John P. Chute

A DNA vaccine encoding the 42 kDa C-terminus of merozoite surface protein 1 of Plasmodium falciparum induces antibody, interferon-γ and cytotoxic T cell responses in rhesus monkeys: immuno-stimulatory effects of granulocyte macrophage-colony stimulating factor

We have constructed a DNA plasmid vaccine encoding the C-terminal 42-kDa region of the merozoite surface protein 1 (pMSP1(42)) from the 3D7 strain of Plasmodium falciparum (Pf3D7). This plasmid expressed recombinant MSP1(42) after in vitro transfection in mouse VM92 cells. Rhesus monkeys immunized with pMSP1(42) produced antibodies reactive with Pf3D7 infected erythrocytes by IFAT, and by ELISA against yeast produced MSP1(19) (yMSP1(19)). Immunization also induced antigen specific T cell responses as measured by interferon-gamma production, and by classical CTL chromium release assays. In addition, immunization with pMSP1(42) primed animals for an enhanced antibody response to a subsequent boost with the recombinant yMSP1(19). We also evaluated Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) as an adjuvant for pMSP1(42.) We tested both rhesus GM-CSF expressed from a DNA plasmid, and E. coli produced recombinant human GM-CSF. Plasmids encoding rhesus GM-CSF (prhGM-CSF) and human GM-CSF (phuGM-CSF) were constructed; these plasmids expressed bio-active recombinant GMCSF. Co-immunization with a mixture of prhGM-CSF and pMSP1(42) induced higher specific antibody responses after the first dose of plasmid, but after three doses of DNA monkeys immunized with or without prhGM-CSF had the same final antibody titers and T cell responses. In comparison, rhuGM-CSF protein did not lead to accelerated antibody production after the first DNA dose. However, antibody titers were maintained at a slightly higher level in monkeys receiving GM-CSF protein, and they had a higher response to boosting with recombinant MSP1(19). The GM-CSF plasmid or protein appears to be less potent as an adjuvant in rhesus monkeys than each is in mice, and more work is needed to determine if GM-CSF can be a useful adjuvant in DNA vaccination of primates.

Outcome of Patients With Small-Cell Lung Cancer During 20 Years of Clinical Research at the US National Cancer Institute

OBJECTIVE To determine the outcome of all patients with small-cell lung cancer (SCLC) treated at the US National Cancer Institute between April 1973 and April 1993. DESIGN We retrospectively analyzed a series of 594 consecutive patients with SCLC treated at a single institution during a 20-year period to assess changes in duration of survival and toxicity related to various treatment regimens. MATERIAL AND METHODS For analysis, patients were grouped by decade, and the duration of survival of patients with limited- and extensive-stage SCLC was examined to assess whether patients treated during the first decade of the study (1973 through 1983), when cyclophosphamide-based regimens were used, had different outcomes than those treated during the second decade (1983 through 1993), when cisplatin-based regimens were used. Patients had a minimal follow-up of 2 years. RESULTS No significant difference was found in the survival of patients with limited- or extensive-stage SCLC treated during the second decade in comparison with during the first decade of the study. Among patients with extensive-stage SCLC, performance status 3 or 4 and metastatic lesions of the liver and central nervous system had a significant adverse effect on survival in both the first and the second decade. Among patients with limited-stage disease, performance status 3 or 4 had the most significant adverse influence on survival during the overall study period. In addition, in a multivariate analysis, etoposide-cisplatin plus twice-daily chest radiotherapy was significantly associated with prolonged survival (P = 0.003). CONCLUSION We noted no significant change in the duration of survival of patients with either limited-or extensive-stage SCLC treated at our institution during a 20-year period. A multivariate analysis showed that patients with limited-stage SCLC given a cisplatin-based regimen plus chest radiotherapy lived modestly longer than similar patients given cyclophosphamide regimens at our institution. No evidence was found of changes in pretreatment factors that would affect survival.

A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures

Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34+CD38- cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34+CD38- cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34+CD38- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34+ cells and the CD34+CD38- subset before and after ex vivo expansion on PMVECs. Purified human CD34+ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34+ population and the CD34+CD38- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34+CD38+ subset were found to be in G1, and 51% were in G2/S/M phase of the cell cycle. More remarkably, 53% of the CD34+CD38- subset were in G1, and 17% were in G2/S/M phase after 7 days of PMVEC coculture. In contrast, only 22% of the CD34+CD38- subset remaining after 7 days of stroma-free culture were in G1, and 6% were in G2/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin) increased after ex vivo expansion. In contrast, CD34+ cells expanded on stroma-free cultures showed lower and more variable expression of CD62L and CD15s. These findings demonstrate that the primitive CD34+CD38- subset of marrow progenitor cells can be induced to enter cell cycle and can be significantly expanded ex vivo on a hematopoietic supportive microenvironment (PMVECs) while preserving the expression of cell adhesion molecules that may be important in stem cell homing and engraftment.

A Metabolic Study of Patients with Lung Cancer and Hyponatremia of Malignancy

Purpose: One-third of patients with lung cancer and hyponatremia have no evidence of ectopic arginine vasopressin (AVP) production and the cause of their hyponatremia is not conclusively established. We sought to distinguish patients with hyponatremia caused by elevated AVP versus those with ectopic atrial natriuretic peptide (ANP) via this detailed metabolic study. Experimental Design: We enrolled 24 patients recently diagnosed with lung cancer in a metabolic study in which patients were placed on sodium and fluid restriction for 4 days. Serum electrolytes, osmolality, urine electrolytes and osmolality, plasma AVP, ANP, aldosterone, urinary cyclic AMP and cyclic guanosine 3′,5′-monophosphate were measured daily and tumor tissue was obtained to measure ectopic hormone production. We attempted to characterize the pathophysiology of hyponatremia caused by ectopic ANP production in patients with small cell lung cancer (SCLC) and to determine its effect on the aldosterone axis. Results: Seven of the nine patients with SCLC presented with hyponatremia and three had elevated ANP levels at presentation without elevation of AVP. All three patients who presented with hyponatremia and elevated ANP showed a decline in serum sodium following fluid restriction, whereas two patients with SCLC and elevated AVP had normalized serum sodium levels. The combination of hyponatremia and elevated ANP was associated with a persistent natriuresis and inappropriately low aldosterone levels despite sodium restriction, suggesting ANP suppression of the aldosterone axis. Conclusions: Management of patients with hyponatremia and SCLC should be guided by the knowledge that some patients with SCLC have ectopic production of ANP as the cause of their hyponatremia.

Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells.

Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.

Hematopoietic cytokine production by human brain endothelial cells (Hubec) Is associated with the ex vivo expansion of CD34+CD38− cells

Abstract We have recently demonstrated that primary human brain endothelial cells (HUBEC) supplemented with GMCSF+IL-3+ IL-6+SCF+Flt-3 ligand support a 400-fold increase in CD34 + CD38 − cells in 7 day cultures. In order to identify soluble factors which might contribute to this hematopoietic effect, we performed ELISAs on supernatant collected from HUBEC monolayers and compared this with supernatant from human umbilical vein endothelial cells (HUVEC) which do not support the expansion of CD34 + CD38 − cells. In unconcentrated HUBEC supernatant, we detected high levels of VEGF (1951 pg/mL±561), IL-11 (1832 pg/mL±142), IL-6 (2625 pg/mL±39), and SCF (172 pg/mL±2), and surprisingly low or nonmeasurable levels of GMCSF (nm), GCSF (22 pg/mL±1), Epo (nm), IL-3 (nm), and Flt-3 ligand (18 pg/mL±0.1). In contrast, supernatant collected from HUVEC contained no measurable VEGF or IL-11, but had very high levels of GCSF (2278 pg/mL±76) and PDGF (2369 pg/mL±235). In addition, HUVEC supernatant contained IL-6 (767 pg/mL±21), GMCSF (67 pg/mL±3), and SCF (340 pg/ml±6), but Flt-3 ligand and Epo were not detectable. In summary, HUBEC secrete high levels of IL-11 and VEGF which have both been identified to induce the proliferation of hematopoietic stem cells (HSC) in vitro. In contrast, HUVEC produce neither IL-11 nor VEGF but do secrete high levels of GCSF which likely contributes to the differentiation of progenitor cells in vitro. We are currently studying the capacity of HUBEC supernatant to induce HSC proliferation in the absence of cell-to-cell contact and the contribution of known or novel cytokines toward this effect.