Plasticity, not genetic variation, drives infection success of a fungal parasite.

Hosts strongly influence parasite fitness. However, it is challenging to disentangle host effects on genetic vs plasticity-driven traits of parasites, since parasites can evolve quickly. It remains especially difficult to determine the causes and magnitude of parasite plasticity. In successive generations, parasites may respond plastically to better infect their current type of host, or hosts may produce generally 'good' or 'bad' quality parasites. Here, we characterized parasite plasticity by taking advantage of a system in which the parasite (the yeast Metschnikowia bicuspidata, which infects Daphnia) has no detectable heritable variation, preventing rapid evolution. In experimental infection assays, we found an effect of rearing host genotype on parasite infectivity, where host genotypes produced overall high or low quality parasite spores. Additionally, these plastically induced differences were gained or lost in just a single host generation. Together, these results demonstrate phenotypic plasticity in infectivity driven by the within-host rearing environment. Such plasticity is rarely investigated in parasites, but could shape epidemiologically important traits.

Increased level of MSU crystal-bound protein apolipoprotein A-I in acute gouty arthritis.

BACKGROUND: Gout is a common form of inflammatory arthritis that is triggered by the crystallization of monosodium urate (MSU). We investigated the potential proteins that relate to the pathogenesis or the spontaneous resolution of acute gouty arthritis. METHOD: We screened for differentially expressed proteins in the plasma of patients with acute gouty arthritis using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) identification. We confirmed these findings in a population study of 209 subjects, and further determined the protein profile of the synovial fluid (SF) from 24 gouty patients during acute attack by liquid chromatography coupled with tandem MS (LC/MS/MS). RESULTS: The highly expressed apolipoprotein A-I (apoA-I) was identified in the plasma of acute gouty patients compared with healthy controls. Moreover, we detected high levels of SF apoA-I in 83.3% of acute gouty patients during attack. From the population study, apoA-I was increasingly associated with normouricaemia, hyperuricaemia, and acute gouty arthritis (ptrend < 0.001), and plasma uric acid (UA) and apoA-I were positively correlated (p = 0.0156). We used a human liver cell model and found that UA enhanced the hepatic apoA-I mRNA expression level (ptrend < 0.01) and apoA-I secretion level (ptrend = 0.002) in a dose-dependent manner. An elevated MSU concentration caused the endogenous apoA-I to deplete gradually. CONCLUSIONS: Based on the role of apoA-I in anti-inflammation, our observational data in acute gout support the hypothesis that apoA-I expression can be induced under the condition of a high concentration of UA and its elevated level may be implicated in the spontaneous resolution of acute gouty arthritis.

Methylprednisolone worsening neuropathic pain in non-traumatic thoracic myelopathy.

Methylprednisolone (MP) is the only neuroprotective medication currently in widespread use for the treatment of spinal cord injury. Increasingly, published studies challenge its clinical effects in view of its serious side-effects including wound infection, pneumonia, sepsis and steroid myopathy. Most cases with spontaneous spinal epidural haematoma (SSEH) need emergency evacuation, and typically show good neurologic recovery. Some patients with SSEH given preoperative or postoperative MP within hours of the onset of symptoms, and have had good motor recovery, although no mention was made of sensory function. Severe, intractable neuropathic pain has not been reported in patients with SSEH. We present a case of SSEH treated with a high-dose MP 16 h after onset of symptoms. Surgical decompression was performed 1 h after MP treatment. Motor recovery was good; however, intractable neuropathic pain developed 5 weeks postoperatively. We discuss the factors contributing to intractable pain. We speculate that the severe, intractable pain might be due to misuse of large-dose steroids in this case of non-traumatic spinal myelopathy, and not because of the injury per se.

Use of oseltamivir during an outbreak of influenza A in a long-term care facility in Taiwan.

We studied the effectiveness of oseltamivir during an outbreak of influenza A among previously vaccinated patients and staff in a long-term care facility. Seven of 14 staff members and 14 of 41 residents developed either influenza-like illness (ILI) or other respiratory symptoms during a 14-day period from late January to 8 February 2004. On 9 February, therapeutic oseltamivir (75 mg twice daily for five days) was administered to one staff member and seven residents who had developed ILI within the previous 48 h (treatment group). Prophylactic oseltamivir (75 mg once daily for seven days) was administered to 12 staff members and 30 residents who were asymptomatic or whose respiratory symptoms did not meet the diagnosis of ILI (prophylaxis group). The remaining four residents and one staff member had had ILI for more than two days (with subsiding symptoms) and did not receive oseltamivir ('no-oseltamivir' group). None of the 42 subjects in the prophylaxis group developed ILI. Presence of influenza A virus was demonstrated in 24 subjects: seven out of eight in the treatment group, 12 of 42 in the prophylaxis group and all five in the no-oseltamivir group. For confirmation of diagnosis, real-time reverse transcription-polymerase chain reaction was more sensitive than antigen detection and virus isolation. In-time therapeutic and prophylactic oseltamivir successfully interrupted an outbreak of influenza A in a long-term care facility.

Mutations at the Darkener of apricot locus modulate transcript levels of copia and copia-induced mutations in Drosophila melanogaster.

Mutations of the Doa locus of Drosophila melanogaster darken the eye color of the copia-induced white(apricot) (wa) allele and increase the accumulation of white promoter-initiated transcripts encoding functional mRNA. We show here that quantities of transcripts initiated in both long terminal repeats (LTRs) of the specific wa-copia element are increased, and those initiating in the 5' LTR of the element are structurally altered, yielding a slightly shortened transcript. Accumulation of host-initiated transcripts of a copia-induced mutation within the achaete-scute complex, Hairy-wing Ua (HwUa), are reduced by Doa mutations. Finally, we show that homozygosity for Doa mutations increases the accumulation of copia transcripts from the population of elements in the genome. These results suggest that Doa modulates the severity of copia-induced mutations while functioning as a dosage-sensitive modulator of copia transcription.

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Multicentric giant cell tumours in an adolescent with haemophilia.

''Multicentric giant cell tumour (GCTs) of the extremity is prone to be distributed over the age range of 20-40 years, but is rare in haemophilia and in the age before 20. We report a case of a 15-year-old haemophilia boy who presented initially with two radiolucent loci in the right femur and tibia revealed from the X-ray films and then another lesion in the posterior femoral shaft shown from MRI by one year. Differential diagnosis of GCTs should be appraised in various aspects. Radiological diagnostic pitfall was avoided by the pathology disclosed GCTs without malignancy. The early diagnosis of GCTs in haemophilia may be delayed unless appearance of symptoms of pathologic fracture. Coincident multicentric GCTs do occur in haemophilic patients and their incidence might be underestimated, as it might not be judged because immediate symptoms of pain would resolve with appropriate factor replacement."

Construction of a Vibrio cholerae vaccine candidate using transposon delivery and FLP recombinase-mediated excision.

Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism, Vibrio cholerae. The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients. We therefore converted a motile Inaba(+) vaccine candidate, Peru-2, to a nonmotile Ogawa(+) phenotype using a mariner-based transposon carrying rfbT, the gene required for expression of the Ogawa phenotype. Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa(+), and both of these strains had insertions in the flgE gene. It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon. The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes.

Use of signature-tagged transposon mutagenesis to identify Vibrio cholerae genes critical for colonization.

The pathogenesis of cholera begins with colonization of the host intestine by Vibrio cholerae. The toxin co-regulated pilus (TCP), a fimbrial structure produced by V. cholerae, is absolutely required for colonization (i.e. the persistence, survival and growth of V. cholerae in the upper intestinal milieu), but many other aspects of the colonization process are not well understood. In this study, we use signature-tagged transposon mutagenesis (STM) to conduct a screen for random insertion mutations that affect colonization in the suckling mouse model for cholera. Of approximately 1100 mutants screened, five mutants (approximately 0.5%) with transposon insertions in TCP biogenesis genes were isolated, validating the use of STM to identify attenuated mutants. Insertions in lipopolysaccharide, biotin and purine biosynthetic genes were also found to cause colonization defects. Similar results were observed for mutations in homologues of pta and ptfA, two genes involved in phosphate transfer. Finally, our screen identified several novel genes, disruption of which also caused colonization defects in the mouse model. These results demonstrate that STM is a powerful method for isolating colonization-defective mutants of V. cholerae.

rfb mutations in Vibrio cholerae do not affect surface production of toxin-coregulated pili but still inhibit intestinal colonization.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is essential for colonization. It was recently reported that rfb mutations in V. cholerae 569B cause the translocation arrest of the structural subunit of TCP, raising the possibility that the colonization defects of lipopolysaccharide mutants are due to effects on TCP biogenesis. However, an rfbB gene disruption in either V. cholerae O395 or 569B has no apparent effect on surface TCP production as assessed by immunoelectron microscopy and CTX phage transduction, and an rfbD::Tn5lac mutant of O395 also shows no defect in TCP expression. We conclude that the colonization defect associated with rfb mutations is unrelated to defects in TCP assembly.

In vivo genetic analysis of bacterial virulence.

In vitro assays contribute greatly to our understanding of bacterial pathogenesis, but they frequently cannot replicate the complex environment encountered by pathogens during infection. The information gained from such studies is therefore limited. In vivo models, on the other hand, can be difficult to use, and this has to some extent diminished the incentive to perform studies in living animals. However, several recently developed techniques permit in vivo examination of many genes simultaneously. Most of these methods fall into two broad classes: in vivo expression technology and signature-tagged mutagenesis. In vivo expression technology is a promoter-trap strategy designed to identify genes whose expression is induced in a specific environment, typically that encountered in a host. Signature-tagged mutagenesis uses comparative hybridization to isolate mutants unable to survive specified environmental conditions and has been used to identify genes critical for survival in the host. Both approaches have so far been used exclusively for investigating pathogen-host interactions, but they should be easily adaptable to the study of other processes.

CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor.

In Vibrio cholerae, the genes encoding cholera toxin (ctxAB) are located on a segment of DNA (termed the "core" region) that is flanked by two or more copies of a repeated sequence called RS1. Together these DNA units comprise the CTX genetic element. Evidence presented here suggests that RS1 sequences encode a site-specific recombination system, which allows integration of a suicide plasmid carrying RS1 into an 18-base-pair sequence (attRS1) located on the chromosome of nontoxigenic V. cholerae strains. Strains of V. cholerae with large deletions removing attRS1 and the entire CTX genetic element no longer undergo site-specific recombination with the RS1 sequence. Additionally, these deletion strains show a defect in intestinal colonization. Recombination experiments localize the gene responsible for enhancing colonization to a portion of the core region of the CTX element. The identified gene encodes a peptide that is highly similar in amino acid sequence to the flexible pilin of Aeromonas hydrophila. These results have important implications in the construction of stable, live attenuated cholera vaccines.

The collagen receptor alpha 2 beta 1, from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide.

Several receptors for the extracellular matrix protein collagen have been described which belong to the superfamily of receptors collectively known as integrins. Although several integrins have been shown to interact with extracellular matrix molecules via a common recognition site, arginine-glycine-aspartic Acid (RGD), within the beta 1 integrin subfamily, only the fibronectin receptor (alpha 5 beta 1) has been convincingly shown to interact with RGD. In the present study, we tested whether a collagen receptor could interact with RGD. Adhesion of an osteosarcoma cell line, MG-63, to immobilized collagen I was inhibited by the cyclic RGD-containing peptide, C*GRGDSPC* (where C* indicates that Cys participates in disulfide), and not by the linear GRGDSP or the non-RGD-containing cyclic peptide, C*GKGESPC*. Similarly, using collagen-Sepharose affinity chromatography, a heterodimeric protein could be specifically eluted from the column by the cyclic RGD peptide. Immunoprecipitations of the eluted material with monoclonal antibodies showed reactivity with the collagen receptor alpha 2 beta 1 and not alpha 3 beta 1. Our data demonstrate that RGD peptides can interact with the collagen receptor, and the differences seen with the linear and cyclic peptide suggest that the cyclic C*GRGDSPC* has a higher avidity for the receptor than the more flexible linear GRGDSP. In this paper, we provide supportive evidence that one possible mode of collagen interaction with alpha 2 beta 1 is via the RGD recognition sequence.

Single amino acid substitutions in the N-terminus of Vibrio cholerae TcpA affect colonization, autoagglutination, and serum resistance.

The toxin-coregulated pilus (TCP) of Vibrio cholerae O1 is required for successful infection of the host. TcpA, the structural subunit of TCP, belongs to the type IV family of pilins, which includes the PilE pilin of Neisseria gonorrhoeae. Recently, single amino acid changes in the N-terminus of PilE were found to abolish autoagglutination in gonococci. As type IV pilins demonstrate some similarities in function and amino acid sequence, site-directed mutagenesis and allelic exchanges were used to create corresponding mutations in TcpA. All four mutant strains demonstrated autoagglutination defects, and all were highly defective for colonization in the infant mouse model. These results support the previously proposed correlation between autoagglutination and colonization. Finally, all four mutants are serum sensitive, indicating that TcpA plays a role in serum resistance, a phenotype previously attributed to TcpC. As the mutations have similar effects in N. gonorrhoeae and V. cholerae, our results support the idea that type IV pilins have similar functions in a variety of pathogenic bacteria.

A novel cyclic pentapeptide inhibits alpha 4 beta 1 and alpha 5 beta 1 integrin-mediated cell adhesion.

Lymphocytes and monocytes initiate and modulate inflammatory and immune responses for host defense. This process is dependent upon extravasation of leukocytes from the circulation to sites of antigenic challenge and is controlled, in part, by various integrins, including alpha 4 beta 1 and alpha 5 beta 1. A small cyclic pentapeptide that inhibits, in vitro, both alpha 4 beta 1 and alpha 5 beta 1 activity is described. This peptide, Arg-Cys-Asp-Thioproline-Cys (RC*D[ThioP]C*), is cyclized by a disulfide bond through the cysteine residues (the asterisks denote cyclizing residues). RC*D(ThioP)C* inhibits alpha 5 beta 1-mediated leukocyte adhesion to the 120-kDa Arg-Gly-Asp (RGD)-containing binding site of fibronectin. Two different adhesion activities of alpha 4 beta 1 are also inhibited: alpha 4 beta 1-mediated cell adhesion to the alternatively spliced CS-1 site of fibronectin and the alpha 4 beta 1-dependent binding of leukocytes to cytokine-activated endothelial cells. Both alpha 4 beta 1 and alpha 5 beta 1 can be purified by affinity chromatography using the immobilized pentapeptide. The peptide does not inhibit adhesion to other extracellular matrix proteins including laminin and vitronectin. The specificity of the RC*D(ThioP)C* peptide for alpha 4 beta 1 and alpha 5 beta 1 suggests potential therapeutic utility for inhibiting inflammatory disease.

Cyclic RGD peptide inhibits alpha 4 beta 1 interaction with connecting segment 1 and vascular cell adhesion molecule.

The integrin supergene family includes receptors for a variety of extracellular matrix as well as cell surface proteins. Integrin alpha 4 has been shown to play an important role in leukocyte adhesion and extravasation during immune and inflammatory reactions. One recognition sequence known to interact with alpha 4 is the Leu-Asp-Val (LDV) site contained in the connecting segment 1 region of fibronectin. Here we present evidence that shows that a conformationally restricted RGD-containing peptide is a potent inhibitor of cell adhesion mediated by alpha 4 beta 1, a receptor not convincingly documented to interact with RGD peptides. This peptide, 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8), blocks Jurkat cell adhesion to connecting segment 1-containing peptides as well as cell adhesion to cytokine-activated endothelial cells. Adhesion of Jurkat cells to either vascular cell adhesion molecule-expressing cells or recombinant vascular cell adhesion molecule-coated plates was likewise inhibited by this peptide. Furthermore, alpha 4 beta 1 can bind directly to a cyclic RGD peptide immobilized to Sepharose. Integrins, alpha 5 beta 1, alpha v beta 3, alpha IIb/beta IIIa, alpha 2 beta 1, alpha v beta 1, alpha v beta 5, alpha v beta 6, and alpha 3 beta 1, have been shown to recognize the Arg-Gly-Asp (RGD) sequence present in a variety of extracellular matrix proteins, and our data support the addition of alpha 4 beta 1 to this group. Further studies using molecular modeling of such cyclic RGD peptides could help in the design of more potent peptides or nonpeptide mimetics that could effectively block alpha 4-mediated activity and have potential application in a number of inflammatory diseases.