Next generation sequencing reveals a novel HLA null allele, HLA-C*14:93 N.

HLA-C*14:93 N differs from 14:02:01 by a single nucleotide substitution at position 502 in Exon 3.

Detection of a novel HLA allele, HLA-B*50:01:09, identified by next generation sequencing.

HLA-B*50:01:09 differs by a synonymous nucleotide exchange in codon 17 from B*50:01:01.

A novel HLA variant, HLA-C*03:376, detected by next generation sequencing.

HLA-C*03:376 differs from HLA-C*03:04:01 in exon 6 by a single nucleotide substitution.

A novel HLA-C variant, HLA-C*04:01:85, detected by next generation sequencing.

HLA-C*04:01:85 differs in exon 1 from C*04:01:01 by a single nucleotide substitution in codon 3.

LBP, CD14, TLR4 and the murine innate immune response to a peritoneal Salmonella infection.

In mice, defense against an intraperitoneal Salmonella infection depends on a vigorous innate immune response. Mutations which lead to an inadequate early response to the pathogen thus identify genes involved in innate immunity. The best studied host resistance factor, NRAMP-1, is an endosomal membrane protein whose loss leads to an inability of the animals to hold the infection in check. However, innate defense against Salmonella is not restricted to mechanisms which directly attack the pathogen within macrophages. Here we have examined the contribution of the LBP, CD14 and TLR4 gene products to innate defense against Salmonella. To this end, we have generated mice which carry a wild-type allele of NRAMP-1, but which are deficient for the LBP, CD14 or TLR4 genes. Loss of any of these genes leads to a susceptibility to Salmonella as dramatic as that seen in animals lacking functional NRAMP-1 protein. This indicates that LBP, CD14 and TLR4 are all critical elements required in the proper induction of this innate defense system.

The essential role of lipopolysaccharide-binding protein in protection of mice against a peritoneal Salmonella infection involves the rapid induction of an inflammatory response.

Acute and chronic hyperinflammation are of major clinical concern, and many treatment strategies are therefore directed to inactivating parts of the inflammatory system. However, survival depends on responding quickly to pathogen attack, and since the adaptive immune system requires several days to adequately react, we rely initially on a range of innate defenses, many of which operate by activating parts of the inflammatory network. For example, LPS-binding protein (LBP) can transfer the LPS of Gram-negative bacteria to CD14 on the surface of macrophages, and this initiates an inflammatory reaction. However, the importance of this chain of events in infection is unclear. First, the innate system is redundant, and bacteria have many components that may serve as targets for it. Second, LBP can transfer LPS to other acceptors that do not induce inflammation. In this study, we show that innate defense against a lethal peritoneal infection with Salmonella requires a direct proinflammatory involvement of LBP, and that this is a major nonredundant function of LBP in this infection model. This emphasizes that blocking the LBP-initiated inflammatory cascade disables an essential defense pathway. Any anti-inflammatory protection that may be achieved must be balanced against the risks inherent in blinding the innate system to the presence of Gram-negative pathogens.

Mutation of amino acids 39-44 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli.

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.

Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide.

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9-13 and 91-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91-94,96)A, and sCD14(97-101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9-13 and 91-101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.

The myeloid differentiation antigen CD14 is N- and O-glycosylated. Contribution of N-linked glycosylation to different soluble CD14 isoforms.

The myeloid differentiation antigen CD14 acts as the major receptor for bacterial lipopolysaccharide (LPS). A soluble form of the protein (sCD14) is present in human serum which functions as a soluble LPS receptor. We have compared the isoform patterns of soluble CD14 derived from human serum and of the recombinant proteins produced by CHO cells transfected with either the wild-type CD14 gene or with a cDNA coding for a truncated protein which lacks the C-terminal 21 amino acids [sCD14-(1-335)-peptide]. Using SDS/PAGE, two dominant isoforms (53 and 50 kDa) and two minor forms (46 and 43 kDa) can be detected in serum as well as in the supernatants of both transfectants. sCD14 is a glycoprotein which carries N- and O-linked carbohydrates. The different isoforms of sCD14-(1-335)-peptide are due to differences in the content of N-linked sugars. However after the removal of N- and O-linked carbohydrates from serum- and CHO-derived wild-type proteins, two isoforms are still present. These results indicate that N-linked glycosylation contributes to but does not fully explain the different forms of soluble CD14. We further examined whether the mutation of individual N-linked glycosylation sites influences the expression of membrane-bound and soluble CD14 forms and the ability of the membrane-bound molecule to bind LPS. As with the wild-type proteins, the different isoforms of the soluble mutants are partially due to differences in N-linked glycosylation. A truncated mutant which lacks the two N-terminal glycosylation sites {[Asp18, Asp132]CD14-(1-335)peptide} does not give rise to multiple forms on SDS gels. Like CD14-(1-335)-peptide, this mutant is not expressed on the cell surface suggesting that a smaller isoform present in the wild-type preparations results from proteolytic cleavage of the membrane-bound molecule. N-linked carbohydrates do not seem to be important for the binding of LPS to membrane-bound CD14.

The molecular basis for therapeutic concepts utilizing CD14.

The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.