Fast specific field detection of RHDVb.

This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross-reactivity with other RHDV types giving a specific result in under 10 min using rabbit liquid exudates or liver homogenate samples taken at necropsy.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts.

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

A specific and ultrasensitive chemiluminescent sandwich ELISA test for the detection and quantitation of pneumolysin.

A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml(-1), with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively. The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains, a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.

Circulating IgG response to stromelysin-3, collagenase-3, galectin-3 and mesothelin in patients with pharynx/larynx squamous cell carcinoma.

With the aim of identifying tumor-associated antigens that could be potential markers and/or targets of diagnostic and/or therapeutic approaches, we studied the occurrence of circulating IgG antibodies to human stromelysin-3, collagenase-3, galectin-3 and mesothelin, by Western blot against their purified recombinant forms, in the sera of 50 patients with pharynx/larynx squamous cell carcinoma (PLSCC), as well as in the sera of 50 healthy blood donors. Overall, antibodies to collagenase-3 were detected in 50% of all the cancer patients and 16% of the blood donors examined; this percentage difference was statistically significant (p = 0.00066). With respect to anti-galectin-3 antibodies, the percentages were 32% and 18%, respectively, but they were not statistically different (p = 0.16). Low levels of antibodies to stromelysin-3 and to mesothelin were detected in sera from only two cancer patients. No significant correlations were found in the present study between the presence of antibodies to these proteins and tumor site, clinical and T stages, lymph node involvement, DNA ploidy and histological grade of differentiation of the primary tumors. To our knowledge, this is the first report on the detection of circulating IgG to collagenase-3 in cancer patients. Some of the percentages found here in certain groups of patients are among the highest reported of circulating antibodies to any tumor component studied so far. The monitoring and the use of human antibodies to collagenase-3 could be of diagnostic and therapeutic interest.

Quantitative immunohistochemical analyses of the expression of E-cadherin, thrombomodulin, CD44H and CD44v6 in primary tumours of pharynx/larynx squamous cell carcinoma and their lymph node metastases.

The quantitative expression of E-cadherin, thrombomodulin, CD44H and CD44v6 in 32 specimens of primary tumours of pharynx/larynx squamous cell carcinoma and their lymph node metastases was studied by immunohistochemistry. With the aim of obtaining comparative and objective data, image acquisition conditions were kept unaltered for all the measurements and the immunostaining intensity was quantified by applying an image processing system. On the one hand, correlations were only observed between CD44H and CD44v6, both in primary tumours and metastases, and between E-cadherin and TM in metastases. On the other hand, statistical analyses of paired data did not show significant differences in the expression of these markers between the two tumour sites. In agreement with previous reports, E-cadherin expression was rather low or negative in primary tumours and metastases of the three poorly differentiated specimens we studied, as well as that of TM, but otherwise some of these samples showed intermediate immunostaining levels of CD44H/CD44v6. It may be concluded from the present study that the quantitative expression of these adhesion molecules in well established lymph node metastases of pharynx/larynx squamous cell carcinoma is essentially unaltered in relation to their primary sites.

Cell proliferation activity and kinetic profile in the prognosis and therapeutic management of carcinoma of the pharynx and larynx.

Prognosis and management of carcinoma of the pharynx and larynx is now based on the morphologic analysis of the tumor spreading, differentiation grading, and type of microscopic invasion. The DNA ploidy status and the cell proliferation activity analyzed by flow cytometry give us complementary information about the prognosis and the management and support of the patients. We performed a study of 91 cases of carcinoma of the larynx and pharynx by means of flow cytometry. Forty-three patients were treated by surgery alone, and 48 patients also received radiotherapy. Fifty-five were aneuploid (60%); this percentage increased to 74% in the pharynx area and fell to 47% at the larynx level. The aneuploid tumors showed worse behavior in the patients treated by surgery alone compared with those who also received radiotherapy. The S-phase fraction was high in aneuploid tumors, in positive lymph nodes, and in advanced stages. The S-phase fraction was higher in poorly differentiated tumors. In patients treated by surgery alone, we noticed that by combining both cytometric variables two different kinetic profiles could be defined related to the patients' behavior. The diploid tumors with a low S phase had the greatest rates of survival, whereas diploid tumors with a high rate of S phase and aneuploids had a lower rate.

The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

Performance of thiophilic adsorption chromatography in the purification of rat IgG2b monoclonal antibodies from serum- and protein-free culture supernatants.

The usefulness of thiophilic adsorption chromatography for the purification of rat IgG2b monoclonal antibodies has been evaluated. This approach has not shown specificity for immunoglobulins; therefore, to minimize potential interferences, the purification was carried out from supernatants of hybridomas grown in serum- and protein-free conditions. The protein purity of the six final antibody preparations assayed was always >/= 97%. In addition to the easiness of this procedure, which enables one-step antibody purification, the materials employed are rather inexpensive and milligram amounts of antibody can be recovered from 1 L of supernatant. Overall, the purification of rat IgG2b monoclonals under the conditions reported here offers an advantageous alternative to other more expensive and cumbersome methods.

Rapid and reliable identification of Streptococcus pneumoniae isolates by pneumolysin-mediated agglutination.

A pneumolysin-based agglutination test which allows an easy, rapid, cost-effective, and accurate (100% specific and 95% sensitive) discrimination between pneumococci and other related human and animal pathogenic bacterial strains has been assayed.

Immunofluorometric assay of pepsinogen C and preliminary clinical applications.

We developed mouse monoclonal antibodies (Abs) against pepsinogen C with highly purified antigen isolated from gastric mucosa. The Abs were used to construct a two-site sandwich-type assay for pepsinogen C with time-resolved fluorometry as a detection technique. The assay has a detection limit of 0.1 microgram/L and is precise (within-run and day-to-day CVs < 11%). We used this assay to measure pepsinogen C in seminal plasma, breast cyst fluid, amniotic fluid, male and female serum, serum from patients with prostate cancer, urine, breast tumor cytosolic extracts, breast milk, and cerebrospinal fluid. Highest pepsinogen C concentrations were in seminal plasma, followed by breast cyst fluid and amniotic fluid. We found no correlation between prostate-specific antigen concentrations and concentrations of pepsinogen C in serum of prostate cancer patients, and concluded that this marker is not useful for either diagnosing or monitoring prostatic carcinoma. The availability of a highly sensitive, reliable, and convenient method for quantifying pepsinogen C will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including benign breast diseases, breast cancer, fertility, and pregnancy.

Comparative flow cytometric analysis of DNA-bound PCNA and DNA content as estimators of S-phase cells in cell cultures.

Flow cytometric estimations of S-phase cells were carried out on cultures from three different cell lines and in frozen aliquots. A PCNA-extraction protocol was applied. Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA non-bound PCNA were compared with those obtained from total DNA histograms (Vindelöv and Christensen's technique, methanol-fixed whole cells and PCNA-extracted nuclei). No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells. Nevertheless, nuclei yield following PCNA extraction was highly variable, ranging from 63% to 10% (mean: 26%). In some cases, the extraction was not complete and samples had to be discarded. Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined. Because of these shortcomings, and the fact that is more costly and time consuming, the estimation of the S-phase fraction by means of bivariate DNA-bound PCNA/total DNA flow cytometric studies does not seem to surpass that obtained from standard DNA cell cycle analyses.

Functional analysis of pneumolysin by use of monoclonal antibodies.

We have produced a panel of monoclonal antibodies to pneumolysin, the membrane-damaging toxin from Streptococcus pneumoniae. We have used these antibodies to identify three regions of the toxin sequence that are involved in the lytic mechanism of this toxin. Two of these sites probably form the cell binding site of this toxin. Antibodies to the third site inhibit the lytic action of this toxin but not the binding of this toxin to cells. This site is engaged in the oligomerization process involved in the formation of pores in cell membranes. Two of these epitopes are also present in the related toxin perfringolysin O.

Yersinia enterocolitica serotype 0:3-induced arthritis in mice: microbiological and histopathological information.

Gross anatomical and histopathological changes in arthritic joints resulting from oral challenge with Yersinia enterocolitica serotype 0:3, upon pretreatment with desferrioxamine, were always more severe than those induced by intravenous infection of immunized animals. In all the acute inflammation episodes studied, live Yersiniae were isolated from the arthritic region. Invariably, a heavy mixed infiltration of synovia, joint spaces and soft tissues was observed at this stage. Concurrent fibrous thickening and vascular proliferation, along with erosion of articular cartilages and anomalous bone regeneration, were also apparent. In spite of these significant facts, the bacterium could be histopathologically identified only in bone marrow where it developed microcolonies and caused significant necrosis as well. The live bacterium was also retrieved from two- and six-month-old arthritic ankles/paws examined, but it could not be seen in histological sections of joints. By this time, no cellular infiltration was evident, but there was extensive fibrosis. Bones were at times greatly enlarged, showing a spongeous-like structure. Additionally, articular cartilages could be completely lost and were substituted by an anomalous ossification filling the joint spaces. This situation led to bone fusion, resembling articular ankylosing traits. In summary, we present the first experimental evidence that Y. enterocolitica serotype 0:3 is a causal agent of osteoarthritis and osteomyelitis, and that it may survive for prolonged periods of time in osseous structures.

Protection against the lethal effect and arthritogenic capacity of Yersinia enterocolitica serovar O:3 for mice.

Protection against the intravenous lethal effect of Yersinia enterocolitica serovar O:3 for mice can be achieved by oral immunization with bacteria expressing the O:3 lipopolysaccharide (LPS). Under similar experimental conditions, Ca(2+)-dependent cells are more protective than their Ca(2+)-independent counterparts, live vaccines are more efficacious than killed ones, and parenteral immunization is more efficient than the oral route. Antibodies induced by enzyme-treated LPS from an O:3 strain are able to mediate protection against challenge with one homologous strain, but they do not prevent induction of arthritis.

Yersinia enterocolitica serotype O:3 is arthritogenic for mice.

It is shown, for the first time, that Yersinia enterocolitica serotype O:3 is experimentally arthritogenic. Moreover, it is arthritogenic for the mouse, an optimal model for human yersiniosis. This arthritis can be induced by the oral route, the most common route in man. The pattern of joint disease closely parallels that of human reactive arthritis associated with this pathogen.

Flow cytometric analysis of the Hermes homing-associated antigen on human lymphocyte subsets.

The homing of lymphocytes is controlled by interactions with high endothelial venules (HEV), specialized vessels that define sites of lymphocyte extravasation into lymph nodes and inflamed tissues. In humans, lymphocyte-HEV binding involves a lymphocyte surface glycoprotein (GP) of 85 to 95 kd (CD44, H-CAM), defined by monoclonal antibody (MoAb) Hermes-1. To define the expression of this homing-associated adhesion molecule during human lymphocyte development, we performed two-color immunofluorescence analyses of human bone marrow (BM), thymus, peripheral blood (PB), and tonsillar lymphocytes. The highest levels of Hermes-1 antigen are displayed by circulating B and T cells in the blood, which are uniformly positive and bear roughly twice the level of antigen present on mature lymphocytes within organized lymphoid tissues and BM. "Immature" (CD4+, CD8+) T cells in the thymus are Hermes-1lo to-, whereas thymocytes of mature phenotype (CD4+ or CD8+) are positive. The Hermes-1 antigen is present at high levels on the same population of thymocytes that bears high surface levels of CD3, a component of the T-cell antigen receptor complex, suggesting that levels of T-cell homing and antigen receptors characteristic of mature peripheral T cells appear coordinately during thymocyte maturation/selection. Essentially all T cells in the periphery are Hermes-1hi, including T blasts, and the homing-associated antigen is maintained at high levels on T cells stimulated in vitro by phytohemagglutinin (PHA) and on interleukin-2 (IL-2) maintained T-cell clones and lines. In contrast, although most resting IgD+ B cells are positive a significant fraction of B cells in tonsils are Hermes-1lo to-; these cells are predominantly PNAhi, IgD-, and CD20hi, a phenotype characteristic of sessile, activated B cells in germinal centers. In all lymphocyte populations examined, there is a linear correlation in staining for Hermes-1 and for Hermes-3, an antibody that defines a distinct functionally important epitope on this molecule. The results demonstrate a precise regulation of this homing-associated antigen during lymphocyte differentiation.

Monoclonal antibodies against the CD44 [In(Lu)-related p80], and Pgp-1 antigens in man recognize the Hermes class of lymphocyte homing receptors.

An 85- to 95 kDa class of lymphocyte surface molecules, defined in man by antibodies of the Hermes series, is involved in lymphocyte binding to high endothelial venules and is likely of central importance in the process of lymphocyte homing. In this report, we have examined the relationship between these Hermes-defined "homing-receptors" and two other 80 to 95 kDa lymphocyte surface molecules that have been extensively studied--CD44 [In(Lu)-related p80] defined by mAb A1G3 and A3D8, and Pgp-1 defined by antibody IM7. Our findings indicate that, in man, similar or identical glycoprotein(s) are recognized by these independently and diversely obtained antibodies. All antibodies showed identical immunohistologic patterns of reactivity on a variety of lymphoid and nonlymphoid human tissues, and demonstrated similar bands on Western blots of both crude tonsil lymphocyte lysates and highly purified Hermes-1 Ag preparations. Similarly, purified CD44/p80 Ag from RBC and human serum bound Hermes-1. Preclearing of tonsil lysates with the Hermes-1 antibody removed antigenic activity for all antibodies. Cross-blocking experiments demonstrated that A3D8, IM7 (anti-Pgp-1), and Hermes-2 antibodies recognize overlapping epitopes. Finally, expression of the epitopes defined by the Hermes-1, Hermes-3, H2-7, and H3-61 antibodies on RBC was shown to be regulated by the In(Lu) gene. These findings unify several different lines of investigation, and suggest the possibility that the CD44/Pgp-1/Hermes class of molecules may serve as cell-cell or cell-substrate adhesion/recognition elements for both hematolymphoid and non-hematolymphoid cell types.

Human lamina propria lymphocytes bear homing receptors and bind selectively to mucosal lymphoid high endothelium.

It has been hypothesized that the selective recognition of tissue-specific endothelial cell molecules helps determine the in vivo distribution of lymphoid effector cells by controlling the extravasation of their circulating precursors. Here we report (a) immunofluorescence studies of the cell surface phenotype of human lamina propria lymphocytes (LPL), including staining with monoclonal antibody Hermes-1, which defines a 90-kDa lymphocyte surface glycoprotein involved in recognition of high endothelial venules (HEV); and (b) functional analyses of the ability of LPL to bind to HEV in frozen sections of mucosal lymphoid tissues (appendix or Peyer's patch) vs. peripheral lymph nodes. Essentially all LPL bear the Hermes-1 antigen, over 90% at levels comparable to those of circulating PBL. As a population, LPL display a quantitative preference for adherence to mucosal HEV, binding 0.8-1.5 times as well as PBL to mucosal HEV, but only 0.1-0.5 times as well to HEV in peripheral lymph nodes. Of particular interest was the behavior of the lymphoblast fraction, which typically constituted 3-7% of LPL. These cells, defined by size, consisted of a mixture of T cells and surface IgA+ blasts. One hundred percent were Hermes-1 bright, and they bound 4-8 times more efficiently to mucosal HEV than PBL while failing to bind detectably to lymph node HEV. LPL binding to mucosal HEV involves the gp90 Hermes, since the monoclonal anti-gp90 antibody, Hermes-3, and a polyclonal anti-gp90 antiserum inhibit the binding of small LPL and of LP blasts. The remarkable efficiency and specificity of binding by LP blasts may reflect retention of homing properties of the blood-borne precursors of these blasts and is discussed in relation to the capacity of immunoblasts in mesenteric nodes and in thoracic duct lymph to traffic selectively to mucosal lymphoid and extralymphoid sites. The demonstration of organ-specific endothelial cell recognition by LP lymphoblasts provides considerable support for the concept that selective interactions with endothelium play an important role in directing the distribution of activated lymphocyte subsets in vivo.

Lymphocyte recognition of high endothelium: antibodies to distinct epitopes of an 85-95-kD glycoprotein antigen differentially inhibit lymphocyte binding to lymph node, mucosal, or synovial endothelial cells.

The tissue-specific homing of lymphocytes is directed by specialized high endothelial venules (HEV). At least three functionally independent lymphocyte/HEV recognition systems exist, controlling the extravasation of circulating lymphocytes into peripheral lymph nodes, mucosal lymphoid tissues (Peyer's patches or appendix), and the synovium of inflamed joints. We report here that antibodies capable of inhibiting human lymphocyte binding to one or more HEV types recognize a common 85-95-kD lymphocyte surface glycoprotein antigen, defined by the non-blocking monoclonal antibody, Hermes-1. We demonstrate that MEL-14, a monoclonal antibody against putative lymph node "homing receptors" in the mouse, functionally inhibits human lymphocyte binding to lymph node HEV but not to mucosal or synovial HEV, and cross-reacts with the 85-95-kD Hermes-1 antigen. Furthermore, we show that Hermes-3, a novel antibody produced by immunization with Hermes-1 antigen isolated from a mucosal HEV-specific cell line, selectively blocks lymphocyte binding to mucosal HEV. Such tissue specificity of inhibition suggests that MEL-14 and Hermes-3 block the function of specific lymphocyte recognition elements for lymph node and mucosal HEV, respectively. Recognition of synovial HEV also involves the 85-95-kD Hermes-1 antigen, in that a polyclonal antiserum produced against the isolated antigen blocks all three classes of lymphocyte-HEV interaction. From these studies, it is likely that the Hermes-1-defined 85-95-kD glycoprotein class either comprises a family of related but functionally independent receptors for HEV, or associates both physically and functionally with such receptors. The findings imply that related molecular mechanisms are involved in several functionally independent cell-cell recognition events that direct lymphocyte traffic.