The host response to Leishmania infection.
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Animals↗
Biomedical subjects
W Solbach
Publications and source records attributed to W Solbach.
Prolyl isomerases in a minimal cell. Catalysis of protein folding by trigger factor from Mycoplasma genitalium.
Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds. Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known. Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor. Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame. Therefore, we studied the PPIase activity in crude extracts of M. genitalium. We showed that this is solely attributed to a single enzyme activity, the trigger factor. Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain. Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not. The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain. Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology. Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs. Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding. In contrast, the isolated PPIase-domain lacks any association with chaperones from E. coli. In summary, trigger factor of M. genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs. Its additional domains confer its ability to be an efficient catalyst of protein folding. The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase.
Amino Acid Sequence↗
Detection of Chlamydia pneumoniae within peripheral blood monocytes of patients with unstable angina or myocardial infarction.
Because individual diagnoses of vascular infection with Chlamydia pneumoniae depend entirely on surgically removed tissues, a better assay to predict vascular infection is needed. Polymerase chain reaction detection of chlamydial DNA was applied to CD14-positive cells collected from 238 patients with angiographically identified unstable angina or acute myocardial infarction. C. pneumoniae was detected in 52 (28%) of 188 persons with unstable angina and in 13 (26%) of 50 persons with myocardial infarction. Differences between groups were not significant. C. pneumoniae is present in monocytes/macrophages of a significant proportion of persons with progressive coronary artery disease. Infarction is not accompanied by a rise in chlamydial detection rates. The potential role of chlamydiae in coronary atherosclerosis may therefore be more related to acceleration of disease or systemic effects by persistent infection than to sudden initiation of infarction by acute infection.
Adult↗
Cardiovascular infection by Chlamydia pneumoniae is not related to apolipoprotein E genotype.
Chlamydia pneumoniae is detectable in the blood vessels of patients suffering from arteriosclerosis. Risk for arteriosclerosis is modulated by the apolipoprotein E (apoE) allele. We assessed the significance of the apoE genotype as a risk factor for vascular C. pneumoniae infection by determining the genotype of 30 coronary heart disease patients with PCR-proven C. pneumoniae infection of coronary artery tissue. The apoE genotype is not distinctly associated with an increased risk for vascular C. pneumoniae infection.
Apolipoproteins E↗
Failure to detect Chlamydia pneumoniae in brain sections of Alzheimer's disease patients.
A recent North American study detected Chlamydia pneumoniae in 17 of 19 brains of Alzheimer's patients and supposed a C. pneumoniae infection to be a risk factor for Alzheimer's disease (AD). In this study, we analyzed paraffin-embedded tissue samples of 20 AD patients by nested PCR and immunocytochemistry with a panel of antichlamydial antibodies and could detect neither C. pneumoniae-specific DNA nor chlamydial antigens. From our data, the presence of C. pneumoniae in the brains of Alzheimer's patients is not a common phenomenon; an association remains questionable.
Aged↗
Reduced bacterial dissemination and liver injury in CD14-deficient mice following a chronic abscess-forming peritonitis induced by Bacteroides fragilis.
The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3x10(8) CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD 14-/-) and normal C57BL/6J (CD 14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These observations were indistinguishable between CD14-/- and CD14+/+ mice. Although no differences were seen in abscess formation, CD14-/- mice were able to clear B. fragilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14-/-compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P<0.05). Furthermore, although the infection resulted in hepatocellular necrosis and severe hepatitis in both groups, at day 14 the liver cell damage was more severe in CD14+/+ than in CD14-/- mice (P<0.05). These results show that the chronic abscess formation induced by B. fragilis capsular polysaccharides is CD14 independent; however, bacterial clearance and/or dissemination and liver cell damage are at least partially influenced by CD14-dependent mechanisms.
Abscess↗
Early gene expression of NK cell-activating chemokines in mice resistant to Leishmania major.
Susceptibility of mice to Leishmania major is associated with an insufficient NK cell-mediated innate immune response. We analyzed the expression of NK cell-activating chemokines in vivo during the first days of infection in resistant and susceptible mice. The mRNA expression of gamma interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), and lymphotactin was upregulated 1 day after infection in the draining lymph nodes of resistant C57BL/6 mice but not in those of susceptible BALB/c mice. In vivo local treatment of BALB/c mice with recombinant IP-10 shortly after infection resulted in an enhanced NK cell activity in the draining lymph node. The data suggest that although the recruitment of NK cells is normal in susceptible mice, the lack of NK cell-activating chemokines is a factor resulting in a suboptimal NK cell-mediated defense.
Animals↗
Systemic infection of an immunocompromised patient with Methylobacterium zatmanii.
We describe the identification of Methylobacterium zatmanii as the causative agent of bacteremia and fever in an immunocompromised patient. The patient, a 60-year-old man, had a 5-month history of acute myeloid leukemia and had been on chemotherapy throughout this period. Seven days after the onset of neutropenia, the patient developed fever. The combination of ciprofloxacin, co-trimoxazole, imipenem, amikacin, and vancomycin led to a complete defervescence. On subculture from six positive blood cultures, the organism grew only on buffered charcoal yeast extract agar and not on standard agars. Identification by universal PCR and subsequent sequence analysis of the amplified 16S rRNA gene segment was achieved. This identification by molecular biology techniques was confirmed by conventional biochemical tests. To our knowledge, this is the first description of M. zatmanii isolated from patient material.
Acute Disease↗
Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin.
The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA-Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.
Amino Acid Sequence↗
Poor correlation between microimmunofluorescence serology and polymerase chain reaction for detection of vascular Chlamydia pneumoniae infection in coronary artery disease patients.
Chlamydia pneumoniae has been associated to coronary artery disease by various methods including recovery of viable bacteria from plaques. The pathogenetical relevance of this is unclear but investigation of antichlamydial therapy in coronary arteriosclerosis is already in progress. The microimmunofluorescence test (MIF), the only species-specific serological assay available, might be considered useful in identifying patients with vascular chlamydial infection. However, this has never been systematically examined. We compared levels of C. pneumoniae antibodies in sera using MIF with direct detection of C. pneumoniae in coronary artery segments from 158 patients undergoing myocardial revascularization. A polymerase chain reaction (PCR) protocol, recently evaluated for use with vascular materials, detected C. pneumoniae infection in 34 patients. Correlation of serology and PCR was poor: in relation to PCR. MIF-IgG analysis had 21% sensitivity, 90% specificity, 37% positive predictive value, and 81% negative predictive value for detection of chlamydial presence. Thus, the MIF test currently appears not suitable to predict individual vascular C. pneumoniae infection.
Aged↗
In vitro susceptibilities of Chlamydia pneumoniae strains recovered from atherosclerotic coronary arteries.
Chlamydia pneumoniae strains have been recovered from arteriosclerotic coronary arteries, but their antibiotic susceptibility profiles have not yet been examined. We report in vitro susceptibility data for five cardiovascular C. pneumoniae isolates. These strains did not differ significantly from respiratory strains in their patterns of susceptibility to azithromycin, erythromycin, roxithromycin, ofloxacin, doxycycline, rifampin, and penicillin G. Roxithromycin was the most active macrolide, and rifampin was the most effective drug overall.
Chlamydophila pneumoniae↗
Coiling phagocytosis of trypanosomatids and fungal cells.
Coiling phagocytosis has previously been studied only with the bacteria Legionella pneumophila and Borrelia burgdorferi, and the results were inconsistent. To learn more about this unconventional phagocytic mechanism, the uptake of various eukaryotic microorganisms by human monocytes, murine macrophages, and murine dendritic cells was investigated in vitro by video and electron microscopy. Unconventional phagocytosis of Leishmania spp. promastigotes, Trypanosoma cruzi trypomastigotes, Candida albicans hyphae, and zymosan particles from Saccharomyces cerevisiae differed in (i) morphology (rotating unilateral pseudopods with the trypanosomatids, overlapping bilateral pseudopods with the fungi), (ii) frequency (high with Leishmania; occasional with the fungi; rare with T. cruzi), (iii) duration (rapid with zymosan; moderate with the trypanosomatids; slow with C. albicans), (iv) localization along the promastigotes (flagellum of Leishmania major and L. aethiopica; flagellum or posterior pole of L. donovani), and (v) dependence on complement (strong with L. major and L. donovani; moderate with the fungi; none with L. aethiopica). All of these various types of unconventional phagocytosis gave rise to similar pseudopod stacks which eventually transformed to a regular phagosome. Further video microscopic studies with L. major provided evidence for a cytosolic localization, synchronized replication, and exocytic release of the parasites, extending traditional concepts about leishmanial infection of host cells. It is concluded that coiling phagocytosis comprises phenotypically similar consequences of various disturbances in conventional phagocytosis rather than representing a single separate mechanism.
Animals↗
In vivo blocking of L-selectin rescues BALB/c mice from fatal Leishmania major infection.
Susceptibility and resistance to experimental Leishmania major (L. major) infection in mice are associated with a Th2- or Th1-type response, respectively. We have previously shown that immunological events occurring within the first 24 h after infection in the lymph node (LN) draining the site of parasite challenge are critical for the development of either type of T-cell responses. In the present study we manipulated these events by preventing the entry of naive lymphocytes into the draining LN by injecting BALB/c mice with a single dose of the anti-L-selectin mAb MEL-14 one day prior to infection with L. major. In contrast to control BALB/c mice, in MEL-14 treated animals the primary lesion healed 12 weeks after infection. The parasite load in the spleen and lymph nodes of MEL-14 treated mice was significantly reduced. The healing was found to be associated with an increased production of IFN-gamma and with a decrease in IL-4 production by LN cells. We observed a dramatic decrease in cellularity in the draining LN in Mel-14 treated L. major-infected mice within the first week of infection. Moreover, the cells in the LN of MEL-14 treated mice were highly enriched in activated cells as well as in cell influx in the draining LN after local L. major infection of BALB/c mice prevents fatal disease. The data suggest the MEL-14-induced enrichment of the draining LN in memory and activated cells is fundamental for the initiation of a protective Th1-type response.
Animals↗
Improved technique for cannulation of the murine thoracic duct: a valuable tool for the dissection of immune responses.
Several experimental approaches have been used in the past for the cannulation of the thoracic duct in mice. Most, however, are characterized by a modest reproducibility and a low lymph yield. Here, we describe a cannulation technique modified with respect to the anesthesia, the use of a silicone cannula and a simple and efficient intraabdominal fixation of the drain. Surgery averaged 45 min with an intra- and postoperative mortality rate of zero. Postoperatively, mice were given access to an exercise wheel allowing increased mobility and consequently a good lymph flow, thus maintaining the function of the cannula. The mice yielded a mean of 29.3 ml/24 h (range 8-40 ml) thoracic duct lymph, which contained a mean of 2.2 x 10(6) lymphocytes/ml during the first 24 h, decreasing to 0.1 x 10(6) lymphocytes/ml on the 2nd day after cannulation. Patency of the cannulae was 100% after 3 days. Interestingly, we have detected strain dependent differences in the anatomy of the thoracic duct in the mouse and these need to be considered when cannulation procedures are attempted.
Animals↗
Control of Leishmania major infection in BALB/c mice by inhibition of early lymphocyte entry into peripheral lymph nodes.
A single i.p. injection with the anti-CD62L (anti-L-selectin) mAb Mel-14 before parasite challenge protected BALB/c mice from the otherwise lethal infection with Leishmania major. The Mel-14 mAb treatment resulted in a significant (>90%) decrease in cellularity of the popliteal lymph node (PLN) with a decrease in the proportion of CD4+ cells and an increase of the proportion of B220+ cells. Furthermore, both activated cells (CD25+ and CD69+) and cells of the memory phenotype (CD45RBdull CD44high) were significantly enriched in PLN from Mel-14-treated BALB/c mice. After infection with L. major, the otherwise massive cellular infiltration in the draining PLN was completely blocked in the Mel-14-treated mice, and in these animals the high representation of both activated and memory cells in PLN remained characteristic for the first days of infection. The protective effect was found to be associated with a markedly increased production of IFN-gamma and with a decrease in IL-4 production upon restimulation of PLN and spleen cells with L. major Ag in vitro. The cured mice were found to be resistant against a secondary challenge with the parasites. These data suggest that the induction of a nonprotective Th2 response to L. major is associated with the entry of lymphocytes from the recirculating pool into the draining LN. The Mel-14-induced changes in the lymphoid microenvironment of the draining peripheral LN appear to favor the development of a protective Th1 cell-mediated immune response against the parasite.
Animals↗
Host-cell cyclophilin is important for the intracellular replication of Leishmania major.
The antiparasitic effects of cyclosporin A were examined in leishmanial infection by analysing the role of CsA-binding proteins (cyclophilins) in the host-parasite interaction. We hypothesized that the leishmanicidal effects of CsA on Leishmania major infected macrophages might be mediated through a cyclophilin of either the parasite or the host cell. Two cyclophilins (20 and 22 kDa) were purified from L. major parasites and N-terminally sequenced. Although enzyme activity of these cyclophilins was inhibited by CsA, pretreatment of L. major parasites with CsA did not result in reduction of a subsequent macrophage infection, arguing against a role of L. major cyclophilins as infectivity potentiators. However, host-cell cyclophilin A (CypA) was found to be critically involved in the intracellular replication of L. major parasites in murine macrophages. An antisense oligonucleotide to murine CypA was constructed and added to cultures of peritoneal macrophages prior to infection with L. major parasites. This treatment strongly reduced the expression of CypA in macrophages and resulted in the inhibition of the intracellular replication of L. major amastigotes. These data indicate that interaction of amastigotes with host-cell cyclophilin is an important part of the intracellular replication machinery of L. major and define, for the first time, a direct involvement of a cyclophilin in the survival strategies of an intracellular parasite.
Amino Acid Isomerases↗
Systemic infection with Mycobacterium genavense following immunosuppressive therapy in a patient who was seronegative for human immunodeficiency virus.
We describe, to our knowledge, the first case of disseminated Mycobacterium genavense infection in a patient who was seronegative for human immunodeficiency virus. The patient, a 47-year-old woman, had been previously treated with immunosuppressive drugs for 9 months to control an unclassified immunologic disorder characterized by intermittent fever and inflammatory pulmonary, hepatic, and dermal infiltrates. Antemortem and postmortem examinations revealed the presence of numerous mycobacteria in the bone marrow, spleen, kidneys, and lungs; these organisms failed to grow in vitro and were identified as M. genavense by 16S rRNA gene sequencing. This case illustrates that systemic M. genavense infections are not restricted to patients with AIDS but can also occur in otherwise immunocompromised patients.
Female↗
IL-4-deficient Balb/c mice resist infection with Leishmania major.
Mice with a genetically engineered deficiency for either IL-4 or IFN-gamma R1 (single mutants), and IL-4/IFN-gamma R1 (double mutants) on the Balb/c and 129Sv background were used to study the course of infection with Leishmania major. In contrast to genetically resistant 129Sv wildtype mice, IL-4/IFN-gamma R1 double mutant mice developed fetal disease with parasite dissemination to visceral organs similar to mice lacking IFN-gamma R1 only. Balb/c mice, which are exquisitely susceptible to L. major, were rendered resistant to infection by disruption of the IL-4 gene. As compared to homozygous IL-4+/- mice, heterozygous IL-4+/- mice, heterozygous IL-4+/- animals consistently developed smaller lesions with less ulceration and necrosis, indicating the likelihood of gene-dosage effects. This implicates that the magnitude of the IL-4 response determines the severity of disease. CD4+ T cells of IL-4-deficient mice showed impaired Th2 cell development, as assessed by quantitative RT-PCR of characteristic cytokines. Development of resistance is not explained by default Th1 development, because this was observed only at very late stages of infection. Moreover, the induction of inflammatory cytokines (e.g., IL-1 alpha, IL-1 beta, TNF-alpha, IL-12) together with iNOS in the lesion and draining lymph nodes was not altered in the absence of IL-4.
Animals↗