[Peritoneal dialysis and kidney transplantation]. / Peritoneumska dijaliza i transplantacija bubrega.

The results of pretransplantation preparation of patients undergoing peritoneal dialysis program before the kidney transplantation at our clinic have been presented. Residual kidney function, and bladder function, respectively, as well as the incidence of the hepatotropic viruses B and C infections and cytotoxic antibodies percentage following blood transfusion have been particularly analyzed. Obtained results have been correlated with those found in 40 patients on hemodialysis and to whom kidneys were transplanted at our clinic. Satisfactory bladder function, the absence of urologic posttransplantation complications, non-existence of hepatotropic viral infections and cytotoxic antibodies resulted in an introduction of a new strategy based on the peritoneal dialysis as the first method of the dialysis treatment prior to kidney transplantation.

A highly specific one-step PCR - assay for the rapid discrimination of enteropathogenic Yersinia enterocolitica from pathogenic Yersinia pseudotuberculosis and Yersinia pestis.

Based on differences within the yopT-coding region of Yersinia. enterocolitica, Y pseudotuberculosis and Y pestis, a rapid and sensitive one-step polymerase chain reaction assay with high specificity for pathogenic Y enterocolitica was developed. By this method pathogenic isolates of Y enterocolitica can be easily identified and discriminated from other members of this genus. The entire coding sequence of the yopT effector gene of Y. pseudotuberculosis Y36 was determined.

In vivo labeling of endothelin receptors with [(11)C]L-753,037: studies in mice and a dog.

UNLABELLED: Endothelin (ET) is a potent mammalian vasoconstrictive peptide and a pressor agent. Its 3 isoforms, ET-1, ET-2, and ET-3, mediate several physiologic actions in several organ systems, binding to 2 major receptor subtypes: ET(A) and ET(B). This study was undertaken to evaluate [(11)C]L-753,037 [(+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxy-propyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno [1,2-beta]pyridine-6-carboxylate), a new mixed ET receptor A and B antagonist, as a tracer for in vivo labeling of ET receptors in mice and a dog. METHODS: [(11)C]L-753,037 was synthesized, purified, and formulated from a normethyl precursor, L-843,974, and [(11)C]H(3)I. The tracer was studied for its in vivo kinetics, biodistribution, and ET receptor binding characteristics in mice. In the dog, PET imaging was performed to evaluate binding of [(11)C]L-753,037 to ET receptors in the heart. Specificity of binding was studied in the heart with the selective ET(A) antagonist L-753,164. RESULTS: Kinetic studies in mice showed highest tracer uptake at 5 min after injection in liver (25.0 percentage injected dose per gram [%ID/g]), kidneys (18.7 %ID/g), lungs (15.2 %ID/g), and heart (5.6 %ID/g). Initial high uptake in liver, lungs, and kidneys was followed by rapid washout during the next 10 min and a very slow clearance during the time of observation (2 h after injection). By contrast, the radioactivity in the heart remained constant over 2 h. Administration of both ET(A) (L-753,164) and mixed ET(A)/ET(B) (L-753,137) receptor antagonists resulted in dose-dependent inhibition of [(11)C]L-753,037 binding in mouse heart, lungs, and kidneys but not in the liver. Radioactivity in the brain was very low, indicating that the tracer does not cross the blood-brain barrier. In the dog, a dynamic PET study of the heart showed high tracer accumulation at 55-95 min after injection. Injection of L-753,164 at 30 min before [(11)C]L-753,037 administration led to a significant reduction in tracer binding. [(11)C]methyl triphenyl phosphonium was used as a tracer for reference images of the dog heart muscle. CONCLUSION: The results suggest that [(11)C]L-753,037 binds to ET receptors in vivo and is, therefore, a promising candidate for investigation of these receptors and their occupancy by ET receptor antagonists using PET.

Peripheral circulating insulin-like growth factor-I and -II in cattle.

Interrelationships between circulating concentrations of the insulin-like growth factors (IGF-I and IGF-II) were investigated in 235 blood samples taken from 145 healthy beef or dairy calves, bulls and cows of different breeds and ages. Autoradiography of Western ligand blots indicated different IGF binding protein (IGFBP) profiles between sera from different categories of cattle. Each IGF radioimmunoassay was validated by determining the effects of IGFBPs, ligand and contraligand, as well as serial dilution and comparison with results obtained after molecular sieve chromatography in acid. In female cattle mean values for IGF-I varied from 5.1 nmol/l in postparturient Holstein cows to 18.5-20.5 nmol/l in growing beef heifers, while mean IGF-II concentrations ranged from 30.0 nmol/l in the cows to 14.7-15.7 nmol/l in the beef heifer calves. In male cattle mean serum IGF-I ranged widely from 8.2 nmol/l in 1-day-old Holstein calves to 67.4 nmol/l in 16-month-old Simmental-type bulls. Mean IGF-II concentrations decreased from 22.9 nmol/l in 1-day-old Holstein bull calves to 11.9 nmol/l in 12-month-old beef bulls. Thus, total molar IGF concentrations were fairly stable in female cattle (24.7-35.1 nmol/l) but extended from 27.3 nmol/l to 81.8 nmol/l in the male cattle. The tendency for a reciprocal relationship between serum concentrations of these growth factors was most obvious in the periparturient cows.

[Outcome of kidney transplantation in patients on peritoneal dialysis]. / Ishod transplantacije bubrega kod bolesnika na peritoneumskoj dijalizi.

The initial experience suggested that kidney transplantation could be hazardous for patients on peritoneal dialysis due to the high risk of peritonitis and a possible high incidence of acute rejection. In this paper we have presented our experience with kidney transplantation in these patients. During the last four years kidney transplantation was performed in 9 patients on peritoneal dialysis. The average time spent on peritoneal dialysis was 20.6 +/- 7.6 months. In all patients peritoneal catheter was removed during the surgery. During the posttransplantation period a triple immunosuppressive therapy including steroids, cyclosporin and azathioprineor mycophenolate mofetil was administered in all patients. In comparison to patients on hemodialysis no significant difference in the incidence of acute rejection episodes, delayed graft function, graft arterial thrombosis and graft function recovery was observed. Patients on peritoneal dialysis had significantly greater and longer wound drainage in comparison to patients on hemodialysis. It was concluded that peritoneal dialysis had no negative influence on short-term outcome of kidney transplantation.

A miniaturised semiautomated system for the identification of Yersinia species within the genus Yersinia.

Commercially available identification systems based on biochemical reactions of bacteria are not suited for typing the species of the genus Yersinia (Y.) or the biovars (BV) of the species Y. enterocolitica. This failure is caused by the limited number of biochemical reactions applied, resulting in the absence of important discriminatory key reactions. The MICRONAUT identification system (Merlin, Bornheim-Hersel) makes use of dried substrates/enzymes reactions in the wells of a 96-well microtitration plate, reading of the results by a scanner device and typing of the isolate by the calculation of probabilities according to a data base. For this study a special identification panel was designed on which 38 substrates and enzyme reactions were configurated including 20 reactions for the identification of the species of the genus and the Y. enterocolitica biovars. The database was calculated using the results obtained from a total of 250 Yersinia strains of the eleven species of the genus. Reevaluation of the results of these strains revealed an overall sensitivity of 98%, as only four strains were not identified satisfactorily. Considering also questionable results the sensitivity was still 85%. The system was also used to identify Y. pestis isolates, but in this case reading was done visually. The printouts usually cite species designation, identification quality and probabilities. The sealing of the plates in an aluminium bag guarantees long life and long lasting quality. However, an evaluation of the system with a considerable number of strains has to be done in a next step. The 'Yersinia identification set' can replace time-consuming tube testing in the future and is a big step forward towards a sensitive identification of Yersinia isolates in the routine laboratory.

Specific detection of plasmid bearing Yersinia isolates by PCR.

A total of 210 isolates belonging to 9 different species of the genus Yersinia (Y.) was investigated with three different PCR assays targeting two plasmoidal genes, the Yersinia adhesin gene (yadA) and the V-antigen gene. The yadA PCR assay described in 1995 by Blais and Phillipe, targeting a Y. enterocolitica specific gene region and a newly designed assay targeting the gene region functionally responsible for autoagglutination, were compared. Both assays identified the same Y. enterocolitica strains. To exclude the possibility that false negative results were obtained due to mutations that had occurred in parallel in both gene regions, a third PCR assay by Neubauer et al. (2000) targeting a conserved region of the V-antigen gene was used as control. Again, DNA of the same Y. enterocolitica strains was amplified. In contrast to the yadA PCR assay described by Blais and Phillipe, the newly established yadA and the V-antigen PCR assays amplified DNA from Y. pseudotuberculosis strains. Therefore, by using the PCR technique as a molecular tool spontaneous mutations could be excluded as the cause of anomalous reactions in PCR assays targeting genes of the Yersinia virulence plasmid. Based on these results, it can be assumed that all presumptive pathogenic Yersinia isolates can be identified on the basis of PCR analysis. These molecular assays may also produce fewer false positive reactions in comparison to phenotypic tests such as the autoagglutination test which depend heavily on the handler's experience. It has to be stressed that the PCR assays used in this study have not been evaluated for routine use. Therefore, standardization of the PCR methodology including sample preparation, primer target sequences and PCR reagents is needed for the reliable and safe diagnosis of pathogenic Yersinia spp. in future.

A combination of different polymerase chain reaction (PCR) assays for the presumptive identification of Yersinia pestis.

A combination of four polymerase chain reaction (PCR) assays targeting the Yersinia pestis-specific plasmoidal genes of the fraction 1 capsular antigen and plasminogen activator/coagulase, the gene of the V antigen of the Yersinia virulence plasmid, and the chromosomal 16S rRNA gene was evaluated for the identification of Y. pestis isolates. All four assays were subjected to the same sample preparation technique, reagents and cycling conditions. Eighteen Y. pestis, 66 Y. pseudotuberculosis, 40 Y. enterocolitica strains, the type strains of the other Yersinia species, and 20 other pathogenic bacterial strains were investigated. By using the proposed combination of PCR assays all Y. pestis strains were identified correctly. The applicability of this combination of PCR assays was demonstrated by the detection of Y. pestis DNA in spiked tissues from Rattus norwegicus and fleas (Xenopsylla cheopis and Ctenocephalides spp.). As little as 60 genome equivalents were detected. This system is applicable for monitoring Y. pestis and its vectors in enzootic natural foci and in the diagnosis of plague in humans and animals.

Yersinia enterocolitica 16S rRNA gene types belong to the same genospecies but form three homology groups.

The species Yersinia (Y.) enterocolitica consists of biochemically and serologically heterogeneous strains. A vernacular nomenclature divides these strains in 'European' and 'American' bioserotypes. We investigated six strains of each group by DNA-DNA hybridization, determination of G + C mol% content and sequence alignment studies. Based on different DNA-DNA hybridization values and the 16S rRNA gene sequences a division into two Yersinia enterocolitica subspecies is justified. We propose the names Yersinia enterocolitica subsp. enterocolitica for strains belonging to the 16S rRNA gene type represented by the Type strain ATCC 9610 and Yersinia enterocolitica subsp. palearctica for strains belonging to the 16S rRNA gene type of strain Y11 (DSMZ13030).

Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany--a ten-year study.

Two hundred and ten E. coli O157:H7/H- strains isolated from single cases and outbreaks of diarrhoea and haemolytic uraemic syndrome (HUS) in Germany between 1988 and 1998 were characterised by a range of molecular subtyping methods and phage typing in order to analyse their clonal nature. A high clonal heterogeneity, together with a considerable clonal stability, has been identified among the bacterial isolates and no single clonal type appeared to be geographically dominant. It is recommended to apply pulsed-field gel electrophoresis (PFGE) together with P gene profile determination (number and genomic positions of lambdoid bacteriophages) as laboratory tools for an extended epidemiological surveillance of E. coli OOFF phage typing will remain helpful as a first line of analysis, particularly in outbreak situations.

Identification of Yersinia enterocolitica within the genus Yersinia.

In this report we describe a PCR strategy for the unambigous identification of biochemically presumptive typed Yersinia (Y.) enterocolitica. A total of 269 isolates belonging to ten species of the genus Yersinia were investigated. In a first PCR only isolates classified as Y. enterocolitica (n = 113) gave rise to a specific amplification resulting in a sensitivity and a specificity of 100%. By sequencing the 269 amplicons of a second pan-Yersinia PCR spanning a distinct 16S rRNA gene region, 20 different sequence clusters could be identified within the genus. By this, Y. enterocolitica isolates of American and European origin could be distinguished safely and already described sequence clusters of the species Y. frederiksenii were confirmed. New 16S rRNA gene sequence clusters were detected for the species Y. frederiksenii, Y. intermedia, Y. mollaretii, Y. aldovae, Y. kristensenii, and Y. rohdei.

Evaluation of a Yersinia adhesion gene (yadA) specific PCR for the identification of enteropathogenic Yersinia enterocolitica.

A total of 101 Yersinia enterocolitica strains was investigated with a PCR assay [Blais and Phillipe, Food Control, 6 (1995) 211-214] targeting the Yersinia adhesin gene (yadA) responsible for autoagglutination. Compared to the autoagglutination test the PCR assay has a specificity of 100% but a sensitivity of only 70%. This failure might be caused by the sequence heterogeneity of yadA.

Serodiagnosis of human plague by an anti-F1 capsular antigen specific IgG/IgM ELISA and immunoblot.

Plague is a re-emerging disease endemic in at least 24 countries. Non-endemic countries should be able to confirm plague to prevent outbreaks due to imported cases. We established a combination of a IgG/IgM screening ELISA and a confirmation immunoblot employing F1 capsular antigen (CA) for the serodiagnosis of plague in countries where yersiniosis is present. The ELISA and the immunoblot assay showed a specificity of 96.1% and 100% among sera from healthy German blood donors. This group had a seroprevalence of 39% of anti-yersinia outer protein (YOP) antibodies obviously caused by previous Y. enterocolitica infection. The ELISA detected anti-F1 CA antibodies in 22 and the immunoblot in 20 out of 26 sera of plague vaccinees. Five control sera from bacteriologically confirmed plague cases from Madagascar reacted positively. It can be concluded that anti-YOP antibodies do not affect assays based on purified F1 CA.

SPECT brain perfusion findings in mild or moderate traumatic brain injury.

BACKGROUND: The purpose of this manuscript is to present the findings in the largest series of SPECT brain perfusion imaging reported to date for mild or moderate traumatic brain injury. PATIENTS AND METHODS: This is a retrospective evaluation of 228 SPECT brain perfusion-imaging studies of patients who suffered mild or moderate traumatic brain injury with or without loss of consciousness (LOC). All patients had no past medical history of previous brain trauma, neurological, or psychiatric diseases, HIV, alcohol or drug abuse. The patient population included 135 males and 93 females. The ages ranged from 11-88 years (mean 40.8). The most common complaints were characteristic of the postconcussion syndrome: headaches 139/228 (61%); dizziness 61/228 (27%); and memory problems 63/228 (28%). LOC status was reported to be positive in 121/228 (53%), negative in 41/228 (18%), and unknown for 63/228 (28%). RESULTS: Normal studies accounted for 52/228 (23%). For abnormal studies (176/228 or 77%) the findings were as follows: basal ganglia hypoperfusion 338 lesions (55.2%); frontal lobe hypoperfusion 146 (23.8%); temporal lobes hypoperfusion 80 (13%); parietal lobes hypoperfusion 20 (3.7%); insular and or occipital lobes hypoperfusion 28 (4.6%). Patients' symptoms correlated with the SPECT brain perfusion findings. The SPECT BPI studies in 122/228 (54%) were done early within 3 months of the date of the accident, and for the remainder, 106/228 (46%) over 3 months and less than 3 years from the date of the injury. In early imaging, 382 lesions were detected; in 92 patients (average 4.2 lesions per study) imaging after 3 months detected 230 lesions: in 84 patients (average 2.7 lesions per study). CONCLUSIONS: Basal ganglia hypoperfusion is the most common abnormality following mild or moderate traumatic brain injury (p = 0.006), and is more common in patients complaining of memory problem (p = 0.0005) and dizziness (p = 0.003). Early imaging can detect more lesions than delayed imaging (p = 0.0011). SPECT brain perfusion abnormalities can occur in the absence of LOC.

Detection of antibodies against Campylobacter jejuni serogroup PEN O:19 purified flagellar protein in a patient with Guillain-Barré syndrome.

C. jejuni serogroup PEN O:19 was isolated from a stool specimen from a patient with Guillain-Barré syndrome (GBS). Flagellar protein was isolated and purified from reference strain C. jejuni PEN O:19, ATCC 43,446, as well as from a homologous patient strain. Antibodies against flagellar protein were detected by means of immunoblotting, enzyme-linked immunosorbent assay (ELISA) and tube agglutination test. The antibody titres were found to be directly correlated at the beginning and in the recovery phase of GBS. Antibodies of IgG and IgA classes were present from the very onset of the disease as well as 5 months later, but with a lower titre population. However, antibodies of the IgM class were persistent only at the onset of the infection and disappeared during the following 5 months. Our results strongly support the hypothesis that in GBS patients, antiflagellar antibodies are induced during C. jejuni infection and can be used in the diagnosis of C. jejuni-associated GBS.

Variations in the 16S rRNA gene sequence of Yersinia enterocolitica isolates influence the specificity of molecular identification systems.

Four identification systems were used to type Yersinia enterocolitica strain Y11 and Yersinia enterocolitica sensu strictoT. Two systems based on biochemical reaction patterns identified both strains as Yersinia enterocolitica. Two molecular assays targeting the 16S rRNA gene failed to identify either strain Y11 or the type strain. Therefore, both strains were typed by the classical taxonomical approach requiring a determination of the overall base composition and the base sequence similarity using hybridization. Again both strains were classified as Yersinia enterocolitica isolates. Consequently, the sequences of the 16S rRNA gene of both strains were determined and compared. The strains differed in a region where nucleotide changes between species of the genus Yersinia had been described earlier. These differences may explain the failure of the molecular assays to identify the strains. They also demonstrate an independent evolution of the 16S rRNA genes in the species Yersinia enterocolitica sensu stricto suggesting an amendment to the nomenclature to be used in the future.

SPET brain perfusion imaging in mild traumatic brain injury without loss of consciousness and normal computed tomography.

We present SPET brain perfusion findings in 32 patients who suffered mild traumatic brain injury without loss of consciousness and normal computed tomography. None of the patients had previous traumatic brain injury, CVA, HIV, psychiatric disorders or a history of alcohol or drug abuse. Their ages ranged from 11 to 61 years (mean = 42). The study was performed in 20 patients (62%) within 3 months of the date of injury and in 12 (38%) patients more than 3 months post-injury. Nineteen patients (60%) were involved in a motor vehicle accident, 10 patients (31%) sustained a fall and three patients (9%) received a blow to the head. The most common complaints were headaches in 26 patients (81%), memory deficits in 15 (47%), dizziness in 13 (41%) and sleep disorders in eight (25%). The studies were acquired approximately 2 h after an intravenous injection of 740 MBq (20.0 mCi) of 99Tcm-HMPAO. All images were acquired on a triple-headed gamma camera. The data were displayed on a 10-grade colour scale, with 2-pixel thickness (7.4 mm), and were reviewed blind to the patient's history of symptoms. The cerebellum was used as the reference site (100% maximum value). Any decrease in cerebral perfusion in the cortex or basal ganglia less than 70%, or less than 50% in the medial temporal lobe, compared to the cerebellar reference was considered abnormal. The results show that 13 (41%) had normal studies and 19 (59%) were abnormal (13 studies performed within 3 months of the date of injury and six studies performed more than 3 months post-injury). Analysis of the abnormal studies revealed that 17 showed 48 focal lesions and two showed diffuse supratentorial hypoperfusion (one from each of the early and delayed imaging groups). The 12 abnormal studies performed early had 37 focal lesions and averaged 3.1 lesions per patient, whereas there was a reduction to--an average of 2.2 lesions per patient in the five studies (total 11 lesions) performed more than 3 months post-injury. In the 17 abnormal studies with focal lesions, the following regions were involved in descending frequency: frontal lobes 58%, basal ganglia and thalami 47%, temporal lobes 26% and parietal lobes 16%. We conclude that: (1) SPET brain perfusion imaging is valuable and sensitive for the evaluation of cerebral perfusion changes following mild traumatic brain injury; (2) these changes can occur without loss of consciousness; (3) SPET brain perfusion imaging is more sensitive than computed tomography in detecting brain lesions; and (4) the changes may explain a neurological component of the patient's symptoms in the absence of morphological abnormalities using other imaging modalities.

Identification of Yersinia species by the Vitek GNI card.

The Vitek GNI card was used to identify 212 isolates of 10 Yersinia species. Identification was correct for 96.3% of the isolates (156 of 162) to the genus level and for 57.4% of the isolates (93 of 162) to the species level for Yersinia spp. listed in the Vitek database. We recommend additional identification methods for isolates assigned to the genus Yersinia by the Vitek system.

[Serum levels of beta 2-microglobulin in various types of hemodialysis]. / Serumska koncentracija beta 2-mikroglobulina u razlicitim vrstama hemodijalize.

beta 2-microglobulin (beta 2 m) is the major constituent of amyloid fibrils in dialysis-related amyloidosis (DRA), which is considered to be one of the most severe adverse effect of long-term dialysis. In this study we evaluated the efficiency of beta 2 m removal during different dialysis procedures. A total of 45 patients undergoing hemodialysis were divided in five groups: cuprophane dialysis (n = 10), high-flux polysulphone dialysis (n = 10), postdilutional hemodiafiltration (n = 10), conventional postdilutional hemofiltration (n = 10) and predilutional on-line hemofiltration (n = 5). Serum level of beta 2 m was determined before and after different procedures using ELISA. In the group of patients on cuprophane dialysis was registered an elevation of beta 2 m and of 16.8 +/- 11.4% on the average. Serum level of beta 2 m was decreased following all other procedures on the average of 40.7 +/- 16.4% after high-flux polysulphone dialysis, 42.0 +/- 13.7% after conventional hemofiltration, 64.7 +/- 9% after hemodiafiltration and 67.9 +/- 10.1% after predilutional hemofiltration. The best removal of serum beta 2 m was realized by predilutional hemofiltration. Also, we have noticed that patients treated with high-flux synthetic membranes in the longer time-period have lower predyalisis value of beta 2 m compared to patients treated with cuprophane membrane. Further long-term studies will be necessary to conclude whether these procedures could be successful prophylactic and/or therapeutic regimen for dialysis-related amyloidosis.

Genetic variation of Yersinia enterocolitica serotype O:9 strains detected in samples from western and eastern countries.

We made use of H-serotyping, ribotyping and restriction endonuclease analysis of virulence plasmid DNA (REAP) to differentiate Yersinia enterocolitica serotype O:9 strains. A close correlation between ribotypes/REAP patterns and the geographical and chronological distribution of serotype O:9 strains was apparent. In European countries, variant clones of serotype O:9 have rapidly increased among humans and swine since the late 1980s.