Human monocyte-released cytotoxic factor: effect on various cellular functions, and dependency of cytolysis on various metabolic processes.

The ability of the human monocyte-released cytotoxic protein factor(s) (CF) to mediate cytolysis when various metabolic processes in target cells were inhibited, and its effect on various cell functions have been studied. Cytolysis was reduced when target cells were exposed to either chloroquine or colchicine, indicating that lysosome function and the formation of microtubule are of importance with respect to the interaction between CF and target cells, possibly by effecting internalization and processing CF and its receptor. A decrease in the energy charge of the target cells did not affect the ability of CF to mediate cytolysis, since the 60-70% reduction of the cellular ATP content by uncoupling oxidative phosphorylation had little effect on CF-induced lysis. It was unnecessary for target cells to be actively growing and dividing for CF to induce lysis because inhibition of cellular protein, RNA, and DNA synthesis potentiated the cytolytic effect of CF. CF-induced cell lysis of Walter and Eliza Hall Institute (WEHI) 164 target cells was first detected between 3.0 and 4.5 hr after addition of CF. Target cell DNA synthesis nearly terminated within 3-4 hr, about the time of first cell lysis detection. Compared to the effect on DNA synthesis, the CF effect on protein synthesis was negligible and only a slight reduction was detected in the RNA synthesis and in the cellular ATP content. The results indicated that the DNA-synthesizing machinery may either be a primary target site of CF or a rapid influence of CF action on its primary target site; however, neither the protein- or RNA-synthesizing machinery nor the cellular ATP generating systems are likely to be primary target sites.

Production and characterization of cytostatic protein factors released from human monocytes during exposure to lipopolysaccharide and muramyl dipeptide.

The effect of activating human monocytes in vitro with lipopolysaccharide (LPS) and muramyl dipeptide (MDP) on the production of cytostatic protein factor(s) (CF) has been investigated, and an antiserum against CF has been raised and tested. Upon incubation for 7 hr with LPS, in vitro differentiated human monocytes released CF. During LPS exposure, the presence of the protein synthesis inhibitor cycloheximide, at concentrations which reduced the overall protein synthesis by 60 and 80%, reduced the amount of CF released by only 20 and 40%, respectively. This indicates that the released CF was to a large extent already present in the monocytes before exposure to LPS. Compared to LPS, MDP induced only modest CF release. However, when lymphokine-activated monocytes were exposed to MDP, an increased CF release was observed. By immunizing a rabbit with CF purified by ion-exchange chromatography, chromatofocusing, and gel filtration, an antiserum was raised which neutralized the cytostatic activity released from monocytes exposed to LPS or lymphokines/LPS in sequence on the fourth day of culture. The cytostatic activity obtained by incubating freshly isolated monocytes with LPS was inhibited by the antiserum to a lesser extent, indicating the presence of other cytotoxins or cytotoxic cellular products in addition to CF in supernatants from freshly isolated monocytes. Various CF preparations were tested for IL-1 activity; no correlation between IL-1 activity and cytostatic activity was observed. Moreover, upon gel filtration the CF and IL-1 activities could be separated from each other and are consequently associated with different proteins.

The role of monocyte cytotoxic factor (CF) in cytostasis mediated by IFN-gamma-activated monocytes.

The role of monocyte cytotoxic factor (CF) in cytostasis mediated by lymphokine- and interferon gamma (IFN-gamma)-activated monocytes was investigated. Recombinant IFN-gamma as well as lymphokines enhanced monocyte-mediated cytostasis concomitantly with an increase in monocyte CF content. The incubation of lymphokines with monoclonal antibody against human IFN-gamma inhibited cytostasis mediated by lymphokine-exposed monocytes, indicating that the main monocyte-activating factor in the lymphokine supernatant was IFN-gamma. A neutralizing antiserum raised against purified CF inhibited monocyte-mediated cytostasis in a dose-dependent manner. When CF antiserum was added to monocytes previously exposed to lymphokines treated with a monoclonal antibody against IFN-gamma, all cytostatic activity was completely abrogated. No inhibitory effect on cytostasis was observed with preimmune serum. The results indicate that CF is an effector molecule in cytostasis mediated by lymphokine- or IFN-gamma-activated monocytes.

[Myelomatosis and low level of vitamin B12]. / Myelomatose og lavt serum-vitamin B12-nivå.

Many patients with multiple myeloma tend to have low serum cobalamin. The cause of this remains unclear. The important issue is whether cobalamin therapy should be used or not. We describe one case of megaloblastic erythropoiesis and multiple myeloma, and refer to some of the few studies describing the subject. Most of the patients with multiple myeloma are elderly, and the frequency of hypo- and achlorhydria is therefore increased. It has been demonstrated that cobalamin uptake and consumption is higher in myeloma cells than in normal bone marrow cells, and that cobalamin may be required for paraprotein synthesis. These facts may suggest that patients with multiple myeloma are more vulnerable to developing megaloblastic anemia than others. Our patient received cobalamin therapy in addition to cytostatic therapy for multiple myeloma without complications. However, we cannot exclude that cobalamin therapy may accelerate multiple myeloma; this should be considered when such therapy is given. However, accurate guidelines require more studies.

Activation of human monocytes by free muramyl dipeptide (MDP).

Activation of human monocytes with MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) 1-100 micrograms per ml for 48 h in vitro enhanced the cytostatic activity against the target cell line K-562, while cytolysis remained unchanged. Catalase, 600 SU per ml, had no inhibitory effect on the cytostasis mediated by MDP-activated monocytes. The optimal MDP concentration for activation was in the range 3-10 micrograms per ml. Supernatants from monocytes activated with MDP 1-30 micrograms per ml for 48 h exerted no cytostatic activity. MDP 1-100 micrograms per ml had no direct cytostatic or cytolytic effect on the target cells in a 24 h assay. When added to monocytes cultured in vitro for four days immediately prior to the chemiluminescence (CL)-assay, MDP 10-100 micrograms per ml enhanced both the zymosan and phorbol myristate acetate-triggered lucigenin-dependent CL. Monocytes pre-activated with MDP for 48 h did not demonstrate any enhanced CL-response. MDP-activation 30 micrograms per ml for 48 h increased the zymosan-triggered generation of H2O2 moderately. The enhanced cytostatic activity induced by MDP-activation is probably not mediated by hydrogen peroxidase or production of cytostatic factors.

Purification of cytostatic protein factors released from human monocytes.

Human monocytes release cytostatic protein factors (CF) upon activation with lymphokines and lipopolysaccharide. CF has been purified from monocyte supernatants by ion-exchange chromatography, chromatofocusing and gel filtration, and this resulted in a 10,000-fold reduction in the amount of protein in the purified CF preparation compared to the amount in the monocyte supernatant. About 5% of the cytostatic activity was recovered after purification. CF constitutes a population of proteins heterogeneous with respect to ionic charge and differing in their isoelectric points, since CF eluted as a broad peak both upon ion-exchange chromatography and chromatofocusing. The isoelectric points of the CF proteins were determined by chromatofocusing to be between 6.0 and 5.0. The molecular weight of CF as determined by gel filtration was in the range of 45,000 to 35,000 daltons. Only one monocyte-secreted protein with a molecular weight of 40,000 was detected upon sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins in the purified CF preparations obtained after the final gel filtration step, and this protein may consequently be CF. If the 40,000-dalton protein is CF, one may estimate that about 10(6) monocytes produced roughly 0.1 microgram CF upon activation and that significant cytostasis may be detected with less than nanogram quantities of CF.

Effect of lymphokines on the production of cytostatic protein factors from human monocytes.

The effect of activating human monocytes in vitro with lymphokines on the production of cytostatic protein factor(s) (CF) was investigated. Upon exposing the monocytes to either lymphokines or lipopolysaccharide (LPS) the amount of CF released was increased approximately fivefold compared to the amount released from unexposed monocytes. With sequential lymphokine and LPS treatment CF release increased nearly 10-fold. Even 10 min lymphokine activation before LPS exposure enhanced CF production significantly. The enhanced CF production was detected between 5 and 10 hr after lymphokine activation. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide reduced the lymphokine-induced CF production in a dose-dependent manner, indicating that lymphokines augment both CF mRNA and CF protein synthesis. When monocytes were exposed to LPS on both Day 2 and Day 4 of culture, the amount of CF obtained on Day 4 was reduced compared to that obtained on Day 2. A significant increase in CF production, however, was observed when the monocytes were activated with lymphokines before the second exposure to LPS on Day 4, supporting the view that lymphokines initiate synthesis of CF in monocytes. Upon ion exchange chromatography, chromatofocusing, and gel filtration the same elution profiles of CF were obtained irrespectively of whether the monocytes had been activated with lymphokines or not. This indicates that lymphokines induce an increased production of the same factor(s) which was obtained in the absence of lymphokines.

IFN-gamma-induced production of monocyte cytotoxic factor.

The effect of activating human monocytes in vitro with recombinant Interferon-gamma (IFN-gamma) and lymphokines on monocyte-mediated cytotoxicity and on the production of cytotoxic protein factor(s) (CF), has been investigated. Lymphokines and IFN-gamma enhanced both cytotoxicity and CF production in a dose-dependent manner. A monoclonal antibody against human IFN-gamma abrogated the lymphokine-induced cytotoxic activity and CF production completely, indicating that the actual monocyte-activating factor in the lymphokine supernatant was IFN-gamma. At concentrations of 10(3)-10(4) U/ml, IFN-gamma induced an enhanced release of CF from the monocytes. However, IFN-gamma at concentrations (less than 100 U/ml) present in our lymphokine preparation was not sufficient to induce enhanced CF release. Since IFN-gamma at concentrations less than 100 U/ml activated monocytes for cytotoxicity, CF as an extracellular factor in the supernatant does not appear to be essential for monocyte-mediated cytotoxicity. However, using neutralizing antiserum raised against purified CF, indirect immunofluorescence microscopy revealed that IFN-gamma induced a marked accumulation of CF on the monocyte membrane. Taken together, the data shows that IFN-gamma is both necessary and sufficient at concentrations less than 100 U/ml for inducing monocyte-mediated cytotoxicity and production of CF as a membrane-associated component, but not as a released factor, suggesting that CF may function as a membrane-associated cytotoxin in monocyte-mediated cytotoxicity.

The role of monocyte cytotoxic factor in monocyte-mediated lysis of tumour cells.

The role of a monocyte cytotoxic factor (CF) in monocyte-mediated lysis of leukaemia cells (K562) has been investigated using a polyclonal rabbit antiserum raised against purified CF. The CF antiserum inhibited K562 cell lysis mediated by interferon gamma (IFN-gamma)-activated monocytes. CF antiserum also inhibited monocyte-mediated lysis of antibody-coated K562 cells (AbK562). Preimmune serum at the same concentration as CF antiserum did not affect monocyte-mediated lysis, and the CF antiserum did not inhibit binding between effector and target cells, indicating that inhibition of monocyte-mediated lysis by CF antiserum was not merely a result of toxic components present in the rabbit serum, or a result of a decrease in monocyte-target cell binding. Taken together, the data suggest that CF is involved in monocyte-mediated lysis of uncoated as well as antibody-coated K562 cells.

WEHI 164 sarcoma cells rendered resistant to monocyte-released cytotoxin are also resistant to monocyte-induced cytolysis.

WEHI 164 sarcoma cells cultured with monocyte-released cytotoxin (CF) for 4 weeks became resistant to CF-induced cytolysis and were concomitantly rendered resistant to monocyte-induced cytolysis. The resistant cell line (R-WEHI 164) has been stable with respect to resistance to monocyte- and CF-induced lysis for more than 7 months. WEHI 164 and R-WEHI 164 cells adsorbed CF and no significant difference in CF adsorption was observed. The results indicate that CF may be an effector molecule in monocyte-mediated cytolysis.

Monocyte-mediated drug-dependent cellular cytotoxicity: effects on different WEHI 164 target cell lines.

The contribution of monocyte cytotoxic protein factor (CF) to monocyte-mediated drug-dependent cellular cytotoxicity (DDCC) has been investigated. Cell lines which have been derived from murine WEHI 164 cells (termed WEHI 164 parental) by selecting for high (WEHI 164 clone 3) and low (R-WEHI 164) sensitivity to CF-mediated cytotoxicity were used as target cells in DDCC. By comparing the CF doses which produced 50% dead cells (LD 50) we found that WEHI 164 clone 3 was approximately 30 times more sensitive than WEHI 164 parental which in turn was 70 times more sensitive than R-WEHI 164. Actinomycin D (Act D) treatment of WEHI 164 parental and R-WEHI 164 greatly increase susceptibility to CF-mediated cytotoxicity. The susceptibility of WEHI 164 clone 3 was apparently somewhat increased at low dilutions of CF, whereas no significant increase was observed at high dilutions. The susceptibility to DDCC of the three target cell lines (WEHI 164 parental, WEHI 164 clone 3, and R-WEHI 164) correlated with the sensitivity pattern obtained in CF-mediated cytotoxicity of Act D-treated target cells. Monocyte- and CF-mediated cytotoxicity against Act D-treated WEHI 164 clone 3 and R-WEHI 164 was inhibited by neutralizing CF antiserum. These data indicate that CF is an effector molecule in monocyte-mediated DDCC.

[X-ray of small bowel with a probe--indications and diagnostic yield in a 10-year material]. / Røntgen tynntarm med sonde--indikasjoner og diagnostisk utbytte i et tiårsmateriale.

BACKGROUND: We wanted to evaluate the indications for ordering small bowel enema, and whether specific clinical symptoms and signs are associated with the diagnostic yield. MATERIAL AND METHODS: Medical charts and requisition slips for 241 patients examined with small bowel enema at Harstad Hospital from 1986 to 1995 were reviewed. RESULTS: The most common symptom leading to small bowel radiography was pain, registered in almost three fourths of the patients; about one half reported diarrhoea. Elevated sedimentation rate and occult blood in the faeces were reported in one fourth of the patients. For a large proportion of the patients, there was no information about adequate preliminary tests in the medical charts. Normal radiography was reported in three fourths of the patients. Lesions consistent with Crohn's disease were found in one of eight patients. No symptoms or signs, except for elevated sedimentation rate, clearly indicate a positive diagnostic finding. Many requisition slips did not contain available information. INTERPRETATION: Small bowel radiography is performed on wide indications. It is difficult to make a careful selection of patients based on reported symptoms and signs. However, some preliminary tests are helpful and should be done.

[Thrombolytic therapy in acute myocardial infarctions]. / Trombolytisk behandling ved akutt hjerteinfarkt.

Data on all patients with acute myocardial infarction who were treated in Harstad District Hospital in 1995 were analysed. Of the 170 patients, 24% received thrombolytic treatment. Thrombolytics were withheld from 15% of the patients, although there were no contraindications present. Thrombolytics were administered two hours and 18 minutes (mean) after admission to hospital and seven hours after the onset of symptoms. 54% of the patients were admitted to hospital within six hours and 73% within 12 hours. In-hospital delay before the administration of thrombolytics is too long. In Norwegian hospitals this factor has only been analysed to a minor degree. Despite a fairly standardized treatment regimen for thrombolytics, how frequently it is used probably varies from hospital to hospital. There is great potential for improving thrombolytic treatment. The results of our analyses have resulted in an extensive change in routine in our hospital.

[Congenital dyserythropoietic anemia type III. A case report]. / Kongenitt dyserytropoetisk anemi type III. En kasuistikk.

Congenital dyserythropoietic anaemia type III is a rare disorder characterized by mild to moderate anaemia, ineffective erythropoiesis, and morphologic abnormalities of mature red blood cells and their precursors. The most extraordinary feature of this condition is the large number of multinuclear erythroblasts found in the bone marrow, some containing up to 12 nuclei. This type of anaemia is an autosomal dominant inherited disorder, though sporadic cases have been described. Little conclusive is known about the pathogenesis of congenital dyserthropoietic anaemia type III. At present the management of patients consists of observation and supportive care. We describe a 20 year old man who was admitted to a county hospital, showing the typical features of this rare illness. He had a Hb value of 10.2 g/100 ml.

[Infrared tympanic thermometry compared to mercury thermometers]. / Infrarød tympanisk termometri sammenliknet med bruk av kvikksølvtermometer.

Because of conflicting reports we examined the accuracy of infrared tympanic thermometry compared with mercury rectal thermometer and a digital rectal thermometer in a medical ward. We studied prospectively the accuracy of parallel measurements with infrared tympanic thermometer and the correlation between this method and the rectal mercury thermometer. Measurements with digital rectal thermometry are also compared with mercury thermometry. 191 adult inpatients were included. The median difference between infrared tympanic and rectal mercury thermometry in the whole material was -0.5 degree C, but increased to -1.4 degrees C in a selected group with rectal temperature 38 degrees C or more. The median difference between parallel measurements with infrared tympanic thermometer was 0.4 degree C. Digital rectal thermometry, however, was in strict accordance with the rectal mercury method. We found an unacceptable difference in body temperature between measurements with infrared tympanic thermometer and rectal mercury thermometer. In a clinical setting the infrared ear thermometer has a very low sensitivity for detecting fever. Digital rectal thermometry seems to be a good alternative to the rectal mercury thermometer.

Primary sclerosing cholangitis with autoimmune hemolytic anemia and pulmonary infiltrations.

A 39-year-old man presenting with pulmonary infiltrations and hemolytic anemia was diagnosed as having primary sclerosing cholangitis (PSC) without evidence of ulcerative colitis. This constellation of associations is unique to the best of our knowledge. Autoimmune hemolytic anemia has been reported to be associated with PSC on only a few occasions, and pulmonary infiltrations in association with PSC are also quite unusual. Genetic and immunologic mechanisms are major factors in the pathogenesis of these disorders.

Corneal crystalline deposits in multiple myeloma. A case report.

A 70-year-old man with a seronegative rheumatoid arthritis showed some peculiar lamellar crystalline deposits mainly at the Descement/endothelial level in both eyes. Serum protein analysis revealed a monoclonal gammopathy with increased amounts of IgG and K-light chains along with traces of the latter in the urine. Corneal biopsy was not available, but electron microscopy of the conjunctival specimen demonstrated crystalline deposits within the endoplasmic reticulum of plasma cells lying within the connective tissue. This material is supposed to be of protein nature. It seems reasonable that the conjunctival deposits are identical to those seen in the cornea by slit-lamp examination, and it is also suggested that they represents paraprotein aggregates. After treatment with melphalan (Alkeran) and prednisone systemically, the corneal deposits decreased over a period of 9 months with corresponding improvement of the visual acuity.

Human monocyte-released cytotoxic factor. Modulation of cytotoxic activity by inhibitors of cellular DNA, RNA, and protein synthesis.

Human lymphokine-activated monocytes release a cytotoxic protein factor (CF). The cytostatic activity of CF was potentiated by the DNA and RNA synthesis inhibitors actinomycin D, daunomycin, and mitomycin C. These inhibitors increased the sensitivity for detecting CF approximately 10-fold, and this is of great practical value when assaying CF. The CF-potentiating inhibitors had a similar effect on the cell cycle distribution in that they all induced an accumulation of cells in the S and G2 phases of the cell cycle. Cytolytic activity was shown to be associated with CF, and this activity was also greatly potentiated by daunomycin and actinomycin D. The two other inhibitors studied, cycloheximide and 5-fluorouracil (5-FU), had an adverse effect on the cytostatic activity of CF. These two inhibitors reduced the sensitivity of the assay for CF about fivefold. Cycloheximide had no apparent effect on the relative cell cycle distribution, whereas 5-FU induced an accumulation of cells in the G1 phase. Of the inhibitors studied, only those that induced an accumulation of cells in the S and G2 phases of the cell cycle potentiated the cytotoxic activity of CF, suggesting that CF may preferentially act in these parts of the cell cycle.

A comparative study of infrared tympanic thermometry and rectal mercury thermometry.

In this study we compared infrared tympanic thermometry with rectal mercury thermometry and digital rectal thermometry in patients admitted to a medical department. We found that infrared tympanic thermometry has a low sensitivity for detecting fever. Digital rectal thermometry is a good alternative to rectal mercury thermometry.

Prognostic factors in multiple myeloma in a population-based trial.

Prognostic factors have been tested in patients with multiple myeloma treated according to a randomized trial of standard therapy versus 5-drug combination therapy. The following population-based study included 92 patients with a median age of 70 yr. The median survival was 31 months. The Cox regression model was used to search for predictors of survival. The cut-off levels for blood analyses derived in earlier studies tended to select few patients in the high-risk groups, for example only 8% of the patients had hemoglobin (Hb) less than or equal to 7.5 g/dl. Lytic bone lesions in the pelvis or in the long bones, or spontaneous fractures and age greater than 70 yr gave prognostic information in addition to anemia and impaired renal function. The MRC staging system was a better prognostic tool than the Durie & Salmon stages. Palliative treatment regimens which take quality of life into account should be considered carefully in multiple myeloma patients greater than 70 yr.