Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins.

CD14, a myeloid cell-surface receptor and soluble plasma protein, binds LPS and other microbial molecules and initiates the innate immune response to bacterial invasion. The blood concentration of soluble CD14 (sCD14) increases during the systemic response to infection. Although high sCD14 blood levels have correlated with increased risk of dying from severe sepsis, sCD14 can diminish cell responses to LPS. We show here that in human serum, sCD14 increases the rate at which cell-bound LPS is released from the monocyte surface and binds to plasma lipoproteins. This enhanced rate of LPS efflux is associated with a significant reduction in the ability of monocytes to produce cytokines in response to LPS. Serum from septic patients reduced the LPS-monocyte interaction by as much as tenfold, and depletion of sCD14 from the serum restored LPS-monocyte binding and release kinetics to near normal levels. In serum from septic patients, monocyte-bound LPS also moved more rapidly into lipoproteins, which completely neutralized the biologic activity of the LPS that bound to them. In human plasma, sCD14 thus diminishes monocyte responses to LPS by transferring cell-bound LPS to lipoproteins. Stress-related increases in plasma sCD14 levels may help prevent inflammatory responses within the blood.

MD-2 binds to bacterial lipopolysaccharide.

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.

Soluble CD14 enhances membrane CD14-mediated responses to peptidoglycan: structural requirements differ from those for responses to lipopolysaccharide.

The purpose of this study was to identify the functional significance of the binding of soluble CD14 (sCD14) to bacterial peptidoglycan (PGN) and to compare the structural requirements of sCD14 for the binding to PGN and lipopolysaccharide (LPS) and for sCD14-mediated enhancement of PGN- and LPS-induced cell responses. sCD14 did not facilitate the responses of membrane CD14 (mCD14)-negative pre-B 70Z/3 cells to PGN, although it facilitated the responses of these cells to LPS and although mCD14 facilitated the responses of 70Z/3 cells to PGN. sCD14 enhanced mCD14-mediated cell activation by both PGN and LPS, but only the responses to LPS, and not to PGN, were enhanced by LPS-binding protein. Four 4- or 5-amino-acid-long sequences within the 65-amino-acid N-terminal region of sCD14 were needed for binding to both PGN and LPS and for enhancement of cell activation by both PGN and LPS. However, deletions of individual sequences had different effects on the ability of sCD14 to bind to PGN and to LPS and on the ability to enhance the responses to PGN and to LPS. Thus, there are different structural requirements of sCD14 for binding to PGN and to LPS and for the enhancement of PGN- and LPS-induced cell activation.

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide.

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.

MD-2 binds to bacterial lipopolysaccharide.

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.

Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand.

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp. Although a high degree of diversity characterized these isolates, the overall population structure of the parasites associated with patent malaria infections was observed to remain relatively stable over time. The highest degree of polymorphism was observed with msp2, and the mean number of lines per infection (multiplicity of infection) calculated with this marker was higher than that obtained using msp1 or glurp alone, or combined. Infections with > or = 2 parasite lines were seen in 76% of the samples, and were proportionally more numerous at the start and end of the rainy season. Two interesting exceptions to the random distribution were observed and involved 2 allelic variants which in one case were found dissociated (msp1 MAD20-family) and in the other were associated (msp2 FC27-family). The epidemiological significance of these types of data is discussed.

Helicobacter pylori lipopolysaccharide can activate 70Z/3 cells via CD14.

Helicobacter pylori persistently colonizes the human gastrointestinal tract and is associated with chronic gastritis and, in some cases, peptic ulcer disease or gastric neoplasms. One factor in the persistence of this organism may be its inability to elicit a strong inflammatory response. Lipopolysaccharide (LPS) is a proinflammatory substance found in the cell walls of all gram-negative bacteria. H. pylori LPS has been found by several different measures to be less active than LPS from Enterobacteriaceae. This study addresses the role of CD14 and LPS-binding protein in the cellular response to H. pylori LPS. We report that H. pylori LPS activates mammalian cells expressing CD14 at much lower LPS concentrations than those for control cells not expressing CD14. The maximal activation of CD14-70Z/3 cells by H. pylori LPS also requires LPS-binding protein. H. pylori LPS at concentrations as high as 30 microg/ml does not elicit an interleukin-8 (IL-8) response from the epithelial cell line SW620 in the presence of CD14; 10 ng of Escherichia coli LPS per ml elicits a maximal IL-8 response. Furthermore, in contrast to some other types of LPS with little activity, H. pylori LPS does not inhibit the CD14-70Z/3 cell response to E. coli LPS. From these studies, we conclude that H. pylori LPS, though much less active than E. coli LPS, stimulates cells via CD14.

CD14 is a cell-activating receptor for bacterial peptidoglycan.

The hypothesis that CD14 (an endotoxin receptor present on macrophages and neutrophils) acts as a cell-activating receptor for bacterial peptidoglycan was tested using mouse 70Z/3 cells transfected with human CD14. 70Z/3 cells transfected with an empty vector were unresponsive to insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin. 70Z/3-CD14 cells were responsive to both insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin, as measured by the expression of surface IgM, activation of NF-kappaB, and degradation of IkappaB-alpha. Peptidoglycan also induced activation of NF-kappaB and degradation of IkappaB-alpha in macrophage RAW264.7 cells. These peptidoglycan-induced effects (in contrast to endotoxin-induced effects) were not inhibited by polymyxin B. Both peptidoglycan- and endotoxin-induced activation of NF-kappaB were inhibited by anti-CD14 mAb. The N-terminal 151 amino acids of CD14 were sufficient for acquisition of full responsiveness to both peptidoglycan and endotoxin, but CD14 deletion mutants lacking four small regions within the N-terminal 65 amino acids showed differentially diminished responses to peptidoglycan and endotoxin. These results identify CD14 as the functional receptor for peptidoglycan and demonstrate that similar, but not identical sequences in the N-terminal 65-amino acid region of CD14 are critical for the NF-kappaB and IgM responses to both peptidoglycan and endotoxin.

The N-terminal half of membrane CD14 is a functional cellular lipopolysaccharide receptor.

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). It was recently reported that an N-terminal 152-amino-acid fragment of soluble CD14 was an active soluble lipopolysaccharide receptor (T. S. -C. Juan, M. J. Kelley, D. A. Johnson, L. A. Busse, E. Hailman, S. D. Wright, and H. S. Lichenstein, J. Biol. Chem. 270:1382-1387, 1995). To determine whether the N-terminal half of the membrane CD14 was a functional LPS receptor on the cell membrane, we engineered a chimeric gene coding for amino acids 1 to 151 of CD14 fused to the C-terminal region of decay-accelerating factor and expressed it in Chinese hamster ovary cells and 70Z/3 cells. We found that the chimeric, truncated CD14 is a fully functional LPS receptor in both cell lines.

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)

Identificación de genotipos de cepas aisladas de Plasmodium falciparum mediante la reacción en cadena de la polimerasa y sus posibles usos en estudios epidemiológicos / Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies

La epidemiología del paludismo es el resultado de la interacción que tienen entre sí y con el medio circundante tres acervos genéticos: el del parásito, el del ser humano y el del mosquito vector. Actualmente se están elaborando métodos para caracterizar la genética de las poblaciones humanas en riesgo y de los posibles vectores, y a fin de llegar a conocer más a fondo la epidemiología, mecanismos patógenos y biología de este parásito también sería enormemente útil caracterizar las poblaciones naturales de Plasmodium y su distribución en huéspedes humanos y en insectos de zonas de estudio determinadas, especialmente si este enfoque se combina con estudios simultáneos en seres humanos y con los vectores. En este trabajo se describe un ensayo basado en la reacción en cadena de la polimerasa (RCP), que proporciona un método sensible, práctico y reproducible para caracterizar distintas poblaciones de parásitos de una misma especie. Con el fin de ilustrar la idoneidad de este tipo de ensayo, se escogieron y amplificaron con la RCP cuatro dominios polimórficos de los genes de tres proteínas de P. falciparum (los bloques 2 y 4 de la proteína de superficie del merozoito tipo 1 (PSM1), la tipo 2 (PSM2) y la proteína rica en glutamato (PRGLU) y una región casi enteramente conservada (el bloque 17 de la PSM1). Sirvieron de molde para amplificar con la RCP los ADN derivados de 15 líneas de P. falciparum cultivadas in vitro (siete de las cuales fueron clonadas) y de muestras de sangre de pacientes infectados procedentes de Tailandia. Los productos de la amplificación se analizaron por electroforesis en gel para detectar polimorfismos de longitud. Se detectaron siete variantes alélicas de la PRGLU, cinco del bloque 2 de la PSM1, tres del bloque 4 de la PSM1 y nueve de la PSM2. Este alto grado de polimorfismo se puede usar para caracterizar la composición genética de cualquier población de parásitos en un momento dado. En este trabajo se examina la aplicabilidad de esta forma de identificar genotipos en el campo de la epidemiología y se recomienda la adopción de patrones internacionales para su empleo, de tal modo que se puedan comparar los datos obtenidos en distintos lugares y momentos (AU)

Se publica también en inglés en el Bull. WHO. Vol. 73(1), 1995

Knowledge of cellular receptors for bacterial endotoxin--1995.

Septic shock due to infections with gram-negative bacteria remains a major clinical problem for infectious disease specialists, although our understanding of the pathophysiology of this syndrome has improved greatly over the past 5 years. The discovery of lipopolysaccharide (LPS) binding protein, a serum protein that catalyses the transfer of LPS to cellular receptors for LPS, was a major breakthrough. The finding that CD14, a glycophosphatidylinositol-linked membrane protein expressed by macrophages, is a receptor for LPS has made a major difference in our understanding of cellular activation by LPS. We will review studies dealing with LPS's binding to these proteins and its activation of cells. A better understanding of septic shock at the molecular level should lead to the development of new treatments for this lethal disease.

Genotyping of Plasmodium falciparum isolates by the polymerase chain reaction and potential uses in epidemiological studies.

The epidemiology of malaria results from the interactions of three gene pools--parasite, human, and mosquito vector--with one another and with their environment. Methods are being developed for characterizing the genetics of human populations at risk and of potential vectors. The characterization of natural populations of Plasmodium and knowledge of their distribution within the human and insect hosts in any given area under study would also greatly enhance understanding of the epidemiology, pathology and biology of this parasite, particularly when combined with simultaneous human and vector studies. This paper describes a polymerase chain reaction (PCR)-based assay which provides a sensitive, reproducible and practical method by which parasite populations within species can be characterized. In order to illustrate the suitability of the PCR assay, four polymorphic domains on the genes of three P. falciparum proteins (MSP1 blocks 2 and 4, MSP2, and GLURP) and one largely conserved region (MSP1 block 17) were chosen for amplification by PCR. DNA derived from 15 in-vitro cultured lines of P. falciparum (7 of which were cloned) and from blood samples obtained from infected patients in Thailand were used as templates for PCR amplification. The amplification products were analysed by gel electrophoresis for length polymorphisms. Seven allelic variants of GLURP, five of MSP1 block 2, three of MSP1 block 4, and nine of MSP2 were detected. This high degree of polymorphism can be used to characterize the genetic composition of any parasite population, at a given time. The paper discusses the applicability of this type of genotyping to epidemiology and urges the adoption of international standards for its use so that data from different areas and different times can be compared.

A region of human CD14 required for lipopolysaccharide binding.

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). CD14 binding of LPS is enhanced by serum proteins, especially lipopolysaccharide binding protein. The serum-dependent binding of LPS to CD14 stimulates macrophages to make cytokines, which can cause septic shock in humans and animals. Here, we identify a region in human CD14 which is important in serum-dependent LPS binding and LPS-induced cellular activation. Four small regions (4-5 amino acids long) within the N-terminal 65 amino acids of CD14 were deleted singly or in combination. The deletion mutants were stably expressed in Chinese hamster ovary (CHO) cells. The mutants were characterized in three assays: reactivity with anti-CD14 monoclonal antibody, serum-dependent LPS binding, and LPS-induced activation of NF-kappa B. Some of the mutants selectively lost reactivity with the anti-CD14 monoclonal antibody that inhibited serum-dependent LPS binding and cellular activation. All of the mutants bound much less LPS than wild type CD14 in the presence of serum. None of the mutants bound more LPS than control CD14-CHO cells in the absence of serum. CD14-CHO cells respond to LPS by activation of NF-kappa B. All of the deletion mutants were less active LPS receptors than wild type CD14-CHO cells. The delta AVEVE mutant, the delta DDED and delta PQPD double mutant, and the delta DDED, delta PQPD, delta AVEVE, and delta DPRQY quadruple deletion mutants were essentially inactive LPS receptors in CHO cells. These studies suggest that the 65 N-terminal amino acids of CD14 are critical for serum-dependent binding of LPS to CD14 and subsequent signal transduction in CHO cells.

Plasmodium falciparum: selective growth of subpopulations from field samples following in vitro culture, as detected by the polymerase chain reaction.

Analysis of the Plasmodium falciparum parasites circulating in the blood of infected persons frequently reveals the presence of two or more genetically distinct parasite populations. P. falciparum parasites cultured in vitro, from blood specimens collected in the field, are often used for biological, immunological, and drug-resistance investigations relating to the epidemiology in the area concerned or on the assumption that the parasites which grow in vitro are in general representative of all P. falciparum parasites. By using the polymerase chain reaction to detect and characterize a number of parasite polymorphic genes with great sensitivity, the composition of P. falciparum populations from 51 isolates were compared on the day of collection and following 2 months of in vitro culture. It was found that substantial changes in the parasite population profile could be detected in ca. 70% of the samples analyzed. The implications of this observation for studies using parasite isolates cultured in vitro are discussed.