rpoB sequence analysis of cultured Tropheryma whippelii.

Until recently no isolate of Tropheryma whippelii was available, and therefore genetic studies were limited to those based on PCR amplification of conserved genes. In this study we determined the nucleotide sequence of rpoB (encoding the beta-subunit of RNA polymerase) from a cultured strain of T. whippelii using degenerate consensus PCR and genome walking. The T. whippelii rpoB consists of 3,657 bp with a 50.4% GC content and encodes 1,218 amino acids with a calculated molecular mass of 138 kDa. Comparison of T. whippelii RpoB with other eubacterial RpoB proteins indicated sequence similarity ranging from 57.19 (Mycoplasma pneumoniae) to 74.63% (Mycobacterium tuberculosis). Phylogenetic analysis of T. whippelii based on comparison of its RpoB sequence with sequences available for other bacteria was consistent with that previously derived from the 16S ribosomal DNA (rDNA) sequence, indicating that it belongs to the actinomyces clade. The sequence comparison allowed the design of a primer pair, TwrpoB.F and TwrpoB.R, specific for T. whippelii rpoB. When incorporated into a PCR, this primer pair allowed the detection of T. whippelii rpoB in three of three 16S rDNA PCR-positive biopsy specimens and zero of seven negative controls. rpoB could therefore be targeted in PCR-mediated detection and identification of this emerging bacterial species. This approach has previously been shown useful for the identification of related mycobacteria. This study underscores that a method involving isolation and then propagation of emerging bacteria is a useful way to quickly achieve extensive molecular knowledge of these pathogens.

16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

[Primary leiomyosarcoma of bone. Report of 5 anatomo-clinical cases and review of the literature]. / Léiomyosarcome primitif osseux. Présentation de cinq observations anatomo-cliniques et revue de la littérature.

INTRODUCTION: Leiomyosarcoma is a malignant smooth muscle tumor occurring most frequently in uterus or soft tissues and more rarely in bone. MATERIALS AND METHODS: We report the clinicopathologic, immunohistochemical and ultrastructural findings of five cases of primary leiomyosarcoma of bone treated in our Department between 1991 and 1994. The pertinent medical literature is discussed. RESULTS: The tumors were located respectively in the distal tibia (n=2), the distal femur, the sternum and the ilium (n=1). Four lesions were high-grade and one low-grade. All patients (3 women and 2 men) underwent wide surgical resection associated with polychemotherapy in four cases. Two patients died of metastatic disease, two had local recurrence and one is alive with no evidence of disease at the last follow-up. DISCUSSION: Excluding cases which involve the facial skeleton, there are to our knowledge 95 cases of primary leiomyosarcoma of bone reported in the literature. This tumor arises more commonly in adults (mean age: 49 years) with an equal gender distribution and involves predominantly the long bones near the knee. In the majority of cases, plain X-rays exhibit an osteolytic lesion with cortical penetration and indistinct margins. The diagnosis is based on microscopic features demonstrating fusiform tumor cells arranged in interwoven bundles, and the immunohistochemical results of widespread cytoplasmic positivity for smooth muscle actin. The best pronostic parameter is the histologic grade correlated with both the recurrence and metastatic rates as well as the survival rate. Surgery constitutes the main treatment since chemotherapy or radiotherapy did not provide an improved prognosis over a wide resection.

[Palmar localization of epithelioid sarcoma mimicking Dupuytren's disease]. / Localisation palmaire d'un sarcome épithélioïde simulant une maladie de Dupuytren.

A case of epithelioid sarcoma localized in the palm of the left hand in a 32-year-old woman is reported. The tumor evolved for many years, clinically and microscopically simulating palmar fibromatosis. Epithelioid sarcoma is an uncommon malignant tumor, often misdiagnosed by surgeons and pathologists. It occurs in young patients and is often localized at the upper distal extremity. Microscopically, epithelioid sarcoma shows nodules manifesting fibrous hyaline cores with central necrosis. It contains epithelioid and spindle cells immunoreactive to keratin, epithelial membrane antigen and vimentin. Recurrences, lymph node metastases and lung metastases are frequent. Surgical literature tends to recommend wide "en bloc" excision or amputation, combined or not with adjuvant radiation therapy.

[Giants schwannoma of the sacrum. Apropos of 3 cases and review of the literature]. / Schwannomes géants du sacrum. A propos de 3 cas et revue de la littérature.

Giant sacral schwannoma is a very rare tumor (25 cases reported). The authors report 3 cases of giant sacral schwannoma treated by curettage through posterior approach and discuss symptoms and treatment. These tumors were characterized by their minimal symptoms compared to radiographic findings. Magnetic resonance imaging must be performed in order to detect extraosseous tissue component and intradural invasion. A biopsy was performed to confirm the diagnosis before definitive treatment. Wide resection was proposed by many authors because of the high recurrence rate. We believe that a wide resection is too sever as it causes neurologic sacrifices. A curettage through posterior approach preserves nerve function, and if a local recurrence occurs it remains possible to perform a wide resection. When sacroiliac joint instability is detected, a lumboiliac arthrodesis is indicated. Osteosynthesis could be performed with spine device (using pedicular and iliac screws).

[A case of hyalinizing spindle cell tumor with giant rosettes in the presacral region. Immunohistochemical and ultrastructural study]. / Un cas de tumeur à cellules fusiformes hyalinisante avec rosettes géantes de la région présacrée. Etude immunohistochimique et ultrastructurale.

Hyalinizing spindle cell tumor with giant rosettes is a slowly growing tumor with a risk of local recurrence in case of incomplete surgical excision that could be regarded as a distinctive type of low-grade fibroblastic tumor. We report a case involving the presacral area. This tumor was composed of areas of bland spindle cell proliferation with a fascicular pattern in a fibrous or myxoid stroma, intermixed with giant rosettes consisting of rounded cells surrounding a central collagen core. The tumor expressed vimentin, and, for cells comprising the rosettes, S-100 protein, NSE and CD-57. These latter cells exhibited ultrastructural features of Schwann cells, the tumoral cells of fascicular area exhibiting features of fibroblastic cells. Flow cytometry showed DNA-aneuploidy and a very low S-phase fraction. This tumor appeared to be composed of two cellular components, fibroblastic and Schwann cells, located, at the opposite of neurofibroma, in two distinct areas.

PP60c-src expression in osteoclasts from osteopetrotic children and in giant tumor cells.

Malignant infantile osteopetrosis is a severe congenital disease characterized by impaired osteoclast activity. Among the multiple factors that influence bone resorption, the c-src protooncogene product pp60c-src plays an essential role, since mice which lack pp60c-src develop osteopetrosis. To gain insight into the possible role of pp60c-csrc in the pathogenesis of infantile osteopetrosis, we examined the osteoclasts of three children displaying the typical features of the disease, aged respectively one, four and seven months. pp60c-csrc expression and localization, together with the expression of a 80/85-kilodalton pp60c-src substrate, cortactin, were examined by immunoelectron microscopy. Osteoclasts from two giant cell tumors were used as controls. Bone and tumor samples were fixed in 4% paraformaldehyde, included in LR-White resin at -30 degrees C and the sections processed with mAb 327 or mAb anti p80/85 by an immunogold technique. pp60c-src was expressed in the cytoplasm, in nuclear membranes and in nuclei of the osteoclasts of the three osteopetrotic children. The subcellular localization of the kinase was not different from the localization in giant tumor cells. In both cases cortactin was abundant. In conclusion, in three children with malignant osteopetrosis, pp60c-src expression in osteoclasts does not appear to be involved in the pathogenesis of the disease. The presence of this protein, however, does not necessarily reflect normal c-src tyrosine kinase activity, nor normal c-src-dependent intracellular signaling pathways. Moreover the presence of the protein in nuclear membranes, and especially around nuclear pores supports the hypothesis that in osteoclasts, c-src may participate in the regulation of RNA processing.

[Alveolar sarcomas of soft tissues. Immunohistochemical, ultrastructural study and flow cytometry]. / Les sarcomes alveolaires des parties molles etude immunohistochimique, ultrastructurale et cytometrie en flux.

Alveolar soft tissue sarcoma is an unusual tumor, known to have a poor prognosis. Although a muscular origin has been supported by most authors, the histogenesis of such tumors remains unclear. We report a case of alveolar soft tissue sarcoma with histological, ultrastructural, immunohistochemical and flow cytometry study. Pour results support a myogenic origin of these tumors and demonstrate its aneuploid nature. Pulmonary metastasis occurred early in spite of intensive chemotherapy and surgical removal of the tumor.

Histological patterns of bone and articular tissues after orthopaedic reconstructive surgery (artificial joint implants).

Revision surgery after failures of joint replacements leads to histological studies on joint and bone tissues close to the implanted material. Aspectic loosening is the main complication. The surgical pathologist has to identify wear debris (metal, polyethylene, polymethylmethacrylate, chiefly) which promotes a histiocytic granuloma. Some surgical procedures such as cup or resurfacing arthroplasties create a new articular surface and a bone remodeling or necrosis. Cemented joint prostheses show various membrane structures between bone and the cement mantle while there is an association of bone resorption and formation. Non-cemented, porous-coated joint prostheses induce little bone ingrowth, even in satisfactory clinical results. Mechanical factors are predominant in massive limb prostheses. For silicone elastomer implants or artificial ligaments, wear of material promotes many tissular reactions. Often used bone grafts show little creeping substitution process in case of homografts, even well-incorporated on X-rays. More retrieval specimen studies are necessary to delineate precise topographical histological lesions, including non-loosened joint implants.

Polyarthritis in MRL-lpr/lpr mice: mouse type II collagen is antigenic but not arthritogenic.

In addition to a lupus-like syndrome and massive T cell proliferation, MRL-lpr/lpr(MRL/l) mice develop an arthritic process very similar serologically and histologically to human rheumatoid arthritis (RA). Recently, we have developed in DBA/1 mice an experimental model of autoimmune arthritis (EAA) which shares clinical features with RA, by injecting homologous type II collagen (CII). In order to investigate the possible relationship between the spontaneous polyarthritis of MRL/l mice and collagen induced EAA, we immunized MRL/l mice with mouse (M) CII. Our findings revealed that the injection of 100 micrograms M-CII in young or old MRL/l mice did not modify the articular pathology which spontaneously develops in non-injected mice. Circulating autoantibodies to native M-CII were found in the sera of immunized young mice but were not detected in non injected or immunized old mice. Conversely, denatured alpha 1 (II) chains or CB peptides derived from M-CII were recognized by most of the MRL/l sera whether mice had been immunized or not. The incidence of positive sera as well as the intensity of the response evaluated by Western blot analysis increased with the age of the mice. Taken together, our data suggest that, even if the injection of homologous CII in MRL/l mice may accelerate the onset of joint pathology, the spontaneous disease arises independently of an autoimmune response against native CII.

Experimental autoimmune arthritis in mice. II. Early events in the elicitation of the autoimmune phenomenon induced by homologous type II collagen.

Intradermal injection of 100 micrograms of native homologous type II collagen (CII) into DBA/1-susceptible mice induced a progressive and chronic polyarthritis. This experimental autoimmune arthritis (EAA) closely mimicked the clinical evolution of human rheumatoid arthritis (RA) except for the sex linkage. Males were highly susceptible to EAA induction even when the amount of autoantigen injected was reduced to 25 micrograms. Conversely, females remained resistant to the disease even when a booster injection of 50 micrograms was administered. With regard to age, no major difference in the incidence was observed, although younger males developed a more severe arthritis than older ones. Anti-CII autoantibodies were detected in all immunized animals, regardless of the presence or absence of joint pathology. However, in arthritic mice, the onset of the disease was associated with a predominance of IgG2a autoantibodies. Kinetic studies revealed that females as well as males exhibited early histological lesions and detectable humoral responses toward mouse CII as of the second week postimmunization. Moreover, a specific cellular autoreactivity to homologous CII occurred in different lymphoid organs with a higher intensity in females than in males. Taken together, these findings suggest that homologous CII injection induces an early subclinical arthritis that develops progressively in all immunized mice, but would be down-regulated several weeks after priming, exclusively in females.

Iron superoxide dismutase. Nucleotide sequence of the gene from Escherichia coli K12 and correlations with crystal structures.

The nucleotide sequence of the iron superoxide dismutase gene from Escherichia coli K12 has been determined. Analysis of the DNA sequence and mapping of the mRNA start reveal a unique promoter and a putative rho-independent terminator, and suggest that the Fe dismutase gene constitutes a monocistronic operon. The gene encodes a polypeptide product consisting of 192 amino acid residues with a calculated Mr of 21,111. The published N-terminal amino acid sequence of E. coli B Fe dismutase (Steinman, H. M., and Hill, R. L. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3725-3729), along with the sequences of seven other peptides reported here, was located in the primary structure deduced from the K12 E. coli gene sequence. A new molecular model for iron dismutase from E. coli, based on the DNA sequence and x-ray data for the E. coli B enzyme at 3.1 A resolution, allows detailed comparison of the structure of the iron enzyme with manganese superoxide dismutase from Thermus thermophilus HB8. The structural similarities are more extensive than indicated by earlier studies and are particularly striking in the vicinity of the metal-ligand cluster, which is surrounded by conserved aromatic residues. The combined structural and sequence information now available for a series of Mn and Fe superoxide dismutases identifies variable regions in these otherwise very similar molecules; the principal variable site occurs in a surface region between the two long helices which dominate the N-terminal domain.

[Induction of flare-ups of chronic progressive polyarthritis in mice after injection of type II homologous collagen]. / Induction d'une polyarthrite chronique évoluant par poussées chez la souris après injection de collagène de type II homologue.

Immunization by heterologous type II collagen induces in mice a sudden onset of subacute polyarthritis. In order to form a model of auto-immune pathology, we immunized DBA/1 mice with homologous type II collagen extracted from mice xiphoid process; the induced disease corresponds to a progressive chronic polyarthritis with bouts interspersed with remissions, and predominates especially in males. Type II anti-collagen auto-antibodies are found in the serum, with no correlation between the level of mice type II anti-collagen immunoglobulins G and the clinical manifestations of the disease. The histological study demonstrates signs of hyperplastic villous synovitis, with a discrete and infrequent infiltration with mononuclear cells. The model that is obtained is similar to rheumatoid polyarthritis, but also to spondylo-arthropathies.

Experimental autoimmune arthritis in mice. I. Homologous type II collagen is responsible for self-perpetuating chronic polyarthritis.

Immunisation with heterologous type II collagen (CII) induces arthritis in mice of the DBA/1 strain, which is genetically susceptible to this disease. To develop an experimental model of autoimmunity more adequate for the study of human rheumatoid arthritis (RA), DBA/1 mice were injected with 100 micrograms of native CII that had been purified from mouse xiphoid cartilage. About six weeks later the animals developed a chronic progressive polyarthritis involving the four paws but mainly confined to interphalangeal and metatarsophalangeal joints. The evolution of the disease fluctuated between remissions and exacerbations. The initial lesions assessed by clinical observations were more severe when the disease occurred early than in the case of late onset. Interestingly, the incidence of arthritis was clearly preponderant in males, and, moreover, the few female mice which developed arthritis had mild disease states with lower arthritic scores than the males. Varying levels of autoantibodies against mouse CII were found in the sera of immunised animals, regardless of the development of arthritis. These data indicate that the injection of homologous CII into mice caused a polyarthritis that is clinically closer to the human RA than the disease induced with heterologous CII and therefore will represent a useful tool for the study of the self-perpetuating mechanisms that characterise RA.

[Desmoid fibroma of the abdominal wall. Apropos of a case]. / Fibrome desmoïde de la paroi abdominale. A propos d'un cas.

A case of desmoid fibroma of anterior abdominal wall, an exceptional benign tumor, is reported. Based on results of CT scan imaging of this lesion, differential diagnosis of other expansive processes of abdominal wall are discussed. This benign tumor is a potentially serious lesion due to its infiltrating character and the high incidence of recurrence. The need for total surgical excision is emphasized.