Effect of prior cancer chemotherapy on human tumor-specific cytotoxicity in vitro in response to immunopotentiating biologic response modifiers.

Tumor-specific cytotoxicity was measured in fresh human biopsy specimens by a modification of the differential staining cytotoxicity assay. ImuVert, a cytokine inducer derived from Serratia marcescens, which produces broad-spectrum activation of both macrophages and lymphocytes, was dramatically more effective when it was tested in tumors obtained from patients with previously treated, chemotherapy-responsive adenocarcinomas (breast and ovary) than when it was tested in tumors obtained from either previously untreated patients or previously treated patients with chemotherapy-refractory adenocarcinomas (colon, lung, pancreas, stomach, kidney, gallbladder, uterus, and prostate). Similar findings, relating to prior chemotherapy treatment status, were obtained for tumor necrosis factor and interferon gamma, but not for interleukin-2 or interferon alpha. On the basis of these findings and on other evidence in the literature, we speculate that response to chemotherapy produces massive release and processing of tumor antigens. We further speculate that this response leads to a state in which the human immune system is primed (via in situ vaccination) to respond to exogenous macrophage-activation signals with potent, specific antitumor effects.

Murine cytomegalovirus-inhibitory effects of ImuVert.

ImuVert, a sterile preparation composed primarily of Serratia marcescens membrane vesicles and ribosomes, was significantly inhibitory to murine cytomegalovirus (MCMV) infections in BALB/c mice. Antiviral activity was manifested as increased survivor number and decreased recoverable virus titers in spleens, lungs and salivary glands. Treatments were intraperitoneal (i.p.) beginning 24 h pre, 4 h post- or 24 h post-virus inoculation and then repeated 4 days later. Doses of 5, 16 or 50 micrograms/mouse were effective; 160 micrograms/mouse, which caused host weight loss in toxicity controls, was not inhibitory to the infection. A single i.p. treatment of mice substantially augmented natural killer (NK) cell activity and increased total B-cells, while reducing total T- and T-helper cells. A late (48 h) decline in T-cell function and transient increases in B-cell function were observed in the treated animals. Serum interferon was not induced. Mice pretreated with anti-asialo GM1 antibody to reduce their NK cell populations, then infected with MCMV and treated with ImuVert were protected to the same degree as normal animals. Severe combined immunodeficient mice infected with MCMV and treated with ImuVert were not protected from the infection. These data suggest ImuVert to act by a mechanism other than NK cell activation in preventing MCMV infections.

Protection from chemotherapy-induced neutropenia by ImuVert.

Neutropenia is a major cause of chemotherapy-induced morbidity. This study examines the effect of ImuVert on the course of neutropenia from Cytoxan and Adriamycin. In adult rats treated with either Cytoxan or Adriamycin and ImuVert, the absolute neutrophil count remained above 7,000 and 2,000, respectively, for the duration of ImuVert therapy. In contrast, in the control Cytoxan group the absolute neutrophil count reached 300/mm3 on day 5 and remained below 1,000 from day 4 through day 7. In the Adriamycin control group, the nadir absolute neutrophil count reached 600/mm3 on day 7 and remained below 100 from day 6 through day 8. C3H/EJ mice (endotoxin-hyporesponsive) treated with Cytoxan developed an absolute neutrophil count below 1,000 from day 4 through day 7. In contrast, in the group concomitantly treated with ImuVert, the nadir absolute neutrophil count remained below 1,000 only on days 4 and 5. The authors conclude that stimulation of endogenous cytokine production by ImuVert may provide a potentially useful approach to bone marrow rescue from chemotherapy.

Use of a biological extract of Serratia marcescens to decrease doxorubicin-induced myelosuppression in dogs.

Fifteen dogs were given doxorubicin, IV, at a dosage of 30 mg/m2 of body surface. A commercially available biological extract of Serratia marcescens (BESM) was administered SC to 9 of these dogs (0.04 mg/kg of body weight every third day, n = 2; 0.08 mg/kg every other day, n = 2; and 0.08 mg/kg daily, n = 5), beginning the day after administration of doxorubicin, in an attempt to find an optimal dosage and schedule of administration of BESM to reduce the duration and severity of chemotherapy-induced myelosuppression. Nine additional dogs were randomized into 3 groups of 3 dogs to receive 1 of the following dosages of BESM SC: 0.08, 0.16, and 0.32 mg/kg. Serum was harvested immediately prior to treatment and at 2, 4, 6, 8, 12, 24, 48, and 72 hours from this latter group of dogs for subsequent analysis of canine granulocyte colony-stimulating factor (G-CSF) by enzyme immunoassay. Increasing the dosage and schedule of administration of BESM reduced the duration and severity of doxorubicin-induced myelosuppression. Neutrophil counts of the group of dogs given BESM daily at a dosage of 0.08 mg/kg and the controls were evaluated statistically. The neutrophil count increased significantly (P < 0.05) above pretreatment values in BESM-treated dogs after day 7. Median neutrophil counts of the BESM-treated dogs were never significantly lower than pretreatment values, whereas the median counts of the dogs treated with doxorubicin alone were significantly below normal for 6 days (days 7-12).(ABSTRACT TRUNCATED AT 250 WORDS)

The possible role of Limulus-amebocyte-lysate-reactive material in hemodialysis. First-use syndrome.

Hypersensitivity-like reactions are an infrequent but persistent clinical manifestation of hemodialysis. Although a Limulus-amebocyte-lysate-reactive material (LAL-RM) has been demonstrated in some hemodialyzers, its potential role in causing anaphylactoid reactions has not been addressed heretofore. In this chapter, current knowledge about LAL-RM will be presented and integrated with the major mechanisms generally recognized to induce hypersensitivity-type reactions. These mechanisms include: classical induction of allergy by IgE and classical complement activation by IgG and IgM. Alternative complement activation will be not discussed as it has been extensively reported in the literature of hemodialysis and because LAL-RM has not been shown to activate C3a extensively.

The Limulus amebocyte lysate endotoxin assay: current status.

Research conducted during this decade has clearly established the Limulus amebocyte lysate (LAL) test as the most sensitive assay for bacterial endotoxin. In addition, the assay is easily performed, relatively inexpensive, specific for endotoxin, and can be used to test materials that are toxic to rabbits employed in the rabbit pyrogen test. The LAL test has been used in the diagnosis of meningitis, urinary infection, mastitis, and endotoxemia. Other broad applications of the test include quality assurance of parenteral solutions and medical devices as well as the microbial quality of food and water. A review of recent developments in LAL testing is presented and experimental applications of the assay are discussed.

ImuVert activation of natural killer cytotoxicity and interferon gamma production via CD16 triggering.

The effect and mechanism of action of ImuVert, a new biological response modifier consisting of ribosomes and natural membrane vesicles of Serratia marcescens, on endogenous natural killer (NK) cells and activated NK activity has been analyzed. The studies showed that endogenous NK activity of peripheral blood mononuclear cells (PBMC) from normal cell donors was significantly increased (P less than 0.03) against K562, U937, and Molt-4 target cells. PBMC from cord blood of newborn infants lacking NK activity were upregulated (1.5-4 fold over endogenous NK activity) by ImuVert. Other studies showed that the abnormal NK activity of PBMC from patients with the human immunodeficiency virus (HIV) infection was significantly augmented in vitro (P less than 0.01) by ImuVert. ImuVert strongly stimulated interferon gamma production and in combination with interleukin 2 produced synergistically enhanced interferon gamma production and greater cytotoxicity than that induced by either alone. Studies on lymphocyte differentiation antigen expression following treatment with ImuVert indicated that ImuVert triggers interferon gamma production through binding the low affinity IgG Fc receptor, type III, CD16. The studies suggest that ImuVert may trigger interferon gamma production by binding to the Fc receptor and that the amplitude of the ensuing reaction and the ability of ImuVert to induce cytotoxicity in a setting where this activity has been down regulated is based on the absence of suppressor activation or direct contra suppressor activity.

Determination of endotoxin levels and their impact on interleukin-1 generation in continuous ambulatory peritoneal dialysis and hemodialysis.

Endotoxins represent a family of ubiquitous bacterial lipopolysaccharides found in water and raw materials. These substances have the ability to generate interleukin-1 (IL-1) and induce fever, as well as other acute phase phenomena. A study was undertaken to determine levels of background endotoxin in (1) continuous ambulatory peritoneal dialysis solution, (2) spent dialysate subsequent to overnight dwell, (3) hemodialysis solution, and (4) Limulus amebocyte lysate-reactive material (LAL-RM) in hemodialyzers and patient plasma. Levels of endotoxin in all of the above cases were less than thought to be required to induce biological activity, such as pyrogenicity, through IL-1 generation. Although nanogram amounts of LAL-RM are associated with some hollow-fiber membranes as well as the plasma of patients on those membranes, this material per se does not appear to produce IL-1 in vitro.

Selective potentiation of host resistance in mice following treatment with Pyrexol.

The purpose of this study was to examine the effect of treatment with the biologic response modifier Pyrexol on murine host resistance to various infectious organisms. Adult female CD1 mice were treated with a single subcutaneous 100-micrograms injection of Pyrexol at 14, 7, 5, 2, or 1 day prior to infection with various infectious organisms. These organisms included the Herpes simplex type 2 and influenza viruses, as well as the bacteria Listeria monocytogenes and Streptococcus zooepidemicus. Pyrexol treatment was found to significantly potentiate resistance to Listeria organisms, but had no appreciable effect on resistance to any of the other organisms tested. Previous reports have demonstrated that treatment with Pyrexol augments a number of cell-mediated immune parameters, several of which have been shown to be responsible for the elimination of Listeria organisms. These results suggest that Pyrexol is capable of selectively potentiating host resistance to infection.

Dialyzer hypersensitivity syndrome: possible role of allergy to ethylene oxide. Report of 4 cases and review of the literature.

The occurrence of hypersensitivity reactions in patients dialyzed on artificial kidneys is well recognized in the dialysis community, but has received little attention in the medical literature. We report 4 cases of dialyzer hypersensitivity reactions and review the previously reported cases. Dialyzer hypersensitivity syndrome presents as an acute anaphylactoid reaction, the symptoms of which may range from mild to life threatening in severity. The cause of this syndrome is unknown, but affected patients appear to have a high incidence of positive radioallergosorbent tests to a conjugate of human serum albumin and ethylene oxide, suggesting that ethylene oxide, a substance used to dry sterilize artificial kidneys, may be an offending allergen.

Establishment of beta-hydroxy fatty acids as chemical marker molecules for bacterial endotoxin by gas chromatography-mass spectrometry.

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.

Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests.

Four commonly used reference endotoxin standards, Escherichia coli O113:H10:K0, E. coli O55:B5, Salmonella abortusequi, and Shigella dysenteriae were compared by the USP rabbit pyrogen and the Limulus amoebocyte lysate tests. By the rabbit pyrogen test, S. abortus equi was identified as the most potent endotoxin, followed closely by E. coli O113:H10:K0 and E. coli O55:B5.

Detection of endotoxin in the plasma of patients with gram-negative bacterial sepsis by the Limulus amoebocyte lysate assay.

A total of 120 Limulus amoebocyte lysate (LAL) determinations were made on plasma obtained from normal, healthy human blood donors. Results demonstrated a mean endotoxin level in blood of 0.02 to 1.57 pg/ml. The amount of Escherichia coli endotoxin added to human plasma samples can be quantitated by both nephelometry and turbidimetry. Endotoxin-spiked samples were shown to be significantly different from unspiked samples. When plasma samples were collected from 45 patients hospitalized at three centers, a strong association was demonstrated between a positive Limulus amoebocyte lysate assay and a septic condition. Sensitivity, specificity, and false-positive and false-negative rates for the Limulus amoebocyte lysate assay as a diagnostic test for gram-negative bacteremia were estimated.

Allergic reactions in healthy plateletpheresis donors caused by sensitization to ethylene oxide gas.

We observed immediate-type hypersensitivity reactions in 6 of 600 donors who underwent automated plateletpheresis procedures. Ethylene oxide gas, which was used to sterilize plastic components in the disposable apheresis kits, represented a possible source of sensitization. In skin-prick testing, 4 of the 6 donors who had had a hypersensitivity reaction and none of 40 controls who had not had such a reaction had positive tests when an ethylene oxide-human serum albumin reagent was used (P less than 0.001). Radioallergosorbent testing revealed that serum from 4 of the 6 donors who had reactions, but from only 1 of 145 controls, contained IgE antibodies to ethylene oxide-albumin (P less than 0.001). All six of the donors who had reactions had specific ethylene oxide-induced basophil histamine release (mean release [+/- SE], 34.2 +/- 5.6 percent), whereas none of four controls had specific histamine release in response to ethylene oxide (mean release, 5.7 +/- 1.2 percent; P less than 0.005). Repeat plateletpheresis in five of the donors who reacted was accompanied by a recurrence of mild allergic symptoms. These studies demonstrate that sensitization to ethylene oxide gas can occur in healthy plateletpheresis donors and that it may result in immediate hypersensitivity reactions during the donation. The prevalence of such reactions was 1.0 percent in our apheresis donor population.

In vitro susceptibility of selected bacteria to three diphosphonia heterocyclic compounds.

The antibacterial activity of 1,1,4,4-tetraphenyl-1,4-diphosphonia-2,5-diphenylcyclohexadi-2,5-ene dichloride and two of its derivatives were screened for activity using the disk method. All derivatives exhibited antibacterial activity against gram-positive organisms tested, but little inhibition of gram-negative species was noted.

Immunologic studies of hollow-fiber dialyzer extracts.

Testing failed to detect free isocyanates in dialyzer rinses or significant levels of immunoglobulin E antibodies to isocyanate-protein conjugates in the serum of individuals reacting during hemodialysis with dialyzers containing Cuprophan membranes. Extended immunization of rabbits with Limulus lysate-reactive material from dialyzer rinses demonstrated antigenic activity of the rinse that appears to originate from cotton linters used in hollow-fiber membranes. The possibility that an immunologic response to these antigens may be a mechanism in adverse reactions to hemodialysis is discussed.

Allergy to ethylene oxide in chronic hemodialysis patients.

Almost a decade ago, a single chronic hemodialysis patient in Hamilton, Ontario, Canada, who was experiencing recurrent acute reactions during hemodialysis was found to have become allergic to ethylene oxide gas (EO). This observation recently provided a stimulus to examine the possibility that others exhibiting acute allergic-type reactions during hemodialysis might be similarly sensitized. Serum was obtained from 27 reacting patients and tested in a radioallergosorbent test (RAST) for antibodies to EO. The test was positive for 22 of these sera. EO-related specificity of the antibodies was confirmed. However, EO sensitization was not found in other hemodialysis patients with isolated eosinophilia. In a survey of the current Hamilton chronic hemodialysis population for EO-related antibodies, 9% had a positive allergy skin test and 12% had a positive RAST. The sensitized individuals had no distinctive symptoms. The lesser sensitivity of the skin test contrasts with usual findings in allergic individuals; however, the use of histamine and codeine in chronic hemodialysis and peritoneal dialysis patients might well explain a reduced sensitivity of the EO-related allergy skin test. Chronic peritoneal dialysis could not be invoked as a source of sensitization. None of these patients had a positive skin test or RAST for EO-related antibodies that could be attributed to peritoneal dialysis.

Evaluation of papain/chymopapain cross allergenicity.

Recent clinical evidence suggests that papain and chymopapain may share common allergenicity. Patients that become sensitized to papain may subsequently experience an allergic reaction when they are exposed to chymopapain. This study demonstrates a cross antigenicity between the proteolytic enzyme preparations papain and chymopapain. Serum samples from six patients who demonstrated 4+ skin reactions to papain also revealed positive RAST ratios to both papain and chymopapain. In addition, serum samples from 12 clinically nonreactive patients who had discolysis with chymopapain demonstrated positive RAST results to papain as well as to chymopapain.

Characterization of Limulus amoebocyte lysate-reactive material from hollow-fiber dialyzers.

Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amoebocyte lysate test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested before and after treatment with cellulase, using three lysates and four Limulus amoebocyte lysate methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified by chromatographic methods. Results indicate that the Limulus amoebocyte lysate reactive material is cellulose derived and not pyrogenic.

Comparison of chemical analyses of hollow-fiber dialyzer extracts.

Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amebocyte lysate (LAL) test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested using three lysates and four LAL methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified using chromatographic methods. Results indicate that the LAL-reactive material is cellulose derived and is not pyrogenic.