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Background Lipoleiomyoma is a rare, benign variant of the commonplace uterine leiomyoma. Unlike leiomyoma, these tumors are composed of smooth muscle cells admixed with mature adipose tissue. While rare, they are most frequently identified in the uterus, but even more infrequently have been described in extrauterine locations. Case presentation We describe a case report of a 45-year-old woman with a history of in vitro fertilization pregnancy presenting 6 years later with abdominal distention and weight loss found to have a 30-cm intra-abdominal lipoleiomyoma. While cross-sectional imaging can narrow the differential diagnosis, histopathological analysis with stains positive for smooth muscle actin, desmin, and estrogen receptor, but negative for HMB-45 confirms the diagnosis of lipoleiomyoma. The large encapsulated tumor was resected en bloc. The patients post-operative course was uneventful and her symptoms resolved. Conclusions Lipoleiomyoma should be considered on the differential diagnosis in a woman with a large intra-abdominal mass. While considered benign, resection should be considered if the mass is symptomatic, and the diagnosis is unclear or there is a concern for malignancy.

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies.

Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%–10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology.

Tumor necrosis factor alpha (TNF) has been shown to be an essential cytokine mediator of innate immunity in bacterial pneumonia. To augment the expression of TNF within the lung, a recombinant adenoviral vector containing the murine TNF cDNA (Ad5mTNF) has been developed, and the intratracheal administration of this vector resulted in the dose- and time-dependent expression of TNF in the lung, but not systemically. Administration of Ad5mTNF resulted in significant airspace and peribronchial inflammation, with a predominant neutrophil influx by 2 days, and mononuclear cell infiltrates by 4 to 7 days posttreatment. Importantly, the administration of Ad5mTNF at a dose of 1 x 10(8) PFU significantly improved the survival of animals challenged concomitantly with Klebsiella pneumoniae, which occurred in association with enhanced clearance of bacteria from the lung and decreased dissemination of K. pneumoniae to the bloodstream. However, the delivery of higher doses of Ad5mTNF (5 x 10(8) PFU) was not beneficial and in fact the intratracheal administration of a similar dose of control vector (Ad5LacZ) actually enhanced Klebsiella-induced lethality by impairing clearance of K. pneumoniae from the lung. Our studies suggests that the transient transgenic expression of TNF within the lung dose dependently augments antibacterial host defense in murine Klebsiella pneumonia.

A vigorous host response is required to effectively clear pathogenic bacteria from the lungs and is dependent upon the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines, referred to as chemokines, have been shown to participate in this complex protective response. In this study, we assessed the role of the C-X-C chemokine KC in lung antibacterial host defense using wild-type (wt) B6D2 mice or transgenic mice that had been bred on a B6D2 background expressing KC under the control of a Clara cell-specific promoter within the lung. The administration of Klebsiella pneumoniae to both wt and KC-transgenic mice resulted in a time-dependent expression of KC protein within the lung that peaked at 24 to 48 h postinoculation. When infected with K. pneumoniae, the KC-transgenic mice showed a striking improvement in survival compared with wt control mice. This improved survival was due to an increase in bacterial clearance, which occurred in association with a vigorous recruitment of neutrophils in the KC-transgenic mice compared with their wt control counterparts. No differences in the lung levels of the specific cytokines TNF-α, IFN-γ, IL-12, and IL-10 were noted. However, inducible macrophage inflammatory protein-2 levels were significantly decreased in the KC-transgenic mice compared with the wt mice. This study indicates that the compartmentalized overexpression of KC in vivo results in increased lung bacterial clearance and improved survival, which occurs in association with enhanced polymorphonuclear leukocyte influx to the lung.

An effective host defense against lung bacterial infection is dependent primarily upon the rapid clearance of the organism from the respiratory tract, which is mediated by the influx and/or activation of phagocytic cells, including neutrophils (polymorphonuclear leukocytes [PMN]) and macrophages (27). The recruitment and activation of leukocytes in the setting of bacterial challenge is a complex and dynamic process which involves the coordinated expression of both pro- and anti-inflammatory cytokines (10–12, 14, 15). Tumor necrosis factor alpha (TNF) is a 17-kDa cytokine which is a critical component of an effective antibacterial host defense (2, 9, 13, 16). Specifically, TNF is a potent activator of both PMN and macrophages, leading to enhancement of protease release, stimulation of the respiratory burst, and induction of leukocyte and vascular adhesion molecule expression, which are essential for transmigration of these cells into sites of infection (7, 19, 24). Moreover, PMN and macrophage microbicidal activity is augmented by endogenous or exogenous TNF (22, 26). Finally, TNF is expressed in increased amounts in the airspaces of humans with bacterial pneumonia (21) and in the lungs of mice challenged with bacterial pathogens (9, 18). The inhibition of TNF has been shown to impair lung bacterial clearance and in some but not all studies to attenuate lung PMN influx in response to aerosolized or intratracheally (i.t.) administered Pseudomonas aeruginosa and Klebsiella pneumoniae (2, 9, 15, 18), resulting in markedly decreased survival in animals with bacterial pneumonia (18). However, TNF can mimic many of the detrimental pathophysiologic events that occur in sepsis and sepsis syndrome (1, 28), making it difficult to obtain the beneficial therapeutic effects of TNF without its cytotoxic consequences. Recently, a TNF agonist peptide composed of the 11 amino acids that constitute the site of binding of native human TNF to its receptors (referred to as TNF70-80) has been characterized (17, 23). Binding of TNF70-80 to TNF receptors (both p55 and p75) has been shown to mediate many leukocyte-activating effects of native TNF. Specifically, this peptide directly stimulates and primes neutrophils for enhanced protease release and respiratory burst, enhances neutrophil phagocytic activity, and augments neutrophil killing of Plasmodium falciparum in vitro and clearance of Plasmodium chabaudi in mice in vivo (17). In that study, TNF70-80 was associated with minimal toxicity when administered systemically, due in part to the fact that this peptide did not alter adhesive properties of the endothelium (17). The emergence of multidrug-resistant microbes in the immunocompromised host has made the treatment of bacterial infections of the lung increasingly difficult (3, 4, 6, 20, 25), underscoring the importance of the immune host defense in determining the eventual outcome of severe pneumonia. Given these clinical dilemmas, we performed this study to determine if the intrapulmonary and/or systemic administration of TNF70-80 was capable of augmenting host innate immunity in the setting of murine gram-negative pneumonia.

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. In this study we assessed the role of endogenously produced interleukin (IL)-12 in murine models of endotoxemia and Gram-negative peritoneal sepsis. Initial studies indicated that intraperitoneal lipopolysaccharide (LPS) administration to mice induced a significant time-dependent increase in plasma, lung, and liver IL-12 levels. Passive immunization with anti-IL-12 serum intraperitoneally before LPS resulted in a marked reduction in plasma levels of tumor necrosis factor and interferon-gamma. Furthermore, we observed an increase in endotoxin-induced mortality in mice transiently overexpressing murine IL-12 using a recombinant adenoviral vector (Ad5 mIL-12) administered intraperitoneally. Neutralization of tumor necrosis factor or interferon-gamma in animals overexpressing IL-12 resulted in significant reductions in LPS-induced mortality, suggesting that the mechanism whereby IL-12 increases LPS-induced mortality is primarily mediated by the enhancement of these cytokines. In contrast, we observed no survival benefit in animals passively immunized with anti-IL-12 serum before the intraperitoneal administration of 2 x 10(8) live Escherichia coli. Interestingly, there was an approximately 70-fold increase in peritoneal fluid E. coli colony-forming units and the early onset of bacteremia in animals treated with anti-IL-12 serum, as compared with control animals. These results indicate that IL-12 is produced in response to LPS exposure, and the neutralization of this cytokine improves survival in endotoxin-challenged animals. However, IL-12 represents an essential component of antibacterial host defense, as anti-IL-12 therapy results in significant impairment in the host's ability to clear Gram-negative bacterial infection.

The overzealous production of proinflammatory cytokines in sepsis can result in shock, multiorgan dysfunction, and even death. In this study, we assessed the role of monocyte chemoattractant protein-1 (MCP-1) as a mediator of sepsis in endotoxin-challenged mice. Intraperitoneal administration of LPS to CD-1 mice induced a substantial time-dependent increase in MCP-1 in plasma, lung, and liver. The passive immunization of mice with rabbit antimurine MCP-1 antiserum 2 h before endotoxin administration resulted in a striking increase in LPS-induced mortality from 10% in control animals to 65% in anti-MCP-1-treated animals. Importantly, the administration of anti-MCP-1 antibodies to endotoxin-challenged mice resulted in increases in peak TNF-alpha and IL-12 levels, and also in a trend toward decreased serum levels of IL-10. Conversely, the administration of recombinant murine MCP-1 intraperitoneally significantly protected mice from endotoxin-induced lethality, and resulted in an increase in IL-10 levels, a decrease in IL-12 levels, and a trend toward decreased levels of TNF. In conclusion, our findings indicate that MCP-1 is a protective cytokine expressed in murine endotoxemia, and does so by shifting the balance in favor of antiinflammatory cytokine expression in endotoxin-challenged animals.

Nitric oxide (NO) has been associated with protection against various parasitic and viral infections and may play a similar role in bacterial infections. We studied the role of NO in host defense against Klebsiella pneumoniae infection in the lung. Initial studies demonstrated a time-dependent increase in NO production of the lungs of CBA/J mice following the intratracheal administration of K. pneumoniae (7 x 10(2) CFU). To assess the role of NO in Klebsiella pneumonia, mice were treated intraperitoneally with either L-NAME (N-omega-nitro-L-arginine methylester), a competitive inhibitor of NO synthesis, or D-NAME, an inert enantiomer. The treatment of Klebsiella-infected mice with L-NAME resulted in a 10- and 46-fold increase in K. pneumoniae CFU in lungs and blood, respectively, at 48 h post-K. pneumoniae inoculation compared to treatment of mice with D-NAME. In addition, a greater-than-twofold increase in mortality was evident in L-NAME-treated mice compared to the mortality in control animals. No significant difference in bronchoalveolar lavage inflammatory cell profiles was noted between L-NAME- and D-NAME-treated mice with Klebsiella pneumonia. Interestingly, increased levels of tumor necrosis factor, gamma interferon, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 mRNA and protein were noted in infected mice treated with L-NAME compared to the levels in mice treated with D-NAME. Importantly, the in vitro incubation of murine alveolar macrophages with L-NAME, but not with D-NAME, resulted in a significant impairment in both the phagocytosis and killing of K. pneumoniae. In total, these results suggest that NO plays a critical role in antibacterial host defense against K. pneumoniae, in part by regulating macrophage phagocytic and microbicidal activity.

Epigenetic control of gene expression is a consistent feature of differentiated mammalian cell types. Epigenetic expression patterns are mitotically heritable and are stably maintained in adult cells. However, unlike somatic DNA mutation, little is known about the occurrence of epigenetic change, or epimutation, during normal adult life. We have monitored the age-associated maintenance of two epigenetic systems—X inactivation and genomic imprinting—using the genes Atp7a and Igf2, respectively. Quantitative measurements of RNA transcripts from the inactive and active alleles were performed in mice from 2 to 24 months of age. For both genes, older animal cohorts showed reproducible increases in transcripts expressed from the silenced alleles. Loss of X chromosome silencing showed cohort mean increases of up to 2.2%, while imprinted-gene activation increased up to 6.7%. The results support the hypothesis that epigenetic loss of gene repression occurs in normal tissues and may be a contributing factor in progressive physiological dysfunction seen during mammalian aging. Quantitatively, the loss of epigenetic control may be one to two orders of magnitude greater than previously determined somatic DNA mutation.

The prolonged and excessive consumption of alcohol has been shown to predispose the host to a variety of infectious complications, which may be due, in part, to the inability to produce important activating cytokines. In this study, we assessed the effect of chronic alcohol ingestion on bacterial clearance, survival, and cytokine mRNA and protein expression in mice with Klebsiella pneumonia. Two-week ethanol feeding resulted in substantial impairment in the clearance of K. pneumoniae and decreased survival, compared with CD-1 mice receiving an isocaloric diet without ethanol. No differences were noted between control and ethanol groups in the total numbers or percent of bronchoalveolar lavage fluid neutrophils or macrophages at 24 and 48 hr post-intratracheal K. pneumoniae. Importantly, the lungs of alcohol-fed mice with Klebsiella pneumonia displayed a decrease or delay in the expression of interleukin (IL)-12 p35 and p40 mRNA and interferon-gamma mRNA, respectively, as well as reduced IL-12 and interferon-gamma protein levels, compared with controls. Conversely, a time-dependent increase in lung IL-10 mRNAand protein was noted in ethanol-fed animals, compared with control animals challenged with K. pneumoniae. In summary, our studies indicate that ethanol ingestion results in a profound suppression of lung bacterial clearance and decreased survival in Klebsiella pneumonia, which occurs in association with a shift in the balance of lung cytokine mRNA and protein expression favoring Th2- rather than Th1-phenotype cytokines.

Microtubule-associated proteins (MAPs) regulate microtubule stability and play critical roles in neuronal development and the balance between neuronal plasticity and rigidity. MAP1a (HGMW-approved symbol MAP1A) stabilizes microtubules in postnatal axons. We describe human MAP1a's genomic organization and deduced cDNA and amino acid sequences. MAP1a is a single-copy gene spanning 10.5 kb. MAP1a coding sequence is contained in five exons. Translation begins in exon 3. Human MAP1a contains 2805 amino acids (predicted molecular weight 306.5 kDa) and is slightly larger than rat MAP1a (2774 amino acids). Like rat and bovine MAP1a, human MAP1a contains conserved tubulin binding motifs in the amino-terminal region. The carboxy-terminal portion contains a conserved pentadecapeptide that is present in the light chain portion of rat and bovine MAP1a/LC2 polyprotein. We show that human MAP1a gene expression occurs almost exclusively in the brain and that there is approximately 10-fold greater gene expression in adult brain compared to fetal brain. Strong, interspecies conservation between human and rat MAP1a cDNA and amino acid sequences indicates important relationships between MAP1a's function and its primary amino acid sequence.