Three-dimensional image analytical detection of intussusceptive pillars in murine lung.

A variety of diseases can lead to loss of lung tissue. Currently, this can be treated only symptomatically. In mice, a complete compensatory lung growth within 21 days after resection of the left lung can be observed. Understanding and transferring this concept of compensatory lung growth to humans would greatly improve therapeutic options. Lung growth is always accompanied by a process called angiogenesis forming new capillary blood vessels from preexisting ones. Among the processes during lung growth, the formation of transluminal tissue pillars within the capillary vessels (intussusceptive pillars) is observed. Therefore, pillars can be understood as an indicator for active angiogenesis and microvascular remodelling. Thus, their detection is very valuable when aiming at characterization of compensatory lung growth. In a vascular corrosion cast, these pillars appear as small holes that pierce the vessels. So far, pillars were detected visually only based on 2D images. Our approach relies on high-resolution synchrotron microcomputed tomographic images. With a voxel size of 370 nm we exploit the spatial information provided by this imaging technique and present the first algorithm to semiautomatically detect intussusceptive pillars. An at least semiautomatic detection is essential in lung research, as manual pillar detection is not feasible due to the complexity and size of the 3D structure. Using our algorithm, several thousands of pillars can be detected and subsequently analysed, e.g. regarding their spatial arrangement, size and shape with an acceptable amount of human interaction. In this paper, we apply our novel pillar detection algorithm to compute pillar densities of different specimens. These are prepared such that they show different growing states. Comparing the corresponding pillar densities allows to investigate lung growth over time.

Sialophorin, a surface sialoglycoprotein defective in the Wiskott-Aldrich syndrome, is involved in human T lymphocyte proliferation.

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.

Site-directed mutagenesis of class I HLA genes. Role of glycosylation in surface expression and functional recognition.

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.

Abnormal cytolytic activity of lymphocyte function-associated antigen-1-deficient human cytolytic T lymphocyte clones.

The involvement of the lymphocyte function-associated antigen-1 (LFA-1) membrane molecule in cytolytic T lymphocyte (CTL) interactions with lymphoid target cells was investigated using CTL clones derived from two patients with a heritable deficiency of LFA-1. LFA-1 surface expression on the CTL clones was 1% of the normal level of LFA-1, unchanged with prolonged culture, and identical on 14 different CTL clones. The function of the LFA-1 molecule was addressed using the LFA-1-deficient CTL clones and LFA-1-deficient lymphoid target cells. The lytic activity of the LFA-1-deficient CTL clones was 43% of control when tested against a target cell line expressing normal levels of LFA-1 and less than 10% of control when tested against an LFA-1-deficient target cell line. These results demonstrate a direct involvement of LFA-1 in CTL-mediated cytolysis and suggest a more general dependence on LFA-1 in lymphoid cell-cell interactions.

Tumor sponge implantation: an in vivo method for studying syngeneic, primary antitumor lymphocyte responses.

Sponge matrices surgically implanted in the s.c. space of the back of normal BALB/c mice were injected with a "regressor" dose of Moloney virus-induced BALB/c tumor cells. The kinetics of the generation of cytotoxic cells within the sponge was studied over a 22-day period in a short-term 51Cr release assay. Cytotoxic activity peaked on Day 16 and then declined to negligible levels by Day 22. No cytotoxicity was detectable when nontransformed BALB/c blast cells, Moloney leukemia virus-induced tumor (LSTRA) cells, or unrelated chemically induced tumor (EL4) cells were used as targets. When the cellular composition of implanted tumor sponges was examined on Day 16, it was found to be 30 to 40% myeloperoxidase-positive cells, 15 to 25% surface immunoglobulin-positive cells, and 40 to 50% theta-positive cells. Treatment with anti-Thy 1.2 plus complement eliminated the cytotoxic response on Day 16. The ratio of T-cells to tumor cells within the sponge was determined by immunofluorescence. Kinetic studies showed that the number of theta-positive cells increased well before cytolytic activity was detected, possibly reflecting increasing numbers of amplifier T-cells or cytotoxic cell precursors. A later decline in theta-positive cells correlated directly with decreased cytotoxicity. Furthermore, onset of cytotoxic activity also correlated with a decline in the percentage of Moloney murine sarcoma virus tumor cells within the sponge. Sponge cells isolated on Day 16 (peak cytotoxicity), mixed with lethal dosages of moloney murine sarcoma virus tumor cells, successfully neutralized the lethal challenge demonstrating the in vivo antitumor efficacy of these effector cells. Sponges were also implanted in mice which had been immunized with single injection of Moloney murine sarcoma virus cells. Inoculation of the sponge with tumor cells resulted in a second set response in which cytotoxic cells appeared much earlier than in unsensitized animals. Cells from spleen, lymph node, or peritoneal cavity of normal or presensitized animals with tumor sponge implants were not cytotoxic, suggesting a highly localized response.

Node status has prognostic significance in the multimodality therapy of diffuse, malignant mesothelioma.

PURPOSE: We studied a multimodality approach using extrapleural pneumonectomy, chemotherapy, and radiotherapy in patients with malignant pleural mesothelioma. PATIENTS AND METHODS: From 1980 to 1992, 52 selected patients, underwent treatment. Median age was 53 years (range, 33 to 69). Initial patient evaluation was performed by a multimodality team. Pathologic diagnosis was reviewed and confirmed before therapy. Patients with no medical contraindication and potentially resectable mesothelioma on computed tomography (CT) (magnetic resonance imaging [MRI] when it became available) received extrapleural pneumonectomy, cyclophosphamide, doxorubicin, and cisplatin (CAP) chemotherapy, and radiotherapy. RESULTS: Perioperative morbidity and mortality rates were 17% and 5.8%, respectively. The overall median survival duration is 16 months (range, 1 month to 8 years). The 32 patients with epithelial histologic variant had 1-, 2-, and 3-year survival rates of 77%, 50%, and 42%, respectively. Patients with mixed and sarcomatous cell disease had 1- and 2-year survival rates of 45% and 7.5%; no patient lived longer than 25 months (P < .01). At resection, positive regional mediastinal lymph nodes were found in 13. Positive lymph nodes were associated with poorer survival than were negative nodes (P < .01). Patients with epithelial variant and negative mediastinal lymph nodes had a survival rate of 45% at 5 years. CONCLUSION: Multimodality therapy including extrapleural pneumonectomy has acceptable morbidity and mortality for selected patients. Prolonged survival occurred in patients with epithelial histologic variant and negative mediastinal lymph nodes. These data provide a rationale for a revised staging system for malignant pleural mesothelioma; furthermore, they permit stratification of patients into groups likely to benefit from aggressive multimodality treatment.

Soluble and surface-bound immunoglobulin triggers human monocyte activation and hydrogen peroxide release.

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.

o-phthaldehyde fluorescence microassay for the determination of antibody concentration.

o-Phthaldehyde (OPA) reagent reacts with primary amines in aqueous solution at room temperature. When the reaction occurs in the presence of 2-mercaptoethanol, a bright blue fluorescence is produced. We investigated the application of the OPA reagent in the routine determination of monoclonal antibody concentration. The OPA reagent produced maximal fluorescence within 30 min and was stable for 2 h. Buffers containing primary amines gave high background fluorescence; however, the OPA reagent gave reliable results with the most commonly used antibody solutions. The OPA reagent provided a rapid and simple measure of protein concentration from 1 microgram to 1 mg and was readily adapted for use with a 96-well fluorescence reader.

Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes.

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

Biphasic response of the regional lymphatics in the normal lymphocyte transfer reaction.

BACKGROUND: Initially developed for histocompatibility testing, the normal lymphocyte transfer (NLT) reaction involves the intradermal injection of allogeneic lymphocytes from one individual to another. Because of the unique kinetics of the immunological response to allogeneic lymphocytes, the NLT reaction has been considered an informative system for the analysis of transplant immunity. METHODS: In this study, we used bilateral efferent lymph duct cannulations in sheep to examine the regional lymphatic response to the NLT reaction. Our studies used monoclonal antibodies to define lymphocyte population dynamics and DNA flow cytometry to reflect lymphocyte proliferative responses. RESULTS: The results confirmed a biphasic NLT reaction. An unexpected finding was the marked differences between the early and late NLT responses. The early response was characterized by T-lymphocyte proliferation, as reflected by S-phase DNA, which was comparable in both the NLT-stimulated and contralateral control efferent lymphocytes. This bilateral proliferative response was observed in both CD4+ and CD8+ lymphocytes. In contrast, the late response was restricted to the efferent lymph from the NLT-stimulated lymph node. Dual-parameter flow cytometry demonstrated that the dominant component of this unilateral NLT response was CD8+ lymphocytes. CONCLUSIONS: These results suggest important functional distinctions between systemic and regional lymphatic responses to intradermal alloantigens.

Immunoblastic lymphoma of donor origin in the allograft after lung transplantation.

Posttransplant lymphoproliferative disorders (PTLD) are EBV-associated lymphoid neoplasms that are caused by the uncontrolled growth of EBV-infected B lymphocytes. The clinical presentation of PTLD can range from benign polygonal lymphoproliferative disorders to aggressive monoclonal immunoblastic lymphomas. In this report, we describe a seronegative lung transplant recipient who developed an immunoblastic lymphoma 4 months after lung transplantation from a seropositive donor. The neoplastic cells expressed B lymphocyte markers (CD19+, CD20+, sIgM+, kappa+) as well as the EBV antigen EBNA-2. A cell line with similar cytologic features spontaneously grew from in vitro cultures of the patient's peripheral blood mononuclear cells. The cell line and the lymphoma were EBV+, expressed a similar spectrum of B cell surface proteins, and had the donor's HLA haplotype. Analysis of immunoglobulin gene rearrangements and viral terminal repeat sequences revealed that the cell line and the tumor represented distinct B cell clones. Cultured peripheral blood mononuclear cells were restimulated in vitro with the EBV transformed cell line and tested for cytolytic activity. The host T cells demonstrated high levels of cytolytic activity against the tumor cell line that was abrogated by the addition of a anti-monomorphic HLA class I monoclonal antibody (mAb) (W6/32). These studies indicate that cells of donor origin can persist in the transplanted organ and may lead to an EBV-associated posttransplant lymphoma.

Membrane glycoconjugates expressed on sheep airway epithelium.

In mammalian lung, selective airway inflammatory reactions have been associated with viral infections, transplant rejection, and autoimmune diseases. Although the molecular basis for this selective reactivity is unknown, the importance of carbohydrates in immunologic processes suggests a potential role for membrane glycoconjugates in tissue-specific inflammatory reactions. In the present work we examined a panel of 39 lectins for their pattern of reactivity in the peripheral airways of the sheep lung. The size of the panel facilitated a comprehensive description of the glycoconjugate localization on the airway epithelium. Four lectins (agglutinins for Helix aspersa, Psophocarpus tetragonolobus, Trichosanthes kirilowii, and Griffonia simplifolia II) revealed selective reactivity with the small airway epithelium. On lectin Western blotting, these four lectins demonstrated a common low molecular weight banding profile that was distinct from control lectins. The histochemical staining patterns and Western blotting profiles provided evidence for the selective expression of membrane glycoconjugates in the peripheral airways of the sheep lung.

Aldehyde fixation of thiol-reactive fluorescent cytoplasmic probes for tracking cell migration.

Tracking of cell migration plays an important role in the study of morphogenesis, inflammation, and metastasis. The recent development of probes that exist as intracellular peptide-fluorescence dye adducts has offered the possibility of aldehyde fixation of these dyes for detailed anatomic studies of lymphocyte trafficking. To define the conditions for fixation of these cytoplasmic fluorescent probes, we compared fixation conditions containing formaldehyde, glutaraldehyde, paraformaldehyde, zinc formaldehyde, and glyoxylate, as well as fixation by quick-freezing in liquid nitrogen-cooled methylbutane. The efficacy of aldehyde fixation of the cell fluorescence was assessed by quantitative tissue cytometry and flow cytometry. We studied cytoplasmic fluorescent dyes with discrete emissions in the green [5-chloromethylfluorescein diacetate (CMFDA); 492 ex, 516 em] and orange [5-(and-6)-(4-chloromethyl(benzoyl)amino) tetramethylrhodamine (CMTMR); 540 ex, 566 em] spectra. The results demonstrated that aldehyde fixation preserved cell fluorescence for more than 6 months. The primary difference between the aldehyde fixatives was variability in the difference between the yield of the cell fluorescence and the relevant background fluorescence. Formaldehyde and paraformaldehyde were superior to the other fixatives in preserving cell fluorescence while limiting background fluorescence. With these fixatives, both the CMFDA and CMTMR fluorescent dyes permitted sufficient anatomic resolution for reliable localization in long-term cell tracking studies.

Mapping functional epitopes of the human LFA-1 glycoprotein: monoclonal antibody inhibition of NK and CTL effectors.

Anti-LFA-1 monoclonal antibody (MoAb) was originally identified by screening antibodies for their ability to inhibit cytolysis in the absence of complement. Anti-LFA-1 MoAb has been shown to inhibit both natural killer (NK) and cytolytic T lymphocyte (CTL) mediated cytolysis. To further define the utilization of this molecule in cell-mediated cytolysis, we used a panel of MoAb to functional epitopes on both the alpha and beta chains of the LFA-1 heterodimer. The panel was used to compare OKT3- NK effectors and OKT3+ CTL clones. As expected, function-associated MoAb to CTL antigens (T3, T8, LFA-2) and target cell antigens (HLA, LFA-3) blocked only CTL clones and not NK effectors. In contrast, anti-LFA-1 MoAb blocked both NK effectors and CTL clones. In addition, the panel of anti-LFA-1 MoAb demonstrated an identical hierarchy of functionally relevant LFA-1 epitopes. Given the similar utilization of LFA-1 in NK and CTL mediated cytotoxicity assays, we explored the ability of MoAb to different epitopes on LFA-1 to inhibit conjugate formation. Anti-LFA-1 MoAb inhibition of NK-target binding paralleled the inhibition of CTL-target binding. Thus, functional epitopes on the LFA-1 molecule have been defined for NK and CTL effectors. The identical hierarchy of functional epitopes indicates that the LFA-1 molecule is similarly utilized in NK and CTL mediated cytotoxicity and that the relevant epitopes are involved in effector-target conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell adhesion molecule expression in the sheep thymus.

Cell adhesion molecules are potential regulating factors in both prethymic and intrathymic T cell development. An experimental challenge has been the development of a large animal model that facilitates in vivo studies of both intrathymic development and lymphocyte migration. To extend earlier studies of thymic development, we have developed a panel of monoclonal antibodies (mAb) to a variety of sheep cell adhesion molecules. Immunohistochemistry was used to define mAb reactivity and flow cytometry was used to quantify expression of cell adhesion molecules within the thymus. To facilitate flow cytometry definition of cortical thymocytes, mAbs were developed to the sheep CD1 antigen. Dual parameter flow cytometry provided a phenotypic characterization of cell adhesion molecule expression on both CD1(+) and CD1(-) sheep thymocyte populations. These studies demonstrated significantly enhanced cortical thymocyte expression of three cell adhesion molecules: beta1 integrin (CD29), ICAM-2 and LFA-3. The beta1 integrin cell adhesion molecule was also expressed at higher levels on CD1(+) thymocytes in post-natal lambs as compared to adult sheep. These studies of thymocyte membrane molecule expression should facilitate future investigations of sheep intrathymic development and T lymphocyte immigration.

Treatment of patients with lung cancer and severe emphysema.

The development of lung cancer and emphysema is associated with the destructive chemical milieu that occurs with smoking. The recent interest in lung volume reduction surgery (LVRS) has stimulated a reassessment of the indications for surgery in patients with early stage lung cancer or emphysema. For patients with both diseases, the issues surrounding LVRS are simplified. The major concern is that the lung cancer can be surgically removed without the need for postoperative ventilation or mortality. A secondary consideration is the potential for long-term postoperative respiratory morbidity. These risks can be estimated by evaluating the anatomic location of the tumor, as well as the physiology of the underlying emphysema. Early results of combined LVRS and lung cancer resections suggest a favorable outcome in carefully selected patients.

Surgical resection in the management of small-cell carcinoma of the lung.

Surgery has had little impact on long-term survival in patients with small-cell lung cancer (SCLC). With the evolution of modern techniques, however, surgery may play an increasingly valuable role in SCLC. Surgery may potentially cure a select minority of SCLC patients. Patients with peripheral nodules and those with regional disease achieving a complete response to chemotherapy may benefit from adjuvant surgical resection for removal of residual disease. In some cases, surgery may also be preferred over adjuvant radiotherapy, since the latter necessitates lowering the total chemotherapy dose administered to SCLC patients.

Preoperative assessment of patients undergoing pulmonary resection.

A careful preoperative assessment of patients with lung cancer is essential for identifying those at high risk for morbidity and mortality related to the surgical procedure. The clinician must assess the risk associated with such treatment, decide whether the risk is prohibitive, and institute therapy to reduce such risk. Testing modalities used in the preoperative evaluation include spirometry, full pulmonary function tests, measurement of arterial blood gases, radionuclide lung scanning, exercise testing, invasive measurement of pulmonary artery pressure, and a variety of studies involving lobar occlusion or lateral position testing. Studies evaluating the utility of these procedures are reviewed. Additionally, the impact of advanced age on postsurgical outcome is evaluated, as are the possibility of operating on high-risk patients and the use of preoperative interventions.

Sleeve lobectomy, segmentectomy, and thoracoscopy in the management of carcinoma of the lung.

In patients with lung cancer, the goals of limited resection procedures of the lung and major airways are to provide an adequate cancer operation while preserving functioning lung tissue. Discussed in this article are sleeve lobectomy, an alternative to pneumonectomy in patients with cancer in a lobar orifice; segmentectomy or wedge resection, an alternative to lobectomy in those with a peripheral lung cancer; and thoracoscopy, an alternative to open thoracotomy for various chest malignancies.

Extrapleural pneumonectomy in the setting of a multimodality approach to malignant mesothelioma.

The use of extrapleural pneumonectomy in a multimodality treatment setting for malignant pleural mesothelioma is described, presenting first the right-sided approach and then the left-sided. This technique used in a multimodality approach with CAP chemotherapy (cyclophosphamide 600 mg/m2, doxorubicin 60 mg/m2, cisplatin 75 mg/m2) 5 cycles at 3-week intervals, and radiotherapy (55 Gy radiation to sites of previous bulky disease or residual disease) to treat 44 patients with malignant pleural mesothelioma resulted in improved operative mortality and decreased length of hospital stay.