Avian cathepsin B cDNA: sequence and demonstration that mRNAs of two sizes are produced in cell types producing large quantities of the enzyme.

Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.

RS3PE syndrome: no evidence for retroviruses.

OBJECTIVE: To determine whether human T cell lymphotrophic virus (HTLV) infection is associated with remitting seronegative symmetrical synovitis with pitting edema (RS3PE) syndrome. METHODS: Three patients presenting with RS3PE syndrome and 7 controls were examined for the presence of HTLV crossreactive antigens. RESULTS: Our patients were elderly men who presented with typical symptoms of abrupt onset of synovitis of the wrist, carpal joints, metacarpophalangeal, proximal interphalangeal, distal interphalangeal joints, and flexor tendons, associated with remarkable pitting edema of the hands. Transmission electron microscopy and immunohistochemical analysis for HTLV-1 P19 and P20 related antigens failed to detect retroviral presence in the sample specimens or controls. CONCLUSION: Our investigation suggests there is no evidence for HTLV synovial infection associated with RS3PE.

Overexpression of zinc-finger transcription factor Z-225/Egr-1 in synoviocytes from rheumatoid arthritis patients.

In rheumatoid arthritis (RA) the proliferation of synovial lining cells appears to be one of the initial pathologic changes that contributes to the destruction of articular joints. To understand the pathomechanisms involved in these functional changes, we analyzed the transcriptional regulation of the zinc-finger gene 225 (Z-225/Egr-1), a transcription factor expressed in the immediate early events of cellular activation. We found that Z-225 transcripts were significantly up-regulated in RA synoviocytes. In primary and long term culture Z-225 was spontaneously transcribed at elevated levels. In situ hybridization of zinc-finger probe showed characteristic Z-225 transcripts in RA synovial tissues. Identity of these signals to the Z-225 gene product were confirmed in freshly isolated synovial tissue by enzymatic amplification of cDNA by the PCR technique. Z-225 transcripts were also detected and characterized in a cDNA library established from a RA synovial explant. We therefore conclude that RA synoviocytes spontaneously produce Z-225 gene products at elevated levels. Because early growth response gene Z-225 is involved in the regulation of expression of other genes such as proto-oncogenes c-ras and c-sis, which are also up-regulated in the RA synovium, activation of Z-225 transcription in RA may represent a key event in articular joint destruction.

A method for the quantification of the size of liver metastases.

A method for the quantification of the size of liver metastases based on stereologic principles is presented. This evaluation procedure was applied retrospectively to routine computerized tomography (CT) scans of the liver and allowed reliable estimation of the volume of liver metastases. Furthermore, the data were used to create three-dimensional (3D) representations of the examined organ by computer reconstruction.

Virus-like particles in synovial fluids from patients with rheumatoid arthritis.

Based on the elevated expression of oncogenes in proliferating transformed-appearing synoviocytes we searched for the possible involvement of a viral agent in the pathogenesis of RA. We report the detection of virus-like particles with retroviral C type morphology in SF, which lack the typical morphologic as well as immunohistochemical features of the human T-lymphotropic and immunodeficiency viruses.

Detection of p24 in HIV-1 infected cells embedded in LR White and Lowicryl K4M.

In this study we present a postembedding on-grid immunogold labelling procedure for the ultrastructural localization of the HIV-1 core protein p24. HIV-1 infected cells were fixed in 0.1% glutaraldehyde, incompletely dehydrated and embedded in LR White or in Lowicryl K4M. Antigenic sites were detected by incubation of ultrathin sections with primary mouse monoclonal antibody anti-HIV-1 p24, followed by the secondary antibody goat anti-mouse IgG coupled to 10nm gold particles. Antigenicity of p24 was found to withstand the applied fixation and was shown to be preserved in LR White as well as in Lowicryl. The described procedure permits the uncomplicated and easy detection of p24 in HIV-1 infected cells and tissues.

Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling.

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.

Collagen dysplasia in idiopathic carpal tunnel syndrome.

Surgical biopsies of dissected transverse carpal ligaments of patients with idiopathic carpal tunnel syndrome were examined with an electron microscope revealing collagen fibrils with extremely varying diameters. Morphometric analysis was performed on electron micrographs exhibiting fibrils with a small diameter comparable to that in control tissue as well as fibrils with a far larger diameter than could be observed in control tissue. Morphometric parameters were evaluated in order to analyse the relation between the number of and the area covered by collagen fibrils in the electron micrographs. In control tissue the numerical density per image area was twice the numerical density in carpal tunnel syndrome. However, the area fraction of the electron micrographs occupied by collagen fibrils in carpal tunnel syndrome and controls were equal.

Ultrastructural and autoradiographic investigations of cell cultures derived from tendons or ligamentous material from patients with fibromatous disorders.

Cell cultures were derived from tendons or ligamentous material from patients with carpal tunnel syndrome (CTS), Dupuytren's contracture (DP), tendopathia nodosa (TN) and hallux valgus (HV). The ultrastructure of the operation specimens as well as of the cell monolayers was investigated, using a floating sheet method in order to preserve both cell-to-cell contacts and the orientation of the monolayers. The histologic features of the tissues obtained in the operations were correlated with the ultrastructure of the cells in culture derived from these specimens. In DP, above all in the nodules, an activation of the capillary endothelium in the vicinity of myofibroblasts and mast cells was observed. In CTS the collagen fibrils varied extremely in diameter. In DP and TN biopsies a splicing process of helicoidly arranged fibrils could be seen. A disintegration of elastic fibers in the fibrillar and amorphous components was found in DP nodules, HV and TN tissues. Transitional forms between fibroblasts and myofibroblasts were observed not only in DP but also-though in a smaller percentage--in the cultures derived from the other patients. The cells showed organelles for active protein synthesis and transport. Autophagocytosis and the formation of multilamellated bodies took place in TN and HV cultures. In CTS, DP and TN cultures cells were connected via gap junctions. In some cultures, above all in those derived from CTS, monocilia were found. In CTS cultures the formation of intracellular collagen occurred. Growth parameters were rather low in HV cultures. PLmax (maximal pulse labelling index) values were higher in TN cultures than in DP and HV cultures. Plating efficiency (PE) values were higher in cultures derived from cell-rich and capillarized tissues than in biopsies with few cells.

Morphometric analysis of collagen fibrils in idiopathic carpal tunnel syndrome: Part 2.

Morphometric parameters were evaluated in order to analyze the relation between number and covered area of collagen fibrils in normal and carpal tunnel syndrome tissue. This analysis revealed that in normal tissue twice as many collagen fibrils as in pathological tissue occupy an equal area. Taking these facts into account, some hypotheses are advanced.

Morphometric analysis of collagen fibrils in idiopathic carpal tunnel syndrome.

Surgical biopsies of dissected transverse carpal ligaments of patients with idiopathic carpal tunnel syndrome were examined with an electron microscope revealing collagen fibrils with varying diameters. Morphometric analysis of transversely cut collagen fibrils was performed on photomicrographs exhibiting fibrils with a small diameter comparable to that in normal tissue as well as fibrils with a large diameter that could not be observed in normal tissue.

Time course of alterations in muscle transfers with microneurovascular anastomoses. An experimental study in the rectus femoris muscle of the rabbit.

The time course of alterations in muscle transfers with microneurovascular anastomoses was studied in 17 rabbits. The left rectus femoris muscle was transferred to the right side. For comparison, in some animals the right rectus femoris muscle was transferred from the right to the left side, but without vascular repair. Two, seven, 14, 21, and 30 days after transfer, the electric excitability, macroscopic appearance, histology, histochemistry, ultrastructure, and activity of muscle enzymes were assessed. In the transfers with microneurovascular anastomoses, almost all muscle fibers survived. Alterations were limited to those typical of a denervation-reinnervation process. Contrary to this, only a few atrophic fibers survived in the periphery of the transfers without vascular repair. By far, the greater central part underwent necrosis. A considerable amount of connective tissue developed. These results clearly show the functional superiority of microsurgically vascularized muscle transfers over those without vascular anastomoses. Excellent functional recovery is possible because the process of complete degeneration and consecutive regeneration, with inevitable augmentation of connective tissue, is prevented by the performance of vascular anastomoses.