Soluble TNF receptor fusion protein (etanercept) for the treatment of myelodysplastic syndrome: a pilot study.

Blockade of tumor necrosis factor (TNF)alpha by a soluble TNF receptor fusion protein (etanercept; Enbrel) improved in vitro hemopoiesis from the marrow of patients with myelodysplastic syndrome (MDS). Therefore, we enrolled 14 MDS patients (4 RA, 2 RARS, 6 RAEB, 2 CMML), 44-80 (median 60) years old, in a pilot trial. Etanercept, 25 mg, was given twice a week s.c. for 16 weeks (increased to three times a week if no response at 8 weeks). Among 12 evaluable patients, four had rises in hemoglobin by 1-1.5 gm/dl (three) or decreased transfusion requirements (one). Two patients had increased platelet counts (54% and 73%), and two increased neutrophils (63% and 120%). Baseline TNFalpha levels, determined in all patients, did not correlate with responses. Among eight marrows available for sequential in vitro assays, four showed increases in CFU-GM of 1.5- to 5-fold at 8 weeks, whereas three showed 3- to 10-fold decrements relative to baseline. Thus, etanercept treatment resulted in moderate improvements of cytopenias in some patients, while cell counts declined in others. Additional trials are needed to evaluate its clinical efficacy in MDS.

Human and mouse IFN-beta gene therapy exhibits different anti-tumor mechanisms in mouse models.

Previously, we suggested that local human interferon-beta (IFN-beta) gene therapy with replication-defective adenoviral vectors can be an effective cancer treatment. Clinical trials to treat cancers with adenovirus expressing the human IFN-beta gene (IFNB1) has been planned. As a continued effort to explore the mechanisms of action of human IFN-beta gene therapy that can occur in the clinical setting, we tested mouse IFN-beta gene therapy in human xenograft tumors in both ex vivo and in vivo models. Delivery of the mouse IFN-beta gene (Ifnb) caused tumor inhibition; this effect was dependent on the indirect anti-tumor activities of IFN-beta, notably a stimulation of natural killer cells. IFN-beta does not show cross-species activity in its anti-proliferative effect and mouse IFN-beta does not cause as significant an anti-proliferative effect on mouse tumor cells as human IFN-beta causes on human tumor cells. Therefore, we believe that mouse models using either human IFN-beta or mouse IFN-beta gene transfer do not capture all aspects of the action of adenovirus-mediated human IFN-beta gene therapy that may be present in the clinical setting. Due to its multiple mechanisms of action, human IFN-beta gene therapy may be effective in treating human cancers that are either sensitive or resistant to the direct anti-proliferative effect of IFN-beta.

Negative regulators of hemopoiesis and stroma function in patients with myelodysplastic syndrome.

The mechanism that leads to hemopoietic failure in patients with myelodysplastic syndrome (MDS) is not well understood. There is evidence, however, that regulatory molecules such as tumor necrosis factor (TNF)-alpha, Fas (CD95), and Fas-ligand, which negatively affect hemopoiesis by way of apoptosis are upregulated. Here we analyzed marrow samples from 80 patients with MDS in regard to TNF-alpha and Fas-ligand levels and a possible correlation with various disease parameters and risk factors. TNF-alpha levels were elevated in comparison to samples from normal marrow donors, however, no significant correlation with FAB subtype, cytogenetic risk group or score by the International Prognostic Scoring System (IPSS) was observed. However, there was an inverse correlation between the cytogenetic risk category (low, intermediate, high) and levels of soluble Fas-ligand. The major source of TNF-alpha were mononuclear (non-stromal) cells which appeared to produce TNF-alpha at maximum levels. Limiting dilution analysis of CD34+ precursor cells showed that individually assayed cells, removed from companion cells that presumably provided negative signals such as TNF-alpha or Fas-ligand, were able to generate progressively increasing numbers of colonies. Stromal layers derived from MDS marrow did not have an inhibitory effect. In fact, higher colony numbers were obtained from both normal and MDS marrow derived hemopoietic precursors propagated on irradiated stromal layers from MDS marrow than on stromal layers from normal marrow. These results show that substantial numbers of normal hemopoietic precursors persist in MDS marrow. However, differentiation into mature cells is inhibited by negative signals originating from accessory or abnormal hemopoietic precursors in the non-adherent marrow fraction.

Recognition of major histocompatibility complex class II antigens by two anti-HLA-DR monoclonal antibodies on canine marrow cells correlates with effects on in vitro and in vivo hematopoiesis.

BACKGROUND: The role of major histocompatibility complex class II antigens in hematopoiesis is not well defined. We have shown that in vitro depletion of HLA-DR+ cells from canine marrow (e.g., by anti-HLA-DR monoclonal antibody [mAb] H81.9 and complement) prevents hematopoietic recovery. In vivo administration of the same mAb H81.9 after transplantation of unmanipulated autologous marrow results in graft failure. In vitro mAb H81.9 inhibited colony formation from short-term and long-term marrow cultures. METHODS AND RESULTS: We investigated the effect of another mAb, Ca1.41, which also recognizes nonpolymorphic determinants on human (HLA-DR) and canine major histocompatibility complex class II antigens but is reactive with a narrower spectrum of cells in both canine peripheral blood and marrow than mAb H81.9 (and other anti-HLA-DR mAbs). In contrast to all other anti-HLA-DR mAbs tested, Ca1.41 did not interfere with colony formation in short-term or long-term marrow cultures and spared a population of small mononuclear cells with low forward light scatter that was eliminated via apoptosis by exposure to mAb H81.9. These target cells included lymphocytes and CD34+ hemopoietic precursors that expressed MHC class II molecules as determined by mAb H81.9 but not by mAb Ca1.41. In addition, transmembrane signaling and up-regulation of interleukin-1beta mRNA occurred with mAb H81.9 but not with Ca1.41. Transplantation of autologous marrow treated in vitro cytolytically with mAb Ca1.41 allowed for complete hematopoietic reconstitution. Further, in vivo administration of Ca1.41 posttransplant did not lead to autologous graft failure as had been observed with mAb H81.9. CONCLUSIONS: These results support the notion that major histocompatibility complex class II is expressed on early hematopoietic precursor cells but recognition is dependent upon the mAb used. Preliminary studies show that mAb H81.9 triggered transmembrane signaling, resulting in up-regulation of interleukin-1beta and apoptosis, although mAb Ca1.41 did not. The fact that Ca1.41 binding was modified in the presence of exogenous invariant chain-derived peptide suggests that both binding and signaling are peptide dependent.

Stability study on specimens mailed to a state laboratory and tested with the Gen-Probe PACE 2 assay for chlamydia.

BACKGROUND: Although specimen collection is acknowledged to be a critical factor in the testing of chlamydia, rarely do studies examine the effects of specimen transport on laboratory results. GOAL: To compare the results on specimens shipped in a controlled environment with duplicate specimens exposed to environmental conditions such as heat or extended time in transit. STUDY DESIGN: Duplicate specimens were collected from 1,017 women tested at South Carolina public health clinics. One specimen from each woman was shipped by courier and the other by U.S. mail. The results, swab collected first, method of transport, and temperature during shipment were compared for each set of specimens. Specimens were tested with the Gen-Probe PACE 2 test. RESULTS AND CONCLUSIONS: Mailed specimens were exposed to longer transport times and elevated temperatures; despite this, 99% of the results using courier specimens agreed with the mailed specimen results. Eighty-eight women tested positive and 891 women tested negative for chlamydia on both specimens. When the 11 specimens with discrepant results were retested by polymerase chain reaction, 10 were positive for chlamydia, with 9 concurring with the mailed specimen results. Results of specimens in this study were not adversely affected by heat and extended transit times when transported by U.S. Mail.

A role for tumour necrosis factor-alpha, Fas and Fas-Ligand in marrow failure associated with myelodysplastic syndrome.

Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), Fas-Ligand (Fas-L) and TNF-alpha factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34-positive cells and on cells co-expressing HLA-DR. In addition, Fas-L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas-L mRNAs as well as mRNA for TNF-alpha, known to increase Fas expression in normal marrow, were up-regulated in patients with MDS. TNF-alpha protein and sTNF-R1 levels in marrow plasma were higher in MDS patients than in controls (P<0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF-alpha levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-T) (P=0.043). Conversely, the proportion of Fas-L-positive cells was lowest in patients with RA (P=0.037). In marrow cultures, Fas-Ig, rhuTNFR:Fc or anti-TNF-alpha antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P<0.001, P<0.003, P<0.001, respectively). These results show significant dysregulation in the expression of TNF-alpha, Fas and Fas-L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.

HLA-DR-triggered inhibition of hemopoiesis involves Fas/Fas ligand interactions and is prevented by c-kit ligand.

The function of MHC class II (HLA-DR) Ags in hemopoiesis is not well defined. Here we investigated the effect of anti-HLA-DR mAb H81.9 on human marrow cells. mAb H81.9 inhibited colony formation from purified CD34+ marrow cells in long term culture-initiating cell assays. Inhibition was prevented, however, if c-kit ligand (stem cell factor (SCF)) was added to cultures concurrently with H81.9. DNA histograms from cultured untreated marrow mononuclear cells showed 2+/-1.2% apoptotic nuclei, whereas 14.1+/-5.4% were apoptotic after 12-h exposure to mAb H81.9. The apoptotic peak was reduced to 1.2+/-0.8% when SCF was added to cultures concurrently with mAb H81.9. The addition of Fas-Ig, a fusion protein that neutralizes Fas ligand (Fas-L), also prevented mAb H81.9-induced apoptosis. As determined by terminal deoxynucleotidyl transferase assays, agonistic anti-Fas mAb also induced apoptosis (in 13+/-4% of cells), and combined treatment with anti-Fas mAb and H81.9 was additive (27% apoptotic nuclei). The extent of apoptosis induced by anti-Fas mAb was significantly reduced by SCF. After H81.9 exposure, Fas was up-regulated on CD34+ cells, and Fas-L expression was 2.5-fold higher than in controls or CD34- cells, particularly within a small cell window with low orthogonal scatter (lymphocyte gate). These findings show that HLA-DR-mediated signals inhibit hemopoiesis in human marrow by a mechanism involving Fas/Fas-L-dependent signals that are blocked by c-kit ligand. These data suggest a possible role for MHC class II molecules in the regulation of hemopoiesis.

HLA-DR-mediated signals for hematopoiesis and induction of apoptosis involve but are not limited to a nitric oxide pathway.

Cross-linking of major histocompatibility complex (MHC) class II antigens by anti-HLA-DR monoclonal antibody (MoAb; H81.9; IgG2a) results in inhibition of hematopoiesis in canine and human models. Inhibition of hematopoiesis is associated with apoptosis in a proportion of marrow cells. Since in murine macrophages class II cross-linking triggers nitric oxide (NO) production, and NO is thought to affect regulation of hematopoiesis, we investigated whether NO was involved in our models. In murine J774 monocytes/macrophages, MoAb H81.9 did induce NO. NO production was blocked by N(G)-monomethyl-L-arginine (NMMA), an inhibitor of NO synthase (NOS), and by the antioxidant N-acetylcysteine (NAC). In human and canine long-term marrow cultures (LTMCs) and in enriched marrow monocytes, however, no measurable increase in NO production was noted after H81.9 exposure. Nevertheless, NAC protected LTMCs against H81.9 induced inhibition of hematopoiesis. Therefore, we determined the effect of an exogenous NO donator, sin-1 (3-morpholinosydnonimine), on canine and human LTMCs and on apoptosis. Sin-1 at concentrations > or =100 microg/mL inhibited LTMCs and induced apoptosis; at low concentrations (1 microg/mL), however, sin-1 stimulated the generation of colony-forming unit granulocyte-macrophage. Combined treatment with sin-1 at 100 microg/mL and MoAb H81.9 resulted in profound inhibition of hematopoiesis in both canine and human LTMCs, and had an additive effect on apoptosis. At 1 microg/mL sin-1 counteracted the effect of H81.9 on hematopoiesis. The effect of sin-1 on apoptosis and hematopoiesis in LTMC was largely prevented by NAC. These results are consistent with the hypothesis that HLA-DR mediated apoptosis and inhibition of hematopoiesis involve oxidative stress. However, the biphasic response of hematopoiesis to sin-1 suggests a complex regulatory network possibly related to differences in NO sensitivity of distinct subpopulations of cells. Signals in addition to NO appear to be involved in the effect of anti-HLA-DR MoAb on hematopoiesis.

Neutralization of stem cell factor in vitro and in vivo: dose-dependent inhibition of stromal cells and induction of granulocyte/monocyte differentiation.

Stem cell factor (SCF), also termed mast cell growth factor or c-kit ligand, plays a central role in the regulation of hematopoiesis and maintenance of viability of hematopoietic cells. We used a new murine monoclonal antibody (MAb) specific for canine SCF to further dissect the role of SCF in vitro and in vivo. This neutralizing MAb, RG7.6 (IgG1), recognizes the soluble form as well as the membrane-bound form of SCF on marrow-derived stromal cells. Treatment of long-term bone marrow cultures (LTMC) with RG7.6 suppressed or stimulated the production of CFU-GM, depending on the MAb concentration and the time of addition to cultures. At concentrations of 0.1-10 micrograms/ml given on the day of recharge of the LTMC, RG7.6 resulted in sustained suppression of CFU-GM grown from nonadherent cells. In contrast, higher doses of RG7.6 (20-100 micrograms/ml) led to a two- to threefold increase in CFU-GM formation from nonadherent cells after 3 days of RG7.6 exposure; after longer RG7.6 exposure there was a rapid decline in the number of CFU-GM. The early increase of CFU-GM was even more distinct when RG7.6 addition to LTMCs was delayed until 1 day before cells were plated for the CFU-GM assay. The early increase of CFU-GMs in the presence of high-dose RG7.6 was mimicked by the addition of granulocyte colony-stimulating factor (G-CSF) to cultures containing suboptimal concentrations of RG7.6, suggesting the possibility that the "positive" response to high-dose RG7.6 was due to an overriding effect of other growth factors, e.g., G-CSF. In stromal cells expressing the membrane-bound form of SCF, the presence of MAb RG7.6, even at low concentrations, interfered with thymidine uptake and proliferation. RG7.6 was also tested in vivo. RG7.6 was given intravenously immediately (days 0-4) after total body irradiation and autologous bone marrow transplantation, and granulocyte counts were followed. The post-irradiation nadir of peripheral blood granulocytes was indistinguishable from controls at low doses of RG7.6 but became more shallow as higher doses of RG7.6 were infused, again suggesting a positive effect on granulocyte differentiation. Thus, the SCF-specific MAb appears to interfere with both stromal and hematopoietic cell function. While only inhibition was observed at lower concentrations, a transient increase in granulocyte production was seen at higher MAb concentrations.

Preparative separation of pyrrolizidine alkaloids by high-speed counter-current chromatography.

We have applied a high-speed counter-current chromatography (CCC) technique to the separation and purification of pyrrolizidine alkaloids from Amsinckia tessellata, Symphytum spp., Trichodesma incanum (Boraginaceae), and Senecio douglasii var. longilobus (Asteraceae). Alkaloidal fractions were separated in a solvent system composed of a chloroform mobile phase and 0.2 M potassium phosphate buffer, of an optimum pH, as the stationary phase. Up to 800 mg of sample could be successfully separated in a single run, with excellent resolution of alkaloids. Lycopsamine and several of its acetylated derivatives were resolved from alkaloidal fractions of Amsinckia and Symphytum. However, diastereomeric pairs such as 7-acetyl-lycopsamine and 7-acetyl-intermedine, could not be separated. The presence of diastereoisomers was determined by gas chromatography-mass spectrometry. Trichodesma contained predominantly trichodesmine, which was resolved from a small quantity of incanine. we report the electron impact mass spectrum of incanine for the first time. Resolving power of CCC was sufficient to separate the closely related alkaloids senecionine and seneciphylline from Senecio, in addition to florosenine and retrorsine, Pyrrolizidine alkaloid compositions of the four species, determined by mass spectral techniques, were consistent with literature, except for the lack of riddelliine and the presence of the otonecine-based florosenine in Senecio douglasii var. longilobus.

Major histocompatibility complex class II-mediated inhibition of hematopoiesis in long-term marrow cultures involves apoptosis and is prevented by c-kit ligand.

Expression of major histocompatibility complex (MHC) class II molecules is developmentally regulated and lineage dependent. Their role in hematopoiesis is not well defined. Previous studies in a canine model showed that dogs given 920 cGy of total body irradiation, transplanted with autologous marrow, and treated with anti-MHC class II monoclonal antibody (MoAb) immediately posttransplant experienced only a transient granulocyte recovery that was followed by graft failure. In the present study, the effect of anti-MHC class II MoAbs on canine in vitro hematopoiesis was investigated. Anti-MHC class II MoAb H81.9 or B1F6 (both recognizing nonpolymorphic determinants) had no inhibitory effect when added directly to colony-forming unit-granulocyte-macrophage (CFU-GM) grown in agar. However, the addition of intact MoAb or as F(ab')2 fragments to long-term marrow cultures (LTMCs) resulted in a dose-dependent inhibition of the generation of CFU-GM among nonadherent cells. Inhibition was most profound with MoAb added at the time of initiation of culture. However, even if MoAb was added 3 weeks after recharging LTMCs, CFU-GM generation rapidly decreased. In addition, the number of adherent cells in LTMCs decreased; predominantly fibroblast-like cells with prominent cytoplasmic vesiculation remained. Acridine orange/ethidium bromide staining and TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) tests showed an increase in the proportion of apoptotic cells in both the nonadherent and adherent compartments. Binding of anti-MHC class II MoAb to unfractionated marrow cells resulted in an increase in free (Ca2+)i; no changes in tyrosine phosphorylation pattern were observed. The addition of stem cell factor (SCF), but not granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, to LTMCs prevented apoptosis, and the generation of CFU-GM was indistinguishable from controls. Similarly, a supportive adherent layer was maintained. Thus, anti-MHC class II MoAbs interfere with hematopoiesis both in vitro and in vivo. The mechanism involves programmed cell death in subpopulations of adherent and nonadherent cells. Inhibition of hematopoiesis is abrogated by exogenous SCF.

Ultrastructural localization of stem cell factor in canine marrow-derived stromal cells.

Stromal cell lines derived from canine long-term bone marrow cultures (LTBMC) were characterized regarding the expression of growth factors and especially the localization of stem cell factor (SCF) (c-kit ligand). One cell line (DO64) was immortalized by transformation with a retroviral vector containing the open reading frames (ORFs) E6 and E7 of the human papilloma virus type 16 (HPV-16). Transfection did not change cellular characteristics but rendered the cell line more independent from culture conditions. The transformed line DO64 consisted mainly of fibroblast-like cells. In addition, some cells showed endothelial and some smooth-muscle cell features. Stromal cells expressed a broad spectrum of surface markers, including low levels of major histocompatibility-complex (MHC) class-II antigens. A new murine monoclonal antibody (MAb), RG7.6 (IgG1), specific for canine SCF, recognized the majority of fibroblast-like stromal cells. The staining pattern for SCF showed perinuclear and intracytoplasmic dense areas. Immunoelectron microscopy revealed the localization of SCF in secretory vesicles, the perivesicular cytoplasm, and bound to the cytoplasmatic membrane. RNA analysis showed that stromal cells transcribed, in addition to SCF, messages for granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF (GM-CSF), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta). In summary, we have established and characterized canine marrow-derived stromal cell lines, and using the new MAb RG7.6, we have localized SCF to cytoplasmatic vesicles as well as the membrane of stromal cells.

Major histocompatibility complex class II expression is required for posttransplant immunological but not hemopoietic reconstitution in mice.

We had previously shown in a canine model that the administration of anti-MHC class II monoclonal antibody (MAB) immediately after autologous marrow transplantation prevented hemopoietic reconstitution. Since MHC class II expression in mice differs from that in dogs we were interested in determining the effect of MHC class II manipulation on posttransplant hemopoietic and immunological recovery in mice. Three murine models including MHC class II knock-out mice were studied. BALB/c mice (I-E+, I-A+) given anti-MHC class II MAB H81.9 (anti-I-E; 1 mg/kg/day, days 0-4) after TBI and infusion of syngeneic marrow or infused with anti-I-E purged marrow both showed normal hemopoietic reconstitution. Similarly, C57B1/6 mice (I-E-, I-A+) transplanted with M5/114 (anti-I-A) purged marrow recovered normal hemopoiesis. MHC class II knock-out (C2D) mice, which lack class II completely, also recovered normal hemopoiesis after TBI and transplantation with either normal or class II-deficient (C2D) marrow, although the kinetics of platelet recovery as determined by megakaryocytopoiesis and platelet counts on day 14 were slightly delayed. C57B1/6 mice transplanted with C2D marrow recovered normally. Immunologic recovery, however, was abnormal both in C2D recipients and in normal mice transplanted with C2D marrow: While CD8+ T lymphocytes recovered normally, no (or only very few) CD4+ T cells were identified posttransplant. Treatment of normal mice with anti-MHC class II MAB in vivo or transplantation of MHC class II-purged marrow, however, did not interfere with complete immunological recovery, although T cell maturation was slightly delayed. Thus, complete immunological reconstitution requires the expression of MHC class II on marrow-derived precursor cells, while the expression of MHC class II antigens is not a requirement for hemopoietic reconstitution in mice.

Anti-IL-4 monoclonal antibody and IFN-gamma administration retards development of immune dysfunction and cytokine dysregulation during murine AIDS.

This study was designed to determine if administration of anti-interleukin-4 (IL-4) monoclonal antibody (mAb), interferon-gamma (IFN-gamma) and their combination after LP-BM5 retrovirus infection of female C57BL/6 mice would prevent retrovirus-induction of immunosuppression and cytokine dysregulation. Splenic natural killer (NK) cell activity, T- and B-cell proliferation, and T-helper type 1 (Th1) and Th2 cytokine (IL-2, IFN-gamma, IL-5 and IL-10) and monokine [IL-6 and tumour necrosis factor-alpha (TNF-alpha)] secretions were monitored, as they are usually altered dramatically after murine retrovirus infection. Administration of IFN-gamma and anti-IL-4 significantly prevented retrovirus-induced suppression of splenic NK cell activity, and splenic T- and B-cell proliferation. They also significantly slowed retrovirus-induced elevation of Th2 cytokine (IL-5 and IL-10) release and monokine (IL-6 and TNF-alpha) secretion by splenocytes. They prevented the loss of Th1 cytokine (IL-2 and IFN-gamma) release by splenocytes, and alleviated splenomegaly and hypergammaglobulinemia, precursor signs of development of acquired immune deficiency syndrome (AIDS). These findings could provide insight into the roles of immunomodulator in AIDS treatment as well as the mechanisms by which retrovirus infection induces cytokine dysregulation, facilitating immunodeficiencies in AIDS.

Rescue from anti-MHC class II antibody-mediated marrow graft failure by c-kit ligand.

Dogs given 920 cGy of total body irradiation (TBI) followed by autologous marrow infusion uniformly achieve sustained hematopoietic reconstitution. We have previously shown that administration of the anti-MHC class II monoclonal antibody (MoAb) H81.98.21 (IgG2a) at 0.6 mg/kg/d immediately after transplantation results in delayed graft failure. A second noncrossblocking anti-MHC class II MoAb, B1F6, of the same isotype, at the same dose, did not interfere with sustained engraftment, suggesting that the observed effect was epitope dependent. Although higher concentrations of B1F6 were required, in the present study both MoAbs interfered with the propagation of long-term marrow cultures. When MoAb B1F6 was given in vivo at 1.2 mg/kg/d, ie, twice the dose used previously, dogs so treated also developed delayed marrow graft failure. Marrow failure with either MoAb involved myeloid, erythroid, and megakaryocytic lineages. Administration of recombinant canine c-kit ligand/stem cell factor (SCF) for 7 or 21 days posttransplant resulted in reversal of graft failure. Although the short course did induce a broad transient early peak of granulocytes, the longer course of SCF was accompanied by earlier sustained recovery than the short course. In conclusion, therefore, marrow graft failure induced by anti-MHC class II MoAb does not appear to be epitope dependent, involves all hematopoietic lineages, and is overcome by the administration of c-kit ligand.

Value of day 100 screening studies for predicting the development of chronic graft-versus-host disease after allogeneic bone marrow transplantation.

We prospectively evaluated 169 patients with a number of screening studies performed between 71 to 121 days after allogeneic marrow transplantation to detect the development of chronic graft-versus-host disease (GVHD). Group 1 patients (n = 78) were asymptomatic and had normal physical examinations at the time of screening and, with a minimum of 8 years follow-up, have not developed chronic GVHD. Group 2 patients (n = 38) had signs and symptoms of chronic GVHD at time of testing. Group 3 patients (n = 53) were similar to those in group 1 in having no clinically evident GVHD at the time of testing, but later developed clinical chronic GVHD. Using time to an event analysis, we compared patients in groups 1 and 3 to determine which of 17 clinical and laboratory factors evaluated at screening accurately predicted the development of subsequent chronic GVHD. Multivariate analyses showed several factors to have independent predictive value. In the first model, results of oral biopsies were excluded since these were done only in one half of the patients. Predictive factors in this analysis included: (1) histologic findings of GVHD on skin biopsy, relative risk 3.23 (95% confidence interval 1.75 to 5.94), P = .0002; and (2) history of grade II through IV acute GVHD, relative risk 3.12 (95% confidence interval 1.72 to 5.64), P = .0002. When oral biopsy results were included in the second model, independent risk factors included: (1) histologic findings of GVHD on skin biopsy, relative risk 5.96 (95% confidence interval 1.95 to 18.19), P = .0017; and (2) low numbers of immunoglobulin A (IgA)-bearing plasma cells detected by direct immunofluorescence in salivary gland areas on oral biopsy, relative risk 11.53 (95% confidence interval 2.51 to 52.03), P = .0017. Our study demonstrates the value of day 100 screening studies for predicting subsequent development of clinical chronic GVHD.

Immediate and delayed neurotoxicity after mechlorethamine preparation for bone marrow transplantation.

Twenty-four patients, including two with aplastic anemia and 22 with malignancy, underwent marrow transplantation after preparation with mechlorethamine, 0.3 to 2.0 mg/kg body weight. Fourteen of the 21 neurologically evaluable recipients developed immediate neurotoxicity a median of 4 days after treatment (range, 0 to 34 days). Confusion and disorientation were observed in six patients, headache in six, hallucinations n four, lethargy in four, tremors in three, paraplegia in one, seizure in one, and vertigo in one. Whereas acute symptoms cleared in 11 patients, three remained symptomatic until death. Twelve evaluable patients survived more than 60 days; all six with previous acute toxicity subsequently developed delayed onset of new neurologic findings (personality change, confusion, seizure, diplopia, or dementia) a median of 169 days (range, 70 to 248 days) after treatment. Cerebrospinal fluid analysis was usually normal but cerebral computed tomographic scans showed ventricular enlargement and electroencephalograms showed diffuse slowing. Postmortem histologic examination of brain showed neuronal degenerative changes with increased vascularity, gliosis, and perivascular fibrosis. Neurotoxicity appeared to increase with age and mechlorethamine dose and was commoner in patients given additional procarbazine or cyclophosphamide.