[Rupture of lateral ligaments of the ankle joint: MR imaging before and after functional therapy]. / Aussenbandrupturen des Sprunggelenkes--Darstellung mit der MRT vor und nach funktioneller Therapie.

PURPOSE: Documentation via MRI of the healing of ruptured lateral collateral ankle ligaments after functional therapy. PATIENTS AND METHODS: 35 patients with ankle sprain were examined by MRI and stress radiographs, 13 were operated afterwards, 22 patients underwent a functional conservative therapy and were examined by MRI and stress radiographs and second time after three months. RESULTS: MRI reports were correct in 12 of 13 operated cases. After conservative therapy we did not find any disrupted ankle ligament. MRI showed intact ligaments thickened by scar. CONCLUSION: MRI is able to show injuries of the lateral collateral ankle ligaments and demonstrates the healing by scar after conservative therapy.

The effect of treating with anti-interleukin-1 receptor antibody on the course of experimental murine cutaneous leishmaniasis.

To assess the role of interleukin-1 (IL-1) in cutaneous leishmaniasis, Leishmania major-infected mice were treated with an anti-IL-1 receptor monoclonal antibody, LA-15.6. MoAb LA-15.6 prevents binding of IL-1 to both the T cell and B cell/macrophage forms of the IL-1 receptor. We found that treating with LA 15.6 inhibited the development of cutaneous lesions of L. major in both genetically-susceptible and resistant mice. Interestingly, this treatment had little or no effect on parasite numbers in the lesions or on the cytokines (interferon-gamma, interleukin-4) that the animals produced in response to infection with the parasite. These results suggest that although IL-1 plays a detrimental role in cutaneous leishmaniasis, it does not mediate this effect by altering the parasite-specific T cell response.

The detection of proline isomerase activity in FK506-binding protein by two-dimensional 1H NMR exchange spectroscopy.

1H NMR assignments of the trans and cis isomers of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were accomplished by two-dimensional NMR techniques. Conformational exchange between the cis and trans isomers was not detected in the two-dimensional exchange spectra (NOESY) until catalytic amounts of FK506-binding protein (FKbp) were added. The addition of FK506 to the enzyme-substrate solution inhibited the enzyme and removed the substrate exchange peaks.

Endotoxin-macrophage interaction: post-translational regulation of tumor necrosis factor expression.

Thioglycollate-elicited murine peritoneal macrophages produce significant quantities of TNF 2 to 4 h after induction with bacterial endotoxin, LPS. However, macrophages exposed to a second LPS stimulus are refractory and the amount of TNF detected in these supernatants is reduced 10- to 50-fold. The acquisition of the refractory state in vitro or in vivo requires the continued presence of LPS for a minimum of 6 to 8 h, is optimal by 20 h, and is reversible. Refractory macrophages incubated for an additional 48 h in the absence of LPS produce significant quantities of TNF after reexposure to endotoxin. Although LPS refractory macrophages do not release TNF in response to a secondary endotoxin challenge, riboprobe ribonuclease protection assays demonstrated amplification of TNF message, suggesting that post-transcriptional events are involved in the regulation of TNF production in endotoxin refractory macrophages. Immunoprecipitation studies revealed the accumulation of the 26-kDa TNF precursor in lysates of refractory macrophages, thus demonstrating a post-translational regulatory process. Although LPS refractory macrophages do not release TNF in response to a second LPS stimulus, ingestion of zymosan by these cells results in the release of significant quantities of TNF. Furthermore, LPS-refractory macrophages do not demonstrate a reduction in other effector functions including Fc-mediated erythrophagocytosis. Therefore, the LPS refractory state is a metabolically dependent post-translational regulatory process, which requires continuous LPS exposure, is specific in which macrophage effector functions are inhibited, and is reversible with further incubation or by non-LPS-related stimuli.

A new murine CD4+ T cell subset with an unrestricted cytokine profile.

CD4+ T cell clones were derived from mice immunized to keyhole limpet hemocyanin to characterize the cytokine profiles of newly isolated clones. Surprisingly, several of the clones had an unrestricted profile, producing IL-2, IL-3, IL-4, IFN-gamma, and TNF after either Con A or Ag stimulation. The coproduction of IL-2 and IL-4 was confirmed at the mRNA level. Subclones were derived which contained RNA transcripts for, as well as secreted, both IL-2 and IL-4 thus confirming the clonality of the original T cell clones. CD4+ T cell clones that expressed an unrestricted cytokine profile upon Con A stimulation were also isolated from mice immunized to other Ag (hen egg lysozyme, OVA, or type II collagen). These data indicate that CD4+ T cell clones newly isolated from immunized mice do not necessarily segregate into the Th1 and Th2 subsets. We propose this new murine CD4+ cell subset with an unrestricted pattern of cytokine production be called Th0.

Murine B lymphoma cell lines release functionally active interleukin 2 after stimulation with Staphylococcus aureus.

In this study it is shown that an IL-2-like functional lymphokine activity derived from the murine B cell lines 2PK-3 and L10A2J after stimulation with Staphylococcus aureus can be blocked with anti-IL-2 mAb such as 9B11, DMS-1, and S4B6. Experiments demonstrate that the stimulation of the IL-2-sensitive cell line CTLL-2 by the IL-2-like functional activity derived from murine B cell tumors can also be blocked with the anti-IL-2R mAb PC61. Additionally, in RNA-RNA hybridization experiments with radiolabeled SP6-derived ssRNA probes developed from murine IL-2 genomic DNA sequences and specific for IL-2 mRNA, quantitatively significant amounts of IL-2-specific mRNA in both 2PK-3 and L10A2J are shown subsequent to stimulating the cells with S. aureus. These results suggest the murine B cell tumor lines 2PK-3 and L10A2J synthesize and release IL-2 after stimulation with selected polyclonal activators such as S. aureus.

Quantitative molecular hybridization with unfractionated, solubilized cells using RNA probes and polyacrylamide gel electrophoresis.

Molecular hybridization with RNA probes was performed on unfractionated cells solubilized in guanidine thiocyanate solutions. Unhybridized probe was digested with ribonuclease, and protected probe fragments were resolved by polyacrylamide gel electrophoresis (PAGE). Since the same medium was used both for solubilization of the cells and as the hybridization buffer, RNA purification was not required and the analysis of large numbers of samples was facilitated. Using this method, specificity is superior to dot blot analysis because the size of hybridized fragments is determined and the signal of the probe hybridized to target RNA is separated from the background by PAGE.

Mouse lymphotoxin and tumor necrosis factor: structural analysis of the cloned genes, physical linkage, and chromosomal position.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are cytotoxic and immunoregulatory lymphokines which have similar activities but are produced by different cell types. We have cloned the murine LT and TNF genes from a lambda:mouse DNA recombinant library, using as probes synthetic oligonucleotides defined by portions of the human LT or TNF cDNA sequences. Analysis of the genomic clones indicates that the LT and TNF genes are physically linked, i.e., approximately 1.2 kb separates the 3' end of LT from the 5' end of TNF genes. By using, first, a series of recombinant inbred lines, and second, a series of H-2-recombinant congenic strains, we determined that the LT/TNF gene cluster lies on chromosome 17, closely linked to the H-2D end of the murine H-2 complex. Comparison of the primary sequence of murine and human LT revealed that the intron-exon structure of murine LT is similar in these two species. Comparison of the predicted amino acid sequences of murine and human LT indicates that the proteins are about 72% homologous with much greater sequence conservation in regions encoding the COOH-terminal portion. Comparison of the 5' flanking sequence of LT to a number of genes that are specifically expressed in activated T cells reveals a number of conserved sequences that may play a role in control of these genes.

Recognition of exon-shuffled class II molecules by T helper cells.

Exon shuffled I-A beta genes transfected into the B lymphoma cell line A20-2J were used to localize the epitope recognized by the monoclonal antibody 10.2.16 to the carboxy terminal portion of the beta 1 domain. In addition, several T helper cell hybrids were tested against these novel I-A molecules and the following observations were made: the beta 1 domain of A beta plays a dominant role in the restricted recognition by T helper cells; there appear to be multiple restriction epitopes on the I-A molecule; these epitopes can consist of conformational epitopes created by specific alpha and beta chains or consist of the polymorphic determinants encoded on the beta chain alone, and these novel I-A molecules serve as restriction elements in the antigen-specific recognition by T cells and in one case stimulate an alloreaction in the absence of antigen.

The role of DNA rearrangement and alternative RNA processing in the expression of immunoglobulin delta genes.

We have established the exon-intron structure of the gene coding for the constant (C) region of the mouse immunoglobulin delta heavy chain, using DNA clones isolated from BALB/c embryos and the delta mRNA extracted from two delta-producing hybridomas, B1-8. delta 1 and GCL2.8. At least three types of C delta gene structures are identified. A 2.7 kb delta mRNA reveals six exons. This delta mRNA may code for a membrane-bound delta chain. A second delta mRNA of 1.8 kb shares the first (5' side relative to direction of transcription) three exons with the 2.7 kb delta mRNA and in addition contains a fourth exon unique to this mRNA species. This delta mRNA most likely codes for a secreted delta chain. A third delta mRNA, also of 1.8 kb, shares the first four exons and a part of the fifth exon with the 2.7 kb mRNA. Its function, if any, remains unclear. We investigated the question of how a lymphocyte can produce the mu and delta heavy chains simultaneously, using the hybridoma GCL 2.8, which makes both IgM and IgD. Results of Southern gel blot analysis and gene cloning experiments indicate that this cell utilizes the same rearranged VH gene for the synthesis of the mu and delta chains, and yet maintains the embryonic configuration for the C mu and C delta genes and for the intervening region. Based on these results, we conclude that the VH sequence is spliced alternatively to the C mu or C delta sequence during processing of the primary RNA transcript. An alternative mechanism for the expression of the delta gene is found in hybridoma B1-8. delta 1, which actively secretes delta chains and synthesizes no mu chain. This mechanism involves deletion of the C mu gene, which brings the complete VH gene closer to the C delta gene.

Linkage of the four gamma subclass heavy chain genes.

The genes for the heavy-chain constant regions of the four gamma subclass immunoglobulins were identified in a set of overlapping mouse DNA fragments representing about 100 kilobase pairs (kb) of the mouse genome that was cloned from bacteriophage lambda libraries of BALB/c mouse embryo DNA. R-loop mapping studies show that the genes are located 5'-C gamma 3-34 kb-C gamma 1-21 kb-C gamma 2b-15 kb-C gamma 2a-3' and lie in the same transcriptional orientation. Two DNA segments, one of 19 kb and another of 15 kb, that surround the C gamma 2b and C gamma 2a genes, respectively, show considerable homology and implicate a tandem duplication mechanism in the evolution of this gene cluster.

Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes.

At least two types of somatic recombination are necessary for the generation of a complete immunoglobulin gamma 2b gene from germ-line DNA sequences. The first type of recombination consists of the assembly of three separate DNA segments, each encoding a different part of the variable region. The second type of recombination replaces the exons coding for the constant region of the mu chain with those coding for the same region of the gamma 2b chain. The DNA sequencing studies suggest that the two types of recombination operate by different mechanisms.

Exon shuffling generates an immunoglobulin heavy chain gene.

From endonuclease EcoRI partial libraries of DNAs from mouse embryo and MOPC 141, a gamma 2b-producing myeloma, clones were isolated by using a DNA fragment carrying the gamma 2b constant (C) region gene as a hybridization probe. One clone from MOPC 141 contained a heavy chain variable (V) gene and the C gamma 2b gene, as demonstrated by R-loop mapping. The V gene and C gene in this clone were separated by a 3.9-kilobase intron. The characterization of this clone as well as the embryonic clones suggest that at least two recombination events occurred to create the gamma 2b gene in MOPC 141. One of the events is analogous to the V-J joining previously demonstrated in the light chain genes, which brings the major part of the V gene next to a short coding sequence (J). The other event we refer to as "C mu-C gamma 2b switch recombination" because a portion of the intron between the V gene and C gene of the rearranged gamma 2b gene is derived from the 5' flanking sequence of the embryonic C mu gene. A model suggesting how the phenomenon of switch seen in lymphocytes may occur is presented.

DNA sequence of the E. coli trpR gene and prediction of the amino acid sequence of Trp repressor.

A DNA sequence of 1041 base pairs from a BamHI fragment containing the E. coli trpR gene has been determined. With this sequence and other experimental evidence, the primary structure (88 amino acids) of the Trp repressor can be predicted. Additional features of the DNA sequences include a 22 base pair region upstream from the proposed structural gene which exhibits striking homology with the trp operator, thus implying that expression of the trpR gene may be under autogenous regulation.

Cloning the trpR gene.

In Escherichia coli, the structural gene for purine nucleoside phosphorylase, deoD, is subject to insertional inactivation by prophage lambda. From one such secondary site lambda lysogen, strain SP265, one may isolate deletions that remove all or part of the trpR gene and other genes in the deo-thr sector of the E. coli chromosome. Specialized transducing phages harboring serB+ and trpR+ were liberated following induction of SP265. All such phages were N-defective, bio-type pseudolysogens whose DNA persisted in the form of plasmids. A collection of transducing phages, differing in their complement of bacterial DNA, was used to locate cleavage sites for BamHI, SalI, and PvuI within the deoD-trpR region of the E. coli genome. The trpR gene lies within a specific 950 base pair BamHI-PvuI segment. A 1250 base pair BamHI fragment carrying a functional trpR gene was cloned into the amplifiable plasmid pBR322. A single SalI site in this fragment was shown to lie within the TrpR gene. In two situations where increased gene dosage might generate elevated amounts of Trp repressor (N-defective trpR+ pseudolysogens and strains harboring pBR322 trpR+ plasmids) neither tryptophan auxotrophy, enhanced sensitivity to DL-5-methyl-tryptophan, nor super repression of the tryptophan biosynthetic enzymes was observed.