bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular immune response to Mycobacterium leprae infection in human immunodeficiency virus-infected individuals
Authors: Sampaio E P , Caneshi J R , Nery J A , Duppre N C , Pereira G M , Vieira L M , Moreira A L , Kaplan G , Sarno E N
Published: January 2020

The immune responses to Mycobacterium leprae and other mycobacterial antigens were studied in 11 leprosy patients with concurrent human immunodeficiency virus type 1 (HIV-1) infection. Three patients manifested borderline lepromatous leprosy, and eight patients had borderline tuberculoid (BT) leprosy. Despite the low CD4+ T-cell count in the peripheral blood, no histologic or phenotypic change in the cellular infiltrate in either the lepromatous or tuberculoid lesions was observed when compared with HIV-1-negative patients. Lepromatous lesions contained heavily parasitized macrophages and few CD8+ T cells. Lesions from the patients with BT leprosy showed extensive CD4+ T-cell infiltration despite a significant reduction in CD4+ T-cell counts in the peripheral blood. No acid-fast bacilli were detected in the tuberculoid lesions. HIV-1 infection did not alter the lack of response in lepromatous leprosy to M. leprae antigens either in vitro or in vivo. In contrast, the skin test response to M. leprae antigens as well as the in vitro lymphoproliferative responses to mycobacterial antigens that are usually seen in patients with tuberculoid leprosy were abrogated in the BT HIV-1+ patients. However, production of gamma interferon in response to the same stimuli was preserved in most of the patients. Analysis of cytokine gene expression showed activation of additional cytokine genes in the unstimulated peripheral blood cells of patients with both leprosy and HIV-1 infections as compared with cells from patients with leprosy alone. These results suggest that granuloma formation in leprosy can be independent of the impaired CD4+ T-cell response of the HIV-1 infection. Furthermore, in HIV-1+ individuals with M. leprae infection, activation of cytokine genes is observed even when the circulating CD4+ T-cell count is significantly reduced.
Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis
Authors: De Bernardis F , Cassone A , Sturtevant J , Calderone R
Published: January 2020

Several strains of Candida albicans were compared for their ability to cause vaginal infection in a rat model, and their vaginopathic potentials were correlated with the expression of two aspartyl proteinases genes (SAP1 and SAP2) and adherence in vivo to the vaginal epithelium. Dot blot reactions and Northern blot analysis with RNA extracted from the vaginal fluid of rats infected with the highly vaginopathic strains H12 and 10261 demonstrated the expression of both SAP1 and SAP2 during the first week of infection. In contrast, neither gene was expressed during infection by a nonvaginopathic strain (N), even though the organism could be recovered during the first 24 h postinfection. A moderately vaginopathic strain (P) also expressed both genes, but the level of SAP1 mRNA appeared to decrease prior to that of SAP2. Neither gene was expressed, even by the highly vaginopathic strains, after the first week of infection, concomitant with a decrease in the number of organisms recovered from the vaginas. Analysis of in vivo adherence showed that the nonvaginopathic strain (N) adhered to vaginal epithelial cells less readily than the highly vaginopathic strain (H12) and moderately vaginopathic strain (P). Thus, in addition to its inability to express SAP1 and SAP2 in vivo, the nonvaginopathic strain does not colonize host cells to the same extent as the other strains tested. Our results demonstrate the early in vivo expression of two aspartyl proteinase gene during candidal vaginitis and suggest its association with the establishment of a vaginal infection.
Dependence of vascular permeability enhancement on cysteine proteinases in vesicles of Porphyromonas gingivalis
Authors: Imamura T , Potempa J , Pike R N , Travis J
Published: January 2020

Infection with Porphyromonas gingivalis is strongly associated with adult periodontitis, and proteinases are considered to be important virulent factors of the bacterium. In order to investigate the function of proteinases in disease development we examined vesicles, a biological carrier of these enzymes, for the generation of vascular permeability enhancement (VPE) activity, believed to correlate with the exudation of gingival crevicular fluid. The vesicles generated VPE activity from human plasma in a dose-dependent manner which could be inhibited 90% by antipain, a specific inhibitor of the Arg-specific cysteine proteinases (Arg-gingipains [RGPs] from P. gingivalis. Incubation of vesicles with high-molecular-weight-kininogen (HMWK)-deficient plasma did not result in VPE activity. On this basis, RGPs associated with vesicles were assumed to be responsible for most of the VPE activity generation via plasma prekallikrein activation and subsequent bradykinin production. The secondary pathway for VPE activity production was dependent on the direct release of bradykinin from HMWK by the concerted action of RGP and a Lys-specific cysteine proteinase (Lys-gingipain [KGP]), also associated with vesicles. These results indicate that RGP and KGP are biologically important VPE factors acting either via prekallikrein activation (RGP) and/or HMWK cleavage (RGP and KGP) to release BK and, thereby, contributing to the production of gingival crevicular fluid at periodontal sites infected with P. gingivalis.
Molecular cloning of Proteus mirabilis uroepithelial cell adherence (uca) genes
Authors: Cook S W , Mody N , Valle J , Hull R
Published: January 2020

Proteus mirabilis bacteria are a common cause of hospital-acquired urinary tract infection. In a previous study, we described a P. mirabilis fimbrial protein, UCA, that adhered to human uroepithelial cells. Genes sufficient for expression of UCA adherence were cloned into Escherichia coli K-12. E. coli bacteria that contained the uca recombinant plasmid adhered to human uroepithelial cells. In addition, the ucaA gene encoding the structural component of UCA pili was subcloned, and its DNA sequence was determined. Amino acid sequence homology (30 to 50%) was found between mature UcaA protein and pilins from pathogenic bacteria representing several genera, including E. coli F17, G, and type 1C pilins, Haemophilus M43 pilin, and a Bordetella pilin.
Comparative study of the C3d receptor and 58-kilodalton fibrinogen-binding mannoproteins of Candida albicans
Authors: López-Ribot J L , Martínez J P , Chaffin W L
Published: January 2020

Using polyclonal antibodies (PAbs) raised against the Candida albicans C3d receptor (CR2; PAb anti-CR2) and the 58-kDa fibrinogen-binding mannoprotein (mp58; PAb anti-mp58) as well as ligand interactions, we have studied the relationship between these two receptors. In an indirect immunofluorescence assay with germ tubes, greater intensity was observed on the mother blastoconidium when PAb anti-CR2 was used, whereas greater intensity was localized to the hyphal extension when PAb anti-mp58 or binding of soluble fibrinogen was used. No competition or change in the fluorescence pattern was observed in dual-labeling experiments with PAb anti-CR2 and either fibrinogen or PAb anti-mp58. Binding competition also was not observed in an enzyme-linked immunosorbent assay using the components present in a beta-mercaptoethanol extract from the cell wall of germ tubes. In immunoblots, PAb anti-CR2 recognized three different discrete bands with apparent molecular masses of 21, 40, and 66 kDa in the beta-mercaptoethanol extracts from the cell wall, whereas a different, single, broader band with an apparent molecular mass of 58 kDa was detected with PAb anti-mp58. However, when nondenaturing conditions were used to separate the materials present in the cell wall extracts, no reactivity could be detected on Western blots (immunoblots) with PAb anti-mp58. When PAb anti-CR2 was used for analysis, a single band migrating in the area corresponding to approximately 40 kDa was detected. These observations suggest a higher molecular weight for mp58 and one or more of the components detected with PAb anti-CR2 in their native state.
A lipooligosaccharide-binding site on HepG2 cells similar to the gonococcal opacity-associated surface protein Opa
Authors: Porat N , Apicella M A , Blake M S
Published: January 2020

The lacto-N-neotetraose-containing lipooligosaccharide (LOS) present on the surface of most Neisseria gonorrhoeae organisms may serve many important functions in gonococcal pathogenesis. This surface glycolipid contains the cross-reactive epitope to human paragloboside and can be sialylated by gonococci grown in the presence of CMP-N-acetylneuraminic acid. Another possible role for this glycolipid could be to mimic human asialocarbohydrates and act as a ligand for asialoglycoprotein receptors contained on numerous human cells. The most noted of this large family of receptors is that expressed on the surface of hepatic cells. In a model cell system, using the hepatoma tissue culture cell line HepG2, we wanted to investigate if the presence of this asialoglycoprotein receptor influenced the adherence and/or invasion of gonococci expressing the lacto-N-neotetraose structure. Piliated variants of the gonococcal wild-type strain 1291 and its isogeneic LOS mutant 1291E were used in adherence-invasion assays. This gonococcal strain is somewhat unusual in that it expresses large amounts of predominantly one species of LOS, thus reducing the complexity of interpreting the data. The data from these assays suggested that the Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc carbohydrate structure on the wild-type LOS affected the adherence-invasion of gonococci into the HepG2 cells. In studies to determine whether the major hepatic asialoglycoprotein receptor was involved in these interactions, we found that the HepG2 cells contained two receptors which bound gonococcal LOS. One of these was the asialoglycoprotein receptor, and the data concerning this receptor will be reported elsewhere. The data on the second receptor are reported here. Purified, 125I-labeled gonococcal LOS was used to identify specific high-affinity LOS-binding sites. These binding experiments revealed one major binding site corresponding to a protein with a molecular mass of 70 kDa (p70). Several lines of evidence in this study suggested that the oligosaccharide region of LOS played an important role in LOS binding to the p70 of HepG2 cells. In addition, we show that this human LOS receptor has some similarities to the gonococcal Opa proteins.
Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi
Authors: Elkins C , Chen C J , Thomas C E
Published: January 2020

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.
Products of enteropathogenic Escherichia coli inhibit lymphocyte activation and lymphokine production
Authors: Klapproth J M , Donnenberg M S , Abraham J M , Mobley H L , James S P
Published: January 2020

The aim of this study was to determine whether products of enteric bacteria are able to regulate lymphocyte activation and cytokine production. Whole bacteria and bacterial lysates from different strains of Escherichia coli were tested for their ability to inhibit cytokine production by peripheral blood mononuclear cells as determined by reverse transcription-PCR, Northern (RNA) blotting of cellular RNA, or enzyme-linked immunosorbent assay for cytokine protein. Lysates from two pathogenic strains of E. coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli, inhibited mitogen-stimulated expression of interleukin-2 (IL-2), IL-4, IL-5, and gamma interferon. IL-1 beta, IL-6, IL-8, IL-10, IL-12, and Rantes mRNA expression was not affected. The inhibitory activity was dose dependent, protease and heat sensitive, nondialyzable, and not due to cellular toxicity. The inhibitory activity remained in EPEC strains having mutations in known virulence factors. Nonpathogenic E. coli HB101 transformed with a 22-kb cosmid clone derived from EPEC chromosomal DNA expressed the inhibitory activity. Thus, certain strains of pathogenic E. coli express a protein or proteins encoded by chromosomal genes that selectively inhibit lymphocyte activation and lymphokine production. Therefore, immunosuppressive factors produced by pathogenic bacteria could be important in modifying gastrointestinal immune responses in enteric bacterial infections or gastrointestinal autoimmune diseases.
Reactivation of arthritis induced by small bowel bacterial overgrowth in rats: role of cytokines, bacteria, and bacterial polymers
Authors: Lichtman S N , Wang J , Sartor R B , Zhang C , Bender D , Dalldorf F G , Schwab J H
Published: January 2020

Arthritis is often associated with intestinal diseases, but the etiology is not known. We developed a rat model whereby arthritis was reactivated by experimental small bowel bacterial overgrowth (SBBO). Self-limited monoarticular arthritis was induced by intra-articular injection of 2 micrograms of rhamnose peptidoglycan-polysaccharide derived from group A streptococci into the ankle joints in female Lewis rats. Eleven days after intra-articular injection, when swelling was resolving, experimental SBBO induced by surgical creation of jejunal self-filling blind loops reactivated arthritis, but SBBO induced by creation of self-emptying blind loops, which minimally increases luminal bacteria, and sham operation did not (P < 0.001). Increased joint diameters in rats with self-filling blind loops persisted for at least 56 days after surgery. Reactivation of arthritis due to SBBO was prevented by anti-tumor necrosis factor alpha antiserum and interleukin 1 receptor antagonist (P < 0.001), indicating that these cytokines mediate joint swelling secondary to intestinal injury. Recombinant bactericidal/permeability-increasing protein, an agent which neutralizes endotoxin, and metronidazole, which is active against anaerobic bacteria, prevented arthritis (P < 0.001), but polymyxin B (which also neutralizes endotoxin) and gentamicin had no effect. Mutanolysin, an enzyme which degrades peptidoglycan-polysaccharide from group A streptococci, exacerbated arthritis for the first 6 days but then diminished joint swelling from 12 to 21 days after surgery (P < 0.001). These studies introduce a reproducible animal model of reactivation of arthritis secondary to intestinal injury and demonstrate a role for bacterial products from endogenous enteric organisms.
Occurrence of gastric ulcers in gnotobiotic piglets colonized by Helicobacter pylori
Authors: Krakowka S , Eaton K A , Rings D M
Published: January 2020

Archival gastric tissues from 10 of 39 gnotobiotic piglets colonized with Helicobacter pylori 26695 contained stromal leukocytic inflammation along with ulcers and erosions. In contrast, gastric tissues from 54 archival control piglets were devoid of both inflammation and ulcers. These data confirm the hypothesis that H. pylori infection alone is ulcerogenic.
Sex-determined resistance to Toxoplasma gondii is associated with temporal differences in cytokine production
Authors: Roberts C W , Cruickshank S M , Alexander J
Published: January 2020

Examination of a wide range of inbred mice of diverse genetic backgrounds and major histocompatibility complex haplotypes revealed a dramatic difference in the susceptibilities of males and females to Toxoplasma gondii infection. Female mice were found to be more susceptible to acute infection, as determined by higher mortality levels, than male mice, while those female mice surviving to have chronic infections harbored more cysts in their brains than did surviving males. This phenomenon was therefore investigated in greater depth immunologically in the BALB/K mouse, a strain showing moderate susceptibility to infection with T. gondii. Plasma tumor necrosis factor alpha (TNF-alpha) levels were elevated in both male and female BALB/K mice on days 8 and 10 postinfection, but not thereafter, with males producing significantly higher levels than females. However, it was not until day 12 postinfection that the first deaths occurred, and these were among female mice, indicating that TNF-alpha production was not responsible for mortality. In vitro examination of T. gondii-specific T-cell proliferative responses from day 15 postinfection onwards revealed significantly higher stimulation indices in male mice than in their female counterparts. This difference was most apparent in splenocyte cultures initiated at day 15 postinfection, where complete suppression of proliferation was noted in the splenocytes from female mice but not from male mice. Analysis of tissue culture supernatants from these cultures revealed distinct differences in the kinetics of production as well as the quantities of gamma interferon (IFN-gamma) and interleukin 10 (IL-10) produced. Spleen cells from male mice produced higher levels of IFN-gamma in the early stages of infection than those from female mice. IFN-gamma levels were highest in the supernatants from male splenocyte cultures initiated at day 15 postinfection. Similar levels of IFN-gamma were not obtained from the supernatants of female splenocyte cultures until day 22 postinfection. IL-10 production, on the other hand, peaked at maximal levels in the cell cultures from both sexes initiated at day 22 postinfection. These results suggest that, in male mice, a rapid response to infection with high levels of TNF-alpha and IFN-gamma helps to control parasite multiplication, after which IL-10 production may be important in down regulating these potentially harmful inflammatory mediators. The failure of female mice to respond quickly in terms of T-cell proliferation and IFN-gamma production compared with their male counterparts may account for their poor survival rates and higher cyst burdens.
A Mycobacterium leprae gene encoding a fibronectin binding protein is used for efficient invasion of epithelial cells and Schwann cells
Authors: Schorey J S , Li Q , McCourt D W , Bong-Mastek M , Clark-Curtiss J E , Ratliff T L , Brown E J
Published: January 2020

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen. M. leprae can infect a variety of cells in vivo, including epithelial cells, muscle cells, and Schwann cells, in addition to macrophages. The ligand-receptor interactions important in the attachment and ingestion of M. leprae by these nonmacrophage cells remains unknown. Fibronectin (FN) significantly enhances both attachment and ingestion of M. leprae by epithelial and Schwann cell lines. We cloned an M. leprae FN binding protein (FN attachment protein [FAP]) distinct from the 85ABC complex which has been shown previously to bind FN. The FAP open reading frame predicts a protein of 29.5 kDa with a 39-amino-acid signal peptide and was previously described as an antigen in leprosy patients. M. leprae FAP has homologies in M. vaccae, M. avium, and M. tuberculosis, as determined by Southern blotting and direct peptide analysis. Both anti-FAP antibodies and an Escherichia coli-expressed recombinant protein significantly blocked M. leprae attachment and internalization by T-24, an epithelial cell line, and JS1, a Schwann cell line. These data suggest that FN can be a bridging opsonic ligand for attachment of mycobacteria to nonphagocytes and that FAP plays an important role in this process. This may be an important step in the initiation of M. leprae infection in vivo.
Directed genomic integration in Actinobacillus actinomycetemcomitans: generation of defined leukotoxin-negative mutants
Authors: Kolodrubetz D , Phillips L H , Ezzo P J , Kraig E
Published: January 2020

To develop targeted gene integration in the periodontal pathogen Actinobacillus actinomycetemcomitans, a ColE1-based, spectinomycin-resistant plasmid containing a segment of the leukotoxin gene was electroporated into strain JP2. In all of the stable spectinomycin-resistant transformants that arose, the plasmid had recombined into the genomic leukotoxin locus since ColE1-based vectors cannot replicate extrachromosomally in A. actinomycetemcomitans. Directed genomic integration was then used to construct a leukotoxin-negative strain by transforming the leukotoxin-producing strain JP2 with a ColE1-based plasmid containing an internal fragment of the leukotoxin gene. Cytotoxicity assays proved that these transformants had < 0.1% of the leukotoxin activity of the parental strain. These results demonstrate that integration-based approaches can be used for generating isogenic mutants in specific virulence genes in A. actinomycetemcomitans.
A natural mutation of the amino acid residue at position 60 destroys staphylococcal enterotoxin A murine T-cell mitogenicity
Authors: Mahana W , al-Daccak R , Lévéillé C , Valet J P , Hébert J , Ouellette M , Mourad W
Published: January 2020

A variety of techniques have been used to identify the amino acid residues of bacterial superantigens involved in their interactions with major histocompatibility complex (MHC) class II and T-cell receptor (TCR). In this study, we isolated a naturally mutated staphylococcal enterotoxin A (SEA) from three different Staphylococcus aureus strains, in which the amino acid at position 60 has been changed from aspartic acid (D) to asparagine (N). We then studied the influence of this change on the immunological activities of SEA. Our results demonstrated that this mutation does not affect the capacity of SEA to bind MHC class II molecules and consequently activates human monocytes and peripheral blood lymphocytes. In contrast, mutated SEA failed to stimulate the proliferation of murine splenic lymphocytes of two different strains, and when presented by human MHC class II molecules, it also failed to activate murine cell line 3DT, which expresses the SEA-specific TCR V beta element (V beta 1). These results indicate that this mutation alters the interaction between SEA and murine TCR. The reactivity patterns of the mutated SEA with two specific anti-SEA monoclonal antibodies suggested that the observed effect of the isolated mutation in the murine system might be due to certain conformational changes in the SEA molecule introduced upon changing the D at position 60 to N. Site-directed mutagenesis of the N residue to D or to glycine reconstituted the ability of SEA to stimulate murine splenic lymphocytes. The different effects of this natural mutation at position 60 on the immunological activities of SEA with murine and human cells highlight the relevance of the affinity and avidity in SEA-TCR interactions in the function of different species or may reflect a difference in epitope specificity.
Fine specificity of the genetically controlled immune response to native and recombinant gp15/400 (polyprotein allergen) of Brugia malayi
Authors: Allen J E , Lawrence R A , Maizels R M
Published: January 2020

Polyprotein allergens are a family of structurally homologous molecules from parasitic nematodes which induce specific immunoglobulin E in infected individuals. We show here that both H-2 and non-H-2 factors determine the ability of mice to generate T- and B-cell responses to the filarial polyprotein allergen (Brugia malayi gp15/400). Further, H-2 and non-H-2 genes can complement one another to overcome nonresponsiveness to this molecule. However, these genetic restrictions govern only responses to the native glycoprotein and all strains of mice respond equivalently when immunized with a recombinant polypeptide. Overlapping fragments of gp15/400 were constructed to compare the T-cell and antibody responses to native versus recombinant gp15/400 in responder (BALB/c H-2d) and nonresponder (B10.D2 H-2d, CBA H-2k, and BALB.K H-2k) strains. BALB/c mice generated T-cell responses to the same fragment (positions 89 to 133 and 1 to 21) whether immunized with native or recombinant material, although the antibody responses differed in fine specificity, H-2k mice, unresponsive to the native molecule, generated T cells responsive to the centrally located peptide (positions 57 to 100) only when immunized with the recombinant. Antibody responses in H-2k mice were directed at the peptide (positions 11 to 67) which is glycosylated in the native molecule. Our findings suggest that recognition of gp15/400 is affected by modifications that occur in the parasite but are absent when the molecule is produced in bacteria. This study provides a detailed evaluation of the immune response to an important nematode antigen as a start to the unraveling of the complex interaction of these multicellular parasites with mammalian hosts.
Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge
Authors: Hensel A , Stockhofe-Zurwieden N , Petzoldt K , Lubitz W
Published: January 2020

A dose-defined aerosol infection of pigs was used to study the immunogenic and protective potentials of oral immunization with dead or live Actinobacillus pleuropneumoniae serotype 9 reference strain CVI 13261 against an aerogenic challenge. Pigs were vaccinated with a single dose of 10(11) CFU of viable (n = 8) or inactivated (n = 8) A. pleuropneumoniae given orally in a gelatin capsule. After 3 weeks, vaccinated pigs and nonvaccinated controls were challenged aerogenically with a dose of 10(8) CFU of A. pleuropneumoniae CVI 13261. The protective efficacy of oral immunization was evaluated by clinical and postmortem examinations. Bronchoalveolar lavage in pigs was performed during the experiment to obtain lavage samples for assessment of local antibodies. Isotype-specific antibody responses in sera and in bronchoalveolar lavage fluids were determined by enzyme-linked immunosorbent assays based on whole-cell antigen. Oral immunization did not induce clinical side effects. After aerosol challenge, two animals of both vaccinated groups (25% in each case) showed a moderate fever for 2 days, whereas all four pigs (100%) of the nonvaccinated control group developed severe fever. In contrast to the controls, which developed severe pleuropneumonia, the vaccinated pigs had only mild pulmonary lesions. Three weeks after challenge, 13 of 16 vaccinated pigs (81%) were found to be free of pathomorphological changes of the lungs. From two of these pigs immunized with live bacteria we were able to reisolate A. pleuropneumoniae. A significant systemic and pulmonary increase in the concentrations of immunoglobulin A (IgA), IgM, and IgG antibodies reactive with A. pleuropneumoniae was detectable after aerosol challenge in both vaccinated groups. Immunization with viable bacteria was found to induce significantly higher concentrations of each Ig isotype in bronchoalveolar lavage fluids and sera than immunization with inactivated A. pleuropneumoniae. These serological findings were not reflected in the reduction in clinical disease after challenge in comparison to the case for the pigs vaccinated with inactivated bacteria. We concluded that a single oral administration of A. pleuropneumoniae provides partial clinical protection against aerosol challenge infection in the respiratory tract.
Protection of C3H/He mice from experimental Borrelia burgdorferi infection by immunization with a 110-kilodalton fusion protein
Authors: Bey R F , Larson M E , Lowery D E , Lee B W , Knutson K S , Simonson R R , King V L
Published: January 2020

A 110-kDa Borrelia burgdorferi fusion protein, Escherichia coli expressing the fusion protein, transformed E. coli lacking the fusion protein insert, and lyophilized whole B. burgdorferi bacteria were compared for immunogenicity in C3H/He mice. Immunized mice were challenged with a variety of isolates from the United States or the European isolate P/Gau 3 weeks following the last inoculation. An average of 76.7% of the mice immunized with 25 micrograms of lyophilized whole B. burgdorferi cells were protected from infection, while 60% of the mice immunized with the 110-kDa fusion protein were protected. Whole E. coli bacteria expressing the fusion protein protected 57.7% of immunized mice against experimental challenge. Lower levels of protection occurred in mice challenged with the European isolate than in those challenged with isolates originating from the United States. These results demonstrate the potential of the 110-kDa fusion protein for use as a component of a subunit vaccine for prevention of Lyme borreliosis.
Characterization of recombinant tetanus toxin derivatives suitable for vaccine development
Authors: Figueiredo D , Turcotte C , Frankel G , Li Y , Dolly O , Wilkin G , Marriott D , Fairweather N , Dougan G
Published: January 2020

Recombinant derivatives of tetanus toxin (TeTx) were isolated and used to immunize mice. Recombinant TeTx light chain, a derivative of fragment C that had lost the ability to bind neurons, and a recombinant TeTx holotoxoid that could protect mice against TeTx challenge were identified.
The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin
Authors: Betsou F , Sebo P , Guiso N
Published: January 2020

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.
Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552
Authors: Jensen L T , Ladefoged S , Birkelund S , Christiansen G
Published: January 2020

Three mutants of Mycoplasma hominis PG21 were isolated and shown to contain alterations in the size of a repeat-containing gene encoding a surface-localized 135-kDa antigen designated Lmp1. The mutants were isolated by cultivating M. hominis for a 3-month period in the presence of Lmp1-specific monoclonal antibody (MAb) 552. The epitope for MAb 552 was localized at the repeated part of the protein. The gene encoding Lmp1 is part of a transcriptional complex that contains 9.5 direct repeats of 471 bp each. Pure cultures of mutant strains were obtained by subcloning, and three mutants were characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells in culture medium was, however, increased, indicating that the repeated elements may be of importance for repulsion of the cells.
Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis
Authors: Gillaspy A F , Hickmon S G , Skinner R A , Thomas J R , Nelson C L , Smeltzer M S
Published: January 2020

To examine the role of the accessory gene regulator (agr) in staphylococcal osteomyelitis, we compared a Staphylococcus aureus osteomyelitis isolate (UAMS-1) with a derivative of the same strain (UAMS-4) carrying an inactivated agr locus. Virulence was assessed with a rabbit model of acute, exogenous osteomyelitis. Bacteria were delivered by microinjection into the midradial region of the forelimb. After 4 weeks, UAMS-1 was identified in the bone of 12 of 13 rabbits infected with > or = 2 x 10(6) CFU and 5 of 6 infected with < or = 2 x 10(5) CFU. In contrast, UAMS-4 was found in 6 of 13 infected with the higher dose and 1 of 6 infected with the lower dose. Additionally, on the basis of a five-point scale assessing radiographic evidence of disease, rabbits infected with UAMS-1 had average scores of 2.64 +/- 0.30 (high dose) and 1.43 +/- 0.39 (low dose) while rabbits infected with UAMS-4 had average scores of 0.95 +/- 0.23 (high dose) and 0.63 +/- 0.20 (low dose). Uninfected controls had an average score of 0.53 +/- 0.08. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.047). The results obtained with UAMS-4 were not significantly different from those obtained with the controls at either dose of UAMS-4 (P > or = 0.150). On the basis of a similar five-point scale assessing histopathological evidence of disease, rabbits infected with UAMS-1 had average scores of 2.31 +/- 0.22 (high dose) and 1.96 +/- 0.36 (low dose) while rabbits infected with UAMS-4 had average scores of 1.58 +/- 0.29 (high dose) and 0.83 +/- 0.32 (low dose). Controls had an average score of 0.33 +/- 0.05. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.040). However, the results obtained with UAMS-4 were significantly different from the controls only at the high dose of UAMS-4 (P = 0.025). We conclude that mutation of agr reduces the incidence and severity of disease but does not eliminate the ability to colonize bone and cause histopathological evidence of osteomyelitis.
Effect of hypertriglyceridemia on endotoxin responsiveness in humans
Authors: van der Poll T , Braxton C C , Coyle S M , Boermeester M A , Wang J C , Jansen P M , Montegut W J , Calvano S E , Hack C E , Lowry S F
Published: January 2020

Triglyceride-rich lipoproteins can inhibit endotoxin activity in vitro and in rodents. We sought to determine whether Intralipid, a triglyceride-rich fat emulsion which in contact with plasma functions similarly to endogenous lipoproteins, can alter the human response to endotoxin. Intralipid inhibited endotoxin-induced cytokine production in human whole blood in vitro in a dose-dependent manner, with maximal inhibition (up to 70%) being achieved at a concentration of 10 g/liter. In healthy men, a bolus intravenous injection of endotoxin (lot EC-5; 20 U/kg of body weight) was given midway through a 4-h infusion (125 ml/h) of either 5% glucose (n = 5) or 20% Intralipid (n = 5). The infusion of Intralipid led to an increase in triglyceride levels in serum from 95 +/- 16 to 818 +/- 135 mg/dl prior to endotoxin administration, i.e., levels that importantly reduced cytokine production in endotoxin-stimulated whole blood. However, in vivo hypertriglyceridemia did not influence inflammatory responses to endotoxin (fever, release of tumor necrosis factor and soluble tumor necrosis factor receptors, and leukocytosis) or even potentiated endotoxin responses (release of interleukins 6 and 8 and neutrophil degranulation). Hypertriglyceridemia does not inhibit the in vivo responses to endotoxin in humans.
Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice
Authors: Dunn P L , North R J
Published: January 2020

Three virulent strains of Mycobacterium tuberculosis (H37Rv, Erdman, and NYH-27) and two virulent strains of M. bovis (Ravenel and Branch) were compared in terms of their growth rates in the livers and the lungs of mice, their ability to cause lung pathology, and the time taken for them to cause death. In immunocompetent mice, all strains caused an infection that progressed for 20 days or more and then underwent resolution in the liver but not in the lungs. In the lungs, infection persisted and induced progressive pathology. According to host survival time, Ravenel was the most virulent strain, followed, in decreasing order of virulence, by Branch, H37Rv, Erdman, and NYH-27. The much longer survival times of mice infected with M. tuberculosis strains allowed time for lung histopathology to change from a histiocytic alveolitis to a chronic fibroblastic fibrosis that eventually obliterated most of the lung architecture. By contrast, in mice infected with M. bovis strains, the alveolitis that developed during early infection was rapid and expansive enough to cause death before chronic lung pathology became evident. In mice depleted of CD4+ T cells, increased growth of all virulent strains induced necrotic exudative lung lesions that rapidly filled most of the alveolar sacs with inflammatory cells. These mice died much earlier than infected control mice did. Attenuated strains had longer population doubling times in vivo and failed to cause progressive disease or pathology in the lungs or livers of immunocompetent mice.
Binding of human plasminogen to Borrelia burgdorferi
Authors: Hu L T , Perides G , Noring R , Klempner M S
Published: January 2020

We studied the binding of plasminogen to Borrelia burgdorferi, a spirochete which causes Lyme disease and produces no endogenous proteases which digest extracellular matrix proteins. Using 125I-labeled plasminogen, we demonstrated that B. burgdorferi bound human plasminogen and that this binding was inhibitable with unlabeled plasminogen. 125I-labeled plasminogen binding by B. burgdorferi was also inhibited by the lysine analog epsilon-aminocaproic acid. There was no significant difference in the binding of Glu- or Lys-plasminogen to B. burgdorferi. Binding of plasminogen was similar in low-passage (infectious) and high-passage (noninfectious) isolates of B. burgdorferi. Plasminogen bound to the surface of B. burgdorferi could be converted into plasmin by a human urokinase-type plasminogen activator. 125I-labeled plasminogen ligand blots of borrelial membrane proteins demonstrated two prominent binding proteins at approximately 70 and approximately 30 kDa. By Western blot (immunoblot), the 30-kDa protein was found to be outer surface protein A (Osp A) of B. burgdorferi. 125I-labeled plasminogen binding to both the 70-kDa protein and Osp A was inhibited by approximately 90% with a 1,000-fold excess of unlabeled plasminogen. By scanning densitometry, the 70-kDa band bound > 10 time more 125I-labeled plasminogen than did Osp A. An Osp A-deficient mutant of B. burgdorferi and wild-type B. burgdorferi bound equal amounts of 125I-labeled plasminogen. Ligand blots of membrane proteins from an Osp A-deficient mutant showed association of 125I-labeled plasminogen at only the 70-kDa protein. Two-dimensional gel electrophoresis showed that the 70-kDa protein had a pI of approximately 5.3, clearly separable from Osp A. The association of host plasmin(ogen) with borrelial surface proteins provides a mechanism by which B. burgdorferi can digest extracellular matrix and disseminate.
Roles of gamma interferon and interleukin-4 in genetically determined resistance to Coccidioides immitis
Authors: Magee D M , Cox R A
Published: January 2020

The profiles of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production were evaluated during the course of coccidioidomycosis in two inbred mouse strains which differ in their susceptibility to Coccidioides immitis. Cytokine responses, measured at the molecular and protein levels, showed increased levels of IFN-gamma in lung extracts from mice of the resistant DBA/2 strain after a pulmonary challenge, whereas the susceptible BALB/c strain manifested a predominant IL-4 response. The importance of these cytokines in host defense against C. immitis was established by treating the mice with recombinant cytokines or neutralizing anticytokine monoclonal antibodies. Treatment of the susceptible BALB/c mice with recombinant murine IFN-gamma significantly protected mice against systemic challenge, and in the reciprocal experiment, the administration of an anti-IFN-gamma monoclonal antibody to the resistant DBA/2 mice significantly decreased their capacity to control disease. Although the treatment of DBA/2 mice with recombinant IL-4 did not alter the disease, neutralization of endogenous IL-4 in infected BALB/c mice by administration of a neutralizing anti-IL-4 antibody led to a significant reduction in the fungal load in their tissues. These results, taken together, establish that IFN-gamma plays a pivotal role in resistance to C. immitis, whereas IL-4 down-regulates protective immunity against C. immitis.
Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi
Authors: Chang Y F , Appel M J , Jacobson R H , Shin S J , Harpending P , Straubinger R , Patrican L A , Mohammed H , Summers B A
Published: January 2020

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.
Enteral human serum immunoglobulin treatment of cryptosporidiosis in mice with severe combined immunodeficiency
Authors: Kuhls T L , Orlicek S L , Mosier D A , Crawford D L , Abrams V L , Greenfield R A
Published: January 2020

The anti-cryptosporidial immunoglobulin G antibodies in two commercially available human serum immunoglobulin (HSIG) products were quantified and characterized. The mean level of Cryptosporidium parvum-specific immunoglobulin G in HSIG was eightfold higher than the antibody level found in the sera of three immunocompetent individuals convalescing from cryptosporidiosis. However, HSIG products displayed no reactivity to cryptosporidial antigens in immunoblot analyses, while convalescent-phase sera demonstrated characteristic banding patterns. When HSIG was given to newborn severe combined immunodeficiency (scid) mice before and shortly after experimental infection, a decreased intensity of infection was observed in the intestines of the mice compared with that of control mice. However, there was no difference in mortality or histopathologic findings in the intestines of HSIG-treated and control mice when treatment was not started until 22 days of age. These results indicate that HSIG may be beneficial when given prophylactically; however, HSIG cannot eradicate cryptosporidia from mucosal surfaces in an established infection.
Pasteurella haemolytica lipopolysaccharide-associated protein induces pulmonary inflammation after bronchoscopic deposition in calves and sheep
Authors: Brogden K A , Ackermann M R , Debey B M
Published: January 2020

The lipopolysaccharide (LPS)-associated protein (LAP) was extracted from Pasteurella haemolytica serotype A1 strains L101 (bovine origin) and 82-25 (ovine origin). Extracts contained 0.017% total LPS and appeared as only two bands at 14 and 16.6 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To determine the extent of pulmonary inflammation induced by LAP and its possible role in the pathogenesis of pneumonic pasteurellosis, LAP (500 micrograms in pyrogen-free saline [PFS]) was deposited by fiber-optic bronchoscopy into the dorsum of the caudal portion of the cranial lobe of the right lung of calves (strain L101 LAP) and sheep (strain 82-25 LAP). LPS (500 micrograms in PFS), 3-h P. haemolytica cultures (1.6 x 10(8) to 1.9 x 10(8) CFU in PFS), and PFS alone were deposited similarly as controls. At necropsy, 24 h after deposition, gross and histologic pulmonary lesions of calves and sheep given LAP, LPS, and P. haemolytica were similar and consisted of various degrees of acute bronchopneumonia (relative severities of lesions induced: LAP < LPS < live organisms). By subjective histologic interpretation and semiquantitative morphometry, animals given LAP had the highest percentage of macrophages per alveolar lumen and the lowest percentage of neutrophils. The lesions from animals given LPS were more severe than those given LAP, but the morphometric cell counts were similar. In contrast, animals inoculated with P. haemolytica had lesions typical of this agent, consisting of many neutrophils, proteinaceous exudate, and a few macrophages. Morphometrically, these lesions had the highest numbers of neutrophils and the lowest numbers of macrophages. These studies show that LAP can induce an inflammatory response in the alveolar lumens and may play a role in the pathogenesis of pneumonic pasteurellosis.