Rapid detection, enrichment and propagation of specific T cell subsets based on cytokine secretion.

T cell lines with defined cytokine profiles are an invaluable tool for assessing the control of immune responses both in vitro and in vivo. Production of such cell lines can be complex and time-consuming. Here we present a powerful technique to assay the cytokines produced by T cells activated polyclonally or with specific antigens. This paper presents a detailed methodology for the identification and isolation of cytokine-producing T cells activated with the artificial superantigen, CytoStim, or viral and fungal antigens. These cells can be analysed for different cytokines simultaneously, or cultured further to rapidly establish T cell lines making known cytokine types. We highlight the enumeration, isolation and phenotype of interleukin-17-producing T cells, and the rapid generation of virus-specific Th1 T cell lines.

[Serum erythropoietin levels in several diseases]. / Serumerythropoietinwerte bei verschiedenen Krankheitszuständen.

The accurate radioimmunological measurement of serum erythropoietin (EPO) levels has only been possible since the development of highly specific antibodies directed against recombinant human EPO. In the present study, we determined the serum EPO levels in 100 healthy volunteers and in over 300 patients with anemias and hyperglobulinemia of various causes. In the healthy group, the females had levels of 11.3 +/- 3.4 mU/ml, while the males had levels of 8 +/- 3.2 mU/ml. The serum EPO concentrations were inversely related to the degree of anemia in patients with nonrenal anemias, while predialysis patients with renal anemias showed only partially such a tendency. Hemodialysis patients exhibited EPO-levels that were inadequately low relative to the degree of anemia. Patients with hyperglobulinemia had significantly higher serum EPO-levels than healthy individuals and polycythemia vera patients, the latter having particularly low serum EPO levels. Our results show that the determination of serum EPO levels can be of value in the differential diagnosis of hyperglobulinemia. Finally, sequential measurements document fluctuating serum EPO-levels after gastrointestinal hemorrhages and in patients with iron deficiency anemias receiving iron substitution. The probable reason for this phenomenon seems to be the intermittent utilisation of the hormone by EPO-sensitive erythropoietic precursor cells.

[Polycythemia vera: clinical aspects and course in 86 patients]. / Polycythaemia vera: Klinik und Verlauf bei 86 Patienten.

We analyze symptoms, clinical course, and survival time of 86 patients with polycythemia vera treated between 1966 and 1987 at the medical polyclinic of the University Hospital of Zürich. The mean age of disease onset in 40 men and 46 women studied was 59 years. Most commonly the first symptoms were vertigo and headache (49%) and pain in the extremities (42%). Clinically, plethora was found in half of the cases and 56% showed signs of abnormal arterial and venous circulation. Two thirds of the patients had thromboembolic complications and 40% had hemorrhages chiefly occurring in the gastrointestinal tract. 48% of the patients died after an average survival time of 10 years. The most common cause of death (46%) was acute myelogenous leukemia, followed in 32% by thrombosis and/or embolism and in 7% by death due to hemorrhage. 18 of the 19 leukemia patients were treated with myelosuppressive agents. Patients treated with 32P showed a substantially higher incidence of malignancy than the group of patients not receiving 32P (p less than 0.001). The development of malignancies also seems to be related to the dosage of 32P. Patients who developed malignancies generally received higher doses of 32P (30 vs 20 mCi). Therefore, consistent phlebotomy therapy and restrictive chemotherapy combined with low dosage salicylates appears to be superior to 32P-therapy.

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.

lac repressor forms loops with linear DNA carrying two suitably spaced lac operators.

Tetrameric lac repressor may bind to two lac operators on one DNA fragment and induce the intervening DNA to form a loop. Electron microscopy, non-denaturing polyacrylamide gel electrophoresis, and DNase I protection experiments were used to demonstrate such DNA loops, where the distance between the centres of symmetry of the two lac operators varies between 63 and 535 bp. Formation of a DNA loop is favoured by correct phasing of the two lac operators and a low concentration of both components of the reaction. When a large excess of lac repressor over DNA is used, a 'tandem' structure is observed, in which both lac operators are occupied independently by two repressor tetramers. When the concentrations of both lac repressor and lac operator are high, a 'sandwich' structure is observed, in which two DNA molecules are connected by two lac repressor tetramers in trans.