Use of O-antigen gene cluster-specific PCRs for the identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis

Yersinia pestis is a very recently evolved clone of Yersinia pseudotuberculosis serotype O:1b. This close relationship causes potential difficulties in DNA-based diagnostic methods. Analysis of the O-antigen gene clusters in these two organisms identified two regions that were used to specifically identify Y. pestis-Y. pseudotuberculosis as a group or Y. pestis alone. Both PCR assays were found to be 100% specific when tested on a large collection of Yersinia species and other Enterobacteriaceae. Furthermore, advantage was taken of the different setups of the O-antigen gene clusters of the 21 known Y. pseudotuberculosis serotypes to develop a multiplex PCR assay to replace the conventional serotyping method of Y. pseudotuberculosis by O-genotyping. The multiplex PCR assay contained nine sets of specific PCRs in a single tube and when used on Y. pseudotuberculosis reference strains allowed the distinction of 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13). Serotype O:7, O:9, and O:10 strains required additional PCRs for O-genotyping. Once applied to Y. pseudotuberculosis strains of various origins, a very good correlation between classical serotypes and O-genotypes was observed, although some discrepancies were found. O-genotyping also proved useful to correct misidentification of some strains and to type Y. pseudotuberculosis isolates that had lost the expression of the O-antigen. The PCR-based O-genotyping can easily be applied in conventional laboratories, without the need for tedious preparation of a large set of specific antisera.     

Role of YadA, Ail, and Lipopolysaccharide in Serum Resistance of Yersinia enterocolitica Serotype O:3

Complement attack is a host strategy leading to elimination of pathogens. Yersinia enterocolitica expresses several potential complement resistance factors: the outer membrane proteins YadA and Ail as well as lipopolysaccharide (LPS). To study the contribution of these factors to the survival of Y. enterocolitica serotype O:3 in nonimmune human serum, we constructed 23 mutant strains of Y. enterocolitica O:3 expressing different combinations of YadA, Ail, LPS O antigen, and LPS outer core. Survival of bacteria was analyzed in normal serum (with functional classical, lectin, and alternative complement activation pathways) and EGTA-Mg-treated serum (only alternative pathway functional). Kinetic killing tests revealed that the most potent single-serum resistance factor needed for long-term survival was YadA; Ail was also indispensable, but it provided short-term survival and delayed the bacterial killing. On the contrary, the LPS O antigen and outer core, when in combination with YadA, Ail, or both, had a minor and often negative effect on serum resistance. Bacteria in the exponential phase of growth were more resistant to serum killing than stationary-phase bacteria. After exposing bacteria to EGTA-Mg-treated serum, O antigen could prevent deposition of covalently bound C3b on bacteria at 3 min of incubation, even as a single factor. At later time points (15 and 30 min) it had to be accompanied by YadA, Ail, and outer core. In normal serum, the bacteria were less resistant to C3b deposition. However, no direct correlation between the C3 deposition pattern and bacterial resistance was observed.     

Whisker trimming begun at birth or on postnatal day 12 affects excitatory and inhibitory receptive fields of layer IV barrel neurons

In rats, whisker trimming during development leads to persistent alterations in the function of cortical barrel circuits and to behavioral deficits later in life. Here we examined how whisker trimming begun either at birth (P0) or on postnatal day 12 (P12), around the onset of whisking behavior, affects receptive fields of layer IV barrel neurons. All whiskers on the left face were trimmed for 40-45 days and then allowed to regrow fully. Extracellular single-unit recordings and controlled deflections of principal and adjacent whiskers (PW and AW, respectively), individually or in paired combinations, were used to assess excitatory and suppressive effects of neighboring whiskers on barrel neurons. Results indicate that whisker trimming both from P0 and P12 leads to enlarged excitatory and weakened inhibitory receptive fields in layer IV neurons. PW- and AW-evoked responses are larger in magnitude in trimmed than in control animals; AW-evoked responses are disproportionately affected, decreasing the spatial focus of barrel neurons. Deprivation after P12 accounts for approximately 50% of the total effect observed in P0 trimmed animals. Suppressive interactions, evoked by two whiskers deflected in succession, are weaker in trimmed than in control animals. Suppressive caudal/rostral and ventral/dorsal gradients, however, seem unaffected by sensory deprivation. Thus the developmental period during which experience persistently modifies maturing barrel circuitry extends up to and likely beyond the onset of whisking behavior. Sensory deprivation during this time affects development of both excitatory and inhibitory receptive fields of barrel neurons and likely impairs cortical integration of sensory information from multiple whiskers.     

The Chinese hamster V79 cell mutant V-H4 is phenotypically like Fanconi anemia cells

It has been shown by genetic complementation analysis that a mitomycin C-sensitive mutant (V-H4) of Chinese hamster V79 cells is the first rodent equivalent of Fanconi anemia (FA) group A. The V-H4 mutant shows many typical characteristics of cells derived from FA patients. V-H4 cells exhibit increased sensitivity towards cross-linking agents as MMC (30-fold), cis-DDP (10-fold), DEB (10-fold), and PUVA (1.6-fold), but an only slightly increased sensitivity to monofunctional alkylating agents (EMS and MMS) and actinomycin D. V-H4 cells are also moderately sensitive to adriamycin (1.6-fold), and not sensitive to H₂O₂. The levels of chromosomal aberrations induced by MMC and cis-DDP treatment are higher (4- to 6-fold) in V-H4 cells than in the wild-type V79 cells. Genetic complementation analysis with other Chinese hamster mutants hypersensitive to MMC (irs1, irs1SF, UV20 and UV41) indicates clearly that V-H4 belongs to a different, new complementation group. This unique mutant is very stable and can serve as a vehicle to isolate the complementing FA-A gene from normal human DNA.     

A role for hyaluronan in macrophage accumulation and collagen deposition after bleomycin-induced lung injury

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

To err is autonomic: error-related brain potentials, ANS activity, and post-error compensatory behavior

A two-component event-related brain potential consisting of an error-related negativity (ERN/Ne) and positivity (Pe) has been associated with response monitoring and error detection. Both the ERN and Pe have been source-localized to the anterior cingulate cortex (ACC)--a frontal structure implicated in both cognitive and affective processing, as well as autonomic nervous system (ANS) modulation. The current study sought to examine the relationships among the ERN, the Pe, two autonomic measures, and behavior. Electroencephalogram (EEG), heart rate (HR), and skin conductance (SC) were recorded while subjects performed a two-choice reaction-time task. In addition to the characteristic ERN-Pe complex, errors were associated with larger SCRs and greater HR deceleration. The ERN correlated with the number of errors, but was unrelated to ANS activity and compensatory behavior. Pe, on the other hand, was correlated significantly with SCR, and both SCR and Pe were significantly correlated with post-error slowing.     

Detection of a novel repeated sequence useful for epidemiological typing of pathogenic Yersinia enterocolitica

Strains (n = 203) of Yersinia species were used in genotyping and PCR experiments in order to evaluate the genotyping potential of the YeO:3RS probe. This probe comprises a 12.5 kb genomic fragment of the Y. enterocolitica O:3 lipopolysaccharide O-antigen gene cluster cloned into plasmid pBR322. The genotyping potential of YeO:3RS was shown to reside in the region upstream of the O-antigen gene cluster, i.e., in the first 1.65 kb of the cloned genomic fragment that contains a repeated sequence (RS) present in multiple copies in the genome. In genotyping, the YeO:3RS probe was hybridised to DNA of Yersinia enterocolitica isolates (n = 112) from humans, animals and food, along with strains of other Yersinia species (n = 5) and Salmonella enterica strains (n = 3). The YeO:3RS probe efficiently detected and subtyped all European pathogenic Yersinia enterocolitica isolates of the serobiotypes O:3/4, O:9/2 and O:5,27/2 studied (n = 87), whereas it hybridised only weakly or not at all with the other strains. Within Yersinia enterocolitica serobiotype O:3/4 strains, YeO:3RS genotyping was as discriminatory as genotyping by pulsed-field gel electrophoresis (PFGE) of XbaI-NotI digested genomic DNA. When these two methods were combined, YeO:3RS genotyping divided both of the two predominant PFGE types into six subtypes, thus increasing the discrimination. In PCR screening of additional 86 Yersinia strains, the 1.65 kb region was detected in European pathogenic serotypes O:1 and O:2 in addition to serotypes O:3, O:5,27 and O:9, indicating that it can be exploited in detecting and typing of European pathogenic serotypes in general.