The GGDEF-domain protein GdpX1 attenuates motility, exopolysaccharide production and virulence in Xanthomonas oryzae pv. oryzae.

AIMS: Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that is synthesized by diguanylate cyclase (DGC) with the GGDEF-domain, regulates diverse virulence phenotypes in pathogenic bacteria. Although 11 genes encoding GGDEF-domain proteins have been shown in the genome of Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99(A) , the causal pathogen of bacterial blight of rice, however, little is known about their roles in the c-di-GMP regulation of virulence in the pathogen. GdpX1, one of the GGDEF-domain proteins in Xoo was investigated in this study to reveal its regulatory function of bacterial virulence expression through genetic analysis. METHODS AND RESULTS: GdpX1 was functionally characterized in virulence expression through deletion and overexpression analysis. Bioinformatics analysis revealed the GGDEF-domain in GdpX1 was well conserved, indicating it is a putative DGC. Deletion of gdpX1 resulted in significant increases in virulence, exopolysaccharide (EPS) production and flagellar motility. In contrast, overexpression of gdpX1 dramatically reduced these virulence phenotypes. qRT-PCR analysis showed genes related to the type III secretion system (T3SS), EPS synthesis, and flagellar motility, were up-regulated in ∆gdpX1 and down-regulated in the gdpX1-overexpressed strains. In addition, overexpression of gdpX1 promoted biofilm formation and xylanase activity. CONCLUSION: GdpX1 is the first GGDEF-domain protein functionally characterized in Xoo, which functions as a negative regulator of bacterial virulence via suppression of virulence-related gene transcription. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification and functional characterization of GdpX1 provided additional insights into molecular mechanisms of c-di-GMP regulation of bacterial virulence expression.

D-4F, an apoA-1 mimetic, decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma.

Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DWFKAFYDKVAEKFKEAFNH(2) (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)ß-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michael's adducts in lungs of OVA-sensitized mice. 4F decreased TGFß-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.

On the pathway of an animal model for restless legs syndrome...

Restless legs syndrome (RLS) is a chronic sleep motor disorder that affects up to 10% of the general population. Except for periodic leg movements (PLM), which can be found in the great majority of RLS patients, no objective hematochimic or neurophysiological markers are available to prove the diagnosis, which is based on clinical standard criteria. Nowadays, the aetiopathogenesis of the syndrome is unknown. In a consistent sample of patients affected by the idiopathic form, the disease is inherited as an autosomal dominant trait related to an unidentified locus, while each symptomatic form is probably linked to a specific cause. Although of possible different origins, both the primary and secondary forms may share the same pathogenetic mechanism, which, even if unclear, could be characterised by a neurological dysfunction of the dopaminergic system. Several issues, including strong efficacy of dopamine-agonist treatments, support this theory, which is currently considered the main pathogenetic hypothesis. Most of the past studies tried to clarify the RLS mechanism using the neurophysiological, biochemical and neuroimaging techniques applied to the field of human research. Now the time has come to accept the challenge in creating an animal model of RLS, which may emerge as a decisive step in understanding RLS pathogenesis, and to develop and test new therapies. Even though there have been a few significant efforts, a valid animal model of RLS still does not exist. In past pioneering studies, the authors attempted to induce restless motor behaviour in animals by different strategies: antidopaminergic pharmacological interventions, spinal or cerebral lesions of specific regions involved in the motor control and in dopamine regulation, and selective deletion of genes coding for dopamine receptors. Rodents (mice and rats) were always chosen by the authors as the animals for their experiments. The current tendency in achieving an RLS model is generally represented by simulation of a symptomatic condition of RLS or by a direct interference of the dopaminergic system. In this regard, the pharmacological method had the intention to reproduce the neuroleptic-induced acathisia, the spinal lesional model was based on the hypothesis of myelopathic- related PLM, and the hypothalamic lesion tested the motor consequence of A11 dopaminergic neurons. Preliminary studies are underway to replicate the pregnancy-related form of RLS by using a hormonal intervention, and the iron-deficiency secondary form by using specific iron-free diets. Today, modern technologies are available to easily replicate in animals most of the symptomatic RLS conditions. In addition, more than a few well validated animal models of different diseases known to be related to RLS or PLM, for instance, Parkinson's disease, rheumatoid arthritis and renal failure, could also be exploited in addressing this topic. The real obstacle in achieving an RLS model is the absence of a certain diagnostic marker to recognise if the animal that underwent the different experimental procedures has developed the RLS condition or not. Concerning this issue, possible specific endpoints are represented by the increase in locomotor activity, which are ascertainable by different techniques, such as openfield or run-wheel activity, or by sleep fragmentation, in which the circadian shift can be verified by applying polysomnography on the animal. PLM are probably the only specific and reliable markers available to recognise and quantify experimentally induced RLS. Despite a few authors who reported the presence of limb-phasic, pseudoperiodic activity during sleep in old or in lesioned rats, the existence of spontaneous or provoked PLM in animals is still debated. Eventually, the PLM features in an animal could be markedly different compared to human ones. To recognise and characterise PLM in animals, three more essential steps are required: a method to record directly, as in humans, the activity of the tibialis anterior (TA) muscles, a consistent amount of normative control data on the TA activity in healthy animals, and reliable analysis to distinguish the generic phasic muscular activity to a possible unambiguous PLM pattern. This review includes a summary and a critical discussion of the previous tentative RLS models, proposals for other possible animal models, and firstly the preliminary normative data on TA activity during sleep in normal rodents.

Development and characterization of anti-idiotype based peptide and DNA vaccines which mimic the capsular polysaccharide of Neisseria meningitidis serogroup C.

Anti-idiotypic antibodies and peptides which mimic antigens may offer an alternate strategy for converting a thymus-independent (TI) antigen to a thymus dependent (TD) antigen. We have developed an anti-idiotype based peptide mimic of the capsular polysaccharide of N. meningitidis serogroup C (MCPS) which induces a T-dependent protective immune response in mice. Subsequent studies have demonstrated that immunization of severe combined immunodeficient mice reconstituted with human B cells respond to immunization with the MCPS peptide mimic with bactericidal anti-polysaccharide directed antibody response. We hypothesized that administration of a DNA vaccine resulting in endogenous expression of this carbohydrate peptide mimic would induce anti-MCPS antibodies.

Protective capacity of the Pasteurella haemolytica transferrin-binding proteins TbpA and TbpB in cattle.

The transferrin-binding proteins TbpA and TbpB from Pasteurella haemolytica biotype A serotype 1 were tested for their ability to confer protection against experimental P. haemolytica infection when administered to calves in vaccine formulations containing one or both antigens. Vaccine groups included TbpB (single immunization), TbpB (two immunizations), TbpA, TbpA+TbpB and a placebo. All animals that received TbpB had measurable antibody titres against the antigen at the time of challenge, while those that received TbpA did not show an antibody response. The TbpA+TbpB group showed the best protection against experimental challenge. Protection correlated with anti-TbpB antibody levels. The enhanced protection in the TbpA+TbpB group suggests TbpA contributed to protection through the induction of a non-antibody-mediated immune response. Sera from the TbpB-immunized animals was cross-reactive with TbpBs from other P. haemolytica serotypes.

Human immune response to a peptide mimic of Neisseria meningitidis serogroup C in hu-PBMC-SCID mice.

An anti-idiotype-based peptide mimic vaccine for Neisseria meningitidis serogroup C polysaccharide (MCPS) has been developed and shown to induce a response in mice that is specific, functional, and T-dependent. In this study, the immunogenicity of the MCPS peptide mimic vaccine preparation, as a potential vaccine for use in humans, is shown using the hu-PBMC-SCID mouse model. The human antibody response to the MCPS peptide mimic vaccine is specific and functional as shown by inhibition enzyme-linked immunoadsorbent assay (ELISA) and bactericidal assay. These data support the usefulness of the peptide mimic vaccine strategy for humans.

Molecular analysis of the heavy chain of antibodies that recognize the capsular polysaccharide of Neisseria meningitidis in hu-PBMC reconstituted SCID mice and in the immunized human donor.

The severe combined immunodeficient (SCID) mouse model, engrafted with human peripheral blood mononuclear cells (hu-PBMC) has proven to be useful in studying the human immune response. A major limitation of the hu-PBMC-SCID model has been the failure to consistently demonstrate a primary human immune response. Previously we developed a hu-PBMC-SCID mouse model in which we addressed both issues of adequate human lymphocyte engraftment and impaired differentiation. We demonstrated that a primary human immune response to the T-independent (TI-2) meningococcal group C capsular polysaccharide (MCPS) can be obtained in hu-PBMC-SCID mice by the administration of human cytokines. In this study we compared the V(H) sequence of the MCPS response generated by B cells derived from a volunteer in the SCID mouse model to those generated by the donors' B cells in vivo. Human peripheral blood mononuclear cells were recovered from MCPS immunized hu-PBMC-SCID mice and immunized donor. B cells with specificity for MCPS were isolated from these cell preparations using an anti-idiotypic monoclonal antibody which mimics MCPS. Immunoglobulin mRNA was isolated from single cells, amplified by the polymerase chain reaction, cloned and sequenced. We analysed a total of 15 V(H) regions from B cells obtained from SCID mice and a total of 13 V(H) regions from B cells obtained from the immunized donor. The response differed between SCID and in vivo cells, when studied at the genetric level. V, D and J gene usage was markedly different, however canonical structures of the hypervariable loops were conserved. The complementary determining region 3 (CDR3) varied, such that SCID-derived sequences encoded longer CDR3 s than those of the donor. However all CDR3 s were rich in hydrophobic amino acids, most notably tyrosine and tryptophan, a characteristic common to many carbohydrate binding antibodies.

Elderly immune response to a TI-2 antigen: heavy and light chain use and bactericidal activity to Neisseria meningitidis serogroup C polysaccharide.

Previous studies of the elderly immune response to TI-2 antigens failed to correlate specific antibody levels with function and to compare responses with those of young adults. Neisseria meningitidis serogroup C capsular polysaccharide (MCPS) was used as a model TI-2 antigen. Anti-MCPS antibody levels were determined in elderly individuals and correlated with bactericidal activity. The anti-MCPS response in most persons was characterized by predominant IgG usage, with IgG2>IgG1. No light chain or IgA subclass predominated, but some responses showed a particular chain type. Bactericidal activity correlated best with IgG2 levels. Elderly subjects had lower anti-MCPS responses than the young adults did in all chain-specific anti-MCPS levels, and levels declined more rapidly. Bactericidal activity following immunization was significantly lower in the elderly persons. These results suggest the anti-MCPS antibody repertoire in the elderly is likely maintained, and the lower level of function is related to the lower antibody levels.

Peptide mimicry of carbohydrate epitopes on human immunodeficiency virus.

Cancer-related, mucin-type carbohydrate epitopes, principally mannose and sialo-syl residues, are expressed on the envelope protein gp 160 of the human immunodeficiency virus (HIV). Anticarbohydrate antibodies directed toward these and other carbohydrate epitopes are known to neutralize HIV-1 infection by cell-free virus. Carbohydrates, however, being T cell-independent antigens, typically elicit diminished immune responses. To overcome this potential draw back, we have examined the ability of peptides that mimic such epitopes to elicit immune responses that cross-react with carbohydrate structures. We report that mouse polyclonal antisera generated against peptides that mimic mucin-related carbohydrate epitopes have anti-HIV-1 activity. Generation of antibodies was not lr-gene restricted, as at least two different strains of mice. Balb/c (H-2d) and C57Bl/6 (H-2b), responded equally to the peptides. The antipeptide sera displayed neutralizing activity against HIV-I/MN and HIV-I/3B viral strains. This neutralization was as good as human anti-HIV sera. These results indicate that peptide mimics of carbohydrates provide a novel strategy for the further development of reagents that elicit immune responses to carbohydrate epitopes associated with many infectious organisms and tumor cells.

Peptide mimicry of adenocarcinoma-associated carbohydrate antigens.

Carbohydrate antigens have been identified as significant antigens in many human tumors either by analyzing antibodies in patients' sera or by using monoclonal antibodies of either mouse or human origin. Three carbohydrate epitopes present on cancer-associated mucins [sialyl-Lewis A (SLA), sialyl-Lewis X (SLX), and sialyl-Tn (STn)] may have functional significance in metastasis. Subsequently, these antigens are considered as targets for active specific immunotherapy. Carbohydrates, as T-cell-independent antigens, often elicit diminished immune responses. To overcome this drawback, carbohydrates are typically coupled to protein carriers to elicit immunoglobulin G (IgG) responses as opposed to low-affinity IgM responses, which often times accompanies carbohydrate-based immunizations. In addition, some complex carbohydrates are difficult to synthesize. This latter aspect is further magnified if one considers that clustering of epitopes on neoglycoproteins must be emulated in the synthesis process, leading to multiple presentation or tandem repeats of the synthetic carbohydrate immunogen. Here, we examine the hypothesis that peptides that mimic carbohydrates might be developed to induce immune responses that target and mediate the killing of tumor cells, particularly breast cancer cells in an adjuvant-type setting. We have found that carbohydrate-mimicking peptides retain carbohydrate-like conformations, inducing anti-carbohydrate immune responses against breast tumor cells and mediating their killing by a complement-dependent mechanism.

Primary human immune response to Neisseria meningitidis serogroup C in interleukin-12-treated severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes.

Lack of primary immune response in severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL) has limited the applicability of this model. Use of human cytokines, in particular interleukin (IL)-12, was studied in the hu-PBL-SCID model. SCID mice were treated with IL-12 and reconstituted with hu-PBL in T replacement factor. The hu-PBL-SCID mice were immunized with serogroup C meningococcal polysaccharide (MCPS). The MCPS-specific antibody response was determined by ELISA. Thirteen of the 15 immunized, IL-12-treated hu-PBL-SCID mice demonstrated a primary human antibody response to MCPS ranging from 0.25 to 3.3 microg/mL, while no MCPS-specific antibody response was detectable in the 18 controls. Expression of cross-reactive idiotypic markers found on human anti-MCPS antibodies in the immunized hu-PBL-SCID mice was similar to that observed in immunized volunteers.

Molecular characterization of a monoclonal antibody produced in response to a group C meningococcal polysaccharide peptide mimic.

We have developed a monoclonal antibody, designated anti-anti-Id Ab3-2C4 which reacts with Neisseria meningitidis serogroup C polysaccharide (MCPS). Anti-anti-Id Ab3-2C4 was produced by immunizing Balb/C mice with a peptide mimic of MCPS. This monoclonal antibody reacts with native polysaccharide and its anti-idiotype antibody Ab2-6F9 by ELISA. The synthetic peptide mimic was constructed based on the sequence of the VHCDR3 region of the anti-idiotype Ab2-6F9. We compared the cDNA sequence of Ab3-2C4 to the sequence of idiotype antibody Ab1-1E4 produced in response to native MCPS. The predicted amino acid sequence of the unique VHCDR3 of anti-anti-Id Ab3-2C4 is similar to that of idiotype Ab1-1E4. Also the VHCDR3 of both antibodies is similar to some of the known or suggested carbohydrate binding motifs. A different VH gene family was utilized by Ab3-2C4 than by Ab1-1E4. These results suggest that immunization with the anti-idiotype-derived peptide mimic of the MCPS antigen stimulates the production of antibodies with a binding site structurally related to idiotype antibodies, even though the antibodies Ab1-1E4 and Ab3-2C4 are not related in gene families. Our results support the premise that the use of peptide antigens which are mimics of carbohydrates is an alternate vaccine strategy for polysaccharide antigens and results in an appropriate response.

Dynamic CT in the evaluation of physiologic status of renal transplants.

Dynamic computed tomographic scans were performed in 23 patients to evaluate perfusion and function of renal transplants. Using corticomedullary junction and corticoarterial junction attenuation/time curve analysis, we were able to differentiate normal renal transplants from those undergoing rejection. Corticomedullary junction time was used to assess function and was significant in distinguishing normal renal transplants from those with rejection (p less than 0.001). Similarly, corticoarterial junction time was used to assess perfusion and had a significance of p less than 0.05. The study demonstrates the ability of computed tomography to yield excellent physiologic information.

Differentiation of tumor from syringohydromyelia: intraoperative neurosonography of the spinal cord.

Real-time ultrasonography was useful in differentiating tumor from syringohydromyelia as the cause of cervical cord widening in four patients. Those with syringohydromyelia had a smooth, regular, cystic space located symmetrically in the spinal cord. Tumor appeared as an echogenic, hypoechoic, or cystic expansile lesion that both narrowed the subarachnoid space and obliterated the spinal canal. When intraoperative sonography detects cystic lesions of the spinal cord, careful search for the presence of these signs is necessary to exclude tumor. Intraoperative spinal cord ultrasonography is simple and rapid, and it provides the surgeon with valuable information not readily available previously.

Utilization of temporary muscle paralysis to eliminate CT motion artifact in the critically ill patient.

The intubated, critically ill patient often cannot suspend respiration long enough to eliminate severe computed tomographic (CT) motion artifact, even with current generation CT scanners. High quality, motion-free CT images were safely and easily obtained in two such patients using pancuronium bromide (Pavulon) to achieve muscle paralysis.

CT demonstration of pneumatosis intestinalis from bowel infarction.

A case is presented that illustrates the value of CT in detecting pneumatosis intestinalis prior to plain film findings.

Repair of thelateral ligaments of the ankle.

A brief review of the literature has been accomplished. A report is given on 32 ankles in 31 patients who had reconstruction of their lateral ligaments. The operative technique is described. A criteria for selection of patients is given.