Spastin, a new AAA protein, is altered in the most frequent form of autosomal dominant spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Among the four loci causing AD-HSP identified so far, the SPG4 locus at chromosome 2p2-1p22 has been shown to account for 40-50% of all AD-HSP families. Using a positional cloning strategy based on obtaining sequence of the entire SPG4 interval, we identified a candidate gene encoding a new member of the AAA protein family, which we named spastin. Sequence analysis of this gene in seven SPG4-linked pedigrees revealed several DNA modifications, including missense, nonsense and splice-site mutations. Both SPG4 and its mouse orthologue were shown to be expressed early and ubiquitously in fetal and adult tissues. The sequence homologies and putative subcellular localization of spastin suggest that this ATPase is involved in the assembly or function of nuclear protein complexes.

Multi-Instrument Characterization of Magnetospheric Cold Plasma Dynamics in the June 22, 2015 Geomagnetic Storm.

We present a comparison of magnetospheric plasma mass/electron density observations during an 11-day interval which includes the geomagnetic storm of June 22, 2015. For this study we used: Equatorial plasma mass density derived from geomagnetic field line resonances (FLRs) detected by Van Allen Probes and at the ground-based magnetometer networks EMMA and CARISMA; in situ electron density inferred by the Neural-network-based Upper hybrid Resonance Determination algorithm applied to plasma wave Van Allen Probes measurements. The combined observations at L ∼ 4, MLT ∼ 16 of the two longitudinally separated magnetometer networks show a temporal pattern very similar to that of the in situ observations: A density decrease by an order of magnitude about 1 day after the Dst minimum, a partial recovery a few hours later, and a new strong decrease soon after. The observations are consistent with the position of the measurement points with respect to the plasmasphere boundary as derived by a plasmapause test particle simulation. A comparison between plasma mass densities derived from ground and in situ FLR observations during favorable conjunctions shows a good agreement. We find however, for L < âˆ¼3, the spacecraft measurements to be higher than the corresponding ground observations with increasing deviation with decreasing L, which might be related to the rapid outbound spacecraft motion in that region. A statistical analysis of the average ion mass using simultaneous spacecraft measurements of mass and electron density indicates values close to 1 amu in plasmasphere and higher values (∼2-3 amu) in plasmatrough.

High-dose azathioprine pulse therapy as a new treatment option in patients with active Wegener's granulomatosis and lupus nephritis refractory or intolerant to cyclophosphamide.

The objective of this study was to evaluate the feasibility and safety of high-dose azathioprine pulse (HAP) therapy in the induction of remission in patients with active Wegener's granulomatosis (WG) or progressive lupus nephritis (LN) refractory to or intolerant of cyclophosphamide. Four patients with antineutrophil cytoplasmic antibody (ANCA)-associated WG and two patients with progressive LN were treated with HAP (1200-1800 mg) applied monthly as continuous intravenous infusions at 50 mg/h. Patients received a total of 50 courses of intravenous azathioprine (AZA) therapy. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS) and the Systemic Lupus Erythematosus Activity Index (SLEDAI). As only partial remission was induced in patients with progressive LN on this regimen, an additional 18 cycles were applied in these patients in which oral AZA at 100 mg/day in weeks 2 and 3 was added between two intravenous courses. A hereditary defect in thiopurine methyltransferase activity was excluded before initiation of treatment. High-dose azathioprine pulse and the intensified HAP treatment were well tolerated. Complete remission was achieved in two patients with WG suffering from three relapses of disease on application of 2-6 courses of HAP. Remission was maintained for 16-24 months. The remaining two patients with WG were withdrawn after 2-3 courses due to unchanged disease activity. In two patients with LN, partial remission was noted on 6-9 courses of HAP; however, the patients relapsed despite therapy with methotrexate and mycophenolate mofetil. The intensified HAP regimen led to partial or complete remission in both LN patients which was confirmed by sequential renal biopsies. Our results suggest that HAP therapy represents a well-tolerated regimen in patients with active WG and LN intolerant of or refractory to cyclophosphamide. As partial or complete remission was observed in four of six patients, further studies seem warranted to assess clinical efficacy in these patients.

TNF alpha therapy activates human B-lymphoma cells in vivo and may protect myelopoiesis.

In vitro TNF alpha induces proliferation and expression of predominantly type A TNF-receptors on B-lymphoma cells. In a pilot study we treated 2 patients with refractory B lymphomas with two courses of TNF alpha and consecutive aggressive chemotherapy (high dose Ara-C and mitoxantrone). TNF alpha was applied on days 1-4, chemotherapy on days 2-4 (TNF-AraM). The TNF-AraM therapy was repeated on day 43. Both patients responded to therapy. TNF alpha therapy induced expression of the 75 kD TNF receptor and the interleukin 2 receptor (CD25) and weakly the 55 kD TNF receptor on leukemic B lymphocytes. Interleukin 3, interleukin 6 and GM-CSF were induced and measured in the serum of patients. The mean time of severe granulocytopenia (less than 0.5/nl) was 9 days (range 8-10 days), the mean time of thrombocytopenia (less than 20/nl) was 5 days (range 2-6 days). In 7 patients, who were treated with high dose Ara-C and mitoxantrone (AraM) mean time of granulocytopenia (less than 0.5/nl) was 23 days (range 18-34 days), and of thrombocytopenia (less than 20/nl) was 13.3 (range 3-27 days). We conclude that TNF alpha can activate tumor B cells in vivo and may exhibit also myeloprotective effects when applied before aggressive chemotherapy.

Evidence against an increase in capillary permeability in subjects exposed to high altitude.

A potential pathogenetic cofactor for the development of acute mountain sickness and high-altitude pulmonary edema is an increase in capillary permeability, which could occur as a result of an inflammatory reaction and/or free radical-mediated injury to the lung. We measured the systemic albumin escape by intravenously injecting 5 muCi of 125I-labeled albumin and the plasma concentrations of cytokines, F2-isoprostanes (products of lipid peroxidation), and acute-phase proteins in 24 subjects exposed to 4,559 m. Ten subjects developed acute mountain sickness, and four subjects developed high-altitude pulmonary edema. The transcapillary escape rate of albumin was 6.9 +/- 2.0%/h (SD) at low (550 m) and 6.3 +/- 1.9%/h at high (4,559 m) altitude (P = 0.23; n = 24). The subjects with high-altitude pulmonary edema had a modest but insignificant increase in the transcapillary escape rate of albumin (4.6 +/- 1.9%/h at low vs. 5.7 +/- 1.9%/h at high altitude; P = 0.42; n = 4). Plasma concentrations of fibrinogen, alpha 1-acid glycoprotein, C-reactive protein, and interleukin-6 were unchanged in the early phases and significantly increased by the end of the observation period in the subjects with high-altitude pulmonary edema, whereas tumor necrosis factor-alpha and F2-isoprostanes did not change at all. This suggests that the inflammatory reaction was rather a consequence than a causative factor of high-altitude pulmonary edema. In summary, these data argue against a dominant role for increased systemic capillary permeability in the development of acute mountain sickness and high-altitude pulmonary edema.

Eight versus 16-week re-evaluation period in rheumatoid arthritis patients treated with leflunomide or methotrexate accompanied by moderate dose prednisone.

In a step-up approach of DMARD treatment of RA a fast response and an early DMARD switch in the case of non-response is important. Therefore, we performed an open trial in which we compared an 8-week and a 16-week observation period during treatment of RA with MTX or LEF, both given in intensified starting doses and accompanied by moderate dose prednisone. MTX and LEF naïve patients with RA (mean time since diagnosis: 2.3 years) were randomised to receive either LEF in a 3-day-loading dose of 100 mg/day followed by 20 mg/day (n = 19) or MTX intramuscularly in a dose of 25 mg once weekly (n = 21). All patients received concomitant treatment with oral prednisone in an initial dose of 20 mg/day with weekly dose reductions of 5 mg/day. The disease activity was re-evaluated 8 and 16 weeks after the start of the treatment. Mean DAS28 before the start of treatment was 5.36 +/- 0.8 for the MTX-group and 5.46 +/- 0.8 for the LEF-group. After 8 weeks of treatment the DAS28 in the MTX-group was 2.59 +/- 1.0 and 3.16 +/- 0.8 in the LEF group (difference not significant). The mean DAS28 at re-evaluation 16 weeks after the starting of treatment (2.58 +/- 1.5 for the MTX-group and 3.25 +/- 1.16 for the LEF-group) was significantly different neither in between the both treatment groups nor in comparison to the week 8 evaluation. Efficiency of RA treatment with MTX or LEF in intensified doses and in combination with moderate dose prednisone can be sufficiently judged 8 weeks after its initiation.

[Expression of TNF receptors in rheumatoid arthritis and ankylosing spondylitis]. / Expression von TNF-Rezeptoren bei rheumatoider Arthritis und Spondarthropathien.

Tumor necrosis factor is an important mediator of the pathophysiologic events in synovitis. The expression of the p75 and p55-TNF-receptors in rheumatic diseases was investigated. Synovial mononuclear cells (SMNC) of patients with rheumatoid arthritis and spondylarthropathies express p75 TNF receptors in all cases, whereas SMNC of patients with traumatic synovitis do not. In 4/9 patients with rheumatoid arthritis and in 6/11 patients with spondylarthropathies SMNC also expressed the p55 TNF receptor. Differential analysis of lymphocytes and monocytes/macrophages revealed that both predominantly expressed the p75 TNF receptor. The highest concentrations of both soluble TNF receptors which may act as TNF antagonists were found in synovial fluids of rheumatoid arthritis patients.

Surveillance of diagnostic and treatment measures in Bavaria, 1974-1976. Results 2 and 5 years after the start of chemotherapy.

A central surveillance register for all bacillary pulmonary tuberculosis cases reported in Bavaria (population in excess of 10 million) was established from 1974 to 1976. The aim of the study was to discover the quality and efficiency of health services delivery to the population in the field of tuberculosis under routine conditions, and to find out the relapse rate after cessation of chemotherapy in expatients who were found to be negative 2 years after starting chemotherapy. A total of 7850 German patients with bacillary pulmonary tuberculosis were diagnosed in Bavaria from 1974 to 1976 corresponding to an average annual rate of 25.7 per 100,000 population: 71% of them were smear-positive and 29% were positive by culture only. Reactivations formed 25% of all bacteriologically confirmed cases. Most (71%) smear-positive new cases were discovered because of symptoms. Of the 5157 cases of bacteriologically confirmed pulmonary tuberculosis reported during 1975 and 1976, 4% died from tuberculosis, 1% from sequelae of tuberculosis and 9% from causes other than tuberculosis. A further 3% of patients had drug side-effects, 3% were uncooperative, 2% emigrated or had no permanent address and 1% had no chemotherapy or no information on treatment. The overall results of treatment were very satisfactory: sputum conversion among 3991 patients in the group with complete treatment was achieved in 97.4% at 2 years, and in those with incomplete treatment in 96.2%. The duration of chemotherapy was long, i.e. 19 months or more in two thirds of the patients. The average reactivation rate during the 3rd, 4th and 5th follow-up years was 0.8% annually; it was higher among males than females and the rate increased with age. Of the 157 patients found to be bacteriologically positive at 2 years after the start of chemotherapy 46 died during the 3-year observation period and 109 were alive at 5 years, 23 of whom were harbouring tubercle bacilli.

Increased heterogeneity of serum thyroglobulin in thyroid cancer patients as determined by monoclonal antibodies.

We investigated the immunological heterogeneity of plasma Tg in thyroid cancer patients using monoclonal antibodies in an immunoradiometric assay and a conventional RIA system with a polyclonal rabbit antibody. The results were compared with measurements of plasma Tg in patients with nonmalignant disease. We can demonstrate an increased immunological heterogeneity in tumor patients compared with patients with non-malignant thyroid diseases. In one case the Tg value measured by a monoclonal test system exceeded the value obtained by a polyclonal RIA system in the same sample by a factor of 25. It has to be further investigated whether this increase in heterogeneity is of diagnostic value in the follow-up of thyroid cancer patients.

Is soluble CD25 antigen (interleukin-2 receptor) a useful parameter for differential diagnosis of thyrotoxicosis?

Soluble CD25 antigen was measured in 28 patients with Graves' disease and 20 patients with thyroid autonomy in order to address the question of whether this parameter could be used in the differential diagnosis of thyrotoxicosis. Soluble CD25 was significantly elevated in active Graves' disease (2430 +/- 442 U/ml, mean +/- SEM) compared to patients with thyroid autonomy (1295 +/- 225 U/ml, mean +/- SEM). However, compared to normal controls (mean 605 +/- 49 U/ml), both groups of patients had significantly elevated CD25 plasma levels. Investigations in thyroidectomized thyroid cancer patients on and off T4 suppressive therapy showed no influence of T4 on the CD25 level. Soluble CD25 concentrations did not differ in thyroid cancer patients compared to normal controls. We conclude that soluble CD25 may indicate a stimulation of the immune system with high sensitivity; however, due to the low specificity of elevated CD25 levels, its usefulness for differential diagnosis of thyrotoxicosis is limited.

Lymphotoxin-alpha/beta heterodimer is expressed on leukemic hairy cells and activated human B lymphocytes.

The expression of human lymphotoxin (LT) alpha/beta cell-surface complex was studied in human B-cell lines as well as in normal and neoplastic human B lymphocytes. In the absence of TNF receptors, only the human hairy-cell leukemia (HCL)-derived cell line JOK-I revealed constitutive cell-surface expression of LT but not TNF-alpha. Immunoprecipitation experiments with anti-LT monoclonal antibody (MAb) 9B9 from cell-surface radioiodinated JOK-I cells revealed that a cell-surface lymphotoxin molecule (25 kDa) is expressed in association with a 33-kDa molecule. Enzymatic digestion with F/N-glycosidase and O-glycosidase showed that both proteins contained N-linked carbohydrate residues, whereas only the 25-kDa molecule contained O-linked sugar residues. Analysis of mRNA expression revealed specific transcripts of LT-alpha and LT-beta in JOK-I cells. Resting tonsillar B cells did not express cell-surface LT. However, LT-beta mRNA was observable in unstimulated tonsillar B cells, whereas LT-alpha mRNA, cell-surface LT and LT secretion could only be detected upon in vitro activation. Thus LT-beta and alpha appear to be sequentially expressed in human B cells. Neoplastic B cells from chronic lymphocytic leukemia (BCLL), being devoid of constitutive cell-surface LT expression, could be induced to express surface LT by in vitro stimulation with Staphylococcus aureus Cowan I (SAC). Constitutive LT-beta transcripts, however, could also be detected in 4 out of 5 cases of BCLL. In contrast, human HCL cells displayed constitutive cell expression of lymphotoxin-alpha and beta. These findings demonstrate that cell-surface LT-alpha is expressed in association with LT-beta on activated normal B cells and neoplastic B cells representing an activated state.

Development of immunoradiometric assays for human thyroglobulin using monoclonal antibodies and the biotin/avidin system.

Monoclonal antibodies against human thyroglobulin were produced by the hybridoma technique. Using three non-crossreactive monoclonal antibodies, an IRMA system was developed, with a polyclonal rabbit antibody fixed on microtiter plates as first extracting antibody. The monoclonal antibodies were used as second antibody, while anti-mouse IgG-biotin from goat and [125I]streptavidin served as the indicator system. The source of the monoclonal antibodies was diluted culture medium without purification. Sensitivities of 3-4 micrograms/l were obtained with all 3 monoclonal antibodies; interassay variation was about 5%. This test system will be used for further immunological characterization of circulating thyroglobulin in different thyroid diseases.

Comparative plasma thyroglobulin measurements with three non-cross-reactive monoclonal antibodies in metastatic thyroid cancer patients.

In 12 normal individuals and 25 patients with metastases of differentiated thyroid cancer, plasma thyroglobulin (Tg) concentrations were measured simultaneously with three immunoradiometric assays. Each of the three systems used a different, non-cross-reactive monoclonal Tg antibody as second antibody. In general, there was a very close correlation between the results of the three different systems in the cross-sectional study of the 25 cancer patients as well as in longitudinal follow-up studies in selected patients. The monoclonal antibody Tg 40 produced values about 30% higher than the two other systems. The difference was, however, not statistically significant owing to the large scatter. The monoclonal antibody Tg 13 appeared to be very sensitive to interference with thyroglobulin autoantibodies. In conclusion, the monoclonal antibodies against the three epitopes tested produced very similar Tg values in normal individuals and 25 patients with metastatic thyroid cancer; however, before more is known about epitope specificity of Tg autoantibodies and heterogeneity of tumour Tg, monoclonal antibodies should be used for routine measurements only with caution.

[Plasma GM-CSF concentrations in rheumatoid arthritis, systemic lupus erythematosus and spondyloarthropathy]. / GM-CSF-Plasmakonzentrationen bei rheumatoider Arthritis, systemischem Lupus erythematodes und Spondylarthropathie.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent hematopoietic growth factor, which is mainly produced by T-cells and stromal cells. Beside the stimulating effects on mature granulocytes, it induces the expression of HLA class II-antigen on synovial tissue-cells in patients with rheumatoid arthritis. The concentrations of GM-CSF in the plasma of 87 patients with rheumatoid arthritis, 48 patients with spondyloarthropathy, 17 patients with systemic lupus erythematosus (SLE), and 43 healthy control persons were investigated. We used an immunoradiometric assay (IR-MA) with a detection limit of 30 pg/ml to measure the GM-CSF concentrations in plasma. The GM-CSF levels of 29 patients with severe rheumatoid arthritis (366 +/- 61 pg/ml, p less than 0.05), 58 patients with moderate rheumatoid arthritis (376 +/- 44 pg/ml, p less than 0.0001), and of 17 patients with SLE (256 +/- 41 pg/ml, p less than 0.05) were elevated compared to the control group (174 +/- 18 pg/ml). No significant differences in the mean GM-CSF plasma levels between the patients with spondyloarthropathy (190 +/- 32 pg/ml) and the control group were found. GM-CSF concentrations as high as 1300 pg/ml were detected in the synovial fluids of patients with rheumatoid arthritis. GM-CSF concentrations in the plasma of patients with severe rheumatoid arthritis were correlated with the plasma concentrations of the soluble interleukin-2-receptor (sCD25) (R = +0.53).

Evaluation of soluble tumor necrosis factor (TNF) receptors and TNF receptor antibodies in patients with systemic lupus erythematodes, progressive systemic sclerosis, and mixed connective tissue disease.

Two TNF binding proteins have been characterized as soluble fragments of TNF receptors. We measured the plasma concentrations of soluble type A (p75) and type B (p55) TNF receptors in patients with systemic lupus erythematodes (SLE), progressive systemic sclerosis (PSS), and mixed connective tissue disease (MCTD). In SLE and PSS patients plasma concentrations of both types of TNF receptors and in MCTD patients type A TNF receptors were significantly elevated compared to controls. Plasma concentrations of both soluble TNF receptors were highly correlated in SLE, PSS, and MCTD patients, indicating a possible coregulation of both TNF receptors. In contrast, soluble interleukin 2 receptor (sCD 25) plasma concentrations were not correlated and seem to be an independent parameter. The soluble forms of the TNF receptors neutralize TNF in cytotoxicity assays and are functionally active as TNF antagonists. In one patient with SLE, autoantibodies against type A TNF receptors were detected, TNF alpha, and TNF beta did not interfere with the autoantibody binding to the receptor.

Treatment of systemic mastocytosis with interferon-gamma: failure after appearance of anti-IFN-gamma antibodies.

We report a case of a patient with systemic mastocytosis who was treated with interferon-gamma. Because of severe diarrhoea, nausea and weight loss due to mast cell infiltration of the gastric mucosa the patient received 150 micrograms d-1 interferon-gamma subcutaneously for 10 months. During therapy, the plasma concentrations of IL-3, IL-4 and GM-CSF, which seem to play a role in mast cell growth and differentiation were monitored. The patient had good symptomatic relief and the initially very high eosinophil counts in the peripheral blood showed a partial reduction. However, after 4 months of therapy the patient relapsed. In serum obtained after the relapse, but not in stored serum from the beginning of the therapy, neutralizing antibodies against interferon-gamma were found. Therefore an initial response to the therapy and a secondary failure mediated by treatment-induced antibodies against recombinant interferon-gamma might be suggested. Interferon-gamma may be a well tolerated therapeutic option in systemic mastocytosis. However, treatment-induced neutralizing antibodies against recombinant interferon-gamma should be considered if secondary treatment failure occurs.

Evidence for immunological differences between circulating and thyroid tissue-derived thyroglobulin in men.

The expression of six antigenic epitopes on plasma Tg and tissue-derived Tg was investigated using mouse mabs in immunoradiometric assays. The sensitivity of the assays ranged between 0.5 and 1.5 micrograms l-1. Plasma thyroglobulin of 15 normal individuals and 18 patients with metastatic, differentiated thyroid cancer was analysed. Whereas five monoclonals produced values comparable to the conventional RIA system, the monoclonal antibody Tg 158 did not detect any Tg in the normal individuals and only low levels in two of the cancer patients with the highest Tg levels. In contrast, dilutions of extracts of three different euthyroid goitre samples and three different samples of differentiated thyroid cancer revealed no difference in the antigenic expression of all six epitopes, including the Tg 158 epitope. Papain digestion of purified Tg resulted in a 76 kD fragment which showed immunoreactivity for Tg 158 as well as for Tg 11. No interference of the binding of Tg 158 by T3, T4 or DIT could be detected. We conclude that the monoclonal antibody Tg 158 detects an epitope which is present in thyroid tissue-derived Tg but not in circulating thyroglobulin. This antibody might be useful for the investigation of the secretory process of thyroglobulin from the thyrocytes into the circulation.