Improved differentiation of Mycobacterium tuberculosis strains, including many Beijing genotype strains, using a new combination of variable number of tandem repeats loci.

Variable number of tandem repeats (VNTR) typing was done on 230 Mycobacterium tuberculosis strains, including 41 strains isolated from 17 groups of epidemiologically linked patients. By PCR amplification, 185 (80.4%) of the 230 strains were Beijing genotype strains. VNTR typing was performed using the 15 loci proposed as a standard set by Supply et al. [Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M.C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C., van Soolingen, D., 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 44, 4498-4510], and cluster analyses of these data were done. By the VNTR typing with the proposed 15 loci, strains having low similarity values by restriction fragment length polymorphism (RFLP) analysis were clustered. Use of a supplemental9 loci, proposed as a high-resolution tool, with the 15 loci showed that strains with low similarity by RFLP analysis were still clustered. Twelve VNTR loci were selected based on previously reported discriminatory index (DI) values and used with the proposed 15 loci for better differentiation by VNTR typing. When eight loci with higher DI values were used with the 15 loci, there were no clusters, including strains with low RFLP similarity. The15 loci and eight additional loci decreased the numbers of clustered strains isolated from epidemiologically unlinked patients significantly compared to using only the 15 loci. Among all tested loci, obvious differences of DI values were observed for 8 loci (miru10, miru16, miru39, Mtub29, Mtub30, QUB11a, QUB26, and QUB1895) of RD105 lineage strains compared to those of other lineage strains. These results suggest that the proposed VNTR typing method cannot be used as a routine epidemiological tool in areas where Beijing genotype strains are prevalent. Several VNTR loci should be added to the proposed method based on differences in polymorphism of VNTR loci among Beijing genotype lineages.

Cytokine production of RSV/PHA-stimulated tonsillar mononuclear cells: influences of age and atopy.

Links between immune responses to respiratory syncytial virus (RSV), age and atopic sensitisation are poorly understood. This study investigated the induction of target organ type-1, type-2 and proinflammatory cytokine responses to RSV and/or phytohaemagglutinin (PHA) in tonsillar mononuclear cells from children, in relation to age and atopic status. In comparison with the control medium, RSV induced production of the type-1 cytokines interferon (IFN)-gamma and interleukin (IL)-18, the pro-inflammatory cytokines IL-6, -8 and RANTES (regulated on activation, normal T-cell expressed and secreted), but not any of the type-2 cytokines IL-4, -5, -10 and -13. Induction of IL-6, -8 and RANTES, but not IFN-gamma or IL-18, were shown to be dependent on virus replication. PHA induced all except IL-12, -13, and -15. Induction of IFN-gamma, IL-6, -8, and RANTES was significantly increased in atopic children. Induction of both IFN-7 and IL-4 increased in parallel in relation to age, with no change in the IFN-gamma:IL-4 ratio. These data are compatible with the hypothesis that immature type-1 immunity during early childhood plays a role in both respiratory syncytial virus bronchiolitis and in its relationship with atopy.

Development of large pseudocysts adjacent to the knee joint in rheumatoid arthritis. Assessment of radiological and histopathological approaches.

Abstract This study examined the pathogenesis of large pseudocysts adjacent to knee joints in rheumatoid arthritis (RA). The radiological and histopathological features of 17 large subarticular pseudocysts in 12 knee joints of 10 patients were analyzed. Nine of the 10 patients were classified as class 2 according to Steinbrocker's functional class. Eight large pseudocysts were located at the lateral femoral condyle, seven were at the proximal part of the tibia, one was at the medial femoral condyle, and one was at the patella. The large pseudocysts were divided into two groups according to whether they did or did not connect with the joint cavity. Serial radiographs revealed that all large pseudocysts in communication with the joint cavity had enlarged gradually over the past several months. They extended from the subarticular area toward the bone marrow. Histopathological findings confirmed that holes allowing communication were located at a transitional zone between the ligament and the hyaline cartilage, and that rheumatoid granulation tissue invaded the large pseudocyst through these holes. The results of this study indicate that large pseudocysts are formed by the extension of articular inflammation. Moreover, repeated extrinsic mechanical stress due to walking and the aggressive inflammatory nature of rheumatoid arthritis play important roles in the formation of large pseudocysts.

Activated, cytotoxic CD8(+) T lymphocytes contribute to the pathology of asthma death.

This study investigates the presence of CD8(+) T lymphocytes and their possible association with viral infection in bronchi of victims of fatal asthma. Postmortem samples from the peribronchial region of the lung were obtained from seven patients who died an asthma death (AD), seven asthmatic patients who died of unrelated causes (AUC), and seven postmortem cases with no history of lung disease (control subjects). Using immunohistochemical techniques, the CD8(+) cytotoxic T-cell population in peribronchial tissue was characterized in three patient groups. The percentage of CD8(+) cells expressing the activation marker CD25 was higher in the AD group than in both the AUC and control groups (11.91 +/- 1.92% versus 3.93 +/- 1.63% and 1.09 +/- 0.56%, respectively (p < 0.001). Perforin expression, a marker of cytotoxicity, was highest in the AD group (9.16 +/- 1.5%) compared with 1.39 +/- 0.9; 1.8 +/- 0.6% in the AUC and control groups respectively (p < 0.001). Expression of interleukin-4 (IL-4) and interferon gamma (IFN-gamma) by CD8(+) T cells was higher in the AD group than the control group (p < 0.05). Furthermore, the IFN-gamma/IL-4 ratio in the AD group was less than half that of the control group (1.46 +/- 0.2 versus 3.2 +/- 0.1; p = 0.02). Using polymerase chain reaction (PCR), viral genome for rhinovirus (RV) was detected in lung tissue from three of the seven cases in the AD group. Two of these cases also had detectable respiratory syncytial virus (RSV). Viral genome for RSV was detected in five of the AUC group and in one of these cases, RV was also detected. No viral genome was detected in the lungs of the control group. In conclusion, this study provides novel evidence of an aberrant CD8(+) T-cell population, possibly in response to viral infection in subjects who die of acute asthma.

Meniscal tearing after ACL reconstruction.

The knees of 72 patients with unilateral anterior-cruciate- ligament (ACL) injury were analyzed before ACL reconstruction as well as by follow-up arthroscopy on the day of staple removal. At ACL reconstruction 31 lateral menisci and 40 medial menisci were found to be normal. 28 lateral menisci and 24 medial menisci were treated surgically, while 13 lateral menisci and 8 medial menisci with small or incomplete meniscal tearing were not treated. At follow-up arthroscopy there were 3 new cases of lateral meniscal tearing and 3 new cases of medial meniscal tearing in the groups diagnosed as normal prior to surgery. Two of the 13 cases with small or incomplete lateral meniscal tearing required resection, 8 healed and the other 3 demonstrated no progressive change. Four of the 8 cases with small or incomplete medial meniscal tears healed, 3 exhibited no progressive change and one required surgical treatment. There was no correlation between meniscal tearing and knee instability as indicated by a positive Lachman test or a positive pivot shift sign. The results of the present study indicate that ACL reconstruction prevents progressive changes in meniscal tears and will prevent secondary osteoarthritis, and that some small tears of the lateral meniscus require no surgical treatment.

Acute disseminated demyelination due to primary human herpesvirus-6 infection.

UNLABELLED: A previously healthy 19-month-old boy developed acute encephalopathy, thrombocytopenia and hepatic dysfunction. Human herpesvirus-6 (HHV-6) DNA was found in his CSF during the acute stage of the disease by means of the polymerase chain reaction. T2-weighted MRI revealed high signal intensity in the left thalamus and left parieto-occipital deep white matter. The myelin basic protein concentration in the CSF was elevated suggesting acute demyelination. The patient is now 2.5 years old and has no sequelae. CONCLUSION: Since clinical course and neuroimaging after HHV-6 infection are similar to those in acute disseminated encephalomyelitis, clinicians must pay attention to primary HHV-6 infection in patients under 2 years old with white matter lesions.

A case of infantile spasms: epileptic apnea as partial seizures at onset.

We report a 2-month-old boy who presented with apneic attacks as a manifestation of epileptic seizures at onset and eventually progressed to infantile spasms. At onset, at 2 months of age, apneic attacks were the sole symptom of epileptic fits. Although these seizures were accompanied by cyanosis, bradycardia was not noted. An ictal electroencephalogram showed focal paroxysmal discharges in the temporal area. Treatment with sodium valproate was not effective to control his seizures. By 6 months of age, he progressed to infantile spasms. Although his seizures could be completely controlled with the use of zonisamide, vitamin B6 or high-dose immunoglobulin, his mental and behavioral development was retarded severely. There have been no previously published cases with infantile spasms that evolved from epileptic apnea as partial seizures.

[Usefulness of endotoxin-specific limulus test for the measurement of endotoxin in cerebrospinal fluid in diagnosis of bacterial meningitis].

Using a new endotoxin-specific chromogneic limulus assay (Endoscopy test), endotoxin concentrations were measured in 93 specimens of cerebrospinal fluid (CSF) from 66 pediatric patients. Eighteen patients were diagnosed as having menigitios. Of these, 6 cases (group A) with gram-negative meningitis proven by culture had high CSF endotoxin concentrations of 115.3, (82-133) (median, range) pg/ml. Ten cases (group B) with gram-positive or aseptic meningitis had endotoxin concentrations of 2.15 (0.1-3.6) pg ml. Other 2 cases with bacterial meningitis (group C), in whom no pathogen was detected, had CSF endotoxin concentrations of more than 100 pg/ml. Four cases with encephalitis (group D) and 45 cases with non-meningitis or non- encephalitis (group E), had CSF endotoxin concentrations of less than 5 pg/ml. Despite a negative culture after antibiotic treatment in group A patients, endotoxin was cleared slowly from the CSF. A clearing of endotoxin from CSF was followed by alleviation of fever with a more gradual decline in CRP values. In 2 cases of group C, the negative bacterial culture appeared to be attributable to the previous treatment with antibiotics. However, these patients had high CSF endotoxin levels, indicating gram negative bacterial meningitis. In 17 CSF specimens from 5 patients of group A, in whom Haemophilus influenzae was detected on admission, an additional a latex agglutination test for the detection of H. influenzae polysaccharide antigen was performed. Only 3 specimens from 3 patients with CSF endotoxin concentrations of more than 80 pg/ml had a positive agglutination test. These results suggest that quantitation of endotoxin concentrations is useful for the diagnosis of gram-negative meningitis. And also, the clearance of endotoxin from CSF during treatment appears to be useful in determining the timing of when antibiotic should be stopped.

Simultaneous avulsion of patellar apexes bilaterally in a hemodialysis patient.

A case of simultaneous bilateral avulsion of the patellar apexes is reported. Plain radiography showed bilateral patella alta, but MR imaging was diagnostic. Weakness of the tendo-osseous junction due to secondary hyperparathyroidism and additional chronic inflammatory changes due to repeated microtrauma were considered to be the cause of the rupture.

[Effect of synovial extract from patients with rheumatoid arthritis on the proliferation of cultured chondrocytes].

To determine whether or not some growth factor (or factors) plays a significant role in the disease process of rheumatoid arthritis, the effects of pathological synovial extract, cultured synovial cell medium and partially purified extract and medium by fast protein liquid chromatography on cultured rabbit chondrocyte proliferation were studied. The synthesis of DNA was investigated by incorporation of 3H-thymidine into cultured cells and by flow cytometry using propidium iodide. The following results were obtained. The incorporation of 3H-thymidine into the cultured cells was increased by addition of synovial extract and the medium of cultured synovial cells. The molecular weights of active materials from the synovial extract were 13-18, 36-52, and 105-150 K daltons and those from the medium of cultured synovial cells were 13-18, 36-52 K daltons. The increase of S and G2 + M phase cells was observed flow-cytometrically 24-48 hr after addition of the synovial extract.