Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of neurofibromatosis type 1.

Background: Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras-GAP protein causes increased Ras-GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model. Procedures: We studied effects of MEK inhibition using 1.5 mg/kg/day PD-0325901 prior to neurofibroma onset in the Nf1 flox/flox;Dhh-Cre mouse model. We also treated mice with established tumors at 0.5 and 1.5 mg/kg/day dosees of PD-0325901. We monitored tumor volumes using MRI and volumetric measurements, and measured pharmacokinetic and pharmacodynamic endpoints. Results: Early administration significantly delayed neurofibroma development as compared to vehicle controls. When treatment was discontinued neurofibromas grew, but no rebound effect was observed and neurofibromas remained significantly smaller than controls. Low dose treatment of mice with PD-0325901 resulted in neurofibroma shrinkage equivalent to that observed at higher doses. Tumor cell proliferation decreased, although less than at higher doses with drug. Tumor blood vessels per area correlated with tumor shrinkage. Conclusions: Neurofibroma development was not prevented by MEK inhibition, beginning at 1 month of age, but tumor size was controlled by early treatment. Moreover, treatment with PD-0325901 at very low doses may shrink neurofibromas while minimizing toxicity. These studies highlight how genetically engineered mouse models can guide clinical trial design

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform

Evolution of Highly Polymorphic T Cell Populations in Siblings with the Wiskott-Aldrich Syndrome

Population level evolutionary processes can occur within a single organism when the germ line contains a mutation that confers a cost at the level of the cell. Here we describe how multiple compensatory mutations arose through a within-individual evolutionary process in two brothers with the immune deficiency Wiskott-Aldrich Syndrome (WAS). As a result, both brothers have T lymphocyte populations that are highly polymorphic at the locus of the germ line defect, and no single allele achieves fixation. WASP, the gene product affected in this disease, is specific to white blood cells where it is responsible for regulating actin cytoskeleton dynamics in a wide range of cellular responses. The brothers inherited a rare allele predicted to result in truncated WASP lacking the carboxy-terminal VCA domains, the region that directly catalyzes actin filament generation. Although the brothers' T cell populations are highly polymorphic, all share a corrective effect relative to the inherited allele in that they restore the VCA domain. This indicates massive selection against the truncated germ line allele. No single somatic allele becomes fixed in the circulating T cell population of either brother, indicating that a regulated step in maturation of the affected cell lineage is severely compromised by the germ line allele. Based on the finding of multiple somatic mutations, the known maturation pathway for T-lineage cells and the known defects of T cells and precursor thymocytes in mice with truncated WASP, we hypothesize that the presence of truncated WASP (WASPΔVCA) confers an extreme disadvantage in early developing thymocytes, above and beyond the known cost of absence of full-length WASP, and that the disadvantage likely occurs through dominant negative competition of WASPΔVCA with N-WASP, a protein that otherwise partially compensates for WASP absence in developing thymocytes

Cytokine Gene Polymorphisms across Tuberculosis Clinical Spectrum in Pakistani Patients

BACKGROUND: Pakistan ranks 7(th) globally in terms of tuberculosis (TB) disease burden (incidence 181/100000 pop./yr; prevalence of 329/pop./yr). Reports from different populations show variable associations of TB susceptibility and severity with cytokine gene polymorphisms. Tuberculosis clinical severity is multi-factorial and cytokines play a pivotal role in the modulation of disease severity. We have recently reported that the ratio of two key cytokines (IFNgamma and IL10) show significant correlation with the severity spectrum of tuberculosis. The objective of the current study was to analyze the frequency of cytokine gene polymorphisms linked to high and low responder phenotypes (IFNgamma +874 T(hi)-->A(lo) and IL10 -1082 G(lo)-->A(hi)) in tuberculosis patients. METHODS AND FINDINGS: STUDY GROUPS WERE STRATIFIED ACCORDING TO DISEASE SITE AS WELL AS DISEASE SEVERITY: Pulmonary N = 111 (Minimal, PMN = 19; Moderate, PMD = 63; Advance, PAD = 29); Extra-pulmonary N = 67 (Disseminated DTB = 20, Localized LTB = 47) and compared with healthy controls (TBNA = 188). Genotype analyses were carried out using amplification refractory mutation system-PCR (ARMS-PCR) and stimulated whole blood (WB) culture assay was used for assessing cytokine profiles. Our results suggest that the IFNgamma +874 TT genotype and T allele was overrepresented in PMN (p = 0.01) and PMD (p = 0.02). IFNgamma +874 TT in combination with IL10 GG(lo) genotypes showed the highest association (chi(2) = 6.66, OR = 6.06, 95% CI = 1.31-28.07, p = 0.01). IFNgamma AA(lo) on the other hand in combination with IL10 GG(lo) increased the risk of PAD (OR = 5.26; p = 0.005) and DTB (OR = 3.59; p = 0.045). CONCLUSION: These findings are consistent with the role of IL10 in reducing collateral tissue damage and the protective role of IFNgamma in limiting disease in the lung

Contribution of Panton-Valentine Leukocidin in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains typically carry genes encoding Panton-Valentine leukocidin (PVL). We used wild-type parental and isogenic PVL-deletion (Δpvl) strains of USA300 (LAC and SF8300) and USA400 (MW2) to test whether PVL alters global gene regulatory networks and contributes to pathogenesis of bacteremia, a hallmark feature of invasive staphylococcal disease. Microarray and proteomic analyses revealed that PVL does not alter gene or protein expression, thereby demonstrating that any contribution of PVL to CA-MRSA pathogenesis is not mediated through interference of global gene regulatory networks. Inasmuch as a direct role for PVL in CA-MRSA pathogenesis remains to be determined, we developed a rabbit bacteremia model of CA-MRSA infection to evaluate the effects of PVL. Following experimental infection of rabbits, an animal species whose granulocytes are more sensitive to the effects of PVL compared with the mouse, we found a contribution of PVL to pathogenesis over the time course of bacteremia. At 24 and 48 hours post infection, PVL appears to play a modest, but measurable role in pathogenesis during the early stages of bacteremic seeding of the kidney, the target organ from which bacteria were not cleared. However, the early survival advantage of this USA300 strain conferred by PVL was lost by 72 hours post infection. These data are consistent with the clinical presentation of rapid-onset, fulminant infection that has been associated with PVL-positive CA-MRSA strains. Taken together, our data indicate a modest and transient positive effect of PVL in the acute phase of bacteremia, thereby providing evidence that PVL contributes to CA-MRSA pathogenesis

The sixth international RASopathies symposium: Precision medicine—From promise to practice

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS‐mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life‐limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion

Determination of Therapeutic Equivalence of Generic Products of Gentamicin in the Neutropenic Mouse Thigh Infection Model

Background: Drug regulatory agencies (DRA) support prescription of generic products of intravenous antibiotics assuming therapeutic equivalence from pharmaceutical equivalence. Recent reports of deaths associated with generic heparin and metoprolol have raised concerns about the efficacy and safety of DRA-approved drugs. Methodology/Principal Findings: To challenge the assumption that pharmaceutical equivalence predicts therapeutic equivalence, we determined in vitro and in vivo the efficacy of the innovator product and 20 pharmaceutically equivalent generics of gentamicin. The data showed that, while only 1 generic product failed in vitro (MIC = 45.3 vs. 0.7 mg/L, P,0.05), 10 products (including gentamicin reference powder) failed in vivo against E. coli due to significantly inferior efficacy (E max = 4.81 to 5.32 vs. 5.99 log 10 CFU/g, P#0.043). Although the design lacked power to detect differences in survival after thigh infection with P. aeruginosa, dissemination to vital organs was significantly higher in animals treated with generic gentamicin despite 4 days of maximally effective treatment. Conclusion: Pharmaceutical equivalence does not predict therapeutic equivalence of generic gentamicin. Stricter criteri

Regulatory Adaptation of Staphylococcus aureus during Nasal Colonization of Humans

The nasopharynx is the main ecological niche of the human pathogen Staphylococcus aureus. Although colonization of the nares is asymptomatic, nasal carriage is a known risk factor for endogenous staphylococcal infection. We quantified S. aureus mRNA levels in nose swabs of persistent carriers to gain insight into the regulatory adaptation of the bacterium to the nasal environment. We could elucidate a general response of the pathogen to the surrounding milieu independent of the strain background or the human host. Colonizing bacteria preferentially express molecules necessary for tissue adherence or immune-evasion whereas toxins are down regulated. From the analysis of regulatory loci we found evidence for a predominate role of the essential two-component system WalKR of S. aureus. The results suggest that during persistent colonization the bacteria are metabolically active with a high cell surface turnover. The increased understanding of bacterial factors that maintain the colonization state can open new therapeutic options to control nasal carriage and subsequent infections

A Commensal Helicobacter sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells

Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents

Risks of serious complications and death from smallpox vaccination: A systematic review of the United States experience, 1963–1968

BACKGROUND: The United States (US) has re-instituted smallpox vaccinations to prepare for an intentional release of the smallpox virus into the civilian population. In an outbreak, people of all ages will be vaccinated. To prepare for the impact of large-scale ring and mass vaccinations, we conducted a systematic review of the complication and mortality risks of smallpox vaccination. We summarized these risks for post-vaccinial encephalitis, vaccinia necrosum (progressive vaccinia), eczema vaccinatum, generalized vaccinia, and accidental infection (inadvertant autoinoculation). METHODS: Using a MEDLINE search strategy, we identified 348 articles, of which seven studies met our inclusion criteria (the number of primary vaccinations and re-vaccinations were reported, sufficient data were provided to calculate complication or case-fatality risks, and comparable case definitions were used). For each complication, we estimated of the complication, death, and case-fatality risks. RESULTS: The life-threatening complications of post-vaccinial encephalitis and vaccinia necrosum were at least 3 and 1 per million primary vaccinations, respectively. Twenty-nine percent of vaccinees with post-vaccinial encephalitis died and 15% with vaccinia necrosum died. There were no deaths among vaccinees that developed eczema vaccinatum; however, 2.3% of non-vaccinated contacts with eczema vaccinatum died. Among re-vaccinees, the risk of post-vaccinial encephalitis was reduced 26-fold, the risk of generalized vaccinia was reduced 29-fold, and the risk of eczema vaccinatum was reduced 12-fold. However, the risk reductions of accidental infection and vaccinia necrosum were modest (3.8 and 1.5 fold respectively)