A new method for studying the selective adherence of blood lymphocytes to the microvasculature of human retina

PURPOSE: To develop a sensitive and reproducible technique for measuring the adherence of blood lymphocytes to vessel walls exposed in sections of human retina and for examining the role of lymphocyte and vascular adhesion molecules in these events. METHODS: Cryostat sections of human retina were overlaid with blood lymphocytes from healthy subjects, and experimental conditions were sought by which preferential attachment of the cells occurred to blood vessel walls in the retinal sections. Adherent lymphocytes were identified by staining with methyl green-thionine, and transected blood vessels were identified by their structure and by staining of basement membranes with periodic acid-Schiff. The adherence of enriched preparations of CD4+ (T-helper) and CD8+ (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Beh?et's disease was examined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukocyte integrins to lymphocyte binding was studied by blocking experiments with monoclonal antibodies. RESULTS: The optimal selectivity of blood lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf serum and overlaid onto retinal sections for 30 minutes at 23 degrees C with gentle agitation. Under these conditions, 92% of the lymphocytes that adhered to the section were confined to the retinal microvasculature, and CD4+ T cells were more adherent than CD8+ T cells (P < 0.01). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Beh?et's disease showed supranormal vascular adherence (P < 0.005). Many transected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICAM-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion molecules occupied < 20% of the area of the blood vessel walls. Lymphocyte adhesion to the retinal vessels was more dependent on CD29 (the common chain of the beta 1 integrins) expression than either CD11a/CD18 or CD49d. CONCLUSIONS: This technique allows measurements to be made of lymphocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence to sections of pathologic retina may be of clinical and experimental applicability in understanding mechanisms of retinal inflammation.     

Injection drug users' practices and attitudes toward intervention and potential for reducing the transmission of HIV

Miami is one of the major centers of illegal drug activity and has a significant proportion of AIDS cases among injection drug users (IDUs). Since Needle Exchange Programs (NEP) are illegal and therefore do not exist in the state of Florida, other strategies must play a large role in reducing the transmission of HIV among IDUs. In order to effectively communicate with IDUs about needle safety, it is necessary to understand the practices and culture of IDUs, including where and how the needle/syringes are obtained and used. Data from recent studies conducted in Miami and other local sites indicate that IDUs inject frequently, averaging more than 1,000 per year, per person. While the vast majority of IDUs feel it is very important to clean needles and to use a needle only one time, these sentiments are not always practiced. Furthermore, data indicate that the context where shooting takes place must be considered in the planning of HIV risk reduction interventions. These findings suggest the importance of understanding patterns of drug use, attitudes toward intervention, and the cultural context where risky behaviors occur. Although Needle Exchange Programs are illegal in Florida, intervention programs must still stress the importance of using only new needles, but since new needles cannot always be obtained, IDUs should be taught and motivated not to use contaminated drug paraphernalia.     

Isoflurane and halothane do not alter the enhanced afterload sensitivity of left ventricular relaxation in dogs with pacing-induced cardiomyopathy

BACKGROUND: The afterload dependence of left ventricular (LV) relaxation is accentuated in the failing heart. The authors tested the hypothesis that isoflurane and halothane alter the afterload sensitivity of LV relaxation in dogs with pacing-induced cardiomyopathy. METHODS: Dogs (n = 6) were chronically instrumented for measurement of LV and aortic pressures and subendocardial segment length. Hemodynamics were recorded, and LV relaxation was evaluated with a time constant of isovolumic relaxation (tau) under control conditions and during decreases and increases in LV load produced by abrupt inferior vena caval (IVC) occlusion and phenylephrine (intravenous infusion), respectively, in the conscious state and during isoflurane and halothane anesthesia (1.5 MAC) on separate days before and after the development of pacing-induced cardiomyopathy. The slope (R) of the tau versus LV end-systolic pressure (P[es]) relation was also used to determine the afterload sensitivity of LV relaxation. RESULTS: IVC occlusion and phenylephrine produced similar or less profound changes in P(es), regional end-systolic force (an index of LV afterload), and end-systolic segment length in cardiomyopathic compared with healthy dogs. However, IVC occlusion and phenylephrine caused more pronounced alterations in tau in conscious and isoflurane- and halothane-anesthetized dogs after the development of cardiomyopathy. R was also greater in cardiomyopathic compared with healthy dogs (e.g., 0.32 +/- 0.03 before pacing to 1.00 +/- 0.13 ms/mmHg in conscious dogs). No differences in the load dependence of LV relaxation were observed between the conscious and anesthetized states before and after production of LV dysfunction. CONCLUSIONS: The results indicate that isoflurane and halothane do not alter the afterload dependence of LV relaxation in the normal and cardiomyopathic heart. The lack of effect of the volatile anesthetics is probably related to anesthetic-induced reductions in the resistance to LV ejection concomitant with simultaneous negative inotropic effects.     

Breast carcinoma: MR imaging before re-excisional biopsy

PURPOSE: To investigate the role of magnetic resonance (MR) imaging in the examination of patients after excisional biopsy of breast carcinoma before re-excision. MATERIALS AND METHODS: Forty-seven patients underwent contrast material-enhanced MR imaging after initial excisional biopsy of breast carcinoma before further surgery. RESULTS: The positive predictive value of MR imaging for predicting residual disease was 82%; the negative predictive value was 61%. Fourteen patients had multifocal (n = 6) or diffuse (n = 8) carcinoma. The extent of tumor was correctly identified with MR imaging alone in nine of the 14 patients, with both mammography and MR imaging in three patients, with mammography alone in one patient, and with no imaging modality in one patient. In four of the 14 patients, management was altered from re-excision to mastectomy (n = 3) or from breast-conservation therapy to mastectomy (n = 1). CONCLUSION: MR imaging has a high positive predictive value for predicting residual tumor after excisional biopsy. The identification of mammographically and clinically unsuspected multifocal or extensive residual tumor may lend support for mastectomy rather than re-excision. However, false-negative findings due to postsurgical changes and false-positive findings due to enhancement of granulation tissue and benign breast tissue remain limitations.     

Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-beta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV

Transforming growth factor-beta1 (TGF-beta1) is well recognized as a potent mediator of both fibrillar (collagen type I) and basement membrane (collagen type IV) production. However, tissue injury is characterized by the concomitant expression of many cytokines and/or growth factors in addition to TGF-beta1, and the ultimate extent of extracellular-matrix (ECM) deposition may reflect the interacting effects of TGF-beta1 and these other cytokines and/or growth factors. We, therefore, sought to determine whether other cytokines and/or growth factors, known to be produced after tissue injury, are capable either alone or in combination with TGF-beta1 of modulating collagen gene expression. Collagen type I and collagen type IV gene expression was assessed in NIH-3T3 cells, a murine fibroblast-like cell line that responds to TGF-beta1, with increases in both collagen type I and collagen type IV production. TGF-beta1 coordinately induced production of collagen type IV messenger ribonucleic acid (mRNA) to a level 3.8-fold above its baseline value (p < 0.001) and collagen type I mRNA to a level 2.6-fold above its baseline value (p < 0.001). Of the other cytokines and/or growth factors tested, only epidermal growth factor (EGF) had significant effects on collagen mRNA expression. We report the novel observation that EGF significantly induced collagen type IV mRNA (3.0-fold; p < 0.001) but did not alter collagen type I mRNA expression. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and insulin-like growth factor-1 (IGF-1) did not alter the expression of mRNA for collagen type IV or collagen type I. Addition of TGF-beta1 to cytokine- and/or growth factor-treated cells increased both collagen type IV and collagen type I mRNA levels. However, collagen type IV mRNA levels were similar in cultures given TGF-beta1 alone and cultures given TGF-beta1 with other cytokines and/or growth factors; there were no additive, synergistic, or antagonistic effects after coadministration of TGF-beta1 and other cytokines and/or growth factors. With regard to collagen type I mRNA expression, all cytokines and/or growth factors tested, with the exception of TNF-alpha, had no effect on collagen type I mRNA levels in TGF-beta1-treated cultures. Importantly, TNF-alpha antagonized the stimulatory effect of TGF-beta1 on collagen type I mRNA levels. These observations support a dominant role for TGF-beta1 in stimulating coordinate expression of collagen type I and collagen type IV mRNAs by NIH-3T3 cells; EGF and TNF-alpha are capable of inducing divergent expression of the genes for these two types of collagen.     

Clozapine treatment increases serum glutamate and aspartate compared to conventional neuroleptics

To examine whether serum excitatory amino acid concentrations change with clozapine treatment and whether these changes correlate with improvement in negative symptoms, serum excitatory amino acids were measured and clinical scales administered in seven subjects with schizophrenia before and after switching from conventional neuroleptics to clozapine. Clozapine treatment was associated with increased serum glutamate and aspartate concentrations. Clinical improvement was negatively correlated with baseline glycine concentrations. These results support the hypothesis that clozapine acts at least in part by increasing glutamatergic activity.     

Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.     

Prospective study of circulating T-lymphocyte subpopulations and disease progression in colorectal cancer

PURPOSE: It has recently been reported that CD4+ T-lymphocytes are reduced in advanced colorectal cancer patients. However, it is not clear whether such changes in T-lymphocyte subsets are an early or late event in such patients. The aim of this study was to examine the relationship between these subsets and disease progression in colorectal cancer. METHODS: Flow cytometric analysis of T-lymphocyte subsets was performed in 39 patients who, approximately 12 months previously, had undergone surgery for colorectal cancer. These patients were grouped according to whether they developed a recurrence in the following two years. A group of healthy subjects was studied as controls. RESULTS: There was a significant increase in the median neutrophil count (4.3 vs. 3.7 10(6)/ml) and the median numbers of platelets (282 vs. 216 10(6)/ml) of the recurrence group compared with the control group, respectively (P < 0.05). The median numbers (0.28 vs. 0.73 10(6)/ml) and percentage (29 vs. 38 percent) of CD4+ T-lymphocytes of the recurrence group were significantly reduced compared with that of the control group (P < 0.05). There were also reductions in the median percentage of CD3+ cells (67 vs. 74 percent) and the median numbers of CD4+ T-lymphocytes (0.28 vs. 0.46 10(6)/ml) of the recurrence group compared with the no recurrence group (P < 0.05). CONCLUSIONS: Reduction of CD4+ T-lymphocytes occurs before detectable recurrence of colorectal cancer. Results of the present study are consistent with impaired immunity, as measured by such lymphocyte subset populations, being important in tumor recurrence in colorectal cancer.     

Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator

A case-control study of the associations of retinoids and specific carotenoids with breast cancer using concentrations of these nutrients in breast adipose tissue was conducted among women attending a breast clinic in the Boston area in 1989-1992. Breast adipose tissue was collected during breast biopsy. Cases (n = 46) were women whose biopsies revealed invasive or in situ breast cancer; control subjects (n = 63) were women whose biopsies revealed benign disease. We observed inverse associations between breast adipose concentrations of retinoids and carotenoids and risk of breast cancer, although not all were statistically significant. The multivariate-adjusted odds ratio comparing women above the median value of the control group for retinol with those below or equal to the median was 0.71 (95% CI: 0.26, 1.93; NS); corresponding odds ratios were 0.61 (95% CI: 0.23, 1.64; NS) for retinyl palmitate, 0.30 (95% CI: 0.11, 0.85) for beta-carotene, 0.32 (95% CI: 0.11, 0.94) for lycopene, and 0.68 (95% CI: 0.27, 1.73; NS) for lutein/zeaxanthin. There was a nonsignificant positive correlation (r = 0.23, P = 0.15) between breast adipose tissue concentrations of retinol and dietary intake of preformed vitamin A, including supplements measured by using a food-frequency questionnaire. No correlation was found between breast adipose concentrations of carotenoids and intake of dietary carotenoids. These data suggest that higher breast adipose concentrations of retinoids and some carotenoids may be associated with decreased risk of breast cancer and that further examination of these relations is warranted.     

Effect of dopamine receptor antagonists on cocaine subjective effects: a naturalistic case study

Schizophrenic patients on neuroleptic medications abuse cocaine and report cocaine-induced euphoria. This study was undertaken to provide better clinical characterization of these phenomena by administering the POMS and a custom-designed questionnaire. A group of heavy cocaine users who were not mentally ill served as the control group. The results clearly suggest that schizophrenic patients report cocaine-induced euphoria and post-use craving despite being treated with therapeutic doses of haloperidol or fluphenazine. The responses of the control group were similar to that of the schizophrenic group except that the latter subjects reported a greater degree of anxiety. These results suggest that blockade of D2 receptors is not sufficient to block cocaine-induced subjective effects in humans.