The role of endogenous interleukin-12 in resistance to murine cytomegalovirus (MCMV) infection and a novel action for endogenous IL-12 p40.

Biologically active interleukin-12 (IL-12), comprising a 40 kDa subunit (p40) covalently linked to a 35 kDa subunit (p35), is produced in response to a range of infectious stimuli. Here, we demonstrate that mice deficient in either IL-12 p40 (p40-/-) or IL-12 p35 (p35-/-) are susceptible to murine cytomegalovirus (MCMV) infection in terms of survival (Balb/c p35-/-) and viral clearance (Balb/c p35-/- and Balb/c p40-/-), and this susceptibility may be correlated to a deficiency in serum interferon-gamma (IFN-gamma) levels. These data support a role for endogenous IL-12 in controlling MCMV infection. The IL-12 p40 subunit is produced in excess of IL-12 p35, and to date the function of the excess endogenous p40 has been assumed to be one of IL-12 antagonism, as demonstrated by experiments with exogenous p40 both in vivo and in vitro. We show that Balb/c p35-/- alone are significantly compromised in survival of a sublethal infection and in clearance of virus from the spleen. These mice produce a very early IFN-gamma spike (8 h after infection) and an aberrant tumor necrosis factor-alpha (TNF-alpha) spike (day 2 after infection). MCMV infection has revealed an altered Balb/c p35-/- phenotype compared with Balb/c p40-/-, and this indicates that endogenous p40 may have an activity independent of and additional to IL-12 antagonism in vivo.

Colorectal resection in Victoria:a comparison of hospital based and individual audit.

BACKGROUND: The results of personal audit have not been tested against a hospital-based audit previously and the results of two such audits of colorectal resection in the State of Victoria have provided this opportunity. In addition, data reflecting the results of colorectal resection across a range of hospitals and surgeons in the Victorian community have been obtained. METHODS: A total of 535 patients undergoing a colorectal resection, with an anastomosis performed, were studied in two serially conducted prospective audits arranged by the Standards Sub-Committee of the Victorian State Committee. One study was public hospital-based and the second was based on voluntary reporting by individual surgeons. RESULTS: Similar results were obtained in each study, demonstrating the accuracy of individual reporting. The combined results (wound infection rate 12.3%, anastomotic leak rate 3.7% and mortality 4.5%) are compared to previously published data. CONCLUSIONS: In the State of Victoria the results of audit by individual surgeons performing colorectal resection were similar to the hospital-based audit. The results obtained compare favourably with previously published data.

Clinical indicators in colorectal surgery.

Two studies conducted in the state of Victoria have tested potential clinical indicators and the suggested thresholds for resection of colorectal carcinoma where an anastomosis has been performed. These studies involving 535 patients were independent of one another: one hospital based and one surgeon based. Threshold figures for these draft indicators have been compared with the study figures and found to be similar. It is suggested that wound infection (elective operation without formation of a stoma), anastomotic leak (clinically recognized) and mortality (elective operations in patients under the age of 80 years) are the most appropriate clinical indicators of colorectal resection for carcinoma.

The effect of levcromakalim (BRL 38227) on bladder function in patients with high spinal cord lesions.

The effects of the potassium channel opener levcromakalim (BRL 38227) 7.5 micrograms kg-1 were examined on urodynamic variables and blood pressure during inflow and voiding cystometry in six high spinal cord lesion patients. Levcromakalim administration significantly increased the duration of bladder contraction (197 +/- 128 s to 267 +/- 167 s, P < 0.05) and also reduced blood pressure (126 +/- 13/67 +/- 9 mm Hg to 104 +/- 25/52 +/- 12 mm Hg) but was without effect on other urodynamic parameters. Because of concerns about hypotensive responses, further studies involving higher doses of levcromakalim should be considered only if the drug was administered intravesically.

Intravesical bacille Calmette-Guérin in the treatment of superficial transitional cell carcinoma of the bladder.

OBJECTIVE: To determine the efficacy of intravesical bacillus Calmette-Guérin (BCG) in the treatment of patients with superficial transitional cell carcinoma (TCC) of the bladder, and to assess the impact of fibrin clot inhibitors. PATIENTS AND METHODS: A retrospective review of 56 patients with superficial TCC of the bladder, treated with intravesical BCG after initial transurethral resection (TUR) of raised or papillary lesions or cold cup biopsy of areas of carcinoma in situ (CIS), was performed. Patient drug histories were reviewed for evidence of ingestion of medication known to inhibit fibrin clot formation. The impact of such medication was assessed using the Chi-square test. RESULTS: Fifty-six patients were treated between 1987 and 1991 of whom 52 were evaluable. Eighteen patients (35%) had a complete response with a mean follow-up of 19 months. Six patients (60%) in the group with CIS had a complete response rate with a mean follow-up of 28 months. Seven patients (13%) developed local progression and required cystectomy or external beam radiotherapy. Six patients (12%) died from metastatic disease. Three patients (6%) had significant complications. The adverse impact of fibrin clot inhibitors was found to be significant. CONCLUSION: Intravesical BCG is effective in the treatment of superficial TCC, especially CIS. A careful drug history is important to identify fibrin clot inhibitors so that, if possible, they may be withdrawn prior to intravesical BCG treatment.

Bile duct injury during laparoscopic cholecystectomy: a report of the Standards Sub-committee of the Victorian State Committee of the Royal Australasian College of Surgeons.

A survey of Victorian surgeons performing laparoscopic cholecystectomy was carried out. This report discusses the bile duct injuries identified in the survey. Twelve injuries were recorded, a rate of 0.2%. Three of the 12 required formal repair, the other 9 being treated by T-tube alone. Possible mechanisms of these injuries, the experience of the surgeon, the role of operative cholangiography and delays in recognition of the injury are discussed.

Characterization of endothelial cells in murine long-term marrow culture. Implication for hemopoietic regulation.

Establishing the presence of various cell types in long-term marrow culture (LTMC) has an important bearing on understanding the regulation of stromal cell-related hemopoiesis. Controversy has surrounded the identity of the very large cells in murine LTMC; they have been given a variety of designations, including blanket cells. Using dual immunogold labeling with a recently derived monoclonal antibody, H513E3, and anti-human factor VIII antibodies, we have conclusively located endothelial cells in LTMC. Endothelial cells are relatively large, with thinly spread cytoplasm, and they overlie macrophages, granulocytes, and other less differentiated developing hemopoietic cells. Previously, demonstration of endothelial cells in LTMC had been difficult due to lack of specific markers in the mouse. We have demonstrated that LTMC endothelial cells have the exact location and ultrastructural characteristics as the previously described blanket cells. We propose that previous designations should not be continued and that these cells should be referred to as endothelial cells. The known functions of endothelial cells now become relevant to the understanding of stromal regulation of hemopoiesis.

Megakaryocyte maturation in long-term marrow culture.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.

A mouse endothelial cell-specific monoclonal antibody: its reactivity with LTMC endothelium.

The binding of a marrow cell-related monoclonal antibody (H513E3 MAb) has been investigated in long-term marrow cultures (LTMC) and in vivo. Immunogold labeling and electron microscopy revealed that this antibody labeled an endothelial-like cell. Cross-reaction of anti-human Factor VIII confirmed endothelial specificity of the H513E3 MAb. In addition, vessel endothelium (vena cava, aorta, and marrow) exhibited binding of the antibody. This antibody provides a unique tool to study the cellular architecture of LTMC and implicates endothelium as an important component of LTMC. The function of the endothelial cell-specific surface antigen is unknown, although preferential labeling of the upper surface of endothelial cells suggests that it may play a role in cell communication, particularly with the floating population. The H513E3 MAb reacts with an external membrane antigen, a property that makes this antibody particularly useful for fluorescences-activated sorting of endothelial cells.

Use of bone marrow somatic cell hybrid lines to generate monoclonal antibodies specifically reactive with rare marrow cells.

The technique of somatic cell hybridization has been applied to obtain new sources of immunogen for monoclonal antibody production. A marrow population enriched for rare and undifferentiated cells was hybridized with A9 cells. Mature cells were depleted by using mice at 8 days following 5-fluorouracil treatment and by using a sorting procedure. Selection of hybrids was in medium containing hypoxanthine, aminopterin, and thymidine (HAT). Chromosome analysis was used to verify that clones contained hybrid cells. An example of an antibody (H513E3) obtained by immunizing with a hybrid cell line (H5Scl1.4.4) is presented. The H513E3 antibody showed low reactivity with normal marrow (0%-3%), and hemopoietic cell lines of various types exhibited no cross-reaction. However, about 10% of cells from the adherent stromal layer of long-term marrow cultures reacted with the H513E3 antibody. A specific cell type was labeled that appeared to have endothelial-like morphology when observed with immunogold labeling and electron microscopy. The hybridization technique allows a cell of a particular differentiation lineage to be conserved, wholly or partially, in a cloned form, thus overcoming the heterogeneity of a normal marrow population.

Identification of a tumor cell-derived differentiation antigen on mouse colony-forming units in the spleen and progenitor cells.

Surface membrane antigen(s) expressed on a mouse mast cell line (FMP1) have also been shown to occur on hemopoietic spleen colony-forming units (CFU-S) and granulocyte/macrophage colony- and erythroid burst-forming cells, using a xeno-antiserum raised against FMP1 cells. This mast cell model has been used to obtain antiserum and large quantities of antigen for the biochemical identification of CFU-S and progenitor cell antigen(s). Immunoprecipitation of FMP1 membrane antigens with the antiserum and subsequent polyacrylamide gel electrophoresis revealed the presence of five membrane proteins with molecular weights of 28,000, 32,000, 36,000, 50,000, and 70,000. Mouse B-lymphoma cell line W279 which reacted with anti-FMP1 serum was found to possess three immunoprecipitable surface proteins with molecular weights of 32,000, 50,000, and 70,000. Attempts have been made to identify the antigen(s) expressed by CFU-S and progenitors which were revealed by immunoprecipitation from the tumor lines. The three lower-molecular-weight proteins (Mr 28,000-36,000) were chosen for initial study. Membrane extracts of FMP1 cells were fractionated on Sephacryl S-200, and selective pools of these antigens were made. Antisera to these pools exhibited complement-dependent cytotoxicity to FMP1 cells, bone marrow CFU-S, granulocyte/macrophage colony-forming cells, and erythroid burst-forming units. These antisera immunoprecipitated Mr 28,000, 32,000, and 36,000 proteins from FMP1 cell membrane extracts but not the Mr 50,000 and 70,000 antigens. The W279 line has only one antigen (Mr 32,000) in the lower-molecular-weight range and is able to absorb anti-CFU-S and anti-progenitor activity, which suggests that it is this antigen which is expressed on hemopoietic cells. In addition, thymocytes react with anti-FMP1 serum, and the Mr 32,000 antigen was immunoprecipitated from thymus cell extracts. Binding studies with concanavalin A, wheat germ agglutinin, and lentil lectin indicated that the Mr 28,000-36,000 proteins were glycoproteins. The apparent molecular weights of these proteins on polyacrylamide gels were not altered by reduction and alkylation and therefore do not contain disulfide-linked subunits.

Expression of tumor-associated surface membrane antigens on marrow CFU-s, CFC-gm, BFU-e, and brain CFU-s.

Antiserum raised against a mouse mast cell line (FMP1) reacts with 90% to 100% of spleen colony-forming units (CFU-s), granulocyte/macrophage colony-forming cells (CFC-gm), erythroid burst-forming units (BFU-e), and 15% of nucleated marrow cells, using a complement-dependent cytotoxicity assay. We demonstrated that bone marrow, spleen, or thymus cells are able to absorb this activity from the antiserum. Although mouse brain cells have low reactivity with anti-FMP1 serum, the cytolysis level was reduced to background when antiserum was absorbed with brain cells. In addition, colony formation by marrow CFU-s, CFC-gm, and BFU-e was no longer prevented when the cells were incubated with brain-absorbed anti-FMP1 serum and complement. These findings suggest the presence of brain-associated antigens on CFU-s, CFC-gm, and BFU-e. To test whether a CFU-s accessory cell population in marrow is affected by treatment with anti-FMP1 serum and complement, antibody-treated marrow cells were mixed with large numbers of thymocytes and injected into recipient mice. Colony formation was not altered, indicating that the antiserum reacted directly with antigens on CFU-s and not on CFU-s accessory cells.

Tumor-associated surface membrane antigens on CFUs: characterization using the fluorescent activated cell sorter.

Antiserum raised in rabbits against the FMP1.1 mouse tumor cell line is known to cross-react with GM-CFC and BFUE from normal marrow using the complement-dependent cytoxicity assay. It has been found that this antiserum is cytotoxic to CFUs, either with or without complement addition in vitro. Immunofluorescent analysis using the fluorescent activated cell sorter (FACS) has confirmed this immunological cross-reactivity with CFUs. Recovery of antibody-coated CFUs posed a potential problem in FACS experiments due to in vivo destruction by either opsinization or cytotoxic lysis. Experiments involving enzyme digestion (papain) of antibody and preincubation of bone marrow cells to allow antibody capping did not show improved recovery of CFUs. However, incubation of anti-FMP1.1 coated bone marrow cells with specific anti-rabbit IgG serum resulted in up to 67% of CFUs being detected. Dual parameter sorting with FACS, involving forward light scatter and fluorescence, gave about 26 and 19 times enrichment of GM-CFC and BFUE, respectively, whereas CFUs were enriched by only 6 times. These colony-forming cells were found in the cell population with high intensity forward light scatter and with relatively low fluorescence. Bone marrow cells undergoing regeneration 7 days after 5-fluorouracil (5-FU) treatment of mice exhibited more intense immunofluorescence than normal marrow cells. In particular, a distinct population of lymphocytes in 5-FU treated marrow reacted with anti-FMP1.1 serum, which was not observed with normal marrow.

Surface membrane antigens of hemopoietic tumor cell lines and normal marrow progenitor cells.

A xenoantiserum raised against a mast cell tumor line (FMP1.1) was found to have cross-reactivity with surface antigens on primitive hemopoietic precursor cells in normal mouse bone marrow: erythroid burst-forming units; granulocyte-macrophage colony-forming cells; and high-proliferative-potential granulocyte-macrophage colony-forming cells. In the presence of anti-FMP1.1 serum and complement, only 15 +/- 2% (S.E.) (n = 5) of normal nucleated marrow cells were lysed, demonstrating that the majority of mature hemopoietic cells did not express the antigens detected on their primitive counterparts. A variety of hemopoietic and other tumor cell lines were examined with anti-FMP1.1 serum, and all B- and pre-B-lymphomas, one plasmacytoma, one mastocytoma, and a monocyte-macrophage line exhibited significant lysis. Direct typing of the FMP1.1 tumor demonstrated that it did not express the B-cell surface antigens such as Ia, surface immunoglobulin, Fc, and complement (C3) receptors. Although the Ly.6 alloantigen was present on FMP1.1 cells, this antigen was not found on marrow erythroid burst-forming units and granulocyte-macrophage colony-forming units. Absorption of anti-FMP1.1 serum with cross-reacting (WEHI-3 and W-279.1) and a nonreacting (P-815) cell line confirmed the specificity of the antiserum reaction with these cells and marrow progenitors. These experiments indicated that more than one antibody is contained in anti-FMP1.1 serum. Thus, the mast cell tumor (FMP1.1) carries an unusual array of antigens, which are found on bone marrow progenitors and are not expressed on the majority of differentiated cells. It has been demonstrated that tumor cell lines provide an important basis for the analysis of surface membrane antigens expressed on normal hemopoietic progenitors.