4D-Dynamic Contrast-Enhanced MRI for Preoperative Localization in Patients with Primary Hyperparathyroidism.

BACKGROUND AND PURPOSE: Our aim was to test the hypothesis that our recently introduced 4D-dynamic contrast-enhanced MR imaging with high spatial and temporal resolution has equivalent accuracy to 4D-CT for preoperative gland localization in primary hyperparathyroidism without requiring exposure to ionizing radiation. MATERIALS AND METHODS: Inclusion criteria were the following: 1) confirmed biochemical diagnosis of primary hyperparathyroidism, 2) preoperative 4D-dynamic contrast-enhanced MR imaging, and 3) surgical cure with >50% decrease in serum parathyroid hormone intraoperatively. 4D-dynamic contrast-enhanced studies were reviewed independently by 2 neuroradiologists to identify the side, quadrant, and number of abnormal glands, and compared with surgical and pathologic results. RESULTS: Fifty-four patients met the inclusion criteria: 37 had single-gland disease, and 17, multigland disease (9 with double-gland hyperplasia; 3 with 3-gland hyperplasia; and 5 with 4-gland hyperplasia). Interobserver agreement (κ) for the side (right versus left) was 0.92 for single-gland disease and 0.70 for multigland disease. Interobserver agreement for the quadrant (superior versus inferior) was 0.70 for single-gland disease and 0.69 for multigland disease. For single-gland disease, the gland was correctly located in 34/37 (92%) patients, with correct identification of the side in 37/37 (100%) and the quadrant in 34/37 (92%) patients. For multigland disease, the glands were correctly located in 35/47 (74%) patients, with correct identification of the side in 35/47 (74%) and the quadrant in 36/47 (77%). CONCLUSIONS: The proposed high spatial and temporal resolution 4D-dynamic contrast-enhanced MR imaging provides excellent diagnostic performance for preoperative localization in primary hyperparathyroidism, with correct gland localization of 92% for single-gland disease and 74% in multigland disease, superior to 4D-CT studies.

Clinical Utility of a Novel Ultrafast T2-Weighted Sequence for Spine Imaging.

BACKGROUND AND PURPOSE: TSE-based T2-weighted imaging of the spine has long scan times. This work proposes a fast imaging protocol using variable refocusing flip angles, optimized for blurring and specific absorption rate. MATERIALS AND METHODS: A variable refocusing flip angle echo-train was optimized for the spine to improve the point spread function and minimize the specific absorption rate, yielding images with improved spatial resolution and SNR compared with the constant flip angle sequence. Data were acquired from 51 patients (35 lumbar, 16 whole-spine) using conventional TSE and the proposed sequence, with a single-shot variant for whole-spine. Noninferiority analysis was performed to evaluate the efficiency of the proposed technique. RESULTS: The proposed multishot sequence resulted in a 2× shorter scan time with a >1.5× lower specific absorption rate. The variable flip angle sequence was noninferior to the conventional TSE (P < .025) for all image-quality and clinical criteria except signal-to-noise ratio for the lumbar spine protocol. However, mean image scores for the TSE-variable refocusing flip angle were ≥4.3 for all criteria, and concordance analysis showed high agreement (>90%) with the TSE, indicating clinical equivalence. The single-shot sequence resulted in 4× shorter whole-spine scans, and image scores were ≥4.4 for all criteria, attesting to its clinical utility. CONCLUSIONS: We present a fast T2-weighted spine protocol using variable refocusing flip angles, including a single-shot variant. The sequences have better point spread function behavior than their constant flip angle counterparts and, being faster, should be less sensitive to patient motion, often seen in the longer TSE scans.

Epstein-Barr virus infection of renal proximal tubule cells: possible role in chronic interstitial nephritis.

Chronic interstitial nephritis frequently accompanies renal diseases of different etiologies. Far less common is the entity of primary interstitial nephritis wherein the glomerular and vascular structures of the kidney are not the primary focus of the disease process. Using in situ hybridization and the polymerase chain reaction, we detected DNA from the Epstein-Barr Virus (EBV) exclusively in renal tissue of patients with the idiopathic variety of chronic interstitial nephritis. The EBV genome, but not that of cytomegalovirus or adenovirus, was detected primarily in renal proximal tubule cells. Furthermore, the CD21 antigen, which serves as the receptor for EBV in B lymphocytes, was detected by immunocytochemistry primarily on proximal tubule cells and was markedly upregulated in the EBV-infected tissue. Western blot analysis of primary cultures of normal proximal tubule cells identified a 140-kDa protein, confirming the expression of the CD21 antigen. Colocalization experiments using proximal and distal tubule markers confirmed that EBV DNA and the CD21 antigen are found primarily in proximal tubule cells. EBV infection of renal proximal tubular cells may participate in evoking a cellular immune response that results in a damaged renal interstitium.

The Drosophila wing differentiation factor vestigial-scalloped is required for cell proliferation and cell survival at the dorso-ventral boundary of the wing imaginal disc.

Links between genes involved in development, proliferation and apoptosis have been difficult to establish. In the Drosophila wing disc, the vestigial (vg) and the scalloped (sd) gene products dimerize to form a functional transcription factor. Ectopic expression of vg in other imaginal discs induces outgrowth and wing tissue specification. We investigated the role of the VG-SD dimer in proliferation and showed that vg antagonizes the effect of dacapo, the cyclin-cdk inhibitor. Moreover, ectopic vg drives cell cycle progression and in HeLa cultured cells, the VG-SD dimer induces cell proliferation per se. In Drosophila, ectopic vg induces expression of dE2F1 and its targets dRNR2 and string. In addition vg, but not dE2F1, interacts with and induces expression of dihydrofolate reductase (DHFR). Moreover, a decrease in VG or addition of aminopterin, a specific DHFR inhibitor, shift the dorso-ventral boundary cells of the disc to a cell death sensitive state that is correlated with reaper induction and DIAP1 downregulation. This indicates that vg in interaction with dE2F1 and DHFR is a critical player for both cell proliferation and cell survival in the presumptive wing margin area.

Actin gene expression is modulated by ecdysterone in a Drosophila cell line.

The steroid hormone ecdysterone induced characteristic and specific changes of morphology, enzymatic activities and protein synthesis in a Kc 0% Drosophila melanogaster cell line. To study the ecdysterone action at a molecular level, a Drosophila genomic library was screened by differential hybridization to poly(A)+ RNA from control and ecdysterone-treated cells. Two recombinant phages were selected for hybridizing very intensively with poly(A)+ RNA of ecdysterone-treated cells and very weakly with poly(A)+ RNA of untreated ones. These two clones (lambda Dm 1632 and lambda Dm A5A1) mapped at the 5 C locus on polytene chromosomes; they overlap for a 9000 base-pair sequence that contains an abundantly transcribed region in ecdysterone-treated cells of about 2000 base-pairs. This region permits the selection of mRNA that gives, after translation in vitro, two polypeptides identified as cytoplasmic actin II and III. We demonstrated that these two recombinant phages, hybridizing preferentially with poly(A)+ RNA of ecdysterone-treated cells, contain the 5 C actin gene. Poly(A)+ RNA prepared from various times of treatment of cells were electrophoresed on agarose gels, transferred to nitrocellulose paper and then hybridized with the cloned actin probe. Results of these experiments indicate that there is a sharp increase in the level of RNA coding for actin after ecdysterone treatment of the cell, and that there are two forms of actin-specific RNA in the D. melanogaster cells. Using genomic blots with specific probes derived from lambda Dm 1632, we show that there are six actin genes per haploid Drosophila cell genome contained on six EcoRI fragments, as in Drosophila embryos, indicating that there is no rearrangement of these sequences in cultured cells. Our results suggest that the expression of actin genes in D. melanogaster Kc 0% cells is modulated by ecdysterone.

A prognostic model for prolonged event-free survival after autologous or allogeneic blood or marrow transplantation for relapsed and refractory Hodgkin's disease.

There are several prognostic models for Hodgkin's disease (HD) patients, but none evaluating patient characteristics at time of blood and marrow transplantation (BMT). We developed a prognostic model for event-free survival (EFS) post-BMT based on HD patient characteristics measured at the time of autologous (auto) or allogeneic (allo) BMT. Between 1/1991 and 12/2001, 64 relapsed or refractory HD patients received an auto (n=46) or allo (n=18) BMT. A multivariate prognostic model was developed measuring time to relapse, progression or death. Median follow-up was 51.7 months; median EFS for auto and allo BMT was 36 and 3 months, respectively (P=0.001). Significant multivariate predictors of shorter EFS were chemotherapy-resistant disease, KPS <90 and > or =3 chemotherapy regimens pre-BMT. Patients with two to three adverse factors had significantly shorter EFS at 2 years (58 vs 11% in auto; 38 vs 0% in allo BMT patients). Despite a selection bias favoring auto BMT, the model was valid in both auto and allo BMT groups. We were able to differentiate patients at high vs low risk for adverse outcomes post-BMT. This prognostic model may prove useful in predicting patient outcomes and identifying high-risk patients for novel treatment strategies. Validation of this model in a larger cohort of patients is warranted.

Dysregulation of interleukin-6 responses in ectopic endometrial stromal cells: correlation with decreased soluble receptor levels in peritoneal fluid of women with endometriosis.

Endometriosis (EM) is characterized by the aberrant growth of endometrial cells at sites outside the uterus. We showed previously that peritoneal leukocyte interleukin-6 (IL-6) production is altered in women with EM relative to that in normal control women. Because studies suggest that IL-6 may be growth regulatory for endometrial cells, we examined IL-6 and IL-6 soluble receptor (IL-6sR) in the peritoneal fluid of 40 women. In addition, the growth responsiveness of ectopic endometrial stromal cells to IL-6 was evaluated. The severity of EM correlated with increased levels of IL-6 accompanied by decreased IL-6sR in peritoneal fluid (controls, 1.0 +/- 0.1 and 525.4 +/- 53; stage I-II EM, 1.4 +/- 0.2 and 274.6 +/- 26; stage III-IV EM, 19.3 +/- 4.6 and 319.4 +/- 26; adhesions, 1.9 +/- 0.4 and 324.7 +/- 26 pmol/L IL-6 and IL-6sR, respectively). Additional studies revealed that unstimulated endometrial stromal cells from ectopic implants secreted this cytokine in vitro. Furthermore, these cells were resistant to growth inhibition induced by exposure to additional IL-6; this response correlated with weak expression of IL-6 receptor. Taken together, these findings lend further support to the hypothesis that dysregulation of IL-6 responses plays a role in the pathophysiology of EM.

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells: II/relationships between hypoxanthine-guanine-phosphoribosyl transferase and 5'-nucleotidase.

All in vitro cell lines from Drosophila melanogaster do not possess measurable hypoxanthine - guanine - phosphoribosyltransferase (HGPRT) and 5'-nucleotidase activities. Nevertheless these enzymatic activities could be detected by the modification of culture conditions or treatment of crude extracts from Drosophila cells (1). A Drosophila melanogaster hemocyte line enabled demonstration of a relationship between the activities of HGPRT and 5'-nucleotidase. Only the addition of azaserine to the culture medium allows detection of a measurable amount of HGPRT and 5'-nucleotidase in cells devoid of endogenous synthesis of purine nucleotides. This action of azaserine suggests that the simultaneous expression of both enzymes is first directed by the suppression of the 5'-nucleotidase inhibition by pyrimidine nucleotides, and later on by the 5'-nucleotidase activity hydrolyzing the mononucleotides which inhibited the HGPRT, resulting its expression. This regulation system is confirmed by the simultaneous recovery of HGPRT and 5'-nucleotidase activities after heating cell cultures or cell extracts. Under such experimental conditions, 5'-nucleotidase is desensitized towards its negative effectors, while HGPRT regulation remains unchanged.

Role of vitamin B12 in the reduction of ribonucleotides into deoxyribonucleotides in Drosophila cells grown in vitro.

Adenosine was found to inhibit growth of Drosophila melanogaster cells in culture. This toxic effect is prevented by the addition of uridine + deoxyuridine, or uridine + deoxycytidine. In the presence of vitamin B12, uridine alone is sufficient to sustain proliferation of Drosophila cells inhibited by adenosine. Moreover, vitamin B12 increases the incorporation of [3H] uridine into DNA, and decreases the incorporation of [3H] thymidine. No modification of the incorporation of [14C] adenine, [14C] adenonsine or [3H] cytosine into DNA could be found in the presence of vitamin B12. It is concluded that vitamin B12 is involved in an enhanced conversion of uridine ribonucleotides into deoxyribonucleotides derived from uridine.

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells: I.--Conditional activity of hypoxanthine-guanine-phosphoribosyltransferase and 5-nucleotidase.

In cultured cells established from Drosophila melanogaster embryos, and grown in usual medium, no hypoxanthine-guanine-phosphoribosyl transferase (HG-PRT) could be measured, and only traces of 5'-nucleotidase activity were detectable. On the contrary, it was observed that if the same medium is supplemented with purine bases, nucleosides, orotate, glutamine, azaserine or antifolates, de novo purine biosynthesis is inhibited, and HGPRT is detectable, along with an important 5'-nucleotidase activity. Moreover, dialysis or treatment of extracts from cells untreated by purines, with activated charcoal restored HGPRT and 5'-nucleotidase activities. These activities were abolished completely by inosinic acid (IMP) and guanosine 5'-monophosphoric acid (GMP). Similar results were obtained with fly extracts. These results suggest that de novo purine biosynthesis masks HGPRT activity, the endogenous synthesis leading to the accumulation of purine nucleotides which are inhibitors of the HGPRT activity.

A sequence from Drosophila DNA cross-hybridizing with a mouse class I H-2 gene: absence of relevant nucleic acid or amino acid sequence homology.

Following serological data (1) showing cross-reactivity of drosophila surface antigens with anti-H-2 and mouse beta 2-microglobulin (beta 2-m) antisera, we have looked for homologous sequences in the drosophila genome by low stringency hybridization with mouse H-2 and beta 2-m probes. A 240 bp drosophila DNA segment cross-hybridizing with a H-2 probe, was isolated and sequenced. No homology at the nucleotide or amino acid levels was found with the mouse probe which was used, except for a perfectly matching 16-mer containing 11 G-C, which might be responsible for the cross-hybridization. Therefore, our present data do not support the existence of class I H-2 and/or beta 2-m related gene sequences in the drosophila genome.

In situ detection of PCR-amplified HIV-1 and EBV nucleic acids in hyperplastic lymph nodes and in AIDS-related lymphoma.

The purpose of this study was to determine the in situ distribution of PCR-amplified HIV-1 and EBV DNA in hyperplastic lymph nodes and in AIDS-related lymphomas. PCR amplified HIV-1 DNA was detected, on average, in about 30% and 20% of the CD4 and CD21 dendritic cells, respectively, in and around the expanded germinal centers of hyperplastic lymph nodes in seropositive, asymptomatic people. PCR-amplified EBV DNA was noted, on average, in about 20% of L26 B-cells. The amplified HIV-1 DNA was noted in rare non-neoplastic cells in five AIDS-related lymphomas; the other three cases were negative for the viral DNA. Amplified EBV DNA was detected in five of eight lymphomas but in only three of these tissues did the viral DNA localize to the malignant cells. We conclude that although many cells in hyperplastic lymph nodes from people with early HIV-1 infection contain HIV-1 and EBV DNA, these viruses are of ten absent in the malignant cells of AIDS-related lymphoma. This suggests that although infection by these viruses and the concomitant lymphoid hyperplasia may predispose to lymphoma, the viruses are not required for maintenance of the malignant phenotype.

Technetium-99m-sulfur colloid SPECT imaging in infants with suspected heterotaxy syndrome.

UNLABELLED: For the evaluation of a variety of hepatosplenic disorders, SPECT complements planar 99mTc-sulfur colloid liver/spleen imaging. By isolating small, ectopic or poorly functioning spleen(s) from overlying or adjacent liver, SPECT imaging should facilitate identification of splenic tissue in infants with suspected heterotaxy syndrome. METHODS: During a 10-yr period, 10 planar-only and 9 planar-plus-SPECT liver/spleen scans were obtained from 15 infants, 13 of whom were less than 1 mo of age at first examination. Four of the planar-only group had follow-up planar-plus-SPECT imaging. Scintigraphic diagnosis regarding presence of splenic tissue was correlated with clinical diagnosis. RESULTS: Thirteen infants had splenic tissue; two were asplenic. Planar-only imaging provided correct diagnoses in six [four with, two without spleen(s)] but was negative or equivocal in four infants. Planar-plus-SPECT imaging was positive in all in whom it was performed; moreover, in 4/13 infants (31%), splenic tissue was documented only by SPECT imaging. CONCLUSION: Particularly when planar views are inconclusive, SPECT imaging is invaluable for identification and localization of functioning splenic tissue in infants with suspected heterotaxy syndrome.

Immunoresponsiveness in endometriosis: implications of estrogenic toxicants.

Endometriosis is a reproductive disease characterized by the growth of endometrial cells at sites outside the uterus. This disease is a serious disorder associated with chronic pain and infertility, which may be present in 6 million women in this country. Traditional medical therapy has consisted of hormonal regimens that limit the action of endogenous estrogen. The etiology of endometriosis is unknown, but studies suggest that soluble factors known as cytokines play a role in disease pathogenesis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is an environmental toxicant that alters the action of estrogen in reproductive organs and adversely affects immunocompetence. The incidence of endometriosis was determined in rhesus monkeys that were chronically exposed to dioxin for a period of approximately 4 years. Ten years after termination of dioxin treatment, the presence and severity of endometriosis was assessed by surgical laparoscopy. The incidence of endometriosis correlated with dioxin exposure and disease severity was dependent upon the dose administered. Moderate to severe endometriosis was not found in control animals but was documented in three of seven animals exposed to 5 ppt dioxin (43%) and in five of seven animals exposed to 25 ppt dioxin (71%). The frequency of spontaneous disease in the control group was 33%, similar to an overall prevalence of 30% in 304 rhesus monkeys with no history of dioxin exposure. This study indicates that endometriosis may be associated with dioxin exposure in the rhesus. In view of overwhelming evidence that cytokines participate in the mediation of reproductive-endocrine phenomena and regulation of endometrial growth, future assessment of the effects of environmental toxicants on reproductive health may depend upon our understanding of the bidirectional cytokine network between the immune and endocrine systems.

Thalidomide as salvage therapy for VAD-refractory multiple myeloma prior to autologous PBSCT.

Several trials have shown the activity of thalidomide (THAL) in relapsed multiple myeloma (MM) patients failing PBSCT or conventional chemotherapy. PBSCT is considered standard treatment for most patients requiring therapy for MM; however, patients with VAD-resistant disease may not be able to receive PBSCT due to rapidly advancing disease. We report four cases of VAD-refractory MM salvaged with THAL + VAD followed by PBSCT. All patients underwent stem cell mobilization with cyclophosphamide (Cy) (4.5 g/m(2)) and GMCSF. Melphalan (140-200 mg/m(2)) was given as conditioning. All patients engrafted within 12-16 days after PBSCT. Day +100 evaluation showed the following: very good partial response (n = 1) and complete response (n = 3). After a median follow-up to 153 days, two patients continue to take THAL with no signs of disease progression. One patient developed CHF and was taken off THAL while another patient has died of progressive disease while on THAL (MTD 50 mg). In conclusion, VAD-refractory patients were salvaged with the addition of THAL to VAD. They were subsequently able to undergo autologous PBSCT for MM, which will likely improve their overall survival. This suggests that THAL and other related immunomodulatory drugs may prove useful for initial MM therapy in combination with standard chemotherapy followed by PBSCT.

Effect of rituximab on peripheral blood stem cell mobilization and engraftment kinetics in non-Hodgkin's lymphoma patients.

Rituximab is used for in vivo tumor cell purging for non-Hodgkin's lymphoma (NHL) patients prior to autologous peripheral blood stem cell transplantation (PBSCT). However, its effects on PBSC mobilization and function are poorly understood. We compared the mobilization characteristics and engraftment kinetics of 13 NHL patients receiving and 34 NHL patients not receiving rituximab 6 months before PBSC mobilization. In the rituximab group, there was a significantly longer time to neutrophil engraftment (P=0.0466), a trend toward the need for BM harvest to supplement low-yield PBSC collections (31 vs 9%, P=0.08) and a significantly increased rate of bacteremia episodes (62 vs 26%, P=0.025). Median progression-free survival (PFS) and overall survival (OS) were significantly longer in the rituximab compared to the nonrituximab patients (P=0.049 and 0.042, respectively). However, patients in the nonrituximab group were at high risk for recurrence and expected to have shorter survival. Rituximab used within 6 months prior to collection may have a detrimental effect on PBSC mobilization and engraftment. However, rituximab is a major therapeutic breakthrough for NHL treatment and this negative effect may be offset by improved survival. Further studies are warranted in larger populations to determine the impact of rituximab on engraftment, PFS and OS.

Use of nonvolume-reduced (unmanipulated after thawing) umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment.

Volume reduction of umbilical cord blood (UCB) units before infusion is standard in most transplant centers. We examined 26 patients who underwent transplantation from May 1997 to December 2001 with unmanipulated (n=18) or volume-reduced (n=8) UCB units for engraftment. Of 18 unmanipulated UCBT patients, 16 achieved ANC >500/mm(3), a median of 26 days (range, 16-104) post-UCBT; two died before engraftment on days +2 and +14. Of 18 unmanipulated UCBT patients, 10 achieved platelet recovery, a median of 60.5 days (range, 41-144) post-UCBT; eight patients died before platelet recovery +2 to +255 days post-UCBT. These results are similar to several reported studies and our series utilizing volume-reduced UCB units for UCBT. At a median follow-up of 29.5 months, the 100-day and 3-year overall survivals of unmanipulated UCBT were 61.1% (95% CI, 38.6-83.6) and 48.6% (95% CI, 24.8-72.4) and of volume-reduced UCBT were 60% (95% CI, 24.4-95.6) and 22.5% (95% CI, 0-58.7). There was no serious toxicity from UCB infusion using unmanipulated UCB units. We conclude that unmanipulated UCB units may be infused safely into UCBT patients with adequate engraftment and survival.

Aspirin effects on endometrial cancer cell growth.

OBJECTIVE: To find whether aspirin (acetylsalicylic acid, ASA) inhibits the growth of endometrial cancer cells in vitro in a way similar to that in colorectal cancer cells and to investigate the mechanisms by which aspirin might lead to growth inhibition. METHODS: Ishikawa human endometrial tumor cells were grown in the presence of ASA (1-5 mM) for 96 hours. Controls were treated with vehicle (absolute ethanol). Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Analysis of cell-cycle distribution and bcl-2 expression was assessed by flow cytometry. RESULTS: Acetylsalicylic acid induced a dose-dependent inhibition of Ishikawa cells in vitro. The percentage of growth inhibition was 21-88% at concentrations of 1-5 mM. It also induced apoptosis and reduced bcl-2 expression in Ishikawa cells in a dose-dependent manner. Control cells and cells treated with the lowest concentration of ASA exhibited 2% apoptosis and more than 60% of the population expressed bcl-2. Apoptosis levels increased as levels of ASA increased from 2 to 5 mM (7-58%) with a concommitant decrease in bcl-2 expression from 46% at 2 mM to 2% at 5 mM. Acetylsalicylic acid concentrations of 3 mM or greater induced a shift from the resting phase (G0/G1) to S phase of the cell cycle. CONCLUSION: Acetylsalicylic acid inhibited Ishikawa cell growth in vitro in a dose-dependent manner. Apoptosis is one of the mechanisms involved in the response, which can be mediated in part by downregulation of bcl-2.

Altered interleukin-6 production by peritoneal leukocytes from patients with endometriosis.

OBJECTIVES: To investigate the ability of peritoneal leukocytes to produce interleukin-6 (IL-6) in vitro and to determine whether IL-6 is present in the peritoneal fluid (PF) in vivo. DESIGN: Peritoneal leukocytes were assessed for spontaneous versus stimulated IL-6 production. Interleukin-6 in the PF was also quantitated. SETTING: Leukocytes were recovered from PF obtained at the time of diagnostic laparoscopy for pain and infertility or from women undergoing bilateral tubal ligation. PATIENTS: The study population included a total of 24 women. Experimental groups consisted of women undergoing tubal ligations (n = 6), patients with postinflammatory pelvic adhesions unrelated to endometriosis (n = 6), and women with minimal to mild endometriosis (n = 6), or moderate to severe disease (n = 6). RESULTS: Peritoneal leukocytes from normal control women and patients with severe endometriosis spontaneously produced low levels of IL-6. In contrast, cells from women with mild disease or adhesions spontaneously released twofold to fourfold higher levels of this cytokine. Peritoneal leukocytes from patients with both mild and severe endometriosis were refractory to additional cytokine release directly in response to stimulation with endotoxin. Bioactive IL-6 was present in the PF of all patient groups, whereas immunoreactive IL-6 was not detected in this fluid. CONCLUSIONS: These data demonstrate that IL-6 was present in the PF of all patient groups. However, the ability of peritoneal leukocytes to produce IL-6 in vitro differed according to stage of disease. We hypothesize that altered leukocyte IL-6 production in vivo may contribute to the pathophysiology of endometriosis.

Hormonal modulation of Ishikawa cells during three-dimensional growth in vitro.

OBJECTIVES: The Ishikawa endometrial cancer cell line is hormonally responsive, expressing estrogen and progesterone receptors (ER, PR) when grown in traditional monolayer culture. The purpose of this paper is to demonstrate a three-dimensional spheroid culture system for cancer cells. We used this system to determine the response of the Ishikawa cell line to estradiol-17 beta (E), tamoxifen (T), megestrol acetate (MA), and progesterone (P). METHODS: Ishikawa cells were incubated in polyurethane culture bags using phenol red-free media containing ethanol (0.1%, controls), E (1 mumol, or 1 nmol), T (1 mumol, or 10 nmol), MA (1 mumol, or 10 nmol), or P (1 mumol). Cellular morphology was assessed by hematoxylin and eosin staining, and expression of estrogen and progesterone receptors was determined immunohistochemically using an immunoperoxidase technique. RESULTS: Cells in control cultures demonstrated minimal organization and lacked hormone receptors. In contrast, cells exposed to either E or T displayed significant glandular formation, with multicellular, microvilli-rich, columnar epithelia exhibiting polarized nuclear arrangements. Within 4 weeks, E- and T-treated cultures showed upregulated nuclear staining for PR, with little ER present. Cells treated with MA or P showed less glandular organization but expressed ER with PR downregulation. CONCLUSIONS: These data support the use of this novel three-dimensional culture system to study the modulation of tumor cell biologic activity in response to hormonal agents. Future applications of this model include examining in vitro responsiveness of cancer cell lines to additional biologic agents and chemotherapeutic regimens.