Clinical significance of serum cytokine profiles for differentiating between Kawasaki disease and its mimickers.

OBJECTIVES: To investigate the clinical significance of serum cytokine profiles for differentiating between Kawasaki disease (KD) and its mimickers. METHODS: Patients with KD, including complete KD, KD shock syndrome (KDSS), and KD with macrophage activation syndrome (KD-MAS), and its mimickers, including multisystem inflammatory syndrome in children, toxic shock syndrome, and Yersinia pseudotuberculosis infection, were enrolled. Serum levels of interleukin (IL)-6, soluble tumor necrosis factor receptor type II (sTNF-RII), IL-10, IL-18, and chemokine (C-X-C motif) ligand 9 (CXCL9) were measured using enzyme-linked immunosorbent assay and compared them with clinical manifestations. RESULTS: Serum IL-6, sTNF-RII, and IL-10 levels were significantly elevated in patients with KDSS. Serum IL-18 levels were substantially elevated in patients with KD-MAS. Patients with KD-MAS and KD mimickers had significantly elevated serum CXCL9 levels compared with those with complete KD. Area under the receiver operating characteristic curve analysis showed that serum IL-6 was the most useful for differentiating KDSS from the others, IL-18 and CXCL9 for KD-MAS from complete KD, and CXCL9 for KD mimickers from complete KD and KD-MAS. CONCLUSION: Serum cytokine profiles may be useful for differentiating between KD and its mimickers.

Identification of specific sequence motif of YopN of Yersinia pseudotuberculosis required for systemic infection.

Type III secretion systems (T3SSs) are tightly regulated key virulence mechanisms shared by many Gram-negative pathogens. YopN, one of the substrates, is also crucial in regulation of expression, secretion and activation of the T3SS of pathogenic Yersinia species. Interestingly, YopN itself is also targeted into host cells but so far no activity or direct role for YopN inside host cells has been described. Recently, we were able show that the central region of YopN is required for efficient translocation of YopH and YopE into host cells. This was also shown to impact the ability of Yersinia to block phagocytosis. One difficulty in studying YopN is to generate mutants that are not impaired in regulation of the T3SS. In this study we extended our previous work and were able to generate specific mutants within the central region of YopN. These mutants were predicted to be crucial for formation of a putative coiled-coil domain (CCD). Similar to the previously described deletion mutant of the central region, these mutants were all impaired in translocation of YopE and YopH. Interestingly, these YopN variants were not translocated into host cells. Importantly, when these mutants were introduced in cis on the virulence plasmid, they retained full regulatory function of T3SS expression and secretion. This allowed us to evaluate one of the mutants, yopNGAGA, in the systemic mouse infection model. Using in vivo imaging technology we could verify that the mutant was also attenuated in vivo and highly impaired to establish systemic infection.

Novel diagnostic ELISA test for discrimination between infections with Yersinia enterocolitica and Yersinia pseudotuberculosis.

Yersiniosis is a foodborne infection caused by Yersinia enterocolitica or Yersinia pseudotuberculosis. Although yersiniosis is most often self-limiting, some patients develop chronic infections, such as reactive arthritis, glomerulonephritis, or myocarditis, which require an antibiotic treatment. Whereas early infections can be diagnosed by direct detection of bacteria, chronic infections can only be identified by serological tests. At this point, a serological method for differentiation between infections with the two Yersinia species is important since antibiotic susceptibility of these bacteria is different. Traditional immunoassays do not distinguish between infections with Y. enterocolitica and Y. pseudotuberculosis. The only test that allows for this differentiation is Mikrogen's strip test where discrimination between the two types of infection is based on two recombinant bacterial proteins, MyfA and PsaA (specific for Y. enterocolitica and Y. pseudotuberculosis, respectively). Here, we show that Y. enterocolitica and Y. pseudotuberculosis, cultured under the conditions that mimic the natural rout of infection, express surface antigens different from MyfA and PsaA that can also be used in a discrimination test. Further, we describe a new ELISA that is based on the whole bacteria and recombinant MyfA and PsaA as antigens, and that allows the differentiation between infections with Y. enterocolitica and Y. pseudotuberculosis and simultaneous detection of yersiniosis.

Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.

Role of YopK in Yersinia pseudotuberculosis resistance against polymorphonuclear leukocyte defense.

The enteropathogen Yersinia pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type III secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte or neutrophil (PMN) defense. A yopK mutant, which is attenuated in the mouse infection model, where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and reactive oxygen species production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction of PMN death, whereas the mutant caused a necrosis-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism whereby YopK functions to prevent unintended Yop delivery and thereby PMN disruption, resulting in necrosis-like cell death, which would enhance the inflammatory response favoring the host.

Acute encephalopathy and tubulointerstitial nephritis associated with Yersinia pseudotuberculosis.

We report the case of a 28-month-old boy with encephalopathy and acute tubulointerstitial nephritis possibly associated with Yersinia pseudotuberculosis (Yp) infection. He was transferred to our center because of impairment of renal function and altered consciousness. He had fever for 5 days after recurrent vomiting and diarrhea. Computed tomography scan was normal, but electroencephalogram (EEG) analyses showed generalized slow wave patterns. Continuous hemodialysis was undergone and then his renal function was improved, but altered consciousness persisted. Single photon emission computed tomography (SPECT) revealed abnormally low signals at entire field, which suggested that he was suffered from encephalopathy. Phenobarbital administration and post-encephalopathy rehabilitation were started, and he recovered in fully premorbid state with normal EEG and SPECT findings on the 33rd hospital day. Various bacterial cultures were negative, but both Yp antibody and Yp-derived mitogen (YPM) antibody, the antibody of a specific Yp exotoxin, had an extremely high titer. This is the first report of encephalopathy potentially caused by Yp, indicated by the presence of a high Yp and YPM antibody titer.

Mathematical relationship between cytokine concentrations and pathogen levels during infection.

The relationship between concentrations of cytokines and microbial pathogen levels during infection is not clear. In a sub-lethal murine infection model using Gram-negative bacterial pathogen Yersinia pseudotuberculosis, the serum concentrations (C) of pro-inflammatory cytokines tumor necrosis factor α (TNFα), interferon γ (IFNγ), interleukine-1ß (IL-1ß) and interleukine-18 (IL-18) formed a mathematical relationship with the splenic pathogen levels (P) as measured by colony forming unit. Naming parameters "m" and "k" for magnitude and kinetics, respectively, the relationship is depicted as C=mP(k). When reanalyzing the TNFα and IFNγ concentrations and the bacterial levels that were determined by other groups during infection with another strain of Y. pseudotuberculosis or with Yersinia pestis, this relationship was maintained. Interestingly, the changes in the values of "m" and "k" were consistent with the progress of the host immune response during infection; while deviation from this relationship was observed in individuals that seemed to be unable to control infection. Furthermore, in a murine model of ricin intoxication the local concentrations of the cytokine monocyte chemotactic protein 1 (MCP-1) and the concentrations of injected castor bean toxin ricin also conform to this relationship. C=mP(k) could be a general relationship in host cytokine response to pathogens or pathogen-associated molecular patterns. If confirmed, this type of analysis will be very useful in identifying the steps in a host immune response with which a pathogen interferes. It will also help to determine the specific functions of a host factor in the immune response.

The presence of anti-Yersinia pseudotuberculosis immunoglobulins in equine serum.

The research was conducted on clinically healthy mares (n = 40) and foals (n = 78) during Y. pseudotuberculosis associated enzootics. The animals were divided into groups: I to IV--mares, IA to IVA--their offsprings, IB to IVB--foals which mothers were not treated with any medicaments. The animals in group I, IA and IB were injected with PBS; in group II, IIA and IIB--with Y. pseudotuberculosis strain-based vaccine, in group III, IIIA and IIIB--with P. acnes strain-based immunostimulator; in group IV, IVA and IVB--with P. acnes strain-based immunostimulator and (5 days after the immunostimulator injection) Y. pseudotuberculosis strain-based vaccine. The presence of antibodies was determined by means of ELISA. The study revealed anti-Yersinia pseudotuberculosis IgG only in 19 mares before, and in 25 mares and 26 foals 3 weeks after vaccination. The mean extinction 3 weeks after vaccination amounted to: II-0.489, IV-2.578, iiA-0.572, IVA-0.974, IIB-0.312, iVB-0.418. The cut-off extinction value was 0.154. The presence of anti-Yersinia pseudotuberculosis IgG before vaccination in the sera of clinically healthy mares may suggest that Y. pseudotuberculosis infection occurs definitely more often than is expected. Vaccination preceded by immunostimulation appeared to be the most efficient method of treatment against yersiniosis.

Role of Yersinia pseudotuberculosis outer proteins (Yops) in murine humoral immune response.

The infection of mice with the wild-type (WT) strain of Y. pseudotuberculosis did not induce polyclonal activation of B lymphocytes. Suppression in the production of certain isotypes of Ig was observed, provoked mainly by YopH, YopJ and YpkA. The WT strain induced a progressive increase in the serum-specific IgG, which peaked after 4 weeks after infection, IgM being produced only after 1 week. Autoantibodies against phosphorylcholine, myelin, thyroglobulin and cardiolipin could be detected in the serum of mice infected with the WT strain. The infection of mice provoked suppression in the production of immunoglobulins by splenic B cells and that YopH, YopJ and YpkA must be involved here.

Attenuation and preserved immunogenic potential of Yersinia pseudotuberculosis mutant strains evidenced in oral pig model.

Experimental oral infection of pigs with a parental Yersinia pseudotuberculosis strain pIB102, serotype O:3 and two mutant isogenic strains - pIB155,DeltayopK and pIB44,DeltaypkA has been carried out. Clinical findings, microbiological and immunological parameters were examined in dynamics from day 7 to day 60 post-infection (p.i.). All types of infections ran asymptomatically, without hyperthermia, loss of appetite, etc. Experiments on the blood parameters demonstrated a transient leucocytosis with lymphocytosis and monocytosis better expressed after yopK infection. Even though pig is usually known as a reservoir of yersiniae, bacterial colonization was found in mesenterial lymph nodes and tonsils on day 7, respectively 14 p.i. with parental strain, and only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of mutants to the bactericidal effect of leukocytes and blood sera is the characteristic feature of attenuation in their pathogenicity, compared to the parental strain. Comparative in vitro experiments on the immune response and immunostimulating capacity of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential, predominantly in case of yopK. Hyperplasia and strong activation of the lymph tissue of Peyer's patches, mesenterial lymph nodes, tonsils and spleen of pigs challenged with both mutant strains were proved as immunomorphological rearrangements. The results obtained give the reason to claim that the genetically constructed yopK null mutant strain is significantly attenuated but is still immunogenic and has the potential for a live vaccine carrier strain.

Seroepidemiologic study on the occurrence of antibodies against Yersinia enterocolitica and Yersinia pseudotuberculosis in urban and rural population of the Lublin region (eastern Poland).

The aim of this study was to assess the seroprevalence of antibodies against Yersinia in the rural and urban population and to determine the frequency of particular serotypes of Yersinia enterocolitica and Yersinia pseudotuberculosis. 472 sera were examined, 257 of rural inhabitants and 215 of urban inhabitants. The survey was carried out by passive hemagglutination test with the antigens of Yersinia serotypes considered pathogenic for humans: Y. enterocolitica 03, 05, 06, 08, 09 and Y. pseudotuberculosis I and III. In the examined rural population positive reactions to Yersinia antigens were significantly more frequent than in the examined urban population (42% versus 20%, p<0.0001). The most frequent reactions were against Y. enterocolitica serotypes 05 and 08.

[The intensity of cell receptor catabolism in intestinal yersiniosis and pseudotuberculosis]. / Intensivnost' katabolizma kletochnykh retseptorov pri kishechnom iersinioze i psevdotuberkuleze.

The intensity of the catabolism of cell receptors in yersiniosis was evaluated by the level of R-proteins in the blood sera of 95 patients. The study revealed that the degree of an increase in the level of R-protein depended on the severity and the course of the pathological process was the same in both nosological forms of the disease. The high and stable level of R-proteins was indicative of the possibility of the prolonged and relapsing course of yersiniosis.

[Immune complexes and R-proteins in yersinioses]. / Immunnye kompleksy i R-belki pri iersiniozakh.

The intensity of the catabolism of cell receptors in yersiniosis patients has been studied. The dynamics of R-protein level reflects the relapsing character of the pathological process irrespective of the presence of clinically pronounced relapses and the form of the disease. The concentration of circulating immune complexes is elevated during the whole course of the disease and does not depend on its gravity and clinical form. The spectrum of immune complexes in the blood sera of yersiniosis patients is heterogeneous and changes in the course of the disease.

[The mononuclear syndrome in yersiniosis and pseudotuberculosis]. / Mononuklearnyi sindrom pri iersinioze i psevdotuberkuleze.

As many as 95 patients with generalized form of yersiniosis and 66 patients with scarlatiniform pseudotuberculosis were examined. Atypical mononuclears were detected in the peripheral blood of 23.2% of patients with yersiniosis and 13.6% of those with pseudotuberculosis. In 83.9% their numbers varied from 2 to 9% and in 16.1% only their count amounted to 10-11%. Clinical symptoms occurring in patients with yersiniosis (fever, changes in the fauces, exanthema, polyadenopathy, affection of the liver) are revealed with varying frequency in infectious mononucleosis. But as far as the condition under consideration is concerned, exanthema occurs in rare cases only; diarrhea, arthralgia, hypertrophy of the lingual papillae are not characteristic of it. Mononuclear reaction of blood of the patients with yersiniosis and pseudotuberculosis was of short duration and was rather weak.

[The clinical manifestations and differential diagnosis of Yersinia hepatitis and viral hepatitis A and B]. / Klinicheskie proiavleniia i differentsial'naia diagnostika iersinioznykh i virusnykh gepatitov A i B.

Hepatic involvement in yersiniosis is associated with clinical symptoms of hepatitis, sometimes dominant in the clinical picture. Such cases need differentiation of yersiniosis with viral hepatitides A and B. Clinical and biochemical findings were compared for matched patients with yersiniosis-induced hepatic lesions, viral hepatitis A and viral hepatitis B verified at specific laboratory examination. This permitted introduction of differential diagnostic signs able to recognize one disease from the other.

[The interleukin-1 content of the blood in patients with a pseudotuberculosis infection and opisthorchiasis]. / Soderzhanie interleikina-1 v krovi bol'nykh psevdotuberkuleznoi infektsiei i opistorkhozom.

Interleukin-1 levels were measured according to S. B. Mizel in 25 patients with pseudotuberculosis and concomitant chronic opisthorchiasis and in 25 ones with pseudotuberculosis alone over the course of the disease and in 3 months after it. The controls were patients with chronic opisthorchiasis and normal subjects. Hyperproduction of interleukin-1 by peripheral blood monocytes was found in chronic opisthorchiasis patients and in pseudotuberculosis patients with concomitant opisthorchiasis over the entire follow-up period. In patients with pseudotuberculosis alone interleukin-1 production was found elevated only at the peak of the disease and during early convalescence, that was followed by a stable normalization of this characteristic.

Cytometric haematological analysis of experimental Yersinia pseudotuberculosis infection at temperatures of cultivation of 25 degrees C and 37 degrees C.

The dynamics of acute lethal infection with Yersinia pseudotuberculosis (serovar III) grown at 25 degrees C and 37 degrees C, respectively, were studied in guinea-pigs. Using an automated hematology analyzer, the quantity and quality of the host cellular response was determined at the primary site of infection (the peritoneal cavity) and in parallel in the blood. A much more intensive leucocytic response of monocytic type was observed in the animals infected with microorganism grown at 37 degrees C. This type of reaction was associated with a lower mortality rate and a relatively benign progression of the infection.

[The temperature-induced early and late development of resistance to the bactericidal action of blood serum in Yersinia pseudotuberculosis]. / Indutsirovannoe temperaturoi rannee i pozdnee razvitie ustoichivosti k bakteritsidnomu deistviiu syvorotki krovi u Yersinia pseudotuberculosis.

Y. pseudotuberculosis strains were grown at 6 degrees-8 degrees C and then incubated at 37 degrees C. 3-6 hours later serum resistance appeared in the strains having plasmid virulence and producing outer membrane polypeptide with a molecular weight of 120 kD, known as P1. 10-12 hours later serum resistance appeared in the strain having the virulence plasmid, but not producing P1, as well as in strains in which the plasmid was eliminated.