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Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b -/- ) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b -/- mice were unable to clear the infection compared to WT mice; also, miR-378b -/- mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b -/- mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b -/- mice showed protection from Chlamydia -induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-β) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-β signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-β expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-β signaling pathways. Inhibition of either the EGFR or TGF-βR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-βR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-β expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-β during infection. Finally, TGF-βR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-β and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-β expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention., (Copyright © 2020 American Society for Microbiology.)

Genital chlamydia infection in women causes complications such as pelvic inflammatory disease and tubal factor infertility, but it is unclear why some women are more susceptible than others. Possible factors, such as time of day of chlamydia infection on chlamydial pathogenesis has not been determined. We hypothesised that infections during the day, will cause increased complications compared to infections at night. Mice placed under normal 12:12 light: dark (LD) cycle were infected intravaginally with Chlamydia muridarum either at zeitgeber time 3, ZT3 and ZT15. Infectivity was monitored by periodic vaginal swabs and chlamydiae isolation. Blood and vaginal washes were collected for host immunologic response assessments. The reproductive tracts of the mice were examined histopathologically, and fertility was determined by embryo enumeration after mating. Mice infected at ZT3 shed significantly more C. muridarum than mice infected at ZT15. This correlated with the increased genital tract pathology observed in mice infected at ZT3. Mice infected at ZT3 were less fertile than mice infected at ZT15. The results suggest that the time of day of infection influences chlamydial pathogenesis, it indicates a possible association between complications from chlamydia infection and host circadian clock, which may lead to a better understanding of chlamydial pathogenesis.

Background: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed., Results: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity., Conclusions: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations., (Published by Elsevier Inc.)

The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor β (TGF-β), TGF-β receptor 1 (TGF-βR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications., (Copyright © 2017 American Society for Microbiology.)

Chlamydia is an obligate intracellular bacterium that relies on host cells for essential nutrients and adenosine triphosphate (ATP) for a productive infection. Although the unfolded protein response (UPR) plays a major role in certain microbial infectivity, its role in chlamydial pathogenesis is unknown. We hypothesized that Chlamydia induces UPR and exploits it to upregulate host cell uptake and metabolism of glucose, production of ATP, phospholipids, and other molecules required for its replicative development and host survival. Using a combination of biochemical and pathway inhibition assays, we showed that the 3 UPR pathway transducers-protein kinase RNA-activated (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1α (IRE1α), and activating transcription factor-6α (ATF6α)-were activated during Chlamydia infection. The kinase activity of PERK and ribonuclease (RNase) of IRE1α mediated the upregulation of hexokinase II and production of ATP via substrate-level phosphorylation. In addition, the activation of PERK and IRE1α promoted autophagy formation and apoptosis resistance for host survival. Moreover, the activation of IRE1α resulted in the generation of spliced X-box binding protein 1 (sXBP1) and upregulation of lipid production. The vital role of UPR pathways in Chlamydia development and pathogenesis could lead to the identification of potential molecular targets for therapeutics against Chlamydia., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Chlamydia trachomatis genital infection in women causes serious adverse reproductive complications, and is a strong co-factor for human papilloma virus (HPV)-associated cervical epithelial carcinoma. We tested the hypothesis that Chlamydia induces epithelial-mesenchyme transition (EMT) involving T cell-derived TNF-alpha signaling, caspase activation, cleavage inactivation of dicer and dysregulation of micro-RNA (miRNA) in the reproductive epithelium; the pathologic process of EMT causes fibrosis and fertility-related epithelial dysfunction, and also provides the co-factor function for HPV-related cervical epithelial carcinoma. Using a combination of microarrays, immunohistochemistry and proteomics, we showed that chlamydia altered the expression of crucial miRNAs that control EMT, fibrosis and tumorigenesis; specifically, miR-15a, miR-29b, miR-382 and MiR-429 that maintain epithelial integrity were down-regulated, while miR-9, mi-R-19a, miR-22 and miR-205 that promote EMT, fibrosis and tumorigenesis were up-regulated. Chlamydia induced EMT in vitro and in vivo, marked by the suppression of normal epithelial cell markers especially E-cadherin but up-regulation of mesenchymal markers of pathological EMT, including T-cadherin, MMP9, and fibronectin. Also, Chlamydia upregulated pro-EMT regulators, including the zinc finger E-box binding homeobox protein, ZEB1, Snail1/2, and thrombospondin1 (Thbs1), but down-regulated anti-EMT and fertility promoting proteins (i.e., the major gap junction protein connexin 43 (Cx43), Mets1, Add1Scarb1 and MARCKSL1). T cell-derived TNF-alpha signaling was required for chlamydial-induced infertility and caspase inhibitors prevented both infertility and EMT. Thus, chlamydial-induced T cell-derived TNF-alpha activated caspases that inactivated dicer, causing alteration in the expression of reproductive epithelial miRNAs and induction of EMT. EMT causes epithelial malfunction, fibrosis, infertility, and the enhancement of tumorigenesis of HPV oncogene-transformed epithelial cells. These findings provide a novel understanding of the molecular pathogenesis of chlamydia-associated diseases, which may guide a rational prevention strategy.

Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (C. trachomatis) is a major cause. Although TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during C. trachomatis genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type C. trachomatis serovar L2 led to infertility, but the noninflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3 deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation, and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications.

Replacement of damaged cells is a promising approach for treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP); however, availability of donor tissue for transplantation remains a major obstacle. Key factors for successful engineering of a tissue include the identification of a neural cell line that is: homogeneous but can be expanded to give rise to multiple cells types; is nontumorigenic, yet capable of secreting neurotrophic factors; and is able to form three-dimensional (3D), differentiated structures. The goal of this study was to test the feasibility of tissue engineering from a multipotential human retinal cell line using a NASA-developed bioreactor. A multipotential human retinal precursor cell line was used to generate 3D structures. In addition, retinal pigment epithelium (RPE) cells were cocultured with neural cells to determine if 3D retinal structures could be generated in the bioreactor with cells grown on laminin-coated cytodex 3 beads. Cell growth, morphology, and differentiation were monitored by light and scanning electron microscopy, Western blot analysis, and analysis of glucose use and lactate production. The neuronal retinal precursor cell line cultured in a bioreactor gave rise to most retinal cell types seen in monolayer culture. They formed composite structures with cell-covered beads associated with one another in a tissue-like array. The beginning of layering and/or separation of cell types was observed. The neuronal cell types previously seen in monolayer cultures were also seen in the bioreactor. Some of the retinal cells differentiate into photoreceptors in the bioreactor with well-developed outer segment-like structures, a process that is critical for retinal function. Moreover, the neuronal cells that were generated resembled their in vivo phenotype more closely than those grown under other conditions. Outer segments were almost never seen in the monolayer cultures, even in the presence of photoreceptor-inducing growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor (TGF-alpha). Muller cells were occasionally seen when retinal, RPE cells were cocultured with retinal cells in the bioreactor. These have never been seen in this retinal cell line before. Cells grown in the bioreactor expressed several proteins specific for the retinal cell types: opsin, protein kinase C-alpha, dopamine receptor D4, tyrosine hydroxylase, and calbindin.

We characterized bovine aortic endothelial cells (BAEC) continuously cultured in the rotating wall vessel (RWV) bioreactor for up to 30 d. Cultures grew as large tissue-like aggregates (containing 20 or more beads) after 30 d. These cultures appeared to be growing in multilayers around the aggregates, where single beads were covered with confluent BAEC, which displayed the typical endothelial cell (EC) morphology. The 30-d multibead aggregate cultures have a different and smoother surface when viewed under a higher-magnification scanning electron microscope. Transmission electron microscopy of these large BAEC aggregates showed that the cells were viable and formed multilayered sheets that were separated by an extracellular space containing matrix-like material. These three-dimensional cultures also were found to have a basal production of nitric oxide (NO) that was 10-fold higher for the RWV than for the Spinner flask bioreactor (SFB). The BAEC in the RWV showed increased basal NO production, which was dependent on the RWV rotation rate: 73% increase at 8 rpm, 262% increase at 15 rpm, and 500% increase at 20 rpm as compared with control SFB cultures. The addition of l-arginine to the RWV cultures resulted in a fourfold increase in NO production over untreated RWV cultures, which was completely blocked by L-NAME [N(G)-nitro-L-arginine-methylester]. Cells in the SFB responded similarly. The RWV cultures showed an increase in barrier properties with an up-regulation of tight junction protein expression. We believe that this study is the first report of a unique growth pattern for ECs, resulting in enhanced NO production and barrier properties, and it suggests that RWV provides a unique model for investigating EC biology and differentiated function.

We conducted a double retroviral vector (RV) gene marking trial to test for the possible contribution to relapse of follicular non-Hodgkin's lymphoma (FNHL) cells present in bone marrow (BM) and peripheral blood (PB) grafts used for hematopoietic reconstitution of patients undergoing myelaoblative chemotherapy and autologous transplant. CD34 positive selection using the CellPro Ceprate CD34 column was performed on PB mononuclear cells obtained after cyclophosphamide/G-CSF mobilization. CD34 positive cells were exposed for 4-6 hours to the LNL6 or G1 Na RV in the absence of growth factors or stromal monolayers. One week later, BM mononuclear cells were similarly processed. Patients then received total body irradiation (TBI), cyclophosphamide, and etoposide followed by infusion of both PB and BM CD34 positive cells. Semiquantitative Southern blot analysis of DNA t(14;18) amplification products showed approximately a three log reduction in t(14;18) positive cells after CD34 positive selection. The first patient showed evidence of engraftment with RV positive BM and PB cells for 9 months. He relapsed one year after transplant. At relapse, one year after transplant, he had lost evidence of RV positive cells in ficolled mononuclear BM and PB cells as well as in CD19 positive cells. The second and third patients showed evidence of engraftment with RV positive cells up to 9 and 6 months post BMT respectively. The second and third patients are still in clinical remission. Our results demonstrate engraftment of RV transduced hematopoietic cells in the PB and BM for up to 9 months.

Most patients relapse after high-dose chemotherapy (HDCT) with autologous stem-cell transplantation (ASCT) for metastatic breast cancer. Further chemotherapy immediately after hematopoietic recovery from ASCT is not given for fear of irreversibly damaging the newly engrafted stem cells. In a pilot chemoprotection trial, autologous CD34+ cells from patients with metastatic breast cancer were exposed to a replication-incompetent retroviral vector carrying MDR-1 cDNA and then reinfused after HDCT. Immediately on recovery, patients received multiple courses of escalating dose paclitaxel. All of the 10 patients tolerated reinfusion of modified cells without any toxicity and had myeloid engraftment within 12 days (range, 11-14). The bone marrow cells of three patients contained vector MDR-1-positive cells only at the time of the first course of posttransplant paclitaxel, indicating that the MDR-1 vector-modified cells had only short-term engrafting potential. A total of 83 courses of paclitaxel were administered starting at a median of 30 (range, 21-32) days from ASCT. The median dose of paclitaxel was 225 mg/m2 and the median interval between paclitaxel cycles of therapy was 21 (range, 20-41) days. Five of the six CR patients were able to receive all of the 12 courses of paclitaxel. Three patients who had achieved less than a complete response to the HDCT (2 patients) and partial response (1 patient) were converted to complete clinical responses during the 12 cycles of paclitaxel. No delayed toxicity or bone marrow failure was noted in these patients with a median follow-up of 2 years from ASCT. This is the first study of chemotherapy immediately after transplantation with autologous CD34+ cells. These data indicate that paclitaxel can be safely administered immediately after ASCT without any delayed toxicities. Paclitaxel given immediately after ASCT can further improve the response to pretransplant chemotherapy in patients with advanced breast cancer.

We have previously demonstrated that liposome-incorporated methylphosphonate antisense oligodeoxynucleotides (oligos) specific for BCR-ABL can selectively inhibit the expression of p210Bcr-Abl protein and the proliferation of chronic myelogenous leukemia cells in vitro. Here, we show that liposome-entrapment of phosphodiester and phosphorothioate oligos specific for BCR-ABL can also selectively inhibit the proliferation of chronic myelogenous leukemia cells. We have studied the intracellular localization of liposomes by fluorescent microscopy and found that liposomes are readily taken up by leukemic cells and are localized in the cytoplasm, allowing increased access of oligos to target cells intracellularly. Liposomal oligos are not toxic to peripheral blood mononuclear cells nor to bone marrow progenitors isolated from normal hematological donors. These studies strongly suggest that liposomal delivery of oligos may indeed circumvent the major limitations that preclude the clinical development of antisense oligos.

Marrow cells were exposed to the LNL6 or G1N safety-modified variants of the N2 retrovirus, which contain the G418 bacterial resistance gene neo. The frequency of acquisition of the G418 resistance phenotype following exposure to LNL6 or G1N was compared among hematopoietic progenitor cells from the marrow of patients with chronic phase chronic myelogenous leukemia (CML), blast crisis CML, or from nonleukemic individuals. Under the conditions of our experiments, the myeloid committed progenitor cells from 3 of 6 nonleukemic individuals, 9 of 18 chronic-phase CML patients, and 2 of 4 blast crisis CML patients acquired resistance to at least 1 mg/ml G418 following incubation with cell-free supernatants from the PA317 LNL6 or PA317 G1N producer cell lines. Ten of the 32 colonies growing up in 0.8 mg/ml G418 from chronic-phase marrow exposed to LNL6 were shown to contain the neo gene by polymerase chain reaction (PCR) assay of DNA. These results were consistent with estimates of the transduction frequency based on acquisition of resistance to G418 as the number of colonies growing under G418 selection was always greater at 0.8 mg/ml G418 than at higher concentrations of G418 (1.0-1.4 mg/ml). The average transduction frequency at each G418 concentration (1.0, 1.2, and 1.4 mg/ml) in cells from blast crisis CML cells ranged from 2 to 14%, as measured by acquisition of G418 resistance. Chronic-phase CML showed slightly lower average frequencies of transduction (0.6-2.8% of the colonies are G418 resistant). The average transduction frequency of cells from nonleukemic marrow was as high as that seen from the marrow of chronic-phase CML individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed a polymerase chain reaction (PCR) assay for detection of integrated retroviral transgenomes containing the neo G418 resistance gene in colonies (40 cells or more) grown in G418 selection after exposure to the neo-positive retrovirus LNL6. This assay also provides for simultaneous characterization of these colonies as belonging to a chronic myelogenous leukemic (bcr-abl positive) or nonleukemic population (bcr-abl negative). Using these techniques, we assessed transduction of the LNL6 retrovirus into the normal and leukemic cells of a blast-crisis chronic myelogenous leukemia (CML) patient. This work was designed to support the use of the LNL6 retroviral marker to help identify the origin of relapse after autologous marrow infusion. The data from these experiments show that the majority of CML blast crisis cells that, following exposure to the LNL6 virus, produce colonies under rigorous G418 selection are indeed transduced by the virus, as shown by the presence of the neo retroviral gene. Most of these colonies are also shown to be leukemic by PCR detection of the bcr-abl RNA. This demonstrates the feasibility of the study of CML marrow for retroviral marking. These procedures will be of use in establishing if relapse arises from leukemic blasts which contaminate purged autologous bone marrow infused following intensive therapy for leukemia.