Interleukin 1 stimulates fibroblasts to produce granulocyte-macrophage colony-stimulating activity and prostaglandin E2.

Granulocyte-macrophage colony-stimulating activity (GM-CSA) can be produced by a variety of normal cell types including mononuclear phagocytes, activated T lymphocytes, endothelial cells, and fibroblasts. Recent evidence shows that a major role of the monocyte-macrophage is the recruitment of environmental cells, i.e., fibroblasts, to produce GM-CSA. In this study we have identified interleukin 1 (IL-1) as a monokine that stimulates fibroblasts to produce and release GM-CSA and prostaglandin E2 (PGE2). Both purified human monocyte-derived IL-1 and human recombinant IL-1 (10(-10) M) can be substituted for monocyte-conditioned medium in stimulating fibroblast GM-CSA and PGE2 production. Both forms of IL-1 stimulate fibroblasts to produce GM-CSA and PGE2 in a dose-dependent fashion. The fibroblast-stimulating activity found in monocyte-conditioned medium was completely blocked by anti-IL-1. We conclude that monocytes produce IL-1, and that monocyte-derived IL-1 induces fibroblasts to produce GM-CSA and PGE2.

CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris.

Engagement of the B7 family of molecules on antigen-presenting cells with their T cell-associated ligands, CD28 and CD152 (cytotoxic T lymphocyte-associated antigen-4 [CTLA-4]), provides a pivotal costimulatory signal in T-cell activation. We investigated the role of the CD28/CD152 pathway in psoriasis in a 26-week, phase I, open-label dose-escalation study. The importance of this pathway in the generation of humoral immune responses to T cell-dependent neoantigens, bacteriophage phiX174 and keyhole limpet hemocyanin, was also evaluated. Forty-three patients with stable psoriasis vulgaris received 4 infusions of the soluble chimeric protein CTLA4Ig (BMS-188667). Forty-six percent of all study patients achieved a 50% or greater sustained improvement in clinical disease activity, with progressively greater effects observed in the highest-dosing cohorts. Improvement in these patients was associated with quantitative reduction in epidermal hyperplasia, which correlated with quantitative reduction in skin-infiltrating T cells. No markedly increased rate of intralesional T-cell apoptosis was identified, suggesting that the decreased number of lesional T cells was probably likely attributable to an inhibition of T-cell proliferation, T-cell recruitment, and/or apoptosis of antigen-specific T cells at extralesional sites. Altered antibody responses to T cell-dependent neoantigens were observed, but immunologic tolerance to these antigens was not demonstrated. This study illustrates the importance of the CD28/CD152 pathway in the pathogenesis of psoriasis and suggests a potential therapeutic use for this novel immunomodulatory approach in an array of T cell-mediated diseases.

Functional integrity of cryopreserved human monocytes.

Human peripheral blood monocytes collected by semicontinuous flow centrifugation were cryopreserved in varying concentrations of dimethyl sulfoxide (DMSO). Optimum protection from lethal cryoinjury was achieved with 7.5-10% DMSO. Compared to fresh cells, cryopreserved cells retained 80% of their chemotactic responsiveness and fructose-6-phosphate shunt activity after stimulation by zymosan. Fresh and cryopreserved human monocytes had comparable but low levels of cytotoxic activity against P-815 murine mastocytoma cells in vitro.

Limited impact of total parenteral nutrition on nutritional status during treatment for small cell lung cancer.

During a randomized trial of total parenteral nutrition (TPN) in patients with small cell lung cancer, we evaluated the short- and long-term effects of 4 weeks of TPN on nutritional assessment parameters. All 119 patients who were accrued to the study received the same chemotherapy and radiotherapy protocol which extended over a 1-year period: 57 patients received TPN; and 62 served as controls. At base line, patients with greater than 5% pretreatment weight loss had significantly lower levels of serum albumin, total iron-binding capacity, and creatinine/height index. TPN administration led to a significant increase in mean caloric intake and weight compared with controls (P less than 0.0001). In the short-term study, body fat, as measured by triceps skinfold thickness, was maintained, and there was a small increase in arm muscle circumference. Serum albumin and hematocrit decreased but promptly returned to pretreatment levels when TPN was stopped. There were no long-term differences in any of the nutritional assessment parameters between the two groups.

Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases.

Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.

Autologous bone marrow transplant in acute myeloid leukemia in first remission.

PURPOSE: The Eastern Cooperative Oncology Group conducted a prospective study of postremission high-dose chemotherapy and autologous bone marrow transplantation (autoBMT) in a group of uniformly treated adults with de novo acute myeloid leukemia (AML) to evaluate whether intensive, myeloablative therapy in first complete remission (CR) could improve the disease-free survival. PATIENTS AND METHODS: After initial CR was induced by the combination of daunorubicin, cytarabine, and thioguanine, patients not eligible for allogeneic bone marrow transplantation (alloBMT) were offered autoBMT. Within a median of 2 months after CR, and without intervening postremission therapy, bone marrow was obtained, purged by exposure to 4-hydroperoxycyclophosphamide (4-HC), and cryopreserved. High-dose therapy consisted of oral busulfan over 4 days (16 mg/kg total) followed by intravenous (IV) cyclophosphamide 50 mg/kg daily for 4 days. The cryopreserved marrow was then reinfused. RESULTS: Of the 39 patients scheduled for autoBMT, four relapsed before transplantation. Two of the 35 (6%) transplant patients died of transplant-related complications, and 11 (33%) relapsed a median of 8 months after marrow reinfusion. No relapse has occurred after 24 months posttransplant. With a median follow-up of 31 months, the median disease-free survival period for all 39 patients has not been reached; however, 54% +/- 16% of patients are projected to be alive and disease-free at 3 years. CONCLUSION: Long-term, disease-free survival after autoBMT in AML seems to be better than the outcome after conventional-dose postremission therapy and rivals the results of alloBMT.

A phase III trial comparing idarubicin and daunorubicin in combination with cytarabine in acute myelogenous leukemia: a Southeastern Cancer Study Group Study.

PURPOSE: A randomized clinical trial was undertaken to compare the therapeutic effectiveness of idarubicin (IDR) to daunorubicin (DNR), and both were given in combination with cytarabine (CA) in acute myelogenous leukemic (AML) patients. PATIENTS AND METHODS: Newly diagnosed patients were given a daily infusion of CA (100 mg/m2) for 7 days and were assigned randomly to receive DNR (45 mg/m2) or IDR (12 mg/m2) daily for the first 3 days. Those patients who achieved a complete remission (CR) were given three consolidation courses that consisted of CA (100 mg/m2 intravenously [IV]) and thioguanine (TG; 100 mg/m2 orally) every 12 hours for 5 days and either DNR (50 mg/m2) or IDR (15 mg/m2) on the first day of each cycle. After consolidation, patients received late intensification, which consisted of the same drugs used for induction except that the CA was given for 5 days and the anthracycline for 2 days. Four courses were planned at 13-week intervals. RESULTS: The CR rates were 75 of 105 (71%) on the IDR arm and 65 of 113 (58%) on the DNR arm (P = .03). The median survival and median remission durations were 297 and 433 days, respectively, on the IDR arm. The median survival and median remission durations were 277 and 328 days, respectively, on the DNR arm. Six deaths occurred during late intensification, five on IDR and one on DNR; this approach was abandoned after 47 patients were entered. The median survival was significantly longer for patients who received late intensification. CONCLUSION: This trial demonstrated that IDR was more effective than DNR in remission induction in AML.

Effects of intravenous hyperalimentation during treatment in patients with small-cell lung cancer.

One hundred nineteen patients were entered onto a randomized trial of the role of intravenous hyperalimentation (IVH) in patients with small-cell lung cancer. IVH was given during the first 30 days of induction chemotherapy to 54 patients. IVH did not effect any improvement in response or survival from therapy. In view of the lack of benefits from IVH, an analysis was made of the toxicities suffered by the 54 patients receiving IVH as well as any effects IVH might have made on chemotherapy-induced toxicity. Toxicities observed included mechanical difficulties with the catheter leading to temporary or permanent discontinuation of the IVH (11 patients), subclavian vein thrombosis (one patient), sepsis in nine patients v none of the 62 control patients, fluid overload (27 patients), hyponatremia (25 patients), and hyperglycemia requiring insulin (13 patients). Patients receiving IVH had higher granulocyte counts on days 14 and 21 of the first cycle of chemotherapy. Analysis shows that this difference is likely caused by fever and infection associated with IVH rather than any nutritional effect on granulopoiesis. In this population of patients, IVH had significant complications but did not ameliorate chemotherapy-induced toxicity and it did not effect any clinical benefit. Future studies of adjunctive nutritional therapy must consider the significant risk in this older population and must limit IVH volume or exclude patients with even mild compromise in cardiovascular functions. Further, any new trial must have a significant rationale for adjunctive use to justify the potential risks.

Evaluating the financial impact of clinical trials in oncology: results from a pilot study from the Association of American Cancer Institutes/Northwestern University clinical trials costs and charges project.

PURPOSE: Medical care for clinical trials is often not reimbursed by insurers, primarily because of concern that medical care as part of clinical trials is expensive and not part of standard medical practice. In June 2000, President Clinton ordered Medicare to reimburse for medical care expenses incurred as part of cancer clinical trials, although many private insurers are concerned about the expense of this effort. To inform this policy debate, the costs and charges of care for patients on clinical trials are being evaluated. In this Association of American Cancer Institutes (AACI) Clinical Trials Costs and Charges pilot study, we describe the results and operational considerations of one of the first completed multisite economic analyses of clinical trials. METHODS: Our pilot effort included assessment of total direct medical charges for 6 months of care for 35 case patients who received care on phase II clinical trials and for 35 matched controls (based on age, sex, disease, stage, and treatment period) at five AACI member cancer centers. Charge data were obtained for hospital and ancillary services from automated claims files at individual study institutions. The analyses were based on the perspective of a third-party payer. RESULTS: The mean age of the phase II clinical trial patients was 58.3 years versus 57.3 years for control patients. The study population included persons with cancer of the breast (n = 24), lung (n = 18), colon (n = 16), prostate (n = 4), and lymphoma (n = 8). The ratio of male-to-female patients was 3:4, with greater than 75% of patients having stage III to IV disease. Total mean charges for treatment from the time of study enrollment through 6 months were similar: $57,542 for clinical trial patients and $63,721 for control patients (1998 US$; P =.4) CONCLUSION: Multisite economic analyses of oncology clinical trials are in progress. Strategies that are not likely to overburden data managers and clinicians are possible to devise. However, these studies require careful planning and coordination among cancer center directors, finance department personnel, economists, and health services researchers.

Long-term follow-up of a randomized post-induction therapy trial in acute myelogenous leukemia (a Southeastern Cancer Study Group trial).

A phase III clinical trial was designed to determine if more intensive induction and consolidation therapy for acute myeloblastic leukemia increases the remission rate and prolongs survival. A minor objective was to determine if the use of non-cross resistant drugs was more effective than the same drugs used for induction. Patients with untreated leukemia between the ages of 15 and 50 were given daunorubicin 45 mg/m2 for the first 3 days of a 10-day continuous infusion of cytosine arabinoside, initially at a dose of 2000 mg/m2 but reduced to 100 mg/m2 because of toxicity. Those under 36 achieving a complete remission and with an histocompatible donor were assigned to a transplant arm. The rest were randomized to receive one of three consolidation arms: A, cytosine arabinoside, 200 mg/m2 daily for 7 days and daunorubicin 45 mg/m2 daily for 3 days for three courses; B, one course as in Arm A followed by amsacrine, 120 mg/m2 daily for 5 days followed by a 5-day continuous infusion of azacytidine, 150 mg/m2/day; C, thioguanine and cytosine arabinoside, 100 mg/m2 every 12 h and daunorubicin 10 mg/m2 daily for 5 days for three courses followed by four maintenance courses of cytosine arabinoside, 100 mg/m2 daily for 5 days and daunorubicin, 45 mg/m2 for 2 days every 13 weeks. From 1981 to 1986, 398 eligible patients were enrolled and 219 achieved a complete remission. The initial induction dose of cytosine arabinoside was reduced after five of 29 patients exhibited fatal gastrointestinal toxicity. Only 11 patients were assigned to the transplant arm. There were no significant differences in the consolidation arms. The 5 year disease-free survivals were 38, 31 and 27% in arms A, B, and C respectively. Intensive consolidation therapy with the same or different drugs used in induction was as effective as lower dose consolidation followed by maintenance therapy.

HLA-identical sibling bone marrow transplants vs chemotherapy for acute myelogenous leukemia in first remission.

There is controversy whether adults with acute myelogenous leukemia (AML) in first remission are best treated with chemotherapy or an HLA-identical sibling bone marrow transplant. We studied 1097 adults, 16-50 years old, with AML in first remission. Results of transplants from HLA-identical siblings reported to the International Bone Marrow Transplant Registry (IBMTR; n = 901) were compared with results of chemotherapy in comparable persons treated by the German AML Cooperative Group (GAMLCG; n = 196). Preliminary analyses identified subject- and disease-related variables differing between the cohorts and associated with treatment outcome within each cohort. We adjusted for these variables and differences in time-to-treatment in subsequent comparisons of treatment-related mortality, relapse, survival and leukemia-free survival (LFS). Five-year probability of treatment-related mortality was greater for transplants than chemotherapy (43% (95% confidence interval, 37-49%) vs 7% (3-11%); P< 0.0001). Five-year relapse probability was less for transplants than chemotherapy (24% (20-28%) vs 63% (55-71%); P< 0.0001). Five-year probability of survival was similar with transplants and chemotherapy (48% (43-53%) vs 42% (33-51%); P = 0.24). Five-year LFS probability was higher for transplants than chemotherapy (46% (42-50%) vs 35% (28-41%); P= 0.01). These data indicate that bone marrow transplants from HLA-identical siblings result in comparable survival but greater LFS than chemotherapy in adults with AML in first remission.

Differences in Fc receptor expression between small and large guinea pig monocytes.

Guinea pig monocytes have been separated into subpopulations differing in size, morphology, and cytochemistry. We report now that these peripheral blood monocyte subpopulations differ also in native Fc receptor expression. All freshly isolated small monocytes formed Fc rosettes with sensitized sheep erythrocytes, whereas Fc receptor expression was not detectable on large monocytes. However, Fc receptor expression developed rapidly in culture such that by 48 hours all monocytes were positive. This phenomenon was inhibited by 2-deoxy-D-glucose and cycloheximide, suggesting that Fc receptor expression depended upon glycolysis and protein synthesis. Large monocytes participated in antibody-dependent lysis of sheep erythrocyte targets. That large monocytes lacking Fc receptors were cytolytic in this assay was concordant with expression of Fc receptors during the 16-hour test period. We believe that the large number of lymphocytes present in the Fc receptor-positive small monocyte fraction interfered with their measurement of antibody-dependent cytotoxicity. We conclude that Fc receptor expression on guinea pig monocytes is heterogeneous within the peripheral circulation. These differences quickly diminish in vitro with the expression of Fc receptors by all monocytes. Additionally, antibody-dependent lysis of sensitized erythrocytes by guinea pig mononuclear cells is due to monocytes and not lymphocytes or Kurloff cells.

Subfractionation of human blood monocyte subsets with Percoll.

Human monocytes can be separated into subsets of large and small cells by centrifugal elutriation. Small and large monocytes share many characteristics, but only small monocytes demonstrate enhanced cytotoxicity toward heterologous tumor cells. The small monocytes isolated by centrifugal elutriation are contaminated with lymphocytes that may hinder further characterization of this subpopulation. This study reports the application of Percoll in the purification of monocytes. The findings indicate that monocyte subsets isolated by centrifugal elutriation that differ in size are nevertheless equal in buoyant density.

The role of interleukin 3 and interleukin 6 in the protection from 4-hydroperoxycyclophosphamide and the proliferation of early human hematopoietic progenitor cells.

Previous reports have shown that interleukin 1 (IL-1) has radioprotective effects when given to mice 20 h before a lethal dose of irradiation and enhances granulocyte recovery in mice treated with cyclophosphamide. We have recently reported that IL-1 can provide protection for human bone marrow colony-forming cells including blast colony-forming cells (B1-CFC) treated with high doses of 4-hydroperoxycyclophosphamide (4-HC). In view of the recent reports that IL-1 induces interleukin 6 (IL-6) in fibroblasts and macrophages and that IL-6 and interleukin 3 (IL-3) are the main growth factors for B1-CFC, we have examined the ability of these interleukins to protect early human hematopoietic progenitor cells from the cytotoxic effects of 4-HC. In addition, we have also studied the ability of IL-3 to promote colony formation by 4-HC-treated bone marrow cells with or without IL-1 preincubation. In this study, we report that preincubation of bone marrow mononuclear cells with IL-3 or IL-6 prior to 4-HC results in no protection and, in fact, may be detrimental to early hematopoietic progenitor cells. On the other hand, addition of IL-3 to 5637-conditioned medium and erythropoietin enhanced colony formation by early progenitors following 4-HC treatment. These findings suggest that IL-3 and IL-6 are not responsible for the protection of early progenitor cells from 4-HC seen with IL-1, but that IL-3 does promote colony formation following 4-HC treatment.

Evidence for the role of leukotrienes C4 and D4 as essential intermediates in CSF-stimulated human myeloid colony formation.

Products of the lipoxygenation of arachidonic acid have been shown to induce a variety of effects on cells of myeloid lineage. Colony-stimulating factor causes release of arachidonic acid from cell membranes, which then undergoes oxygenation via the cyclooxygenase and lipoxygenase pathways. Nordihydroguaiaretic acid (NDGA) and 3-amino-1-[m(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C), compounds that inhibit both the cyclooxygenase and lipoxygenase pathways, cause dose-dependent inhibition of CSF-induced human granulocyte-monocyte colony formation in vitro, with complete inhibition at 20 and 50 microM, respectively. Indomethacin, which inhibits cyclooxygenase but not lipoxygenase, has no effect on colony growth at 50 microM, which is well in excess of the dose needed for complete inhibition of cyclooxygenase. Leukotrienes (LTs) C4 and D4 (5-100 ng/ml) reverse NDGA inhibition of colony growth. At similar concentrations, neither leukotriene B4 or 5-HETE caused reversal of NDGA inhibition. These results support a role for LTC4 and LTD4 as essential intermediates in CSF-stimulated myeloid colony formation.

Dilution techniques for optimum recovery of cryopreserved bone marrow cells.

Bone marrow granulocyte progenitor cells (CFU-C) were assayed in methyl-cellulose prior to cryopreservation in Dimethylsulfoxide (DMSO) and after thawing and diluting the DMSO. The time of dilution from 10% to 1% DMSO and the temperature of the sample and diluting media were studied. Compared with samples diluted at 0 degrees-4 degrees C, samples which were diluted at 24 degrees C were more viable by Trypan Blue exclusion (p less than .01) and had greater CFU-C growth in vitro (p less than .01). There was no advantage to prolonging dilution time from 10 minutes at a constant rate to 40 minutes using stepwise technique. Recovery of CFU-C at 24 degrees C ranged from 40% to 114% with a mean +/- S.D. of 67% +/- 19.5%. There was evidence that clonogenic cells were selectively preserved under the conditions described.

Clinical results of autotransplantation after nitrosourea therapy.

Bone marrow harvested from two previously treated patients was cryopreserved prior to their first dose of Methyl CCNU. The thawed bone marrow was reinfused 96 hours after a subsequent dose of MeCCNU in one patient after the previous dose caused severe myelosuppression. In the other patient, the bone marrow was given 96 hours after an escalation of the MeCCNU dose. In both patients, myelosuppression was abrogated and viable stem cells were recovered in the peripheral blood.

The reversal of myelofibrosis associated with chronic myelogenous leukemia after allogeneic bone marrow transplantation.

A 20-year-old male with chronic myelogenous leukemia (CML) and severe bone marrow fibrosis underwent splenectomy, then ablative chemotherapy, followed by bone marrow transplantation from a histocompatible sister. The myelofibrosis completely resolved. Prompt marrow engraftment and disappearance of Philadelphia chromosome positive cells were documented. Therefore, the presence of marrow fibrosis which frequently accompanies CML is rapidly reversible following high dose chemotherapy and is not indicative of a hostile microenvironment which could preclude marrow transplantation for patients with CML and myelofibrosis.

Biochemical analyses of proteolytic nicking of the human glycoprotein hormone alpha-subunit and its effect on conformational epitopes.

Conformational features of two epitopes on the glycoprotein hormone alpha-subunit were investigated using two antihuman FSH (anti-hFSH) monoclonal antibodies (mAbs) 3A and 5F that recognize different epitopes and are specific for alpha-subunit. These mAbs were used to investigate whether the conformation of these epitopes was different in heterodimeric hFSH, hTSH, hLH, or hCG. Any differences in the mass of hormone in each preparation were accounted for by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of all hormone preparations used in this study. Rabbit anti-hFSH alpha-(11-27) antipeptide antisera and [125I]protein-G were used in the Western blot analysis. Radioactivity associated with each band was determined and used to normalize the mass of alpha-subunit in each reference preparation used in the displacement assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also performed in order to examine the integrity of each of the hormone reference preparations. hTSH alpha and, to a lesser extent, hLH alpha preparations contained an internal nick in the polypeptide chain. RIA analysis performed using heterodimeric glycoprotein hormones as competitors revealed that an average 100-fold difference in the ED50 values for hFSH compared to the other glycoprotein hormones was seen with mAb 3A. Therefore, the conformation of 3A epitope appeared to be different in hFSH than in hTSH, hLH, or hCG. In comparison, the epitope recognized by mAb 5F only had an average 7-fold difference in reactivity (ED50 values) for hFSH compared to hTSH, hLH, and hCG. Likewise, competition assays using the respective alpha-subunits and mAb 5F revealed a pattern of competition similar to that observed with heterodimers, with an average 4-fold difference in the ED50 values for hFSH alpha compared to those for hTSH alpha, hLH alpha, and hCG alpha. Therefore, the conformation of the 5F epitope appears unaffected by association of alpha-subunit with beta-subunit. Accordingly, any differences in the conformation of the four alpha-subunits, as demonstrated by these small differences in ED50 values, appear to be inherent to each alpha-subunit. In fact, the 5F epitope appears to be quite rigid, since nicked alpha-subunit preparations could compete with [125I]hFSH for binding to 5F with comparable potency to non-nicked alpha-subunits. These findings support the concept that epitopes on heterodimeric hFSH alpha may have different conformational features. Some are specific for heterodimeric hFSH alpha, and we refer to these as conformationally active (flexible). Others are common to the four human glycoprotein hormone alpha-subunits, suggesting that they are conformationally constrained (rigid).

Epitope mapping of human follicle stimulating hormone-alpha using monoclonal antibody 3A identifies a potential receptor binding sequence.

Five monoclonal antibodies (mabs) were generated (3A, 4B, 5F, 2E, 1E) by immunizing BALB/c mice with human (h) FSH. The mabs were used to relate antigenic structures (epitopes) to function (receptor binding). All five mabs could immunoneutralize (inhibit binding to receptor) hFSH and could be placed into two groups based on potency (degree of neutralization). Group I mabs (5F, 2E, 1E) were less potent than group II mabs (3A, 4B) even though group I mabs had a 2-fold higher average affinity constant than group II. Those data suggested that group II mabs recognize an epitope near or in the receptor binding site of hFSH. Immunoradiometric epitope cross-matching demonstrated that group I and group II mabs recognize different epitopes. Further characterization of 5F and 3A (representative of group I and group II, respectively) utilized an enzyme-linked immunosorbent assay (ELISA) and a RIA. In the ELISA, both mabs bound hFSH and hFSH alpha but not hFSH beta. In the RIA, 3A bound [125I]hFSH and [125I]hFSH alpha but not [125I]hFSH beta. In contrast, 5F bound only [125I]hFSH. hFSH effectively competed with [125I]hFSH for 5F and 3A. In contrast, hFSH alpha competed with [125I]hFSH for 5F but not for 3A even though 3A could bind hFSH alpha in the ELISA and the RIA. These results suggest that 3A and 5F recognize different epitopes. The epitope recognized by 3A is unique in that its conformation appears to be dependent on association with hFSH beta. Since 3A was a more potent inhibitor of receptor binding than 5F, its epitope specificity was characterized further by epitope mapping. This was accomplished utilizing a peptide ELISA and by affinity chromatography. The results from epitope mapping demonstrated that 3A recognizes sequences 61-78 and 73-92 with binding to 73-92 being 4-fold greater than to 61-78. Thus, the epitope comprised of sequence 73-92 (and to a lesser extent 61-78) appears to be important for receptor binding.