Expression of an electrically silent voltage-gated potassium channel in the human placenta.

Human placental expression of K(V)9.3, a voltage-gated K channel linked to tissue oxygenation responses, has been suggested at the messenger RNA level but tissue localisation has not been described. We aimed to: (1) produce an antibody to human K(V)9.3 and (2) assess channel expression and distribution in human placental tissue. We determined human placental protein expression and localisation using an antibody to K(V)9.3. Antibody specificity was confirmed by Western blotting. Staining was observed in syncytiotrophoblast microvillous membrane, endothelial cells (in intermediate, stem villi and chorionic plate blood vessels) and vascular smooth muscle cells (large diameter vessels only) by immunohistochemistry. Expression was unchanged in tissue from women with small-for-gestational age babies. It was concluded that K(V)9.3 is localised to human placental vascular tissues and syncytiotrophoblast.

Glibenclamide inhibits agonist-induced vasoconstriction of placental chorionic plate arteries.

BACKGROUND: Preliminary data suggest that K(ATP) channels may be expressed in placental arteries and veins [Wareing M, Turner C, Greenwood SL, Baker PN, Fyfe GK. Expression of mRNA encoding K+ channels in chorionic plate arteries and veins. J Soc Gynecol Investig 2004;11:353A]. However, no data exist on glibenclamide's effects in placental chorionic plate arteries. AIM: To assess the effect of glibenclamide on placental chorionic plate arterial vasoconstriction. METHODS: Arteries were dissected from placental chorionic plate biopsies obtained at term from uncomplicated pregnancies (N=63). Arteries were mounted onto a wire myograph in HCO3- -buffered physiological salt solution (PSS) at 37 degrees C (5% O2/5% CO2 bubbling) and normalised at 0.9 of L5.1 kPa. Constriction viability was assessed with 120 mmol l(-1) potassium solution (KPSS). Dose-response curves were produced with the thromboxane-mimetics U46619 and U44069 (10(-10)-2 x 10(-6)M), arginine vasopressin (10(-10)-5 x 10(-8)M) and endothelin-1 (10(-11)-3 x 10(-7)M) in the presence or absence of 50 micromol l(-1) glibenclamide. The effect of glibenclamide on arginine vasopressin- and U46619-induced constriction was also assessed in the presence of the cyclo-oxygenase inhibitor indomethacin (10 micromol l(-1)). RESULTS: Pre-incubation with 50 micromol l(-1) glibenclamide significantly right-shifted dose-response curves to all vasoconstrictive agonists tested (repeated measures ANOVA). Indomethacin did not modify the inhibitory effect of glibenclamide. CONCLUSION: Glibenclamide's effects on agonist-induced constrictions are unlikely to be via an inhibition of ATP-sensitive K+ channels, and with U46619- and U44069-induced constrictions, glibenclamide may be acting as a competitive antagonist of thromboxane receptors.

Conductive cation transport in apical membrane vesicles prepared from fetal lung.

In order to characterise the apically-located conductive cation pathway of the type II pneumocyte, apical plasma membranes were prepared from mature fetal guinea pig lung. The protocol yielded purified apical membranes that enriched 19-fold with the brush border enzyme marker alkaline phosphatase; there was no significant contamination with other cellular membranes. A technique for imposing an outwardly-directed electrochemical Na+ gradient was used to amplify conductive 22Na+ uptake into vesicles. Uptake of 22Na+ was time-dependent, proportional to the magnitude of the Na+ gradient, specific and sensitive to the amiloride analogues phenamil and EIPA (apparent minimum Ki values of 50 nM and 10 microM, respectively, with maximum uptake inhibition of 42% and 39% at 100 microM). Uptake experiments in which the outwardly-directed Na+ gradient was replaced by outwardly-directed gradients of small monovalent cations and molecular cations were performed. The Na+/K+ permeability ratio was 1.2:1, and over the extended range of small monovalent cations, a permeability sequence of Na+ > K+ > Li+ > Rb+ > Cs+ was observed, indicating the presence of fixed negative charge in or spatially close to the pore. The molecular cation permeability sequence of NH4+ > methylamine+ > dimethylamine+ > choline+ > N-methyl-D-glucamine+ > tetraethylammonium+ > tetramethylammonium+, after transformation, gives an estimate of 8 A for the conducting pore diameter. These data are consistent with the presence in the apical membrane of fetal type II pneumocytes of a cation specific channel with low Na+ selectivity and amiloride sensitivity.

Conductive Na+ transport in fetal lung alveolar apical membrane vesicles is regulated by fatty acids and G proteins.

We have characterised G protein and fatty acid regulation of the Na+ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 microM GTP gamma S or beta gamma-methylene-GTP, irreversible G protein activators, stimulated conductive 22Na+ uptake (ratio of experimental to control 1.35 +/- 0.02 and 1.34 +/- 0.05, respectively). Conversely, the addition of GDP beta S, an irreversible G protein inhibitor, reduced conductive 22Na+ uptake from 1.00 (control) to 0.79 +/- 0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive 22Na+ uptake, by between 1.18 +/- 0.05 to 1.56 +/- 0.13 over the control. Both arachidonic acid and GTP gamma S-dependent stimulation were abolished in the presence of 10 microM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive 22Na+ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na+ channel Since mepacrine (50 microM), a phospholipase A2 inhibitor, did not affect the GTP gamma S-stimulated conductive 22Na+ uptake, and inhibition of G protein turnover by GDP beta S did not attenuate the arachidonic acid response we conclude that these two regulatory pathways modulate alveolar Na+ transport directly and independently of each other.

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.

In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended in NaCl, but did not have a significant effect, 94+/-19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.

Results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy.

PURPOSE: To determine the efficacy and toxicity of a high-dose interleukin-2 (IL-2) regimen in patients with metastatic renal cell carcinoma. PATIENTS AND METHODS: Two hundred fifty-five assessable patients were entered onto seven phase II clinical trials. Proleukin (aldesleukin; Chiron Corp, Emeryville, CA) 600,000 or 720,000 IU/kg was administered by 15-minute intravenous (i.v.) infusion every 8 hours for up to 14 consecutive doses over 5 days as clinically tolerated with maximum support, including pressors. A second identical cycle of treatment was scheduled following 5 to 9 days of rest, and courses could be repeated every 6 to 12 weeks in stable or responding patients. RESULTS: The overall objective response rate was 14% (90% confidence interval [CI], 10% to 19%), with 12 (5%) complete responses (CRs) and 24 (9%) partial responses (PRs). Responses occurred in all sites of disease, including bone, intact primary tumors, and visceral metastases, and in patients with large tumor burdens or bulky individual lesions. The median response duration for patients who achieved a CR has not been reached, but was 19.0 months for those who achieved a PR. Baseline Eastern Cooperative Oncology Group (ECOG) performance status (PS) was the only predictive prognostic factor for response to IL-2. While treatment was associated with severe acute toxicities, these generally reversed rapidly after therapy was completed. However, 4% of patients died of adverse events judged to be possibly or probably treatment-related. CONCLUSION: High-dose IL-2 appears to benefit some patients with metastatic renal cell carcinoma by producing durable CRs or PRs. Despite severe acute treatment-associated toxicities, IL-2 should be considered for initial therapy of patients with appropriately selected metastatic renal cell carcinoma.

Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and long-term safety data.

PURPOSE: Tumor angiogenesis mediated by vascular endothelial growth factor (VEGF) is inhibited by the recombinant humanized (rhu) monoclonal antibody (MAb) rhuMAbVEGF, which has synergy with chemotherapy in animal models. The present study was designed to assess the safety and pharmacokinetics of weekly intravenous (IV) rhuMAbVEGF with one of three standard chemotherapy regimens. PATIENTS AND METHODS: Twelve adult patients were enrolled four on each combination. rhuMAbVEGF, 3 mg/kg IV, was administered weekly for 8 weeks with (1) doxorubicin 50 mg/m(2) every 4 weeks; (2) carboplatin at area under the curve of 6 plus paclitaxel 175 mg/m(2) every 4 weeks; and (3) fluorouracil (5-FU) 500 mg/m(2) with leucovorin 20 mg/m(2) weekly, weeks 1 to 6 every 8 weeks. RESULTS: The median number of rhuMAbVEGF doses delivered was eight (range, four to eight doses). Grade 3 toxicities were diarrhea (one 5-FU patient), thrombocytopenia (two patients on carboplatin plus paclitaxel), and leukopenia (one patient on carboplatin plus paclitaxel). These toxicities were likely attributable to the chemotherapy component of the regimen. The mean (+/- SD) peak serum level of rhuMAbVEGF was 167 +/- 46 microg/mL, and the mean terminal half-life was 13 days. Total (free plus bound) serum VEGF levels increased from 51 +/- 39 pg/mL (day 0) to 211 +/- 112 (day 49) pg/mL. Three responding patients continued treatment with rhuMAbVEGF and chemotherapy, receiving the equivalent of 36, 20, and 40 total rhuMAbVEGF doses with no cumulative or late toxicities. CONCLUSION: rhuMAbVEGF can be safely combined with chemotherapy at doses associated with VEGF blockade and without apparent synergistic toxicity. Its contribution to the treatment of advanced solid tumors should be evaluated in randomized treatment trials.

Subunit composition determines the single channel kinetics of the epithelial sodium channel.

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of alpha with either wild-type beta or gamma subunits and alpha with carboxy-terminal truncated beta (betaT) or gamma (gammaT) subunits in Xenopus laevis oocytes. alphabeta and alphabetaT channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 microM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (Po) for alphabeta was 0.54 and for alphabetaT was 0.50. In comparison, alphagamma and alphagammaT channels exhibited different kinetics: alphagamma channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short openings with long closed times, resulting in a low Po (0. 16). The mean Po for all alphagamma channels was 0.48. alphagammaT (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean Po of 0.6, similar to that of alphagamma. Inclusion of 0. 1 microM amiloride in the pipette solution reduced the mean open time of alphagammaT to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of alphabeta, alphabetaT (1 microM amiloride), and alphagammaT (0.1 microM amiloride) channels. alphabeta and alphabetaT had similar blocking and unblocking rate constants, whereas the unblocking rate constant for alphagammaT was 10-fold slower than alphabetaT. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the beta subunit, whereas a similar deletion in the gamma subunit affects channel kinetics but not Po.

Structure and function of the Mec-ENaC family of ion channels.

The epithelial sodium channel (ENaC) is the prototype of a new family of ion channels known as the Mec-ENaC superfamily. This new family of proteins are involved in a wide variety of functions that range from maintenance of sodium homeostasis to transduction of mechanical stimuli and nociceptive pain by specialized neurons. They show distinct tissue- and cell type-dependent expression and differential sensitivity to inhibition by the diuretic amiloride and its analogs. Despite the very little amino acid identity shared by these proteins, they all have the same common structure that has become a hallmark of the Mec-ENaC superfamily. The efforts to understand the structure and regulation of these ion channels have been stimulated by the recent discovery of severe disturbances in the maintenance of blood pressure caused by gain- or loss-of-function mutations in the genes that encode the subunits of ENaC in humans. Moreover, cloning of the ion channels that mediate pain elicited by tissue injury and inflammation will facilitate the development of new drugs to treat these common ailments.

The superficial musculoaponeurotic system of the face: a model explored.

Regional differences in the integument of the body are explained, at least in part, by differences in fascial arrangements. In the face, where the skin is more mobile due to the action of the underlying facial muscles, fascial organisation is important for support and separation of muscle groups. This study used bequeathed cadaver material to investigate a current model of the SMAS proposed by Macchi et al., the original boundaries of which were explored and extended using both histology and gross dissection. As a clearly identifiable structure spanning the lateral and midface, the SMAS in the specimen supported the model proposed by Macchi et al. The three main findings that support the model were the layered morphological appearance of the SMAS, its progression from fibrous to aponeurotic in a lateral to medial direction, and the enveloping of the zygomaticus musculature. Extension beyond the proposed model into the temporal region was observed, but nasal and forehead regions showed no evidence of SMAS, while its presence in the cervical platysma region remained inconclusive. Fascial and soft tissue variability was considerable within facial regions of the examined specimen, helping to explain the debate around the SMAS in the literature.

K+ channel inhibition modulates the biochemical and morphological differentiation of human placental cytotrophoblast cells in vitro.

Maintaining placental syncytiotrophoblast, a specialized multinucleated transport epithelium, is essential for normal human pregnancy. Syncytiotrophoblast continuously renews through differentiation and fusion of cytotrophoblast cells, under paracrine control by syncytiotrophoblast production of human chorionic gonadotropin (hCG). We hypothesized that K(+) channels participate in trophoblast syncytialization and hCG secretion in vitro. Two models of normal-term placenta were used: 1) isolated cytotrophoblast cells and 2) villous tissue in explant culture. Cells and explants were treated with K(+) channel modulators from 18 h, and day 3, onward, respectively. Culture medium was analyzed for hCG, to assess secretion, as well as for lactate dehydrogenase (LDH), to indicate cell/tissue integrity. hCG was also measured in cytotrophoblast cell lysates, indicating cellular production. Syncytialization of cytotrophoblast cells was assessed by immunofluorescent staining of desmosomes and nuclei. Over 18-66 h, mononucleate cells fused to form multinucleated syncytia, accompanied by a 28-fold rise in hCG secretion. 1 mM Ba(2+) stimulated cytotrophoblast cell hCG secretion at 66 h compared with control, whereas 5 mM tetraethylammonium (TEA) inhibited hCG secretion by >90%. 0.1-1 mM 4-aminopyridine (4-AP) reduced cytotrophoblast cell hCG secretion and elevated cellular hCG; without altering cellular integrity or syncytialization. In villous explants, hCG secretion was not altered by 1 mM Ba(2+) but inhibited by 5 mM 4-AP and 5/10 mM TEA, without affecting LDH release. Anandamide, pinacidil, and cromakalim were without effect in either model. In conclusion, 4-AP- and TEA-sensitive K(+) channels (e.g., voltage-gated and Ca(2+)-activated) regulate trophoblast hCG secretion in culture. If these K(+) channels participate in hCG secretion in situ, they may regulate trophoblast turnover in health and disease.

Long-term survival update for high-dose recombinant interleukin-2 in patients with renal cell carcinoma.

PURPOSE: To update response duration and survival data for patients with metastatic renal cell carcinoma treated with high-dose interleukin (IL)-2. PATIENTS AND METHODS: Two hundred fifty-five assessable patients were entered onto seven phase II clinical trials. Recombinant IL-2 600,000 or 720,000 IU/kg was administered by 15-minute intravenous infusion every 8 hours for up to 14 consecutive doses over 5 days as clinically tolerated with maximal support. A second, identical cycle of treatment was scheduled following 5 to 9 days of rest, and courses could be repeated every 6 to 12 weeks in stable or responding patients. All data were updated as of December 1998, with report forms completed by the clinical investigators. These data had last been updated as part of the Food and Drug Administration reporting requirements in 1996. RESULTS: Objective responses previously have been reported in 37 of 255 patients (15%) with 17 complete responses (7%) and 20 partial responses (8%). These data remain unchanged from previous reports. Median response duration for all objective responders remains unchanged at 54 months, but the range now extends from 3 to > 131 months. Median duration for all complete responses has not yet been reached, but was at least 80 months (range, 7- > 131 mo) at the time of this analysis. Median duration for all partial responses remains 20 months (range, 3- > 126 mo). Median survival time for all 255 patients remains 16.3 months, with 10% to 20% of patients estimated to be alive 5 to 10 years after treatment with high-dose IL-2. CONCLUSION: With prolonged follow-up, treatment with high-dose recombinant IL-2 remains extremely effective for a subset of patients with metastatic renal cell carcinoma.

Biosynthesis and processing of epithelial sodium channels in Xenopus oocytes.

The epithelial sodium channel (ENaC) provides the rate-limiting step in the reabsorption of sodium by many epithelia. The number of channels at the cell surface is tightly regulated; most cells express only a few channels. We have examined the biosynthesis and cell surface expression of ENaC in Xenopus oocytes. The subunits of ENaC are readily synthesized in the endoplasmic reticulum, but most of them remain as immature proteins in pre-Golgi compartments, where they are degraded by the proteasomal pathway without apparent ubiquitination. Even when the three subunits, alpha, beta, and gamma, are expressed in the same cell, only a very small fraction of the total channel population leave the endoplasmic reticulum, acquire complex oligosaccharides, and reach the plasma membrane. Overexpression of subunits does not increase the number of channels in the plasma membrane but results in the appearance of cytoplasmic subunits in a form not membrane bound. The data indicate that maturation and assembly of the subunits are slow and inefficient processes, and constitute limiting steps for the expression of functional ENaC channels in the plasma membrane.

The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels.

The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na(+), block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits alpha, beta, and gamma to the kinetic properties of ENaC. Chimeric subunits were constructed between alpha and beta subunits (alpha-beta) and between gamma and beta subunits (gamma-beta). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from alpha subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li(+)/Na(+) selectivity, and single channel conductance were also affected by this segment.

Structure and regulation of amiloride-sensitive sodium channels.

Amiloride-sensitive Na+ channels constitute a new class of proteins known as the ENaC-Deg family of ion channels. All members in this family share a common protein structure but differ in their ion selectivity, their affinity for the blocker amiloride, and in their gating mechanisms. These channels are expressed in many tissues of invertebrate and vertebrate organisms where they serve diverse functions varying from Na+ absorption across epithelia to being the receptors for neurotransmitters in the nervous system. Here, we review progress made during the last years in the characterization, regulation, and cloning of new amiloride-sensitive Na+ channels.

High-dose aldesleukin in renal cell carcinoma: long-term survival update.

PURPOSE: This article updates response duration and survival data for patients with metastatic renal cell carcinoma receiving high-dose recombinant interleukin-2 (rIL-2). PATIENTS AND METHODS: Two hundred fifty-five assessable renal cell carcinoma patients were entered into seven phase II clinical trials. They were administered 600,000 or 720,000 IU/kg rIL-2 by 15-minute intravenous infusion every 8 hours for up to 14 consecutive doses over 5 days as clinically tolerated with maximal support. A second identical cycle of treatment was scheduled following 5 to 9 days of rest, and courses could be repeated every 6 to 12 weeks in stable or responding patients. All data have been updated as of June 1996 with report forms completed by the clinical investigators. RESULTS: Objective overall responses have now been achieved in 37 of 255 patients (15%) with 17 complete (7%) and 20 partial (8%) responses. Median response duration for all objective responders is 54 months with a range of 3 to 107+ months. Median response duration for all complete responses has not been reached, with a range of 7 to 107+ months. Median response duration for all partial responses is 20 months, with a range of 3 to 97+ months. Median survival for all 255 patients was 16.3 months, and 10% to 20% of patients were estimated to be alive 5 to 10 years following treatment. CONCLUSION: Treatment with high-dose rIL-2 is extremely effective for a subset of patients with metastatic renal cell carcinoma.

Inhibition of alphabeta epithelial sodium channels by external protons indicates that the second hydrophobic domain contains structural elements for closing the pore.

We have examined the effect of extracellular protons on the activity of epithelial sodium channels (ENaCs). We found that alphabeta channels, but not alphabetagamma or alphagamma channels, are inhibited by low extracellular pH. External protons induced short and long closed states that markedly decreased the open probability of alphabeta channels. External protons did not change the single-channel conductance or amiloride binding. Analysis of the proton-induced changes on the kinetics of single channels indicates that at least two protons sequentially bind to the extracellular domain at sites that are not in the ion pathway. Conformational changes induced by protonation of those sites are transmitted to the second hydrophobic domain (M2) of the subunits to induce closure of the pore. The results suggest that elements located in the carboxy-terminal half of M2 participate in the gating mechanism of ENaCs.

Phase II trial of recombinant human macrophage colony-stimulating factor in metastatic soft tissue sarcoma.

This Phase II study was undertaken to access the activity of recombinant human macrophage colony stimulating factor (M-CSF) in metastatic soft tissue sarcoma based on the observation of a partial response in a patient with leiomyosarcoma during an earlier Phase I trial. Fifteen patients with metastatic soft tissue sarcoma (seven males and eight females) were entered on the trial between October 1990 and March 1991. Seven of these patients had leiomyosarcoma. One mg/M2 of M-CSF was administered by rapid intravenous infusion every 8 h on days 1-5 and 15-19. Treatment cycles were repeated at 35-day intervals. Patients were evaluated initially for response after the first cycle, and then following alternate cycles. One partial response was observed in a patient with metastatic small bowel leiomyosarcoma (response rate 7%, 95% confidence interval, 0-33%). Two additional patients had stable disease for 10-15 months on study. Four patients had clinically significant bleeding from tumor sites during M-CSF therapy. No evidence of toxicity directly attributable to M-CSF was observed in any patient. Mean monocyte counts increased in patients during the first 20 days of treatment (p = 0.013). At this dose and schedule, meaningful activity of M-CSF in previously treated patients with soft tissue sarcoma could not be demonstrated. However, the activity observed in patients with leiomyosarcoma in this trial and in the previous Phase I study are intriguing. Further studies of M-CSF in previously untreated patients with leiomyosarcoma may be warranted.

Subpopulations of B lymphocytes in germinal centers.

With two new monoclonal antibodies and flow cytometry, we defined three subpopulations among B cells expressing binding sites for peanut agglutinin (i.e., B cells of the germinal center). On monoclonal antibody (5B5) binds globotriaosyl ceramide. The B lymphocytes binding 5B5 have binding sites for peanut agglutinin on the surface and express only small amounts of sIgD and sIgM. When tested against a panel of B cell lines, only Burkitt's lymphoma cells were 5B5+. Moreover, the 5B5+ cells have larger average sizes and a large fraction of proliferating cells. The other monoclonal antibody (HK23) binds a 90,000 protein. Lymphocytes binding HK23 are 5B5- and include T cells and a subpopulation of B cells. In contrast to 5B5+ cells, the HK23+ and peanut agglutinin positive B cells express a large amount of sIgM. These two subpopulations of germinal centers are distinct from the germinal center B cell subpopulation expressing the CD23 (Blast-2) antigen. The CD23+ B cells are 5B5- and express an intermediate level of HK23 antigen. In addition, CD23+ B cells are highly variable in number, whereas the proportions of HK23+ and 5B5+ cells are relatively stable.