Association between RANTES polymorphisms and asthma severity among Tunisian children

The chemokine known as RANTES (regulated upon activation, normal T cells expressed and secreted) is an important element for the chemotaxis at the site of allergic inflammation. Many studies have made an interesting link between RANTES polymorphisms and asthma, showing that the variant in the promoter region is associated with high risk of asthma and severe airway obstruction. We conducted a case-control and family study aiming at identifying the relationship between polymorphisms (-28 C/G and -403 G/A) and haplotypes in the RANTES gene with asthma and severity. The results of the case control study suggest an association between alleles level of -28 C/G and -403 G/A promoter polymorphism (p = 0.01) (p = 0.00175) and asthma. Univariate analysis of the RANTES polymorphisms show an increased prevalence of the AC and AG haplotypes in asthmatics (p = 0.014) and (p = 0.015) respectively. Our data suggest that -28 C/G and -403 G/A polymorphisms within the RANTES promoter region play an important role in asthma predisposition and in the severity of airway obstruction.     

Natural Tregs, CD4+CD25+ inhibitory hybridomas, and their cell contact dependent suppression

The natural CD4+CD25+ T regulatory (Treg) lymphocyte has emerged as a critical cell for controlling immune responses to self, foreign proteins, and pathogens. Identified initially by the constitutive expression of CD4 and CD25, natural Tregs suppress a variety of immune cells and responses, including CD4+CD25− proliferation and IL-2 production, and CD8 cell proliferation, IFNγ production and CTL activity. Although natural Tregs require activation with specific antigen to attain their suppressive phenotype, once activated they execute inhibition in an antigen specific as well as non-specific (bystander) fashion. Treg suppression depends on IL-2, CD25, and cell:cell contact. The use of live cell imaging in vivo and in vitro to visualize the dynamic cell:cell interactions involving natural Tregs as well as the CD4+CD25+ Treg inhibitory hybridoma RD6 has refined the mechanistic models of contact dependent Treg suppression.     

Human neutrophil peptide-1 inhibits both the classical and the lectin pathway of complement activation

Human neutrophil peptide-1 (HNP-1) is a member of the alpha-defensin family. Defensins are cationic antimicrobial peptides, which play an important role in the antimicrobial response to microorganisms. In addition, recent studies have revealed the involvement of defensins in inflammation, immunity and wound repair. Defensins are present in the azurophilic granules of neutrophils and are released upon neutrophil stimulation. Previous studies showed that HNP-1 binds to C1q and inhibits the classical complement pathway. In view of the structural and functional similarity between C1q and MBL, we have now examined the interactions between HNP-1 and MBL. We observed a dose-dependent binding of HNP-1 to MBL in calcium-free buffer, indicating that HNP-1 binds to MBL most likely via the collagenous domains. To identify the binding sites in HNP-1 involved in the binding to C1q and MBL, we used a series of overlapping synthetic linear peptides that spanned the entire HNP-1 sequence. Both MBL and C1q showed a dose-dependent binding to the same set of peptides, suggesting a similar binding site in HNP-1 for both MBL and C1q. Strongest binding was observed to peptides containing the C- or N-terminal part of the HNP-1 molecule. Using an ELISA based system, we demonstrated that HNP-1 inhibits activation of both the classical pathway and lectin pathway of complement. Furthermore, we demonstrated that C1q and MBL can form complexes with HNP-1 in solution. Together, the data indicate that HNP-1 interacts with both C1q and MBL efficiently resulting in inhibition of both the classical and the lectin pathway of complement. We conclude that HNP-1 may play a role in protection against tissue injury during inflammatory conditions by inhibiting the early phase of complement activation.     

Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin

Stressed cells undergoing necrosis release molecules that acts as endogenous danger signals to alert and activate innate immune cells. Both HMGB1 and HSP70 are induced in activated monocytes/macrophages and also are released from stressed or injured cells. We investigated whether HMGB1 and HSP70 released from necrotic monocytes/macrophages, can act as danger signals to mediate proinflammatory cytokine responses to bacterial endotoxin or lipopolysaccharide (LPS). We show that cell lysate, obtained from necrotic cells directly stimulates the proinflammatory cytokine and chemokine responses in human monocyte/macrophage cell line, THP-1, as revealed by the induction of TNF-alpha, IL-6 and IL-8 mRNA expression and protein production. In the presence of LPS, necrotic cell lysate induced a more robust increase in all three proteins. We found that HMGB1 and HSP70 were indeed present in the necrotic cell lysate and were responsible for the significant induction of the proinflammatory cytokine expression, as neutralization with antibodies against both proteins blocked the increase in the cytokine production seen after incubating LPS-stimulated cells with the necrotic cell lysate. We also found that the newly identified triggering receptor expressed on myeloid cells-1 (TREM-1) was involved in mediating the HMGB1- and HSP70-induced cytokine production. Blocking TREM-1 on THP-1 cells with a recombinant chimera prevented the increase in cytokine production, while simultaneous blocking of TLR4 and TREM-1 completely abolished the proinflammatory response, suggesting that TREM-1 synergizes with TLR4 to mediate the effects of such signals from necrotic cells. In addition, blocking HMGB1 or HSP70 simultaneously with TREM-1 did not decrease the cytokine level further, confirming the involvement of TREM-1 in mediating the effect of HMGB1 and HSP70. Although the interaction of HMGB1 and HSP70 with TREM-1 induced I kappa B alpha and p38 expression, both of which are required for the inflammatory cytokine expression, blockade of TREM-1 did not affect I kappa B alpha expression but markedly reduced p38 activation, as revealed by Western blot analysis. Together, these results demonstrate that HMGB1 and HSP70 released from necrotic cells function as endogenous danger signals to augment the proinflammatory responses in monocytes/macrophage and that TREM-1 relays such signals to the cytokine expression cascade. This mechanism may contribute to the amplification and persistence of the inflammatory response to bacterial infection.     

Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture

The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.     

Myeloperoxidase is critically involved in the induction of organ damage after renal ischemia reperfusion

In this study the role of myeloperoxidase (MPO) in a murine (C57BL/6) model of ischemia and reperfusion (I/R)-induced renal failure was investigated. The renal function after I/R was analyzed in MPO-deficient (Mpo(-/-)) mice and compared with wild-type (WT) controls. A significant reduction in renal function loss (blood urea nitrogen) was observed after 24 hours of reperfusion of ischemically damaged kidneys in Mpo(-/-) mice compared with I/R WT controls (I/R Mpo(-/-) = 31.3 +/- 1.7 mmol/L versus I/R WT = 42.8 +/- 2.1 mmol/L, sham = 7.0 +/- 0.5 mmol/L; P = 0.003). The early reperfusion phase (2 hours of reperfusion) was characterized by a substantial increase in apoptosis and early complement activation, surprisingly similar in Mpo(-/-) and WT mice. Improved renal function in Mpo(-/-) mice after extended reperfusion was accompanied by a reduced neutrophil influx (P = 0.017) compared with WT controls. Activation and deposition of complement was not significantly reduced in Mpo(-/-) mice compared with WT controls after 24 hours of reperfusion, indicating no specific in vivo role for MPO in activating complement after renal I/R. Taken together, these results demonstrated an important contribution of MPO in the induction of organ damage after renal I/R by influencing critical factors such as neutrophil extravasation but not complement activation.     

Effects of JC virus infection on anti-apoptotic protein survivin in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the productive infection of oligodendrocytes by the opportunistic polyomavirus JC virus (JCV). Apoptosis is a host defense mechanism to dispose of damaged cells; however, certain viruses have the ability to deregulate apoptotic pathways to complete their life cycles. One such pathway involves survivin, a member of the inhibitor of apoptosis family, which is abundantly expressed during development in proliferating tissues but should be absent in normal, terminally differentiated cells. Immunohistochemistry performed in 20 cases of PML revealed the presence of survivin in JCV-infected oligodendrocytes and bizarre astrocytes within demyelinated plaques. Survivin up-regulation was also found in oligodendroglial and astrocytic cultures infected with JCV. Cell cycle analysis and DNA laddering demonstrated a significantly lower number of cells undergoing apoptosis on JCV infection compared with noninfected cultures; small interfering RNA inhibition of survivin resulted in a dramatic increase in apoptotic cells in JCV-infected cultures. This is the first report describing the activation of survivin by JCV infection in vitro and in PML clinical cases. These observations provide new insights into the anti-apoptotic mechanisms used by JCV to complete its lytic cycle and may suggest new therapeutic targets for PML.