Neoadjuvant management of locally advanced pancreatic ductal adenocarcinoma - Heading towards a promising change in treatment paradigm.

Traditional chemotherapy-based adjuvant therapies for locally advanced pancreatic ductal adenocarcinoma (PDAC) have been associated with poor clinical outcomes driven partly by its complex anatomy and molecular heterogeneity. Treatment for PDAC is challenged by presence of a dense tumour microenvironment involving an interplay of multiple tumoural and stromal components which promote metastatic oncogenic behaviour. PDAC also involves aberrations in multiple signalling pathways with paucity of treatment options against the most common mutations including KRAS, TP53, CDKN2A andSMAD4. However, recent discovery of new mechanisms implicated in pancreatic carcinogenesis have led to identification of promising mechanistic therapeutic targets such as NET1 and ULK1. Early evidence also suggests the utility of targeting multiple DNA repair processes, modulators of DNA replication and major DNA damage response regulators. We explore the clinical rationale behind a neoadjuvant therapeutic strategy and emerging predictors of survival benefit associated with this approach. We also discuss challenges and opportunities originating from recent clinical trials evaluating neoadjuvant treatments composed of various combinations of radiotherapy, chemotherapy and immunotherapeutic regimens that have aimed to address some of these biological challenges. Selective treatment of patients harbouring specific genomic aberrations with targeted agents and immunotherapy can translate into optimum survival outcomes in PDAC. We also share perspectives on emerging prospective clinical evidence regarding stromal modifying agents, such as Tumour Growth Factor-Beta and Connective Tissue Growth Factor inhibitors along with novel vaccination-based approaches in improving PDAC outcomes.

Molecular integrity and global gene expression of breast and lung cancer stem cells under long-term storage and recovery.

Cryopreservation is a common procedure widely used in biological and clinical sciences. Similar protocols are also applied in preserving cancer stem cells, a field with high promises and challenges. Specific cell surface membrane proteins are considered to be biomarkers of cancer stem cells and they may play a critical role in differentiating stem cells from non stem cells. We have looked at the possible effect of long-term cryopreservation on the molecular integrity of breast MCF7 and lung, A549 and H460, cancer stem cells and to assess if these cells are more sensitive to long-term storage process. We analyzed the expression of CD24 and CD38 as two potent biomarkers of lung cancer stem cells and EpCAM and ALDH that are used as biomarkers of a wide range of cancer stem cells. We also selected three genes essential for the normal functioning of the cells, Fos, MUC1, and HLA. Our results indicate a pattern of down-regulation in the expression of the genes following freezing, in particular among cell surface marker proteins. Global gene expression of the post-thaw breast and lung cancer stem cells also reveals a significant down-regulation in freeze-thaw cells independent from each other. Analyzing the canonical pathways between two populations reveals a significant alteration in the gene expression of the pathways involved in cell cycle, mitosis, and ataxia telangiectasia mutated pathways. Overall, our results indicate that current protocols for long-term storage of lung and breast cancer stem cells may substantially influence the activity and function of genes.

Pamrevlumab, a Fully Human Monoclonal Antibody Targeting Connective Tissue Growth Factor, for Non-Ambulatory Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a neuromuscular disease stemming from dystrophin gene mutations. Lack of dystrophin leads to progressive muscle damage and replacement of muscle with fibrotic and adipose tissue. Pamrevlumab (FG-3019), a fully human monoclonal antibody that binds to connective tissue growth factor (CTGF), is in Phase III development for treatment of DMD and other diseases. METHODS: MISSION (Study 079; NCT02606136) was an open-label, Phase II, single-arm trial of pamrevlumab in 21 non-ambulatory patients with DMD (aged≥12 years, receiving corticosteroids) who received 35-mg/kg intravenous infusions every 2 weeks for 2 years. The primary endpoint was change from baseline in percent predicted forced vital capacity (ppFVC). Secondary endpoints included other pulmonary function tests, upper limb function and strength assessments, and changes in upper arm fat and fibrosis scores on magnetic resonance imaging. RESULTS: Fifteen patients completed the trial. Annual change from baseline (SE) in ppFVC was -4.2 (0.7) (95% CI -5.5, -2.8). Rate of decline in ppFVC in pamrevlumab-treated patients was slower than observed in historical published trials of non-ambulatory patients. MISSION participants experienced slower-than-anticipated muscle function declines compared with natural history and historical published trials of non-ambulatory patients with DMD. Pamrevlumab was well-tolerated. Treatment-emergent adverse events were mild to moderate, and none led to study discontinuation. CONCLUSIONS: nti-CTGF therapy with pamrevlumab represents a potential treatment for DMD. The lack of internal control group limits the results.

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy.

BACKGROUND: Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge. METHODS: To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro. RESULTS: We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses. CONCLUSION: Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

Expression of the epigenetic factor BORIS (CTCFL) in the human genome.

BORIS, or CTCFL, the so called Brother of the Regulator of Imprinted Sites because of the extensive homology in the central DNA binding region of the protein to the related regulator, CTCF, is expressed in early gametogenesis and in multiple cancers but not in differentiated somatic cells. Thus it is a member of the cancer testes antigen group (CTAs). Since BORIS and CTCF target common DNA binding sites, these proteins function on two levels, the first level is their regulation via the methylation context of the DNA target site and the second level is their distinct and different epigenetic associations due to differences in the non-homologous termini of the proteins. The regulation on both of these levels is extensive and complex and the sphere of influence of each of these proteins is associated with vastly different cellular signaling processes. On the level of gene expression, BORIS has three known promoters and multiple spliced mRNAs which adds another level of complexity to this intriguing regulator. BORIS expression is observed in the majority of cancer tissues and cell lines analyzed up to today. The expression profile and essential role of BORIS in cancer make this molecule very attractive target for cancer immunotherapy. This review summarizes what is known about BORIS regarding its expression, structure, and function and then presents some theoretical considerations with respect to its genome wide influence and its potential for use as a vaccine for cancer immunotherapy.

CTLA4 blockade with ipilimumab to treat relapse of malignancy after allogeneic hematopoietic cell transplantation.

Relapse of malignancy after allogeneic hematopoietic cell transplantation (allo-HCT) remains a therapeutic challenge. Blockade of the CTLA4 molecule can effectively augment antitumor immunity mediated by autologous effector T cells. We have assessed the safety and preliminary efficacy of a neutralizing, human anti-CTLA4 monoclonal antibody, ipilimumab, in stimulating the graft-versus-malignancy (GVM) effect after allo-HCT. Twenty-nine patients with malignancies that were recurrent or progressive after allo-HCT, received ipilimumab as a single infusion at dose cohorts between 0.1 and 3.0 mg/kg. Dose-limiting toxicity was not encountered, and ipilimumab did not induce graft-versus-host disease (GVHD) or graft rejection. Organ-specific immune adverse events (IAE) were seen in 4 patients (grade 3 arthritis, grade 2 hyperthyroidism, recurrent grade 4 pneumonitis). Three patients with lymphoid malignancy developed objective disease responses following ipilimumab: complete remission (CR) in 2 patients with Hodgkin disease and partial remission (PR) in a patient with refractory mantle cell lymphoma. At the 3.0 mg/kg dose, active serum concentrations of ipilimumab were maintained for more than 30 days after a single infusion. Ipilimumab, as administered in this clinical trial, does not induce or exacerbate clinical GVHD, but may cause organ-specific IAE and regression of malignancy. This study is registered at (http://clinicaltrials.gov) under NCI protocol ID P6082.

A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation.

Translation of small interfering RNA (siRNA)-based approaches into practical therapeutics is limited because of lack of an effective and cell-specific delivery system. Herein, we present a new method of selectively delivering siRNA to dendritic cells (DCs) in vivo using CD40 siRNA-containing immunoliposomes (siILs) that were decorated with DC-specific DEC-205 mAb. Administration of CD40 siILs resulted in DC-specific cell targeting in vitro and in vivo. On treatment with CD40 siILs, the expression of CD40 in DCs, as well allostimulatory activity was inhibited. In vivo administration resulted in selective siRNA uptake into immune organs and functional immune modulation as assessed using a model antigen. In conclusion, this is the first demonstration of DC-specific siRNA delivery and gene silencing in vivo, which highlights the potential of DC-mediated immune modulation and the feasibility of siRNA-based clinical therapy.

Effect of eszopiclone on sleep, fatigue, and pain in patients with mucositis associated with hematologic malignancies.

PURPOSE: Patients with malignancy sometimes develop painful mucositis and require patient-controlled analgesia (PCA) to treat their pain. Pain disrupts sleep and there is some evidence that analgesic medications also disrupt sleep. This study examined whether treatment with the sedative hypnotic eszopiclone could improve self-reports of sleep, fatigue, and pain as well as decrease opioid self-administered via PCA. METHODS: Inpatients who developed mucositis severe enough to require PCA treatment were randomized double-blind to a 2-day trial on eszopiclone or placebo-administered at bedtime. Patients completed questionnaires which assessed sleep, pain, and fatigue. PCA medication was calculated in terms of morphine equivalents. Data were analyzed with unpaired t tests and repeated measures analysis of variance. RESULTS: Twenty-two patients were randomized to placebo and 23 to eszopiclone. Groups were comparable in age and treatment characteristics. Mean pain scores were lower in the eszopiclone group at all time points (morning p = 0.01, afternoon p = 0.04, evening p = 0.04). The eszopiclone group reported increased sleep time (p < 0.05), fewer nighttime awakenings (p < 0.001), better self-reported sleep quality (p = 0.01), and depth (p = 0.04). There were no significant differences between eszopiclone and placebo in terms of self-reports of fatigue or opioid usage. CONCLUSION: Sedative hypnotic agents improve sleep and analgesia even in the setting of considerable pain and discomfort.

A novel event-free survival endpoint in locally advanced pancreatic cancer.

The treatment paradigm for locally advanced pancreatic cancer (LAPC) is evolving rapidly. The development of neoadjuvant therapies composed of combination therapies and the evaluation of their impact on conversion to borderline resectable (BR) status, resection, and ultimately overall survival (OS) are presently being pursued. These efforts justify re-visiting study endpoints in order to better predict therapeutic effects on OS, by capturing not only the achievement of R0 resection at the end of induction therapy but also the long-term reductions in the rate of local and distal recurrence. The proposed herein event-free survival (EFS) endpoint, with its novel definition specific to LAPC, is formulated to achieve these objectives. It is an analog to disease-free survival (DFS) endpoint in the adjuvant setting applied to the neoadjuvant setting and may be a valuable surrogate endpoint for this patient population.

The search for multiple myeloma stem cells: the long and winding road.

Recent years have brought significant breakthroughs in the understanding of tumor biology, related to discovery of cancer stem cells (CSCs) in acute myelogenous leukemia as well as in a number of solid tumors. This finding revealed that not all tumor cells are able to divide indefinitely, and that the bulk of tumor cells are expanded because of divisions and differentiation of CSC fraction. Although the CSCs identified in acute leukemia have a phenotype of early hematopoietic progenitors, it seems that CSCs in multiple myeloma (MM) may resemble the memory B cell fraction. Previous studies in patients with MM have documented the existence of cells without plasma cell characteristics expressing MM-type immunoglobulin genes--so-called "clonotypic" B cells. These cells have been characterized functionally and phenotypically as chemoresistant recirculating B cells. They have been found to self-renew and to be capable of initiating MM growth in immunocompromised animals. Controversy exists as to whether these cells truly belong to an MM clone, however; they may represent only the remaining clones of premalignant B cells. The identification of MM stem cells responsible for the recurrence of MM is of primary importance in designing targeted therapies to definitely cure this disease. This article summarizes the current state of knowledge on these hypothetical "MM stem cells."

Lasers, stem cells, and COPD.

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a "photoceutical" for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.

Tumor vaccines in 2010: need for integration.

Induction of tumor-specific immunity is an attractive approach to cancer therapy, however to date every major pivotal trial has resulted in failure. While the phenomena of tumor-mediated immune suppression has been known for decades, only recently have specific molecular pathways been elucidated, and for the first time, rationale means of intervening and observing results of intervention have been developed. In this review we describe major advances in our understanding of tumor escape from immunological pressure and provide some possible therapeutic scenarios for enhancement of efficacy in future cancer vaccine trials.

Gemcitabine/nab-paclitaxel with pamrevlumab: a novel drug combination and trial design for the treatment of locally advanced pancreatic cancer.

PURPOSE: Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition. Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer (LAPC) prevents surgical resection. This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor, pamrevlumab, to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient population. METHODS: In this phase I/II trial, 37 patients with LAPC were randomised 2:1 to gemcitabine/nab-paclitaxel plus (Arm A, n=24) or minus (Arm B, n=13) pamrevlumab. Those who completed six cycles of treatment were assessed for surgical eligibility by protocol-defined criteria. Resection rates, progression-free and overall survival were evaluated. RESULTS: Eighteen (75%) patients in Arm A and seven (54%) in Arm B completed six cycles of therapy with similar toxicity patterns. In Arms A and B, carbohydrate antigen 19-9 response, as defined by ≥50% decline from baseline, occurred in 13 (65%) and 5 (42%), respectively. Sixteen (16%) per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria (5 in Arm A (21%) and 1 (8%) in Arm B). Positron emission tomography normalised in 9 (38%) vs 3 (23%) of patients in Arm A vs Arm B, respectively, and correlated with surgical exploration. Eligibility for surgical exploration was 17 (71%) vs 2 (15%) (p=0.0019) and resection was achieved in 8 (33%) vs 1 (8%) of patients in Arm A vs Arm B (p=0.1193), respectively. Postoperative complication rates were not different between arms. CONCLUSIONS: Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity. This combination merits evaluation in a larger patient cohort.

Autologous hematopoietic stem cell transplantation as an intensive consolidation therapy for adult patients in remission from acute myelogenous leukemia.

Autologous peripheral blood stem/progenitor cell transplantation (APBSCT) has been investigated as a potential therapeutic option to improve outcome in patients with acute myelogenous leukemia (AML). However, its optimal role in treatment for adults in remission has not been clearly established. We performed a retrospective analysis on 45 patients aged 21 to 73 years (median 51 years) with de novo AML who underwent APBSCT stratified by age, complete remission status, and cytogenetic risk. The 5-year disease-free survival (DFS) for all patients was 33.9% (95% confidence interval [CI], 20.1%-53.7%) and overall survival (OS) was 43.6% (CI, 29.2%-62.8%). For patients under the age of 60 years, the 5-year DFS for intermediate and high cytogenetic risk was 53.3% (CI, 23.5%-85.6%) and 50.0% (CI, 16.1%-100.0%); the 5-year OS for patients under the age of 60 years with low, intermediate, and high cytogenetic risk was 80.0% (CI, 40.0%-100.0%), 60.0% (CI, 31.2%-90.7%), and 75.0% (CI, 39.0%-100.0%), respectively. For patients over the age of 60 years, the 5-year DFS and OS for intermediate cytogenetic risk was 21.4% (CI, 7.9%-58.4%) and 21.4% (CI, 7.9%-58.4%). The DFS and OS of these patients are comparable to the historic survival of those who underwent allogeneic stem cell transplantation when adjusted by age. In addition, there was no treatment-related mortality (TRM). We conclude that APBSCT is a reasonable and safe intensive consolidation for patients with AML who do not have a suitable HLA-matched donor.

Human embryonic stem cells hemangioblast express HLA-antigens.

BACKGROUND: It has been suggested that the initial differentiation of endothelial and hematopoietic cells during embryogenesis occurs from a common progenitor, called hemangioblast (hB). We hypothesized that these cells with dual hematopoietic/endothelial potential could be used in future regenerative medicine. METHODS: We used the two-step differentiation technology to generate bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in our in vitro EB system followed by differentiation in semisolid culture medium supplemented with mixture of cytokines. RESULTS: The occurrence of blast-colony-forming cells (BL-CFC) during EB differentiation (day 0-6) was transient and peaked on day 3. The emergence of this event was associated with expression of mesoderm gene T, and inversely correlated with expression of endoderm gene FoxA2. Similarly, the highest BL-CFC number was associated with increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The derived colonies were composed of 30-50 blast cells on day 6 in culture. These cells had homogenous appearance in Wright-Giemsa stain, but to a different extent expressed markers of immature hematopoietic and endothelial cells (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). We found that some of them expressed fetal and embryonic globin genes. Interestingly, these cells expressed also HLA class I molecules, however at very low levels compared to endothelial and hematopoietic cells. The blast cells could be successfully differentiated to hematopoietic cells in a CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4 +/- 0.8%) containing macrophages, BFU-E colonies (19.5 +/- 3.5%) containing nucleated red blood cells, and CFU-EM colonies (17.1 +/- 2.7%) composed of macrophages and nucleated erythrocytes. Cells of CFU-EM and BFU-E colonies expressed both epsilon - and gamma- globin genes, but not adult-type gamma-globin. When in endothelial cell culture conditions, blast cells differentiated to endothelial cells which had the ability to take up Dil-Ac-LDL and to form complex vascular networks in Matrigel. CONCLUSION: 1) Hematoendothelial precursors exist transiently in early embryonic development and form single cell-derived colonies; 2) their differentiation can be tracked by the use of chosen molecular markers; 3) blast colonies consist of cells having properties of endothelial and hematopoietic precursors, however the issue of their ability to maintain dual properties over time needs to be further explored; 4) blast cells can potentially be used in regenerative medicine due to their low expression of HLA molecules.

Allogeneic endometrial regenerative cells: an "Off the shelf solution" for critical limb ischemia?

Critical limb ischemia (CLI) is an advanced form of peripheral artery disease which is responsible for approximately 100,000 amputations per year in the US. Trials to date have reported clinical improvement and reduced need for amputation in CLI patients receiving autologous bone marrow or mobilized peripheral blood stem cells for stimulation of angiogenesis. While such treatments are currently entering Phase III trials, practical and scientific pitfalls will limit widespread implementation if efficacy is proven. Hurdles to be overcome include: a) reduced angiogenic potential of autologous cells in aged patients with cardiovascular risk factors; b) invasiveness/adverse effects of bone marrow extraction and G-CSF mobilization, respectively; and c) need for on-site cellular manipulation. The Endometrial Regenerative Cell (ERC) is a mesenchymal-like stem cell derived from the menstrual blood that is believed to be associated with endometrial angiogenesis. We discuss the possibility of using allogeneic ERCs as an "off the shelf" treatment for CLI based on the following properties: a) High levels of growth factors and matrix metalloprotease production; b) Ability to inhibits inflammatory responses and lack of immunogenicity; and c) Expandability to great quantities without loss of differentiation ability or karyotypic abnormalities.

Non-anthracycline based remission induction therapy for newly diagnosed patients with acute myeloid leukemia aged 60 or older.

We assessed remission rates and toxicity in 24 consecutive elderly (age>or=60) patients with untreated Acute myeloid leukemia (AML) who received the anthracycline-free combination of fludarabine, cytosine arabinoside and G-CSF (FLAG) as initial induction chemotherapy at our center. CR was achieved following one cycle of FLAG in 14 patients (58%). Another four patients cleared blasts from their bone marrow by day 30 without complete platelet recovery. Three patients died from infections prior to neutrophil recovery (12%). No other grade 3/4 toxicities and no clinically significant mucositis were seen. No significant association was found between age, WBC and cytogenetic risk group with likelihood of achieving CR. Fifteen patients proceeded to consolidation therapy and seven patients received a stem cell transplant (six autologous, one allogeneic). Primary induction with FLAG in elderly AML patients achieves a high remission rate without prohibitive mucosal or cardiac toxicity and may thus be considered as an alternative to standard anthracycline-based regimens in this setting.

Murine Sca-1(+)/Lin(-) cells and human KG1a cells exhibit multiple pseudopod morphologies during migration.

OBJECTIVE: The migration of primitive hematopoietic cells has been studied mostly via population-based assays while the actual mechanisms of cell motion have been defined by tracking individual mature cells. In this report, we examined individual immature hematopoietic cells to determine if any notable differences in migration mechanisms exist due to the primitive nature of the cells. MATERIALS AND METHODS: Murine cells of the Sca-1(+)/Lin(-) phenotype were isolated from C57BL/6 mice using Miltenyi bead purification and flow cytometric sorting. These cells were then observed for long periods of time with an environmentally controlled time-lapse microscope system in either multiwell plates or micropore transwell chambers. Experiments were also performed with the human KG1a immature hematopoietic cell line. RESULTS: Murine Sca-1(+)/Lin(-) immature hematopoietic cells and human KG1a cells were observed to exhibit a variety of mechanisms/morphologies during migration, which include the classic "hand mirror" shape; broad, flat lamellipodia; trailing uropodia; dynamic filopodia; and retraction fibers. Time-lapse observations of transmembrane assays revealed long, thin magnupodia passing through the pores, while other measurements show magnupods can generate forces capable of accelerating a cell to a velocity of 5 microns/minute. CONCLUSION: Many of these mechanisms have been reported separately for differentiated cells; however, we show that immature hematopoietic cells are capable of exhibiting all of these mechanisms of migration. These data provide insight into the loss of phenotypic functions as stem cells differentiate.

Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: the role of recipient CD4(-) cells.

OBJECTIVE: We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13). MATERIALS AND METHODS: Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance. RESULTS: When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells. CONCLUSION: These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.

Chimerism and tolerance post-in utero transplantation with embryonic stem cells.

BACKGROUND: Clinical application of in utero transplantation (IUT) in human fetuses with intact immune systems resulted in a very low level of donor chimerism. In this study, we examined whether the fetal immune system early in the second trimester of pregnancy (13.5 dpc) can initiate immune tolerance for major histocompatibility complex (MHC)-mismatched embryonic stem (ES) cells. We also examined whether immune tolerance mechanisms respond differently to ontogenetically different stem cells. METHODS: MHC-mismatched ES, fetal liver (FL), and bone-marrow (BM) cells (H-2kd) at 1 x 10(9) cells/kg fetal body weight were injected intraperitoneally into 13.5 dpc BALB/c fetuses (H-2Kd). Peripheral chimerism was determined in blood by flow cytometry (sensitivity< or =0.1%) at monthly intervals. Donor-specific immune responses were determined by cytotoxic lymphocyte (CTL) assay, mixed lymphocyte reaction, and T helper (Th)1 and Th2 cytokine assays. Chimeric mice at the age of 9 months received postnatal boosts (PB) with minimal conditioning of 200 cGy by intravenous injection of 1 x 10(9) of the corresponding cells/kg body weight. RESULTS: After IUT with ES, FL, or BM cells, the level of peripheral chimerism within the first 9 months of life was 0% to 0.4%. PB with 1 x 10(9)/kg of corresponding cells resulted in a decrease in the peripheral chimerism to 0% within 2 weeks of PB. CTL and cytokine assays before and after PB demonstrated a shift toward immunity. CONCLUSIONS: Immunologic tolerance was not achieved after IUT of murine fetuses at 13.5 dpc with MHC-mismatched ES cells, and only a low level chimerism was achieved.