Biomedical subjects

D D Taylor

Publications and source records attributed to D D Taylor.

At least 19 recordsLinked to original sources

PubMed2001

Modulation of TcR/CD3-zeta chain expression by a circulating factor derived from ovarian cancer patients.

In women with ovarian cancer, suppression of components of the immune system may promote tumour development. Previous studies in ovarian cancer have demonstrated that decreased expression and function of the T-cell receptor (TcR)-associated signal transducing zeta-chain correlates with deficient immune responsiveness of T cells. In this study, sera and ascitic fluids obtained from woman with advanced ovarian cancer were found to suppress the expression of TcR-associated zeta chain. This suppression of zeta chain expression was dose-dependent and was not observed with biologic fluids obtained from healthy women. The factor responsible for the loss of zeta chain was purified from ascites and characterized as a protein with an appropriate molecular weight of 14 kD. Suppression of T-cell TcR-zeta was specific, since neither lck nor ZAP-70 expression was affected, while zeta chain was almost completely suppressed. This selective suppression of TcR-zeta expression by the 14 kD ascites-derived factor was shown to operate at the mRNA level. By defining the mechanism through which this protein modulates TcR-zeta chain levels, it might be possible to ultimately prevent the suppressive influences of the tumour microenvironment and restor immune competence in patients with ovarian carcinoma.

Adult↗

PubMed2001

Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.

OBJECTIVES: Cancer patients generally exhibit circulating tumor-reactive immunoglobulins; however, these antibodies fail to eradicate tumors or prevent their progression. This study identifies and characterizes an aberrant tumor-reactive IgG population present in women with ovarian cancer. METHODS: In this pilot study, IgG was isolated from the sera of women with advanced-stage ovarian cancer (stages III and IV, n = 62) and age-matched female volunteers (n = 50) by affinity chromatography. These IgGs were characterized on the basis on their aberrant binding to concanavalin A affinity columns. Subsequently, the concanavalin A-binding moiety was localized following IgG fragmentation, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and characterized by oligosaccharide profiling. RESULTS: The level of concanavalin A-binding IgG in our control population was 8.9 +/- 2.9%, whereas in ovarian cancer patients, the level of concanavalin A-binding IgG was 38.8 +/- 7.4%. In the patients with ovarian cancer, 87.5 +/- 5.7% of the tumor-reactive IgG was demonstrated to be concanavalin A-binding. Based on oligosaccharide profiling of the fragmented concanavalin A-binding IgG, the aberrant lectin binding appeared to be the consequence of altered glycosylation of one of the two Fc chains. CONCLUSIONS: While our previous studies have identified the presence of circulating IgG reactive with specific tumor-associated antigens and its association with poor prognosis, this report demonstrated the presence of an aberrantly glycosylated IgG population in cancer patients. This altered IgG appeared to be the primary class of tumor-reactive antibodies in these women.

Antibodies, Neoplasm↗

PubMed2001

Epitope recognition by anti-cathepsin D autoantibodies in endometrial cancer patients.

OBJECTIVE: This study analyzed a model for the identification of specific epitopes recognized by autologous tumor-reactive humoral responses of endometrial cancer patients as potential markers for the monitoring of cancer. METHODS: The presence of circulating pro- and mature forms of cathepsin D and antibodies reactive with this enzyme were identified by Western immunoblot and quantitated by an enzyme immunoassay. Specific immunoreactivities with 34- and 52-kDa cathepsin D forms were analyzed by Western immunoblot using sera from endometrial cancer patients (n = 40) and normal volunteers (n = 15). Subsequently, reactivities with specific cathepsin D epitopes were defined by a peptide-specific ELISA. RESULTS: Circulating pro-forms of cathepsin D were detected in 31 of 40 endometrial cancer patients tested and none of the control volunteers. Circulating IgG reactive with cathepsin D could be demonstrated in 29/31 patients with circulating procathepsin D, while an anti-cathepsin D response was not detectable in normal controls. This response appeared to be directed against the pro-peptide portion of cathepsin D. Using a peptide-specific ELISA, the frequencies of antibody production against specific epitopes within the pro-peptide were defined. CONCLUSION: There is a demonstrable tumor-reactive immune response elicited in endometrial cancer patients, directed against specific antigenic epitopes, some of which are conserved among these patients. Since these proteins are recognized as non-self, due at least in part to posttranslational processing errors, defining these epitopes will be useful as a means of diagnosis, assessment of therapeutic success, and, ultimately, identification of immunotherapeutic targets.

Amino Acid Sequence↗

PubMed2001

Altered expression of TCR-CD3zeta induced by sera from women with preeclampsia.

OBJECTIVE: To investigate TCR-CD3zeta expression by cultured T lymphocytes exposed to midtrimester sera from pregnant women in whom preeclampsia developed at term compared with normotensive pregnant control subjects. STUDY DESIGN: Sera obtained at 24 to 28 weeks' gestation from 16 nulliparous women in whom preeclampsia developed at term and from 32 gestational age-matched control subjects without preeclampsia were evaluated for TCR-CD3zeta chain expression with use of Jurkat cells. Subsets of serum samples from 6 women with preeclampsia and 6 control subjects were then evaluated for their ability to induce apoptosis and to suppress interleukin-2 production. Groups were compared by use of the Kruskal-Wallis test, and P <.05 was considered significant. RESULTS: TCR-CD3zeta chain expression in cultured T lymphocytes was suppressed in approximately 60% of untreated control subjects after incubation with sera from normotensive pregnant women compared with 30% after incubation with sera from women with preeclampsia (P <.001). T-cell apoptosis was significantly higher after incubation with sera from normotensive control subjects, as was the expression of the proapoptotic regulator Bax, compared with sera from women with preeclampsia. Interleukin-2 levels were higher in T cells incubated with sera from women in whom preeclampsia later developed compared with sera from normotensive pregnant women (27.7 ng/mL versus 72.5 ng/mL; P <.001). CONCLUSIONS: Nulliparous women in whom preeclampsia developed did not suppress TCR-CD3zeta levels to the extent of normotensive control subjects, which may be linked to decreased lymphocyte apoptosis. This occurs remotely from the manifestation of clinical disease and suggests a deficiency in a serum factor in preeclampsia that may induce T cell zeta chain suppression in normal pregnancy.

Adult↗

PubMed2001

Voluntary limb-load distribution in horses with acute and chronic laminitis.

OBJECTIVES: To compare limb-load distribution between horses with and without acute or chronic laminitis. ANIMALS: 10 horses with carbohydrate-induced acute laminitis, 20 horses with naturally occurring chronic laminitis, and 20 horses without foot abnormalities (controls). PROCEDURES: Limb-load distribution was determined, using a custom-designed system that allowed simultaneous quantification of the mean percentage of body weight voluntarily placed on each limb (ie, mean limb load) and the SD of the mean load over a 5-minute period (ie, load distribution profile [LDP]). Load distribution profile was used as an index of frequency of load redistribution. RESULTS: Mean loads on fore- and hind limbs in control horses were 58 and 42%, respectively, and loads were equally and normally distributed between left and right limbs. In addition, forelimb LDP was greater, compared with hind limbs, and was affected by head and neck movement. In comparison, limb-load distribution in horses with chronic laminitis was characterized by an increase in the preferential loading of a forelimb, a decrease in total forelimb load, and an increase in LDP that was correlated with severity of lameness. In horses with carbohydrate-induced acute laminitis, mean limb loads after onset of lameness were not different from those prior to lameness; however, LDP was significantly decreased after onset of lameness. CONCLUSION AND CLINICAL RELEVANCE: Quantification of limb-load distribution may be an applicable screening method for detecting acute laminitis, grading severity of lameness, and monitoring rehabilitation of horses with chronic laminitis.

Animals↗

PubMed2001

Clonal heterogeneity of p53 mutations in ovarian cancer.

We analyzed clonal populations of ovarian cancer cells for heterogeneity in p53 mutations (exons 4-9) and chemosensitivity. UL-3A cells were developed from a patient with stage IIIC ovarian adenocarcinoma. Heterogeneity in p53 mutations was demonstrated, ranging from point mutations to deletions in exons 4, 6 and 7. UL-3A cells contained two point mutations, in codon 248 of exon 7 and in codon 76 of exon 4. Five groups of clones were identified according to the p53 mutations. UL-3A clones with low p53 levels were more sensitive to CDDP (LD50 <8.0 microg/ml). Heterogeneity of p53 mutations may provide growth advantage during disease progression or chemotherapy.

Antineoplastic Agents↗

PubMed2000

Alterations in humoral immune responses associated with recurrent pregnancy loss.

OBJECTIVE: To investigate the reactivity of maternal antibodies with endometrium-derived antigens and to correlate their association with recurrent pregnancy loss (RPL). DESIGN: Prevalence study. SETTING: Academic research center. PATIENT(S): Nulliparous women (n = 10), women with RPL (n = 15), pregnant women (n = 8), and multiparous women with a normal obstetric history (n = 20). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Reactive antibodies were analyzed by Western immunoblot techniques and quantitated by densitometry. RESULT(S): Antibodies from women with RPL and multiparous women recognized antigens ranging from 10-120 kd on normal endometrium and endometrial tumors. Antibodies from most women with RPL (10/15) and from multiparous women (15/20) recognized 65-kd and 80-kd proteins in normal endometrium. Antibodies from women with RPL recognized 21-kd and 28-kd antigens (12/15 and 13/15, respectively) in endometrial tumors at a significantly greater rate (than did antibodies from multiparous women (5/20 and 8/20, respectively). Women with RPL had significantly lower levels of asymmetric IgG compared with controls. CONCLUSION(S): Recurrent pregnancy loss may be linked with the failure to elicit asymmetric IgG and a unique immunologic recognition of endometrial antigens.

Abortion, Habitual↗

PubMed1999

AutoPap system detection of infections and benign cellular changes: results from primary screener clinical trials.

Primary screening devices for cervical cytology must show performance data for the detection of infectious organisms and benign cellular changes (BCC) for cytologists who routinely report these findings. The data on infection and BCC from the AutoPap primary screening clinical trials are presented herein. The presence of infectious organisms (candida, trichomonas, shift in bacterial flora, herpes, actinomyces) and BCC were noted in each of the clinical trial arms (current practice, CP; AutoPap-assisted practice, AP). For the purposes of these analyses, a report of infection or BCC from either arm was considered to be "truth." In 25,124 slides analyzed, there were 2,925 cases of infection identified. Of these, CP identified 2,141, and AP identified 1,985. The overall detection results are statistically equivalent. Of 17 cases of actinomyces, CP detected 8, while AP detected 12. Of 1,282 cases of candida, CP detected 983, and AP detected 865. Of 1,375 cases of shift of bacterial flora, CP detected 897, and AP detected 869. Of 14 cases of herpes, CP detected 9, and AP detected 11. Of 343 cases of trichomonas, CP detected 293, and AP detected 275. There were 5,156 cases of BCC identified in the trial. CP detected 3,431, and AP detected 3,276. The detection rates for BCC are statistically equivalent. The results show that the AutoPap-assisted practice for the primary screening of conventional cervical cytology slides is equivalent to the current practice for the detection of cervical infections and benign cellular changes.

Actinomyces↗

PubMed1999

Modulation of proliferation and chemosensitivity by procathepsin D and its peptides in ovarian cancer.

Since the presence of precursors (pro-forms) of the aspartyl endoprotease, cathepsin D, appears to be linked with tumor progression, their presence was examined in sera and tumor tissues of ovarian cancer patients. The role of cathepsin D pro-forms was further assessed in the dysregulated proliferation and chemoresistance observed in advanced ovarian cancer. Cathepsin D was isolated from sera of ovarian cancer patients (n = 20) and normal volunteers (n = 11), as well as from solubilized normal ovarian epithelium (n = 8) and ovarian epithelial tumor tissue (n = 12). The specific molecular forms of cathepsin D were analyzed in these samples by Western immunoblot. Multiple circulating molecular weight forms of cathepsin D were identified in ovarian cancer patients ranging from 24 to 60 kDa, while in normal controls, a major band was observed at 34 kDa in all samples and minor bands corresponding to 27 and 48 kDa were detected in approximately half of the controls. To assess its consequences on ovarian cancer, the 52-kDa protein was immunoprecipitated from culture medium of an exponentially growing ovarian tumor cell line and was further purified by reverse-phase high-pressure liquid chromatography. Its effect on proliferation was assayed by determining cell doubling times and their chemosensitivity was measured in a standard cytotoxicity assay using cisplatin. In addition, decapeptides corresponding to the pro-portion of cathepsin D were analyzed in parallel. Procathepsin D and one decapeptide, peptide 2, as well as IGF-II (as a known positive) increased cell proliferation, with doubling times of 28.4, 28.8, and 30.3 h, respectively, versus untreated UL-1 cells (36.4 h). Procathepsin D treatment of UL-1 tumor cells significantly increased the cisplatin LD(50) (74.9 microgram/ml) over untreated (33.9 microgram/ml) as well as IGF-II-treated (38.8 microgram/ml) cells. Peptide 2 also showed a significant increase in LD(50) (69.5 microgram/ml) compared to untreated and peptide 1-treated cells (37.1 microgram/ml). There are several unique forms of cathepsin D expressed and accumulated by ovarian tumors and these forms are detectable in the sera of those with ovarian cancer. The presence of these procathepsin D can increase the proliferation of these tumor cells, while decreasing their sensitivity to chemotherapeutic agents. While procathepsin D and IGF-II both enhance proliferation, only procathepsin D (and peptide 2) appears to modulate chemosensitivity, suggesting a separate receptor or pathway for this consequence.

Cathepsin D↗

PubMed1999

Altered cord serum lipid levels associated with small for gestational age infants.

OBJECTIVE: To determine whether small for gestational age (SGA) infants show changes in lipid metabolism that could distinguish growth-restricted subpopulations. METHODS: Sera from the arterial cord blood from 38 SGA infants were analyzed for apolipoprotein A-I level, total lipid content, and distribution of those lipids as triglycerides, diglycerides, free fatty acids, and phospholipids. Comparisons were made between appropriate for gestational age (AGA) controls (n = 25), SGA infants with a ponderal index below the tenth percentile (SGA I, n = 20), and SGA infants with a ponderal index above the tenth percentile (SGA II, n = 18). RESULTS: Total cord serum lipid content was markedly decreased in all SGA infants compared with AGA infants (2.8 times lower). Although SGA infants showed total lipid concentration decreases, SGA I and SGA II infants showed distinct characteristics. Infants in the SGA I group had higher triglyceride levels (1.8 times higher) and lower free fatty acid levels (1.4 times lower), compared with AGA infants (P < .001). The lipid subclass distribution in SGA II infants was not significantly different from that in AGA infants, with the exception of an increase in triglyceride concentrations (1.3 times higher). Although the 22-kD placenta-derived apolipoprotein A-I was similar in all groups, the level of fetal liver-derived 28-kD apolipoprotein A-I was 6.5 times lower in SGA I infants than in AGA or SGA II infants (P < .001). CONCLUSION: The SGA I infants appeared to have impaired utilization of circulating triglycerides, consistent with peripheral adipose depletion. Diminished fetus-derived apolipoprotein A-I levels with normal levels of placenta-derived apolipoprotein A-I levels might indicate a defect in the production or secretion of apolipoproteins associated with growth restriction.

Fetal Blood↗

PubMed1999

Taxol-induced bcl-2 phosphorylation in ovarian cancer cell monolayer and spheroids.

Expression of Bcl-2, Bax, p53 and induction of apoptosis were studied in cisplatin or Taxol treated monolayer and spheroid cultures of ovarian cancer cell lines (SKOV-3, UL-1, UL-3C). While cisplatin (15-75 microg/ml) induced apoptosis in monolayer and spheroid cultures, Taxol (100-800 nM) induced fragmentation in monolayers only. Cisplatin induced up to 5-fold DNA fragmentation in monolayers, while 3-fold (UL-3C, SKOV-3), and 1.5-fold (UL-1) in spheroids. Taxol treatment of monolayers resulted in the characteristic phosphorylation of Bcl-2, which was not demonstrated in spheroid cultures. Bax expression was reduced in spheroids following cisplatin or Taxol treatment, while p53 levels remained unchanged.

Antineoplastic Agents↗

PubMed1998

Immune recognition of endometrial tumor antigens induced by multiparity.

OBJECTIVE: The risk of developing endometrial cancer is reduced with increasing parity. The purpose of this study was to investigate the possibility that maternal immunization against fetal antigens might be elicited during pregnancy and, if so, to characterize antigens reactive with this immune response. METHODS: Sera were obtained from nulliparous (n = 9) and multiparous women (n = 14). Cellular proteins were isolated from normal endometrium and cultured cells from early (HEC-1A) and late (KLE and RL95-2) stage endometrial cancers. These were separated by SDS-PAGE and those proteins reactive with each individual's serum were assessed by Western immunoblot. Reactive proteins were isolated from KLE tumor cells by immunoaffinity columns. Three commonly recognized proteins were identified, separated, and processed for internal microsequencing. RESULTS: Sera from multiparous women, used as primary antibodies, recognized multiple bands on endometrial tumors, ranging from 10 to 120 kDa. Several antigens were commonly recognized by the sera of multiparous women. The three commonly recognized proteins, normally expressed by fetal tissues, were identified as cystatin A (10 kDa), epidermal fatty acid binding protein (18 kDa), and keratin 10 (54 kDa). Nulliparous women failed to recognize these antigens. CONCLUSION: These findings suggest that certain antigens expressed by the fetus and/or the placenta immunize women during pregnancy. This immune response may protect these women from developing endometrial cancer and explain epidemiologic findings. Future studies will explore the utility of these reexpressed fetal antigens as possible targets for active immunotherapy.

Adult↗

PubMed1998

Cathepsin D antigenic epitopes identified by the humoral responses of ovarian cancer patients.

Previous studies implicated cathepsin D as one commonly recognized target of tumor-reactive immunoglobulins from ovarian cancer patients. These immunoglobulins are shown to be immunoreactive with both the 52-kDa procathepsin D and the 32-kDa mature cathepsin D derived from the UL-1 ovarian cancer cell line. Whether the carbohydrate domains or the core protein were associated with its immunogenicity was analyzed with cathepsin D isolated from tunicamycin-treated UL-1 cells. No significant difference was detected in the immunoreactivity of patient serum with the glycosylated and deglycosylated forms of the cathepsin D, suggesting that patient humoral responses are directed primarily against the core protein. To define the antigenic epitopes of cathepsin D, tryptic fragments were prepared from UL-1-derived procathepsin D. The epitopes of the core protein recognized by sera from more than one patient were identified using a peptide-specific enzyme-linked immunosorbent assay and microsequencing of positive immunoreactive peptides. This protocol identified four epitopes: two peptides within the propeptide, a third at the carboxy terminus and the fourth at the glycosylation site of the mature enzyme. This approach to the identification of specific antigenic epitopes may be useful in defining effective targets for directed active immunotherapy against cancer.

Amino Acid Sequence↗

PubMed1998

Identification of a unique form of p53 in human cord blood associated with the development of maternal autoantibodies.

PROBLEM: The possible link between p53-reactive antibodies in multiparous women and exposure to a unique p53 protein during pregnancy was examined. METHOD OF STUDY: p53-reactive antibodies were evaluated in sera from nulligravid and multiparous women and patients with ovarian cancer by Western immunoblot. Furthermore, the presence of p53 protein was assayed in cord blood by enzyme-linked immunosorbent assay. Cord blood-derived p53 was compared structurally by protein fingerprinting and functionally by gel mobility shift assay to other isolates of p53. RESULTS: Antibodies reactive with wild-type p53 were observed in 92% of multiparous women and 42% were reactive with one tumor-derived p53 protein. p53 protein was detected in 27 of 154 samples of cord blood. Structural analysis indicated that the fetal p53 resembled the UL-1 p53. Functionally, the fetal and UL-1 protein failed to bind DNA. CONCLUSIONS: Fetal p53 protein seems to be distinct from wild-type p53, characterized by enhanced stability, structural differences and inability to bind DNA, analogous to alternatively spliced variants. Exposure to fetal p53 protein may form the basis for immunologic protection against cancer induced by multiparity.

Antibodies, Monoclonal↗

PubMed1997

Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in ovarian cancer cell lines.

OBJECTIVE: Cisplatin- and Taxol-induced apoptosis was studied in four human ovarian cancer cell lines to evaluate apoptosis as a measure of chemosensitivity. METHODS: In vitro sensitivities of OVCAR-3, SKOV-3, UL-1, and UL-2 cells to cisplatin or Taxol were determined by the sulforhodamine B assay. Induction of apoptosis was studied by DNA fragmentation following treatment with cisplatin and/or Taxol after 24- and 48-hr exposure. DNA fragmentation was further quantitated by the diphenylamine assay and the proportion of cells in the G1, G2/M, and S phase of the cell cycle was determined by flow cytometry. Presence of the p53 gene product was examined by Western blotting. RESULTS: The four cell lines represent various sensitivities to cisplatin and Taxol (LD50 range for cisplatin, 5-30 microg/ml; Taxol, 30-1000 nM). UL-2 represents a resistant cell line which was 10-30 times resistant to Taxol and 6 times resistant to cisplatin when compared to the others. Demonstration of apoptosis correlated with the sensitivity of the cell lines to both cisplatin and Taxol for OVCAR-3 and UL-2. DNA fragmentation of OVCAR-3 was uniformly present when treated with cisplatin or Taxol, at 24 or 48 hr. UL-2 demonstrated no apoptosis after 24 or 48 hr of treatment with either cisplatin or Taxol. When sequencing experiments were performed with cisplatin and Taxol, DNA fragmentation correlated with the cytotoxicity assays, except in UL-1 cells where no significant difference was observed in different interactions of cisplatin and Taxol. Pretreatment with Taxol generally resulted in enhanced cytotoxicity in a schedule-dependent manner, and increased fragmentation was demonstrated; cisplatin pretreatment consistently resulted in decreased fragmentation. Quantitation of the fragmented DNA correlated with that seen on gel electrophoresis. OVCAR-3 and UL-1 demonstrated the greatest change from baseline at 24 hr (3.8 and 3.7 times baseline, respectively), whereas UL-2 had little change from the baseline following treatment. G1 arrest occurred more readily in OVCAR-3 and SKOV-3 cells. UL-2 cells had very little change in the proportion of cells entering G1 arrest, but had a significant increase in the G2/M proportion. In OVCAR-3, UL-1, and UL-2 cells, we demonstrated the presence of an aberrantly expressed p53 gene product, while no p53 was detected in the SKOV-3 cells. CONCLUSIONS: Our findings indicate that the ability to achieve significant cytotoxicity by cisplatin and Taxol may be directly related to the induction of apoptosis; however, cellular and genetic characteristics determine the eventual outcome of these treatments.

Antineoplastic Agents↗

PubMed1997

Lymphokine-activated killer (LAK)-mediated lysis of sequentially isolated ovarian cancer cell lines.

PROBLEM: Understanding immunologic eradication of cancer is hampered by failure to explore tumor evolution. Cell surface molecules may alter with therapy and disease progression. These alterations can translate into variable susceptibility to immune-mediated cell lysis. METHOD OF STUDY: Cell lines from a patient with ovarian carcinoma isolated at surgical debulking (UL-3A), during chemotherapy (UL-3B), and after progression (UL-3C) were used to study changes in cell lysis by natural killer (NK) and lymphokine-activated killer (LAK) cells. The role of adhesion molecules ICAM-1, LFA-3, and glycoproteins in the demonstrated differential killing was also examined. RESULTS: An inverse relationship between attachment and lysis was demonstrated. UL-3C, the most sensitive to lysis (50%), attached the least lymphocytes (40%), whereas UL-3A, the least sensitive (33%), attached the most lymphocytes (71%). A correlation with ICAM-1 and LFA-3 expression was not demonstrated. CONCLUSION: Ovarian cancer cells evolve throughout the disease course, and this may manifest as differential sensitivity to immune-mediated cell lysis.

Cytotoxicity, Immunologic↗

PubMed1997

Humoral immune responses to cathepsin D and glucose-regulated protein 78 in ovarian cancer patients.

Many cancer patients develop tumor-reactive immune responses against antigens that are either expressed on the surface of tumor cells or released from them into the peripheral circulation. In this study, tumor-reactive immunoglobulins, present in the sera of ovarian cancer patients, were used to identify commonly recognized tumor-associated antigens on ovarian tumor cells. Western immunoblot analysis of cellular proteins, obtained from UL-1 ovarian tumor cell line, demonstrated several commonly recognized immunoreactive proteins. Two of these proteins (Mr 32,000 and 71,000) were selected for further investigation. Cellular proteins isolated from normal human ovarian epithelia, in a similar fashion, failed to exhibit corresponding immunoreactivity to these proteins. As an additional control, sera from normal (nontumor-bearing) individuals failed to identify these proteins on Western immunoblots. Furthermore, the absorption of the ovarian cancer patients' sera with normal ovarian epithelial tissue did not remove the reactivity of these two proteins. The Mr 32,000 and 71,000 proteins were subsequently purified by reverse-phase high-performance liquid chromatography, separated by SDS-PAGE, transferred to the polyvinylidene difluoride membrane, and digested with trypsin. These resulting tryptic fragments were separated by microbore reverse-phase high-performance liquid chromatography, and selected fragments were sequenced by mass spectrometry. This sequence analysis identified the Mr 32,000 protein as cathepsin D and the Mr 71,000 as glucose-regulated protein 78 (member of the heat shock protein family). The identities of cathepsin D and glucose-regulated protein 78 were confirmed by Western blot analysis. Additionally, the presence of cathepsin D was demonstrated in association with immune complexes in vivo. Currently, the common antigenic epitopes of these proteins are being defined.

Adenocarcinoma, Mucinous↗

PubMed1996

Interleukin-2 activation of cytotoxic cells in postmastectomy seroma.

Lymphocytes were isolated from breast seroma fluids and used to study the mechanism of activation of cytotoxic lymphocytes and possible role of immunological potentiation following surgery in breast cancer patients. Single or serial samples were obtained from patients who had undergone mastectomy or lumpectomy with axillary node dissection. Lymphocytes were activated with rIL-2 (interleukin-2) and their cytotoxic activity was studied against Daudi and K562 cells and against a breast tumor line (SKBr-3). All of the patients (21/21) responded to IL-2 stimulation by significant activation of cytotoxic activity. The unstimulated cytotoxic activity of these cells against NK targets was low with less than 10% specific release in cytotoxicity assays. In simultaneous experiments, autologous seroma fluid was included during activation of lymphocytes to study possible regulatory molecules that may be present. In 17/21 patients, the presence of their seroma fluid, during the activation period, enhanced or did not effect the cytotoxic potential of their lymphocytes; inhibition was observed when seroma fluids from 4/21 patients were included. Analysis of the cytotoxic population derived from combined IL-2 and seroma treatments indicates the presence of cells with increased expression of CD56, and CD2, as well as in some cases CD16 expression. Cytotoxic lymphocytes derived from IL-2 and seroma treatments appeared to be more effective killers. Modulation of CD2 expression with seroma alone appeared to result in the generation of this highly cytotoxic population. This study demonstrates the role of CD2 expression in the effectiveness of LAK cell killing and also potential benefit of an immunotherapeutic approach to the postoperative treatment of carcinoma of the breast.

Adult↗