Normal Horse Serum Research Articles

Dynamika klinichnykh oznak i pokaznykiv biomarkera zapalennia neopterynu v syrovattsi krovy krolykiv iz eksperymentalnym neinfektsiinym avtoimunnym uveitom, z urakhuvanniam riznoho stupenia tiazhkosty

Introduction. Uveitis is an inflammatory condition involving iris, ciliary body and choroid of the eyebulb. Uveitis curretntly affects about two million people worldwide and is among five leading causes of blindness in the working population. Diagnosis of uveitis requires a number of investigations. As important biomarker in the diagnosis of uveitis can be used the detection of Neopterin (NP) in blood serum, since it is a stable metabolite and its increased level encompass activation of cellular immunity. It should be also noted that NP is present in blood serum and plasma, urine, tears, aqueous humor, vitreous body, other biological fluids and can be easily detected. According to literature data and our own investigations, the concentration of NP usually depends on the degree of activity of the pathological process, and its high concentration of NP is one of the main predictors of an unfavorable prognosis. The aim of the study. To investigate the dynamics of clinical signs and inflammatory biomarker Neopterin level in blood serum of rabbits with experimental non-infectious autoimmune uveitis (NAU) considering varying degrees of severity. Materials and methods. The experimental study was conducted on 30 rabbits of the “Chinchilla” breed, which were divided into two main groups (I and II), 15 animals in each. NAU was induced by intravitreal injection of an undiluted normal sterile horse serum to group I rabbits, while group II animals received diluted with the physiological solution serum in the proportion of 1:2. General sensitization was previously carried out with the same serum injected intravenously. Results. The average level of NP in rabbits before the start of the experiment was 1.7 ± 0.2 nmol/l. It was considered the norm, since there is no information in the literature about the study of NP in healthy rabbits of the “Chinchilla” breed. In the reported research the severity of experimental NAU was estimated due to the concentration of the injected normal sterile horse serum intravitreally. As an additional proof of the development of NAU clinical picture was used the detection of Neopterin concentration in blood serum of rabbits. In both group animals the development of acute inflammation on days 3, 7, 10 and 14 of experiment the NP level was significantly higher compared to control (p-value less than 0.05). However, in the I group animals with moderate and severe NAU, the level of NP was significantly higher than that in group II, in which uveitis was characterized by a mild course. Namely, on the 3rd day of experiment NP concentration in the I group animals was 5.60 ± 0.70 nmol/l, in the group II – 4.70 ± 0.60 nmol/l (p-value less than 0.05); on day 7th – 6.00 ± 0.20 nmol/l and 4.45 ± 0.30 nmol/l (p-value less than 0.05); on day 10th – 5.90 ± 0.40 nmol/l and 2.60 ± 0.40 nmol/l (p-value less than 0.05); on the 14th – 3.80 ± 0.40 nmol/l and 2.10 ± 0.10 nmol/l (p-value less than 0.05). On the day 21st, the NP concentration in both group animals did not differ statistically – estimated level 2.70 ± 0,30 nmol/l, (p-value more than 0.05) – which apparently encompass the regression of inflammatory changes in the eye. Conclusions. A correlation between the severity of experimental non-infectious autoimmune uveitis with concentration of inflammatory biomarker neopterin in blood serum was investigated in rabbits. NP level in all experimental animals was elevated and statistically different in comparison to NP control levels on days 3rd, 7th, 10th and 14th day of experiment. In the group of animals with a more severe course of uveitis, a significantly higher level of NP was noted compared to animals with a milder course of NAU. This observation confirms the immune response to inflammation and the stability of NP biomarker in blood serum. The dynamics of this biomarker levels in rabbits of two experimental groups has a correlation with the dynamics of clinical changes. Detection of the inflammatory biomarker Neopterinin in blood serum can be used as a criterion for assessement the severity of non-infectious autoimmune uveitis, its course and, probably, prognosis.

ВЛИЯНИЕ ПАРЕНТЕРАЛЬНОГО ВВЕДЕНИЯ N-АЦЕТИЛЦИСТЕИНА, ДЕКСАМЕТАЗОНА НА БИОХИМИЧЕСКИЕ ПАРАМЕТРЫ В ВОДЯНИСТОЙ ВЛАГЕ ПЕРЕДНЕЙ КАМЕРЫ ГЛАЗ КРОЛИКОВ С ЭКСПЕРИМЕНТАЛЬНЫМ ИММУНОГЕННЫМ УВЕИТОМ

Objective. To evaluate the impact and compare the efficiency of parenteral administration of N-acetylcysteine (NAC) and dexamethasone when used as monotherapy or combination therapy in the treatment of experimental immunogenic uveitis (EIU) in rabbits, as well as to explore the changes of biochemical parameters in the aqueous humor of the eyes in experimental animals. Material and Methods. An experimental study was performed on 45 rabbits (90 eyes). Of these 5 healthy intact rabbits (10 eyes) served as a control group. Acute immunogenic uveitis was caused in 40 rabbits by injecting normal horse serum subcutaneously (5 ml) and then intravitreally (0.07 ml). The animals with experimental uveitis were divided into 8 groups (5 animals each). The first 4 groups – control-1, experiment-1, control-3, experiment-3 – received daily intramuscular injections of placebo, NAC, dexamethasone or a combination of NAC and dexamethasone respectively for 3 days, and thereafter they were withdrawn from the experiment. The remaining 4 groups – control-2, experiment-2, control-4, experiment-4 received, respectively, daily intramuscular injections of placebo, NAC, dexamethasone, a combination of NAC and dexamethasone for 7 days, and after that they were also withdrawn from the experiment. The drugs in the aforementioned groups were used from the moment of the horse serum intravitreal injection. When withdrawing animals from the experiment, aqueous humor was taken from the anterior chamber of their eyes, followed by the evaluation of protein (albumin) concentration and the number of leukocytes. Results. A significant elevation of albumin and the number of leukocytes in the aqueous humor of the eyes in the rabbits with experimental immunogenic uveitis was noted. NAC effectively reduced the level of albumin and the number of leukocytes in the aqueous humor. Dexamethasone showed more efficacy in reducing the investigated aqueous humor biochemichal parameters than NAC. Nevertheless, a synergism of the pharmacological action of NAC and dexamethasone was detected, since their combination had the greatest potency in reduction of albumin level and the number of leukocytes in the aqueous humor of the eyes in the rabbits with experimental immunogenic uveitis, even though the dosage of dexamethasone in the groups with combined (NAC and dexamethasone) therapy was reduced by 50% (1 mg / kg body weight). Conclusion. Parenteral administration of NAC significantly reduces inflammation in EIU. Combination of NAC and dexamethasone showed synergy of action in reducing the intensity of inflammatory process in rabbits with EIU, which is an objective rationale for including NAC in the complex therapy of uveitis, which in turn will reduce a single or course dose of dexamethasone and lower the risks of side effects caused by glucocorticoids.

Normal equine serum for staphylomycosis

Studying the effect of normal horse serum in vivo and in vitro on staphylococci, Benedek (Zeit. F. 1mm. U. Exper. Ther., B. 47, H. 2) found that it has antitoxic, bactericidal and tissue immunizing effects.

On the use of normal horse serum for the treatment of diphtheria

The question of treating diphtheria with normal horse serum instead of antitoxic serum, which was much sensational in connection with the observations of Віlіng'a, has been repeatedly decided by both clinicians and bacteriologists in favor of antitoxic serum.

Age-Related Features of Mast Cell Reactivity in Allergies

Introduction: During last few years, a novel scientific sphere has developed and is successfully growing,associated with the study of age-related changes in immune and allergic reactions at the cellular level.Although there is a fact that mast cells are the subject of many researches, many aspects of their physiologyand pathology remain unsolved. Moreover, clarification in the age-related features of mast cell reactivitymakes a great contribution to the expansion of our view of point on the characteristics of the clinic, etiologyand pathogenesis of allergies in ontogenesis.Materials and Method: We used 443 guinea pigs of both sexes of different ages, previously experienced 10-14 day-quarantine control under conditions of vivariums. Animals were constantly monitored, body weightwas periodically determined, and rectal temperature was measured. Sensitization and anaphylactic reactionswere reproduced by conventional method. Normal horse serum was used as an allergen. The resolving doseof a specific allergen was administered once intravenously or intraperitoneally 14-16 days after the startof the first sensitizing dose of the allergen. Collected digital data processed on electronic microcalculatorRokwell international Model 44 (USA).Results: With age, the rate of filling mast cells with granules increases. In guinea pigs of an older age, ascompared to newborns, the rates of filling mast cells with granules are 1.3-2.2 times greater (p <0.05), whichis consistent with the literature. Spontaneous mast cell degranulation was observed in intact animals. Theirfrequency ranged from 5.4 to 8.6% and, in principle, did not depend on age. A comparative analysis of thefrequency of degranulation, depending on age, showed that the frequency of degranulation in sensitizednewborns is slightly lower than in older animals. However, with age in sensitized guinea pigs, the frequencyof degranulated mast cell forms increased and the mast cell reactivity in one month aged pigs becameapproximately the same as in mature animals.Conclusion: In general, the functional activity of mast cells increases with age, and by the age of one monthreaches the level of functional activity. In the development of specific therapy, a decrease in the titer ofhomocytotropic antibodies and the percentage of mast cell degranulation is vital.

Comparison of Peroxidase‐Anti‐Peroxidase and Avidin‐Biotin‐Peroxidase Complex (ABC) Immunohistochemical Staining Procedures for Tryptophan Hydroxylase in Rat Brain

The neurotransmitter serotonin (5‐hydroxytryptamine; 5‐HT) plays a key role in sleep, feeding behavior, motor activity and, importantly, mood and the mechanism of action of antidepressant drugs. Immunohistochemistry for tryptophan hydroxylase (TpOH), the synthetic enzyme for 5‐HT, can be used to identify 5‐HT neurons in the central nervous system. As part of a project examining 5‐HT in brain function in the rat, we compared 2 indirect methods of visualizing primary antibodies against TpOH, a peroxidase‐anti‐peroxidase (PAP) method and the more sensitive avidin‐biotin‐peroxidase (ABC) procedure.A cryostat microtome was used to cut 40 micron sections from brains from rats transcardially perfused with 0.9% saline and 4% p‐formaldehyde in 0.1 M phosphate buffer (PB). Brains were post‐fixed 24 h in p‐formaldehyde and cryoprotected in 20% sucrose in 0.1 M PB. Sections were washed in phosphate‐buffered saline (PBS) before and between all steps. Tissues were treated with 0.4% H2O2 in methanol for 20 min to quench endogenous peroxidase activity and 1% sodium borohydride in 0.1 M PB for 30 min to enhance permeability of the tissue and improve staining quality. Sections were then treated with 1% normal donkey (PAP method) or horse (ABC method) serum in PBS with 0.2% Triton X‐100 detergent for 1 h and then 0.8% bovine serum albumin in PBS for 40 min. Tissues were incubated for 48 h in mouse monoclonal anti‐TpOH antibody (Sigma, 1:1000) in PBS with 0.3% Triton X‐100 and serum. Some sections were incubated without the primary antibody (omit negative controls). For the PAP protocol, tissues were treated with donkey anti‐mouse antibody (Jackson; 1:100) and then mouse PAP (Jackson; 1:400), both in PBS with 0.3% Triton X‐100 for 1 h. For the ABC protocol, tissues were treated with biotinylated horse anti‐mouse antibody and then avidin‐biotin‐peroxidase complex (Vector Elite kit), both in PBS with 0.3% Triton X‐100 and serum for 1 h. All sections were reacted with 0.0005% diaminobenzidine with 0.0001% H2O2 for 45 – 120 sec and mounted on gelatin‐coated slides that were coverslipped with DPX. Stained and negative control sections from both methods were examined using standard light microscopy.Staining using the PAP method was faint, homogenous and only slightly darker than negative controls. Individual neurons could not be identified. Increasing concentrations (1:500, 1:250, 1:100) of anti‐TpOH antibody had no effect on specific labeling but slightly increased background staining. This was surprising because the exact same reagents and PAP procedure has produced outstanding staining of tyrosine hydroxylase in brainstem catecholamine neurons (e.g., Muma et al., Exp. Neurology, 168:135–143, 2001). In contrast, labeling of TpOH using the ABC method was excellent. TpOH immunoreactivity was highly visible in the somata and primary dendrites of neurons in the dorsal and median raphe nuclei. These results add to and amplify the enhanced staining quality of the ABC procedure over the PAP method in formaldehyde‐fixed rat brain sections.Support or Funding InformationSupported by the School of Biological Sciences and the Department of Psychology, Illinois State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

Effect of melatonin instillations on the clinical course of experimental uveitis and biochemical processes in tears and aqueous humor

Acute immunogenic uveitis was modeled in rabbits via the subcutaneous and intravitreal injections of normal horse serum. We studied the effect of instillations of 0.1% melatonin solution on the clinical course of uveitis and biochemical parameters of tear fluid and aqueous humor: antioxi-dant activity, protein concentration and α(2)-macroglobulin level. Melatonin instillations decreased clinical manifestations of uveitis. We found that the antioxidant activity in tears of the rabbits treated with melatonin was substantially higher and the α(2)-macroglobulin level lower than in untreated animals. Antioxidant activity in aqueous humor taken on day 10 of uveitis was also twice higher while protein and α(2)-macroglobulin levels were 1.5-2 times lower than in untreated animals. These data indicate that instillations of melatonin increase the local antioxidant activity and decrease the acuity of inflammation and permeability of hematoophthalmic barrier in uveitis.

Effects of dexamethasone and superoxide dismutase instillations on clinical course of uveitis and local biochemical processes (experimental study)

to evaluate and compare the effect of topical superoxide dismutase (SOD), which is an antioxidant enzyme, dexamethasone, and a combination of these on the course of experimental uveitis in rabbits as well as biochemical parameters of aqueous and vitreous humor. Acute uveitis was induced in 16 rabbits by a double injection (subcutaneous and intravitreal) of normal horse serum. Of them 12 animals, divided into 3 groups of 4 each, received topical SOD, dexamethasone, or both daily for 7 days. The remaining 4 rabbits (8 eyes) were treated with placebo and, thus, constituted the control group. On day 8 the following parameters were measured in aqueous humor: protein concentration, antioxidant activity, SOD activity, α2-macroglobulin level, and leukocyte number. Total protein and albumin levels in vitreous humor were also determined. The effects of SOD and dexamethasone instillations were considered similar in many parameters. However, SOD was associated with a greater increase in antioxidant activity and a greater decrease in aqueous humor leukocytes, while dexamethasone was more effective in decreasing aqueous humor α2-macroglobulin and vitreous humor protein and albumin. The substances had a synergistic effect on iridal edema as well as aqueous humor leukocyte number and α2-macroglobulin level. CONCLUSION. Adding SOD to the complex therapy of uveitis results in lower inflammation intensity and enhanced dexamethasone effect.

Development of novel oncology biomarkers for cancer

According to American Cancer Society projections for 2014, approximately 1.5 million new cancers are expected to be diagnosed and more than several hundred patients are expected to die of cancer, with more than 90% of these cancers being solid tumours (ACS, 2010). Epithelial cell-derived cancers comprise approximately 80-85% of all cancers, and include, amongst others, breast, bladder, lung, pancreatic, thyroid and prostate cancers [1-4]. Presently, breast cancer is the most common cancer among women worldwide as more than 1 in 4 cancers in women (about 28%) are of the breast, and thyroid cancer is the most common epithelial cell-derived malignancy of the endocrine glands [4,5]. These cancers may exhibit no signs in its early stages. Further, the aggressive cases are difficult to detect and when undetected, prognosis deteriorates rapidly. Neutralization antibodies targeted to genes associated with cancer is a good strategy to detect cancer at early stages among many treatment approaches because of its high specificity and affinity. KalGene is developing a multi-protein assay to identify aggressive epithelial cancers, including breast, colon, and prostate, thyroid cancers. We have successfully developed several monoclonal antibodies against important biomarkers. Our monoclonal antibodies have shown excellent affinity and specific reactivity to the recombinant protein and cell lysates. Purified monoclonal antibodies were tested on tissue microarrays (TMA). Tissue microarray technology allows a massive acceleration of studies correlating molecular in situ bindings with clinico-pathological information. In this method, minute tissue cylinders (diameter 0.6 mm) are removed from hundreds of different primary tumour blocks and subsequently brought into one empty 'recipient' paraffin block. Sections from TMA blocks can be used for all different types of in situ tissue analyses including immunohistochemistry. TMA sections were de-paraffinized and heat-mediated antigen retrieval was performed. Non-specific immunoglobulin binding was blocked with 10% normal horse serum (NHS) for 30 min at room temperature. The TMA sections were then incubated with the appropriate diluted Kalgene monoclonal antibody in NHS. Thereafter, the slides were incubated with the appropriate secondary antibody diluted in NHS. Signal from the secondary antibody can be detected, then visualized.

Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes

Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5(mu/mu)) and dysbindin (Bloc1s8(sdy/sdy)). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5(mu/mu) and Bloc1s8(sdy/sdy) mice. Unlike Bloc1s8(sdy/sdy), elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders.

Abstract 231: ABCG1 Null and SR-BI Null Mice Reduced Plasma HDL Uptake by the Liver and Small Intestine

Aim: To reveal cholesterol transport via non-liver excretion pathway of plasma HDL-cholesterol, we focused on small intestinal and kidney in hyper- and hypo-lipoproteinemia model mice. Method: Mouse plasma was collected within EDTA coated tubes. Plasma lipoprotein profiles were measured by size exclusion HPLC and plasma preβHDL level was detected by immno-blotting of 2D PAGE by anti-mouse apoA-I peptide polyclonal antibody. Mouse HDL was co-incubated with 3H-cholesteryl oleyl ether (CEt) presence of CETP containing human plasma proteins for 48 hrs. The (100μL) of 3H-CEt-HDL was injected trough tail vein in anesthetized mice. 30μL of blood was collected each time point to chase the radioactivity, Mice organs were harvested after 3 hrs of the injection. Since lack of cellular catabolic pathway, CEt within the cells remained. For the immunohistochemical study, mouse was anesthetized and perfused thoroughly with PBS/5mM EDTA followed by 4% PFA/0.1M PB for the fixation. Leica CM1850 Cryostat was used for sectioning (thickness 16 μm). The sections were incubated in PBS containing 5% skim milk, 0.5% Triton X-100 and 5% inactivated normal horse serum for 3 hrs at room temperature, then incubated with primary antibodies (anti-SR-BI antibody diluted to 1:10,000) for overnight at 4°C. Secondary antibodies (Alexa fluor594) dissolved in the same blocking solution were applied to the sections for 90 min at room temperature. The fluorescence samples were observed by Nikon A1RSi confocal microscopy system. Results and conclusion: HDL derived CEt in intestinal cells were significantly reduced in ABCG1 null and SR-BI null mice during this experimental period. The other hand, kidney radioactivity showed no difference. Interestingly, preβHDL in SR-BI null mice was reduced by 2D western blotting of anti-mouse apoA-I peptide. Indicates that reduced speed of the plasma apoA-I recycling in SR-BI null mice, it may also cause reduced uptake of HDL-CEt by alternative pathway. Here we show that strong SR-BI-immunoreactivity was also found in the basolateral membrane and even higher intensity in some region. SR-BI located at the basolateral membrane may function as HDL-CE receptor in the small intestine.

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.

Dual Inhibition of Met Kinase and Angiogenesis to Overcome HGF-Induced EGFR-TKI Resistance in EGFR Mutant Lung Cancer

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.

New Molecular Bridge between RelA/p65 and NF-κB Target Genes via Histone Acetyltransferase TIP60 Cofactor

The nuclear factor-κB (NF-κB) family is involved in the expressions of numerous genes, in development, apoptosis, inflammatory responses, and oncogenesis. In this study we identified four NF-κB target genes that are modulated by TIP60. We also found that TIP60 interacts with the NF-κB RelA/p65 subunit and increases its transcriptional activity through protein-protein interaction. Although TIP60 binds with RelA/p65 using its histone acetyltransferase domain, TIP60 does not directly acetylate RelA/p65. However, TIP60 maintained acetylated Lys-310 RelA/p65 levels in the TNF-α-dependent NF-κB signaling pathway. In chromatin immunoprecipitation assay, TIP60 was primarily recruited to the IL-6, IL-8, C-IAP1, and XIAP promoters in TNF-α stimulation followed by acetylation of histones H3 and H4. Chromatin remodeling by TIP60 involved the sequential recruitment of acetyl-Lys-310 RelA/p65 to its target gene promoters. Furthermore, we showed that up-regulated TIP60 expression was correlated with acetyl-Lys-310 RelA/p65 expressions in hepatocarcinoma tissues. Taken together these results suggest that TIP60 is involved in the NF-κB pathway through protein interaction with RelA/p65 and that it modulates the transcriptional activity of RelA/p65 in NF-κB-dependent gene expression.

HIV-1 Tat Protein Promotes Neuronal Dysfunction through Disruption of MicroRNAs

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.

CCAAT/Enhancer-binding Protein δ Mediates Tumor Necrosis Factor α-induced Aurora Kinase C Transcription and Promotes Genomic Instability

Epidemiologic and clinical research indicates that chronic inflammation increases the risk of certain cancers, possibly through chromosomal instability. However, the mechanism of inflammation-dependent chromosomal instability associated with tumorigenesis is not well characterized. The transcription factor CCAAT/enhancer-binding protein δ (C/EBPδ, CEBPD) is induced by tumor necrosis factor α (TNFα) and expressed in chronically inflamed tissue. In this study, we show that TNFα promotes aneuploidy. Loss of CEBPD attenuated TNFα-induced aneuploidy, and CEBPD caused centromere abnormality. Additionally, TNFα-induced CEBPD expression augmented anchorage-independent growth. We found that TNFα induced expression of aurora kinase C (AURKC) through CEBPD, and that AURKC also causes aneuploidy. Furthermore, high CEBPD expression correlated with AURKC expression in inflamed cervical tissue specimens. These data provide insight into a novel function for CEBPD in inducing genomic instability through the activation of AURKC expression in response to inflammatory signals.

Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

The removal of edema from the air spaces is a critical function of the alveolar barrier requiring intact tight junctions. Alveolar fluid clearance contributes to graft function after transplantation and is associated with survival in patients with acute lung injury. Claudin-4 concentrations are known to increase during lung injury and the loss of claudin-4 decreases alveolar fluid clearance in mice. This study was therefore undertaken to evaluate whether differences in lung expression of the tight junction protein claudin-4 are associated with alveolar fluid clearance or clinical measures of lung function. Alveolar fluid clearance rates were measured in ex vivo perfused human lungs not used for transplantation and were compared with histological lung injury and clinical measures of lung injury in the donors. Claudin-4 staining demonstrated a positive correlation with alveolar fluid clearance (Spearman rank correlation [r(s)] = 0.71; P < 0.003); however, claudin-4 staining was not strongly associated with histological measures of lung injury. The expression of other tight junction proteins (including ZO-1) was not associated with alveolar fluid clearance or claudin-4 levels. Claudin-4 staining was lower in lungs from donors with greater impairment in respiratory physiology. These data suggest that claudin-4 may promote alveolar fluid clearance and demonstrate that the amount of claudin-4 expressed may provide specific information regarding alveolar epithelial barrier function that strengthens the link between histological changes and physiological impairment.

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.