Anti-idiotypic monoclonal antibody AB3, reacting with the primary antigen (CEA), can localize in human colon-carcinoma xenografts as efficiently as AB1.

BALB/c mice were immunized with anti-idiotypic monoclonal (MAb) antibody (anti-Id or Ab2) directed against an AB1 MAb anti-carcinoembryonic (CEA) in order to obtain AB3 MAbs (anti-anti-Id). AB3 MAbs were shown to recognise the primary antigen (CEA) and one of them was tested extensively in vitro and in vivo. This AB3 MAb was shown to bind specifically to CEA on frozen sections of a human colon carcinoma by immunoperoxidase. Scatchard plot analyses showed that the affinity of this AB3 was of the same order of magnitude as the AB1. In vivo experiments, in nude mice bearing CEA-producing human colon-carcinoma xenografts showed that up to 30% of the intravenously injected dose of 125I-labelled AB3 were localized per gram of tumour tissue. Furthermore, calculation of the ratios of AB3 concentration in the tumour over those in normal organs such as lung, liver, kidney, spleen and bone gave relatively high values similar to results obtained with AB1. All together our results show that AB3 can localize as efficiently and specifically in the tumour as AB1, despite the fact that the mice from which it was derived were immunized with a mouse MAb (AB2) and had never been exposed to CEA.

Expression of E-Selectin and Vascular Cell Adhesion Molecule-1 in So-Called 'Negative' Appendices: First Results to Support the Pathological Diagnosis in Borderline Cases.

Background: Since the rate of histologically 'negative' appendices still ranges between 15 and 20%, appendicitis in 'borderline' cases remains a challenging disease. As previously described, cell adhesion molecule expression correlates with different stages of appendicitis. Therefore, it was of interest to determine whether the 'negative' appendix correlated with the absence of E-selectin or vascular cell adhesion molecule-1 (VCAM-1). Methods: Nineteen grossly normal appendices from a series of 120 appendectomy specimens from patients with suspected appendicitis were analysed in frozen sections for the expression of E-selectin and VCAM-1. As control, 5 normal appendices were stained. Results: This study showed a coexpression of E-selectin and VCAM-1 in endothelial cells in early and recurrent appendicitis. In patients with symptoms for less than 6 h, only E-selectin was detected. Cases with fibrosis and luminal obliteration were only positive for VCAM-1. In cases of early appendicitis with symptoms of less than 6 h duration, a discordance between histological and immunohistochemical results was found. Conclusions: This report indicates that E-selectin and VCAM-1 expression could be useful parameters in the diagnosis of appendicitis in borderline cases.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma.

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).

Clinical relevance of CD44 cell-surface expression and N-myc gene amplification in a multicentric analysis of 121 pediatric neuroblastomas.

PURPOSE: In contrast to other human tumors, a repression of the cell-surface glycoprotein CD44 on neuroblastoma is a marker of aggressiveness that usually correlates to N-myc amplification. We thus compared the prognostic value of both markers in the initial staging of 121 children treated for neuroblastoma in collaborative institutions. METHODS: Frozen samples were analyzed by a rapid and well-standardized technique of immunostaining with monoclonal antibodies (MoAbs) against epitopes in the CD44 constant region. RESULTS: In this retrospective series, CD44 was expressed on 102 specimens and strongly correlated with favorable tumor stages and histology, younger age, and normal N-myc copy numbers. In univariate analysis, CD44 expression and normal N-myc were the most powerful markers of favorable clinical outcome (P < 10(-6) and chi 2 = 65.40 and P < 10(-6) and chi 2 = 42.56, respectively), but analysis of CD44 affords significant prognostic discrimination in subgroups of patients with or without N-myc-amplified tumors. In the subgroup of stage IV neuroblastomas, CD44 was the only significant prognostic marker (P < .02, chi 2 = 5.76), whereas N-myc status was not discriminant. In multivariate analysis of five factors, ie, N-myc amplification, CD44 expression, age, tumor stage, and histology, the only independent prognostic factors of event-free survival were CD44 expression and tumor stage. CONCLUSION: The analysis of CD44 cell-surface expression must be recommended as an additional biologic marker in the initial staging of the disease.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

Visceral leishmaniasis and hemophagocytic syndrome

A 11 months old female infant from Portugal, free of family history, consults for apathy, weight loss, tachycardia, tachypnea, petechiae, pallor without icterus and hepatoslenomegaly. Seven months earlier, while being in Portugal, she presented a persistent bluish pimple on her buttock. Laboratory results showed anemia (35 g/l), leucopenia (3.3 G/l), thrombocytopenia (13 G/l), impaired coagulation (INR 1.4, PTT 41 sec.), hyponatremia (124 mmol/l), elevated CRP (139 mg/l), high ferritin (34.775 μg/l) and high triglycerides (5.22 mmol/l). After correction of vital parameters, a bone marrow aspiration and biopsy (BMB) revealed both the etiological diagnosis, namely a visceral leishmaniasis (VL) as well as one of its potential complications, the hemophagocytic syndrome (HS). Transfusions of whole blood, platelets and fresh frozen plasma were immediately started. Dexamethasone (10 mg/m2) and amphotericin B (3 mg/kg/day) have also been administrated. Visceral leishmaniasis is caused by a protozoan (Leishmania donovani) transmitted by the female sandfly. It is endemic in the Mediterranean basin (including France, Italy, Spain and Portugal), South America, sub-Saharan Africa as well as in India and Bangladesh. The parasite infects macrophages and, after several weeks of incubation, the disease occurs by affection of bloodlines (anemia, leucopenia, thrombocytopenia), hepatosplenomegaly, cachexia, gastrointestinal damage. The complications of the disease may lead to death. Liposomal amphotericin B is the currently recommended treatment. HS is caused by the proliferation and activation of macrophages in the marrow in response to a cytokine storm. It may be of primary cause. When it is secondary, it may be related to infections such as leishmaniasis. Patients present with fever and laboratory diagnostic criteria include cytopenia, hypertriglyceridemia, high ferritin and hemophagocytosis in the BMB. The treatment consists among other in the administration of high doses corticosteroids and, in secondary cases, in the treatment of the underlying cause. In conclusion, the clinical and biological features of VL may mimic haematological disorders as leukemia, but an enlargement of the liver and especially of the spleen should remind in this parasitic infection and its potential fatal complication, the HS.

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice.

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

Background. Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.Methods. Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9.Results. Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS).Conclusions. Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.

Comparison of aminolevulinic acid and hexylester aminolevulinate induced protoporphyrin IX distribution in human bladder cancer.

PURPOSE: Successful photodynamic therapy of epithelial cancer requires a specific photosensitization of malignant tissue. We evaluate the intensity and localization of protoporphyrin IX (PpIX) in superficial transitional cell carcinoma and nonmalignant cells of the human bladder following topical administration of its precursor, either aminolevulinic acid (ALA) or hexylester aminolevulinate (HAL). MATERIALS AND METHODS: Solutions of ALA or HAL were instilled into the bladder of 18 patients presenting with recurrent transitional cell carcinoma. The distribution of PpIX through the bladder wall was studied on frozen biopsies using fluorescence microscopy and correlated with pathological findings. RESULTS: Topical bladder instillation with 180 mmol (3%) ALA administered for 6 hours or 8 mmol (0.2%) HAL administered for 4 hours gave similar results regarding intensity and tissue distribution of PpIX fluorescence, whereas 8 mmol HAL administered for 2 hours followed by 2 hours of resting time (2+2 hours concept) induced a PpIX fluorescence twice as high. The fluorescence remained limited to cancer cells. Only a trace of PpIX fluorescence was observed in suburothelial connective tissue, that is chorion, but none in the bladder smooth muscle regardless of experiment conditions. CONCLUSIONS: HAL is an excellent precursor for PpIX synthesis in bladder cancer. With the 2+2 hour topical administration condition it yielded the highest PpIX fluorescence intensity and fluorescence contrast between normal and malignant urothelial cells. This approach allows us to optimize PpIX tissue distribution for photodynamic therapy in superficial bladder cancer.

Biomarker analysis of stored blood products: emphasis on pre-analytical issues.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

Ex-Vivo Ultransonographic Control Of Resection Margin During Partial Nephrectomy

Introduction & Objectives: Surgery remains the treatment of choice for localized renal cell neoplasia. While radical nephrectomy was long considered as gold standard, partial nephrectomy (PN) has widened its indications over the past twodecades and has shown oncological results equivalent to radical nephrectomy for small tumors. Moreover, it is considered superior to radical nephrectomy in terms of non-cancer related mortality. The role of negative surgical margin has been widely debated. Intraoperative frozen section analysis has been shown to be unreliable, expensive, time-consuming and not well correlated to final pathology. The goal of the present study was to assess the correlation of intraoperative exvivo ultrasonographic (US) evaluation of resection margin to definitive pathology in patients undergoing PN.Materials & Methods: An observational study was carried out in ours 2 institutions from February 2008 to October 2010. Patients undergoing PN for T1-T2 renal tumors were included. Ex vivo US evaluation was performed. Considering availability of US engine, not all consecutive eligible patients were included. PN was undertaken either by open surgery or laparoscopic access in a standardized technique. The "minimal healthy tissue margin" technique was applied. Once resected, the specimen was kept in a saline solution and US determination of tumor margins was performed. Sequential images were captured in order to evaluate the whole capsule.Results: Twenty-two patients (9 women, age 63±11 years[46-78]) were included in the present analysis. Open or laparoscopic PN was performed in 19 and 3 patients, respectively. Intraoperative ex-vivo US showed negative surgical margin in all cases except one, needing a complementary renal parenchyma resection. US duration ranged from 1 to 4 minutes, with a median time of 1 minute. Definitive histological analysis confirmed the presence of 3 angiomyolipoma, 15 clear cell carcinoma (11 pT1a,3 pT1b,1 pT2), 3 chromophobe carcinoma (1 pT1a,1 pT1b,1 pT2) and 1 pT1a type II papillary tumor. Mean tumor size was 3,4±2.1 cm [0,6-7,2]. Final pathology revealed R0 margins in all cases.Conclusions: Intraoperative ex-vivo US evaluation of resection margin in patients undergoing PN is feasible, time-efficient, well correlated to definitive pathological examination, and should be evaluated in further prospective trials.

Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients.

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin-W, the fourth and last member of the tenascin family remains the least well-characterized one. Our study aimed to evaluate the potential significance of tenascin-W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin-W levels, a sensitive sandwich-ELISA was established. Mean tenascin-W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin-C in the same patient cohort. However, the increase was much more striking for tenascin-W. We also detected elevated tenascin-W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin-W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin-W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin-C, tenascin-W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin-W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin-W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.