Ii Gene Research Articles

Localization of Chicken Rab22a in Cells and Its Relationship to BF or Ii Molecules and Genes.

Rab22a is an important small GTPase protein the molecule that is involved in intracellular transportation and regulation of proteins. It also plays an important role in antigens uptake, transportation, regulation of endosome morphology, and also regulates the transport of antigens to MHC (Major Histocompatibility Complex) molecules. To investigate the role of Rab22a, the intracellular co-localization of chicken Rab22a (cRab22a) molecule and its relationship to BF and chicken invariant chain (cIi) molecules was studied. A 3D protein structure of Rab22a was constructed by using informatics tools (DNASTAR 4.0 and DNAMAN). Based on the model, the corresponding recombinant eukaryotic plasmids were constructed by point mutations in the protein's structural domains. HEK 293T cells were co-transfected with plasmids pEGFP-C1-cIi to observe the intracellular co-localization. Secondly, the DC2.4 Mouse Dendritic Cell and Murine RAW 264.7 cells were transfected with recombinant plasmids of pmCherry-cRab22a and pmCherry-mRab22a respectively. Subsequently, the intracellular localization of cRab22a in early and late endosomes was observed with specific antibodies against EEA1 and LAMP1 respectively. For gene expression-based studies, the cRab22a gene was down-regulated and up-regulated in HD11 cells, following the detection of transcription levels of the BFa (MHCIa) and cIi genes by real-time quantitative PCR (RT-qPCR). The interactions of the cRab22a gene with BFa and cIi were detected by co-immunoprecipitation (Co-IP) and Western blot. The results showed that the protein structures of chicken and mouse Rab22a were highly homologous (95.4%), and both localize to the early and late endosomes. Ser41 and Tyr74 are key amino acids in the Switch regions of Rab22a which maintain its intracellular localization. The down-regulation of cRab22a gene expression significantly reduced (p < 0.01) the transcription of BFa (MHCIa) and cIi in HD11 cells. However, when the expression of the cRab22a gene was increased 55 times as compared to control cells, the expression of the BFa (MHCIa) gene was increased 1.7 times compared to the control cells (p < 0.01), while the expression of the cIi gene did not significantly differ from control (p > 0.05). Western blot results showed that cRab22a could not directly bind to BFa and cIi. So, cRab22a can regulate BFa and cIi protein molecules indirectly. It is concluded that cRab22a was localized with cIi in the endosome. The Switch regions of cRab22a are the key domains that affect intracellular localization and colocalization of the cIi molecule.

Design of Species-Specific PCR Primers That Target the aac(6′)-Ii Gene for the Rapid Detection of Enterococcus faecium

In this study, we used the sequence of the aac(6′)-Ii gene, which is responsible for the intrinsic low-level aminoglycoside resistance of Enterococcus faecium, to design novel species-specific primers. Two oligonucleotide pairs named EF_200 and EF_120 were designed, generating amplification products with sizes of 200 bp and 120 bp, respectively. They were successfully applied for the identification of various isolates of clinical or environmental origins in both pure cultures and complex food samples. The obtained results indicated that both primer pairs permitted the highly specific, simple, fast and inexpensive detection of E. faecium isolates.

The fowl adenovirus serotype 4 (FAdV-4) induce cellular pathway in chickens to produce interferon and antigen-presented molecules (MHCI/II)

FAdV-4 is the major strain of adenovirus that responsible for hydro-pericardial syndrome (HPS) in poultry. In this study, the virus's specific gene fragments were isolated from clinically suspected cases and amplified by PCR. Finally, after a viral infection to investigate the immune response of the host, the gene expression of MHC (major histo-compatible) molecules (MHCIα, MHCIIβ), Ii (Invariant Chain) gene, inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), and transcription factors (MDA5, STING, IRF7, and NF-kB) were detected by real-time PCR (fluorescence technology). The results of sequence comparison showed that the clinically isolated virus was 100% homologous to a virulent strain of avian adenovirus group C serotype 4 (FAdV-4), which were named AH-FAdV-4. The TCID50 and pathogenicity of the virus were determined that was 106.52/0.1 mL with a mortality rate of 100% in chickens and 0% in ducks. Furthermore, results showed that the expression level of MHCIα, MHCIIβ, and Ii genes in chicken embryo kidney cells significantly (P < 0.01) upregulated (increased) after infection, which was 43, 5.2, and 2.5 times higher than the control group. With the addition of PDTC, an inhibitor of NF-kB, then the expression level of MHCIα, MHCIIβ, and Ii was decreased significantly (P < 0.01) than the control group. The transcription levels of these genes were decreased 0.64, 0.27, and 0.26 respectively. Simultaneously, the expression levels of IFN-β, IFN-γ, and IL-1β were also significantly (P < 0.01) up-regulated (increased) 7.8, 22.7, and 5 times higher than the control group. It was found that up-regulation of STING and NF-κB pathways are directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), MHC molecules (MHCIα, MHCIIβ), and Ii gene. The results also showed that the gene regulation pathways consecutively increased the expression levels of MDA5, STING, IRF7, and NF-kB. It is conducted that the expression levels of cytokines, MHC molecules, and li gene were increased by STING and NF-kB pathways.

High-Level Expression of an Allo-MHC II Transgene Can Be Achieved in Mouse Hepatocytes, But Lack of Chaperones and Accessory Molecules May Prevent Tolerance Induction.

Background: Previous studies have shown induction of specific tolerance and long term survival of fully allogeneic cardiac grafts when donor MHC class II genes were transferred to recipient bone marrow prior to transplantation. In our published data, expression of donor MHC class I (Kb) in recipient liver induced tolerance to Kb-expressing skin grafts. Here, we investigate the effect of high level recombinant Adeno-Associated Virus (AAV)-mediated expression of MHC class II in heart transplant setting. Methods: We developed an efficient liver-specific vector encoding H-2IAd (rAAV-IAd). C57BL/6 (B6, H-2b) WT and Foxp3GFPmice were injected with varying doses (5x108-5x1011 vector genome copies, vgc) to produce expression of H-2IAd on hepatocytes. Expression of IAd and costimulatory molecules was assessed by FACS on isolated hepatocytes, whilst expression of Invariant chain (Ii) and H-2M was determined by RT-PCR. Fully-allogeneic hearts from DBA/2 (H-2d) were transplanted into C57BL/6 at d7 or d100 post-transduction. Results: High level expression of H-2IAd on hepatocytes of B6 mice with doses of 5x1010 or 5x1011 rAAV-IAdwas observed.Figure: No Caption available.Foxp3+ CD4+ T cells were increased in the livers of mice receiving 5x1011 rAAV-IAd with 2±0.76% in normal ctrl, 2.4± 0.6% for 5x1010 IAd injected mice and 7.7±2.2% for 5x1011 IAd injected mice (p<0.03).Yet, the survival rate of hearts grafted into rAAV-IAd treated B6 mice was not improved compared to grafts in normal mice (MST 8d and 7d respectively). Hepatocytes from IAd-injected mice did not express CD40, CD80, CD86 or PD-L1. In addition, RT-PCR revealed minimal expression of H2-M and Ii, suggesting an impaired function in peptide editing of expressed IAd. Conclusion: High level expression of MHCII IAd can be achieved in recipient liver using rAAV vectors. However, initiating an immune response and tolerance to grafts bearing IAd might require supplying H2-M and invariant chain (Ii) genes for biosynthesis of fully functional MHCII IAd from hepatocytes.

Selective modulation of MHC class II chaperons by a novel IFN-γ-inducible class II transactivator variant in lung adenocarcinoma A549 cells

Class II transactivator (CIITA) plays a critical role in controlling major histocompatibility complex (MHC) class II gene expression. In this study, two novel alternatively spliced variants of human interferon (IFN)-γ-inducible CIITA, one missing exon 7 (CIITAΔE7), the other with TAG inserted at exon 4/5 junction (CIITA-TAG), were identified and characterized. Both variants are naturally occurring since they are present in primary cells. Unlike CIITA-TAG, CIITAΔE7 is expressed more abundantly in lung adenocarcinoma A549 cells than in the non-transformed counterpart BEAS-2B cells following IFN-γ stimulation. Transfection experiments showed that CIITAΔE7 induced a markedly lower level of surface HLA-DR, -DP, -DQ expression than CIITA-TAG in A549 cells but not in BEAS-2B cells, although both variants elicited similar amounts of total DR, DP, and DQ proteins. This differential effect was correlated with, in A549 cells, decreased expression of Ii and HLA-DM genes, along with increased expression of HLA-DO genes. Ii and HLA-DM are chaperons assisting in HLA class II assembly, while HLA-DO functions to inhibit endosomal peptide loading and HLA class II membrane transport. These findings raise the possibility that CIITAΔE7 interacts with unknown cancer-associated factors to selectively modulate genes involved in the assembly and transport of HLA class II molecules.

Molecular cloning and site-directed mutagenesis of leucine-based sorting motifs of the porcine invariant chain

Invariant chain (Ii) is a transmembrane protein that associates with MHC class II molecules in the endoplasmic reticulum. The cytoplasmic tail of Ii contains two leucine residues able to direct Ii to the endocytic pathway. We obtained the pig Ii gene by RT-PCR. Mutated Ii was prepared via site directed mutagenesis by the PCR Megaprimer method to study the effect of the two leucines on the localization of pig Ii. These mutated fragments were ligated to the vector pmCherry-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine(TM) 2000. Fluorescence of fusion proteins (mCherry-Ii) was observed with a fluorescent microscope. Amino acid sequence alignment showed that pig Ii has domains similar to those seen in other mammalian Ii, including the cytoplasmic, transmembrane, class II-associated Ii-derived peptide, and trimerization domains. Based on observations with the fluorescent microscope, we found that two leucine-based motifs are required for pig Ii intracellular localization, and that both motifs independently mediate this function in Ii.

Exposing the Specific Roles of the Invariant Chain Isoforms in Shaping the MHC Class II Peptidome

The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.

English

Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been extensively studied, less attention is paid to its fish counterpart. In this study, in order to understand the structure and biological function of Chinese Pengze crucian carp (Carassius auratus) Ii, we first cloned Ii gene (Caau-Ii) using RT PCR and rapid amplification of cDNA end techniques. Firstly, a conserved DNA fragment was amplified using a pair of degenerate primers from fish&rsquo;s head kidney and spleen total RNA. Then specific primers for RACE were designed to amplify the 5&#39; and 3&#39; ends of&nbsp;Caau-Ii. Finally, the full-length cDNA of&nbsp;Caau-Ii&nbsp;was amplified, sequenced and analyzed and the results showed that it was 1063 bp in length and contained an open reading frame of 696 bp encoding&nbsp;Caau-Ii&nbsp;precursor protein. Amino acid sequence alignment showed that (1)&nbsp;Caau-Ii&nbsp;had high homology with that of carp and zebrafish (identify &gt;70%) and low homology with mammalian Ii (30% &lt; identity &lt;40%); (2)&nbsp;Caau-Ii&nbsp;had similar domains seen in mammalian Ii, including transmembrane domain, CLIP (class II-associated Ii-derived peptide) and TRIM (trimerization) domain. &nbsp; Key words:&nbsp;Invariant chain,&nbsp;Carassius auratus, RACE, cloning, domain analysis. &nbsp; &nbsp;

Statement on the assessment of the safety of Cylactin® (Enterococcus faecium) used in animal nutrition

EFSA JournalVolume 8, Issue 12 1955 StatementOpen Access Statement on the assessment of the safety of Cylactin® (Enterococcus faecium) used in animal nutrition EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP), EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP)Search for more papers by this author EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP), EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP)Search for more papers by this author First published: 21 December 2010 https://doi.org/10.2903/j.efsa.2010.1955Citations: 5 Panel members: Gabriele Aquilina, Georges Bories, Paul Brantom, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Reinhard Kroker, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Derek Renshaw and Maria Saarela Correspondence: feedap@efsa.europa.eu Acknowledgement: The Panel wishes to thank the members of the Working Group on Microorganisms, including Roland Leclercq, Atte von Wright and Guido Rychen, for the preparatory work on this scientific opinion for the support provided to this scientific opinion. Adoption date: 7 December 2010 Published date: 21 December 2010 Question number: EFSA-Q-2010-01264 On request from: European Commission AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References Bismuth R and P. Courvalin, 2009. Aminoglycosides and Gram-positive bacteria. In: Antibiogram. Eds P. Courvalin, R. Leclercq, L. Rice. 2009, ASM Press, pp. 213– 224. Costa, Y., Galimand, M., Leclercq, R., Duval, J., and Courvalin, P. 1993. Characterization of the chromosomal aac(6′)-Ii gene specific for Enterococcus faecium. Antimicrobial Agents and Chemotherapy, 37, 1896– 1903. DANMAP (Danish Integrated Antimicrobial resistance Monitoring and Research Programme), 2009, online. Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark. Available from www.danmap.org. EC (European Commission), 2001, updated 2003, online. Opinion of the Scientific Committee on Animal Nutrition on the criteria for assessing the safety of micro-organisms resistant to antibiotics of human clinical and veterinary importance. Available from europa.eu.int/comm/food/fs/sc/scan/out108_en.pdf. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2005. Scientific Opinion on the updating of the criteria used in the assessment of bacteria for resistance to antibiotics of human and veterinary importance. The EFSA Journal, 223. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2008a. Technical guidance - Update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. The EFSA Journal, 732, 1– 15. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2008b. Guidance for the preparation of dossiers for zootechnical additives. The EFSA Journal, 776, 1– 17. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2010. Scientific Opinion on the safety and efficacy of Cylactin® (Enterococcus faecium) for chickens for fattening. EFSA Journal, 8(7): 1661. [13 pp.]. Galimand M., Schmitt E., Panvert M., Douthwaite S., Mechulam Y. and Courvalin P., 2010, in press. Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM. RNA. Moellering R.C. Jr., Korzeniowski O.M., Sande M.A. and Wennersten C.B., 1979. Species-specific resistance to antimicrobial synergism in Streptococcus faecium and Streptococcus faecalis. Journal of Infectious Diseases, 140, 203– 208. Citing Literature Volume8, Issue12December 20101955 ReferencesRelatedInformation

Cloning of pigeon invariant chain (Ii) gene by RACE

In order to compare the structure and function of pigeon invariant chain (pIi) gene with other avian's, pIi gene was cloned using a method of RACE (Rapid Amplification of cDNA Ends). Firstly, according to high conservative nucleotide sequence of homologous fragment in avian invariant chain (Ii) gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from pigeon spleen cell RNA by PCR. Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of pIi gene were cloned by RACE respectively. Finally a complete cDNA of pIi was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that pIi gene is 1,050 bp in length (GenBank No. AY904337), which includes an open reading frame of 633 bp encoding a precursor protein with 211 amino acid residues. In comparison with the nucleotide sequences of other species' Ii genes, pIi is similar to chicken's, showing an overall identity of 82.8 with chicken and over 52.0 with human and other mammalian animals. In addition, some amino acid residues in Ii molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.

Silencing invariant chain of DCs enhances Th1 response using small interfering RNA

RNA interference (RNAi), which causes the degradation of any RNA in a sequence specific manner, is a posttranscriptional gene silencing mechanism. Targeting the invariant chain (Ii) in DCs has been used as an approach to enhance antitumor immunity. It is demonstrated in this article that transfection of H-2(K) DCs with siRNA specific for Ii gene can significantly knock down Ii. When exposed to TNF-alpha, immature DCs transfected with Ii siRNA can differentiate into mature DCs without reducing viability or IL-12p70 production. Ii siRNA-treated H-2(K) DCs exhibited an increased allostimulatory capacity in a lymphocyte proliferation assay. Furthermore, Ii siRNA-transfected H-2(K) DCs enhanced Th1 responses by increasing IFN-gamma and decreasing IL-4 production, and much stronger cytotoxic activity was observed when DCs were co-transfected with Ii siRNA and an endogenous tumor antigen in vitro. Our findings indicate that silencing the Ii gene in DCs with siRNA may offer a potential approach to enhancing antitumor immunotherapy.

Cloning of human uroplakin II gene from Chinese transitional cell carcinoma of bladder and construction of its eukaryotic expression vector.

To clone Uroplakin Ii gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GIII / T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin II gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba I and Hind III restrictive sites of eukaryotic expression vector pcDNA3. 0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin II negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin II were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin II cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100% homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP II for Uroplakin II was successfully constructed. After being transferred with pcDNA-UP II for 72 h, cellular Uroplakin II mRNA levels were significantly improved (P < 0.01). It is concluded that human Uroplakin II gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin II gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

Molecular cloning and expression of two chicken invariant chain isoforms produced by alternative splicing

The biosynthesis of distinct forms of the invariant chain (Ii) protein from a unique gene as the result of differential splicing patterns has been observed in humans and mice. However, there have been no reports on the existence of Ii isoforms in avian species. In the present study, we identified two chicken Ii cDNAs by RT-PCR and RACE, and examined the Ii gene copy number, mRNA expression and protein expression by Southern blotting, Northern blotting and immunofluorescence confocal microscopy, respectively. One of the Ii cDNAs, named Ii-1, was 1,151 bp in length, and had an open reading frame (ORF) of 672 nucleotides, in agreement with a previously identified chicken Ii sequence; the other, named Ii-2, was 1,337 bp long and had an ORF of 861 nucleotides. Southern blotting confirmed that these cDNAs were derived from a single copy gene. Northern blotting performed with total RNA from various tissues of 6-week-old chickens revealed high levels of Ii-1 and Ii-2 mRNA expression in the spleen and bursa of Fabricius, and low levels of Ii-1 expression in the thymus, heart and liver, while Ii-2 was not expressed in these tissues. High levels of expression of both Ii isoforms were detected in the spleen and bursa of Fabricius during late embryogenesis. Immunofluorescence staining showed that Ii proteins were expressed in the cell membranes of the splenocytes. These data suggest that chicken Ii exists in two isoforms resulting from alternative splicing, and is strongly expressed in the major immune organs.

Long Distance Control of MHC Class II Expression by Multiple Distal Enhancers Regulated by Regulatory Factor X Complex and CIITA

MHC class II (MHC-II) genes are regulated by an enhanceosome complex containing two gene-specific transcription factors, regulatory factor X complex (RFX) and CIITA. These factors assemble on a strictly conserved regulatory module (S-X-X2-Y) found immediately upstream of the promoters of all classical and nonclassical MHC-II genes as well as the invariant chain (Ii) gene. To identify new targets of RFX and CIITA, we developed a computational approach based on the unique and highly constrained architecture of the composite S-Y motif. We identified six novel S'-Y' modules situated far away from the promoters of known human RFX- and CIITA-controlled genes. Four are situated at strategic positions within the MHC-II locus, and two are found within the Ii gene. These S'-Y' modules function as transcriptional enhancers, are bona fide targets of RFX and CIITA in B cells and IFN-gamma-induced cells, and induce broad domains of histone hyperacetylation. These results reveal a hitherto unexpected level of complexity involving long distance control of MHC-II expression by multiple distal regulatory elements.

Characterization of high-level aminoglycoside-resistant enterococci in Kuwait hospitals.

This study investigated the distribution of genes for aminoglycoside-modifying enzymes (AME) and the genetic relatedness of high-level aminoglycoside-resistant enterococci isolated in Kuwait hospitals. A total of 117 enterococci, consisting of 109 Enterococcus faecalis, seven Enterococcus faecium, and one Enterococcus casseliflavus were studied. The MICs of gentamicin, kanamycin, amikacin, tobramycin, and streptomycin were determined by agar dilution and the genes encoding the AAC(6')- APH(2"), ANT(4'), APH(3'), APH (2")-Ib, APH (2")-Ic, APH (2")-Id, and ANT(6) enzymes were amplified by PCR. They were typed by pulsed-field gel electrophoresis (PFGE). Filter mating was used to transfer gentamicin resistance determinants. They were all resistant to kanamycin (MIC 2000 mg/L). Fifty-five isolates were resistant to gentamicin (MIC 500 mg/L), 72 were resistant to tobramycin (MIC 64 mg/L), 115 were resistant to amikacin (MIC 64 mg/L), and 97 were resistant to streptomycin (MIC 1000 mg/L). The aac(6')-Ie-aph(2")-Ia was detected in all isolates with gentamicin MIC 500 mg/L and in 15 isolates with gentamicin MIC 256 mg/L. The aph(3')-IIIa gene was detected in 101 isolates, whereas the ant(6')-Ia gene was detected in 85 of the 97 streptomycin-resistant isolates with MIC 1000 mg/L. The aac(6')-Ii gene was detected only in the seven E. faecium isolates. None of them contained ant(4')-Ia, aph(2")-Ib, aph(2")-Ic and aph(2")-Id. PFGE revealed heterogeneous patterns with no dominant clone. The results demonstrated that AME are common in aminoglycoside-resistant enterococci isolated in Kuwait. However, the absence of a dominant clone suggests that they acquired high-level aminoglycoside independently.

Induction of protective immunity to Listeria monocytogenes by immunization with plasmid DNA expressing a helper T-cell epitope that replaces the class II-associated invariant chain peptide of the invariant chain.

Listeria epitope-specific helper T (Th) cells were able to be primed and induced in vivo by immunization with a plasmid carrying an invariant chain (Ii) gene whose class II-associated invariant chain peptide (CLIP) region was replaced by a Listeria Th epitope. Immunization of C3H/He mice with an Ii-LLO 215-226 plasmid induced specific interferon-gamma- and interleukin 2-producing Th cells and conferred significant protective immunity against listerial infection.

Association analysis of HLA-Class iI and class iII gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis

HLA-DRB1, -DQB1, TNFα, TNFβ, HSP70-2 and HSP70-hom genetic polymorphisms were analyzed in 156 unrelated patients who developed mediterranean visceral leishmaniasis (MVL) due to Leishmania infantum, and 154 unrelated healthy controls, who have got asymptomatic infection with this parasite and were selected on the basis of a positive leishmanin skin test (LST). A signifi- cantly reduced frequency of HLA-DR2 was observed among MVL patients (16.1%), compared with controls (26.3%) (relative risk = 0.54; p = 0.04). HLA-DR2/DR13 as well as HLA-DQB1∗0201/- genotype frequencies were significantly lower in patients vs controls (relapse rate = 0.17 and 0.46, respectively; p < 0.05). However, using Bonferroni correction, none of these associations remained significant. No association was found, between either the −308 base pair TNFα gene polymorphism or the NcoI polymorphism in the first intron of the TNFβ gene and susceptibility to MVL. Analysis of PstI and NcoI polymorphisms in the coding region of HSP70-2 and HSP70-hom genes, respectively, revealed a significantly higher frequency of homozygotes for the HSP70-2/PstI negative allele, among patients (21.8%) vs controls (12.6%) (relapse rate = 1.94; p = 0.04). Again, this result was not significant after using Bonferroni correction. These results do not support association between susceptibility to MVL and the MHC class II and class III loci analyzed in this study.

Identification of an IFN-gamma responsive region in an intron of the invariant chain gene.

The mechanism by which IFN-gamma up-regulates invariant chain mRNA in antigen-presenting cells has been under intensive investigation. This study shows that in murine monocytic cells the transcriptional up-regulation mediated by the invariant chain (Ii) upstream enhancer only accounts for part of the induction of Ii mRNA by IFN-gamma. We have identified an intronic region in the murine Ii gene that contributes to the transcriptional up-regulation by IFN-gamma. The region has S (H), X/X2 and Y boxes similar to those in MHC class II promoters and the Ii upstream enhancer. Mutations at the putative S, X and Y boxes have abolished the ability of the region to mediate Ii up-regulation by IFN-gamma. Consistent with the functions of these boxes, our findings reveal that the up-regulation of Ii transcription by IFN-gamma mediated by the intronic region is dependent on the induction of class II transactivator by IFN-gamma.

H2-M, a facilitator of MHC class II peptide loading, and its negative modulator H2-O are differentially expressed in response to proinflammatory cytokines.

H2-M is a major histocompatibility complex (MHC) class II-like molecule that catalyzes peptide binding to MHC class II molecules. Recently, the H2-O heterodimer, encoded by H2-Oa and H2-Ob in the MHC class II region, has been shown to be physically associated with H2-M in B cells and to downregulate H2-M function. Examination of H2-O expression in freshly isolated mouse organs revealed that H2-Oa- and H2-Ob-specific transcripts are present in both lymphoid and nonlymphoid tissues. To evaluate the gene regulation and functional impact of H2-O on antigen presentation, we examined the effects on MHCII, invariant chain (Ii), H2-M, and H2-O gene expression of interleukin (IL)-4, IL-10, and interferon (IFN)-gamma in different antigen-presenting cells (APCs). In nonprofessional APCs, e.g., L929 fibroblasts, IFN-gamma-inducible expression of the MHC class II-specific transcription factor CIITA is associated with coordinate expression of MHCII, Ii, H2-M, and H2-Oa genes but without concomitant H2-Ob induction. In contrast, professional APCs, e.g., the macrophage cell line P388D1, exhibit constitutive H2-Oa and H2-Ob expression, which is not inducible by IFN-gamma in contrast to CIITA, MHCII, Ii, and H2-M expression. In B cells, CIITA, MHCII, Ii, and H2-M genes are differentially expressed relative to H2-Oa and H2-Ob genes upon stimulation with IL-4, IL-10, or IFN-gamma. A differential ratio of H2-M to H2-O may represent one mechanism by which professional and nonprofessional APCs bypass H2-O inhibitory activity.