Assessment of oncolytic HSV efficacy following increased entry-receptor expression in malignant peripheral nerve sheath tumor cell lines.

Limited expression and distribution of nectin-1, the major herpes simplex virus (HSV) type-1 entry-receptor, within tumors has been proposed as an impediment to oncolytic HSV (oHSV) therapy. To determine whether resistance to oHSVs in malignant peripheral nerve sheath tumors (MPNSTs) was explained by this hypothesis, nectin-1 expression and oHSV viral yields were assessed in a panel of MPNST cell lines using γ134.5-attenuated (Δγ134.5) oHSVs and a γ134.5 wild-type (wt) virus for comparison. Although there was a correlation between nectin-1 levels and viral yields with the wt virus (R=0.75, P =0.03), there was no correlation for Δγ134.5 viruses (G207, R7020 or C101) and a modest trend for the second-generation oHSV C134 (R=0.62, P=0.10). Nectin-1 overexpression in resistant MPNST cell lines did not improve Δγ134.5 oHSV output. While multistep replication assays showed that nectin-1 overexpression improved Δγ134.5 oHSV cell-to-cell spread, it did not confer a sensitive phenotype to resistant cells. Finally, oHSV yields were not improved with increased nectin-1 in vivo. We conclude that nectin-1 expression is not the primary obstacle of productive infection for Δγ134.5 oHSVs in MPNST cell lines. In contrast, viruses that are competent in their ability to counter the antiviral response may derive benefit with higher nectin-1 expression.

Systematic review: outpatient management of acute uncomplicated diverticulitis.

PURPOSE OF REVIEW: Acute uncomplicated diverticulitis is traditionally managed by inpatient admission for bowel rest, intravenous fluids and intravenous antibiotics. In recent years, an increasing number of publications have sought to determine whether care might instead be conducted in the community, with earlier enteral feeding and oral antibiotics. This systematic review evaluates the safety and efficacy of such an ambulatory approach. METHODS: Medline, Embase and Cochrane Library databases were searched. All peer-reviewed studies that investigated the role of ambulatory treatment protocols for acute uncomplicated diverticulitis, either directly or indirectly, were eligible for inclusion. RESULTS: Nine studies were identified as being suitable for inclusion, including one randomised controlled trial, seven prospective cohort studies and one retrospective cohort study. All, except one, employed imaging as part of their diagnostic criteria. There was inconsistency between studies with regards to whether patients with significant co-morbidities were eligible for ambulatory care and whether bowel rest therapy was employed. Neither of these variables influenced outcome. Across all studies, 403 out of a total of 415 (97 %) participants were successfully treated for an episode of acute uncomplicated diverticulitis using an outpatient-type approach. Cost savings ranged from 35.0 to 83.0 %. CONCLUSION: Current evidence suggests that a more progressive, ambulatory-based approach to the majority of cases of acute uncomplicated diverticulitis is justified. Based on this evidence, the authors present a possible outpatient-based treatment algorithm. An appropriately powered randomised controlled trial is now required to determine its safety and efficacy compared to traditional inpatient management.

Enteroviruses, type 1 diabetes and hygiene: a complex relationship.

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the host's insulin-producing beta cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non-obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long-term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host-virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed.

Human Cortex Spheroid with a Functional Blood Brain Barrier for High-Throughput Neurotoxicity Screening and Disease Modeling.

The integral selectivity characteristic of the blood brain barrier (BBB) limits therapeutic options for many neurologic diseases and disorders. Currently, very little is known about the mechanisms that govern the dynamic nature of the BBB. Recent reports have focused on the development and application of human brain organoids developed from neuro-progenitor cells. While these models provide an excellent platform to study the effects of disease and genetic aberrances on brain development, they may not model the microvasculature and BBB of the adult human cortex. To date, most in vitro BBB models utilize endothelial cells, pericytes and astrocytes. We report a 3D spheroid model of the BBB comprising all major cell types, including neurons, microglia and oligodendrocytes, to recapitulate more closely normal human brain tissue. Spheroids show expression of tight junctions, adherens junctions, adherens junction-associated proteins and cell specific markers. Functional assessment using MPTP, MPP+ and mercury chloride indicate charge selectivity through the barrier. Junctional protein distribution was altered under hypoxic conditions. Our spheroid model may have potential applications in drug discovery, disease modeling, neurotoxicity and cytotoxicity testing.

Histone H2A.Z has a conserved function that is distinct from that of the major H2A sequence variants.

Saccharomyces cerevisiae contains three genes that encode members of the histone H2A gene family. The last of these to be discovered, HTZ1 (also known as HTA3), encodes a member of the highly conserved H2A.Z class of histones. Little is known about how its in vivo function compares with that of the better studied genes (HTA1 and HTA2) encoding the two major H2As. We show here that, while the HTZ1 gene encoding H2A.Z is not essential in budding yeast, its disruption results in slow growth and formamide sensitivity. Using plasmid shuffle experiments, we show that the major H2A genes cannot provide the function of HTZ1 and the HTZ1 gene cannot provide the essential function of the genes encoding the major H2As. We also demonstrate for the first time that H2A.Z genes are functionally conserved by showing that the gene encoding the H2A.Z variant of the ciliated protozoan TETRAHYMENA: thermophila is able to rescue the phenotypes associated with disruption of the yeast HTZ1 gene. Thus, the functions of H2A.Z are distinct from those of the major H2As and are highly conserved.

Spectrally Tunable Sources for Advanced Radiometric Applications.

A common radiometric platform for the development of application-specific metrics to quantify the performance of sensors and systems is described. Using this platform, sensor and system performance may be quantified in terms of the accuracy of measurements of standardized sets of source distributions. The prototype platform consists of spectrally programmable light sources that can generate complex spectral distributions in the ultraviolet, visible and short-wave infrared regions for radiometric, photometric and colorimetric applications. In essence, the programmable spectral source is a radiometric platform for advanced instrument characterization and calibration that can also serve as a basis for algorithm testing and instrument comparison.

Cell surface properties associated with malignancy of metastatic large cell lymphoma cells.

The highly malignant and metastatic RAW117-H10 cell line was developed by in vivo selection from the Abelson leukemia virus induced parental RAW117-P lymphoma. In this study we have characterized these cell lines with regard to their expression of lymphocyte and macrophage differentiation antigens, adherence, phagocytic properties, binding of various lectins, binding of antibodies to glycolipid asialo-monoganglioside, and the role of butanol extractable cell surface molecules to determine if any of these cell surface properties are associated with the malignant potential of RAW117-H10 cells. The only major difference in immunological phenotypes between RAW117-P and RAW117-H10 cells was an increased expression of Thy-1 molecules by the latter. However, the highly malignant RAW117-H10 cells bound significantly less concanavalin A, Ricinia communis agglutinin, succinylated wheat germ agglutinin, and particularly anti-asialomonoganglioside than their parental counterpart and were resistant to natural killer cell mediated cytolysis. Removal of butanol extractable cell surface molecules significantly decreased the malignancy of RAW117-H10 cells and increased their susceptibility to natural killer cell mediated cytolysis. The butanol treated RAW117-H10 cells regained high in vivo malignancy when recultured for 3 days to permit regeneration of their cell surface components. The butanol extracted RAW117-H10 cells still expressed high levels of Thy-1 indicating that this most probably represented "inappropriate" antigen expression. Since the expression of lymphocyte differentiation antigens did not correlate with the malignant behavior of the cells, we postulate that these antigenic differences merely represent phenotypic variation. The decreased malignant potential of the butanol treated RAW117-H10 cells did correlate with increased cell surface anti-asialomonoganglioside binding (glycolipid) and increased natural killer cell susceptibility.

Phase I trial of recombinant fusion protein PIXY321 for mobilization of peripheral-blood cells.

PURPOSE: Mobilization of peripheral-blood cells (PBC) with cytokines alone results in rapid hematopoietic recovery and avoids the potential morbidity associated with mobilization by chemotherapy. PIXY321, a fusion protein that consists of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), has enhanced hematopoietic colony-forming activity as compared with individual or equimolar combinations of the two cytokines. A phase I trial of PIXY321 for mobilization of PBC in patients with malignant lymphoma was performed. PATIENTS AND METHODS: Thirteen patients with malignant lymphoma who were eligible for high-dose therapy (HDT) were enrolled onto the trial. All patients were ineligible for autologous bone marrow transplantation due to overt metastatic disease in the marrow or to severe marrow hypocellularity. PIXY321 was administered at three dose levels of 250, 500, and 750 micrograms/m2/d by continuous infusion until completion of PBC collections. Collections were initiated when the WBC count was greater than 10 x 10(9)/L or 4 days after the initiation of PIXY321, whichever came first. Collections were continued until a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg patient weight were obtained. RESULTS: PIXY321 was generally well tolerated. Side effects associated with PIXY321 administration did not exceed grade 2 and included fever (85%), chills/sweats (54%), myalgias (38%), fatigue (31%), nausea/vomiting (31%), headache (31%), edema (23%), and rhinorrhea (23%). The median numbers of colony-forming units-granulocyte/macrophage (CFU-GM) in the graft products for the three dose levels were 0.31, 2.94, and 2.88 x 10(4)/kg, respectively; the median numbers of burst-forming units-erythroid (BFU-e) were 0.20, 6.94, and 12.78 x 10(4)/kg, and the median numbers of CD34+ cells were 2.30, 0.74, and 0.39 x 10(6)/kg. Following transplantation, the median times to an absolute neutrophil count (ANC) > 0.5 x 10(9)/L were 12, 15, and 12 days, respectively, and the median times to platelet transfusion independence were 30, 19, and 15 days. CONCLUSION: PIXY321 can be safely administered and effectively mobilizes PBC in patients with bone marrow defects. PIXY321-mobilized PBC autotransplants result in rapid and sustained hematopoietic recovery.

Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colony-stimulating factor.

PURPOSE: The optimal dose of granulocyte colony-stimulating factor (G-CSF) for mobilization of allogeneic-blood stem cells (AlloBSC) has yet to be determined. As part of a prospective trial, 41 related human leukocyte antigen (HLA)-matched donors had blood cells mobilized with G-CSF at 5 micrograms/kg/d by subcutaneous administration. The purpose of this trial was to monitor adverse effects during G-CSF administration and stem-cell collection, to determine the optimal timing for stem-cell collection, and to determine the cellular composition of stem-cell products following G-CSF administration. PATIENTS AND METHODS: The median donor age was 42 years. Apheresis began on day 4 of G-CSF administration. At least three daily 12-L apheresis collections were performed on each donor. A minimum of 1.0 x 10(6) CD34+ cells/kg (recipient weight) and 8.0 x 10(8) mononuclear cells/kg were collected from each donor. All collections were cryopreserved in 5% dimethyl sulfoxide and 6% hydroxyethyl starch. RESULTS: Toxicities associated with G-CSF administration and the apheresis process included myalgias/arthralgias (83%), headache (44%), fever (27%), and chills (22%). The median baseline platelet count of 242 x 10(4)/ mL decreased to 221, 155, and 119 x 10(6)/mL on days 4, 5, and 6 of G-CSF administration, respectively. Median numbers of CD34+ cells in collections 1, 2, and 3 were 1.99, 2.52, and 3.13 x 10(6)/kg, respectively. The percentage and total number of CD4+, CD8+, and CD56+/CD3- cells remained relatively constant during the three collections. Median total numbers of cells were as follows: CD34+, 7.73 x 10(6)/kg; and lymphocytes, 6.93 x 10(8)/kg. CONCLUSION: Relatively low doses of G-CSF can mobilize sufficient numbers of AlloBSC safely and efficiently.

Hematopoietic recovery after allogeneic blood stem-cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies.

PURPOSE: To compare hematopoietic recovery, duration of hospitalization, and 100-day survival in patients who received allogeneic-blood stem cells (BSC) or conventional allogeneic bone marrow transplantation (BMT). PATIENTS AND METHODS: From December 1994 to August 1995, 21 patients participated in a phase II study of allogeneic BSC transplantation. Cells mobilized with granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg/ d) were collected from human leukocyte antigen (HLA)-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate. G-CSF (10 micrograms/kg/d) was administered posttransplant. The outcomes were compared with 22 identically treated historical patients who received allogeneic BMT. RESULTS: The median infused CD34+ cell and granulocyte-macrophage colony-forming unit (CFU-GM) content were 7.73 x 10(4)/kg and 41.6 x 10(4)/kg, respectively. The median time to a neutrophil count greater than 500/ microL was 11 days after BSC and 16.5 days after BMT (P = .0003). A trend toward faster platelet and RBC recovery after BSC was observed. BSC patients received fewer platelet transfusions: 10 versus 19 (P = .015). The median length of hospitalization was shorter after BSC transplantation: 25 versus 31.5 days (P = .0243). The 100-day survival rates were similar: 83% after BSC and 75% after BMT (P = .3585). The incidence of acute GVHD grade II to IV was 57% and 45% for BSC and BMT, respectively (P = .4654). CONCLUSION: In comparison to BMT, allogeneic BSC transplantation may result in faster hematopoietic recovery, shorter hospital stay, and similar early survival. Whether allogeneic BSC are superior to bone marrow needs to be determined in randomized trials.

Activity of acetyl-n-ser-asp-lys-pro (AcSDKP) on hematopoietic progenitor cells in short-term and long-term murine bone marrow cultures.

The tetrapeptide AcSDKP is a potent inhibitor of hematopoietic stem cell proliferation. Its activity was systematically examined in murine long-term bone marrow cultures (LTBMC) and short-term liquid cultures in the presence or absence of exogenous cytokines. The effects of AcSDKP on the production of granulocyte-macrophage colony-forming cells (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in LTBMCs were examined. AcSDKP was added daily to LTBMCs at various concentrations (10-3--10-16M) for up to 5 weeks. AcSDKP inhibited the entry of progenitor cells into S phase as measured by 3H-thymidine suicide assay and the absolute number of progenitor cells with peak activity at 10-12 M with less activity seen at higher or lower concentrations. The number of nonadherent CFU-GM per LTBMC was unchanged from control values at 1 week of treatment with AcSDKP but was significantly depressed at weeks 3 and 5. In contrast, HPP-CFC progenitor cells were decreased throughout the treatment period, and the numbers of CFU-GM and HPP-CFC in S phase were significantly decreased throughout the treatment period. Maximum S-phase inhibitory activity was observed at 10-12 M AcSDKP. AcSDKP had no effect on the number of adherent CFU-GM or HPP-CFC, cellularity per culture or percent of adherent progenitor cells in S phase. Murine short-term bone marrow cultures were also treated with AcSDKP in the presence or absence of cytokines (interleukin-3 [LI-3], stem cell factor [SCF], or granulocyte colony-stimulating factor [G-CSF]) for various time periods. Dose-response studies showed maximum effects at 10-12 M AcSDKP when no cytokines were added and 10-14 M AcSDKP when exogenous cytokines were added. These studies indicate that the concentration of the tetrapeptide critical in obtaining an effect on hematopoietic progenitor cells, and furthermore, we report that the presence of cytokines or stromal cells also affects the response of progenitor cells to AcSDKP.

Circulating factors may be responsible for murine strain-specific responses to mobilizing cytokines.

OBJECTIVE: To determine if circulating factors influence strain-specific responses to administration of hematopoietic stem-cell mobilizing cytokines, a murine model was employed. METHODS: Plasma aliquots from intact DBA2, Balb/c, and C57Bl/6 mice were injected into intact Balb/c mice prior to delivery of mobilizing cytokines. Control Balb/c mice were injected with mobilizing cytokines alone. Plasma from hemi-body irradiated Balb/c mice, known to inhibit mobilization, was also injected into Balb/c mice. Twenty-four hours later, spleen cells were harvested and assayed for granulocyte-macrophage colony-forming cells (GM-CFC) and high-proliferative-potential colony-forming cells (HPP-CFC). Simultaneously harvested blood aliquots were assayed for CD45(+)/CD34(+) cells using flow cytometric techniques. RESULTS: Mice receiving plasma from any source demonstrated significant inhibition of mobilization of HPP-CFC and GM-CFC to the spleen as compared to mobilized controls; for HPP-CFC, plasma from C57Bl/6 mice was more inhibitory than plasma from Balb/c (p = 0.001) or from DBA2 mice (p = 0.01), while for GM-CFC, plasma from C57Bl/6 mice was more inhibitory than Balb/c plasma but not more inhibitory than DBA2 plasma. Mice injected with plasma from previously irradiated Balb/c mice exhibited the expected HPP-CFC and GM-CFC mobilization inhibition, which was not statistically different from the inhibition seen in animals that received C57Bl/6 plasma. Mobilization of CD34(+)/CD45(+) cells to the blood also appeared to be inhibited by pretreatment with C57Bl/6 plasma, but not DBA2 plasma. CONCLUSION: These data suggest that strain-specific patterns of mobilization may be influenced by a circulating mobilization inhibitor(s).

Cell adhesion molecule expression on CD34+ cells in grafts and time to myeloid and platelet recovery after autologous stem cell transplantation.

The relationship between cell adhesion receptor expression on CD34+ cells in stem cell grafts and the time to neutrophil and platelet recovery after autologous stem cell transplantation (ASCT [n = 25]) was studied with granulocyte/monocyte-colony stimulating factor (GM-CSF)-mobilized peripheral blood stem cells (PBSCs) and steady-state bone marrow (BM) harvests. Cell adhesion receptor expression was analyzed using flow cytometry after CD34+ cell enrichment. Significantly higher expression of L-selectin and CD44, and significantly lower expression of VLA-4, LFA-1, ICAM-1, Sialyl Lewis(x), Sialyl Lewis(A), and Thy-1 were observed on PBSCs compared with BM CD34+ cells. The log of the number of reinfused CD34+ cells, colony forming units granulocyte/macrophage (CFU-GM), and CD34+ cells coexpressing VLA-4, VLA-5, LAF-1, Mac-1, LFA-3, or CD38 but not ICAM-1, Sialyl Lewis(x), Sialyl Lewis(A), or Thy-1 correlated with the time required to reach an absolute neutrophil count (ANC) of > or =0.5 x 10(9)/L. In addition, the log of the number of CD34+ L-selectin+ and CD34+CD44+ cells reinfused after ASCT correlated better with the time required to reach an ANC of > or =0.5 x 10(9)/L than did the log of the number of CD34+ cells or CFU-GM reinfused. The log of the number of reinfused CD34+ cells, CFU-GM, and CD34+ cells coexpressing CD44, L-selectin, VLA-5 Mac-1, or CD38, but not VLA-4, LAF-1, ICAM-1, LAF-3, Sialyl Lewis(X), Sialyl Lewis(A), or Thy-1, correlated with the time required to reach a platelet count of >20 x 10(9)/L. Thus, L-selectin or CD44 may play an important role in the homing of progenitors after ASCT. In addition, the higher proportion of CD34+L-selectin+ or CD34+CD44+ cells in leukapheresis products may provide one explanation for the more rapid hematologic reconstitution observed after PBSC transplantation.

In vivo protective effects of tetrapeptide AcSDKP, with or without granulocyte colony-stimulation factor, on murine progenitor cells after sublethal irradiation.

Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) demonstrated hemato-protective activity in mice after sublethal irradiation (7 GY). Bone marrow interleukin-3 (IL-3)-responsive colony-forming cells (CFC and high proliferative potential colony-forming cells (HPP-CFC) were significantly (p < 0.05) increased by day 10 after irradiation in mice receiving a continuous infusion of 1000 ng/day of AcSDKP compared to irradiated control mice. The maximum protective effect for bone marrow progenitors was achieved when AcSDKP was administered for 3 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days beginning 24 hours before irradiation. Other dosages and schedules in relationship to irradiation were less active. Further, when granulocyte colony-stimulating factor (G-CSF) was administered for 10 days after AcSDKP infusion in irradiated mice, significantly increased numbers of IL-3 responsive CSF-only control mice. In addition, platelets were significantly (p < 0.05) increased in mice receiving AcSDKP and G-CSF on days 18 and 21 after irradiation compared with mice receiving G-CSF alone. We conclude that ACSDKP has a radioprotective effect in vivo for progenitor cells, and that time of initiation and duration of AcSDKP administration relative to irradiation are crucial for these effects. Further, AcSDKP has a significant additive protective effect not only for progenitor cells but also for platelets when given in combination with G-CSF. We suggest that these in vivo observations provide a basis on which to design optimal clinical hypothesis and protocols.

Erythropoietin for mobilization of circulating progenitor cells in patients with previously treated relapsed malignancies.

A trial to determine the usefulness of recombinant human erythropoietin (rhEpo) as a mobilizing cytokine for patients with previously treated relapsed malignancies was performed. An initial peripheral stem cell apheresis collection was conducted during steady-state hematopoiesis for each patient to provide baseline data. rhEpo, 200 U/kg/day, was administered subcutaneously until the last apheresis procedure was completed. Immediately after the fourth daily dose of Epo, apheresis procedures were resumed and continued beyond five collections, when necessary, to accrue a total of 6.5 x 10(8) mononuclear cells (MNCs)/kg. Eight female and four male patients (median age = 44 years) were evaluated. Five to 14 (median = 8) apheresis procedures were performed for each patient. Toxicity attributable to Epo administration was negligible. Mobilization effects, as determined by an increase in the number of colony-forming units granulocyte/macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E) in the apheresis products after Epo administration, were observed in all patients. Nine patients received high-dose chemotherapy and Epo-mobilized peripheral stem cell transplantation (PSCT). Beginning the day of the transplant, GM-CSF was administered until neutrophil recovery was satisfactory. The median time to recover 0.5 x 10(9)/L granulocytes was 16 days after PSCT. Epo appears to have mobilization properties. Further studies are needed to determine the clinical usefulness of Epo as a mobilizing cytokine. The addition of Epo to other mobilizing cytokines may provide increased effectiveness without adding toxicity.

Inappropriate TSH secretion. Paradoxical rise in serum TSH in response to thyroxine therapy.

Serum thytropin (TSH) in a 45-year-old woman suffering from Hashimoto's thyroiditis and hypothyroidism rose over a period of 8 months from 23 to 98 muU/ml while on 0.15 mg of thyroxine daily. A significant pituitary tumor was excluded and the response of other anterior pituitary hormones to provocative stimuli were normal. The TSH response to thyrotropin releasing hormone (TRH) carried out after a year of therapy and while on 0.15 mg T4 was exaggerated and required 100 mug of triiodothyronine (T3) daily in addition to the thyroxine replacement to suppress it. After the completion of the T3 suppression test and while on 0.2 mg thyroxine, serum TSH rose from less than 0.5 to 27 muU/ml when seen last. It is postulated that in this patient there exists a partial selective resistance of the thyrotrophs to T4 and that the paradoxical increase in serum TSH associated with thyroxine therapy results from T4 dependent increase in the synthesis and secretion of endogenous TRH. Other possibilities include an as yet undetected pituitary microadenoma or a pituitary defect in the deiodination of T4.

Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Fatal eosinophilic disease following autologous bone marrow transplantation.

A 15-year-old girl developed massive, fatal eosinophilic disease following autologous bone marrow transplantation (BMT) for Hodgkin's disease (HD). Prior to autologous BMT, the erythrocyte sedimentation rate (ESR) was elevated, with active HD, but eosinophilia was absent. Post-autologous BMT, ESR and peripheral eosinophilia were observed to correlate with respiratory symptoms. Initial evaluation revealed no recurrent tumor, infection or other identifiable etiology. A diagnosis of chronic eosinophilic pneumonia was made following lung biopsy. A complete response was initially achieved with steroid therapy; however, when steroid therapy was tapered, the eosinophilia and elevated ESR recurred with worsening respiratory symptoms. Terminally, severe pulmonary disease developed and recurrent HD was found in lung, lymph nodes and bone marrow. During episodes of eosinophilia, the patient's serum stimulated her bone marrow as well as control marrow to produce predominantly eosinophilic colonies. Eosinophilic colony production was not observed with patient's sera obtained prior to or during autologous BMT or with control sera. This patient died of eosinophilic inflammatory disease following autologous BMT. The etiology of this disease was not definitely identified but appeared to be due to an eosinophilic-stimulating factor which developed after autologous BMT.

Immunologic attributes of cytokine mobilized peripheral blood stem cells and recovery following transplantation.

The immunologic attributes of cytokine mobilized peripheral blood stem cell (PSC) products (n = 52) and the resulting reconstitution of the hematopoietic and immunologic system following autologous transplantation were examined in a consecutive population of non-Hodgkin lymphoma (NHL), or solid tumor patients at the University of Nebraska Medical Center. Granulocyte-monocyte colony stimulating factor (GM-CSF)-mobilized PSC products had a high frequency of monocytes (31%) and bands (15%) as compared to normal peripheral blood (PB) cells. The phenotypic analysis of the mobilized PSC product revealed that they had normal levels of CD4+ cells, an increased frequency of CD8+ cells and a corresponding decrease in the CD4+:CD8+ cell ratio as compared to the peripheral blood leukocytes (PBL) of normal individuals. PSC products also had an increase in CD34+ cells as compared to PB. Natural killer (NK) and T cell activity in the PSC products were also lower than that observed in PB. Post-transplantation there was an accelerated reconstitution of NK-cell function in the PB as compared to T cell function (PHA (phytohemagglutinin) mitogenesis) which did not return to normal by day 100 post-transplantation. We also report for the first time high levels of an irradiation resistant suppressor cell activity in the PSC product and in the PB post-transplantation. There was also a concomitant increase in CD4-, CD8-, TCR alpha/beta+ cells (phenotypic homolog of 'natural suppressor' (NS) cells) in the PB post-transplantation. The number of months of prior chemotherapy correlated with PHA response but the NS activity and frequency of CD4-, CD8- and TCR alpha/beta+ cells did not. Further, cytokine mobilization and apheresis appears to contribute to the loss of PHA responsiveness and the increased levels of suppressor cell activity.

Cell cycle-based purging of lymphoma cells from bone marrow harvests using radioiodinated 5-iodo-2'-deoxyuridine.

Minimal residual disease in lymphoma patients is a major problem in the clinical management of their cancer. High-dose chemotherapy followed by autologous bone marrow transplantation has been used to treat the disease. However, residual lymphoma may be reintroduced along with the marrow if it is present in the bone marrow harvest. In this report we describe results of experiments testing the efficacy of 5-[125I]-iodo-2'-deoxyuridine (125IdU) for purging murine RAW117 large cell lymphoma cells (Joshi et al., Oncology 44, 180-185, 1987; Cancer Res. 47, 3551-3557, 1987) from bone marrow in a relevant animal model. Donor BALB/c mice were injected with murine RAW117 cells and euthanized on day 13, and their bone marrow that had been contaminated with tumor cells was harvested and treated in vitro with 125IdU or nonradioactive 127IdU (control). Nine of 10 mice receiving 127IdU-treated bone marrow contaminated with tumor cells died at an average of 17 days after injection. In comparison, 9 of 10 mice injected with 125IdU-treated bone marrow contaminated with tumor cells were still alive after 82 days. In addition, the 125IdU treatment did not diminish the formation of hematopoietic progenitor cell colonies in normal mouse and human peripheral blood stem cells.