Allergens and irritants transcriptionally upregulate CD80 gene expression in human keratinocytes.

The human CD80 costimulatory molecule is an important signal between professional antigen-presenting cells and T helper cells. The immunobiology of CD80 expression by keratinocytes, especially during allergic and irritant contact dermatitis, however, is less well understood. CD80 cell surface expression and gene transcription by keratinocytes was increased when keratinocytes were exposed to certain allergens (chemicals that induce inflammation via hapten-specific T cells) and irritants (chemicals that are toxic to epidermal cells). Therefore, the human CD80 promoter was cloned and luciferase reporter constructs containing various promoter fragments were engineered. Promoter mapping of these CD80 constructs in transiently transfected keratinocytes showed that a construct containing the proximal 231 bp immediately upstream of the transcription start site of the CD80 promoter was most active in keratinocytes and was inducible to a level ranging from 2- to 10-fold higher in keratinocytes treated with certain allergens and irritants, compared with untreated keratinocytes. This pattern of promoter fragment activity in keratinocytes is identical to that found in professional antigen-presenting cells. This is the first demonstration that the CD80 promoter is active in keratinocytes and that this activity is further increased in keratinocytes treated with certain allergens and irritants. These data suggest that allergens and irritants may, in part, break peripheral tolerance by their direct effects on keratinocyte costimulatory molecule expression, thereby facilitating interactions with epidermotropic T helper cells via the CD80-CD28 or CTLA-4 pathways.

Isoform diversity and tandem duplication of the glycoprotein A gene in ferret Pneumocystis carinii.

Two ferret P. carinii gpA cDNA clones were identified that reacted identically with a panel of anti-gpA monoclonal antibodies, although their nucleotide sequences were 22% divergent. Each clone hybridized to a single mRNA species of 3,600 nucleotides only in P. carinii-infected lung mRNA, but RT-PCR analysis demonstrated that these cDNA clones were derived from two distinct gpA mRNA transcripts. Further PCR analysis demonstrated that the ferret P. carinii genome contains at least two gpA genes lying in tandem on a single chromosome separated by a 329-bp intergenic region. Based on the terminal gene sequences of this tandem repeat and the cDNA clones, a composite full-length ferret P. carinii gpA coding sequence was constructed. The intergenic region immediately downstream of the stop codon of the first gpA gene contains three putative polyadenylation signals, and constitutes the 3' untranslated region (UTR) of the gpA mRNA. Primer extension of the gpA mRNA resulted in products extending 74 and 244 nucleotides into the 5' UTR. However, the intergenic region lying greater than 25 nucleotides upstream of the first methionine of the second gpA gene was found to be absent from the 5' UTR.

Molecular characterization of mouse Pneumocystis carinii surface glycoprotein A.

Since the mouse offers an easily manipulated experimental animal model for the study of the immunopathogenesis of pneumonia caused by the opportunist Pneumocystis carinii, we cloned and characterized cDNAs encoding an abundant, immunogenic surface antigen termed glycoprotein A (gpA) from mouse P. carinii. A cDNA library was constructed in bacteriophage lambda gt11 from P. carinii-infected mouse lung poly(A+) RNA. Using a nucleic acid probe derived from a conserved region of the mouse P. carinii gpA structural gene, cDNAs encoding gpA were identified. A composite full-length gpA coding sequence was assembled from two overlapping cDNA clones. A DNA element homologous to the rat P. carinii upstream conserved sequence (UCS) was identified at the 5' end of several of the mouse P. carinii gpA cDNA clones, just upstream of the sequences encoding gpA structural gene isoforms. Using primer extension analysis, two neighboring putative transcriptional start sites were located on UCS-gpA mRNAs approximately 25 and 30 nt, respectively, upstream of the most 5' gpA cDNA clone isolated, suggesting a 5' UCS of 489 or 494 nucleotides in mouse P. carinii gpA. A comparative alignment of the composite mouse P. carinii gpA deduced amino acid sequence with gpA homologs from rat, human and ferret P. carinii demonstrated 156 identical residues, including 46 cysteines, further supporting the hypothesis for conserved secondary structure, as well as function, for gpA from all P. carinii.

Cloning and characterization of a conserved region of human and rhesus macaque Pneumocystis carinii gpA.

Although the genes encoding Pneumocystis carinii (Pc) glycoprotein A (gpA) display a high degree of host species-specific genotypic diversity, the Pc gpA derived from different host species share defined regions of significant homology in their primary amino acid (aa) structure. Using two degenerate oligodeoxyribonucleotide (oligo) primers corresponding to a conserved Cys region (Cys-primers) of the ferret (F), rat (R) and mouse (M) PcgpA, a 306-bp portion of the human (H) PcgpA was amplified from only one of three known HPc-infected lung samples using PCR. The deduced aa sequence of the HPc PCR product was 72% similar to the corresponding region of a published HPc gpA aa sequence. Because the conserved Cys-primers amplified only one of three samples of HPcgpA, a primer-pair was designed from sequences internal to the Cys-primer sequences of the HPcgpA PCR product (hPc). The hPc primers amplified the expected 254-bp product from each of the three HPc-infected lung DNA samples, suggesting that the Cys-primers may have either amplified a HPcgpA present in fewer copies in the genome of HPc or, alternatively, amplified a gene from an uncommon strain of Pc encoding an isoform variant of gpA not present in the other human isolates analyzed in this report. Restriction analysis of the amplified products demonstrated heterogeneity in the internal sequence, confirming that more than one gpA exists in HPc as well. To determine the relationship of HPcgpA to the gpA of Pc from another primate, the hPc primers were used successfully to amplify a 261-bp product from Pc-infected Rhesus macaque (Rm) lung genomic DNA. These results are consistent with our earlier findings that closely related host species are infected with Pc organisms encoding similar gpA, suggesting that the evolutionary divergence of Pc followed that of the mammalian host species.

Molecular characterization of KEX1, a kexin-like protease in mouse Pneumocystis carinii.

Expression screening of a Pneumocystis carinii-infected mouse lung cDNA library with specific monoclonal antibodies (mAbs) led to the identification of a P. carinii cDNA with extensive homology to subtilisin-like proteases, particularly fungal kexins and mammalian prohormone convertases. The 3.1 kb cDNA contains a single open reading frame encoding 1011 amino acids. Structural similarities to fungal kexins in the deduced primary amino acid sequence include a putative proenzyme domain delineated by a consensus autocatalytic cleavage site (Arg-Glu-Lys-Arg), conserved Asp, His, Asn and Ser residues in the putative catalytic domain, a hydrophobic transmembrane spanning domain, and a carboxy-terminal cytoplasmic domain with a conserved tyrosine motif thought to be important for localization of the protease in the endoplasmic reticulum and/or Golgi apparatus. Based on these structural similarities and the classification of P. carinii as a fungus, the protease was named KEX1. Southern blotting of mouse P. carinii chromosomes localized kex1 to a single chromosome of approximately 610 kb. Southern blotting of restriction enzyme digests of genomic DNA from P. carinii-infected mouse lung demonstrated that kex1 is a single copy gene. The function of kexins in other fungi suggests that KEX1 may be involved in the post-translational processing and maturation of other P. carinii proteins.

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens.

A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.

Purification and characterization of stage-specific glycoproteins from Trypanosoma cruzi.

Four developmentally regulated glycoproteins were purified from detergent solubilized cell membranes of Trypanosoma cruzi. Three trypomastigote specific glycoproteins each migrated as single bands under denaturing conditions with approximate Mr of 90,000, 85,000, and 55,000 and pI values of 4.3-5.0, 8.5-9.1, and 8.2, respectively. The fourth, epimastigote specific, protein had an approximate Mr of 72,000 and a pI of 4.8-5.1. The Mr of all four glycoproteins changed by 5-50% upon endoglycosidase F treatment. The Mr 72,000 antigen was the only one that reacted strongly with anti-epimastigote sera raised in rabbits. Sera from a Chagasic patient reacted strongly with the three trypomastigote specific glycoproteins and very weakly with the Mr 72,000 glycoprotein.

Efficacy of combined immunostimulation and chemotherapy in experimental visceral Leishmaniasis.

A regimen of combined immunostimulation and chemotherapy for the elimination of Leishmania donovani amastigotes was evaluated. An in vitro experimental model utilized cultured peritoneal macrophages from C57B1/6 mice infected with L. donovani tissue forms. Partial or complete activation of macrophages as judged by killing of tumor cells significantly enhanced the efficacy of sodium antimony gluconate (Pentostam). The quantity of drug required for elimination of parasites from immunostimulated cells was considerably lower than that required to achieve comparable amastigote killing in thioglycolate-elicited macrophages. In contrast, amphotericin B cleared infected cells of amastigotes at comparable drug levels when tested with immunostimulated and unstimulated macrophages. Several drugs tested inhibited the conversion of amastigotes to promastigotes in vitro but were ineffective in killing of intracellular tissue forms. Allopurinol and difluoromethylornithine (DMFO) blocked amastigote conversion significantly. These drugs at high concentrations, however, exerted only minimal toxicity for amastigotes residing within macrophages. Efficacy of combined therapy was also demonstrated in vivo. Immunoenhancement of L. donovani-infected mice with Corynebacterium parvum vaccine combined with a regimen of sodium antimony gluconate was significantly more effective than was immunotherapy or drug therapy alone.

Antigenic properties of recombinant glycosylated and nonglycosylated Pneumocystis carinii glycoprotein A polypeptides expressed in baculovirus-infected insect cells.

Since a continuous culture system is not yet available for the opportunistic fungal pathogen Pneumocystis carinii, obtaining suitable amounts of purified P. carinii antigens free of mammalian-host lung contaminants is difficult. Hence, production of recombinant antigen possessing epitopes found in native P. carinii antigens is critical for immunological studies. We utilized the baculovirus expression vector system (BEVS) in insect cells to determine whether B-cell epitopes present in the protein core of a native P. carinii surface glycoprotein were conserved in the recombinant polypeptide, and to investigate its glycosylation by insect cells. B-cell epitopes were retained, but the insect cells appeared to hyperglycosylate the recombinant protein.

The influence of morphological variation on Candida albicans adhesion to denture acrylic in vitro.

Using denture acrylic pieces coated with either whole human stimulated saliva or oral streptococci, the binding ability of three different Candida albicans strains was investigated. The C. albicans strains include a clinical isolate with the commonly observed, smooth, round colonial morphology (strain 613p), a morphological variant spontaneously derived from the clinical isolate strain 613p (strain 613m1BK) and a clinical isolate from an oral lesion that was also a morphological variant upon primary isolation (strain 228). Levels of adhesion to the acrylic pieces were determined radiometrically using C. albicans cells metabolically labelled with [35S]-methionine. Whole stimulated saliva significantly increased the binding of all strains compared to uncoated acrylic. However, the level of binding of strain 613p to saliva-coated acrylic was significantly greater than the levels observed for the morphological variant strain 613m1BK. Coating acrylic pieces with either Streptococcus sanguis NCTC 10904, Strep. mutans GS-5 or Strep. sobrinus ATCC 27352 instead of saliva resulted in significantly greater binding by strain 613p compared to uncoated acrylic. Pre-coating the acrylic with the oral streptococci did not significantly increase the binding of morphological variant strains 613m1BK and 228 compared to uncoated acrylic. In general, preincubation of adherent streptococci with sucrose to induce the synthesis of extracellular carbohydrate polymers did not significantly increase the binding levels of the C. albicans strains above those observed using streptococci in buffer alone. Compared to its parental strain 613p, morphological variant strain 613m1BK adhered poorly to denture acrylic coated with either salivary constituents or oral streptococci, while strain 228 adhered to the same substrates at an intermediate level. Furthermore, physical disaggregation of clusters of the morphological variant strain 613m1BK did not appear to increase its binding capacity to saliva-coated denture acrylic. The effect of whole stimulated saliva on the adherence of C. albicans 613p to a variety of plastic substrates in addition to denture acrylic was examined. Overall, saliva pre-coating of the various plastics promoted C. albicans 613p adhesion. The adhesion of strain 613p to denture acrylic coated with whole stimulated saliva from each of five different donors or with parotid and submandibular/sublingual saliva from each of two donors was also examined. Regardless of donor, a coating of whole stimulated saliva significantly increased the binding of strain 613p to denture acrylic compared to uncoated acrylic. In addition, a coating of parotid saliva significantly increased the binding of strain 613p to denture acrylic compared to submandibular/sublingual saliva.

Effect of anaerobiosis and nitrate on gene expression in Pseudomonas aeruginosa.

DNA microarrays were used to examine the transcriptional response of Pseudomonas aeruginosa to anaerobiosis and nitrate. In response to anaerobic growth, 691 transcripts were differentially expressed. Comparisons of P. aeruginosa grown aerobically in the presence or the absence of nitrate showed differential expression of greater than 900 transcripts.

Identification and characterization of a Candida albicans-binding proteoglycan secreted from rat submandibular salivary glands.

A previously identified Candida albicans-binding glycoprotein secreted from rat submandibular glands (RSMG) has been further purified from an aqueous RSMG extract by ion-exchange chromatography and gel filtration. Biochemical analysis of the glycoprotein revealed high levels of uronic acid and sulfate, suggesting that it was a proteoglycan. Its amino acid and carbohydrate compositions were similar to those observed for other proteoglycans and differed significantly from those of RSMG mucin, the major secretory glycoprotein of RSMG. In addition, the apparent molecular weight of the glycoprotein was reduced following treatment with either chondroitinase ABC or heparitinase, demonstrating the presence of chondroitin sulfate and heparan sulfate. On the basis of its structure and anatomical source, the glycoprotein is referred to as submandibular gland secreted proteoglycan 1 (SGSP1). SGSP1 also binds monoclonal antibody 1F9, which recognizes the human blood group A carbohydrate epitope found on RSMG mucin. Hence, SGSP1 appears to be a hybrid molecule with carbohydrate structures found in both proteoglycans and RSMG mucin. Enzymatic digestion of SGSP1, followed by its interaction with a radiolabelled C. albicans strain in a filter-binding assay, demonstrated that binding to this strain appears to be mediated primarily via the heparan sulfate side chains of SGSP1 and not via the blood group A oligosaccharide.

Analysis of Candida albicans adhesion to salivary mucin.

Clearance of Candida albicans from the oral cavity is thought to be mediated via specific receptor-ligand interactions between salivary constituents and the fungus. Since surfaces in the oral cavity are normally coated with a saliva-derived pellicle, specific interactions between salivary constituents and C. albicans may also contribute to adhesion of C. albicans to the oral mucosa and dental prostheses. Therefore, the purpose of this study was to identify salivary constituents to which C. albicans is capable of binding. A solid-phase overlay assay was used in which electrophoretically separated rat and human salivary constituents bound to membrane filters were incubated with radiolabelled C. albicans cells. C. albicans adhered to a single salivary component from each host. Correlation of cell-binding activity with specific monoclonal antibody (MAb)-binding activity indicated that the constituent bound by C. albicans in human saliva was low-molecular-weight mucin (MG2) and that in rat saliva was rat submandibular gland (RSMG) mucin. Further studies showed an identical cell hybridization signal and MAb colocalization by using RSMG ductal saliva and an aqueous RSMG extract in the solid-phase overlay assay. Analysis of cell binding to the aqueous extract of RSMG fractionated by anion-exchange chromatography demonstrated that C. albicans binding was restricted to an acidic subfraction of the RSMG extract, which also bound the RSMG mucin-specific MAb. The Candida-binding fraction contained predominantly RSMG mucin glycoprotein and also a noncovalently associated, chloroform-extractable material. Furthermore, we identified two strains of C. albicans which differed severalfold in the ability to bind RSMG mucin in the overlay assay. These results suggest that C. albicans binds to only a specific subfraction of RSMG mucin and that the two C. albicans strains tested differ in the ability to bind RSMG mucin subfractions.

Experimental studies of the pathogenesis of infections due to Pseudomonas aeruginosa: extracellular protease and elastase as in vivo virulence factors.

The effects on mortality of supplemental injections of protease and elastase were determined in burned mice infected with non-lethal inocula of a toxin-producing but non-proteolytic-enzyme-producing strain of Pseudomonas aeruginosa. When a variety of solutions containing proteolytic enzyme were injected under these conditions, the mortality increased significantly. This did not occur when organisms other than P. aeruginosa were used. Injections of the enzyme solutions alone were non-lethal. Injection of a solution of alpha 2-macroglobulin, which was shown to inhibit proteolytic activity, together with a proteolytic enzyme--toxin producing strain of P. aeruginosa caused a significant delay in mortality when compared with controls. It was concluded that protease, elastase, and toxin production were necessary for P. aeruginosa to express full virulence in the burned mouse model.

Antigenic characterization of Pneumocystis carinii.

Studies of Pneumocystis carinii have been limited by our inability to propagate it in continuous culture. In this context, studies of P. carinii antigens have provided significant insight into the biology of this organism. The mannose-rich surface major surface glycoprotein of P. carinii termed glycoprotein A (gpA) is the best studied of these P. carinii antigens. Significant genetic and immunologic diversity exists between the gpA molecules expressed by P. carinii derived from different mammalian sources. The molecular and biochemical nature of gpA and other P. carinii antigens including p55 are reviewed. In addition, available information concerning the role of P. carinii gpA and other antigens in host-organism interactions are also discussed.

A role for oxygen-dependent mechanisms in killing of Leishmania donovani tissue forms by activated macrophages.

Leishmania donovani, the causative agent of visceral leishmaniasis, infects macrophages (M phi ) of susceptible vertebrates. Immunologically activated M phi are leishmanicidal, but the mechanisms involved in the killing process are not well defined. We sought to investigate the role of reactive oxygen intermediates in the killing of L. donovani. Both the free-swimming promastigote and the intracellular amastigote forms were found to be susceptible to killing in vitro by hydrogen peroxide and other oxygen intermediates. Upon phagocytosis by mouse peritoneal M phi, promastigotes elicited a significantly stronger respiratory burst compared with amastigotes as measured by release of superoxide anion. Although amastigotes do not elicit a strong burst of M phi oxidative metabolism during the initial phagocytic event, immunologically activated M phi that acquired leishmanicidal capacity could be triggered to release substantial amounts of H2O2. Hence, the development of leishmanicidal capacity was correlated temporally with enhanced H2O2 generation by the M phi. In contrast, M phi that lost their ability to release significant amounts of H2O2 after several days in culture were unable to eliminate their parasite burden. Catalase markedly inhibited the elimination of amastigotes by lymphokine-stimulated M phi. In toto, the results implicate reactive oxygen intermediates in killing of the tissue form of L. donovani by its host cell, the mononuclear phagocyte.

Elimination of Leishmania donovani amastigotes by activated macrophages.

Tissue macrophages are the obligatory host cells for Leishmania donovani, the causative agent of visceral leishmaniasis. In this study we sought to determine whether activated macrophages, as defined by the functional criterion of tumor cell cytotoxicity, were also able to exert a microbicidal effect on ingested L. donovani amastigotes. We found that mouse peritoneal macrophages activated by a variety of means exerted a cytotoxic effect on tumor cell targets but were not able to kill L. donovani amastigotes unless the infected macrophages were exposed continually to an activating stimulus. Corynebacterium parvum, Mycobacterium tuberculosis H37Ra, and lymphokine-activated peritoneal macrophages from C57BL/6J mice were cytotoxic for EMT6 tumor cell targets. However, L. donovani Sudan strain 1S amastigotes were not killed by these macrophages unless the activated state was maintained by daily addition of lymphokine to the infected monolayers for several days postinfection. The killing of amastigotes was dependent on the time of exposure to lymphokine, as well as on the concentration of lymphokine added to the culture.

Intracellular localization of a Trypanosoma cruzi kDNA minicircle transcript using RNA: RNA in situ hybridization.

Using RNA: RNA in situ hybridization, the intracellular location of a transcript encoded by and spanning the entire length of a Trypanosoma cruzi kinetoplast DNA minicircle was determined. In axenically cultured T. cruzi epimastigotes, the hybridization signal was restricted to the kinetoplast, which was situated in the perinuclear region of the cell. Following conversion of epimastigotes to culture-derived metacyclic trypomastigotes, the kinetoplast moved to an acentric position in the metacyclic trypomastigote. Again, the hybridization signal co-localized with the position of the kinetoplast. These results suggested that the transcript remained closely associated with the T. cruzi kinetoplast within the mitochondrion in each of the morphological forms. Using specific oligonucleotide probes derived from a cDNA encoding the transcript, the entire native kDNA minicircle encoding the transcript was cloned and its nucleotide sequence was determined. The nucleotide sequence of the intact native minicircle was identical to that of the full-length cDNA corresponding to the minicircle transcript, indicating that the transcript was not modified prior to the time of cDNA synthesis and cloning.

Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi.

A 1.3 kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.