Lymphocytes stimulated with recombinant human interleukin-2: relationship between motility into protein matrix and in vivo localization in normal and neoplastic tissues of mice.

Murine splenic lymphocytes nonspecifically stimulated with recombinant human interleukin-2 in vitro were fractionated according to their ability to migrate through type I collagen gel during a 24-hour period. After labeling with 111In and adoptive transfer into hosts of subcutaneous mammary tumors, motile fractions exhibited approximately twofold greater tumor localization and approximately twofold lower lung localization than nonmotile fractions. The results suggest that lymphocyte properties associated with motility through extracellular matrix also influence patterns of lymphocyte localization in vitro. It should be possible to identify these properties by comparative analysis of motile and nonmotile fractions obtained from various matrices.

Motility and tumoricidal activity of interleukin-2-stimulated lymphocytes.

The motility of murine splenic lymphocytes stimulated nonspecifically by recombinant interleukin 2 (RIL-2) was studied in a three-dimensional collagen-gel system. Nonadherent BALB/c splenic lymphocytes were cultured in medium containing Cetus RIL-2 (700 to 1000 units/ml) or excipient control. They were then allowed to locomote randomly for 16 to 18 h into slabs of type I rat tail collagen gel. The gels were digested with collagenase, and total lymphocyte populations and motile subpopulations were collected and compared with respect to their lymphokine-activated killer activity (measured as 4-h cytotoxicity against the natural killer-resistant mammary adenocarcinoma line 410.4), their natural killer activity (measured as 4-h cytotoxicity versus lymphoma YAC-1), and their subset distribution (defined by immunofluorescence). Some of the slabs were not digested but fixed for measurement of leading-front distance. RIL-2-stimulated lymphocyte populations displayed greater motility than unstimulated populations; the mean leading front distance was 2.4 times greater, and the percentage of cells exhibiting motility was approximately doubled. The most motile RIL-2-stimulated cells, however, were not the most tumoricidal. Motile subpopulations displayed approximately 25 to 60% lower lymphokine-activated killer activity than did the total populations from which they were derived. Natural killer activity followed a similar pattern. Motile subpopulations contained a lower proportion of asialo-GM1+ and T-null cells than did total populations and a higher proportion of L3T4+ cells. Chemokinetic stimulation with alpha-interferon increased overall motility, but the lymphokine-activated killer activity of the motile subpopulation was still lower than that of the total population. Lymphocyte motility is important in the infiltration of tumors and other inflammatory lesions. The results indicate that the most tumoricidal lymphocytes in RIL-2-stimulated populations may not be the best tumor infiltrators, and that the tumoricidal activity of circulating lymphocytes may be a misleading indicator of the effectiveness of immunotherapy.

Inflammatory infiltrates of experimental mammary cancers.

The purpose of this review was to summarize observations on the type and function of inflammatory infiltrates of mouse mammary tumors and to speculate on the underlying mechanisms and the significance of infiltrates to mammary tumor biology. Although the major conclusion is that much more work is needed, certain themes seem to be emerging. The number of infiltrating cells can be very high but is unrelated to biological behavior of the tumors. What seems to be important is the relative contributions of inflammatory cell subsets. In the case of T-cell subsets and NK cells, the infiltrates from tumors of long-term cell lines so far seem uninformative. The general characteristics are similar to those of infiltrates from rapidly proliferating, normal mammary tissues. These characteristics do not correlate with diverse biological behavior or malignant potential. A more informative model appears to be one in which the development of tumors from preneoplastic tissue can be observed. Here our attention is currently focused on NK cells. By contrast, the correlation between activated TAM and metastatic behavior suggests that our transplantable MMT lines may be biologically relevant in the study of infiltrating macrophages. We are especially interested in the role of TAM in the generation of tumor cell variability. Overall, our data indicate that the host infiltrate is another manifestation of both inter- and intra-tumor heterogeneity and, as such, is not simply a response to, but, rather, a part of the tumor ecosystem. Unraveling the cellular and molecular mechanisms that govern the inflammatory cell component of tumors should provide insight into the types of cellular interactions that result in tumor development and progression.

Assessment of right ventricular anatomy and function by quantitative radionuclide ventriculography.

Determination of right ventricular ejection fraction and volumes from radionuclide studies is cumbersome and is subject to considerable methodologic error. Further, assessment of regional wall motion has only infrequently been approached in a systematic way. A system of right ventricular ejection fraction and volume measurements is described that utilizes the previously validated single plane geometric method applied to first pass radionuclide angiocardiograms. Five right ventricular chords were defined and used to assess regional wall motion; normal values were obtained from 14 patients who were without demonstrable cardiac disease. Among 23 patients with anterior myocardial infarction, the right ventricular ejection fraction was within 2 SD of normal in 16; however, 3 of these patients showed regional wall motion abnormalities in the right ventricle. Of 21 patients with inferior myocardial infarction, right ventricular ejection fraction was reduced in 15; of the 6 with normal values, 3 had regional wall motion abnormalities as demonstrated by the chord shortening method. Of 21 patients with dilated cardiomyopathy, right ventricular function was abnormal in 20; the presence of a wall motion abnormality in the conus segment separated these patients from patients with right ventricular dysfunction after recent myocardial infarction. Thus: 1) right ventricular ejection fraction, volumes and wall motion can be assessed by a simple, geometric technique; 2) analysis of chord shortening by this method provides information unavailable from global ejection fraction data alone; and 3) the clinical correlates of these data will require further investigation.

Regulation of L-selectin expression by a dominant negative Ikaros protein.

Ikaros family members play critical roles in hematopoietic development, yet molecules regulated by Ikaros proteins remain incompletely characterized. To determine the requirements for functional Ikaros proteins, we overexpressed Ik7, a dominant negative Ikaros protein, in human cell lines and hematopoietic progenitor cells. Ik7 is known to block the normal function of other Ikaros family members in human and mouse cells. Retroviral-mediated overexpression of Ik7 affected two distinct, migratory properties of the CEM T-cell line. Ik7 down-regulated L-selectin cell-surface expression, an effect not a result of increased shedding but of a decrease in L-selectin mRNA levels. Ik7 also reduced the spontaneous migration of CEM T cells in 3-D collagen gels. A reduction in L-selectin, cell-surface expression was also induced by Ik7 in CD34+ hematopoietic progenitor cells. In contrast, the Reh B cell line showed an up-regulation of L-selectin, cell-surface levels when expressing Ik7. For the first time, this study defines an effect of Ikaros proteins in the control of migration-related properties and shows that intact Ikaros proteins are important in a cell type-specific manner for the normal regulation of L-selectin expression.

Sarcoidosis and reactive pulmonary hypertension.

Despite diffuse disease of the lungs (often with widespread inflammation or obliteration of blood vessels) in sarcoidosis, pulmonary hypertension is uncommon, occurring in 1% to 4% of cases. We report a case of sarcoidosis and severe pulmonary hypertension that, in striking contrast to other reports, occurred in the absence of obliterative pulmonary vascular disease. We therefore examined the possibility of whether an abnormality in pulmonary vascular tone might be a cause of the pulmonary hypertension. In pharmacologic studies, we demonstrated pulmonary vasodilatation and, in response to increased pulmonary blood flow, the elaboration of the pulmonary vasoconstricting eicosanoid, thromboxane.

In vivo localization of lymphocytes labelled with low concentrations of Hoechst 33342.

Hoechst 33342 (HO 33342) is a fluorescent dye which binds specifically to DNA and can be used to label lymphocytes for in vivo migration studies. Lymphocytes were treated with varying concentrations of HO 33342 and assayed in vitro for effects on viability, mitogen-stimulated proliferation, and motility. In vivo traffic studies were performed to determine a dye concentration with minimal toxicity for lymphocytes, but sufficient fluorescence for detection of cells in frozen sections. The concentration reported to yield quantitative staining of nuclear DNA (10.7 microM, or 6 micrograms/ml) reduced motility and proliferative response, and resulted in an altered lymphocyte migration pattern compared to untreated lymphocytes. A concentration of 0.25 microM, however, produced no toxicity in the in vitro assays, and an in vivo migration pattern similar to that of untreated cells; lymphocytes stained with 0.25 microM HO 33342 for 30 min were readily observable in histological sections. This study indicates that the concentration of HO 33342 optimal for DNA staining may exert deleterious effects on in vivo lymphocyte traffic studies, and that far lower dye concentrations are more suitable for such studies.

A new assay to measure monoclonal antibody-dependent complement or macrophage-mediated tumor growth inhibition.

A new in vitro assay has been developed to measure the inhibition of tumor growth by antibody and complement or by antibody and macrophages. Tumor cells (1 X 10(5) cells) or a mixture of tumor cells (1.5 X 10(4) cells) and macrophages (1.5 X 10(5) cells) are immobilized in a 1 microliter agarose droplet. Antibody is added to the medium bathing the agarose droplet. Complement is also added to wells containing droplets with tumor cells alone. The area covered by tumor cell monolayer is measured non-destructively from day 0 to day 7 with a split image tracing device. Cell growth is expressed by the increase in the square root of the measured area. This assay does not require isotopes and can be used to test tumor cells freshly dissociated from solid tissues. It permits cell-cell interactions which may change the sensitivity of tumor cells to various treatments. The extended period of observation allows the testing of multiple treatments. Surviving cells can also be recovered for further study. This assay may be useful for testing the efficacy of monoclonal antibody treatment on solid tumors.

Foreign antigenic peptides delivered to the tumor as targets of cytotoxic T cells.

Cytotoxic T cells (CTL) are readily activated by immunogenic peptides and they exert potent anti-tumor activity if the same peptides are displayed on class I major histocompatibility complex (MHC) of the tumor cells. A handful of tumor-associated antigens have been identified and many of them are weak antigens. As an alternative strategy, strongly antigenic foreign peptides are delivered to the tumor, marking them for CTL recognition. To establish the principle of this new strategy, in vitro and in vivo tumor destruction was tested with BALB/c CTL to L(d)-associated beta-galactosidase (beta-gal) peptide p876. In vitro, anti-p876 CTL destroyed tumor cells in a single-cell suspension or in 3-D tumor boluses when exogenous p876 was added. Exogenous IL-2 was required to sustain CTL activity for complete destruction of tumor boluses. In vivo, BALB/c mice were immunized with p876 and a CD4 activating Pan DR reactive epitope (PADRE). PADRE, which binds to several different MHC class II antigen and activates CD4 T cells, induced delayed-type hypersensitivity and stimulated T cell proliferation. Immunized mice were injected with tumor cells loaded with p876 and mixed with PADRE. Starting from the day after tumor injection, mice received five rounds of peptide injection at the tumor sites and all tumors were rejected. Injection with saline had no effect. Injection with PADRE had minor anti-tumor activity. Immunization and treatment with p876 alone was not protective. Therefore, by delivering CD4 and CD8 reactive foreign peptides to the tumor, peptide-specific T cells rejected the tumors as demonstrated by the in vitro and in vivo tests.

Utility of endomyocardial biopsy in the diagnosis of cardiac sarcoidosis.

Cardiac involvement in sarcoidosis can be demonstrated in about 25 percent of autopsied cases, but antemortem diagnosis is uncommon. To evaluate the usefulness of the endomyocardial biopsy in detecting cardiac sarcoid disease, the medical records of ten patients with sarcoidosis who underwent endomyocardial biopsy for routine clinical indications were reviewed. The patients fell into two groups: patients with known sarcoidosis and presumed cardiac involvement (n = 8), and patients in whom the biopsy finding of sarcoid disease was unexpected (n = 2). Four patients in the first group had positive endomyocardial biopsy results (granulomas and/or marked mononuclear cell infiltrate) and were treated with glucocorticoid therapy with improvement in three; the fourth was disabled with lung disease. The diagnoses of three other patients were revised on the basis of the biopsy results; their therapy was tailored accordingly. The remaining patient may represent a false-negative biopsy result, based on clinical criteria. The two patients in the second group presented with symptomatic ventricular tachycardia and restrictive cardiomyopathy respectively, and in neither case was sarcoidosis considered prior to biopsy results. Overall, a change in treatment strategy based on biopsy results occurred in eight of ten cases. Thus, endomyocardial biopsy is useful for the diagnosis of cardiac sarcoidosis; treatment strategies may be affected by biopsy findings; and rarely, endomyocardial biopsy can provide the first clinical evidence of sarcoid disease that is otherwise occult.

Genetic markers in thyroid tumors.

Tissue from nine patients with malignant tumors and two with benign tumors was cultured briefly before cytogenetic analysis. The tumors included one goiter and one Hürthle cell adenoma, one lymphoma, one medullary carcinoma, two Hürthle cell cancers, and five papillary cancers, varying widely in clinical staging and histologic differentiation. When assessed, DNA content was aneuploid in two of six malignant tumors. Various culture conditions (oxygen levels, dissociation methods, and media) were evaluated; the end points were growth, cell differentiation, and time to first harvest. Clonal aberrations were detected in one of four successfully harvested papillary cancers: they consisted of trisomy 7 and a rearrangement of chromosome 10. The rea (10) seen in 22 of 27 cells involved bands q11-21. Two other papillary tumors and a medullary cancer (a family member with multiple endocrine neoplasia type IIA) showed tetraploidy and nonclonal numerically aberrant cells. A lymphoma and two benign lesions showed no cytogenetic abnormality. The tumor with rea (10) is of special interest because abnormalities of 10q have been reported repeatedly in thyroid tumors, including two cases of papillary thyroid tumors with a structural aberration similar to that of the presented case. This rearrangement could affect the ret-proto-oncogene, localized to 10q11.2 which is activated in some papillary thyroid carcinomas.

Meclofenamate enhances blood oxygenation in acute oleic acid lung injury.

To assess the role of vasoactive prostanoids in acute lung injury, we studied 16 dogs after intravenous injection of oleic acid (OA; 0.08 ml/kg). Animals were ventilated with 100% O2 and zero end-expiratory pressure. Base-line hemodynamic and blood gas observations were obtained 90-120 min following OA. Observations were repeated 30 min after infusion of meclofenamate (2 mg/kg; n = 10), or after saline (n = 6). Resistance to pulmonary blood flow was assessed using the difference between pulmonary arterial diastolic and left atrial pressures (PDG). Ventilation-perfusion (VA/Q) distributions were derived with the multiple inert gas technique. Prior to infusion, there were no significant differences between the two groups. PDG was elevated mildly above normal levels, and shunt flow was the principal gas exchange disturbance. Saline induced no significant changes in hemodynamics or gas exchange. Meclofenamate enhanced PDG to a small, significant degree and effected a 32% reduction in shunt flow (P less than 0.01). Perfusion was redistributed to normal VA/Q units with little change in low VA/Q perfusion or in overall flow. Arterial PO2 rose from 75 +/- 36 to 184 +/- 143 Torr (P less than 0.05). At autopsy, there were no significant differences in wet to dry lung weights. Prostaglandin inhibition redistributes perfusion from shunt to normal VA/Q units, thereby improving arterial PO2, without altering lung water acutely.

Chromosomal aberrations in two adrenocortical tumors, one with a rearrangement at 11p15.

Adrenocortical tumors are detected with increasing frequency, but symptomatic cases with excessive hormone production are rare. We investigated cytogenetically one benign aldosterone-producing tumor (Conn Syndrome)(case 1) and one malignant cortisol-producing tumor (Cushing Syndrome)(case 2). Radioimmunoassay of cell culture supernatant of case 2 detected cortisol secretion during 2 months in culture. Flow cytometry of spill-out cells from case 2 showed a bimodal pattern (DNA Index 1.0, 1.4). Case 1 revealed a marker chromosome in 4/25 cells analyzed; the marker was a long acrocentric partially derived from chromosome 2,der(2q). In case 2, a cytogenetic harvest was achieved after prolonged culture time (6 weeks) and a marker chromosome, add(11)(p15), was detected in 16/22 cells. A breakpoint of 11p13, as well as loss of heterozygosity of alleles on 11p15, has been reported in the literature for other malignant adrenocortical cancers.

Chromosomal aberrations in two sporadic gastrinomas.

Results of cell culture and cytogenetic analysis (standard and fluorescent in situ hybridization, FISH) of two sporadic gastrinomas are reported. Maintenance of hormonal activity was assessed by detection of gastrin levels during the first 3 months in culture. Case 1 showed clonal aberrations consisting of two marker chromosomes: marker 1 is a large metacentric chromosome and marker 2 is a small acrocentric chromosome. Case 2 showed a constitutional polymorphism with chromosome 15p+ and a clone in the tumor cell culture with trisomy for chromosome 3. To our knowledge, this is the first cytogenetic report of sporadic gastrinomas (Zollinger-Ellison syndrome).

Mechanisms of lymphocyte traffic in neoplasia.

The composition of tumor infiltrating lymphocyte populations is often reported to be different from that of the lymphocyte pool of peripheral blood. This suggests that infiltration-regulating mechanisms reside in or near the tumor microenvironment. Available evidence indicates that these mechanisms exert their effects on lymphocyte traffic. Two models of regulation are proposed. In the selective immigration model, different lymphocyte types display different tendencies to extravasate into the tumor. Selective immigration could reflect heterogeneity of such lymphocyte properties as binding to vascular endothelium, response to chemotactic factors, or spontaneous locomotion. In the other model, selective entrapment, different types of lymphocytes exit from the tumor microenvironment at different rates. Entrapment could be regulated by selectively acting adhesive, locomotion-inhibiting, or negatively chemotactic factors. Available information supporting each model is presented. The composition of infiltrate may also be influenced by the composition of the circulating lymphocyte pool. Evidence is presented that this pool in turn may be influenced by tumor-induced systemic changes in lymphocyte traffic. Deliberate manipulation of lymphocyte traffic should improve the effectiveness of immunotherapeutic regimes. One promising method of traffic manipulation is the modulation of spontaneous motility. A collagen gel assay for spontaneous lymphocyte motility is described.

Diagnosis of miliary tuberculosis by examination of middle ear discharge.

Extrapulmonary foci of tuberculosis appear more frequently than in previous years, while the incidence of tuberculous otitis, a neglected and often difficult to diagnose entity, is unknown. We describe a case of an adult with miliary tuberculosis not initially suspected on clinical criteria, in whom the diagnosis of miliary tuberculosis was made by inspection of a modified Kinyoun stain of occult ear discharge. The significance and diagnostic problems associated with tuberculous otitis are briefly reviewed.

Incidence of exertional right ventricular wall motion abnormalities in patients with coronary artery disease.

To evaluate the role of analysis of right ventricular function with exercise in patients with presumed coronary artery disease referred for radionuclide ventriculography, the records of 55 patients referred to our laboratory over a 19-month period were reviewed. All underwent rest and exercise first-pass radionuclide stress testing and cardiac catheterization within a period of four months. Three groups were identified: (1) patients with normal exercise right ventricular function (n = 24); (2) patients with exercise-induced right ventricular regional wall motion abnormalities (n = 15); and, (3) patients with abnormal resting right ventricular function without new exercise abnormalities (n = 16). Patients in each group were similar in age, sex, baseline left ventricular function, medication usage, and indication for study. The incidence of right coronary artery disease was identical in the three groups, as was the incidence of left ventricular functional abnormalities with exercise. Patients with proximal right coronary artery disease were more likely to have reduced left ventricular ejection fraction and more extensive coronary artery disease than those without disease at this site. We conclude that: (1) analysis of rest and exercise right ventricular function does not allow prediction of coronary anatomy in an unselected group of patients; (2) normal right ventricular function with exercise is compatible with extensive coronary artery disease, including proximal right coronary artery disease; and (3) abnormal exercise right ventricular function may be due to exertional left ventricular dysfunction in the absence of proximal right coronary artery disease.

Dental fellowships in developmental disabilities help broaden care of disabled.

Continuation of the national trend toward deinstitutionalization and community placement for persons with developmental disabilities, physical handicaps and other medical problems will mean increased demand for dentists trained to care for this segment of the population. The New York State Office of Mental Retardation and Developmental Disabilities now offers dental fellowships in developmental disabilities to help fill the learning gap.