Tissue inhibitor of metalloproteinases 1 regulation of interleukin-10 in B-cell differentiation and lymphomagenesis.

Tissue inhibitors of metalloproteinases (TIMPs), first described as specific inhibitors of matrix metalloproteinases, have recently been shown to exert growth factor activities. It was previously demonstrated that TIMP-1 inhibits apoptosis in germinal center B cells and induces further differentiation. Interleukin-10 (IL-10) is reported as a vital factor for the differentiation and survival of germinal center B cells and is also a negative prognostic factor in non-Hodgkin lymphoma (NHL). However, the mechanism of IL-10 activity in B cells and the regulation of its expression are not well understood. IL-10 has been shown to up-regulate TIMP-1 in tissue macrophages, monocytes, and prostate cancer cell lines, but IL-10 modulation of TIMP-1 in B cells and the effect of TIMP-1 on IL-10 expression has not been previously studied. It was found that TIMP-1 expression regulates IL-10 levels in B cells and that TIMP-1 mediates specific B-cell differentiation steps. TIMP-1 inhibition of apoptosis is not IL-10 dependent. TIMP-1 expression in B-cell NHL correlates closely with IL-10 expression and with high histologic grade. Thus, TIMP-1 regulates IL-10 expression in B-cell NHL and, through the inhibition of apoptosis, appears responsible for the negative prognosis associated with IL-10 expression in these tumors.

Diagnosis of unexpected acute myeloid leukemia and chronic lymphocytic leukemia: a case report demonstrating the perils of restricted panels in flow cytometric immunophenotyping.

We report on the flow cytometric identification of concomitant acute myeloid leukemia and chronic lymphocytic leukemia in cytology specimens submitted with minimal clinical information. A 64-year-old man presented with fever and progressive dyspnea on exertion. Chest X-ray and computed tomography scan showed a left upper lobe pulmonary mass. Pulmonary capillary pullback specimens were collected to determine infectious verses neoplastic etiology. The pulmonary capillary pullback specimens showed atypical mononuclear cells with enlarged, slightly irregular nuclei; visible nucleoli; and basophilic cytoplasm. Flow cytometric analysis of the specimen for lymphoma was requested. Flow cytometric immunophenotypic studies showed that 78% of the cells were CD34 positive, CD45 dim positive and CD11c positive, consistent with acute myeloid leukemia. About 0. 75% of the cells expressed CD5 as well as dim CD20 and were monoclonal for kappa light chains: consistent with chronic lymphocytic leukemia/small lymphocytic lymphoma. At this time the clinician communicated a history of myelodysplastic syndrome of refractory anemia subtype. Peripheral blood was obtained for further immunophenotyping and the patient was immediately treated for his acute myeloid leukemia. This case demonstrates that a diagnostic antibody panel should allow evaluation of all cell types as per the U.S./Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry (Stewart et al.: Cytometry 30:231-235, 1997). Published 2000 Wiley-Liss, Inc.

Radiation and the Apo2L/TRAIL apoptotic pathway preferentially inhibit the colonization of premalignant human breast cells overexpressing cyclin D1.

The role of cyclin D1 overexpression in human breast premalignancy was investigated using immortal, nontumorigenic MCF-10A cells. Previous work documented that cyclin D1 overexpression promoted in vitro anchorage-independent colonization. We now report that the colonization of MCF-10A cyclin D1 transfectants was preferentially inhibited by gamma-radiation and specific classes of apoptosis inducers [Apo-2 ligand (Apo-2L), but not tumor necrosis factor alpha]. Antibody inhibition studies and semiquantitative PCR indicated that radiation inhibition of colonization was partially mediated via the Apo2L/TRAIL pathway. The apoptotic removal of cyclin D1-overexpressing, colonization-competent premalignant breast cells by Apo2L/TRAIL or other biologicals may represent a novel approach to the prevention of breast cancer.

Cyclin D1 overexpression in a model of human breast premalignancy: preferential stimulation of anchorage-independent but not anchorage-dependent growth is associated with increased cdk2 activity.

Cyclin D1 is frequently overexpressed in human breast ductal carcinoma in situ (DCIS) specimens, which confer a high risk for the development of infiltrating ductal carcinoma. If causally involved in the genesis of human breast malignancy, cyclin D1 may represent an interesting target for chemopreventive approaches, as it sits at the junction of many growth factor and hormonal pathways. We have used the MCF-10A human breast cell line, derived from a mastectomy containing a low risk premalignant lesion, as a model system. Three cyclin D1 transfectants exhibited physiologically relevant levels of transgene overexpression, but no coordinate overexpression of other cell cycle related genes. Proliferation assays, flow cytometry, and cdk enzymatic assays of anchorage-dependent proliferation indicated only a minimal and transient effect of cyclin D1. In contrast, cyclin D1 overexpression significantly stimulated anchorage-independent colonization in soft agar or methylcellulose, accompanied by greater Gl-S progression. The cdk4 activity of the control- and cyclin D1 transfectants in colonization assays was comparable, but the cdk2 activity was higher in the latter. Injection of control- and cyclin D1 transfected MCF-10A cells in matrigel into nude mice failed to produce tumors within 1.5 years. The data suggest that cyclin D1 overexpression is an early feature of breast neoplastic progression, and can contribute to cancer development through the promotion of colonization.

Development of lymphoproliferative disorder of granular lymphocytes in association with hairy cell leukemia.

Lymphoproliferative disorder of granular lymphocytes (LDGL) is a low grade T-cell disease characterized by clonal expansion of large granular lymphocytes of either T cell or natural killer (NK) cell lineage that express the cytotoxic T-cell/NK cell antigens CD16, CD56 and/or CD57. LDGL has been described in association with other malignancies, leading to theories of a common abnormal stem cell as well as development of the LDGL as an immune response to a primary tumor. We have studied 32 patients with hairy cell leukemia (HCL). In 15 patients (47%) we detected an increase in cells expressing cytotoxic T-cell/NK cell antigens. In 10(31%) patients these cells were of T cell lineage, while 5 patients (16%) had increased NK-cells. T cell clonality was detected by PCR in all cases with increased cytotoxic T-cells in which adequate DNA was obtained from peripheral blood. Since in 2 patients the LDGL was not present at diagnosis but developed during follow up, our data suggests that clonal LDGL may develop in response to the HCL. The significance of LDGL in the setting of HCL and flow cytometric evaluation of HCL versus LDGL will be discussed.

Increased peripheral blood gamma delta T-cells in patients with lymphoid neoplasia: A diagnostic dilemma in flow cytometry.

We have observed increased numbers of non-neoplastic gammadelta-T-cells in the peripheral blood of a series of patients with non-Hodgkin's lymphoma not of gammadelta-T-cell origin. The majority of normal gammadelta-T-cells are negative for surface CD4 and CD8 and a subpopulation does not express CD5, two immunophenotypic findings strongly suggestive of neoplasia in alpha beta T-cells. In addition, they express cytotoxic T-cell/Natural killer cell antigens. In this study, up to 22% of PBLs were CD4 and CD8 negative gammadelta-T-cells and up to 33% PBLs were CD5 negative gammadelta-T-cells. In addition, as high as 42% of PBLS were gammadelta-T-cells expressing cytotoxic T-cell/Natural killer cell antigens, suggestive of a large granular lymphoproliferative disorder. Failure to recognize that these are normal gammadelta-T-cells could lead to the erroneous diagnosis of peripheral blood involvement with a T-cell neoplasm, especially in the setting of a history of non-Hodgkin's lymphoma. Cytometry (Comm. Clin. Cytometry) 38:280-285, 1999. Published 1999 Wiley-Liss, Inc.

In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1.

Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.

Role of p53 and apoptosis in sensitization of cis-diamminedichloroplatinum antitumor activity by interleukin-1 in ovarian carcinoma cells.

We have previously reported that interleukin-1 (IL-1 ) sensitized cisplatin cytotoxicity against human ovarian NIH:OVCAR-3 tumor cells. We have further examined inter-actions of IL-1 with cisplatin in these ovarian cells. Treatment of cells with either IL-1 or CDDP or combinations resulted in a significant accumulation of cells in G1 phase and a concomitant decrease in the S phase of the cell cycle. IL-1 and CDDP treatment induced p53 protein in NIH:OVCAR-3 tumor cells. CDDP and IL-1 treatment decreased the steady-state expression of c-myc RNA and induced significant degradation of the genomic DNA into internucleosomal sized DNA fragments which was further increased in the presence of both agents in these cells. Taken together, these studies suggest that IL-1 may kill ovarian NIH:OVCAR-3 tumor cells by inducing a blockade at G1/S of the cell cycle, down-regulating c-myc gene and inducing p53-dependent apoptosis. The synergistic interactions of IL-1 with CDDP may involve the enhancement of p53-dependent apoptosis.

The activated anaplastic lymphoma kinase increases cellular proliferation and oncogene up-regulation in rat 1a fibroblasts.

More than 60% of anaplastic large-cell lymphomas (Ki-1 lymphoma) are associated with a t(2;5)(p23;q35) translocation that produces an 80 kDa hyperphosphorylated chimeric protein (p80) derived from the fusion of the anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM). The NPM-ALK chimeric gene is an activated tyrosine kinase that has been shown to be a potent oncogene. We have developed a cellular model for the study of p80 action in rat 1a fibroblasts. Expression of cDNA's encoding NPM-ALK (p80) in rat 1a fibroblasts induces anchorage-independent growth in soft agar and promotes foci formation in culture. Cells expressing exogenous p80 showed significantly increased proliferation characterized by accelerated cell cycle entry into S-phase. Consistent with increased G0/G1 to S-phase transition, there is also marked up-regulation of cyclin A and cyclin D1 expression. In addition, p80 transformed cells showed elevated expression of several immediate early genes involved in cellular proliferation, including fos, jun, and c-myc. DNA binding analysis of nuclear extracts prepared from p80 transformed cells reveal marked up-regulation of AP-1 DNA binding activity. Functional AP-1-specific transfection assays also show up-regulation of AP-1-dependent transcriptional activation. These finding demonstrate that p80 transformed rat 1a fibroblast can be a highly useful model system for the molecular and biochemical characterization of the mechanisms of action of this interesting new oncogene.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells.

We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage. Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method. TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells. In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil. Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies. Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas. TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.

Flow cytometric analysis of kappa and lambda light chain expression in evaluation of specimens for B-cell neoplasia.

Analysis of light chain expression is one of the most important determinations in flow cytometric immunophenotyping of patient specimens. Numerous technical factors, such as antibody choice and cytophilic antibody artifact, impact a laboratory's ability to perform this test. There have been conflicting reports concerning the efficacy of polyclonal versus monoclonal antibodies, as well as methods of circumventing cytophilic antibodies, indicating that a consensus has not been reached on optimal methods for light chain determination. The authors have investigated methods for light chain analysis in 104 normal donors and 366 patient specimens, comparing different anti-light chain antibodies as well as strategies for analysis of specimens with low numbers of monoclonal B cells, admixed polyclonal B cells, or cytophilic antibodies. The patient specimens were either part of the initial diagnostic evaluation of patients with suspected lymphoma, or were performed for staging or assessment of treatment of patients with known B-cell neoplasia. No monoclonality was detected in control specimens, and there was no significant difference in staining with monoclonal verses polyclonal anti-light chain antibodies. In addition, cytophilic antibody did not obscure results in normal controls. Monoclonality was detected in 106 patient specimens, with 89 showing gross involvement with a predominant monoclonal B-cell process. However, in 43% of the grossly monoclonal specimens, there was failure to detect monoclonality with at least one light chain antibody set, with 8% of these cases showing failure with two anti-light chain sets. This indicates the importance of antibody choice in light chain analysis. Cytophilic antibody artifact in monoclonal specimens was easily overcome by appropriate antibody combinations, obviating the need for cytophilic antibody-shedding by incubation at 37 degrees C in fetal calf serum. In 27 patient specimens with low numbers of B cells or admixed polyclonal B cells, a clonal search based on FSC and CD19 or CD20 expression was performed. In 17 of the 27 cases (63%), a small monoclonal population was detected among admixed polyclonal B cells. The authors conclude that multiple strategies are necessary in flow cytometric analysis for B-cell monoclonality.

Targeted T-cell therapy for human leukemia: cytotoxic T lymphocytes specific for a peptide derived from proteinase 3 preferentially lyse human myeloid leukemia cells.

Proteinase 3 is present in high concentration in the primary granules of acute and chronic myeloid leukemia blasts, and may represent a potential T-cell target antigen. We screened proteinase 3 against the binding motif of HLA-A2.1. Based on its high predicted binding, a 9-mer peptide, "PR-1," was synthesized and tested for binding to HLA-A2.1 using the T2 cell line. PR-1 at 100 micrograms/mL significantly increased expression of HLA-A2.1, with median channel of fluorescence increasing from 22 to 294. Binding half-life was determined to be 1,460 minutes by I125-labeled beta 2-microglobulin incorporation. HLA-A2.1+ peripheral blood mononuclear cells from a normal donor were used to generate a T-cell line specific for PR-1. The line demonstrated 85% PR-1-specific lysis at an E:T ratio of 50:1, compared with 20% lysis without PR-1, using T2 cells as targets. It also showed 79% specific lysis to fresh chronic myelogenous leukemia blasts, 54% to fresh acute myelogenous leukemia blasts, and only background lysis (< 20%) to HLA-A2.1+ normal allogeneic marrow cells. The amount of lysis of HLA-A2.1+ myeloid cells was proportional to cytoplasmic proteinase 3 expression. Thus, HLA-A2.1-restricted cytotoxic T cells, raised against a peptide contained in proteinase 3, preferentially lysed fresh human leukemic cells.

TNF-alpha and IL-1 alpha induce mannose receptors and apoptosis in glomerular mesangial but not endothelial cells.

The macrophage mannose receptor, a carbohydrate-binding membrane protein, mediates endocytosis and phagocytosis. This study was undertaken to determine whether mannose receptors were expressed in resting glomerular mesangial and endothelial cells and whether their level was affected by cytokines. Neither mannose receptor mRNA nor proteins were found in resting mesangial or endothelial cells. Mannose receptor mRNA was induced in a dose- and time-dependent manner in mesangial cells by interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) but not by platelet-derived growth factor-B or IL-6. Cell surface receptors were found by fluorescence-activated cell sorter analysis. Binding to stimulated mesangial cells was saturable and inhibited by excess mannose-bovine serum albumin (BSA) but not by galactose-BSA. TNF-alpha and IL-1 alpha also induced apoptosis in mesangial cells. Mannose receptor expression was not restricted to apoptotic stimulated mesangial cells. Neither agonist induced mannose receptor expression or apoptosis in endothelial cells. Because immunoglobulin A, M, and G contain mannose residues, immune aggregates may be removed from the mesangium through cytokine-induced mannose receptors.

Flow cytometric detection of neoplastic T cells in patients with mycosis fungoides based on levels of T-cell receptor expression.

The authors report the flow cytometric detection of neoplastic T cells in the peripheral blood of four out of five (80%) patients with peripheral blood involvement with mycosis fungoides (Sezary syndrome) based on the levels of T-cell receptor expression as measured by CD3 and TCR-alpha beta staining. Antigen receptor expression was abnormal in terms of increased density of surface CD3 or TCR-alpha beta per cell. Other immunophenotypic abnormalities were present in three of these patients. However, in one patient abnormal T-cell receptor expression was the only immunophenotypic evidence of neoplasia, although morphologically abnormal lymphocytes were present and a T-cell clone was detected by polymerase chain reaction (PCR). In another patient, the authors were able to detect development of a new, more aggressive neoplastic T-cell population based on levels of T-cell receptor expression. Levels of T-cell receptor expression may be of diagnostic utility in the evaluation of peripheral blood for the presence of neoplastic T-cell populations.

Prevalence of measles susceptibility in hospital staff. Evidence to support expanding the recommendations of the Immunization Practices Advisory Committee.

BACKGROUND: In 1989 and 1990, measles reached epidemic proportions in the United States, including several areas of California. Children's Hospital Oakland (Calif), a major health care provider for children in a measles epidemic area of California, reported 131 cases between 1989 and 1991, the largest number ever reported by that institution. In February 1990, four cases of measles were reported among hospital staff. Continued risk of nosocomial infection prompted the development of a program to ensure that all hospital staff were adequately protected against measles. METHODS: All hospital employees who were unable to document proof of measles immunity were required to be serologically screened for measles antibody and to be vaccinated against measles if they were determined to be nonimmune. Serologic screening was performed in-house with a commercially available enzyme-linked immunosorbent assay measles antibody test. Dates of birth were recorded for all employees screened. Individuals with negative or repeatedly equivocal results were considered to be nonimmune and were vaccinated with trivalent measles-mumps-rubella vaccine. RESULTS: Between March and June 1990, 1694 staff were serologically tested for measles antibody. Eighty-nine (5.3%) of the employees were considered to have inadequate immunity. Forty (45%) of these susceptible individuals were born before 1957. CONCLUSIONS: We conclude that the recommendations of the Immunization Practices Advisory Committee should be expanded to include serologic screening or vaccination of hospital personnel who were born before 1957. Serologic screening of hospital staff may be a reasonable alternative to vaccination under certain circumstances.

Further characterization of "promptly" and "delayed" human serum-sensitive strains of Serratia marcescens.

The kinetics of the bactericidal activity of 80 vol% of fresh human serum against representative 'delayed serum-sensitive' (DSS) and 'promptly serum-sensitive' (PSS) strains of Serratia marcescens were further examined with regard to various chemical and absorption procedures known to affect various components of the alternative and classical pathways of human complement activation. Inulin treatment of fresh human serum failed to diminish serum bactericidal activity against DSS and PSS assay strains. Fresh human serum that had been depleted of properdin (factor P) through absorption with zymosan, was as active as control serum against DSS strains of S. marcescens; however, PSS strains were killed in a 'delayed' fashion. Human serum that had been heat-inactivated at 50 degrees C for minutes (depletion of factor B), no longer killed DSS strains, whereas PSS strains of S. marcescens and the PSS control strain Escherichia coli C were killed in a slightly delayed fashion. Hydrazine-hydrate treatment (inactivation of C3 of the complement system) and exposure of fresh human serum to dithiothreitol completely abolished serum bactericidal activity. Bentonite-absorbed fresh human serum no longer killed DSS strains of S. marcescens; some PSS strains of S. marcescens were killed in a delayed manner, whereas control strain E. coli C was as PSS as before. Addition of Seitz-filtered fresh human serum, that lacked beta-lysin and was deficient in lysozyme, to bentonite-absorbed human serum restored bactericidal activity against DSS and PSS strains of S. marcescens; addition to heat-inactivated or Seitz-filtered, heat-inactivated human serum failed to do so. Therefore, bentonite absorption removed to a heat-labile component from fresh human serum clearly different from beta-lysin and lysozyme. Furthermore, human serum beta-lysin and lysozyme were not required for serum-mediated killing of S. marcescens strains of either serum susceptibility category.

Serotyping of Serratia marcescens: simplified tube O-agglutination test and comparison with other serological procedures.

A simplified tube O-agglutination test was developed and evaluated for the determination of somatic serogroup (O) antigens of Serratia marcescens. Use was made of Tryptic Soy broth (TSB)-grown O-cells that had been boiled for 1 hour; 0.145 M NaCl proved a satisfactory diluent. Various technical parameters of this test were examined as well. Rabbit anti-O immune sera, that had been elicited with 5 x concentrated, TSB-grown, 1 hour-boiled O-cells of all 15 currently employed O-antigen reference strains of S. marcescens yielded satisfactory O-agglutinin titers. The tube O-agglutination test compared favorably with the indirect hemagglutination technic, although the latter technic yielded significantly higher O-agglutinin titers with merely 7 of the 15 O-antigens of S. marcescens. The tube O-agglutination test permitted detection of higher O-agglutinin titers than a microtiter O-agglutination test utilizing O-cells that had been stained with safranin O. Conversely, titers obtained with TTC-stained O-cells in a microtiter agglutination procedure approximated those yielded by the tube O-agglutination test, but O-cells of the various S. marcescens strains were stained nonuniformly by triphenyltetrazolium chloride (TTC). As before, there existed marked serologic cross-reactivity between O-antigens O6 and O14. A new O6 candidate strain, S. marcescens isolate S 1i, serotype O6:H20, was proposed. Contrary to O-agglutinins of human control serum, the O-agglutinins of rabbit anti-O immune sera proved refractory to treatment with 0.1 M of 2-mercaptoethanol (2-ME) and 0.01 M dithiothreitol (DTT) respectively. Dual absorptions of rabbit anti-O immune sera with killed cells of Staphylococcus aureus Cowan I (protein A), failed to significantly reduce O-agglutinin titers, although human IgG and IgM was bound by protein A. It was tentatively concluded that the 2-ME- and DTT-refractory rabbit anti-S. marcescens O-agglutinins resided in the IgM immunoglobulin class.

Neutralization of human serum beta-lysin by sodium polyanetholsulfonate and sodium amylosulfate.

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.

Variable neutralization of several nonspecific antibacterial systems in fresh, defibrinated human blood by sodium polyanetholsulfonate and sodium amylosulfate.

Fresh, defibrinated human blood (80 vol%, i.e., 80% [vol/vol] of a 2-ml final assay volume) from two healthy adult donors killed "delayed serum-sensitive" (DSS) and "promptly serum-sensitive" (PSS) strains of Serratia marcescens, PSS control strain Escherichia coli C, Bacillus subtilis strain ATCC 6633, and Micrococcus lysodeikticus ATCC 4698 in a kinetic manner comparable to that of fresh human serum (80 vol%). However, heat-inactivated (56 degrees C, 30 min), defibrinated human blood revealed markedly reduced or a total lack of beta-lysin activity against the B. subtilis assay strain. Similarly, lysozyme activity of defibrinated blood was diminished somewhat by heat treatment, as determined with the M. lysodeikticus assay strain. Addition of 500 mug of sodium polyanetholsulfonate (SPS) per ml to 80 vol% of fresh, defibrinated human blood completely neutralized blood bactericidal activity against all assay strains of S. marcescens, E. coli C, and B. subtilis; however, SPS at this concentration failed to abolish lysozyme activity for prolonged periods of incubation. Addition of 500 mug of sodium amylosulfate (SAS) per ml to 80 vol% of fresh defibtinated human blood resulted in protection of cell inocula of DSS strains of S. marcescens only; SAS failed to protect cell inocula of the PSS strains of S. marcescens, E. coli C, B. subtilis, and M. lysodeikticus for extended periods of observation. Based on these data, it is recommended that blood culture specimens that are first drawn into specimen containers (such as Vacutainer tubes or the like) at the patient's bedside, and which contain >/=250 mug of SPS per ml, be diluted into suitable broth media with at least >/=250 mug of SPS per ml by the receiving laboratory within 2 to 4 h after procurement of the specimen. This procedure would ensure continued, adequate neutralization of the specimen's inherent beta-lysin, lysozyme, and complement- and antibody-mediated bactericidal activities.

Serotyping of Serratia marcescens: current status of seven recently described flagellar (H) antigens.

The slightly revised, current scheme of 20 flagellar (H) antigens of Serratia marcesens was examined. The seven new H antigens were demonstrated to be antigenically distinct as determined with Le Minor's H-immobilization test. The H-immobilization antibodies of rabbit anti-H immune sera proved resistant to treatment with 2-mercaptoethanol and dithiothreitol, respectively. On the other hand, dual absorptions of rabbit anti-H immune sera with killed cells of Staphylococcus aureus strain Cowan I, i.e., protein A, failed to reduce significantly H-immobilization titers of rabbit sera, although human immunoglobulins G and M were bound by protein A. It was tentatively concluded that the 2-mercaptoethanol- and dithiothreitol-refractory H-immobilizing rabbit antibodies belonged to the immunoglublin M class. H-antigen (phase) variation was not demonstrable in several extramural, clinical isolates of S. marcescens for which this phenomenon had been claimed. Rather, four of these six isolates were found to consist of cell populations of two distinct serotypes, as also borne out by bacteriocin typing; the flagellar H-antigens of the remaining two isolates were stable, with minor, hterologous H-antigen cross-reactivity.