muramidase and Carcinoma--Renal-Cell

To study the morphologic features, immunophenotypes, differential diagnoses and prognosis of histiocytic sarcoma (HS).. The clinical and pathologic findings of 6 cases of HS were reviewed. Immunohistochemical assay (Elivision staining) was also performed. Follow-up information was available in 4 patients.. There were altogether 3 males and 3 females. The age of patients ranged from 12 to 81 years old (median = 54.6 years). The sites of involvement included lymph node (number = 2 cases) and skin or soft tissue (number = 4 cases). The tumor was composed of sheets of large epithelioid cells with abundant eosinophilic cytoplasm, oval to irregular nuclei, vesicular chromatin and large nucleoli. Binucleated form was not uncommon. Two of the cases showed increased pleomorphism with multinucleated tumor giant cell formation. Focal cytoplasmic with foamy appearance was identified in 3 cases. One case demonstrated foci of spindly sarcomatoid appearance. Hemophagocytosis was identified in 2 cases. Mitotic figures were readily identified. The tumor cells were often accompanied by various numbers of inflammatory cells. Immunohistochemical study showed that all cases were diffusely positive for leukocyte common antigen, CD4, CD68 and CD163. Four of the 5 cases studied also expressed lysozyme. Amongst the 4 patients with follow-up information available, 3 died of the disease at 6 to 11 months interval after diagnosis. One patient, whose lesion was localized at the skin and soft tissue, survived for 3 years, with no evidence of tumor recurrence.. Accurate diagnosis of the HS is based on the combination of morphologic examination and immunohistochemical assay. HS often presents with clinically advanced disease and pursues an aggressive clinical course, with a poor response to therapy. However, a subset of cases presenting with clinically localized lesion may carry a relatively favorable long-term outcome.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Carcinoma, Renal Cell; Child; Diagnosis, Differential; Female; Follow-Up Studies; Histiocytic Sarcoma; Humans; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Large-Cell, Anaplastic; Male; Melanoma; Muramidase; Prognosis; Receptors, Cell Surface; Skin Neoplasms; Soft Tissue Neoplasms; Young Adult

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

Topics: ADP Ribose Transferases; Amino Acid Substitution; Anti-Bacterial Agents; Arginine; Bacterial Toxins; Carcinoma, Renal Cell; Cell Line, Tumor; Chromatography, Gel; Cloning, Molecular; Escherichia coli; Exotoxins; Genetic Vectors; Glutamine; Humans; Inclusion Bodies; Interleukin-13; Isopropyl Thiogalactoside; Kanamycin; Muramidase; Plasmids; Promoter Regions, Genetic; Protein Renaturation; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Time Factors; Transformation, Genetic; Virulence Factors