HLA and T-cell receptor gene polymorphisms in Guillain-Barré syndrome.

OBJECTIVE: We examined a possible involvement of genetic factors influencing the development of Guillain-Barré syndrome (GBS). METHODS: We studied T-cell receptor (TCR), alpha-chain constant (AC), and beta-chain variable (BV) gene polymorphisms using microsatellite markers and serologic HLA class I antigens, HLA-DRB1, and HLA-DQB1 alleles in 81 Japanese patients with GBS and 87 controls. RESULTS: There were no significant differences in these genetic markers between GBS patients and controls. Subgrouping of GBS patients according to recent Campylobacter jejuni infection, the presence of anti-GM1 antibody in the sera, or their combinations also failed to reveal significant associations with these genetic markers. There was, however, a tendency for an increased frequency of HLA-DRB1*0803 in the C. jejuni + GM1 + GBS group, when compared with controls. CONCLUSIONS: The data suggest that the roles of TCRAC, T-cell receptor beta-chain variable (TCRBV), HLA class I or class II in the development of GBS are not critical, and further research is necessary to clarify other genes encoded within the HLA region for genetic susceptibility to GBS.

Antibodies to gangliosides and galactocerebroside in patients with Guillain-Barré syndrome with preceding Campylobacter jejuni and other identified infections.

The relationship between preceding infections and antibodies to glycolipids was investigated in 205 Japanese patients with Guillain-Barré syndrome (GBS). Serological evidence of recent Campylobacter jejuni (C. jejuni) infection was found in 45% of the patients, compared with 1% in healthy controls. In contrast, recent infection of cytomegalovirus (CMV), Mycoplasma pneumoniae (M. pneumoniae) and Epstein-Barr virus (EBV) was detected in only 5%, 2% and none of the patients, respectively. C. jejuni-associated GBS was more frequent in early spring than in other seasons. All stool specimens positive for C. jejuni isolation were obtained within 10 days after the onset of GBS symptoms. Of 13 C. jejuni isolates from GBS patients, 10 (77%) belonged to Penner serotype 19 (heat-stable, HS-19). Elevated titers of anti-GM1 antibody were found in 8 (80%) of 10 GBS patients whose C. jejuni isolates belonged to HS-19 and in none of those infected with non-HS-19 C. jejuni (P = 0.04), and in 49% of 92 patients with C. jejuni infection and 25% of patients without infection of C. jejuni, CMV, EBV, or M. pneumoniae (P = 0.0007). The frequencies of elevated antibody titers to GD1a, GD1b and GQ1b were also significantly higher in GBS patients associated with C. jejuni than those not associated with C. jejuni, CMV, EBV, and M. pneumoniae. GBS in Japan seems to be associated more frequently with C. jejuni and less frequently with CMV than in Europe and North America.

Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin.

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.

Detection of neutral sugars in purified type G botulinum progenitor toxin and the effects of some glycolytic enzymes on its molecular dissociation and oral toxicity.

Arabinose and galactose were detected in purified type G botulinum toxin (Mr about 500,000) of Clostridium argentinense. The i.p. LD50/mg N of type G progenitor toxin was one-tenth, but the oral LD50/mg N twice that of type A-L toxin. The lysozyme-, endo-beta-galactosidase-, and N-glucanase-treated toxins each had a molecular mass of about 300,000. The oral toxicity of the endo-beta-galactosidase or N-glucanase-treated toxin was one-fifth that of untreated progenitor toxin. On DEAE-Sephadex chromatography, the N-glucanase-treated toxin dissociated into two fractions, nontoxic and toxic. SDS-PAGE of the toxic fraction showed a single band with a Mr of about 150,000, and after dithiothreitol treatment, two bands with Mr of 100,000 and 50,000.

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan.

A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A (flaA) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy (1980) J. Clin. Microbiol. 12, 732-737). Six AfaI, seven MboI, and five HaeIII RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-1 strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni, and may also be useful for the analysis of C. jejuni subtypes from GBS patients.

Effect of bursectomy and thymectomy on Pasteurella multocida infection in chickens.

The effects of various treatments on the immune response of chickens to vaccine prepared from Pasteurella multocida strain P1059 were investigated. The treatments, given on or before the day of hatching, consisted of surgical bursectomy (SB), hormonal bursectomy (HB), surgical thymectomy (ST) and X-irradiation (X). In chickens subjected to SB, SBX or HB before vaccination, the production of agglutinin and immunoglobulin was impaired, but the production of resistance against challenge was not. In chickens subjected to ST or STX before vaccination, the production of agglutinin and immunoglobulin was unimpaired, but the production of resistance against challenge was diminished. Protection was therefore thymus-dependent.

Characterization of Campylobacter jejuni isolates from patients with Guillain-Barré syndrome.

Campylobacter jejuni is a major pathogen preceding Guillain-Barré syndrome (GBS), and most C. jejuni isolates from GBS patients belong to Penner serotype 19 (heat-stable; HS-19). We analyzed sixteen independent clinical isolates from GBS patients, twelve of which belonged to HS-19, three to HS-2, and one to HS-4, using PCR-based RFLP analysis of a flagellin-A (flaA) gene. Two isolates from patients with Miller Fisher syndrome (MFS), and 27 from patients with uncomplicated enteritis were also examined. All HS-19 isolates, regardless of GBS, showed an identical pattern (Cj-1) by RFLP typing and were distinguishable from those of the other Penner serogroups. In contrast, HS-2 and HS-4 isolates were divided into several different RFLP groups, suggesting HS-19 strains are genetically distinctive among C. jejuni isolates. A DNA fingerprinting method also failed to detect any specific band pattern for GBS-related C. jejuni isolates. We examined relationships among anti-GM1 antibody titres in the sera of GBS patients, clinical forms of GBS, serotype of C. jejuni, and the presence of GM1-like structures in lipopolysaccharide (LPS) components from C. jejuni isolates by immunoblotting. HS-19 related GBS was significantly associated with elevated anti-GM1 antibody titers in the sera of the patients, but not associated with any clinical pattern of GBS. No significant correlations were found between anti-GM1 antibody and the pattern of disease, or between GBS-related C. jejuni strains and the presence of GM1-like structures. HS-19 strains seem to be unique among C. jejuni isolates, and HS-19-related GBS may provide an excellent model for clarification of the pathogenesis of GBS.

Two interchangeable forms of ciguatoxin.

Ciguatoxin, the principal toxin responsible for ciguatera, isolated from moray eel viscera, has been separated into two distinct components by alumina chromatography. A series of chromatographies showed that the two components are interchangeable. This proves that ciguatoxin is a single molecular entity. Both forms were crystallized and their 1H NMR spectra were recorded.

Three patients with ophthalmoplegia associated with Campylobacter jejuni.

Cranial polyneuropathy is idiopathic in most patients. Idiopathic cranial polyneuropathy is an acute postinfectious syndrome, along with Guillain-Barré syndrome and Miller Fisher syndrome, in which the common preceding pathogen is Campylobacter jejuni. Serum anti-GQ1b antibodies are elevated in Miller Fisher syndrome and Guillain-Barré syndrome with ophthalmoplegia. Three patients with idiopathic cranial polyneuropathy with predominant ocular involvement are presented. C. jejuni isolated from stool specimens belonged to Penner serotypes O:4, O:23, and O:33. Serum anti-GQ1b antibodies were elevated in all patients but demonstrated rapid reduction concomitant with clinical recovery. All patients recovered completely. Because both preceding C. jejuni infection and elevated anti-GQ1b antibodies decreasing with time were seen in all patients, the pathogenesis of idiopathic cranial polyneuropathy with ophthalmoplegia may be similar to that of Miller Fisher syndrome.

[Surveillance of various enteropathogenic bacteria from diarrheal cases during 1989-1999 in Kobe City].

Incidence of various enteropathogenic bacteria was examined from diarrheal faecal samples that were collected from the patients of Kobe City General Hospital and some station hospitals (23,862), and from overseas travelers (2,855) over a period of decade (1989-1999) in Kobe. A total of 1,580 strains were isolated from domestic and 331 strains from overseas travelers. The results are as follows. 1) Thirteen kinds of enteropathogenic bacteria were isolated from domestic diarrheal cases (6.6%). Salmonella was the most predominant bacteria followed by Campylobacter, Vibrio parahaemolyticus, enteropathogenic Escherichia coli and Shigella. 2) Eleven kinds of enteropathogenic bacteria were isolated from overseas diarrheal travelers (11.6%). The most frequently isolated species was Salmonella, followed by Vibrio parahaemolyticus, Shigella and Plesiomonas shigelloides. 3) Of Salmonella strains isolated from domestic and overseas diarrheal cases, serovar Enteritidis was the most predominant. Other frequent serovars in both cases were Typhimurium, Tennessee, Hadar, Infantis, Blockley and Montevideo. 4) Antibiotics resistant rate of the isolated Salmonella strains was 42.6% for domestic samples and 29.3% for overseas diarrheal cases. In domestic cases. Enteritidis was resistance to streptomycin only and the multiple antibiotic resistance was observed in Typhimurium serovars. In overseas samples, the multiple antibiotic resistance was seen in a few Typhimurium, Anatum and Blockley strains. 5) Among Shigella, S. sonnei was isolated from both domestic and overseas cases. The frequency of acquiring infection was the highest in India, followed by Indonesia, Thailand and Nepal. 6) With reference to the incidence of the members of the genus Vibrio, Aeromonas and Plesiomonas, V. parahaemolyticus were abundant from domestic samples where as V. parahaemolyticus, P. shigelloides, Vibrio cholerae non-O1 and Vibrio cholerae O1 were isolated more frequently from overseas samples. The frequency of acquiring infection was the highest in Thailand, followed by Indonesia and India.

[A case report of infant botulism without a history of honey ingestion].

A 66-day-old female with infant botulism is reported. She was admitted to our hospital with respiratory failure. Laboratory examinations detected botulinal toxin type A in her feces. This was the first case with no history of honey ingestion among the 13 cases of infant botulism reported in Japan. It is possible that other cases, in which honey had not been consumed, remain undiagnosed. Further studies may be needed to clarify the factors necessary to cause infant botulism.

[T-serotype distribution of group A hemolytic streptococci in a rapidly expanding residential area in Sanda City, Hyogo Prefecture, with reference to an outbreak of serotype T-28 in autumn, 1988].

In the fall of 1988, an outbreak of streptococcal infections was observed at 2 pediatric clinics in Sanda City, Hyogo prefecture. The 2 clinics were independent of each other; one (clinic A) was located in the down town area, in the older part of the city, where there is little population turn over, while the other (clinic B) was in a newly developed-fast growing residential district. The strains and the distribution of T-serotypes isolated at each location are as follows: clinic A; 58 strains (34.5% serotype T-4, 31.0% serotype T-12, 10.3% serotype T-1, and 23.3% serotype T-28), clinic B; 43 strains (48.8% serotype T-28, 23.3% serotype T-12, 11.6% serotype T-4 and 7.0% serotype T-1). According to the data from Kobe City infectious disease surveillance center, there were a total of 102 group A hemolytic streptococci strains isolated in 1988, the T-serotypes distribution of which was as follows: 47.0% serotype T-4, 15.7% serotype the T-12, 10.8% serotype T-1 and 9.8% serotype T-28. Serotype T-4 was dominant here, as it was in location A of Sanda City. The epidemic proportion of serotype T-28 found at location B is considered to be due to the fact that location B has very little social interaction with location A and other areas. No difference was observed among the different serotypes on the drug susceptibility test: all strains showed a sensitivity to ampicillin and penicillin G, but were resistant to tetracycline and chloramphenicol.

[A strain of Pseudomonas vesicularis isolated from shower hose which supports the multiplication of Legionella].

In Japan, a fatal case due to Legionella micdadei was first recognized in our laboratory in 1986. On the epidemiological study just after the case, no Legionella was detected from the environmental samples of the patient's residence, such as shower water, tank water and so on. In the course of prospective investigations, no Legionella was isolated, but many organisms were grown on BCYE alpha and MWY agar plates. In the retrospective study, one of these organisms was found to support satellite growth of Legionella on BCYEagar without L-cysteine. This was the isolate from the shower hose and identified as Pseudomonas vesicularis with the biochemical and DNA-DNA hybridization test. And P. vesicularis type strain ATCC11426 also supported satellite growth of Legionella. Especially in the water supply system, the existence of P. vesicularis seemed to be effective on the growth of Legionella. It must be taken into consideration that efforts made to isolate the nutrient produced organisms as well as Legionella are needed.

[The relationship between hemorrhagic gastric ulcers often appearing among in the great Hanshin-Awaji earthquake sufferers and Helicobacter pylori infections].

Hemorrhagic gastric ulcer were often found in the population who suffered from the Great Hanshin-Awaji earthquake. Gastric biopsy samples taken from 136 patients in the population were examined for Helicobacter pylori by culture and PCR methods. Furthermore, production of CagA protein from isolates was investigated. Number of positive cases with culture and PCR method were 20 (50.0%) and 26 (65.0%) in hemorrhagic ulcer, 62 (86.1%) and 65 (90.3%) in other ulcers, respectively. Number of CagA positive strains from the patients of the hemorrhagic ulcer and other ulcers were 20 (76.9%) and 21 (32.3%), respectively. The significance in both ulcers was observed (p < 0.001). From the present results, it was considered that the stress of the earthquake, the infection of H. pylori and the production of CagA protein of the organism were combined and this caused hemorrhagic gastric ulcers.

[Usefulness of Helicobacter pylori detection from feces specimens of the patients with peptic ulcer by polymerase chain reaction].

Detection of Helicobacter pylori was studied on the feces and biopsy specimens of 91 patients with gastric ulcer by using cultured and polymerase chain reaction methods. Number of samples from feces and biopsy specimens were 1 (1.1%) and 56 (61.5%) by culture method, on the other hands 49 (53.8%) and 70 (76.9%) in polymerase chain reaction method, respectively. Sensitivity of polymerase chain reaction applied to feces and biopsy specimens were 68.1 and 97.2, respectively. Noninvasive diagnosis such as detection of organisms from feces is effective for patients who have difficulty in collecting the gastric biopsy specimens. Infection route was not clarified, however, fecal-to-oral transmission was strongly suggested by the fact that the organisms were detected from feces samples in this study.

[Resuscitation from the viable but nonculturable state of Helicobacter pylori].

It is well known that the change of state from the culturable to not culturable on pathogenic organisms could easily occurred. These states of bacteria were so called viable but nonculturable (VNC). The coccoid form of H. pylori is seems to be in this state. The possibility of resuscitation of H. pylori was examined in vitro. The coccoid form bacteria was treated with Ammonium Sulfate and heat shock before culture, and cultured in Brucella broth containing, sodium pyruvate, laked human erythrocyte and serum. The growth of the rod spiral form was found in the coccoid form bacteria, and further the bacteria could grow on blood agar and Skirrow agar as well as the original strain. It was strongly suggested that the resuscitation from VNC state of cells to culturable form consists of two processes, stimulation and supplemented of appropriate nutrition.

[Sensitivity of group B Streptococcus to various antibiotics (author's transl)].

Group B Streptococcus was isolated from the clinical materials of the patients examined in the Kobe Central Municipal Hospital, in 1974 to 1979. 1. Clinical isolates were all resistant to bacitracin. 2. Serotypes of 19 isolates were type III in 12 strains, Ia in 3, Ic in 2 and untypable in 2. 3. The MICs were determined to 15 antibiotics, and the results showed that penicillin G was the best and in the order of decreasing potency, cefotaxime, ampicillin, ceftizoxime and cefoperazone. 4. Compared with group A Streptococcus, the sensitivity of group B Streptococcus to penicillin G and ampicillin was inferior by 2 to 3 tubes. 5. Based on the above results, the significance of the cephalosporin antibiotic of the third generation, particularly cefotaxime, was evaluated in the treatment of infectious diseases of the newborn infants.

[The antibacterial activity of new cephem antibiotics against clinical isolates. A comparison of the antibacterial activity of cefotaxime with 6 other antibiotics].

During the period from May through July 1981, a comparative study was carried out on the antibacterial activities of cefotaxime (CTX) and ceftizoxime (CZX), cefoperazone (CPZ), latamoxef (LMOX), cefotiam (CTM), cefmetazole (CMZ) and cefazolin (CEZ). CTX and these other cephem antibiotics were tested against fresh clinical isolates which had been obtained from clinical materials by the laboratories of 14 participating medical institutions. 1. The clinical isolates were obtained from various clinical materials in the following decreasing order: urine, sputum and pus/discharge; 85.7% of the isolates came from these materials. 2. Concerning the sources of each species of clinical isolates, it was found that P. aeruginosa was isolated from the greatest number -9- of different clinical materials. This was followed by E. coli and E. cloacae, each isolated from 8 different clinical materials, and C. freundii and E. aerogenes, each found in 7 different clinical materials. 3. In relation to S. pyogenes, S. agalactiae and S. pneumoniae, CTX showed the best antibacterial activity; the second most potent antibiotic was CZX. CMZ and LMOX were found to show relatively high MIC values for those species. Against S. aureus, CEZ showed the best antibacterial activity, but 3 resistant strains had MICs of greater than 100 micrograms/ml. 4. With regard to Gram-negative bacteria, CTX and CZX showed the best antibacterial activities for all of the species, except for P. aeruginosa. These were followed, in order, by LMOX and CPZ. Compared with these 4 antibiotics, CTM, CMZ and CEZ were found to have inferior antibacterial activities against these bacteria. In relation to P. aeruginosa, the peak of the MIC distribution for CPZ was 6.25 micrograms/ml, and this was the best antibacterial activity detected with the various antibiotics tested. This was followed by CTX (25 micrograms/ml) LMOX (25 micrograms/ml) and CZX (50 micrograms/ml). CTM had an MIC of 100 micrograms/ml for 1 strain, and MICs of greater than 100 micrograms/ml for all of the other strains of P. aeruginosa, indicating them to be resistant to this antibiotic. All of the strains were resistant to CMZ and CEZ, showing MICs of greater than 100 micrograms/ml. 5. For each of the tested antibiotics, no correlation was found between the MIC and the serogroup for either P. aeruginosa or S. marcescens.

[Present status and advances in detecting Shiga toxin-producing Escherichia coli O157].

Currently, detection of Shiga toxin-producing Escherichia coli(STEC) in stool samples is based on the isolation method in most clinical laboratories. The procedures are as follows: i) isolation with selective agar plates, ii) biological test with differential media, iii) serological test of anti-O antisera, iv) detection of toxin or toxin gene. These procedures take 4 days, therefore more rapid method is required. In the near future, a rapid detection method that detects STEC directly from stool samples will be introduced. Polymerase-chain reaction (PCR), enzyme-linked immuno-sorbent assay (ELISA), detection of serum anti-O157 antibodies are now available in clinical laboratories. Result of PCR for detection Shiga toxin gene and serum anti-O157 antibodies are described. Fifteen stool and serum samples from patients suspected of STEC infection were examined. With the culture and PCR method, 2 patients were positive by both methods and the results were confirmed in both cases. Six patients were positive by the antibodies detection method. From these results, the PCR method using stool samples was useful as a rapid detection method in clinical laboratories. Detection of serum antibodies has been simplified and is not an expensive method. Therefore, the method is useful for clinical diagnosis of STEC infection, especially, for diagnosing HUS or after antimicrobial agents have been administered to patients.

[Studies on a serum factor inhibiting lactate dehydrogenase (LDH) activity in a patient's serum].

We found extremely low activity of serum lactate dehydrogenase (LDH, EC. 1.1.1.27) in a 70-year-old female patient. The decrease of LDH activity was observed when the normal serum was incubated with the patient's serum. Inhibition rate of LDH activity by the patient's serum was higher at 4 degrees C than at 37 degrees C. The patient's serum inhibited both subunits of LDH, and inhibited more strongly the M-subunit than the H-subunit LDH isoenzymes. IgG (lambda type) in the patient's serum was found to be responsible for the inhibition of LDH activity. The mechanism why IgG inhibiting LDH activity developed in the patient's serum remain undetermined.