Passively Administered Pooled Human Immunoglobulins Exert IL-10 Dependent Anti-Inflammatory Effects that Protect against Fatal HSV Encephalitis

HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1–3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b+ Ly6Chigh monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45+ Ly6Clow monocytes but not for inhibiting development of Ly6Chigh monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3+ CD4+ T regulatory cells (Tregs) and FoxP3− ICOS+ CD4+ T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS+ CD4+ T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases

Molecular cloning and differential IgG responses to a histidine-rich antigen (OvL3.C1) of Onchocerca volvulus by selected residents of onchocerciasis endemic regions in Cameroon and Ecuador

In order to further investigate host-parasite interactions in onchocerciasis, a major Onchocerca volvulus histidine rich antigen termed OvL3.C1 was isolated from an O. volvulus cDNA library using antibodies from putatively immune subjects living in onchocerciasis endemic communities in Cameroon. Analysis of its sequences predicted the protein to be helix-rich with a single transmembrane region. Recombinant OvL3.C1 antigen induced from pBAD-TOPO/Thio vector in Escherichia coli was purified as inclusion bodies and further by a combination of Ni2+ chelate chromatography and electroelution. Anti-OvL3.C1 immunoglobulin G (IgG) subclass levels were assessed by ELISA in 15 pairs and 18 pairs of selected and cross-matched infected and putatively immune subjects from Cameroon and Ecuador, respectively. IgG3 and IgG4 levels were shown to be significantly higher in putatively immune (immune protected) subjects. A higher IgG3 level in endemic normal subjects is implicated in parasite killing and the development of the putative immune status while IgG4 has been shown to block onchocercal pathology. OvL3.C1 is a dominant antigen in onchocerciasis which elicits strong responses in subjects expose to both African and South American forms of onchocerciasis. It is therefore an important player in mechanisms of resistance or allergy attenuation in onchocerciasis.Keywords: Onchocerciasis, immunoglobulin G, putative immunit

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing

Molecular cloning and differential IgG responses to a histidine-rich antigen (OvL3.C1) of Onchocerca volvulus by selected residents of onchocerciasis endemic regions in Cameroon and Ecuador

In order to further investigate host-parasite interactions in onchocerciasis, a major Onchocerca volvulus   histidine rich antigen termed OvL3.C1 was isolated from an O. volvulus cDNA library using antibodies from putatively immune subjects living in onchocerciasis endemic communities in Cameroon. Analysis of its sequences predicted the protein to be helix-rich with a single transmembrane region. Recombinant OvL3.C1 antigen induced from pBAD-TOPO/Thio vector in Escherichia coli   was purified as inclusion bodies and further by a combination of Ni2+ chelate chromatography and electro-elution. Anti-OvL3.C1 immunoglobulin G (IgG) subclass levels were assessed by ELISA in 15 pairs and 18 pairs of selected and cross-matched infected and putatively immune subjects from Cameroon and Ecuador, respectively. IgG3 and IgG4 levels were shown to be significantly higher in putatively immune (immune protected) subjects. A higher IgG3 level in endemic normal subjects is implicated in parasite killing and the development of the putative immune status while IgG4 has been shown to block onchocercal pathology. OvL3.C1 is a dominant antigen in onchocerciasis which elicits strong responses in subjects expose to both African and South American forms of onchocerciasis. It is therefore an important player in mechanisms of resistance or allergy attenuation in onchocerciasis

Sedimentation profiles of U1-70K (Usp101p), U1H (Usp107p), U1J (Usp108) and U1L (Usp109p) complexes

<p><b>Copyright information:</b></p><p>Taken from "Proteomic analysis of the U1 snRNP of reveals three essential organism-specific proteins"</p><p></p><p>Nucleic Acids Research 2007;35(5):1391-1401.</p><p>Published online 30 Jan 2007</p><p>PMCID:PMC1865046.</p><p>© 2007 The Author(s).</p> () Protein extract (5 mg) of cells expressing U1-70K-TAP, U1H-TAP and U170-HA or U1J-TAP and U1-70K-HA or U1L-TAP and U1-10K-HA were separated on a 10–30% glycerol gradient. The gradient fractions () were precipitated with IgG antibodies and separated by SDS-PAGE, immunoblotted and probed with αTAP antibodies to determine the distribution of U1-70K-TAP, U1H-TAP, U1J-TAP and U1L-TAP, as indicated. () The gradient fractions 3–8 and 10–15 of a gradient-containing U1L-TAP and U1-70K-HA were pooled bound to IgG-Sepharose. The bound material was separated on SDS-PAGE, immunoblotted and probed with IgG and HA antibodies. An aliquot (50%) of the bound material was used to isolate RNA and hybridized with a labeled U1 probe to visualize U1 snRNA as indicated. Mo, pooled gradient fractions 10–15 of a protein extract from a wild-type strain. The pooled gradient fractions 3–8 from this gradient also showed no signals (not shown). The gradient was calibrated with small (30S) and large (50S) ribosomal subunits from