Arif Hameed

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.

Exfoliative cytology of neuroendocrine small cell carcinoma of the endometrium : A report of two cases

BACKGROUND In the female genital tract, neuroendocrine small cell carcinoma can occur in the endometrium as well as the cervix, ovary and vagina. This tumor has a high propensity for systemic spread and a poor prognosis. Small cell carcinoma of the endometrium is cytologically identical to its counterparts in the lung and other sites. Its characteristic appearance in a cervicovaginal smear should raise concern about small cell carcinoma. Other tumors of the uterus should be considered in the differential diagnosis, including adenocarcinoma with neuroendocrine features, small cell nonkeratinizing squamous cell carcinoma, endometrial stromal sarcoma, rhabdomyosarcoma, primitive neuroectodermal tumor, non-Hodgkins lymphoma and metastatic breast carcinoma. CASES Case 1 was a 59-year-old, white female, and case 2 was a 47-year-old, white female. Both patients presented with vaginal bleeding. The Papanicolaou smears in both cases had similar, characteristic exfoliative cytology. The tumor cells were small and either single or arranged in groups and files. They had barely visible cytoplasm, darkly staining nuclei with finely stippled chromatin, and inconspicuous nucleoli. The characteristic molding of the nuclei was also present. Immuno-histochemical staining for neuron-specific enolase and synaptophysin was positive in tissue sections. Pancytokeratin, vimentin, muscle-specific actin, desmin, alpha-fetoprotein, S-100, glial fibrillary acid protein, common leukocyte antigen and chromogranin were negative. CONCLUSION When a uterine small cell carcinoma is suspected in a cervicovaginal smear, the similarity of cervical and endometrial small cell carcinoma requires a differential curettage and immunohistochemical demonstration of neuroendocrine differentiation in order to arrive at the final diagnosis.

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.

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.

Exfoliative cytology of atypical polypoid adenomyoma. A case report.

BACKGROUND An atypical polypoid adenomyoma (APA) is a well-defined entity. It occurs in the endometrium, lower uterine segment and endocervix. It is usually composed of atypical complex glands with squamous metaplasia admixed with myofibromatous stroma. CASE A 35-year-old female presented with one-year history of irregular menstrual periods. A diagnosis of adenocarcinoma in situ was rendered on her cervicovaginal smear. Pelvic examination revealed an enlarged uterus due to a leiomyoma. Colposcopic examination revealed a 0.6-cm, sessile, polypoid mass at the junction of the endocervix and ectocervix. A cone biopsy of the lesion showed irregular, endometrial-type glands embedded in a prominent myofibromatous stroma. The atypical glandular component of the mass demonstrated varying degrees of architectural complexity, ranging from simple to complex hyperplasia. In tissue sections the lesion was diagnosed as APA of the cervix. The patient underwent a hysterectomy for the leiomyoma. The hysterectomy specimen showed an 8.5-cm leiomyoma. The cervix and uterine corpus revealed no residual APA. CONCLUSION APA of the cervix should be considered among the differential diagnoses of atypical glandular cells of undetermined significance. The diagnosis of APA cannot be made on cytology; the final diagnosis requires histologic confirmation.

Human T-lymphocyte serine proteases (granzymes) 1, 2, and 3 mediated DNA fragmentation in susceptible target cells

We reported the characterization of three serine proteases (granzymes 1, 2, and 3) from human cytotoxic T lymphocytes. In this study, human granzymes 1, 2, and 3 were purified from the cytoplasmic granules of lymphokine activated killer (LAK) cells by gel filtration and cation exchange chromatography. Human perforin was purified by phenyl superose and heparin-agarose chromatography. Each purified granzyme was used with purified perforin to study DNA fragmentation in target cells of both human and murine origin. As measured by agarose gel electrophoresis and [125I]dUrd assay, the granzymes induced oligonucleosomal DNA fragmentation and [125I]dUrd release respectively from various target cells. Murine target cells were generally more susceptible to nuclear DNA release than were human targets. Both enzyme activity and nuclear DNA breakdown were significantly inhibited by 3,4-dichloroisocoumarin (DCI) or by heat inactivation of each granzyme. Perforin alone or granzyme alone failed to fragment nuclear DNA in various target cells. We conclude that human granzymes are an important family of effector molecules that with perforin induce DNA fragmentation in susceptible target cells.

3,4-DICHLOROISOCOUMARIN SERINE PROTEASE INHIBITOR INDUCES DNA FRAGMENTATION AND APOPTOSIS IN SUSCEPTIBLE TARGET CELLS

Abstract 3, 4-Dichloroisocoumarin (DCI) inhibition of serine proteases generates reactive intermediates that have been theorized to affect apoptosis. To examine this possibility various target cells were treated with different concentrations of DCI and assessed for intracellular nuclear DNA fragmentation and apoptosis. DCI treatment caused oligonucleosomal DNA fragmentation in cell lines expressing high levels of protease activity (LAK cells, NK-92, CTLL-2, L929, 3T3). This DNA breakdown characteristic of apoptosis occurred in a dose-dependent fashion within 4-6 hr of treatment and was confirmed by electron microscopy. In cell lines expressing low levels of protease activity (unstimulated human peripheral blood mononuclear (PBMN) cells, YAC-1 cells), DCI effectively inhibited protease activity without inducing oligonucleosomal DNA fragmentation. ZN+ ions significantly inhibited DCl-induced DNA degradation. The mixture of DCI and BLT esterase active NK cell lysate triggered DNA fragmentation in isolated YAC-1 nuclei. Degree of DNA fragmentation in YAC-1 nuclei was proportional to the level of BLT esterase activity. Cell lysate protease activity, initially inhibited by DCI acylation, was restored by hydroxylamine deacylation, thus preventing DCi-mediated DNA fragmentation. Our results suggest that DCI treatment of cells expressing high levels of protease activity generates toxic levels of acylenzyme intermediates. These intermediates may trigger nuclear DNA breakdown and apoptosis by activating endogenous endonucleases. This effect may compromise the analysis of apoptosis in experimental systems using high concentrations of DCI for extended periods.

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.