Systemic PPMO-mediated dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease that arises due to the loss of dystrophin expression, leading to progressive loss of motor and cardiorespiratory function. Four exon-skipping approaches using antisense phosphorodiamidate morpholino oligomers (PMOs) have been approved by the FDA to restore a DMD open reading frame, resulting in expression of a functional but internally deleted dystrophin protein, but in patients with single-exon duplications, exon skipping has the potential to restore full-length dystrophin expression. Cell-penetrating peptide-conjugated PMOs (PPMOs) have demonstrated enhanced cellular uptake and more efficient dystrophin restoration than unconjugated PMOs. In the present study, we demonstrate widespread PPMO-mediated dystrophin restoration in the Dup2 mouse model of exon 2 duplication, representing the most common single-exon duplication among patients with DMD. In this proof-of-concept study, a single intravenous injection of PPMO targeting the exon 2 splice acceptor site induced 45% to 68% exon 2-skipped Dmd transcripts in Dup2 skeletal muscles 15 days post-injection. Muscle dystrophin restoration peaked at 77% to 87% average dystrophin-positive fibers and 41% to 51% of normal signal intensity by immunofluorescence, and 15.7% to 56.8% of normal by western blotting 15 to 30 days after treatment. These findings indicate that PPMO-mediated exon skipping is a promising therapeutic strategy for muscle dystrophin restoration in the context of exon 2 duplications.

Splice modulating antisense oligonucleotides restore some acid-alpha-glucosidase activity in cells derived from patients with late-onset Pompe disease.

Pompe disease is caused by mutations in the GAA gene, resulting in deficient lysosomal acid-α-glucosidase activity in patients, and a progressive decline in mobility and respiratory function. Enzyme replacement therapy is one therapeutic option, but since not all patients respond to this treatment, alternative interventions should be considered. One GAA mutation, c.-32-13T > G, impacts upon normal exon 2 splicing and is found in two-thirds of late-onset cases. We and others have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients with one c.-32-13T > G allele. We designed 20 oligomers and treated fibroblasts derived from five patients to identify an oligomer sequence that maximally increased enzyme activity in all fibroblasts. The most effective splice correcting oligomer was chosen to treat forced-myogenic cells, derived from fibroblasts from nine patients carrying the c.-32-13T > G mutation. After transfection, we show increased levels of the full-length GAA transcript, acid-α-glucosidase protein, and enzyme activity in all patients' myogenic cells, regardless of the nature of the mutation in the other allele. This data encourages the initiation of clinical trials to assess the therapeutic efficacy of this oligomer for those patients carrying the c.-32-13T > G mutation.

Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy.

OBJECTIVE: To report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping. METHODS: Part 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry. RESULTS: Twelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%-4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry. CONCLUSION: Golodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization. CLINICALTRIALSGOV IDENTIFIER: NCT02310906. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500-5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production.

OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

Utility of PET-CT to evaluate retroperitoneal lymph node metastasis in advanced cervical cancer: Results of ACRIN6671/GOG0233 trial.

OBJECTIVE: To assess if FDG PET combined with diagnostic CT improves diagnostic CT accuracy to detect lymph node (LN) metastasis in advanced cervical cancer. METHODS: A prospective HIPAA compliant ACRIN/GOG multicenter trial was conducted. Patients underwent concurrent diagnostic contrast-enhanced CT (DCT) and PET and pelvic/abdominal lymphadenectomy. Seven independent blinded readers reviewed PET-DCT and DCT one-month apart. Reference standard was surgically removed LN pathology. Accuracy values were calculated at participant level, correlating abdominal (right and left para-aortic/common iliac) and pelvic (right and left external iliac/obturator) LN regions with pathology, respecting laterality. Reader average sensitivities/specificities of PET-DCT vs. DCT were compared with generalized linear mixed models, and AUCs with Obuchowski's method. RESULTS: One hundred fifty-three patients had PET-DCT and pathology. Forty-three of 153 patients had metastasis to abdominal LNs. Sample size calculation required review of the first 40 abdominal positive and 40 randomly selected abdominal negative studies. Patients were 24 to 74years (48.9±10.6) old. Mean sensitivities of PET-DCT/DCT for detection of LN metastasis in abdomen were 0.50 (CI: 0.44, 0.56) and 0.42 (CI: 0.36, 0.48) (p=0.052) and in pelvis 0.83 (CI: 0.78, 0.87) and 0.79 (CI: 0.73, 0.83) (p=0.15). Corresponding specificities were 0.85 (CI: 0.80, 0.89) and 0.89 (CI: 0.84, 0.92) (p=0.21) and 0.63 (CI: 0.54, 0.70) and 0.62 (CI: 0.53, 0.69) (p=0.83). Mean AUC values were 0.70 (CI: 0.61, 0.79) and 0.68 (CI: 0.59, 0.77) (p=0.43) and 0.80 (CI: 0.71, 0.88) and 0.76 (CI: 0.67, 0.85) (p=0.21) respectively. CONCLUSION: Addition of PET to DCT resulted in statistically borderline increase in sensitivity to detect LN metastasis in abdomen in advanced cervical cancer.

Randomized, Double-Blind, Phase III Trial of Enzastaurin Versus Placebo in Patients Achieving Remission After First-Line Therapy for High-Risk Diffuse Large B-Cell Lymphoma.

PURPOSE: To compare disease-free survival (DFS) after maintenance therapy with the selective protein kinase C ß (PKCß) inhibitor, enzastaurin, versus placebo in patients with diffuse large B-cell lymphoma (DLBCL) in complete remission and with a high risk of relapse after first-line therapy. PATIENTS AND METHODS: This multicenter, phase III, randomized, double-blind, placebo-controlled trial enrolled patients who were at high risk of recurrence after rituximab-cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients (N = 758) with stage II bulky or stage III to IV DLBCL, three or more International Prognostic Index risk factors at diagnosis, and a complete response or unconfirmed complete response after 6 to 8 cycles of R-CHOP were assigned 2:1 to receive oral enzastaurin 500 mg daily or placebo for 3 years or until disease progression or unacceptable toxicity. Primary end point was DFS 3 years after the last patient entered treatment. Correlative analyses of biomarkers, including cell of origin by immunohistochemistry and PKCß expression, with efficacy outcomes were exploratory objectives. RESULTS: After a median follow-up of 48 months, DFS hazard ratio for enzastaurin versus placebo was 0.92 (95% CI, 0.689 to 1.216; two-sided log-rank P = .541; 4-year DFS, 70% v 71%, respectively). Independent of treatment, no significant associations were observed between PKCß protein expression or cell of origin and DFS or overall survival. CONCLUSION: Enzastaurin did not significantly improve DFS in patients with high-risk DLBCL after achieving complete response to R-CHOP. Achievement of a complete response may have abrogated the prognostic significance of cell of origin by immunohistochemistry.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

Racial disparities in breast cancer diagnosis in Central Georgia in the United States.

BACKGROUND: Mortality rates in breast cancer are worse for African Americans than for whites. OBJECTIVE: To investigate the presence of racial disparities in clinical staging in women diagnosed with breast cancer and understand whether such disparities exist in Central Georgia in the United States. METHODS: We retrospectively reviewed records from the Tumor Registry of the Medical Center Navicent Health in Macon, Georgia, of women who had been diagnosed with breast cancer during 2011-2013. The chi-square test was used to assess statistically significant differences between whites and African Americans. We also assessed the patients' health insurance status and age at diagnosis. RESULTS: A total of 578 participants were identified. Statistically significant differences existed in the clinical stage between the races (𝑃 = .0003). Whites were more often clinical stage I at diagnosis, whereas African Americans had a greater percentage of stages II, III, or IV. African Americans were more than twice as likely to be diagnosed at clinical stage IV than were their white counterparts. Statistical differences also existed with age at diagnosis (𝑃 = .0066) and insurance coverage (𝑃 = .0004). A greater percentage of white patients were aged 65 years or older at diagnosis, whereas a greater percentage of African American patients were aged 49 years or younger. A greater percentage of African Americans had Medicaid insurance, whereas a greater percentage of whites had private health insurance. LIMITATIONS: As a single-center study, it is difficult to generalize these results elsewhere. Furthermore, this study focused on association and not on causation. It is difficult to pinpoint why such disparities exist. CONCLUSIONS: The etiology of racial disparities between African American and white women with breast cancer seems to be multifaceted. Screening mammography remains an important tool for identifying breast cancer. Low socioeconomic and educational status as well as a lack of a primary care physician may play a role in these disparities. Other factors that may have a role include biological factors and possible mistrust of the health care system.

Alternative splice forms of CTLA-4 induced by antisense mediated splice-switching influences autoimmune diabetes susceptibility in NOD mice.

Activated and regulatory T cells express the negative co-stimulatory molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) that binds B7 on antigen-presenting cells to mediate cellular responses. Single nucleotide polymorphisms in the CTLA-4 gene have been found to affect alternative splicing and are linked to autoimmune disease susceptibility or resistance. Increased expression of a soluble splice form (sCTLA-4), lacking the transmembrane domain encoded by exon 3, has been shown to accelerate autoimmune pathology. In contrast, an exon 2-deficient form lacking the B7 ligand binding domain (liCTLA-4), expressed by diabetes resistant mouse strains has been shown to be protective when expressed as a transgene in diabetes susceptible non-obese diabetic (NOD) mice. We sought to employ an antisense-targeted splice-switching approach to independently produce these CTLA-4 splice forms in NOD mouse T cells and observe their relative impact on spontaneous autoimmune diabetes susceptibility. In vitro antisense targeting of the splice acceptor site for exon 2 produced liCTLA-4 while targeting exon 3 produced the sCTLA-4 form in NOD T cells. The liCTLA-4 expressing T cells exhibited reduced activation, proliferation and increased adhesion to intercellular adhesion molecule-1 (ICAM-1) similar to treatment with agonist α-CTLA-4. Mice treated to produce liCTLA-4 at the time of elevated blood glucose levels exhibited a significant reduction in the incidence of insulitis and diabetes, whereas a marked increase in the incidence of both was observed in animals treated to produce sCTLA-4. These findings provide further support that alternative splice forms of CTLA-4 affects diabetes susceptibility in NOD mice and demonstrates the therapeutic utility of antisense mediated splice-switching for modulating immune responses.

Gene-silencing antisense oligomers inhibit acinetobacter growth in vitro and in vivo.

BACKGROUND: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA/RNA analogues that silence expression of specific genes. We studied whether PPMOs targeted to essential genes in Acinetobacter lwoffii and Acinetobacter baumannii are active in vitro and in vivo. METHODS: PPMOs were evaluated in vitro using minimum inhibitory concentration (MIC) and viability assays, and in vivo using murine pulmonary infection models with intranasal PPMO treatment. RESULTS: MICs of PPMOs ranged from 0.1 to 64 µM (approximately 0.6-38 µg/mL). The most effective PPMO tested was (RXR)4-AcpP, which is targeted to acpP. (RXR)4-AcpP reduced viability of A. lwoffii and A. baumannii by >10(3) colony-forming units/mL at 5-8 times MIC. Mice treated with ≥0.25 mg/kg of (RXR)4-AcpP survived longer and had less inflammation and bacterial lung burden than mice treated with a scrambled-sequence PPMO or phosphate-buffered saline. Treatment could be delayed after infection and still increase survival. CONCLUSIONS: PPMOs targeted to essential genes of A. lwoffii and A. baumannii were bactericidal and had MICs in a clinically relevant range. (RXR)4-AcpP increased survival of mice infected with A. lwoffii or A. baumannii, even when initial treatment was delayed after infection. PPMOs could be a viable therapeutic approach in dealing with multidrug-resistant Acinetobacter species.

Shared vision, collective impact, and persistent challenges: the first decade of Georgia's oncology research network.

Ten years ago, Georgia was lauded for dedicating a portion of tobacco settlement funds to the Georgia Cancer Coalition (GCC). The plan championed by then-Governor Roy E. Barnes was designed to make Georgia a leader in prevention, treatment, and research. This plan called for the expansion of clinical trials to ensure Georgians had access to the highest quality care based on the most current treatments and discoveries. As a result, oncologists in the state were engaged in a planning process that resulted in a shared vision to improve the quality of cancer care through research and the formation of a new organization: the Georgia Center for Oncology Research and Education.

Development of novel bioanalytical methods to determine the effective concentrations of phosphorodiamidate morpholino oligomers in tissues and cells.

Phosphorodiamidate morpholino oligomers (PMO) are neutrally charged, sequence-specific antisense agents that interfere with targeted gene expression. PMO have been shown to be highly specific and potent therapies after cellular uptake, yet methods to detect PMO in tissue and inside the cell are limited. We offer in this report novel methods for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) and a sandwich hybridization technique to quickly and sensitively quantify tissue resident PMO. These methods rely on oligonucleotide probes complementary to a PMO to specifically detect and quantify cell-associated and tissue resident PMO after in vitro or in vivo administration. Using the sandwich hybridization technique, we show that intranasally delivered PMO demonstrates zero-order clearance kinetics from the lung. Furthermore, PMO was detected in nonhematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, although an increase in PMO uptake in infected hematopoietic cells was observed. Coincident measurement of target knock-down to cell-associated influenza A PMO concentration allowed for the calculation of an EC50.

Lymphocytic choriomeningitis virus infection in FVB mouse produces hemorrhagic disease.

The viral family Arenaviridae includes a number of viruses that can cause hemorrhagic fever in humans. Arenavirus infection often involves multiple organs and can lead to capillary instability, impaired hemostasis, and death. Preclinical testing for development of antiviral or therapeutics is in part hampered due to a lack of an immunologically well-defined rodent model that exhibits similar acute hemorrhagic illness or sequelae compared to the human disease. We have identified the FVB mouse strain, which succumbs to a hemorrhagic fever-like illness when infected with lymphocytic choriomeningitis virus (LCMV). FVB mice infected with LCMV demonstrate high mortality associated with thrombocytopenia, hepatocellular and splenic necrosis, and cutaneous hemorrhage. Investigation of inflammatory mediators revealed increased IFN-γ, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Depletion of T cells at time of infection prevented mortality in all treated animals. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVB×C57BL/6 mating exhibit disease signs and mortality concomitant with the FVB challenged mice, extending this model to more widely available immunological tools. This report offers a novel animal model for arenavirus research and pre-clinical therapeutic testing.

CD8+ T cell responses to a viral escape mutant epitope: active suppression via altered SHP-1 activity.

One mechanism viruses use to subvert immune surveillance is through mutation of MHC contact residues of antigenic epitopes that weaken T cell recognition to the point that the immune system is ignorant of the infection. However, in contrast to ignorance, results presented herein demonstrate that intracellular signaling does occur upon stimulation with a lymphocytic choriomeningitis virus-derived escape mutant as demonstrated by the sustained activation of Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1). In addition to the increased SHP-1 activity, we found that the mutated epitope failed to induce oxidation of SHP-1, further enhancing enzymatic activity. Sustained activation of SHP-1 in a reduced form correlated with ERK and early growth response gene 1 activation and failure of T cells to commit to the effector lineage. Thus, instead of immune ignorance, these studies demonstrate the activation of a negative signaling pathway that actively suppresses T cell responses and limits recognition of viral escape mutants.

Murine pregnancy leads to reduced proliferation of maternal thymocytes and decreased thymic emigration.

During mammalian pregnancy the maternal thymus undergoes significant involution, and then recovers in size after birth. The mechanism behind this involution is not known, but it has been suggested that elevated levels of hormones during pregnancy induce the involution. We have recently shown that injection of 17beta-oestradiol into mice causes loss of early thymocyte precursors and inhibits proliferation of developing thymocytes. This suggests that elevated oestrogen in pregnancy may contribute to thymic involution. We have investigated this idea by examining the fate of thymocytes during mouse pregnancy in much greater detail than has been previously reported. Looking over a broad time-course, we find that pregnancy does not affect thymocyte precursor populations in the bone marrow, but induces a profound loss of early thymic progenitors in the thymus as early as day 12 x 5 of pregnancy. This loss is accompanied by decreased thymocyte proliferation, which returns to normal 2-4 days postpartum. No enhancement of apoptosis is detectable at any stage of pregnancy. We also find that there is a reduction in recent thymic emigrants after oestrogen treatment and at day 17 x 5 of pregnancy, suggesting that thymic involution during pregnancy influences the peripheral T-cell repertoire. The similarities between oestrogen-mediated involution and pregnancy-mediated involution suggest that oestrogen is a significant contributor to loss of thymocyte cellularity during pregnancy, and probably functions primarily by reducing thymocyte proliferation.

Palifermin reduces the incidence of oral mucositis in patients with metastatic colorectal cancer treated with fluorouracil-based chemotherapy.

PURPOSE: To characterize the efficacy and safety of palifermin in reducing the incidence of oral mucositis (OM) and diarrhea when administered to patients with metastatic colorectal cancer (CRC) receiving fluorouracil/leucovorin (FU/LV) chemotherapy. PATIENTS AND METHODS: Patients (N = 64) were randomly assigned to receive either placebo or palifermin (40 microg/kg for 3 consecutive days) before each of two consecutive cycles of chemotherapy with FU/LV. The incidence of OM and diarrhea, safety, disease progression, and survival were evaluated. RESULTS: Thirty-six patients received placebo and 28 patients received palifermin. The incidence of WHO grade 2 or higher OM was lower in patients who received palifermin compared with placebo (29% v 61% in cycle 1; 11% v 47% in cycle 2). FU dose reductions in the second chemotherapy cycle were more frequent in the placebo group (31%) than in the palifermin group (14%). Investigators reported lower mucositis scores and patients reported less severe symptoms with palifermin. There were no statistically significant differences in the incidence or severity of diarrhea or in overall survival between the groups. Overall, palifermin was safe and well tolerated. CONCLUSION: Palifermin administered at the indicated dosing regimen (40 microg/kg for 3 consecutive days) before chemotherapy was well tolerated and resulted in a statistically significant and clinically meaningful reduction in the incidence of WHO grade 2 or higher OM in patients with metastatic CRC.