Analysis of chromosomal insertion sites for Bacteroides Tn4555 and the role of TnpA

Tn4555, a mobilizable transposon carrying cefoxitin resistance, is directed to a preferred target site in the Bacteroides fragilis chromosome by a transposon-encoded targeting protein TnpA. In an effort to characterize target site selection for Tn4555, the existence of preferred target sites in other species of Bacteroides and in Escherichia coli was examined. For these analyses a Tn4555 mini element, pFD660, was transferred from E. coli donors to Bacteroides thetaiotaomicron or Bacteroides ovatus recipients and the resulting sites of insertion analyzed. A similar construct, pFD794 was used to determine insertion sites in E. coli, and preferred sites were found in all bacteria tested. Also the ability of TnpA to bind to various targets was examined in mobility shift assays. Although TnpA bound to all tested sequences, it displayed higher affinity for the target sites. The binding characteristics of TnpA and the lack of significant base sequence homology between targets suggested that secondary structure of the sites was important for TnpA binding. Circular permutation tests supported the idea that TnpA targets bent DNA.     

Toll-like receptor-7 agonist administered subcutaneously in a prolonged dosing schedule in heavily pretreated recurrent breast, ovarian, and cervix cancers

Background

The primary objective was to study the antitumor activity of prolonged subcutaneous dosing of systemic 852A, a Toll-like receptor-7 agonist (TLR-7), in recurrent breast, ovarian and cervix cancer. Secondary objectives included assessment of safety and immune system activation.

Methods

Adults with recurrent breast, ovarian or cervix cancer failing multiple therapies received 0.6 mg/m² of 852A subcutaneously twice weekly for 12 weeks. Doses increased by 0.2 mg/m²/week to a maximum of 1.2 mg/m². Serum was collected to assess immune activation.

Results

Fifteen patients enrolled: 10 ovarian, 2 cervix and 3 breast. Three completed all 24 injections. There were two grade 2 (decreased ejection fractions), nine grade 3 (1 cardiovascular, 1 anorexia, 3 dehydration, 2 infections, 2 renal) and two grade 4 (hepatic and troponin elevation) unanticipated toxicities. Cardiac toxicities included three cardiomyopathies (2 asymptomatic) and one stress-related non-ST elevated myocardial infarction. Five patients discontinued therapy due to possibly associated side effects. One who had stable disease (SD) following 24 doses received 17 additional doses. A cervix patient with SD following 24 doses received chemotherapy after progressing 3 months later, and remains disease free at 18 months. Immune activation, as evidenced by increased IP-10 and IL-1ra, was observed.

Conclusions

In this first human experience of a TLR-7 agonist delivered subcutaneously using a prolonged dosing schedule, 852A demonstrated sustained tolerability in some patients. Clinical benefit was modest, but immune activation was seen suggesting further study of antitumor applications is warranted. Because of cardiac toxicity; 852A should be used cautiously in heavily pretreated patients.     

Ligand Independence of the T618I Mutation in the Colony-stimulating Factor 3 Receptor (CSF3R) Protein Results from Loss of O-Linked Glycosylation and Increased Receptor Dimerization

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.     

Molecular epidemiology of ESbetaL producing P. mirabilis strains from a long-term care and rehabilitation facility in Italy

We report the detection of multidrug resistant ESbetaL producing Proteus mirabilis isolates from a long-term care and rehabilitation facility (LTCRF) in Northern Italy. 53% of the collected P. mirabilis strains were ESbetaL producers. PCR and sequencing techniques confirmed the presence of the bla(TEM-92) and bla(CMY-16) resistance genes in 23/26 (88.5%) and 3/26 (11.5%) of the ESbetaL producers respectively. PFGE showed that the TEM-92 beta-lactamase producing isolates were not clonally related, indicating the presence of at least four different clonal lineages (A, B, C, D), whereas all the CMY-16 enzyme producers belonged in the same lineage. The bla(TEM-92) and bla(CYY-16) determinants were distributed in seven different wards, but in three of them they coexisted. Our results show that the most patients are co-colonized by ESbetaLs producing P. mirabilis strains at the time of admission to an LTCRF. An effective strategy to curtail the spread of ESbetaLs mediated resistance in LTCRFs could be to activate sourveillance programs to monitor routinely the entry of resistant bacteria.     

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.     

Maximum likelihood estimation of ancestral codon usage bias parameters in Drosophila

We present a likelihood method for estimating codon usage bias parameters along the lineages of a phylogeny. The method is an extension of the classical codon-based models used for estimating dN/dS ratios along the lineages of a phylogeny. However, we add one extra parameter for each lineage: the selection coefficient for optimal codon usage (S), allowing joint maximum likelihood estimation of S and the dN/dS ratio. We apply the method to previously published data from Drosophila melanogaster, Drosophila simulans, and Drosophila yakuba and show, in accordance with previous results, that the D. melanogaster lineage has experienced a reduction in the selection for optimal codon usage. However, the D. melanogaster lineage has also experienced a change in the biological mutation rates relative to D. simulans, in particular, a relative reduction in the mutation rate from A to G and an increase in the mutation rate from C to T. However, neither a reduction in the strength of selection nor a change in the mutational pattern can alone explain all of the data observed in the D. melanogaster lineage. For example, we also confirm previous results showing that the Notch locus has experienced positive selection for previously classified unpreferred mutations.     

A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer

BackgroundNatural killer (NK) cells derived from patients with cancer exhibit diminished cytotoxicity compared with NK cells from healthy individuals. We evaluated the tumor response and in vivo expansion of allogeneic NK cells in recurrent ovarian and breast cancerMethodsPatients underwent a lymphodepleting preparative regimen: fludarabine 25 mg/m² × 5 doses, cyclophosphamide 60 mg/kg × 2 doses, and, in seven patients, 200 cGy total body irradiation (TBI) to increase host immune suppression. An NK cell product, from a haplo-identical related donor, was incubated overnight in 1000 U/mL interleukin (IL)-2 prior to infusion. Subcutaneous IL-2 (10 MU) was given three times/week × 6 doses after NK cell infusion to promote expansion, defined as detection of ≥100 donor-derived NK cells/μL blood 14 days after infusion, based on molecular chimerism and flow cytometryResultsTwenty (14 ovarian, 6 breast) patients were enrolled. The median age was 52 (range 30–65) years. Mean NK cell dose was 2.16 × 10⁷cells/kg. Donor DNA was detected 7 days after NK cell infusion in 9/13 (69%) patients without TBI and 6/7 (85%) with TBI. T-regulatory cells (Treg) were elevated at day +14 compared with pre-chemotherapy (P = 0.03). Serum IL-15 levels increased after the preparative regimen (P = < 0.001). Patients receiving TBI had delayed hematologic recovery (P = 0.014). One patient who was not evaluable had successful in vivo NK cell expansionConclusionsAdoptive transfer of haplo-identical NK cells after lymphodepleting chemotherapy is associated with transient donor chimerism and may be limited by reconstituting recipient Treg cells. Strategies to augment in vivo NK cell persistence and expansion are needed.     

Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.