Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism.

Tick-borne pathogens would appear to be vulnerable to vertebrate host immune responses during the protracted duration of feeding required by their vectors. However, tick salivary components deposited during feeding may inhibit hemostasis and induce immunosuppression. The mode of action and the nature of immunosuppressive salivary components remains poorly described. We determined that saliva from the main vector of the agent of Lyme disease, Ixodes dammini, profoundly inhibited splenic T cell proliferation in response to stimulation with concanavalin A or phytohemagglutin, in a dose-dependent manner. In addition, interleukin 2 secretion by the T cells was markedly diminished by saliva. Tick saliva also profoundly suppressed nitric oxide production by macrophages stimulated with lipopolysaccharide. Finally, we analyzed the molecular basis for the immunosuppressive effects of saliva and discovered that the molecule in saliva responsible for our observations was not PGE2, as hypothesized by others, but rather, was a protein of 5,000 mol wt or higher.

A new member of the immunoglobulin superfamily that has a cytoplasmic region homologous to the leukocyte common antigen.

A human gene (LAR) that hybridizes to mouse leukocyte common antigen cDNA under relaxed hybridization conditions was isolated. The LAR gene is expressed in a broad range of cells, including T lymphocytes, kidney, and prostate cells. The structure of the protein encoded by the LAR gene was deduced by determining the nucleotide sequences of a 7.7-kb LAR cDNA. The putative LAR protein is composed of a 1,234 amino acid extracellular region, a 24 amino acid transmembrane segment, and a 623 amino acid cytoplasmic region. The cytoplasmic region contains two homologous domains that have extensive sequence similarity to the cytoplasmic region of the leukocyte common antigens. The NH2-terminal region of the extracellular segment of the LAR protein contains three tandem Ig-like domains and nine non-Ig-like domains. Among the known Ig-like proteins, the LAR protein has the highest degree of similarity to neural-cell adhesion molecule. The non-Ig-like domains of the LAR protein are also similar to the non-Ig-like domains of neural-cell adhesion molecule.

Differential usage of three exons generates at least five different mRNAs encoding human leukocyte common antigens.

Leukocyte common antigens (LCAs, also known as T200 and CD 45) are integral membrane proteins expressed exclusively on hematopoietic cells. These molecules exhibit varying molecular masses and epitopes when expressed in different cell types. To determine the genetic bases for the generation of this diversity, three classes of human LCA cDNA clones that are different near their 5' ends have been isolated. These differences arose as a result of differential usage of three exons as determined from an analysis of a genomic DNA clone. Furthermore, Northern blot analysis with LCA exon-specific probes demonstrates the existence of at least two more LCA mRNA forms that are generated by differential splicing. A comparison of the human and mouse LCA protein sequences revealed a marked difference only in the extracellular domain.

Reversion to virulence in Leishmania major correlates with expression of surface lipophosphoglycan.

An attenuated clone of Leishmania major was produced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and was biochemically characterized to determine the reason(s) for its loss of virulence. We found that the degree of virulence of L. major did not correlate with either the level of expression of promastigote surface protease (PSP) or with the enzymatic activity of the molecule. In contrast, the levels of lipophosphoglycan (LPG) expressed by the attenuated clone were found to be at least 6-fold less than those of virulent L. major. When the attenuated L. major was injected into BALB/c mice and allowed to revert to virulence, the degree of reversion to virulence that the parasites underwent correlated directly with the amount and form (metacyclic) of LPG expressed by the parasites. Thus, these results further implicate LPG as an important Leishmania virulence factor.

The role of eosinophils and neutrophils in helminth-induced keratitis.

PURPOSE: Intrastromal injection of mice with antigens from the parasitic helminth that causes river blindness (Onchocerca volvulus) induces eosinophil recruitment to the corneal stroma at the time of maximum corneal opacification and neovascularization. The present study was conducted to examine the role of eosinophils and neutrophils in onchocercal keratitis in control C57Bl/6 mice and in interleukin-5 gene knockout (IL-5(-/-)) mice. METHODS: C57Bl/6 and IL-5(-/-) mice were immunized subcutaneously and injected intrastromally with soluble O. volvulus antigens. Mice were killed at various times thereafter. Development of keratitis was assessed by slit lamp examination, and inflammatory cells in the cornea were identified by immunohistochemistry. RESULTS: A biphasic recruitment of inflammatory cells was observed in C57Bl/6 mice; neutrophils predominated during the first 72 hours after intrastromal injection and subsequently declined, whereas eosinophil recruitment increased as time elapsed and comprised the majority (90%) of cells in the cornea by day 7. In contrast, neutrophils were the predominant inflammatory cells in IL-5(-/-) mice at early and late time points and were associated with extensive stromal damage and corneal opacification and neovascularization. Eosinophils were not detected in these mice at any time. CONCLUSIONS: In the absence of eosinophils, neutrophils can mediate keratitis induced by helminth antigens. Together with the early neutrophilic infiltrate in control animals, these observations indicate that neutrophils have an important role in onchocercal keratitis.

Impaired eosinophil recruitment to the cornea in P-selectin-deficient mice in Onchocerca volvulus keratitis (River blindness).

PURPOSE: A murine model of helminth-induced keratitis (river blindness) that is characterized by a biphasic recruitment of neutrophils (days 1-3) and eosinophils (days 3+) to the cornea has been developed. The purpose of this study was to determine the relative contribution of P- and E-selectin in recruitment of these inflammatory cells from limbal vessels to the corneal stroma. METHODS: P- and E-selectin gene knockout (-/-) mice were immunized with antigens extracted from the parasitic helminth Onchocerca volvulus. One week after the last immunization, parasite antigens were injected directly into the corneal stroma. Mice were killed on days 1 and 3 postchallenge, and eyes were immunostained with either anti-eosinophil major basic protein (MBP) or with anti-neutrophil Ab. The number of cells in the cornea was determined by direct counting. RESULTS: Recruitment of eosinophils to the cornea was significantly impaired in P-selectin(-/-) mice (63.9% fewer eosinophils on day 1 [P: = 0.0015], and 61% fewer on day 3 [P: < 0.0001]) compared with control C57BL/6 mice. In contrast, P-selectin deficiency had no effect on neutrophil recruitment to the cornea. There was no inhibition of eosinophil and neutrophil migration to the corneas of E-selectin(-/-) mice, indicating that there is no direct role for this adhesion molecule in helminth-induced keratitis. CONCLUSIONS: The present study demonstrates that P-selectin is an important mediator of eosinophil recruitment to the cornea. P-selectin interactions may therefore be potential targets for immunotherapy in eosinophil-mediated ocular inflammation.

Temporal recruitment of neutrophils and eosinophils to the skin in a murine model for onchocercal dermatitis.

The parasitic helminth Onchocerca volvulus causes ocular onchocerciasis (river blindness) and onchocercal skin disease. To understand the immunologic basis for early stage skin disease, we developed a model in which C57B1/6 mice were immunized subcutaneously and injected intradermally (in the ear) with soluble O. volvulus antigens (OvAg). We found that ear thickness increased significantly after intradermal injection of OvAg and remained elevated for at least 7 days. Dermatitis was dependent on prior immunization, and was associated with an intense cellular infiltrate in the dermis. Neutrophils were the predominant inflammatory cells in the dermis 12 hr after intradermal injection, with only occasional eosinophils present. Conversely, increased ear thickness at later time points was associated with eosinophils, and neutrophils were only rarely detected. Both cell types were present at intermediate time points. These data indicate that recruitment of neutrophils and eosinophils to the skin is temporally regulated.

Identification of a novel receptor tyrosine kinase expressed in acute myeloid leukemic blasts.

Using the polymerase chain reaction with degenerate oligonucleotides derived from conserved motifs within the catalytic kinase domain of protein tyrosine kinases, and RNA extracted from embryonic stem cells, sequences that encode a segment of the kinase domain of several potentially novel receptor tyrosine kinases (RTKs) have been identified. One of these was selected for further study because in Northern analysis it hybridized to RNA from multipotential hematopoietic cell lines, but not from lines representative of lineage-committed cells. A cDNA for this receptor, designated developmental tyrosine kinase (DTK), was isolated and encodes a protein with structural similarities to AXL. Together these receptors form a new class of RTK. DTK is expressed in a number of human leukemic cell lines, and in the blasts of 6 of 11 patients with acute myeloid leukemia (AML) analyzed. The structure of DTK suggests that it may function as a cell adhesion molecule, and mediate cell-to-cell or cell-matrix interactions between hematopoietic cells and their respective microenvironments.

Determination of pentobarbital and certain other barbiturates by capillary gas-liquid chromatography.

A rapid capillary gas-liquid chromatographic method for the determination of barbiturates in plasma at concentrations of clinical importance is presented. This method employs a rapid and simple extraction procedure and direct flame ionization detection with no derivatization.

Quality of life and functional status among homeless men attending a day shelter in Louisville, Kentucky.

The homeless present important social and medical problems in most parts of the United States, including urban areas of Kentucky. While some studies have examined the health status of the homeless, very little is known about their quality of life or functional status. In the present study, 188 homeless men attending St. John's Day Shelter in Louisville completed an interviewer-assisted instrument that assessed self-perceived quality of life using the Perceived Quality of Life (PQOL) scale and self-perceived functional status using the Dartmouth COOP charts. The instrument also included questions on demographics and health risk behaviors of the homeless men. The results show that the typical homeless male attending St. John's is an unmarried, white, middle-aged high school graduate who is unemployed. In terms of quality of life, these men indicate general satisfaction with their physical and cognitive abilities, but significantly lower satisfaction with the social aspects of their lives. With regard to functional status, the homeless men report that they function best in terms of physical fitness and ability to perform daily activities. They report lower assessments of overall health, feelings, quality of life, and social support. These results suggest that poor social role functioning among homeless men is a major contributor to their poor quality of life. Thus, improving their health and quality of life may require an emphasis on social services in addition to routine medical care.

Immune mechanisms in Onchocerca volvulus-mediated corneal disease (river blindness).

Infection with the parasitic nematode Onchocerca volvulus can lead to severe visual impairment and blindness. In an effort to characterize the molecular basis for the inflammatory response in the cornea, we have developed a murine model for O. volvulus-mediated keratitis in which parasite antigens are injected into the corneal stroma of sensitized mice. This model reproduces the two main clinical features of human disease, corneal opacification and neovascularization. Histological analysis of corneas from these mice reveals a biphasic recruitment of neutrophils and eosinophils to the central cornea, along with a small, but persistent number of CD3+ cells. In this review, we present evidence that production of antigen-specific T cell and antibody responses are essential for development of O. volvulus keratitis, and we propose a sequence of molecular and cellular events that lead to migration of inflammatory cells to the cornea and to loss of corneal clarity.

Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450.

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.

N-dealkylation of tertiary amides by cytochrome P-450.

1. N-Methyl-N-alkyl-p-chlorobenzamides (alkyl = Me, Et, nPr, nBu, PhCH2, isoPr and cylcoPr) underwent mono-N-dealkylation exclusively with phenobarbital-induced rat liver microsomes; with each compound both demethylation and dealkylation occurred. 2. The time-courses showed bilinear kinetics, but there was no evidence for general suicide-substrate activity with the cyclopropyl amide, and product ratios did not vary with time. 3. The demethylation/dealkylation ratio varied from 0.3 to 2.0 among the primary alkyl groups but was ca. 40 when the alkyl group was isoPr or cylcoPr. Dealkylation of the benzyl substituent was 2-3 times more favourable than for any other primary alkyl group. Despite wide variations in the demethylation/dealkylation ratios, at near-saturating concentrations of substrates the rates of total oxidation (demethylation plus dealkylation) varied little across the entire series. 4. The results of this study are consistent with a kinetic mechanism involving significant commitment to catalysis, substituent-induced metabolic switching at the product-determining step, a non-catalytic step which is partly rate-limiting in turnover, and a chemical mechanism involving H-atom abstraction as opposed to electron abstraction.

Pathogenesis of onchocercal keratitis (River blindness).

Onchocerciasis is a major cause of blindness. Although the World Health Organization has been successful in reducing onchocerciasis as a public health problem in parts of West Africa, there remain an estimated 17 million people infected with Onchocerca volvulus, the parasite that causes this disease. Ocular pathology can be manifested in any part of the eye, although disease manifestations are frequently characterized as either posterior or anterior eye disease. This review focuses on onchocerca-mediated keratitis that results from an inflammatory response in the anterior portion of the eye and summarizes what is currently known about human disease. This review also describes studies with experimental models that have been established to determine the immunological mechanisms underlying interstitial keratitis. The pathogenesis of keratitis is thought to be due to the host inflammatory response to degenerating parasites in the eye; therefore, the primary clinical symptoms of onchocercal keratitis (corneal opacification and neovascularization) are induced after injection of soluble O. volvulus antigens into the corneal stroma. Experimental approaches have demonstrated an essential role for sensitized T helper cells and shown that cytokines can regulate the severity of keratitis by controlling recruitment of inflammatory cells into the cornea. Chemokines are also important in inflammatory cell recruitment to the cornea, and their role in onchocerciasis is being examined. Further understanding of the molecular basis of the development of onchocercal keratitis may lead to novel approaches to immunologically based intervention.

Sand fly vector saliva selectively modulates macrophage functions that inhibit killing of Leishmania major and nitric oxide production.

The saliva of Phlebotomus papatasi, a sand fly vector for Leishmania major, contains a factor that exacerbates leishmaniasis and may be required for the establishment of infection with Leishmania in nature. We have examined the effect of sand fly saliva on various macrophage functions in vitro. Our data demonstrate that although saliva does not alter uptake of L. major by macrophages, it inhibits the ability of IFN-gamma to activate macrophages to kill the intracellular parasite. This inhibition of parasite killing is observed when both the promastigote and amastigote forms of the parasite are used for infection. Furthermore, this inhibition of parasite destruction correlates with reduction of nitric oxide (NO) production, suggesting that the ability of sand fly saliva to reduce nitrogen oxidation in response to IFN-gamma may be responsible for the inhibitory effect of saliva on intracellular killing of L. major. Finally, despite the fact that saliva inhibits NO production in IFN-gamma-activated macrophages, it does not prevent IFN-gamma from up-regulating class II MHC expression on macrophages. This suggests that the immunosuppressive effect of sand fly saliva on the macrophage is targeted to certain critical, but not all, functions of the cell.

CXC chemokine receptor 2 but not C-C chemokine receptor 1 expression is essential for neutrophil recruitment to the cornea in helminth-mediated keratitis (river blindness).

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2(-/-) and CCR1(-/-) mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1 alpha was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2(-/-) mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2(-/-) mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2(-/-) mice, which was similar to BALB/c mice. Furthermore, although MIP-1 alpha production was lower in CXCR2(-/-) mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.

A dominant role for Fc gamma receptors in antibody-dependent corneal inflammation.

Although production of specific Ab is a critical element of host defense, the presence of Ab in tissues leads to formation of immune complexes, which can trigger a type III Arthus reaction. Our studies on a mouse model of river blindness showed that Ab production is essential for recruitment of neutrophils and eosinophils to the cornea and for development of corneal opacification. In the current study, we determined the relative contribution of complement and FcgammaR interactions in triggering immune complex-mediated corneal disease. FcgammaR(-/-) mice, C3(-/-) mice, and immunocompetent control (B6/129Sj) mice were immunized s.c. and injected intrastromally with Onchocerca volvulus Ags. Slit lamp examination showed that control mice, C3(-/-) mice, and control mice injected with cobra venom factor developed pronounced corneal opacification, whereas corneas of FcgammaR(-/-) mice remained completely clear. Furthermore, recruitment of neutrophils and eosinophils to the corneal stroma was significantly impaired in FcgammaR(-/-) mice, but not in C3(-/-) mice or cobra venom factor-treated mice. We therefore conclude that FcgammaR-mediated cell activation, rather than complement activation, is the dominant pathway of immune complex disease in the cornea. These findings demonstrate a novel role for FcgammaR interactions in mediating ocular inflammation.

Complete exon-intron organization of the human leukocyte common antigen (CD45) gene.

Ten genomic DNA clones encoding the human leukocyte common Ag (LCA, CD45) gene were isolated by screening human genomic DNA libraries with LCA cDNA probes. One genomic DNA clone contains the promoter region and the first two exons, as determined by primer extension analyses and S1 nuclease protection studies as well as nucleotide sequence determination. The first exon does not encode a peptide, while the second exon contains the initiation ATG codon and encodes the signal peptide. The other nine genomic DNA clones, which are separated from the first genomic clone by an unknown distance, are connected and span a total of 73 kb. The nine connected genomic clones encode a total of 31 exons. The 33 exons encoded by these 10 genomic clones account for the entire cDNA sequences including the 5' and 3' untranslated sequences. Exon 3 and exons 7 through 15 encode the extracellular domain sequences that are common to all LCA isoforms. Differential usage of exons 4, 5, and 6, generates at least five distinct LCA isoforms. Exon 16 encodes the transmembrane peptide. The cytoplasmic region of the leukocyte common antigens is composed of two homologous domains. Exons 17 through 24 encode the first domain, and exons 25 through 32 encode the second domain. The comparison of these exons indicated that the homologous domains were generated by duplication of several exons. The most 3' exon (exon 33) encodes the carboxy terminus of the LCA molecules and includes the entire 3' untranslated sequence.

Structure of a cytotoxic T-lymphocyte-specific gene shows a strong homology to fibrinogen beta and gamma chains.

Using a subtractive cDNA cloning method, we isolated a number of T-lymphocyte-specific genes. One of these genes, represented by the cDNA clone pT49, is expressed in cytotoxic T lymphocytes but not in helper T lymphocytes or B lymphocytes. The protein structure deduced from the nucleotide sequence showed a high degree of homology to fibrinogen beta and gamma subunits. This might indicate that evolutionarily fibrinogen has its origin in lymphocytes. In spite of the strong homology of pT49 protein to the fibrinogen subunits, the positions of the introns in the pT49 gene are totally different from those of the fibrinogen genes.

Expression and functional analysis of two isoforms of the human GM-CSF receptor alpha chain in myeloid development and leukaemia.

The human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha chain RNA is alternatively spliced to yield receptor isoforms. Two of these, alpha 1 and alpha 2, differ in their cytoplasmic domains. Because the GM-CSFR beta chain (beta c) is shared with the receptors for interleukins 3 and 5 it is possible that the alpha chain confers specificity on the GM-CSF response and that the different isoforms might refine this response further. Studies have been directed at determination of the respective biological roles of the alpha 1 and alpha 2 isoforms. Expression of the isoforms was examined by RNase protection analysis in normal granulocytes and a variety of cell lines of haemopoietic origin, at different stages of differentiation and activation. Expression was also analysed in cells from patients with a variety of leukaemic subtypes. Results demonstrated that the relative abundance of the isoforms was similar in all cell populations examined. The human GM-CSFR alpha 1 or alpha 2 receptors were independently expressed in the murine factor-dependent cell line FDC-P1, so that the properties of the receptors could be compared. Cell lines that expressed either receptor could be converted to growth in response to human GM-CSF and assumed a more differentiated phenotype when compared with the parental cell line. However, the morphology, expression of cell surface antigens and dose-growth response characteristics did not differ significantly between cells that expressed either the alpha 1 or alpha 2 receptor. These studies demonstrate that the alpha 1 and alpha 2 subunits of the GM-CSF receptor are co-ordinately regulated in both normal and malignant haemopoiesis. Furthermore, each receptor is able to deliver both proliferative and differentiative signals to myeloid cells.