Loss of the ex vivo but not the reinducible CD8+ T-cell response to Tax in human T-cell leukemia virus type 1-infected patients with adult T-cell leukemia/lymphoma.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). In asymptomatic carriers and HAM patients, HTLV-1 infection leads to a vigorous cytotoxic T-cell (CTL) response mainly directed to the regulatory Tax protein. In contrast, initial studies showed that anti-HTLV-1 CTL activities were not reproductively detected in ATLL patients, neither ex vivo, nor after in vitro restimulation. To better understand this discrepancy, we explored the anti-HTLV-1 CD8+ T-cell response of eight ATLL patients by using in vitro restimulated or freshly isolated CD8+ T cells. In all the ATLL patients, we found that mitogenic activation allowed the induction of CD8+ T cells able to lyse autologous HTLV-1-infected cells and/or to produce IFNgamma in response to Tax peptides. In contrast, only a minority of the patients possessed CD8+ cells able to respond ex vivo to the same epitopes. These findings indicate that although a restimulatable anti-HTLV-1 CTL activity persists during ATLL, the specific ex vivo response is not constantly maintained. This provides definitive evidence that the CD8+ T-cell response to HTLV-1 is affected by ATLL development and reveals that a major defect concerns the generation and/or the functionality of CD8+ effectors.

High frequency ultrasound in the preoperative staging of primary melanoma: a statistical analysis.

High frequency sonography has been shown to be a useful tool in the preoperative staging of malignant melanoma. In the present study sonometric and histometric data concerning tumour thickness were compared, using appropriate statistical methods, in order to assess the accuracy of ultrasonography. From December 1997 all pigmented lesions suspected of being melanoma were preoperatively assessed by a 20 MHz ultrasound B scan. The results of these ultrasound examinations were compared with histometric data. Pearson's correlation coefficient and absolute and relative differences were used for statistical analysis. Of the 261 examined lesions, 193 were malignant melanoma. A high correlation between sonometry and histometry was computed (r = 0.95), with an absolute difference of 0.32 +/- 0.03 mm (mean +/- SEM) and a mean relative difference of 27.2% (95% confidence interval 23-31.4%). The highest correlation was found in melanoma > or = 1.51 mm thick and the lowest correlation in melanoma < or = 0.75 mm. In conclusion, the high accuracy of this technique in the preoperative staging of malignant melanoma would offer a basis for defining the surgical margins of > or = 0.76 mm thick lesions. The limited accuracy of sonometry in the preoperative staging of thin melanoma < or = 0.75 mm has emerged by applying adequate statistical methods.

Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979 to 2000.

The molecular fingerprints of 1,349 isolates of Mycobacterium bovis received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain Mycobacterium bovis BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of M. bovis across national borders.

Mycobacterium avium and Mycobacterium intracellulare infection in mammals.

Mycobacterium avium subsp. avium and M. intracellulare are ubiquitous organisms in the environment. The reservoir of M. avium subsp. avium is generally accepted to be environmental, in particular, water and soil are sources of the organism. In contrast to M. avium infection in wild and domestic birds, M. avium infection in mammals occurs only sporadically and is rarely transmissible. Generalised disease is usually uncommon, owing to the non-progressive, chronic character of the infection. However, some cases of disseminated disease have been reported, e.g. in captive non-domestic hoofed animals as well as in immunosuppressed dogs and cats. The majority of M. avium and M. intracellulare infections in livestock are detected at slaughter and the diagnosis is confirmed by bacteriological procedures. Condemnation of affected portions of the carcass can result in significant economic losses, although gross lesions are mostly restricted to lymph nodes close to the alimentary tract. Successful treatment with antibiotics in combination with surgery has been reported in some affected domestic cats, but is not considered to be effective or economical in other species. In the past, differentiation of M. avium bacteria from the closely related M. avium subsp. paratuberculosis was based on the mycobactin dependence and prolonged incubation period of the latter. More recently, amplification of the genomic insertion sequence IS900 has proved to be a powerful tool for identification of M. avium subsp. paratuberculosis. The potential zoonotic importance of M. avium infections has been indicated, but requires clarification.

Molecular typing of Mycobacterium bovis isolates from Cameroon.

In order to gain a better understanding of the molecular epidemiology of Mycobacterium bovis isolates in Cameroon, 75 isolates of M. bovis collected in three provinces of northern Cameroon were studied by spoligotyping. For 65 of these isolates, typing was also carried out by pulsed-field gel electrophoresis (PFGE) with DraI, and 18 of the isolates were also typed by restriction fragment length polymorphism (RFLP) analysis with probe IS6110-RHS. Molecular typing of the isolates by these techniques revealed a high degree of homogeneity, with 10 spoligotypes for 75 isolates, four PFGE profiles for 65 isolates, and three RFLP types for 18 isolates. Some types were present in the three different provinces, while some were confined to one or two areas. These results suggest that geographical mapping of M. bovis strains could be helpful for the control of bovine tuberculosis at the regional level. An interesting feature of all the spoligotypes was the absence of spacer 30, suggesting a common origin for all of the Cameroon isolates tested; an evolutionary scenario for the isolates is discussed. In addition, a comparison of the three techniques showed that for M. bovis strain differentiation in Cameroon and in surrounding countries, spoligotyping would be a more discriminating and practical tool for molecular typing than the other two techniques used in this study.

Molecular characterization of environmental mycobacterium strains by PCR-restriction fragment length polymorphism of hsp65 and by sequencing of hsp65, and of 16S and ITS1 rDNA.

Fifteen mycobacterial strains from the environment, not clearly identifiable by biochemical properties, were analyzed with molecular markers: PCR-restriction enzyme analysis of hsp65 and sequencing of hsp65, and of the internal transcribed spacer 1 (ITS1) and 16S rDNA. The 16S rDNA sequencing closely related the strains to a slow-growing mycobacterial group including Mycobacterium simiae, Mycobacterium lentiflavum, Mycobacterium genavense, Mycobacterium triplex and Mycobacterium heidelbergense. A stretch of T bases at the level of 16S rDNA enabled the separation of M. simiae and M. lentifiavum from M. genavense, M. triplex and M. heidelbergense; hence the attribution of some environmental strains to the first or second group. But the distances between the two clades were very short and the relative positions of environmental strains and of reference strains were not resolved in terms of node robustness (low bootstrap values) in the distance tree. However, the hsp65 restriction profiles suggested assigning six strains to the M. lentiflavum species, although these strains had been found closely related to M. genavense and M. triplex from 16S rDNA nucleotide signatures. The clustering of environmental strains into the same three clusters was deduced from analysis of three sequence data (hsp65, and ITS1 and 16S rDNA), but the taxonomic affiliation of environmental strains to reference strains remained tentative. Among environmental strains and reference strains, the distances found from hsp65 sequences had the same amplitude as those found between different strains of Mycobacterium gordonae. From ITS1 rDNA sequences, the distances found between the strains of the Mycobacterium avium complex also had the same amplitude as those found between environmental strains and reference strains. From our results, it appears that the environmental strains and the reference strains could constitute a complex of subspecies or closely related species. Their taxonomic status must be confirmed by DNA/DNA hybridization experiments.

Toxicity and activity of docetaxel in anthracycline-pretreated breast cancer patients: a phase II study.

Docetaxel has proven effective in advanced breast cancer. Myelosuppression and cumulative fluid retention syndrome are troublesome, potentially avoidable toxicities. In this consecutive cohort study, docetaxel (100 mg/m2 by 1 hour i.v. infusion, q3 weeks) activity and toxicity was explored in 56 anthracycline-pretreated patients (eligible: 55: median age: 51 years [range: 28-68 years]; median performance status: 0 [range: 0-3]) with metastatic breast cancer, using two different granulocyte colony-stimulating factor and steroid pre- and postmedication schedules. Twenty-nine patients (group A) received a 5-day oral prednisone premedication, and 26 (group B) received 4-day low-dose i.m. dexamethasone; group B patients also received prophylactic granulocyte colony-stimulating factor. All patients were evaluable for toxicity and 53 for response. Prophylactic granulocyte colony-stimulating factor significantly lowered the incidence of grade III-IV neutropenia and neutropenic fever (p = 0.0001 and 0.01, respectively). The incidence of moderate-severe fluid retention syndrome was lower in patients receiving i.m. dexamethasone (p = 0.08). Overall response rate was 53% (4 complete responses/24 partial responses, 95% confidence interval 39.4-66.2%); 32% have stable disease and 15% progressive disease. In 21 anthracycline-refractory/resistant patients, as well as in 10 paclitaxel-pretreated patients, the overall response rate was 50%. Docetaxel is highly active in anthracycline- and paclitaxel-pretreated metastatic breast cancer, with manageable toxicity. Optimal use of both granulocyte colony-stimulating factor support and steroid premedication deserves further investigation.

[Plantar fibromatosis: ultrasonography results]. / La fibromatosi plantare: risultati ecografici.

INTRODUCTION: Plantar fibromatosis, or Ledderhose's disease, is a fibrous proliferation arising within the plantar fascia end exhibiting typical clinical nodular features. The lesion appears as a firm single thickening or a nodule, occasionally painful, localized to the medial portion of the sole of the foot. This unusual condition has not been extensively studied with US and very little has been written about it. MATERIAL AND METHODS: In 1998-99, six patients with plantar fibromatosis were submitted to US with 13 MHz linear array and 20 MHz mechanical annular array probes. All patients were examined in prone recumbency with the probe positioned on the sole of the foot. Only some of them were subsequently submitted to surgery. RESULTS: Plantar fibromatosis exhibited an almost pathognomonic pattern and US proved to be a quick, noninvasive and cost-effective technique to confirm clinical diagnosis. The nodule is typically single and isoechoic, with maximum diameter of about 1 cm, inhomogeneous internal structure and few thin hyperechoic septa. The nodular fibrous proliferation adheres with the major axis along the plantar fascia; it exhibits clear-cut margins and US beam transmission is good. No calcifications or fluid collections are seen within the nodule. Color and power Doppler show no flow inside. CONCLUSIONS: US is an adequate tool for the study of plantar fibromatosis.

Isolation of Mycobacterium bovis from baboons, leopards and a sea-lion.

This study reports on two series of cases of Mycobacterium bovis infection in zoo animals. The first was in a captive population of baboons (Papio hamadryas) and the second in a mixed group of wild mammals, including four leopards (Panthera uncia and Panthera pardus) and a sea-lion (Otaria byrona). The isolation and identification of strains of M. bovis confirmed the presence of M. bovis infections in both zoos. The epidemiological study using genetic markers such as the IS6110-based DNA fingerprinting system made it possible to differentiate between M. bovis strains. The M. bovis strains isolated from baboons were shown to contain a single IS6110 copy, as usually do cattle isolates, whereas the M. bovis strains isolated from the other exotic animals presented multiple copies. This finding suggests that the origin of the contamination for the baboons in zoo A could be related to cattle. The origin of the contamination for the leopards and sea-lion in zoo B is more difficult to determine. In conclusion, the authors suggest some recommendations for avoiding outbreaks of tuberculosis infections in zoos.

[Role of high-definition and high frequency ultrasonography in determining tumor thickness in cutaneous malignant melanoma]. / Ruolo dell'ecografia con alta definizione ed elevata frequenza nella determinazione dello spessore tumorale del melanoma maligno cutaneo.

PURPOSE: We investigated the predictive capabilities of high frequency, high resolution ultrasound (US) in the preoperative assessment of tumor thickness in cutaneous malignant melanomas. This evaluation is a valuable help for correct surgical planning. MATERIAL AND METHODS: December 1997 to August 1998, we studied with US over 120 pigmented skin lesions and collected the data, including the final histologic diagnosis, of 78 of them, which make our series. Sixty-six of 78 lesions were histologically defined as cutaneous malignant melanomas with superficial spread and the other 12 as melanocytic nevi. All examinations were carried out with an Esaotebiomedica AU 5 Harmonic scanner equipped with a 20-MHz annular array probe and a linear 13-MHz probe; B-mode, color and power Doppler images were always acquired. RESULTS: The statistical analysis of the comparative US and histologic measurements of tumor thickness showed very good agreement, with a high Pearson's coefficient (R: .93). However, US frequently underestimated the actual thickness relative to histology, which is in contradiction with previous literature reports. We could study with US the melanoma in situ, which has never been described before. Last but not least, low-thickness melanomas had very few color and power Doppler signals. CONCLUSIONS: US is the correct tool for the preoperative assessment of the thickness of cutaneous malignant melanomas because it provides high agreement with histologic data even in lesions thinner than 1 mm.

Mycobacterium avium infections in animals. Literature review.

Mycobacterium avium causes tuberculosis in chickens and other fowls but can also infect an extensive range of different animal species. The authors reviewed the available literature on this organism to show the importance of M avium infection.

[Pathogenic organisms in milk and milk products: the situation in France and in Europe]. / Les germes pathogènes dans le lait et les produits laitiers: situation en France et en Europe.

Milk and dairy products harbour a natural microbial flora and/or other micro-organisms, which vary within the wide range of products available on the French market. The origin of contamination by pathogenic bacteria varies with the type of product and the mode of production and processing. Contamination of milk and dairy products by pathogenic micro-organisms can be of endogenous origin, following excretion from the udder of an infected animal. Contamination may also be of exogenous origin, through direct contact with infected herds or through the environment (e.g. water, personnel). Treatment and processing of milk can inhibit or encourage the multiplication of micro-organisms. The authors describe the relevant aspects of bacterial physiology and ecology, the occurrence of bacteria in dairy products, and the public health significance for each of the principal micro-organisms found in such products. Bacteria most frequently involved are mycobacteria, Brucella sp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella). At present, systems of testing and surveillance are required for the control of pathogenic bacteria in milk and dairy products, as specified by regulations currently being developed for all countries in the European Union. Preventive measures should take into account the well-established facts concerning the potential microbiological impact of pathogenic bacteria on milk and dairy products. There should be increased recourse to risk analysis methods to assess the threat to the consumer with regard to the presence of pathogenic bacteria in food.

Glycolipid antigen for use in diagnostic assays for bovine tuberculosis.

A glycolipid antigen, was isolated, purified and characterized from Mycobacterium bovis An5. Chemical analysis (thin-layer chromatography, nuclear magnetic resonance and infrared spectra) showed that this glycolipid was a 2,3-di-O-acyl trehalose (DAT), similar to the DAT of M. tuberculosis. This antigen was used to establish ELISA-based serodiagnostic tests for M. bovis-infected cattle. The sensitivity and specificity of the assay were investigated using sera of cattle from tuberculosis-free herds and from tuberculosis-infected herds. No correlation was found between DAT-ELISA and the skin test, nor between DAT-ELISA and interferon-gamma with bovine purified protein derivative. The antibody titres were not related to cell-mediated immunity. Although the antigen was highly specific (95.9%), the sensitivity of DAT-ELISA, as judged from assays in bacteriologically confirmed tuberculosis, was low (29 to 36.8%). The low sensitivity of ELISA might also be attributed to a reciprocal relationship between B-cell proliferation and T-cell protective immunity.

[Use of the in vitro enzymatic amplification method for the detection of Mycobacterium paratuberculosis in feces]. / Utilisation de la méthode d'amplification enzymatique in vitro pour la détection de Mycobacterium paratuberculosis dans les fèces.

A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.

Isolation and characterization of serologically reactive lipooligosaccharides from Mycobacterium tuberculosis.

Two major highly polar antigenic glycolipids were isolated from recent isolates of Mycobacterium tuberculosis from a wide range of geographical origins. The occurrence of these polar glycolipids was demonstrated by isolation, purification and chromatographic characterization and/or serological procedures in 12 strains. Based on their chromatographic properties, these polar glycolipids belong to the lipooligosaccharide family. Preliminary data on the use of these newly described antigens in the serodiagnosis of tuberculosis is presented.

[Experimental paratuberculosis in sheep after intravenous or oral inoculation: pathogenicity and biologic diagnosis]. / Paratuberculose expérimentale chez le mouton après inoculation par voie veineuse ou par voie orale: pouvoir pathogène et diagnostic biologique.

An experimental paratuberculosis study was performed in sheep. One group of 6 lambs was inoculated intravenously with the equivalent of 50 mg (wet weight) of live bacilli, another group of 6 lambs was inoculated orally by placing 500 mg (wet weight) of live organisms in milk feed and a group of 3 lambs was used as controls. The degree of cellular immunity was followed by examining delayed hypersensitivity using 3 allergens (bovine tuberculin PPD, avian tuberculin PPD and johnine PPD) and that of humoral immunity using complement fixation test, agar gel immunodiffusion test and ELISA. The elimination of bacilli in the faeces was examined simultaneously. After 2 years no macroscopic or microscopic lesion was observed in intravenously inoculated lambs and in those exposed orally to M paratuberculosis; cultures were negative. It appears that domestic sheep were able to control the infection. Nevertheless, most of them developed cellular and humoral immunity against paratuberculosis antigen. The best results were obtained in intravenously inoculated lambs.