited further dissemination of the virus. These results support the hypothesis that the progressive disease produced by Shope fibroma virus in neonatal rabbits may be due to the inability of the reticuloendothelial system to clear infectious virus.

Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.

Cell-mediated immune response to Shope fibroma virus-induced tumors in adult rabbits.

The parameters of cell-mediated immune responses of adult rabbits infected with Shope fibroma virus (SFV) were characterized by measurement of the size of local draining nodes, number of cells per lymph node, mitogen responses of lymphocytes, and kinetics of virus-specific cell-mediated lymphocytotoxicity (CML). In addition, the cytolytic effector population was characterized. After intradermal injections, tumors appeared within 3-4 days, reached maximum size in 10-12 days, and then regressed completely with 24 days. The size of local popliteal lymph nodes, in particular the diffuse cortex (paracortex), and the number of cells per node increased during tumor growth but then declined as the tumor regressed. Maximum specific CML to SFV-infected kidney cell monolayers (RK-13) occurred 10 days after inoculation of SFV and correlated with the initiation of tumor regression. Adult cytotoxic lymphocytes passed through nylon wool, and most of their activity was removed by treatment with antithymocyte globulin plus complement. Cytotoxic T-cells from SFV tumor-bearing rabbits killed only targets infected with SFV and not targets uninfected or infected with vaccinia virus. Therefore, T-cell-mediated virus-specific CML appeared as a major immune effector mechanism that correlated with tumor regression. However, antibody-dependent cell-mediated and NK cytotoxicity were also demonstrable. The presence of different cell-mediated cytotoxic mechanisms suggested a heterogeneity of effector mechanism.