Treatment of hairy cell leukemia with granulocyte colony-stimulating factor and recombinant consensus interferon or recombinant interferon-alpha-2b.

Patients with hairy cell leukemia and neutropenia (absolute neutrophil count less than 1.5 x 10(9)/L) were treated with recombinant granulocyte colony-stimulating factor (G-CSF) at doses of 3.6 and 7.2 micrograms/kg by daily subcutaneous injection, until normalization of neutrophil counts occurred. Patients then received either recombinant interferon-alpha-2b (r-IFN-alpha-2b) or a unique IFN, recombinant consensus IFN (rIFN-con-1), each given at doses of 10 micrograms/m2 subcutaneously three times a week, coupled with continued daily G-CSF therapy, for 3 months. After 3 months the G-CSF was discontinued; patients continued to take IFN for 1 year. All 10 patients responded to G-CSF with normalization of neutrophil counts within 2 weeks; the increase in neutrophil counts was greater in previously splenectomized patients. Four patients were treated with r-IFN-alpha-2b, and six were treated with rIFN-con-1. No patients developed recurrent neutropenia with the initiation of IFN therapy. Nine patients are evaluable for response to IFN. Five of six patients demonstrated hematologic improvement with rIFN-con-1, with two patients obtaining complete responses. All three patients receiving r-IFN-alpha-2b demonstrated hematologic improvement; one complete response was observed. Toxicities of both IFNs included influenza-like symptoms. We conclude that G-CSF can abrogate the myelosuppressive effects of IFN, and may be a useful adjunct to this therapy in neutropenic patients. We conclude that rIFN-con-1, the product of a synthetic gene, has activity in the treatment of hairy cell leukemia, and merits clinical investigation in other settings.

Reversibility of acid denaturation of recombinant interferon-gamma.

It has been shown that interferon-gamma (IFN-gamma) loses activity after acid treatment and this property can be used to distinguish it from other types of interferons. Therefore, reversibility of acid denaturation of IFN-gamma was examined using the recombinant human protein. The fluorescence spectra showed that conformation of the protein is similar before and after acid treatment, suggesting reversibility of the acid denaturation. The antiviral activity of the protein was also identical in the same treatment. However, the antiviral activity was significantly reduced when it was determined by directly diluting the acidic samples into the assay medium containing high salts and serum proteins. Similar results were obtained with the recombinant murine IFN-gamma. This observation demonstrates that acid denaturation of the IFN-gamma is dependent on the way the protein is renatured, and hence that the difference in response to acid treatment between IFN-gamma and other interferons is quantitative rather than qualitative.

Enhanced treatment of Pneumocystis carinii pneumonia in rats with interferon-gamma and reduced doses of trimethoprim/sulfamethoxazole.

Interferon-gamma (IFN-gamma) was used to treat rats with steroid-induced Pneumocystis carinii pneumonia (PCP). Treatment with 427,000 U/day prophylactically prevented infection in this model. Treatment with 200,000 U, three times/week for 2 weeks caused a significant reduction in the number of cysts in the lungs and prolonged survival of the rats. In addition, IFN-gamma and trimethoprim/sulfamethoxazole behaved synergistically in the treatment of PCP in rats. Reduced dosages of each drug, when given together, caused an almost complete eradication of the infection. This may be a useful approach in patients with acquired immune deficiency syndrome in whom anti-Pneumocystis drugs are often toxic.

The effect of C-terminal processing on the activity of human interferon-gamma.

Homogeneous recombinant human interferon-gamma (IFN-gamma) obtained from Escherichia coli (E. coli) was treated with a protease-containing fraction prepared from mechanically lysed E. coli cells. Polyacrylamide gel electrophoresis of the resulting product revealed two major components of molecular weight less than that of intact IFN-gamma. These were purified by ion exchange chromatography in the presence of 7 M urea and shown to have intact IFN-gamma N-terminal sequences, suggesting that they resulted via C-terminal cleavages of IFN-gamma. Amino acid analysis indicated that 4 C-terminal residues of IFN-gamma were lacking in one, and 15 in the other. The species lacking 4 C-terminal residues had activities virtually indistinguishable from those of IFN-gamma in antiviral and growth inhibitory assays using Encepharomyocarditis-treated HeLa or T98G cells and in a macrophage activation assay using macrophage-like U937 cells. The species lacking 15 C-terminal residues had markedly decreased activities in each of these assays, and had decreased binding affinity for IFN-gamma cell surface receptors. These observations define the C-terminal residues important for IFN-gamma's biological activity--information which should be useful in designing analogs of IFN-gamma with enhanced or altered biological activities.

Role of single disulfide in recombinant human tumor necrosis factor-alpha.

Two analogs of tumor necrosis factor-alpha (TNF-alpha) were produced by in vitro site-directed mutagenesis. In these analogs, cysteine residues at positions 69 and 101, which form a disulfide bond, were changed to alanine or leucine. CD spectra showed that the analogs are apparently similar in secondary and tertiary structure to the natural sequence TNF-alpha. In addition, the molecular size of the analogs was identical to that of the natural sequence TNF-alpha as determined by gel filtration. However, fluorescence spectra and quenching indicated that the removal of the disulfide bond alters the local conformation around tryptophan residues. The cytolytic, macrophage activation, and lipogenic activities decreased in the order of the natural sequence TNF-alpha greater than the alanine analog greater than the leucine analog, suggesting that the surface involving the disulfide bond plays a role in these biological functions and the introduced modifications decrease the activity. Differential effect of the modifications was suggested in the antiviral activity, since in this assay only the leucine analog showed significantly lower activity.

Conformation and stability of two recombinant human interferon-alpha analogs.

Structural features of a recombinant E. coli derived interferon-alpha analog, interferon consensus1, was studied by circular dichroism and fluorescence spectroscopy. Circular dichroic spectra of the purified protein showed that it has about 70% alpha-helix and a distinct tertiary structure. These structural features are similar to those for a natural interferon-alpha subtype, interferon-alpha 2, indicating that the amino acid substitutions in interferon consensus1 apparently did not alter the protein structure. Another analog, interferon consensus5, which has Ser instead of Cys at residues 1 and 99 but is otherwise identical to interferon consensus1, was prepared to study the role of the disulfide bond between Cys 1 and 99. Circular dichroic and fluorescence spectra indicated similarity in the structure of these two analogs. However, interferon consensus1 was significantly more stable than interferon consensus5 against denaturation. pH unfolding experiments indicated that the former protein is more stable in the transition region by about 1.6 kcal/mol, which was interpreted in terms of the increased free energy of the denatured state due to an extra disulfide bond in interferon consensus1.

Structure of human tumor necrosis factor alpha derived from recombinant DNA.

Recombinant DNA derived tumor necrosis factor alpha, when expressed at a high level in Escherichia coli, appeared in the pellet and soluble fractions of disrupted cells. The protein was purified from the pellet fraction by solubilizing it in urea and reducing agent and was refolded into a buffer without these additives. The structure of the protein was identical with that purified from the soluble fraction without exposure to both reducing and denaturing agents, as demonstrated by circular dichroism, gel filtration, and sulfhydryl titration. As a reflection of the structural similarity, both purified proteins showed identical cytolytic activity on mouse L929 cells. The protein was characterized as an essentially nonhelical and beta-sheet-rich structure and possibly as a noncovalently associating oligomer. Two cysteine residues form an intrapolypeptide disulfide bond.

Structure and activity of recombinant human interferon-gamma analogs.

We have prepared interferon-gamma (IFN-gamma) analogs to study the structural role of particular amino acids in relation to their effects on antiviral activity. Three IFN-gamma analogs were prepared on the basis of predicted secondary structure. In two of the analogs, [Gln25]IFN-gamma and [Thr45]IFN-gamma, changes were made at residue 25 (Asn to Gln) and at residue 45 (Met to Thr), respectively. [Gln25Lys78]IFN-gamma had two changes, at residue 25 (Asn to Gln) and residue 78 (Asn to Lys). Another analog, [Cys-Tyr-Cys]IFN-gamma, incorporated Cys-Tyr-Cys at the amino terminus. Comparison of the structure and activity of these analogs with that of the natural sequence protein suggested that residues 25 and 78 are at the protein surface and play an important role in antiviral activity. The residue at position 45 was found to be important for maintaining the protein structure, as assessed by circular dichroism spectroscopy. The addition of Cys-Tyr-Cys resulted in a small perturbation of protein structure and a small decrease in antiviral activity.

The giant (gt) mutants of Drosophila melanogaster alter DNA metabolism.

Abnormalities in DNA metabolism have been found in third-instar females of Drosophila melanogaster that are heteroallelic or homoallelic for X-chromosomal giant (gt) mutations. Analysis of DNA metabolism in larval brain ganglia was carried out using alkaline sucrose gradient centrifugation, incorporation assays and a neutral filter elution assay. These analyses show that gt stocks synthesize DNA of a reduced molecular weight, have an unusually high frequency of spontaneous single and double-strand breaks, and exhibit a reduction in the normal inhibition of DNA synthesis following treatment with UV and the carcinogen AAAF. These phenomena are not associated with a defect in the repair of X-ray induced DNA breaks nor are they accompanied by any alterations in chromosome stability. Analysis of homozygous 1(2)gl larvae also reveal that these phenomena are specific to the gt locus and are thus not attributable solely to an extended developmental program. These findings strengthen the suggestion that the genetic instability associated with gt is related to perturbations in chromosome metabolism (Green 1982).