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M Carl

Publications and source records attributed to M Carl.

At least 55 records · Page 3Linked to original sources
PubMed1990

Detection of murine typhus infection in fleas by using the polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.

Animals↗
PubMed1989

The importance of the crystalline surface layer protein antigens of rickettsiae in T-cell immunity.

Studies in animal models have demonstrated that solid immunity to typhus rickettsiae is dependent on immune T cells. In addition, the surface protein antigen (SPA) of typhus rickettsiae has been shown to be an effective immunogen, protecting vaccinated animals against subsequent challenge with virulent organisms. In the present studies we describe three classes of human lymphocytes which are capable of lysing cells infected with typhus rickettsiae. The first class is CD3,8-positive and is capable of specially lysing both HLA-matched and mismatched targets infected with typhus rickettsiae. Since this cytotoxic effector can be generated with IL-2 as well as with SPA it appears to be a lymphokine-activated killer (LAK). The second class of lymphocytes is CD3,4-positive and is capable of producing gamma interferon in response to the SPA of typhus rickettsiae. Gamma interferon in turn can cause the lysis of cells infected with typhus rickettsiae as well as inhibit intracellular rickettsial growth. A third cytotoxic effector which is CD3,4-positive and which is capable of lysing only HLA-matched targets infected with typhus rickettsiae was generated with a sonicated antigen, and its fine antigenic specificity is not known at present. We conclude that the SPA is an immunologically important protein for the human host and represents an outstanding candidate for a subunit vaccine against typhus.

Antigens, Bacterial↗
PubMed1989

Heterogeneity of CD4-positive human T-cell clones which recognize the surface protein antigen of Rickettsia typhi.

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.

Antigens, Bacterial↗
PubMed1988

Recognition of typhus group rickettsia-infected targets by human lymphokine-activated killer cells.

Lymphokine-activated killer cells caused significant lysis of target cells infected with typhus group rickettsiae (TGR) but not cells infected with Rickettsia tsutsugamushi. Monoclonal and polyclonal antibodies against TGR or TGR-infected cells failed to bind specifically to infected targets, as determined by flow cytometry or by immunoprecipitation of radiolabeled cell surface proteins. Major unaltered antigenic determinants of TGR therefore cannot be detected on the surface of infected targets.

Cytotoxicity, Immunologic↗
PubMed1988

Incorporation of bacterial lipopolysaccharide by human Leu-11a+ natural killer cells. Ultrastructural and functional correlations.

Lipopolysaccharides (LPS) of gram-negative bacteria are known to augment the ability of macrophages and natural killer (NK) cells to lyse susceptible target cells. In the present studies, we sought correlations between the ultrastructural changes and function of Leu-11a+ cells from the peripheral blood mononuclear cells which occurred as a result of their incorporation of LPS. We also studied the effect of LPS on the NK activity of purified Leu-11a+ cells. LPS samples from E. coli and Pseudomonas aeruginosa were utilized. A double-immunolabeling technique employing immunogold and immunoperoxidase markers was used to identify the Leu-11a+ cells with incorporated LPS. Pinocytosis, phagocytosis, and direct insertion into membrane bilayers appeared to be the routes for incorporation of LPS by Leu-11a+ cells. Subsequent ultrastructural effects included dilation of intracellular membrane compartments, formation of tubuloreticular inclusions and increased acid phosphatase activity. Opsonized Staphylococcus aureus were ingested both by control and LPS-treated Leu-11a+ cells; however, the number of Leu-11a+ cells with bacteria increased significantly as a result of LPS treatment. Autoradiography combined with immunolabeling showed no [3H] thymidine incorporation by either control or LPS-stimulated Leu-11a+ cells. NK cell-mediated cytotoxicity was significantly increased as compared with the control (p less than or equal to 0.02) when isolated Leu-11a+ cells were treated with LPS for 24 hours. This result suggests that LPS may have direct effect on the NK cell activity. Tests of peripheral blood mononuclear cell samples after LPS treatment showed up to a 2-fold increase in NK cytotoxicity and a dose-related increase of in vitro interferon production. This latter finding together with the ultrastructural observations of tubuloreticular inclusions in Leu-11a+ cells suggests that, in addition to any direct effect of LPS, cytotoxic activity could be indirectly augmented as a result of autocrine or paracrine interferon, or lymphokine production.

Acid Phosphatase↗
PubMed1988

Ultrastructural and functional effects of lipopolysaccharide and interleukin-2 on human NK cells.

Bacterial endotoxin (lipopolysaccharide, LPS) and interleukin-2 (IL-2) are known to stimulate NK cell mediated cytotoxicity against tumor cells. In the present report we sought to correlate the stimulatory effect of LPS and IL-2 on NK cell activity with ultrastructural changes which occurred as a result of such stimulation. Peripheral blood mononuclear cells (PBMC) were purified from healthy donors by a Ficoll-Hypaque density gradient technique. Leu-11a+ NK cells were isolated by flow microfluorometry using a monoclonal FITC conjugated anti-Leu-11a antibody and a FACS II cell sorter. The PBMC were incubated, respectively, with E. coli LPS or recombinant IL-2 (IL-2) for various time periods. Sorted Leu-11a+ NK cells were incubated with LPS for 24 hours. The NK cytotoxicity in the PBMC and sorted Leu-11a+ cells was assessed by a 51Cr release technique using K562 tumor cells as targets. Leu-11a+ NK cells were identified by immunoelectron microscopy using anti-Leu-11a antibody and labeling with horseradish peroxidase or colloidal gold. Results showed that both LPS and IL-2 significantly enhanced the cytotoxic activity of PBMC. The cytotoxicity of sorted Leu-11a+ cells was augmented by LPS. Recombinant IL-2 induced a significant increase in the number of dense granules, hypertrophy of Golgi apparatus and rough endoplasmic reticulum, and mitosis of Leu-7+ cells and Leu-11a+ cells 4 or 7 days after stimulation. These data indicate that: (1) the effect of LPS on the enhancement of NK cytotoxicity in PBMC may be a direct and/or indirect process involving production of lymphokines; (2) LPS has a direct effect on sorted Leu-11a+ cells; (3) IL-2 stimulates mitosis of Leu-7+ cells and Leu-11a+ cells; and (4) the LPS or IL-2 induced ultrastructural changes in Leu-11a+ cells are consistent with the enhanced NK cytotoxicity.

Cell Separation↗
PubMed1987

Lysis of cells infected with typhus group rickettsiae by a human cytotoxic T cell clone.

Cytolytic human T cell clones generated in response to the intracellular bacterium Rickettsia typhi were characterized. Growing clones were tested for their ability to proliferate specifically in response to antigens derived from typhus group rickettsiae or to lyse targets infected with R. typhi or Rickettsia prowazekii. Two clones were able to lyse targets infected with typhus group rickettsiae. One of these clones was more fully characterized because of its rapid growth characteristics. This cytolytic clone was capable of lysing an autologous infected target as well as a target matched for class I and II histocompatibility leukocyte antigens (HLA). It was not capable, however, of lysing either a target mismatched for both class I and II HLA or a target partially matched for class I HLA. In addition, the clone exhibited specificity in that it was able to lyse an autologous target infected with typhus group rickettsiae, but did not lyse an autologous target infected with an antigenically distinct rickettsial species, Rickettsia tsutsugamushi. These results demonstrate, for the first time, that cells infected with intracellular bacteria can be lysed by human cytotoxic T lymphocytes.

Antigens, Bacterial↗
PubMed1987

Immunoultrastructural studies of human NK cells: I. Ultracytochemistry and comparison with T cell subsets.

Human NK activity is known to be associated with a population of large granular lymphocytes (LGL) exhibiting several immunophenotypic surface markers including Leu-11a (NKP-15), Leu-7 (HNK-1), Leu-3a (T4), and Leu-2a (T8). Based upon correlation with cytolytic activity, Leu-11a is now considered the most specific antigenic marker for human NK cells. Present investigation compared the ultrastructure of cells expressing Leu-11a, Leu-7, Leu-3a, and Leu-2a, both in human peripheral blood lymphocytes (PBL) and the purified LGL fraction. Subcellular cytochemical reactions were investigated in Leu-7+ or Leu-11a+ PBL or LGL and in cells conjugated with K562 targets (indicating NK cytolytic potential). The surface markers, localized with monoclonal antibodies, were detected by immunoelectron microscopy by using direct or indirect avidin-biotin-peroxidase (ABC) or colloidal gold methods. A peroxidase-colloidal gold double-labeling system was used to identify subsets of Leu-7+ or Leu-11a+ cells. Previously described ultrastructural features of LGL including a villous surface, reniform nuclei, low nuclear/cytoplasm ratios, and abundant cytoplasm with vesicles, vacuoles, electron-dense granules, parallel tubular arrays (PTA), or paracrystalline inclusions were associated with Leu-7+, Leu-11a+, Leu-7+/Leu-11a+, Leu-7+/Leu-11a-, and Leu-7-/Leu-11a+ PBL or LGL. Results showed that the Leu-7+/Leu-11a+ cells were the most abundant NK cells in PBL. Lymphocyte subsets with Leu-3a or Leu-2a surface marker showed some ultrastructural features including PTA similar to Leu-7+ cells and Leu-11a+ cells, and their subsets. These T-cells appeared ultrastructurally more similar to the Leu-7+/Leu-11a- subset. Cytochemical studies showed that electron-dense cytoplasmic granules and PTA typical of the Leu-11a+ cells and Leu-7+ cells contained glycoprotein, acid phosphatase, and arylsulfatase. Large cytoplasmic vacuoles were heterogeneous and typically contained electron-dense material with DAB reactivity, membranous material, PTA, and/or paracrystalline inclusions. Glycoprotein, acid phosphatase, and arylsulfatase, and peroxidase reactive material were also found in these vacuoles. These features suggested that the vacuoles could be secondary lysosomes. The coexistence of intact PTA or degenerating PTA in the same vacuoles with paracrystalline inclusions suggested that the latter are possibly derived from PTA.

Cytoplasm↗
PubMed1987

Immunoultrastructural studies of human NK cells: II. Effector-target cell binding and phagocytosis.

The binding of NK cells to a target cell appears to be a necessary step for NK cell-mediated cytolysis. In this report, we demonstrated effector-target binding by immunoelectron microscopy by using monoclonal antibodies against NK cells (Leu-7, Leu-11a) and T-cell subsets (Leu-2a/T8, Leu-3a/T4). The surfaces of NK and K562 cells were characterized by antitransferrin receptor antibody and various lectins. In addition, the controversial phagocytic activity of NK cells was studied by incubation of peripheral blood mononuclear cells with opsonized Staphylococcus aureus and labeling with anti-Leu-7 or anti-Leu-11a antibody. Results showed that only Leu-11a+ cells displayed a broad cell-to-cell contact with the target by a shallow intercellular interdigitation of cytoplasmic projections, while Leu-7+, Leu-2a+, or Leu3a+ cells showed only a partial contact with target without interdigitation. The Leu-11a+ cells were frequently observed in small clusters and in close association with monocytes. Cluster formation and association with monocytes were not observed in other NK and T-cell immunophenotypes. In Leu-11a+ cells conjugated with target cells, membrane-bound granules, small vesicles, parallel tubular arrays, Golgi apparatus, endoplasmic reticulum, and small vacuoles were evident and concentrated toward the target. The surface of NK cells was intensely stained for glycoprotein by chromic acid-phosphotungstic acid, whereas target cells were not stained. Transferrin receptors were stained only on the surface of target cells. Only the lectins RCA and UEA labeled the surfaces of both NK and target cells. Phagocytic vacuoles containing cell debris or fragments and ingested bacteria were found in the cytoplasm of Leu-11a+ cells but not in Leu-7+ cells. NK cells were also found within the cytoplasm of K562 target cells. All these findings suggest that Leu-11a+ cells are the true functional NK cells involved in NK cell-mediated cytolysis, phagocytosis, and emperipolesis. Therefore, the NK cell is probably "a phagocyte in lymphocyte's clothing." The presence of peroxidase in the small vesicles of NK cells and endocytotic vesicles of target cells at the effector-target contact area indicates that cytolytic enzymes or factors derived from NK cells may be transported into the target by endocytosis.

Antibodies, Monoclonal↗
PubMed1986

Characterization of human cytotoxic lymphocytes directed against cells infected with typhus group rickettsiae: evidence for lymphokine activation of effectors.

An in vitro culture and assay system was used to determine whether cytotoxic lymphocytes are generated in humans after rickettsial infection. Peripheral blood mononuclear cells (PBMC) were obtained from six individuals with serologic evidence of prior infection with typhus group rickettsiae and from six nonimmune individuals. After PBMC from immune individuals were stimulated in vitro for 7 days with rickettsial antigen, they were capable of lysing typhus group rickettsia-infected, autologous phytohemagglutinin (PHA)-induced blasts, but not uninfected PHA-blasts. No cytotoxic effector cells were generated when either PBMC from immune individuals were placed in culture for 7 days without antigenic stimulation, or when PBMC from nonimmune individuals were stimulated in vitro with antigen for 7 days. Freshly isolated PBMC from immune donors were also unable to lyse typhus group rickettsia-infected autologous PHA-blasts or an autologous rickettsia-infected lymphoblastoid cell line (LCL). Neither supernatants from antigen-stimulated cultures of PBMC from immune donors nor recombinant human interferon-gamma were capable of significantly lysing typhus group rickettsia-infected PHA blasts by this assay. Populations of cytotoxic effector cells depleted of OKT3, OKT4, or OKT8-positive cells by treatment with the respective monoclonal antibodies and complement were assayed for their cytotoxic capacity. The results suggest that the cytotoxic effector cell population is predominantly OKT3 and OKT8-positive, but OKT4-negative. Positive selection with the use of a fluorescence-activated cell sorter also suggested that most of the cytotoxic effector cells are OKT8-positive. PBMC from immune donors after in vitro stimulation with rickettsial antigen were capable of significantly lysing infected autologous LCL or infected HLA-mismatched LCL as compared with the respective uninfected controls. In addition, PBMC from either immune donors or nonimmune donors after stimulation in vitro for 7 days with media containing purified lymphokines were capable of significantly lysing autologous infected LCL as compared with the uninfected autologous control. We conclude that lysis of cells infected with typhus group rickettsiae is mediated by a lymphokine-activated killer.

Antigens, Bacterial↗
PubMed1986

Human T helper cells specific for antigens of typhus group rickettsiae enhance natural killer cell activity in vitro.

The peripheral blood mononuclear cells (PBMC) from 5 individuals immune to typhus group rickettsiae and from 13 nonimmune individuals were stimulated in vitro for 7 days with typhus group rickettsial antigen (TGRA). At the end of day 7, lysis of the natural killer (NK)-susceptible target K562 by these PBMC was determined. As controls, PBMC from both groups of donors were cultured in vitro for 7 days without antigen or were freshly isolated, and lysis of the K562 target was determined. There was no significant difference between the level of NK activity in freshly isolated PBMC from immune and nonimmune donors. PBMC from immune donors which were stimulated with antigen for 7 days exhibited significantly greater NK activity than did the control population, which was cultured for 7 days without antigen. PBMC from immune donors which were stimulated with TGRA demonstrated significantly higher NK activity than the same PBMC stimulated with antigen derived from an antigenically unrelated rickettsia, Coxiella burnetii. There was no significant difference, however, in the level of NK activity of nonimmune antigen-stimulated PBMC compared with that of the same PBMC population cultured without antigen. Most of the antigen-stimulated NK activity was mediated by Leu-11-positive cells as determined by electronic cell sorting. The ability of TGRA to sustain the NK activity of PBMC from immune donors was abolished when the T4/Leu-3-positive population of lymphocytes was eliminated by positive or negative selection prior to antigen stimulation. The ability of TGRA to sustain the NK activity of PBMC from immune donors was also significantly decreased in the presence of antibodies against human interleukin-2. The results suggest that the activity of human NK cells can be sustained in vitro by antigen-specific T helper cells and that the effect of the T helper cell is mediated, at least in part, by interleukin-2.

Antibodies, Monoclonal↗
PubMed1985

Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies.

Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.

Adult↗
PubMed1982

Interruption of hepatitis B transmission by modification of a gynaecologist's surgical technique.

Between January, 1979, and January, 1980, hepatitis B developed in three women within 6 months of their undergoing gynaecological surgery at a community hospital in Mississippi. Subsequent investigation revealed another case of hepatitis B associated with gynaecological surgery. The gynaecologist who carried out all four operations (gynaecologist A) was a chronic carrier of hepatitis B. Hepatitis B surface antigen subtyping on serum from gynaecologist A and from one of the hepatitis B patients gave identical results. Patients of gynaecologist A who underwent total abdominal hysterectomies or bilateral salpingo-oophorectomies were at a greater risk of acquiring hepatitis B than were patients in whom the same procedures were carried out by a control group of gynaecologists. After gynaecologist A modified his surgical technique and began wearing two pairs of gloves during surgery, there was no further hepatitis B transmission to his surgical patients.

Carrier State↗
PubMed1982

Excretion of hepatitis A virus in the stools of hospitalized hepatitis patients.

A study was carried out to determine whether hepatitis A virus (HAV) can be detected in the stools of patients hospitalized for HAV infection. Acute phase samples of whole blood and stool, as well as completed questionnaires, were obtained from 31 patients hospitalized at any of 13 hospitals in the Phoenix metropolitan area. Blood specimens were tested for hepatitis B surface antigen (HBsAg), IgG antibody to HAV (IgG anti-HAV), and IgM antibody to HAV (IgM anti-HAV). Stools were tested for HAV by radioimmunoassay. Five patients (16.1%) had acute hepatitis B, five (16.1%) had acute non-A/non-B hepatitis, and 21 (67.7%) had acute hepatitis A. Of these 21 patients with acute hepatitis A, 11 (52.4%) were found to have HAV in their stools. These results confirm the potential for infectivity of stools of patients hospitalized for hepatitis A and emphasizes the need for caution when dealing with such stools.

Feces↗