Biomedical subjects
Publications and source records attributed to S J Mentzer.
A technique for extrapleural pneumonectomy in diffuse, malignant, pleural mesothelioma is described. The technique used in a protocol at Brigham and Women's Hospital has resulted in improved operative mortality figures and length of hospital stay. The right-sided procedure is presented followed by differences in technique required by the left-sided approach.
The use of stapling instruments is common in thoracic surgery, but there is a continued reluctance to use them on hilar vascular structures. We have developed a technique that satisfies the major objections to stapler use on hilar vascular structures.
Congenital cystic adenomatoid malformations (CAMs) are rare lung lesions characterized by the presence of a multicystic mass of pulmonary tissue. To clarify the surgical management of CAM of the lung, we reviewed our institutional experience with 34 patients with histologically documented CAM. Symptoms developed in the first 2 days of life in 20 patients; 14 patients developed symptoms a median of 69 days (range, 22 days to 15 years) after birth. A diagnosis of CAM was confirmed at thoracotomy in all patients. Four patients were treated with a limited pulmonary resection for disease confined to one lobe. Seven patients underwent a composite resection involving a lobectomy plus limited resection of the second lobe. One patient underwent pneumonectomy for a severely malformed lung. The remainder of the patients were treated with anatomic lobectomy. The only recurrence was a patient who had a partial cystectomy performed at the initial operation. We conclude that in the appropriate malformation subtype, limited pulmonary reactions can preserve lung tissue and may prevent subsequent complications of CAM.
Alginate is a transparent polymer of guluronic and mannuronic acids that provide a favorable microenvironment for cell growth. Alginate gelation is calcium dependent and temperature independent. To facilitate the isolation of stable and productive antibody-producing hybridomas, we have developed a technique of cloning hybridomas in the three-dimensional alginate matrix. To provide cavities for hybridoma growth, we encorporated 10-15% (v/v) gelatin into the alginate prior to gelation. We have cloned more than 90 monoclonal antibody-producing hybridomas using the alginate matrix. The alginate matrix is readily reversible with the addition of a calcium chelator. The alginate matrix permits efficient cloning in limited incubator space, without the use of a feeder layer, and with minimal amount of medium. The transparent matrix also permits easy screening for clonality and growth.
Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.
Recent studies indicate that the relative expression of HLA class I molecules can play an important role in regulating the cellular immune response. In this report, we used cloned human cytolytic T lymphocytes (CTL) and transfected HLA class I molecules to investigate the effect of HLA class I induction on CTL reactivity. CTL clones with identical antigen specificities but different antigen affinities were compared for their ability to lyse target cells with varying levels of HLA class I expression. The cytolytic reactivity of the high-affinity clone was independent of the level of HLA class I expression. In contrast, cytolytic reactivity of the low-affinity CTL clone was directly related to the level of HLA expression. These data suggest that the induction of HLA class I surface expression has a disproportionate effect on the reactivity of low-affinity CTL and may provide a mechanism for the recruitment of these cells in vivo.
Human mononuclear phagocytes (MO) can mediate the destruction of a variety of foreign and tumor target cells. In most circumstances, however, peripheral blood MO are noncytotoxic and must acquire cytotoxic activity. To investigate the cell surface molecules that participate in the acquisition of MO-mediated cytotoxicity, we used a panel of monoclonal antibodies (MAbs) recognizing a variety of membrane molecules. MAbs to eight different MO surface molecules did not trigger the killing of tumor target cells. To determine if these cell surface molecules triggered an activated but incomplete lytic mechanism, the ability of these molecules to trigger hydrogen peroxide (H2O2) release was assessed. Several MAbs triggered H2O2 release from MO isolated from the peripheral blood. The pattern of MAb-triggered H2O2 release correlated not with the MO surface antigen, but with the immunoglobulin (Ig) isotype. Isotype-specific H2O2 release was abrogated with the enzymatic removal of the Fc portion of the Ig and enhanced by interferon-gamma pretreatment, indicating that the membrane signal was mediated by cell surface Fc receptors. H2O2 release was independent of the presentation of the Ig molecule. Comparable H2O2 release was observed whether the Ig was in surface-bound or soluble form. These data support an important role for Fc receptors in the acquisition of cytotoxic potential by human MO.
Antibody reactive with the CD3 complex on the surface of T lymphocytes can either: inhibit CTL lysis of target cells expressing Ag; or redirect CTL to lyse target cells expressing FcR in the absence of Ag expression. To investigate these phenomena we examined the effect of anti-CD3 mAb on two indicators of CTL activation, the release of esterase and target cell lysis. Esterase release by long term allo-reactive human CTL in response to target cells (JY or HLA transfected K562 cells) was found to be Ag specific and correlate with target cell lysis. Addition of anti-CD3 to either JY targets or K562 cells expressing FcR resulted in a high level of esterase release. Triggering of esterase release was found with both soluble intact and Fab fragment of anti-CD3 in the absence of cells expressing measurable FcR. This apparent FcR-independent triggering of esterase release occurred at 37 degrees C but not at 24 degrees C. In contrast esterase activity was released from CTL at both 24 and 37 degrees C in response to intact target cells, JY or K562 cells plus intact anti-CD3 mAb. Addition of anti-CD3 mAb, at a level capable of blocking target cell lysis by greater than 50%, resulted in an initial velocity of esterase release almost twice that found in response to JY target cells. With a low level of anti-CD3 mAb, able to block JY lysis by approximately 10%, the initial rate of esterase release was much slower than that found in response to target cells. In contrast when FcR+ cells, K562, were added along with a low level of anti-CD3 the initial velocity of esterase release was about twofold more than the velocity of esterase release triggered by soluble anti-CD3 alone. These results indicate that soluble antibody can trigger long term active CTL and the velocity of this triggering correlates with anti-CD3-mediated inhibition as well as redirected lysis.
Based on the crystal structure of HLA-A2.1 and the recognition of a panel of mutant HLA-A2.1 molecules by a large number of alloreactive cytotoxic T lymphocyte clones, a model to explain alloreactivity is described. In this model recognition of an allogeneic major histocompatibility complex molecule by a self-restricted T-cell receptor occurs as the result of accommodation by the receptor of a few amino acid differences in the major histocompatibility complex molecule--i.e., cross-recognition. Alloreactivity is the result of the presence in the foreign antigen binding site of the allogeneic major histocompatibility complex molecule of unusual self-peptides, reactivity to which could not have been eliminated by negative thymic selection.
Cytolytic T lymphocytes (CTLs) are an efficient immune mechanism for the destruction of foreign or pathogenic cells. Attempts to use CTLs in human cancer therapy have focused on the cell-surface molecules that regulate CTL function. An important molecule in CTL function is the CD3 antigen. Biochemical characterization has suggested that the CD3 antigen may function as a "trigger" for T-lymphocyte activation. To investigate this possibility, we used monoclonal antibody (MAb) to the CD3 antigen to trigger activation of long-term CTL lines. The anti-CD3 MAb was able to trigger killing of a variety of human and mouse tumor cell lines; however, not all tumor cells were lysed by the CTL. The susceptibility of the tumor cells to CTL-mediated lysis appeared to correlate with the binding of the anti-CD3 MAb to the tumor cell surface. The requirement for surface binding of the MAb was tested by covalently cross-linking the anti-CD3 MAb to the tumor cell membrane. Membrane-bound anti-CD3 MAb triggered high levels of CTL-mediated tumor cell killing. Similar results were obtained when anti-CD3 MAb was cross-linked to phosphatidylethanolamine and inserted into the cell membrane. These results indicate that the attachment of anti-CD3 MAb to the tumor cell surface provides a powerful new approach to the in vitro activation of human killer T cells and the in vivo treatment of human cancer.
The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.
Monocyte cell surface molecules play an important role in the regulation of monocyte function. To investigate the molecular basis of monocyte-mediated cytotoxicity, we tested the ability of a variety of mediators to stimulate human monocyte-mediated cytotoxicity. Phorbol myristic acetate (PMA) stimulated significant monocyte-mediated killing of tumor cells in an 18-hr indium-111 release assay. Five other cytoactive substances did not induce monocyte-mediated cytotoxicity. The acquisition of monocyte cytotoxicity was associated with nearly a twofold increase in surface expression of three CD18-bearing cell surface molecules (CD11a, CD11b, CD11c). The direct involvement of the CD18-bearing molecules in monocyte-mediated cytotoxicity was investigated using monoclonal antibody (MAb) inhibition. MAb recognizing the CD18 subunit significantly inhibited monocyte-mediated killing. The inhibition by anti-CD18 MAb could not be attributed to LFA-1 (CD11a) alone, suggesting that CR3 (CD11b) and p150,95 (CD11c) may also participate in monocyte-mediated cytotoxicity. In contrast, seven of eight other cell surface structures were not affected by PMA treatment, and MAb to all eight cell surface structures did not inhibit killing. These findings suggest that CD18-bearing molecules are upregulated with monocyte activation and may play a functional role in monocyte-mediated killing.
The human T-cell leukemia virus type I (HTLV-I) is capable of chronically infecting various types of T cells and nonlymphoid cells. The effects of chronic infection on the specific functional activities and growth requirements of mature cytotoxic T lymphocytes (CTL) have remained poorly defined. We have, therefore, investigated the results of HTLV-I infection of both CD4+ and CD8+ human CTL clones. HTLV-I infection resulted in the establishment of functional CTL lines which propagated indefinitely in culture many months longer than the uninfected parental clone. The infected cells became independent of the need for antigen (target cell) stimulation as a requirement for proliferation and growth. Like their uninfected counterparts, however, these HTLV-I-infected clones remained strictly dependent on conditioned medium from mitogen-stimulated T lymphocytes for their growth. This growth factor requirement was not fulfilled by recombinant interleukin-2 alone. Furthermore, the infected lines remained functionally identical to their uninfected parental CTL clones in their ability to specifically recognize and lyse the appropriate target cells. Our findings indicate that the major effects of HTLV-I infection on mature CTL consist of (i) the capacity for proliferation in the absence of antigen stimulation and (ii) a prolonged or immortal survival in vitro, but they also indicate that the fine specificity and cytolytic capacity of these cells remain unaffected.
Although the genes for four hematopoietic colony-stimulating factors (CSFs) have been cloned, neither the mechanism of the regulation of their production nor their cellular origins have been established with certainty. Monocytes are known to produce colony-stimulating and burst-promoting activities, as well as several monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). These monokines indirectly stimulate other mesenchymal cells to produce certain colony-stimulating factors such as granulocyte-macrophage CSF (GM-CSF). To determine whether monocytes produce other CSFs and if so, to compare the mechanism of regulation of production with that of endothelial cells and fibroblasts, we investigated the synthesis of CSFs by monocytes, human umbilical vein endothelial cells, and fibroblasts. We used total cellular RNA blot analysis to determine interleukin-3 (IL-3), GM-CSF, granulocyte CSF (G-CSF), and monocyte CSF (M-CSF) messenger RNA (mRNA) content and immunoprecipitation or bioassay to confirm the presence of the specific secreted proteins. The results indicate that M-CSF mRNA and protein are produced constitutively by all three cell types and their level of expression does not increase after induction. In contrast, GM-CSF and G-CSF mRNAs are barely detectable in uninduced monocytes and show an increase in expression after lipopolysaccharide treatment. Retrovirus-immortalized endothelial cells, unlike primary endothelial cells or both primary and immortalized fibroblasts, produce IL-1 constitutively; this correlates with their constitutive production of GM-CSF and G-CSF. IL-3 mRNA was not detectable in any of these cells either before or after induction. The results indicate that these mesenchymal cells can produce three CSFs: GM-CSF, G-CSF, and M-CSF; furthermore, the data suggest that the mechanism of regulation of M-CSF production is different from that of GM-CSF and G-CSF, and that the latter two inducible CSFs are regulated by different factors in monocytes compared with the other mesenchymal cells.
To study the functional role of the CD11a cell surface molecule in antigen nonspecific cytolysis, a homogenous population of human lymphokine activated killer (LAK) cells were obtained after more than two months of culture in recombinant interleukin-2 (IL-2). Dual parameter immunofluorescence analysis demonstrated that LAK cells from long-term cultures were predominantly CD3+ and CD8+ cells. Monoclonal antibodies (MAb) to six function-associated cell surface molecules were used to inhibit the LAK-target cell interaction. MAb inhibition indicated that the CD11a was involved in the LAK-target cell interaction. None of the other function-associated MAb inhibited killing. A panel of MAb to both the alpha and beta subunits of the CD11a molecule confirmed the anti-CD11a inhibition and demonstrated a hierarchy of functional epitopes. The dominant utilization of CD11a did not appear to depend on IL-2 induction of CD11a surface expression. CD11a surface expression on CD3+ cells did not increase after incubation with IL-2 or other mediators. These findings indicate that CD11a is an important regulatory molecule in LAK-target cell interactions and may play a role in the ability of LAK cells to effectively interact with potential target cells.
Non-Hodgkin's lymphomas may involve a variety of abdominal organs, including the liver, spleen, gastrointestinal tract, and retroperitoneum. The number of organs potentially involved and the noncontiguous mode of spread make non-Hodgkin's lymphoma a difficult tumor to evaluate at the time of laparotomy. To clarify the surgical management of patients with this tumor, we retrospectively reviewed the medical records of 202 patients with histologically proven abdominal lymphomas. Within this group, 36 patients underwent laparotomy before they had chemotherapy or radiation therapy. Ten patients were explored to establish a histologic diagnosis of lymphoma. The remaining 26 patients underwent laparotomy because of presumed benign disease. Twenty patients were found to have localized disease at laparotomy. Patients with localized disease demonstrated significantly better survival than patients with extranodal and nodal involvement (p less than 0.05). Four patients with local resection received no adjuvant therapy and were free of disease a median of 50 months after surgery.
We have investigated the role of the carbohydrate moiety on the HLA-B7 molecule in mAb and CTL recognition using oligonucleotide-directed mutagenesis and gene transfer techniques. A conservative substitution of asparagine to glutamine at amino acid 86 in HLA-B7 was created to abolish the unique glycosylation site present on all HLA molecules. A second mutant B7 molecule was made by substituting asparagine-aspartic acid-threonine for the resident lysine-aspartic acid/lysine tripeptide at amino acids 176-178, thus creating an N-linked glycan at amino acid 176, which is additionally present on all known murine H-2 class I antigens. Upon gene transfer into mouse and human cell recipients, the HLA-B7M176+ mutant and normal HLA-B7 expressed identical levels of surface protein. However, the binding of two mAbs (MB40.2 and MB40.3) thought to recognize different epitopes of the HLA-B7 molecule was completely eliminated. In contrast, the HLA-B7M86- mutant displayed no surface expression (mouse L cells) or minimal surface expression (human RD cells or mouse L cells coexpressing human beta 2 microglobulin [beta 2m]) after indirect immunofluorescence (IIF) and flow cytometric analysis with a panel of 12 HLA-B7 mAb reactive with monomorphic and polymorphic determinants. Immunoprecipitation analysis demonstrated that intracellular denatured mutant protein was present. Tunicamycin treatment did not rescue the expression of HLA-B7M86- antigens to the cell surface; while interferon did induce higher levels of surface expression. Tunicamycin treatment also did not allow binding of the mAbs MB40.2 or MB40.3 to HLA-B7M176+ mutant antigens, suggesting that the carbohydrate moiety itself was not directly involved in the recognition or conformation of these mAb epitopes. Further mutation of the B7M86- molecule to create a glycan moiety at amino acid position 176 (B7M86-/176+) did not rescue normal levels of surface expression. Finally, neither mutation was seen to affect recognition by a panel of 12 allospecific CTL clones. The low expression of HLA-B7M86- on the surface of human cell transfectants was sufficient to achieve lysis, albeit at a reduced efficiency, and lysis could be increased by interferon induction of higher levels of expression. Thus, the carbohydrate moiety on HLA antigens plays a minimal or nonexistent role in recognition by available mAb and allospecific CTL clones.
The mAb L10 was used to determine the distribution and the function of sialophorin, the heavily glycosylated surface molecule that is deficient/defective in lymphocytes of patients with the X-linked immunodeficiency Wiskott-Aldrich syndrome. Dual-parameter FACS analysis indicated that sialophorin is expressed on CD4+ and CD8+ lymphocytes, on a subpopulation of peripheral blood B lymphocytes, on all thymocytes, and on a subpopulation of bone marrow cells. Functional studies demonstrated that L10 mAb stimulates the proliferation of peripheral blood T lymphocytes as measured by stimulation of [3H]thymidine incorporation. The time course and magnitude of increased [3H]thymidine incorporation by T lymphocytes in response to L10 mAb paralleled that induced by anti-CD3 mAb. Effective stimulation was dependent on the presence of monocytes and the Fc portion of L10 mAb. Stimulation of lymphocytes by L10, like stimulation by anti-CD3 mAb, involves increased expression of 4F2, HLA-DR, and IL-2-R. These observations suggest that sialophorin functions in T cell activation.