Bilateral Morgagni hernia: operative discovery of appendix lying on superior pulmonary vein.

A patient presented having an acute abdomen on a background of a twelve-month history of worsening asthma. Computed tomography showed giant bilateral intrathoracic hernias extending to both thoracic apices. Our case was unusual as the defect was bilateral and left-sided. Surgical repair revealed each hernia sac measuring >20 cm and to contain the entirety of the small bowel and colon (including retroperitoneal bowel). The appendix was discovered adjacent to right superior pulmonary vein. Both sacs were excised and the defects dissected and transfixed in a single stage operation. In the post-operative stage, he developed a 6.3 cm fluid collection anterior to the right atrium and a left-sided pleural effusion. Morgagni hernias can escape detection and be attributed to other diagnoses courtesy of false localising signs on clinical examination and symptoms in the history.

Systemic local anaesthetic toxicity from continuous thoracic paravertebral block.

Continuous paravertebral block is commonly used for post-thoracotomy analgesia and compares favourably with other systemic and regional methods with regard to safety and efficacy. No major complications of continuous paravertebral block for post-thoracotomy analgesia have been reported previously. We report here a case of systemic local anaesthetic toxicity from continuous paravertebral block administration after thoracotomy and lobectomy leading to seizure, aspiration, and ultimately, death. Potential contributing factors in this case included small patient size, concomitant antifungal therapy, extensive surgical disruption of the pleurae, and inappropriate paravertebral bolus administration. Postoperative delirium was a diagnostic confounder. We discuss the potential causes and means of avoiding similar complications in the future.

Consequent intrafamilial immunization for DL-A haplotyping in canines.

A procedure of intrafamilial immunization is described for production of antisera recognizing DL-A haplotypes. In a colony consisting of 1 sire, 6 bitches, and 67 offspring all haplotypes could be accurately allocated. In the colony the observed reaction frequencies of the antisera are in agreement with mendelian codominant inheritance. Mixed lymphocyte culture tests confirmed the accuracy of the serologic typing and the presence of homozygous individuals within the colony. Further evidence is presented supporting the presence of two or more subloci within the DL-A system. Colonies of canines such as the one described should provide a sensitive system for evaluating interaction between serologic DL-A typing, MLC reactivity, and immune response genetics in a nonrodent species which is not highly inbred.

BPA and NP removal from municipal wastewater by tropical horizontal subsurface constructed wetlands.

It has been recognized that numerous synthetic compounds like Bisphenol A (BPA) and nonylphenols (NP) are present in effluents from wastewater treatment plants (WWTP) at levels of parts per billion (µg L(-1)) or even parts per trillion (ng L(-1)) with a high potential to cause endocrine disruption in the aquatic environment. Constructed wetlands (CW) are a cost-effective wastewater treatment alternative with promising performance to treat these afore mentioned compounds. This research was aimed to evaluate the efficacy of CW treatment of WWTP effluent for mitigating the effects endocrine disrupting compounds (EDCs). This research goal was accomplished by (1) quantifying the removal of BPA and NP in CWs; (2) isolating CW fungal strains and testing for laccase production; and (3) performing endocrine disruption (reproduction) bioassays using the fruit fly Drosophila melanogaster. Three pilot scale horizontal subsurface flow constructed wetlands (HSSF-CW) were operated for eight weeks: one planted with Phragmites australis; one planted with Heliconia psitacorum; and one unplanted. The Heliconia CW showed a removal efficiency of 73.3(± 19%) and 62.8(± 20.1%) for BPA and NP, respectively; while the Phragmites CW demonstrated a similar removal for BPA (70.2 ± 27%) and lower removal efficiency for NP 52.1(± 37.1%).The unplanted CW achieved 62.2 (± 33%) removal for BPA and 25.3(± 37%) removal for NP. Four of the eleven fungal strains isolated from the Heliconia-CW showed the capacity to produce laccase. Even though complete removal of EDCs was not achieved by the CWs, the bioassay confirmed a significant improvement (p < 0.05) in fly viability for all CWs, with Heliconia sp. being the most effective at mitigating adverse effects on first and second generational reproduction. This study showed that a CW planted with a native Heliconia sp. CW demonstrated a higher removal of endocrine disrupting compounds and better mitigation of reproductive disruption in the bioassay.

Red blood cell homeostasis: recognition of distinct types of damaged homologous red blood cells by a mouse macrophage cell line.

The mouse macrophage (M phi) cell line IC-21 preferentially ingests a subpopulation of homologous red blood cells (MRBC) from normal mice. This subpopulation presumably bears the so-called transfusion lesion, a consequence of damage acquired during the drawing and processing of blood. To determine if all damaged MRBC were recognized by a common receptor site on IC-21 M phi, we prepared suspensions of MRBC damaged in vitro by treatment with tannic acid and compared the phagocytic uptake of these cells with those bearing the transfusion lesion. Trypsin treatment of IC-21 M phi rendered them unable to recognize MRBC bearing the transfusion lesion; but it had no effect on the uptake of tannic acid-damaged MRBC, showing that IC-21 M phi have separate recognition sites for these two populations of damaged MRBC.

Expression of Ia antigen in colonies of bone-marrow-derived macrophages.

Cells in colonies of culture-derived mouse and rat bone marrow macrophages were examined for membrane Ia antigen to determine if the steady-state expression of the antigen was restricted to macrophages derived from a distinct population of progenitor cells. Multiple subcultures of macrophages derived from single soft-agar colonies were tested for Ia+ cells on three different days of culture. About one-half of the colonies gave rise to subcultures that never contained Ia+ cells, about 40% yielded subcultures that contained some Ia+ cells on at least 2 of the 3 assay days, and about 10% of the colonies produced subcultures that contained Ia-bearing cells on all 3 assay days. Thus, when cultures were assayed at any one time for their content of Ia-bearing cells, the results raised the possibility of phenotypically distinct subpopulations of progenitors. However, sequential analyses of the subcultures revealed the variable expression of Ia on cells from at least one-half of the colonies. We conclude that, under steady-state conditions, the presence of Ia antigen on bone marrow-derived macrophages is not clonally restricted. That a T-cell-derived lymphokine induced Ia antigen on essentially all the cells in most of the colonies of macrophages confirms their potential to express the antigen.

Mouse macrophages contain a truncated CD4 transcript.

Mouse macrophages do not express CD4 on their surfaces. We used the polymerase chain reaction to investigate CD4 gene transcription in individual clones of primary mouse splenic macrophages and cell lines of spleen and bone marrow macrophages. The results show only the presence of CD4 mRNA transcripts that are truncated in the 3' coding sequence, thus explaining the lack of expression of a mature CD4 gene product by these cells.

Establishment of mononuclear phagocyte cell lines.

Macrophage cell lines have been derived from cells residing in a number of tissues from a variety of species. In this report, the methods used to obtain these cell lines are reviewed and a simple and efficient method for generating nonvirus-transformed lines from individual cloned progenitors located in mouse tissues is described.

In vivo activation of quiescent B cells by anti-immunoglobulin. II. Production of lysozyme-specific monoclonal antibodies of desired isotype.

We wished to determine whether injection of mice with anti-isotype antibody would be a means to regulate in vivo isotype expression and to obtain hybridomas secreting monoclonal antibodies (mAbs) of desired antigen specificity and isotype. Treatment with a rat mAb (7D2) reactive with both the IgG2a and IgG2b isotypes of mouse Ig resulted in large increases in the serum concentrations of mouse IgG2(a + b). Moreover, injection of antigen-7D2 conjugates had a profound effect on the isotype distribution of hybridomas subsequently obtained from these animals. Thus, while greater than 95% of anti-hen eggwhite lysozyme (HEL) mAbs prepared from mice immunized with HEL alone were of the IgG1 isotype, 12/15 (80%) of the mAbs from mice injected with HEL-7D2 conjugates were of the IgG2a or IgG2b isotype. When tested for effector functions using HEL-coated erythrocytes, the mAbs showed the expected activities, i.e., the IgG2, but not IgG1 anti-HEL mAbs were able to fix complement, bind protein A, and mediate antibody-dependent cytotoxicity and phagocytosis. These results indicate that in vivo immunization with anti-isotype-antigen conjugates can be used to produce hybridomas of predetermined antigen and isotype specificities.

Use of the Pannell-Milstein roller bottle apparatus to produce high concentrations of the CSF-1, the mouse macrophage growth factor.

A simple roller bottle apparatus for growing B cell hybridomas in dialysis tubing yields high concentrations of monoclonal antibodies (Pannell and Milstein, J. Immunol. Methods (1992) 146, 43-48). Here we describe an adaptation of this apparatus for the simple production of high concentrations of the mouse macrophage specific growth factor, CSF-1. This apparatus appears to have general applicability for the production of other secreted cytokines.

Immortalization of growth factor-dependent mouse splenic macrophages derived from cloned progenitors.

We describe a nonviral transformation strategy for the establishment of permanent cell lines derived from the progeny of individual mouse splenic macrophage (M phi) progenitors. These colony stimulating factor-1 (CSF-1)-dependent cell lines possess many features of mature M phi s, including antibody-dependent phagocytic and cellular cytotoxic activities, ability to secrete lysozyme, and expression of the Mac-1 antigen and mRNA for the CSF-1 receptor. It was also possible to immortalize selected clones of splenic M phi s differing in their constitutive antigen-presenting activities with the retention of the antigen-presenting phenotype in the resultant cell lines. The approach described in this report should be useful in obtaining additional cell lines of M phi s expressing other phenotypes of interest.

Some fixation reagents reduce or abolish the detectability of Ia-antigen and HLA-DR on cells.

The effect of paraformaldehyde (PF), glutaraldehyde (GT), methanol (ME), ethanol (ET) and acetone (AC) fixation on the detectability of Ia antigens on murine and rat peritoneal exudate (PE) and resident peritoneal (RP) macrophages (M phi), and on detectability of HLA-DR antigens on human blood leukocytes (HBL) and human splenic M phi (HSM phi) was examined. Ia-antigen on Mø from H-2k mice was detected by a rosetting assay using erythrocytes (E) to which a monoclonal antibody (MoAb) reactive to Ia.2 (E anti-Ia.2) had been coupled, and by the direct binding of 125I-labeled anti-Ia.2. The antigen was detected on Wistar/Furth (W/Fu) rat RPMø splenocytes (SC) by rosetting with E coupled with a MoAb to the murine determinate Ia.17, which cross-reacts with an Ia-like molecule on cells from the W/Fu strain. HLA-DR framework determinants were detected on HBL and HSMø by the binding of 125I-labeled MoAb and by an avidin-biotinylated peroxidase procedure. Exposure of murine PEMø or RPMø to 1% PF or 0.5% GT for 15 min at room temperature reduced 125I-anti-Ia.2 binding and E anti-Ia.2 rosetting by at least 60%; the radioimmunoassay was more affected by the fixatives than was the rosetting assay. Further, PEMø were more sensitive to the effect of PF fixation than were RPMø. Treatment of freshly isolated RPMø with 1% PF reduced the proportion of Ia-bearing cells detected by the rosetting assay by greater than 50%. Culturing alone did not affect the detectability of Ia on RPMø as assessed by the rosetting test, but cultured RPMø were more sensitive to the effects of FX fixation than fresh cells except when lymphokine from Con A-stimulated murine SC was included in the culture medium. Similar losses of HLA-DR were recorded when HBL and HSMø were exposed to PF, GT, ME or ET, but brief (less than 20 s) treatment with cold AC did not appreciably reduce antigen detectability. Procedures in which fixation takes place after the primary antibody binding step did not result in an appreciable loss of detectable Ia. Thus, commonly used fixatives affect the detectability of Ia and Ia-like antigens on a variety of cells. Results obtained from assays on cells treated prior to the primary antibody binding step, therefore, must be interpreted with caution.

Isolation of human splenic macrophages and lymphocytes by countercurrent centrifugal elutriation.

Certain tissues, such as the spleen, are rich sources of mononuclear phagocytes (MP); however, separating the phagocytes from tissues and removing the contaminating cells have been difficult. We report here a method for the extraction and purification of human splenic MP that employs gentle homogenization of splenic fragments with a Tenbroeck tissue homogenizer, controlled digestion with purified collagenase to free MP from splenic stroma, incubation with DNase to dissociate cell clumps and purification by countercurrent centrifugal elutriation (CCE). With homogenization and enzymatic digestion most of the splenic nonspecific-esterase-positive cells are freed into suspension as determined by morphometric analysis of 2 micron sections from plastic embedded spleen stained for alpha-naphthyl butyrate esterase (ANB). Overall cell recovery after homogenization and enzyme treatment is 56 +/- 7%; no selective cell loss occurs as determined by differential cell counts at each purification step. CCE of up to 3 X 10(9) treated spleen cells results in recovery of 63 +/- 6% of the elutriated cells and separates nearly 50% of the recovered MP into enriched fractions. These MP are morphologically intact as determined by light and electron microscopy and are actively phagocytic. Highly purified (96%) autologous splenic lymphocytes are a useful by-product of this separation technique.

Separate precursor cells for macrophages and microglia in mouse brain: immunophenotypic and immunoregulatory properties of the progeny.

The brain contains two populations of macrophages: the microglia of brain parenchyma, and the central nervous system (CNS) macrophages located in the perivascular spaces, the leptomeninges and the choroid plexus. The microglia are characterized, in part, by their paucity of major histocompatibility complex (MHC) molecules and lack of constitutive antigen (Ag)-presenting activity for naïve CD4+ T-cells. Some CNS macrophages, on the other hand, constitutively express MHC molecules and present Ag to naïve CD4+ T-cells. We have reported that mouse brain contains precursor cells that, in the presence of colony-stimulating factor-1, the macrophage growth factor, give rise to clones of cells that differ in their ability to constitutively present Ag to naive CD4+ T cells. Here we report that this population of precursor cells can be separated into two discrete subpopulations based on differences in cell density and that the two cell populations give rise to progeny that differ in their content of cells constitutively expressing MHC class II and CD86 molecules, and the ability to present Ag to naïve CD4+ T-cells. A comparison of the level of CD45 staining of the progeny, an indication of a microglial or a CNS macrophage origin, suggests that one population of precursor cells yields immunologically immature microglia and the other CNS macrophages.

Phenotypes and alloantigen-presenting activity of individual clones of microglia derived from the mouse brain.

To clarify the origin and function of the microglia residing in the central nervous system, we cloned brain cells from newborn and adult mice in soft agar containing the macrophage-specific growth factor, colony-stimulating factor-1 and expanded the cells from individual colonies in liquid culture medium. The results of molecular, immunophenotypic and functional analyses showed that the clones consisted of microglia derived from the macrophage family of cells. For instance, the microglia contain mRNA transcripts for the receptor for colony-stimulating factor-1 and truncated CD4 transcripts similar to those found in mouse macrophages but not T helper cells. About a third of the microglial progenitors gave rise to progeny that constitutively induced the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ T cell-independent manner, a response that was inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I molecules on the microglia. Since all microglia expressed similar levels of MHC class I molecules, the basis for the alloantigen presentation likely resides in the ability of some clones of microglia to synthesize co-stimulator molecules that are required for CD8+ T cell proliferation. Thus, at least some microglia in mouse brain arise from endogenous progenitors and appear capable of specialized functions.

Mouse microglial cell lines differing in constitutive and interferon-gamma-inducible antigen-presenting activities for naive and memory CD4+ and CD8+ T cells.

We developed a panel of non-virus transformed cell lines derived from individual microglial precursors residing in the brains of normal mice. These colony stimulating factor-1-dependent cell lines are B7-1+ (CD80), Mac-1+, Mac-2+, Mac-3+, CD45+, MHC class I+, colony stimulating factor-1 receptor+, and they ingest antibody-coated particles. However, the cell lines differ in their expression of B7-2 (CD86), F4/80, Ly-6C and MHC class II molecules. They also differ in their ability to constitutively process and present antigens to naive CD4+ and CD8+ T cells, memory CD4+ and CD8+, and in the manner by which interferon gamma modulates their antigen-presenting activities. These cell lines should be valuable as models for studies on the immunobiology of the microglia.

Thoracoscopic pulmonary lobectomy. Early operative experience and preliminary clinical results.

Thoracoscopic video-assisted lobectomy procedures were performed in 11 patients (7 men, 4 women; age range 40 to 74 years, mean 66 years). Ten patients had peripheral pulmonary opacities: eight of these were bronchogenic carcinomas, one was an atypical carcinoid lesion, and one was a pulmonary infarct. All of these cases had preoperative evaluation by computed tomographic scanning to exclude mediastinal lymphadenopathy. The remaining patient had preoperatively diagnosed lobar bronchiectasis. Surgical access was gained via three stab (1 cm) incisions and one short (7 cm) submammary incision, which was made without rib separation and was used for specimen delivery. Lobes resected were the left upper (n = 4), left lower (n = 2), right upper (n = 2), and right lower (n = 3). All patients survived. Overall mean operative time was 3.3 hours and blood loss 263 ml. For the latter five cases, however, these figures were reduced to 2.3 hours and 100 ml, respectively, indicating improvement with experience. In no cases was ventilatory assistance required. Mean high-dependency unit time was 41 hours. In each case, it was possible to perform a standard dissectional lobectomy with lobar lymph node clearance equal to that obtained at open thoracotomy. Comparison with a series of 33 open lobectomy procedures demonstrated reduced postoperative pain, morphine consumption, and high-dependency unit stay. This preliminary experience supports the development of video-assisted thoracoscopic pulmonary lobectomy for patients with small peripheral opacities or known benign disease.

Primary small-cell cancer of the esophagus.

Primary small-cell cancer of the esophagus is a rare tumor that disseminates early with a uniformly poor prognosis if untreated. Sixteen patients with malignant dysphagia referred to the Thoracic Surgical Unit, City Hospital, Edinburgh, within a 10-year period had a diagnosis of primary small-cell cancer of the esophagus. Seven patients underwent subtotal esophagectomy or esophagogastrectomy, either alone or with adjuvant chemotherapy or radiotherapy, with a mean survival of 20 months (standard deviation 35.4 months, range 2 weeks to 96 months). The remaining nine patients had disseminated disease when they were first seen and were treated symptomatically by intubation alone (1 patient), intubation and palliative chemotherapy or radiotherapy (3 patients), palliative chemotherapy (2 patients), palliative radiotherapy (1 patient), or no therapy (2 patients), with a mean survival of 4.8 months (standard deviation 2.6 months, range 2 to 9 months). Patients seen with this aggressive tumor should be assessed urgently for evidence of metastatic spread and then offered resection in combination with chemotherapy if they are otherwise fit for operation. This treatment regimen has given us one long-term survivor (96 months) who, we believe, is the only patient to have been cured of this condition. Patients seen with disseminated disease should have symptomatic treatment of the dysphagia combined with palliative chemotherapy.

Prognostic significance of tumor deoxyribonucleic acid content in surgically resected small-cell carcinoma of lung.

The prognostic role of deoxyribonucleic acid flow cytometry was investigated in 53 cases of surgically resected small-cell lung cancer. Deoxyribonucleic acid aneuploidy was detected in 26 patients (49.1%), the remaining tumors being either diploid or tetraploid. Patients with aneuploid tumors had a significantly reduced 2-year survival (38.5%) when compared with patients with diploid or tetraploid tumors (70.3%; p less than 0.05). This finding was independent of tumor stage on multiple logistic regression analysis. Diploid or tetraploid deoxyribonucleic acid content was associated with a particularly good 2-year survival (85%) in N0 or N1 disease. Tumor deoxyribonucleic acid ploidy should be taken into account in planning of management and assessment of prognosis in small-cell lung cancer.

A comparison of cobalt (57Co) bleomycin scanning and contrast-enhanced CT scanning for assessment of the mediastinum in lung cancer.

Sixty patients with histologically proven lung cancer who had been accepted for mediastinoscopy or thoracotomy were prospectively entered into a study to evaluate computed tomographic (CT) scanning, 57Co-bleomycin scanning, and barium swallow in preoperative assessment of mediastinal lymph node metastasis. Fifty-six patients had thoracotomy at which all accessible lymph nodes were sampled. Twenty-four patients were found to have mediastinal tumor on histologic analysis of the resected mediastinal lymph nodes. Neither 57Co-bleomycin scanning nor barium swallow were clinically useful, with sensitivities of 21 percent and 11 percent respectively, whereas CT scanning was helpful. However, there was no clear cutoff point of node size to optimize sensitivity and specificity for CT scanning. When nodes greater than or equal to 15 mm were taken to indicate likely malignancy, the sensitivity was 58 percent and the specificity was 87 percent and when greater than or equal to 10 mm was used the sensitivity was 80 percent but the specificity was only 55 percent. There was no clear relationship between the size of the largest resected lymph node in each patient and the presence of malignant lymph nodes. Only 42 percent of patients with resected nodes greater than or equal to 2 cm had histologic evidence of metastases. We conclude that CT scanning should be used to indicate the presence and site of mediastinal lymph nodes, which, when visualized, should always be sampled and histologically examined prior to resection of primary tumor.