The organochlorine contamination history of the Mersey estuary, UK, revealed by analysis of sediment cores from salt marshes.

Sediment profiles in the Banks, Ince and Widnes Warth salt marshes in Northwest England contain a mappable record of historic pollution. For persistent organochlorine compounds this stretches back over 90 years. The PCB and HCH profiles can be successfully rationalised by dating methods, and they can be related to the dates of initial production and subsequent withdrawal from use of these chemicals as a result of restrictive environmental legislation. HCB has a more complex pollution profile as it has been manufactured in Northwest England, both deliberately as a pesticide and accidentally as a by-product of several chlorination processes, dating back to the start of the 20th century. The concentrations of degradation products of DDT are relatively constant through the sediment profile and are dominated by op'- and pp'-DDD with only minor contributions from the most toxic species, pp'-DDT. The quantities of these compounds resident in the reservoir of pollutants under these marshes have been calculated, and have fallen progressively in the last 30-50 years.

Detection of the Human Cytomegalovirus 2.0-kb Immediate Early Gene 1 Transcripts in Permissive and Nonpermissive Infections by RNA in situ Hybridization.

The immediate early gene 1 (IE1) is the first gene to be expressed following the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. Therefore, the IE1 gene is a potential candidate for the development of probes to detect HCMV in various states of infection. Using strand-specific (32)P- or digoxigenin-labeled riboprobes derived from an exon-specific subgenomic fragment of the HCMV Towne IE1 gene, we performed Northern blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By Northern blot analysis and by in situ hybridization, expression of the 2.0-kb IE1 gene was found in permissive as well as in nonpermissive infections. Specific nuclear and cytoplasmic hybridization was found at 5, 10, 24 and 72 h after infection in human fibroblasts. In comparison, hybridization was first detected at 10 h after infection in mouse fibroblasts. Hybridization with the IE1 probe was detected in cells with and without cytopathic changes in the formalin-fixed paraffin-embedded HCMV-infected human tissues. Hybridization patterns of the IE1 riboprobe were compared to those of the HCMV 2.7-kb major early beta-riboprobe which we have previously described [Am J Pathol 141:1247-1254;1992]. Although both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results suggest that RNA in situ hybridization with a probe directed at the IE1 transcripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections. Copyright 1997 S. Karger AG, Basel

Detection of perforin in human peritoneal fluid T-lymphocytes.

OBJECTIVE: Perforin is a specific marker of functionally active cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells. The purpose of this study was to detect perforin-positive lymphocytes in ascites secondary to various gynecologic malignancies and nonneoplastic conditions. STUDY DESIGN: Fifty-three unselected peritoneal fluid specimens submitted for cytopathologic diagnosis were used. A monoclonal antibody to human perforin was used to examine its expression in mononuclear cells from ascites specimens using a standard immunoperoxidase technique. RESULTS: Strong perforin expression by 15-30% of mononuclear cells was detected in 10 of the 13 patients with severe alcoholic hepatitis, 3 of the 5 patients with chronic active hepatitis without cirrhosis and 1 patient with an autoimmune disorder of unknown etiology. The lymphocytes showed variable positivity for T cell markers (CD2, CD4, CD8 and CD56). Perforin was not detected in ascites specimens obtained from patients with such nonneoplastic disorders as cirrhosis or end stage renal or cardiac failure. Similarly, ascites specimens from patients with various gynecologic malignancies were negative for perforin-positive lymphocytes. CONCLUSION: The detection of cytotoxic lymphocytes in peritoneal fluid obtained from patients with liver injury may have implications for the pathogenesis of this disease.

Perforin expression in human cell-mediated luteolysis.

After ovulation and in the absence of fertilization, the human corpus luteum regresses in an orderly sequence of morphological changes. This study demonstrated that luteal regression involved progressive infiltration of lymphocytes and macrophages. The inflammatory infiltrate began in the theca externa and gradually invaded granulosa cells, with maximum accumulation of lymphocytes and macrophages at the time of menstruation. Immunoperoxidase staining showed that the majority of lymphocytes were CD2+, CD3+, CD8+ T lymphocytes, and 15% of these T cells expressed perforin, a cytolytic protein implicated as a mediator of cytotoxicity. The remaining mononuclear infiltrate showed strong reactivity with monocyte/macrophage markers. These findings indicate that (a) a physiologic cell-mediated inflammatory process in the regressing human corpus luteum is mediated mainly by CD8+ T lymphocytes and cells of monocyte/macrophage lineage and (b) perforin expression in T lymphocytes supports a possible role for cytolytic T cells during the physiologic inflammatory response in human luteolysis.

Perforin expression in endometrium during the menstrual cycle.

Perforin is a marker of functionally active cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We examined perforin expression in endometrium throughout the menstrual cycle. In addition, perforin expression was studied in endometrial leukocytes in women with anovulatory cycles with or without progesterone therapy and in postmenopausal women. In the endometrium, perforin-positive endometrial lymphocytes increased in numbers in the middle through late secretory phases. In women with anovulatory cycles, the proliferative endometrium, before estrogen withdrawal endometrial breakdown, contained less perforin-positive lymphocytes compared with the premenstrual endometrium of the normal menstrual cycle. The progestin-induced endometrial decidualization was accompanied by an extensive recruitment of perforin-positive cells. In contrast, the postmenopausal atrophic endometrium showed complete absence of perforin-positive cells. The lymphocytes isolated from secretory endometrium were CD3-, CD56+ (approximately 80%) or CD3+, CD8+ T-cell receptor (TCR)-gamma delta- (approximately 20%). Almost 90% +/- 5.5 of the CD3-, CD56+, and up to 25% +/- 7% of CD8+ T lymphocytes contained perforin. Perforin expression was further confirmed in endometrial lymphocytes by Northern blot analysis. In vitro, isolated endometrial lymphocytes exhibited NK-like cytotoxicity. After cyclic hormone withdrawal during normal menstrual cycle, these perforin-positive cytotoxic cells may be involved in endometrial stromal breakdown during menstruation.

Studies of the mechanism of cytolysis by HIV-1-specific CD4+ human CTL clones induced by candidate AIDS vaccines.

Vaccine-induced, virus-specific CTLs may rapidly eliminate the host cells that first become infected after virus exposure, thereby preventing disseminated infection. Thus, there is much interest in the ability of candidate AIDS vaccines to elicit CTLs. All HIV-1 envelope (env) protein-based vaccines tested to date in seronegative humans induce CTLs from the CD4+ subset. Because the mechanism of cytolysis by CD4+ CTLs is controversial, a detailed study of the cytolytic reactions mediated by vaccine-induced, HIV-1-specific human CD4+ CTL clones was conducted. CD4+ CTL clones induced rapid destruction of Ag-pulsed target cells. Lysis was readily detectable within 15 min. Lysis was not a result of syncytium formation between CD4+ effector cells and env-expressing targets. Target cell destruction was not dependent upon de novo RNA or protein synthesis in either the effector or the target cell. Expression of perforin mRNA was detected by Northern blotting and reverse-transcriptase-PCR in CD4+ CTL clones but not in autologous B lymphoblastoid cell lines. Immunohistochemical studies demonstrated perforin protein in cytoplasmic granules in CD4+ CTL clones. Lysis by CD4+ CTLs was strictly dependent upon extracellular Ca2+ and was highly specific, with no lysis of innocent bystander cells. DNA fragmentation was detectable in target cells, but did not precede 51Cr release. Taken together, these results provide a dramatically different view of cytolysis by human CD4+ CTLs. Target cells are lysed by a rapid and efficient mechanism that involves a preformed mediator and that is functionally similar to the mechanism used by CD8+ CTLs.

Immunohistochemical expression of CD68 antigen in human peripheral blood T cells.

CD68 antigen is expressed by tissue macrophages and cells of myeloid/mononuclear lineage. CD68 is recognized by many monoclonal antibodies (mAbs), including KP1, EMB11, Y1/82A, Y2/131, Ki-M6, and Ki-M7. Using the labeled strept-avidin-biotin (LSAB) immunoperoxidase technique, we examined CD68 antigen expression in human peripheral blood T cells. The anti-CD68 mAbs KP1 and EMB11 stained virtually all fresh isolated gamma/delta T cells and CD3-CD56+ natural killer (NK) cells in a granular cytoplasmic pattern. In contrast, fresh isolated CD4+ and CD8+ T lymphocytes showed no detectable reactivity with these two anti-CD68 mAbs. However, in vitro stimulation with T-cell mitogen or recombinant interleukin-2 (rIL-2) induced expression of CD68 antigen in activated CD4+ and CD8+ T lymphocytes. Similarly, the lymphokine-activated killer (LAK) cells generated after long-term (14 to 21 days) culture of peripheral blood mononuclear cells (PBMC) in rIL-2 also showed strong granular cytoplasmic staining by the anti-CD68 antibodies. This study shows that the CD68 antigen is constitutively expressed in NK cells and gamma/delta T cells and that its expression is strongly induced in activated CD4+ and CD8+ T lymphocytes as well as LAK cells.

Is submission of remaining tissue necessary when incidental carcinoma of the prostate is found on transurethral resection?

We examined 151 entirely submitted transurethral resection of the prostate (TURP) specimens that showed incidental prostate cancer. Fifty-one (34%) showed > 15% involvement of the specimen by tumor; these were not reviewed because submission of more tissue would not convert these cases from stage T1b (> or = 5% of tumor) to stage T1a ( < 5% of tumor). Sixty-six cases (44%) that were totally submitted in < or = nine cassettes also were excluded because most laboratories would totally submit these specimens from the outset. The remaining 34 cases (22%) had < or = 15% tumor and required 10 or more cassettes for total submission. The first eight slides were reviewed and percentage of tumor involvement and grade was calculated. The remaining slides were then reviewed to see if the overall tumor percentage or grade changed. Because in one case the tumor grade was significantly increased in the remaining slides and of the infrequency with which more than eight cassettes needs to be submitted, we recommended submission of all remaining tissue in stage T1a lesions. There is no need to submit additional tissue in stage T1b lesions because the percentage will not decrease with greater sampling.

Selective prothymocyte targeting by prenatal diethylstilbesterol exposure.

Estrogens have been reported to modulate immunologic responses at both physiologic and pharmacologic concentrations. Treatment of experimental animals with the synthetic estrogen, diethylstilbesterol (DES), markedly decreases thymic cellularity, manifested histologically as a progressive loss of cortical thymic lymphocytes. In the present report thymic atrophy after prenatal DES exposure was found to be more severe than has been reported following adult exposure, indicating a possible greater sensitivity of the developing immune system to estrogenic hormones. DES exposure resulted in a limited alteration of cell maturation within the fetal thymus as evidenced by only slight alterations in the expression of CD4 and CD8 cell-surface antigens. To examine the possibility that DES targets hematopoietic stem cells in the fetal liver, cytometric analysis was conducted using a panel of fluorescent antibodies to quantitate the hematopoietic subpopulations present in control and DES-exposed Gestational Day (gd) 18 fetal mouse liver. There were no significant DES-induced alterations in the number of hematopoietic stem cells, or in fetal liver cells expressing CD44 (hematopoietic precursors), Mac-1 (granulocyte-macrophage lineage precursors), or CD45R (B-lineage lymphocytes) surface antigens. However, DES selectively reduced the number of fetal liver precursors containing the lymphocyte stem cell-specific DNA polymerase, terminal deoxynucleotidyl transferase, which suggested that DES may specifically target the fetal liver prothymocyte. Reconstitution of irradiated hosts with gd 18 fetal liver from vehicle and DES-exposed syngeneic donors demonstrated an impaired ability of the DES-treated fetal liver to repopulate the thymus of irradiated hosts. In addition, fetal liver cells enriched for prelymphoid cells contained potentially significant levels of estrogen specific receptors. Taken together these data, in conjunction with the lack of direct thymocyte injury (necrosis, apoptosis, and/or inhibition of cell proliferation) by DES treatment, suggest that estrogen-mediated thymic atrophy may result, at least in part, from a specific alteration in the lymphocyte stem cell population responsible for colonizing the thymus.

Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.

The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.

Perforin expression localizing cytotoxic lymphocytes in the intimas of coronary arteries with transplant-related accelerated arteriosclerosis.

Accelerated arteriosclerosis is the major long-term complication of cardiac transplantation. It has been demonstrated recently that accelerated arteriosclerosis is caused, in part, by rejection-related, cell-mediated immunity. However, the role of cytotoxic T lymphocytes in this process is a subject of controversy. Perforin is a specific marker of functionally active cytotoxic lymphocytes because it is a functional component of the cytotoxic granules of these cells. We examined 11 coronary arteries from seven autopsied and four retransplanted heart transplant recipients for the presence of perforin-containing lymphocytes. Immunohistochemical stains for perforin were performed using a monoclonal antibody against human perforin. Eight of the 11 coronary arteries examined were found to contain perforin-positive cells. These perforin-positive cells were present in subendothelial spaces of the coronary arteries, and the staining seen was cytoplasmic and granular. The granules often were polarized to the endothelial surface. Furthermore, the cells identified were usually in close proximity to, or in direct contact with, coronary artery endothelial cells. These results suggest that cell-mediated endothelial injury by perforin-positive cytotoxic lymphocytes may contribute to the development of accelerated arteriosclerosis in heart transplant recipients.

Methods of radical prostatectomy specimen processing: a novel technique for harvesting fresh prostate cancer tissue and review of processing techniques.

Standard processing techniques for radical prostatectomy specimens uniformly require initial formalin fixation. Fresh prostate cancer tissue is needed for many types of prostate cancer research and may be needed for diagnostic purposes in the future. We describe a method for processing radical prostatectomy specimens that allows pre-fixation sectioning and harvest of fresh prostate cancer tissue. Because of increased difficulty interpreting capsular margin sections, however, we do not recommend it for routine use. We review other methods of radical prostatectomy specimen processing and provide an algorithm for their appropriate use.

Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue.

Comparison of the marginal fit of various ceramic crown systems.

This study evaluated the marginal fit of four ceramic crown systems, (1) metal ceramic crowns with a metal margin, (2) metal ceramic crowns with a porcelain facial margin, (3) Cerestore crowns, and (4) Dicor crowns. Measurements of the marginal adaptation were recorded from the facial and lingual margins by using a video-enhanced microscope with digital micrometer and image intensification in a high resolution television screen. Results indicate that all four crown systems yielded comparable and acceptable marginal fit.