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Objectives: Lucilia sericata maggots are successfully used for treating chronic wounds. As the healing process in these wounds is complicated by bacteria, particularly when residing in biofilms that protect them from antibiotics and the immune system, we assessed the effects of maggot excretions/secretions (ES) on Staphylococcus aureus and Pseudomonas aeruginosa biofilms, the clinically most relevant species. Methods: We assessed the effects of ES on biofilms using microtitre plate assays, on bacterial viability using in vitro killing and radial diffusion assays, and on quorum sensing systems using specific reporter bacteria. Results: As little as 0.2 μg of ES prevented S. aureus biofilm formation and 2 μg of ES rapidly degraded biofilms. In contrast, ES initially promoted P. aeruginosa biofilm formation, but after 10 h the biofilms collapsed. Degradation of P. aeruginosa biofilms started after 10 h and required 10-fold more ES than S. aureus biofilms. Boiling of ES abrogated their effects on S. aureus, but not on P. aeruginosa, biofilms, indicating that different molecules within ES are responsible for the observed effects. Modulation of biofilms by ES did not involve bacterial killing or effects on quorum sensing systems. Conclusions: Maggot ES are differentially effective against biofilms of S. aureus and P. aeruginosa.

In human immunodeficiency virus type 1 (HIV-1) infection, functional activities of T lymphocytes are impaired. Analogous to tumor-infiltrating T lymphocytes from cancer patients, in whom poor proliferative responses are associated with fewer zeta molecules, this study compared expression of CD3zeta molecules by T lymphocytes from HIV-infected persons and healthy controls. Flow cytometry and immunoblotting revealed significantly diminished zeta expression by CD3, CD4, and CD8 T lymphocytes from AIDS patients but not from persons without AIDS. zeta-mRNA levels were also decreased in cells from AIDS patients. CD3zeta expression correlated significantly with CD4 cell counts and HIV-1 RNA levels; impaired expression of CD3zeta molecules appeared to be reversible upon virus load reduction following highly active antiretroviral treatment (HAART). Thus, reduced expression of CD3zeta molecules by T lymphocytes from HIV-infected persons correlates with disease status. Investigations into CD3zeta expression by subpopulations of peripheral T lymphocytes and by T lymphocytes in lymphoid tissues will contribute to the understanding of immune reconstitution of HIV-infected patients following HAART.

Previously we have identified and characterized three murine microbicidal proteins purified from the granule fraction of cells from the murine macrophage cell line RAW264.7. During these studies evidence was obtained for the presence of an additional antimicrobial protein in the cytosolic fraction of RAW264.7 cells that had been activated with interferon-γ (IFN-γ). In this study we have purified this protein, designated ubiquicidin, to apparent homogeneity and demonstrated that it is a cationic, small (Mr 6654) protein. Ubiquicidin displayed marked antimicrobial activity against Listeria monocytogenes and Salmonella typhimurium. Using a gel overlay procedure evidence was obtained that the protein also displays activity against Escherichia coli, Staphylococcus aureus, and an avirulent strain of Yersinia enterocolitica. Amino-terminal amino acid sequencing and mass spectrometry analysis of purified ubiquicidin indicated that it is most likely identical to the ribosomal protein S30. This protein is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of S30 linked to an unusual peptide with significant homology to ubiquitin. The fau gene has been reported to be expressed in a variety of tissues in humans and various animal species. The presence of ubiquicidin in the cytosol of macrophages may serve to restrict the intracellular growth of microorganisms. In addition, because macrophage disintegration will likely lead to release of ubiquicidin into the extracellular environment, it may contribute to host defense after macrophage death. J. Leukoc. Biol. 66: 423–428; 1999.

Monomethylfumarate (MMF), the most active metabolite of the new antipsoriasis drug Fumaderm R , stimulates an anti-inflammatory mediator profile in human leucocytes and inhibits the proliferation of keratinocytes. These effects of MMF on cells in vitro may in part explain the beneficial action of Fumaderm R in patients. In addition, we have reported that MMF stimulates an increase in the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) and the cyclic adenosine monophosphate (cAMP) concentration in granulocytes and keratinocytes. Because Ca 2+ and cAMP control many physiological cellular responses, including cell proliferation and inflammatory mediator production, the present study focused on the intracellular signal transduction pathway which links interaction between MMF and granulocytes with increases in [Ca 2+ ] i and the cAMP concentration. The increase in [Ca 2+ ] i in granulocytes after MMF depended both on extracellular Ca 2+ and Ca 2+ from intracellular stores. Ca 2+ is essential for the increase in the cAMP concentration after stimulation with MMF. The results found for pharmacological inhibitors of various protein kinases suggest that a staurosporine-sensitive protein kinase different from protein kinase C (PKC) and protein kinase A is involved in the MMF-induced increase in [Ca 2+ ] i in granulocytes. As MMF activated protein tyrosine kinase (PTK). and inhibition of this protein kinase partially reduced the increase in [Ca 2+ ] i in granulocytes, PTK activity most likely is involved. In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF-stimulated increase in [Ca 2+ ] i in granulocytes as well. As MMF stimulated an increase in the GTP-ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca 2+ ] i and PKH4 activity of granulocytes stimulated by this compound, it is concluded that MMF activates PTX-sensitive G proteins. Competition binding studies with radiolabelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granulocytes. In summary, MMF binds to specific sites on the plasma membrane of cells. This interaction activates pertussis toxin-sensitive G proteins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca 2+ ] i and the intracellular cAMP concentration. Elevated [Ca 2+ ] i and intracellular cAMP concentration stimulate protein kinases that regulate transcription factors. Activation of these factors results in induction of downstream gene expression and thus controls cell functions, e.g. cell proliferation and production of inflammatory mediators, as has been found for cells incubated with MMF.