Phylogenetically conserved sequences in the promoter of the rabbit sodium-hydrogen exchanger isoform 1 gene (NHE1/SLC9A1).

NHE1 (gene symbol SLC9A1) encodes an isoform of the amiloride-sensitive Na+-H+ exchanger that is present in many cells and the basolateral membrane of renal epithelia. Expression of NHE1 is modulated in response to chronic metabolic acidosis, growth factors, and phorbol esters. To begin examining the molecular basis for this regulation, a rabbit genomic clone that contained 6 kb of 5' flanking region, the first exon, and a portion of the first intervening sequence of NHE1 was obtained. The principal transcription start site in native rabbit tissues was located 798 bp 5' to the initiation codon. The sequence of the proximal 5' flanking region of rabbit NHE1 was similar to the human sequence and contained a TATA-box, (G + C)-boxes, homopyrimidine direct repeats, and a putative AP-1 site. When ligated to luciferase and transfected into porcine renal epithelial cells (LLC-PK1), 708 bp of proximal 5' flanking region exhibited orientation-dependent promoter activity.

[Bouginage of peptic esophageal stenoses. Results of one year with long-term omeprazole medication]. / Bougierung peptischer Osophagusstenosen. Ein-Jahres-Resultate unter Dauermedikation mit Omeprazol.

METHOD: Within the framework of an open prospective study, 47 patients with chronic reflux esophagitis, unresponsive to H2-receptor blockers and complicated by stenosis, underwent endoscopic bougienage. Unsuccessful treatment with H2-receptor blockers was followed in all patients by antisecretion treatment with omeprazole at a dose of 40 mg/day. RESULTS: At the latest after 3 months, stenotic and inflammatory changes had cleared up in all patients and under continued omeprazole over the long-term, remission of at least one year was achieved. CONCLUSION: A combination of endoscopic bougienage and simultaneous treatment with the proton pump blocker, omeprazole, represents effective treatment of chronic reflux disease complicated by stenosis.

Thrombin-based antithrombin assays show overestimation of antithrombin III activity in patients on heparin therapy due to heparin cofactor II influence.

An extensive comparison has been performed on the clinical chemistry automate Hitachi 717 between thrombin- and Factor Xa-based methods for determination of antithrombin III activity. In 460 patients who did not receive any heparin therapy the agreement between assays was in general close although the thrombin-based methods resulted in slightly higher assignments of 0.3-2.6% antithrombin III activity. The discrepancy was, however, substantial in plasmas from patients receiving heparin of > or = 20000 IU/day, resulting in plasma levels of heparin of 0.8-1.2 IU/ml. Thus, analysis of 102 patients showed that the thrombin-based methods resulted in, on average, 7-16% higher assignment of antithrombin III activity as compared to the Factor Xa-based method used. Addition of antibodies to antithrombin III and heparin cofactor II revealed that the discrepancy was primarily due to contribution of heparin cofactor II activity in the thrombin-based methods. The results thus suggest that the Factor Xa-based antithrombin III activity method provides more valid results in patients on heparin therapy.

Correlation between low CSA plasma concentration and severity of acute GvHD in bone marrow transplantation.

Between 1982 and 1986 51 patients were treated with ciclosporin a (CSA) to prevent graft versus host disease (GvHD) after bone marrow transplantation (BMT). Major side effects of the drug were tremor, hypertension, hepatotoxicity and nephrotoxicity. Acute GvHD 0 degree to II degree occurred in 80% of our patients, and GvHD III degree and IV degree in 20% despite the use of CSA. Two to four days before the onset of GvHD, CSA serum levels were significantly lower on the average in patients who developed GvHD III degree and IV degree compared to the others. Our data indicate that plasma CSA concentrations higher than 250 ng/ml should be achieved to reduce the severity of GvHD after BMT.

HLA-DP in rheumatoid arthritis.

Frequencies of HLA-DR, Dw and DPw specificities were compared between rheumatoid arthritis (RA) patients, Felty's syndrome (FS) patients and normal controls. It was confirmed that the frequency of DR4 was increased in RA patients (54% (n = 111) vs 23% (n = 272), relative risk (RR) = 3.98, P less than 0.001). Cellular typing showed a highly significant increase in HLA-Dw14 in the entire RA population (17% (n = 32) vs 2% (n = 242), RR = 11.90, P less than 0.001), and a tendency towards an increase of HLA-Dw14 in DR4+ RA patients compared to DR4+ controls (28% (n = 32) vs 11% (n = 47), RR = 3.29, P less than 0.05). Regarding DPw specificities, the only significance was for a negative association with DPw3 (13% vs 22% (n = 254), RR = 0.51, P less than 0.05), with an additional tendential decrease of DPw1 (11% vs 19%, RR = 0.53, not significant (NS]. The decrease of DPw3 was more marked in DR4- RA patients (RR = 0.33, P less than 0.05) than in DR4+ RA patients (RR = 0.69, NS). In FS patients, 96% of whom were DR4+, decreased DPw1 was very marked, whereas the frequency of DPw3 was unaltered compared to DR4+ normals. These alterations in frequencies were not caused by linkage disequilibria between HLA-DR and -DP alleles. Thus, taken together, these data suggest that, in the presence of the major DR4-associated "susceptibility" gene(s) for RA, DPw1 may have "protective" effects, whereas in the absence of DR4, the presence of DPw3 has significant "protective" activity.

Human leukocyte antigen-DP in leukemia.

Human leukocyte antigen-DP (HLA-DP) typing was performed on patients with chronic myelogenous leukemia (CML, n = 44), acute nonlymphoblastic leukemia (ANLL, n = 34), or acute lymphoblastic leukemia (ALL, n = 41). Frequencies of DPw alleles in CML and ANLL patients were not significantly different from 254 controls, except that in ANNL DPw1 was absent. This was most likely due to the concurrent absence of DR3 with which DPw1 is in linkage. In contrast, in ALL, frequencies of DPw2 and DPw5 were significantly increased (corrected P less than 0.05, relative risk (RR) = 2.19 and corrected P less than 0.01, RR = 6.92, respectively). This was not due to linkage with DR. The frequency of DPw1 also tended to be reduced, but this was not caused by a similar decrease of DR3 in ALL. These results, therefore, demonstrate both positive and negative associations between major histocompatibility complex (MHC) gene products which are in only very weak linkage with the rest of HLA, and acute lymphocytic, but neither acute nor chronic myelogenous, leukemias. The HLA-DP region could thus contain long sought-after genes influencing susceptibility and resistance to leukemogenesis.

Relative cloning efficiencies and long-term propagation capacity for T cell clones of highly purified natural interleukin 2 compared to recombinant interleukin 2 in man.

Highly purified (HP) natural (n) and recombinant (r) interleukin 2 (IL 2) have been compared for their ability to support clonal outgrowth and long-term clonal propagation of alloactivated human T lymphocytes. The frequency of outgrowth of T cell colonies was as high as 1:2 to 1:4 when HPnIL 2 was employed for limiting dilution cloning. Separated CD4+ and CD8+ activated populations produced similar cloning efficiencies. Recloning of established CD4+ lines in HPnIL 2 suggested that a clonable cell frequency of 1:2-1:4 was the maximum possible that could be achieved in this system. In contrast, purified rIL 2 allowed outgrowth of only 1:10-1:20 cells from alloactivated populations. Again, CD4+ and CD8+ fractions generated similar cloning efficiencies. In terms of the fraction of derived clones, which could be propagated to greater than 1 X 10(6) and greater than 1 X 10(8) cells, nIL 2 again proved superior to rIL 2. In either source of IL 2, the proportion of CD4+ clones which could be extensively propagated was greater than in the CD8+ population. Surprisingly, although the addition of PHA to these lectin-free IL 2 preparations reduced the frequency of clonable cells, the proportion of clones which could be extensively propagated was increased. These results suggest that nIL 2, consisting of three differently glycosylated molecular species, may be preferable to rIL 2, which consists only of a single non-glycosylated species, for the cloning and long-term propagation of human T cells. These results may have some bearing on the choice of rIL 2 versus nIL 2 for therapeutic applications.

Lung function changes after allogenic bone marrow transplantation.

The lung function of 21 patients with leukaemia (11 with acute myeloid leukaemia, six with acute lymphatic leukaemia, four with chronic myeloid leukaemia) and of five with severe aplastic anaemia was tested before and after allogenic bone marrow transplantation. Vital capacity (VC) was lowered in patients with leukaemia before transplantation. VC and FEV1 fell significantly after transplantation. Residual volume (RV) and RV as a percentage of total lung capacity (RV % TLC) were already increased and rose significantly after transplantation. Patients with severe aplastic anaemia had noticeably increased RV and RV % TLC, values that did not change after transplantation. In contrast to the patients with aplastic anaemia, the patients with leukaemia had significantly reduced VC, RV, RV % TLC, and FEV1 before and after transplantation. The specific airway resistance (sRaw) was raised significantly before and after transplantation in the leukaemic patients. In addition, transfer coefficient (Kco) fell significantly more after transplantation in the patients with leukaemia than in those with severe aplastic anaemia. In three patients with histologically established obstructive bronchiolitis in conjunction with chronic graft versus host disease after transplantation, VC, FEV1 and FEV1 % VC fell, while RV, RV % TLC, and sRaw rose; Kco was far below normal. On the basis of these findings it is concluded that in patients with leukaemia obstructive disorders of ventilation develop or, if they are already present, worsen. In patients with severe aplastic anaemia lung function was not impaired in the early phase after transplantation. These differences are probably due to the more intensive immunosuppressive and cytotoxic preparatory regimen before transplantation in the leukaemic patients. Obstructive bronchiolitis, a complication of graft versus host disease, first manifests itself in a typical rise in specific airway resistance and must be treated early.

Lung diseases after bone marrow transplantation. Results of a clinical, radiological, histological, immunological and lung function study.

The case histories of 72 subsequently treated patients - 44 with acute leukemia, 10 with chronic myeloid leukemia, 16 with severe aplastic anemia and 2 with neuroblastoma - were analyzed after bone marrow transplantation (BMT) with respect to pulmonary diseases. Thirty-eight patients suffered from a total of 51 pulmonary complications, which led to death in 20. Of 13 patients, 3 died of bacterial pneumonia, all of them during granulocytopenia; 2 of 6 patients died of fungal pneumonia and 2 out of 3 of a mixed bacterial-mycotic infection. Adult respiratory distress syndrome (ARDS) led to death in 2 patients. A granulocyte count under 500/microliter correlated significantly (P less than 0.002) with the fatal outcome of bacterial, fungal and ARDS pneumonia as well as with bronchitis. Viral pneumonia led to death in 8 of 9 patients; in each there was a significant correlation (P less than 0.05) with graft-versus-host disease (GvHD). Patients with repeated episodes of pulmonary illness had significantly more chronic GvHD (P less than 0.05); several of these patients displayed a reduction in helper T cells and an increase in suppressor T cells in the peripheral blood. The natural killer (NK) cells were reduced and the percentage of activated NK cell level lay between 6% and 69%. B-cells were absent or deficient. These findings explain in part the absence of specific antibody reactivity. Five of these patients also contracted GvHD-associated obstructive bronchiolitis, which did not respond to therapy. Pulmonary infiltrates of unknown origin (including idiopathic interstitial pneumonia) occurred in 8 of the patients (11.1%), with a fatal outcome in 3 patients. Significant changes (P less than 0.05) in lung function after BMT appeared in the form of reduced vital capacity (VC) increased residual volume (RV) and an increase in RV expressed as the percentage of total lung capacity. Pulmonary diseases were the most common complication and cause of death in our patients after BMT.

Acute folic acid deficiency after bone marrow transplantation.

After bone marrow transplantation (BMT), megaloblastic bone marrow changes are often observed that can only be partially explained by drug effects. Our goal was to find out whether folic acid deficiency represented an additional factor. The serum folic acid concentrations of 41 patients were determined regularly before and after BMT. A 2nd degree polynomial regression analysis revealed a clear and acute drop in folic acid concentrations within 7-9 days after BMT. In 19 patients the level fell below 3.0 ng/ml, the range of folic acid deficiency. The mean folic acid values without oral administration of folic acid after BMT lay significantly below the mean values with substitution (P less than 0.001). If a case of acute graft versus host disease (GvHD) was more severe than grade I, the mean folic acid levels were significantly lower (P less than 0.01). Patients with megaloblastic bone marrow changes after BMT had significantly lower folic acid values than those without such changes (P less than 0.01). The 18 patients with folic acid deficiency had a significantly higher rate of megaloblasts, binucleate erythropoietic precursors, Howell-Jolly bodies, giant myelocytes, and giant metamyelocytes in bone marrow smears than the remaining 23 patients (P less than 0.05). Folic acid deficiency did not slow down the increase in leukocytes, granulocytes, thrombocytes, or reticulocytes after BMT. There were 8.2%-9.7% hypersegmented neutrophils in the blood (normal 5%) after BMT both with and without folic acid deficiency. Folic acid deficiency after BMT was caused by insufficient intake combined with simultaneous decreased intestinal resorption and increased requirements for the regeneration of bone marrow and intestinal mucosa.

Strong lymphoproliferative suppressive function of PLT clones specific for SB-like antigens.

From a total of 37 different priming combinations between donors matched for HLA-A,B, and/or Dw/DR, but mismatched for SB, antigens, T cell clones strongly restimulated with concordance for SB specificities were isolated from only two. Most of the alloproliferative (PLT) clones obtained were restimulated by determinants not correlated with any currently known HLA product. Nonetheless, their stimulation was inhibited by a monoclonal antibody TU 39, which preferentially blocks stimulation by SB-, rather than by Dw/DR-associated determinants. Despite having an OKT4+, OKT-, Leu8- phenotype, and secreting Interleukin-2 after contact with stimulatory cells, these clones strongly suppressed proliferative responses of cloned PLT reagents as well as unprimed lymphocytes in mixed leukocyte cultures. They may thus represent a novel type of immunoregulatory T cell, stimulated by SB-related antigens, which despite their "helper/inducer" phenotype are able directly to suppress lymphoproliferative responses.

Alloactivated long-term cultured human T lymphocytes express both HLA-DR and SB antigens but lack lymphocyte stimulation capacity.

Cell populations obtained from mixed leukocyte cultures of 6- or 10-day duration were found specifically to restimulate primed lymphocytes detecting HLA-linked SB as well as HLA-D-associated antigens. After expansion in vitro (9-75 days) with medium containing interleukin 2, the cultured cells expressed the T lymphocyte markers detected in indirect immunofluorescence by monoclonal antibodies Lyt-3, OKT3, OKT4, OKT8, and had high levels of HLA-DR antigens. In addition, they were shown in cell-mediated lymphocytotoxicity specifically to express SB antigens of the donor B cell type. Despite their positivity for DR and SB antigens, such cultured T cells failed to restimulate either SB- or D-specific secondary lymphocyte proliferation. Homogeneous cloned populations of cultured T cells also lacked lymphocyte stimulation capacity. In contrast, B cell lines, which also expressed DR and SB antigens, were potent stimulators of both SB- or D-directed proliferation. These data show that the activated T lymphocytes which express both HLA-DR and SB antigens are by themselves unable to stimulate lymphocyte proliferation.

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations.

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).