Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity.

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.

Polyethylene glycol (PEG)-modified granulocyte-macrophage colony-stimulating factor (GM-CSF) with conserved biological activity.

Polyethylene glycol (PEG) modification improves the pharmacological properties of proteins, usually extending plasma half-life and concomitantly increasing in vivo bioactivity, reducing both antigenicity and immunogenicity, and increasing solubility and resistance to proteolysis. Despite these established benefits, few PEG proteins are in use. Current coupling methods are either traumatic for the protein or involve lengthy and difficult procedures to activate monomethoxyPEG (MPEG). We have applied a new coupling method that allows coupling of MPEG directly to proteins under physiological conditions. Using this method with recombinant human (rh)granulocyte-macrophage colony-stimulating factor (GM-CSF) we were able to construct biologically active PEG-GM-CSF. Fast protein liquid chromatography (FPLC) and phase-partitioning confirmed the presence of PEG modification, and the former was used to fractionate modified and unmodified material. Bioactivity was measured in colony assays of normal human bone marrow cells and by tritiated thymidine uptake (of chronic myeloid leukemia cells and TF-1 cells). With both uptake and colony assays, using unfractionated material, we observed only a modest reduction in biological activity. Assays of FPLC-fractionated material confirmed that much of the bioactivity of the PEG-GM-CSF preparations was due to the modified species and any residual unmodified GM-CSF. Species uncontaminated by tresylmonomethoxyPEG (TMPEG; which was somewhat inhibitory in the thymidine uptake assay and eluted over a broad region of the FPLC profile) had no significant reduction in activity, but we cannot rule out the possibility that PEG-GM-CSF species eluting elsewhere in the profile had modest reduction of activity. Subcutaneous injection into mice confirmed the anticipated improved half-life in vivo and demonstrated a longer uptake from the injection site. This is, as far as we are aware, the first successful construction of PEG-GM-CSF with conserved biological activity.

Prolonged interferon-gamma application by subcutaneous infusion in cancer patients: differential response of serum CD14, neopterin, and monocyte HLA class I and II antigens.

This study reports on biological response modification induced by prolonged continuous subcutaneous (s.c.) infusion of recombinant interferon-gamma (rIFN-gamma) with particular attention to changes of soluble CD14. This glycoprotein with an unknown function is derived from myeloid cells carrying membrane CD14, which is the receptor for lipopolysaccharide (LPS)-LPS-binding protein (LBP) complexes. Fifteen metastatic cancer patients received weekly escalating doses of rIFN-gamma starting at either 50 or 100 micrograms/24 h and increasing up to 400 micrograms/24 h for a median duration of 6 weeks. The maximum tolerated dose was higher (200 micrograms/24 h) with the lower (50 micrograms/24 h) starting dose. Biological activity of rIFN-gamma was evaluated by weekly measurements of CD14, neopterin, and beta 2-microglobulin concentrations in serum as well as monocyte HLA class I and II antigen expression and tumor cytotoxicity. Serum IFN-gamma concentrations increased 20-fold within 4 weeks of therapy. The levels were correlated to the mean dose (r = 0.95, p less than 0.05). Among the biological markers, two patterns were observed. First, serum CD14 concentration and expression of monocyte HLA class II antigens increased significantly during the first week, and marker expression correlated with serum IFN-gamma levels (p less than 0.05); CD14 and HLA class II antigens thereafter returned to pretreatment levels within 4 weeks of therapy despite persistently elevated serum IFN-gamma concentrations. Second, serum neopterin and beta 2-microglobulin concentrations as well as monocyte HLA class I expression also increased significantly within the first week, but remained elevated thereafter without any further dose relationship.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of Cutaneous T-cell Lymphoma with Cyclosporin A: Case Report and Literature Review.

A 48 year old Caucasian man with cutaneous T-cell lymphoma, stage IVB is described. Initially he presented with an ichthyosis-like dermatosis and developed lymphadenopathy, hypercalcaemia, hepatosplenomegaly and lymphomatoid papulosis over a period of 7 years. Following splenectomy he developed a leukemic phase which was refractory to polychemotherapy. Therapy with cyclosporin A initially caused deterioration of the skin infiltrations and the peripheral blood T-helper lymphocytosis. However after 3 weeks an improvement of the patient's general condition was noted including decreased skin involvement, reduction of hepatomegaly and a sustained decrease of the absolute lymphocytosis, which lasted for ten months. Lymphomatoid papulosis was not significantly affected by cyclosporin A. In conclusion, this case and several case reports from the literature suggest that cyclosporin A may be valuable in the palliative treatment of selected patients with advanced cutaneous T-cell lymphoma.

[What is the value of tumor debulking and intraperitoneal cytostasis]. / Sind Tumor-Debulking und intraperitoneale Zytostase sinnvoll.

Local recurrences of intraperitoneal malignancies are mainly localized at the primary tumor site and the peritoneal surface. Surgical procedures alone are neither able to further reduce the development of local recurrence nor to treat the already established recurrent disease. In this situation intraperitoneal chemotherapy as an "adjuvant" treatment modality could be thought helpful. Experimental and clinical trials have shown that high doses of cytotoxic agents can be applied to the abdominal cavity with less systemic side effects. While the recent published data are not promising for gastrointestinal malignancies, they seem to be more encouraging for ovarian cancer.

Recombinant interferon gamma up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients.

The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon gamma (IFN gamma) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon gamma (rIFN gamma). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 micrograms/day or 100 micrograms/day and increasing weekly up to 600 micrograms/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 micrograms/day for a median duration of 43 days. Serum IFN gamma concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN gamma concentration was positively correlated with the rIFN gamma dose (P less than 0.05). Therapy induced a dose-dependent enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 micrograms rIFN gamma/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN gamma concentration (starting dose 100 micrograms/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN gamma levels (starting dose 50 micrograms/day). Since rIFN gamma is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN gamma concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN gamma in vivo and in vitro. In vivo, secondary mediators, induced by rIFN gamma and acting on a constantly renewed cell population, may contribute to the enhanced CD14 expression.

[Cost aspects of oncologic chemotherapy]. / Kostenaspekte onkologischer Chemotherapien.

The costs chemotherapy for eight patients with different diagnoses treated between 1983 and 1985 were analyzed, based on case histories and invoices submitted to medical insurance companies. Total monthly costs of therapy included drug costs, diagnostic costs, fees and hospital charges. The total monthly chemotherapy costs ranged between SFr. 379.- and SFr. 6065.- and between SFr. 874.- and SFr. 3476.- for 5 outpatients and two inpatients respectively. Total monthly drug costs ranged between SFr. 103.- (Fluorouracil) and SFr. 1744.- (Adriamycin) for palliative therapies, and between SFr. 620.- (MOPP-ABVD) and SFr. 2490.- (Cis-Platin, Velbe, Bleomycin) for curative therapies. The drug costs (Methotrexate) of a third inpatient exceeded the flat rate paid by insurance, the remaining costs being covered by the state. About 50% of cancer chemotherapy costs are due to drug costs. Drug costs of curative therapies seem to be higher than those of palliative therapies. A reduction of total costs by more than 30% is possible if hospitalisation is avoided, which might be inacceptable for some patients. Our method gives an incomplete picture of total chemotherapy-related costs because it does not consider loss of earnings and costs borne by the state to cover the deficit. Possibilities to reduce chemotherapy costs in tumor patients are discussed.

Intraperitoneal chemotherapy in peritoneal malignancy: impact of intensive system care on practicability.

In a group of 31 patients a port-a-cath-system for intraperitoneal chemotherapy was installed. From 23 evaluable patients 9 showed a tumor remission, but only 1 was a patient with gastrointestinal carcinoma. All others were patients with ovarial carcinoma. The median survival rate of all patients was 9 months after implantation of the intraperitoneal catheter. 50% of all patients with gastrointestinal carcinoma died within the first 6 months, only 1 of 11 patients with ovarial carcinoma died in this period.

Capillary sclerosis of the urinary tract and analgesic nephropathy.

Morphology, frequency and significance of capillary sclerosis (CS) in the ureter and electron microscopic findings in early papillary necrosis are described. CS of the urinary tract is characterized by a thickening of the basement membrane of capillaries lying just underneath the urothelium. The basement membrane changes can be demonstrated by PAS, Sudan stain and autofluorescence with equal reliability. By electron microscopy the thickened basement membranes exhibit a tree ring like pattern permeated by lipid vacuoles. CS is most often present in the renal pelvis and the ureter and only in particularly severe cases also in the urinary bladder. The most severe CS is found in the proximal and middle third of the ureter. In a prospective autopsy study CS was found in 3.5% of autopsies of adults and in 83% of clinically recognized phenacetin abusers. Since there is no association with other renal or metabolic diseases, CS can be considered as specific for phenacetin abuse. This finding is further substantiated by a significant correlation between the degree of severity of capillary sclerosis and the daily dose of phenacetin in grams. In about half of the patients with known analgesic abuse but without CS, possible causes for the lack of CS can be identified, of which the most important is regression of CS after stopping the abuse. Electron microscopic studies of early papillary necrosis show the same BM changes as in the ureter in peritubular capillaries, loops of Henle and similar BM alterations in the collecting ducts. The morphologic findings in the ureter and in the renal papilla suggest that CS in papillary necrosis are the consequence of a toxic damage of endothelial and in the kidney of endothelial and epithelial cells.

Phenacetin abuse and malignant tumors. An autopsy study covering 25 years (1953-1977).

The findings presented in this study are based on 29.226 autopsies performed (between 1953 and 1977) at Basel University on adult inhabitants of Basel, from which 409 urinary tract tumors (UTT) and 513 phenacetin abusers (PA) were discovered. There were 44 (8.6%) PA with UTT which, when compared with the control group (1.27%), represents a statistically significant increased incidence. Of the 50 UTT in PA, 52% occurred in the bladder, 6% in the ureter, and 42% in the renal pelvis. The induction time for tumors of the urinary bladder was about 27 years, and for tumors of the renal pelvis about 20 years. The commonest tumors arising in PA were invasive solid and non-invasive papillary urothelial carcinomas. PA with UTT died earlier than nonabusers but had metastases as frequently as nonabusers. Analgesic nephropathy was not always an accompanying disease. The daily dose of g/phenacetin in tumor patients in general, and in patients with tumors of the urinary bladder in particular, was about 1 g lower than in patients with analgesic nephropathy (without tumors) and in those with tumors of the renal pelvis. Thus, for the localization of the tumors, the daily dose seems to be more important than the total dose. Our investigation proved that not only tumors of the renal pelvis, but also of the ureter and the urinary bladder, are significantly more frequent in PA than in nonabusers. It is suspected that despite restriction of the over-the-counter sale of phenacetin-containing analgesics and even after stopping any analgesic abuse, UTT will further increase due to the longer induction time. Routine cytological screening tests of the urine are recommended for all known PA. A prescription for all phenacetin- and paracetamol-containing analgesics is necessary.

[Phanacetin abuse III. Malignant urinary tract tumors in phenacetin abuse in Basle 1963-1977]. / Phenacetinabusus III. Maligne Harnwegtumoren bei Phenacetinabusus in Basel 1963-1977.

In 442 inhabitants of Basel 451 malignant tumors of the lower urinary tract were found at autopsy or biopsy from 1963 to 1977. 69/442 patients were abusers of phenacetin containing analgesics. Carcinomas and sarcomas of the lower urinary tract were nearly 13 times as frequent in abusers as in non-abusers. Carcinomas of the renal pelvis were 77 times, carcinomas of the ureter 89 times and those of the urinary bladder 7 times as frequent among abusers. The differences in incidence of malignant tumors are statistically highly significant for all localizations and for multiple carcinomas as well, even if smokers are excluded. "Phenacetin tumors" occurred in younger patients and were more common in women than in non-abusers. They were ofen, though not always, accompanied by analgesic nephropathy. Comparison with other etiological factors such as aromatic amines or thorotrast demonstrated that phenacetin abuse is of greatest importance. The significance of smoking cannot be established unequivocally. Because of the occurrence of a large number of malignant tumors in phenacetin abusers it is very important to prohibit by legislation the use of phenacetin or paracetamol containing analgesics without medical prescription. In addition, these drugs should be replaced by other analgesic compounds.