Evaluation of CD44 variant expression in oral, head and neck squamous cell carcinomas using a triple approach and its clinical significance.

Cancer stem cells possess the qualities of self-renewal, tumorigenesis and the ability to recapitulate a heterogeneous tumor. Our group was the first to isolate head and neck squamous cell carcinoma (HNSCC) stem cells using the cell surface marker CD44. CD44 is a trans-membrane glycoprotein with a multitude of key-functions that regulate cancer cell proliferation and metastasis. The variety of CD44 functions is due to tissue-specific patterns of glycosylation of the extracellular portion, and to the multiple protein isoforms (CD44 variants, CD44v) generated by alternative splicing. This study investigates the expression pattern of CD44 variants in HNSCC. Ten cell lines from the most common HNSCC locations and representative of various clinical outcomes were assayed by quantitative realtime PCR, flow cytometry and immunofluorescence comparatively with normal oral keratinocytes. The CD44 v4 and v6 were exclusively abundant in HNSCC while the isoform v1,2 was expressed in normal oral keratinocytes. Of interest, the highest level of CD44v6 expression was detected in advanced metastatic HNSCC, suggesting a link between CD44v6 expression and HNSCC metastasis, while the highest CD44v4 was detected in a stage IV HNSCC refractory to chemotherapy which developed recurrence. Oral-derived HNSCC expressed the highest CD44v4 and v6, and levels corresponded with staging, showing also an increasing tendency with recurrence and metastasis. CD44v were detected predominantly in smaller cells (a characteristic that has been associated with stem cell properties) or cells with mesenchymal morphology (a characteristic that has been associated with the migratory and invasive potential of epithelial tumor cells), suggesting that CD44v differential expression in HNSCC may be representative of the morphological changes inherent during tumor progression towards a more aggressive potential, and thus contributing to the individual tumor biology. The mechanism of CD44 variant involvement in HNSCC progression and metastasis is under investigation.

Real-time feedback on nonverbal clinical communication. Theoretical framework and clinician acceptance of ambient visual design.

INTRODUCTION: This article is part of the focus theme of Methods of Information in Medicine on "Pervasive Intelligent Technologies for Health". BACKGROUND: Effective nonverbal communication between patients and clinicians fosters both the delivery of empathic patient-centered care and positive patient outcomes. Although nonverbal skill training is a recognized need, few efforts to enhance patient-clinician communication provide visual feedback on nonverbal aspects of the clinical encounter. OBJECTIVES: We describe a novel approach that uses social signal processing technology (SSP) to capture nonverbal cues in real time and to display ambient visual feedback on control and affiliation--two primary, yet distinct dimensions of interpersonal nonverbal communication. To examine the design and clinician acceptance of ambient visual feedback on nonverbal communication, we 1) formulated a model of relational communication to ground SSP and 2) conducted a formative user study using mixed methods to explore the design of visual feedback. METHODS: Based on a model of relational communication, we reviewed interpersonal communication research to map nonverbal cues to signals of affiliation and control evidenced in patient-clinician interaction. Corresponding with our formulation of this theoretical framework, we designed ambient real-time visualizations that reflect variations of affiliation and control. To explore clinicians' acceptance of this visual feedback, we conducted a lab study using the Wizard-of-Oz technique to simulate system use with 16 healthcare professionals. We followed up with seven of those participants through interviews to iterate on the design with a revised visualization that addressed emergent design considerations. RESULTS: Ambient visual feedback on non- verbal communication provides a theoretically grounded and acceptable way to provide clinicians with awareness of their nonverbal communication style. We provide implications for the design of such visual feedback that encourages empathic patient-centered communication and include considerations of metaphor, color, size, position, and timing of feedback. CONCLUSIONS: Ambient visual feedback from SSP holds promise as an acceptable means for facilitating empathic patient-centered nonverbal communication.

Multiple interests in structural models of DARC transmembrane protein.

Duffy Antigen Receptor for Chemokines (DARC) is an unusual transmembrane chemokine receptor which (i) binds the two main chemokine families and (ii) does not transduct any signal as it lacks the DRY consensus sequence. It is considered as silent chemokine receptor, a tank useful for chemiotactism. DARC had been particularly studied as a major actor of malaria infection by Plasmodium vivax. It is also implicated in multiple chemokine inflammation, inflammatory diseases, in cancer and might play a role in HIV infection and AIDS. In this review, we focus on the interest to build structural model of DARC to understand more precisely its abilities to bind its physiological ligand CXCL8 and its malaria ligand. We also present innovative development on VHHs able to bind DARC protein. We underline difficulties and limitations of such bioinformatics approaches and highlight the crucial importance of biological data to conduct these kinds of researches.

Identification of mandibular fracture epidemiology in Canada: Enhancing injury prevention and patient evaluation.

BACKGROUND: Mandibular fractures can lead to significant functional and aesthetic sequelae if treated improperly. They may act as an indicator of concomitant trauma and are very demanding on the public health care system. Thus, knowledge of mandibular fracture epidemiology is critical to effective prevention, as well the establishment of accurate trauma evaluation protocols. OBJECTIVES: To identify the epidemiology of mandibular fractures treated at a level 1 Canadian trauma centre, clarify the pathogenesis of these epidemiological patterns and suggest potential targets for preventive efforts. METHODS: A retrospective review of all mandibular fracture patients presenting to the Montreal General Hospital between 1998 and 2003 was performed. Medical records and digitized radiographic imaging were used to collect patient demographics and injury data. RESULTS: The chart review identified 181 patients with 307 mandibular fractures. Fifty-two per cent of the fractures occurred in individuals 21 to 40 years of age, 78% of patients were male, and there was wide ethnic diversity. Sixty percent of patients had multiple mandibular fractures; 29% were symphyseal/parasymphyseal fractures, 25% were condylar fractures and 23% were angle fractures. Assault was the most common mechanism of injury, with 29% of fractures involving alcohol or illegal drug use. Thirty percent of patients had an associated facial fracture, and more than one-third had another major injury. CONCLUSIONS: The present epidemiological review reveals several potential prevention targets as well as significant trends. Further research into the impact of these preventive measures could more objectively identify their impact on mandibular trauma.

Rabies surveillance, trends in animal rabies and human post-exposure treatment in Poland, 1990 -2004.

This paper describes recent changes in the epizootical and epidemiological situation of rabies in Poland. Analysis of routine surveillance data on animal cases and human post-exposure treatment was performed in order to examine the impact of introduction of cell culture vaccine for human use and the implementation of the fox immunisation programme. The success of the immunisation programme for wild animals has become evident during the past 3 years, as a 9-fold decrease in animal rabies cases has been observed. To date, however, the downward trend in animal rabies cases has had no effect on the frequency of administration of the post-exposure treatment for humans. Moreover, two cases of locally acquired human rabies have occurred in patients who did not receive post-exposure vaccination. These cases prove that rabies should be still considered a public health concern in Poland.

Recombinant forms of Gerbich blood group antigens: expression and purification.

Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.

Recombinant forms of glycophorin C as a tool for characterization of epitopes for new murine monoclonal antibodies with anti-glycophorin C specificity.

Glycophorin C (GPC) and glycophorin D (GPD) are minor but important components of human RBC membranes. They carry the high-frequency antigens Ge2, Ge3 and Ge4 of the Gerbich blood group system. The epitopes for five new monoclonal antibodies (MoAbs) with anti-GPC specificity were characterized. Two antibodies (4G11 and 5B11) reacted with glycosylated N-terminal epitopes, and three reacted with internal epitopes of GPC. Pepscan analysis showed that the MoAb RB11 required for binding the EPDP sequence, occurring twice in GPC polypeptide chain. The MoAb 7F11 recognized the sequence 13PLSLEPDP20, and the MoAb RB8 did not react with synthetic peptides. Further characterization of the internal epitopes was performed in fluorescence-activated cell sorter (FACS) with the use of recombinant GPC and its variant forms transiently expressed on COS-7 cells. The results indicated that the MoAb RB11 recognized distinctly its target sequence EPDP only in a normal GPC molecule. The reactivity of the MoAb 7F11 with the PLSLEPDP sequence was confirmed and found to be enhanced by the O-glycan at the Ser15 residue. The MoAb RB8 recognized the glycopeptidic epitope in proximity to the Ser15 residue, requiring the presence of O-glycan. The combination of immunochemical techniques with the use of the recombinant forms of GPC has made it possible to define the role of sugar chains in the recognition of peptidic epitopes in glycosylated antigen and sheds new light on the Gerbich system antigens.

Section 5: Structural/genetic analysis of mAbs to blood group antigens. Coordinator's report.

The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Hydrogen-bonding in ethanol adducts to bis(3-R-penta-2,4-dionato)nickel(II) species.

Ethanol adducts of bis(3-R-penta-2,4-dionato) nickel(II) have been prepared by recrystallization of the corresponding nickel bisacetylacetonate species from ethanol, and their crystal structures have been determined by X-ray diffraction: R = methyl, C16H30NiO6, a = 5.177(1), b = 9.326(1), c = 9.649(1), a = 95.39(1), beta = 100.04(1), gamma = 97.16(1), space group Ponebar, Z = 1; R = hex-5-enyl, C26H46NiO6, a = 5.176(1), b = 9.677(1), c = 14.458(1), a = 92.333(3), beta = 93.945(4), gamma = 96.011(6), space group Ponebar, Z = 1; R = phenyl, C26H34NiO6, a = 27.399(1), b = 5.349(1), c = 19.827(2), beta = 117.410(7), space group C2/c, Z = 4. The compounds show remarkable differences in their ability to form hydrogen bonds in the solid phase and in solution, depending upon the nature of the substituent in the 3-position of the acetylacetone fragment. Analysis of the strength of hydrogen bonds within the limits of supermolecular approximation based on the results of calculations by DFT method has been carried out and were found to correlate with experimental observations.

Light-transmittance predictions under multiple-light-scattering conditions. I. Direct problem: hybrid-method approximation.

Our aim is to present a method of predicting light transmittances through dense three-dimensional layered media. A hybrid method is introduced as a combination of the four-flux method with coefficients predicted from a Monte Carlo statistical model to take into account the actual three-dimensional geometry of the problem under study. We present the principles of the hybrid method, some exemplifying results of numerical simulations, and their comparison with results obtained from Bouguer-Lambert-Beer law and from Monte Carlo simulations.

Light-transmittance predictions under multiple-light-scattering conditions. II. Inverse problem: particle size determination.

Our aim is to present the application of the hybrid method presented in part I to an inverse procedure to determine particle size and concentration under multiple-scattering conditions. The hybrid method is introduced as a combination of the four-flux method with coefficients obtained from Monte Carlo statistical simulations to take into account the actual three-dimensional geometry. Then an inversion scheme is expanded to enable the application of the hybrid method to particle size and concentration determination. We present the inversion method as well as exemplifying results of spectrum inversions.

Expression and binding properties of a soluble chimeric protein containing the N-terminal domain of the Duffy antigen.

The blood group Duffy antigen of human erythrocytes, which exists in two allelic forms, Fy(a) and Fy(b), is a promiscuous chemokine receptor. In this report we describe the expression and purification of a chimeric protein composed of the amino-terminal extracellular domain of the Duffy antigen (aa 3-60), C-terminal intracellular fragment of glycophorin A (GPA, aa 104-131), and the hexahistydyl tag. We obtained two forms of the recombinant protein containing the Fy(a) or Fy(b) epitope, denoted Fy(a)/GPA and Fy(b)/GPA, respectively. These constructs were expressed in Escherichia coli as periplasmic proteins and were purified by affinity chromatography on the Ni-NTA-agarose. Both proteins bound the monoclonal antibodies recognizing the common Fy6 epitope of the Duffy antigen and an epitope of the C-terminal fragment of GPA, and only the Fy(a)/GPA bound anti-Fy(a) antibody. However, binding of IL-8 to the recombinant proteins was not detected, which indicated that an N-terminal domain of the Duffy antigen is not sufficient for an effective chemokine binding. The lack of the chemokine binding was not likely to be due to the lack of glycosylation of the Fy/GPA, since IL-8 was effectively bound to de-N-glycosylated erythrocytes.

A molecular approach for isolating high-affinity Fab fragments that are useful in blood group serology.

BACKGROUND: Multiple mouse hybridoma antibodies recognize the antigens of the MNS blood group system. The Fab fragments of several of these antibodies were expressed on bacteriophage and as soluble proteins. The parental N92 anti-N IgG monoclonal antibody (parental N92 MoAb), but not its monovalent, soluble Fab fragment (N92 Fab fragment), agglutinated antigen-positive red cells by an antiglobulin method. Light-chain shuffling was used to isolate mutant N92 Fab fragments with higher affinity that would function by agglutination. STUDY DESIGN AND METHODS: Light-chain cDNA libraries, constructed from mice immunized with N-type glycophorin A, were inserted into a recombinant pComb3H vector containing the N92 Fd fragment. The N92 Fd fragment:light-chain libraries were panned on N-type glycophorin A or NN red cells, and antigen-binding clones were isolated. Purified parental N92 MoAb and the Fab fragments were evaluated by enzyme-linked immunosorbent assay and agglutination. RESULTS: The novel NNA7, C1, and G11 Fab fragments all bound to N-type glycophorin A with higher affinity than did the N92 Fab fragment. The affinity of the library-derived clones was equivalent to that of the parental N92 MoAb. Although their fine specificity differed slightly from the parental N92 MoAb, the clones functioned equivalently by agglutination using an antiglobulin method. CONCLUSIONS: Light-chain shuffling allowed the isolation of bacterially produced, high-affinity, soluble, monovalent recombinant anti-N Fab fragments that functioned well by agglutination. This approach is useful in obtaining inexpensive serologic reagents that may replace conventional MoAbs produced by tissue culture methods.

The murine monoclonal antibody NaM26-4C6 identifies a common structure on band 3 and glycophorin C.

The murine monoclonal antibody NaM26-4C6 (IgM class), obtained from the splenocytes of a BALB/c mouse immunized with human umbilical cord red blood cells, was characterized by agglutination test and immunoblotting analysis. The structure of the NaM26-4C6 epitope was further elucidated by using a series of peptides synthesized on pins. The antibody agglutinated untreated and chymotrypsin-treated but not trypsin- or neuraminidase-treated human erythrocytes. Agglutination-inhibition test demonstrated that the antibody recognizes an epitope located on the N-terminal trypsin-sensitive portion of glycophorin C. The antibody bound on immunoblots to glycophorin C, and also to the band 3 protein and its 69-kDa N-terminal fragment but did not bind to desialylated and de-O-glycosylated glycophorin C. Peptide mapping allowed localization of the binding site on the 23-kDa N-terminal intracellular peptide of band 3. The antibody binds to the amino-acid sequences 22EDPDIP27 of band 3 protein and 15SLEPDPGM22 of glycophorin C, and residues D and P were found to be essential. The new epitope identified by NaM26-4C6 corresponds to a linear amino acid sequence located on the N-terminal intracellular portion of band 3 and to a more complex structure involving oligosaccharide chains on the N-terminal extracellular domain of GPC.

Phage display selection of P1 mutants of BPTI directed against five different serine proteinases.

The P1 position of protein inhibitors and oligopeptide substrates determines, to a large extent, association energy with many serine proteinases. To test the agreement of phage display selection with the existing thermodynamic data, a small library of all 20 P1 mutants of basic pancreatic trypsin inhibitor (BPTI) was created, fused to protein III, and displayed on the surface of M13 phage. The wild type of displayed inhibitor monovalently and strongly inhibited trypsin with an association constant of Ka = 3 x 10(11) M(-1). The library was applied to select BPTI variants active against five serine proteinases of different specificity (bovine trypsin and chymotrypsin, human leukocyte and porcine pancreatic elastases, human azurocidin). The results of enrichment with four proteinases agreed well with the available thermodynamic data. In the case of azurocidin, the phage display selection allowed determination of the P1 specificity of this protein with the following frequencies for selected P1 variants: 43% Lys, 36% Leu, 7% Met, 7% Thr, 7% Gln.

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Only selected light chains combine with a given heavy chain to confer specificity for a model glycopeptide antigen.

The M and N human blood group glycopeptide Ags are carried on RBCs by glycophorin A. Previous results suggested that the murine humoral immune response against the N, but not the M, Ag is restricted. In addition, these results suggested that particular highly homologous heavy chains might be able to combine promiscuously with various light chains to yield anti-N specificity. To examine this, the current study used Fab phage methodology to couple an array of light chains, obtained from cDNA libraries isolated from immunized mice, to single Fd obtained from N61, N92, and 425/2B hybridomas. Interestingly, for the chimeric Fab to retain M or N specificity, the new light chains needed to belong to the same Vk gene family as the light chain from the parental, hybridoma-derived mAb. In some cases the new light chains modified the Fab affinity and fine specificity. For example, library-derived light chains coupled with the N92 Fd yielded chimeric Fab with increased affinity. In particular, the affinity of these univalent chimeric Fab for the N Ag was equivalent to that of the bivalent parental IgG mAb. Taken together, these results demonstrate that particular structures formed by the light chain V region are required to cooperate with a particular heavy chain V region to create a functional binding site for these glycopeptide Ags. They also demonstrate a lack of heavy chain promiscuity in the formation of murine anti-M and anti-N Abs.

Human CD34+ cells do not express glutathione S-transferases alpha.

The expression of glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+ cells and bone marrow was studied using RT-PCR and immunoblotting. The GSTA1 protein conjugates glutathione to the stem cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body. Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow. Similarly, baboon CD34+ cells and dog bone marrow do not express GSTA1. Human GSTA1 may be useful as a chemoprotective selectable marker in human stem cell gene therapy.

Mapping of peptidic epitopes of glycophorins A (GPA) and C (GPC) with peptides synthesized on plastic pins (Pepscan analysis).

The peptidic epitopes of 12 anti-GPA and 4 anti-GPC antibodies were identified with the use of peptides synthesized on the pins. Most of the antibodies were specific for epitopes located in extracellular portion of glycophorins, and only 2 anti-GPA and 1 anti-GPC recognized epitopes in their C-terminal cytoplasmic tails. The extracellular GPA epitopes were located in two regions of the polypeptide chain, within a.a. residues 38-44 and 49-58.

Fine characterization of a series of new monoclonal antibodies directed against glycophorin A.

OBJECTIVES: Glycophorins A (GPA) and B (GPB) are the major sialoglycoproteins of the human erythrocyte (RBC) membrane. To prepare tools for the analysis of GPA and GPB, we produced a series of new monoclonal antibodies (mAbs) that identified epitopes of GPA. METHODS: Seven murine monoclonal antibodies directed to glycophorin A (GPA) were fully characterized by agglutination of untreated and enzyme-treated human erythrocytes, inhibition of agglutination using chemically modified glycophorins and peptides from GPA, immunoblotting, and binding to synthetic peptides on plastic pins. RESULTS: The antibodies identify epitopes located on four different portions of GPA. (1) NaM13-6D2 binds to the N-terminal portion of GPA and GPB carrying the N blood group antigen; (2) NaM26-3F4 recognizes the homologous portion of GPA and GPB corresponding to their amino acids 6-26; (3) NaM10-2H12, NaM16-IB10 and NaM10-6G4 are specific for the amino acid sequence 38-45 of GPA; and (4) NaM37-5F4 and NaM13-4E4 bind to the amino acid residues 119-124 located on the intracellular ponion of GPA. CONCLUSION: These antibodies represent precise tools to investigate GPA and related molecules in different cells and tissues.