[Radiotherapy of lymphatic fistula and lymphocele]. / Strahlentherapie von Lymphfisteln und Lymphozelen.

BACKGROUND: The treatment of persistent postoperative lymphatic fistulas or lymphoceles is often a problem. Approximately 2% of patients will develop lymphatic fistula after vascular surgery. This can require a long lasting conservative therapy. If spontaneous cure fails, a second operation with wound revision becomes necessary. We studied low-dose percutaneous radiotherapy to be used as an alternative treatment in addition to conservative or surgical therapy. PATIENTS AND METHODS: Between 1989 and 1998 29 patients (25 with lymphatic fistulas, 4 with lymphoceles) received radiation therapy. Depending on the depth of the fistula 27 patients were treated with electrons (7 to 18 MeV). Two other patients suffering of retroperitoneal lymphoceles received a treatment with photons (15 MV). In all patients the fractionation was 4- to 5 x 1.0 Gy/week and the dose ranged from 3 to 12 Gy depending upon the onset of the radiation therapy effect. RESULTS: In 27 of 28 evaluable patients a complete disappearance of the fistula or lymphocele was achieved by radiation during therapy or shortly afterwards. In 1 case no benefit was observed after a dose of 11 Gy. This patient required further surgery with wound exploration. CONCLUSION: Low dose percutaneous radiotherapy (up to 10 to 12 Gy) is effective to heal lymphatic fistulas and lymphoceles without complications. Individual dosage is required because doses even lower than 10 Gy may be effective. Radiation can be effective even after a failed conservative therapy or instead of surgery.

V(D)J recombination activity in human hematopoietic cells: correlation with developmental stage and genome stability.

V(D)J recombinase activity was measured in an array of human cell lines derived from hematopoietic malignancies representing various lineages and developmental stages. The level of recombinase activity was found to vary over a 2000-fold range between different cell lines. Several myeloid cell lines were positive for V(D)J recombinase activity, providing additional insight into the relationship between myeloid and lymphoid differentiation. Despite high levels of V(D)J recombination in two human acute lymphoblastic leukemia cell lines, the cytogenetic karyotype has remained essentially constant over several years of continuous cell culture. Silencing of recombination of chromosomal and minichromosomal targets has been strongly correlated with the replication of CpG methylated DNA in murine cells. Here, in human cells, we show that human minichromosomes bearing V(D)J recombination signals are protected well over 100-fold from recombination if they are CpG methylated, providing a rational basis for the karyotypic stability in cells with high levels of V(D)J recombination activity.

Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.

Crystal structures of binary and ternary complexes of the E. coli Rep helicase bound to single-stranded (ss) DNA or ssDNA and ADP were determined to a resolution of 3.0 A and 3.2 A, respectively. The asymmetric unit in the crystals contains two Rep monomers differing from each other by a large reorientation of one of the domains, corresponding to a swiveling of 130 degrees about a hinge region. Such domain movements are sufficiently large to suggest that these may be coupled to translocation of the Rep dimer along DNA. The ssDNA binding site involves the helicase motifs Ia, III, and V, whereas the ADP binding site involves helicase motifs I and IV. Residues in motifs II and VI may function to transduce the allosteric effects of nucleotides on DNA binding. These structures represent the first view of a DNA helicase bound to DNA.

RAG mutations in human B cell-negative SCID.

Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B- SCID). Although several genetic causes are known for B+ SCID, the etiology of B- SCID has not been defined. Six of 14 B- SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.

Mechanistic constraints on diversity in human V(D)J recombination.

We have analyzed a large collection of coding junctions generated in human cells. From this analysis, we infer the following about nucleotide processing at coding joints in human cells. First, the pattern of nucleotide loss from coding ends is influenced by the base composition of the coding end sequences. AT-rich sequences suffer greater loss than do GC-rich sequences. Second, inverted repeats can occur at ends that have undergone nucleolytic processing. Previously, inverted repeats (P nucleotides) have been noted only at coding ends that have not undergone nucleolytic processing, this observation being the basis for a model in which a hairpin intermediate is formed at the coding ends early in the reaction. Here, inverted repeats at processed coding ends were present at approximately twice the number of junctions as P nucleotide additions. Terminal deoxynucleotidyl transferase (TdT) is required for the appearance of the inverted repeats at processed ends (but not full-length coding ends), yet statistical analysis shows that it is virtually impossible for the inverted repeats to be polymerized by TdT. Third, TdT additions are not random. It has long been noted that TdT has a G utilization preference. In addition to the G preference, we find that TdT adds strings of purines or strings of pyrimidines at a highly significant frequency. This tendency suggests that nucleotide-stacking interactions affect TdT polymerization. All three of these features place constraints on the extent of junctional diversity in human V(D)J recombination.

The mechanism of V(D)J recombination: site-specificity, reaction fidelity and immunologic diversity.

Site-specific recombination reactions in higher eukaryotes are uncommon, perhaps because of the potential genomic instability that they may create. We focus this review on the issues of site-specificity, reaction fidelity and immunologic diversity in the V(D)J recombination reaction.

Unequal signal and coding joint formation in human V(D)J recombination.

Substrates for studying V(D)J recombination in human cells and two human pre-B-cell lines that have active V(D)J recombination activity are described. Using these substrates, we have been able to analyze the relative efficiency of signal joint and coding joint formation. Coding joint formation was five- to sixfold less efficient than signal joint formation in both cell lines. This imbalance between the two halves of the reaction was demonstrated on deletional substrates, where each joint is assayed individually. In both cell lines, the inversional reaction (which requires formation of both a signal and a coding joint) was more than 20-fold less efficient than signal joint formation alone. The signal and coding sequences are identical in all of these substrates. Hence, the basis for these differential reaction ratios appears to be that coding joint and signal joint formation are both inefficient and their combined effects are such that inversions (two-joint reactions) reflect the product of these inefficiencies. Physiologically, these results have two implications. First, they show how signal and coding joint formation efficiencies can affect the ratio of deletional to inversional products at endogenous loci. Second, the fact that not all signal and coding joints go to completion implies that the recombinase is generating numerous broken ends. Such unresolved ends may participate in pathologic chromosomal rearrangements even when the other half of the same reaction may have proceeded to resolution.

The basis for the mechanistic bias for deletional over inversional V(D)J recombination.

V(D)J recombination between recognition sites in the genome is characterized by certain biases. At some loci, proximal sites undergo recombination substantially more frequently than distal ones. The joining of DH/JH is an example of this. Because the DH element bears signal sequences on each side, inversion would be expected as often as deletion in DH/JH recombination. However, the markedly favored outcome is deletion, entailing utilization of the closer recombination site. One model proposed to explain these biases is the tracking model in which the recombinase tracks from one site to the other. Here, we have directly tested for various types of tracking in V(D)J recombination and have found no indication that it occurs. In addition, we have created DH-JH minilocus substrates for analysis of the basis for the preference for deletion. We find that we can reproduce the deletional bias for the system. Moreover, by flipping the orientation of the D segment, we can reverse the bias such that the frequency of inversions can exceed the number of deletions. These results indicate (1) that there is no intrinsic topological preference in this reaction, and (2) that the sequence of the signal and coding ends determines the bias.

[Comparative biochemical and histological studies of the effect of magnesium and profound hypothermia on the preservation of the isolated perfused rat liver (author's transl)]. / Etude comparative biochimique et histologique de l'action du magnésium et de l'hypothermie profonde sur la conservation du foie de rat isolé.

In order to determine, if magnesium aspartate has the same favorable effect on the liver as on the arrested heart, the isolated rat liver preperfused with isotonique Mg-aspartate or saline solution 15 minutes and stored 1 hour at 36 degrees C or 12 hours at 36 degrees C has been reperfused 150 minutes at 37 degrees C. During the storage the concentrations of ATP and ADP decrease significantly whereas AMP, lactate and beta-hydroxybutyrate increase remarkably, accompanied by a pronounced breakdown of glycogen. Upon reperfusion a de novo synthesis of ATP and ADP is resumed. Lactate, beta-hydroxybutyrate and AMP approach normal values whereas a resynthesis of glycogen is missing. A focal distribution of well preserved and disintegrated hepatocytes is being observed and seems rather to be due microcirculatory disorders. No conclusive evidence of a protective Mg-effect as observed in induced cardiac arrest has been obtained. Several concentrations of Mg-aspartate should be studied, in order to elucidate the different acting mechanism of Mg++ on the two organs.

The effect of local testicular irradiation on testicular histology and plasma hormone levels in the male rat.

Adult male rats were subjected to local testicular irradiation, plasma hormone levels and testicular histology being quantified at intervals up to 52 days thereafter. LH and FSH increased coincidently with spermatid but not with spermatocyte or spermatogonia depletion. Testosterone levels seemed to decrease but this effect was not significant. Oestradiol levels showed no significant changes. From the correlations between the various parameters it was concluded that the lack of inhibin was the main cause of the increase in both LH and FSH and that spermatids provide the signal for production of this non-steroidal inhibitor. The site of inhibin production was not definitively established but the results would be consistent with production of inhibin by the Sertoli cells in association with spermatids.

The effect of magnesiumaspartate on preservation of the rat liver.

1. Isolated rat livers reperfused with an oxygenated isotonic Mg-aspartate or saline solution and stored 1 h at 36 degrees C, 4 h at 22 degrees C or 12 h at 6 degrees C were reperfused at 37 degrees C 150 min with a Krebs-Henseleit solution containing bovine albumin and erthrocytes gased with a 95% O2-5% CO2 gas mixture. 2. Light- and electronmicroscopic studies revealed minor changes after storage, whereas in the reperfused livers a focal distribution of well preserved and disintegrated hepatocytes was observed. 3. During storage the sum of adenine nucleotides in general decreased markedly paralleled by a significant rise of the lactate/pyruvate and hydroxybutyrate/acetoacetate ratios and pronounced breakdown of glycogen. Oxidative phosphorylation was resumed upon reperfusion resulting in de novo synthesis of ATP and ADP. The lactatelpyruvate and hydroxybutyrate/acetoacetate ratios normalized, whereas a resynthesis of glycogen was missing. 4. No conclusive evidence of a protective Mg-effect as observed in induced cardiac arrest has been obtained, probably due to structural and metabolic differences between the two organs and microcirculatory disorders as has been demonstrated by measurements of oxygen uptake using the multiwire electrode.