Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.     

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.     

TB Incidence in an Adolescent Cohort in South Africa

Background

Tuberculosis (TB) is a major public health problem globally. Little is known about TB incidence in adolescents who are a proposed target group for new TB vaccines. We conducted a study to determine the TB incidence rates and risk factors for TB disease in a cohort of school-going adolescents in a high TB burden area in South Africa.

Methods

We recruited adolescents aged 12 to 18 years from high schools in Worcester, South Africa. Demographic and clinical information was collected, a tuberculin skin test (TST) performed and blood drawn for a QuantiFERON TB Gold assay at baseline. Screening for TB cases occurred at follow up visits and by surveillance of registers at public sector TB clinics over a period of up to 3.8 years after enrolment.

Results

A total of 6,363 adolescents were enrolled (58% of the school population targeted). During follow up, 67 cases of bacteriologically confirmed TB were detected giving an overall incidence rate of 0.45 per 100 person years (95% confidence interval 0.29–0.72). Black or mixed race, maternal education of primary school or less or unknown, a positive baseline QuantiFERON assay and a positive baseline TST were significant predictors of TB disease on adjusted analysis.

Conclusion

The adolescent TB incidence found in a high burden setting will help TB vaccine developers plan clinical trials in this population. Latent TB infection and low socio-economic status were predictors of TB disease.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Association of Vitamin D Receptor Polymorphism with Susceptibility to Symptomatic Pertussis

Pertussis, caused by infection with the gram negative B. pertussis bacterium, is a serious respiratory illness that can last for months. While B. pertussis infection rates are estimated between 1–10% in the general population, notifications of symptomatic pertussis only comprise 0.01–0.1% indicating that most individuals clear B. pertussis infections without developing (severe) clinical symptoms. In this study we investigated whether genetic risk factors are involved in the development of symptomatic pertussis upon B. pertussis infection. Single-nucleotide polymorphisms (SNPs) in candidate genes, MBL2, IL17A, TNFα, VDR, and IL10 were genotyped in a unique Dutch cohort of symptomatic clinically confirmed (ex-)pertussis patients and in a Dutch population cohort. Of the seven investigated SNPs in five genes, a polymorphism in the Vitamin D receptor (VDR) gene (rs10735810) was associated with pertussis. The VDR major allele and its homozygous genotype were more present in the symptomatic pertussis patient cohort compared to the control population cohort. Interestingly, the VDR major allele correlated also with the duration of reported pertussis symptoms. Vitamin D₃ (VD₃) and VDR are important regulators of immune activation. Altogether, these findings suggest that polymorphisms in the VDR gene may affect immune activation and the clinical outcome of B. pertussis infection.     

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.     

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

 Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10⁻² s⁻¹. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting. Received: 13 July 2000 / Accepted: 12 October 2000