Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. OBJECTIVES: To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. METHODS: We examined the role of the basement membrane proteins laminin subunits α3, ß3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. RESULTS: Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, ß3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. CONCLUSIONS: These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Pancreatic Cancer UK Grand Challenge: Developments and challenges for effective CAR T cell therapy for pancreatic ductal adenocarcinoma.

BACKGROUND: Death from pancreatic ductal adenocarcinoma (PDAC) is rising across the world and PDAC is predicted to be the second most common cause of cancer death in the USA by 2030. Development of effective biotherapies for PDAC are hampered by late presentation, a low number of differentially expressed molecular targets and a tumor-promoting microenvironment that forms both a physical, collagen-rich barrier and is also immunosuppressive. In 2017 Pancreatic Cancer UK awarded its first Grand Challenge Programme award to tackle this problem. The team plan to combine the use of novel CAR T cells with strategies to overcome the barriers presented by the tumor microenvironment. In advance of publication of those data this review seeks to highlight the key problems in effective CAR T cell therapy of PDAC and to describe pre-clinical and clinical progress in CAR T bio-therapeutics.

Mammalian Adaptation of an Avian Influenza A Virus Involves Stepwise Changes in NS1.

Influenza A viruses (IAVs) are common pathogens of birds that occasionally establish endemic infections in mammals. The processes and mechanisms that result in IAV mammalian adaptation are poorly understood. The viral nonstructural 1 (NS1) protein counteracts the interferon (IFN) response, a central component of the host species barrier. We characterized the NS1 proteins of equine influenza virus (EIV), a mammalian IAV lineage of avian origin. We showed that evolutionarily distinct NS1 proteins counteract the IFN response using different and mutually exclusive mechanisms: while the NS1 proteins of early EIVs block general gene expression by binding to cellular polyadenylation-specific factor 30 (CPSF30), NS1 proteins from more evolved EIVs specifically block the induction of IFN-stimulated genes by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially and were rapidly selected for during EIV evolution, highlighting the importance of evolutionary processes in immune evasion mechanisms during IAV adaptation.IMPORTANCE Influenza A viruses (IAVs) infect certain avian reservoir species and occasionally transfer to and cause epidemics of infections in some mammalian hosts. However, the processes by which IAVs gain the ability to efficiently infect and transmit in mammals remain unclear. H3N8 equine influenza virus (EIV) is an avian-origin virus that successfully established a new lineage in horses in the early 1960s and is currently circulating worldwide in the equine population. Here, we analyzed the molecular evolution of the virulence factor nonstructural protein 1 (NS1) and show that NS1 proteins from different time periods after EIV emergence counteract the host innate immune response using contrasting strategies, which are associated with two mutations that appeared sequentially during EIV evolution. The results shown here indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.

The pharmacokinetics and in vitro cyclooxygenase selectivity of deracoxib in horses.

The purpose of this study was to determine the pharmacokinetics of deracoxib following oral administration to horses. In addition, in vitro equine whole blood cyclooxygenase (COX) selectivity assays were performed. Six healthy adult horses were administered deracoxib (2 mg/kg) orally. Plasma samples were collected prior to drug administration (time 0), and 10, 20, 40 min and 1, 1.5, 2, 4, 6, 8, 12, 24, and 48 h after administration for analysis with high pressure liquid chromatography using ultraviolet detection. Following PO administration, deracoxib had a long elimination half-life (t(1/2) k(10) ) of 12.49 ± 1.84 h. The average maximum plasma concentration (C(max) ) was 0.54 µg/mL, and was reached at 6.33 ± 3.44 h. Bioavailability was not determined because of the lack of an IV formulation. Results of in vitro COX selectivity assays showed that deracoxib was selective for COX-2 with a COX-1/COX-2 ratio of 25.67 and 22.06 for the IC(50) and IC(80) , respectively. Dosing simulations showed that concentrations above the IC(80) for COX-2 would be maintained following 2 mg/kg PO q12h, and above the IC(50) following 2 mg/kg PO q24h. This study showed that deracoxib is absorbed in the horse after oral administration, and may offer a useful alternative for anti-inflammatory treatment of various conditions in the horse.

Movement disorders of aged rats: reversal by dopamine receptor stimulation.

When placed in a tank of water, aged rats (24 to 27 months old) showed marked impairments in swimming. Compared with young adult rats (3 to 4 months old), the older animals moved their limbs less vigorously and were less successful in keeping their heads above water. The young, but not old, rats maintained a position nearly horizontal to the water surface and planed across it. These movement dysfunctions of aged rats resemble those seen in young adult animals that have sustained injury to brain dopamine-containing neurons. The swimming impairments of the aged rats were reversed by the dopamine receptor stimulant apomorphine and by the biosynthetic precursor of dopamine, L-dopa. Thus, age-related alterations in brain dopaminergic systems may be responsible for some of the movement disturbances associated with senescence.

Striatal efferent fibers play a role in maintaining rotational behavior in the rat.

Rats in which ascending dopamine-containing neurons have been unilaterally destroyed by injections of 6-hydroxydopamine are known to rotate after being injected with apomorphine or L-dopa. The rotation is markedly reduced by either (i) ipsilateral electrocoagulations of the caudate-putamen or internal capsule or (ii) ipsilateral coronal knife cuts immediately rostral to the substantia nigra. Neostriatal efferent fibers, in particular the strionigral projection, appear to be required for the expression of this dopamine-dependent behavior.

High-grade fuels from biomass farming: potentials and constraints.

The key parameters controlling the productivity and the cost of net high-grade fuel from a system for biomass agriculture and conversion are analyzed. Performance depends sensitively on a "symbiotic" interaction between agronomy and technology. The conditions for obtaining net productivity and costs are explored for U.S. grain alcohol as a reference point. Currently practiced technology consumes more high-grade fuel than it generates. Some potentials and constraints for future systems, including use of other plant species and conversion systems, are explored.

Sensory neglect produced by lateral hypothalamic damage.

Unilateral lateral hypothalamlic lesions in rats produce deficits in orientation to contralateral visucal, olfactory, whisker-toluch, and somatosetnsory stimuli. This syndrome of sensory neglect appears to be involved in some of the deficits in feedinig and attack which follow bilateral lateral hypothalamic lesions.

Brain dopamine and serotonin receptor sites revealed by digital subtraction autoradiography.

Autoradiography combined with image analysis permitted quantitative visualization of dopamine (D2) and serotonin (S2) binding sites in rat brain. Forebrain sections were incubated with tritiated spiroperidol alone or with tritiated spiroperidol plus unlabeled compounds that saturated the D2 or S2 sites. By subtracting the digitized image of an autoradiograph derived from the latter sections from that of the former, the D2 or S2 sites were specifically revealed. The resulting quantitative images demonstrate the differing anatomical distributions of these sites. The D2 site is largely restricted to the striatal complex (caudate-putamen, nucleus accumbens septi, and olfactory tubercle), whereas the S2 site is enriched in layer 5 of motor cortex, the perirhinal and cingulate cortices, and the claustrum.

Proportion of nonsteroidal anti-inflammatory drug prescription in equine practice.

BACKGROUND: There is little knowledge of the prescription of nonsteroidal anti-inflammatory drugs (NSAIDs) and whether their prescription varies between countries. OBJECTIVE: To describe prescription practices of NSAIDs in equids in the United Kingdom (UK), United States of America (USA) and Canada. STUDY DESIGN: Descriptive observational study. METHODS: Free-text electronic medical records from 141,543 equids from 10 equine practices in the UK, 255,777 equids from 7 equine practices with 20 branches from the USA and 2 practices with 7 branches from Canada were evaluated. A validated text-mining technique was used to describe the proportion of equids prescribed NSAIDs at least once in these countries. The choice of NSAIDs in orthopaedic and colic cases was evaluated. RESULTS: The prescription of NSAIDs is more common in the USA (42.4%) and Canada (34.2%) than in the UK (28.6%). Phenylbutazone and flunixin meglumine were the drugs mostly prescribed in all countries. While flunixin meglumine was most prescribed with colic cases in all countries, a proportion received phenylbutazone despite this drug being licensed for use only with musculoskeletal disease. Phenylbutazone was the most commonly prescribed drug in cases with orthopaedic disease followed by flunixin meglumine in all countries. Only a small proportion of cases received meloxicam, ketoprofen or firocoxib. MAIN LIMITATIONS: The retrospective design might have resulted in an unknown number of incomplete records, particularly in the reporting of colic and orthopaedic disease. Although the data set is large, the relatively small number of practices recruited from each country may introduce bias. CONCLUSIONS: Clinical practice can differ between countries although the influence of individual practitioners and practice-specific policy on apparent intercountry differences requires further research. Despite several other NSAIDs being available and a substantial effort being made to evaluate their efficacy, the prescription of NSAIDs other than phenylbutazone and flunixin meglumine remains rather limited.

Running wheel exercise enhances recovery from nigrostriatal dopamine injury without inducing neuroprotection.

Forced use of the forelimb contralateral to a unilateral injection of the dopaminergic neurotoxin 6-hydroxydopamine can promote recovery of motor function in that limb and can significantly decrease damage to dopamine terminals. The present study was conducted to determine (1) whether a form of voluntary exercise, wheel running, would improve motor performance in rats with such lesions, and (2) whether any beneficial effects of wheel running are attributable to ameliorating the dopaminergic damage. In experiment 1, rats were allowed to run in exercise wheels or kept in home cages for 2 1/2 weeks, then given stereotaxic infusions of 6-hydroxydopamine into the left striatum. The rats were replaced into their original environments (wheels or home cages) for four additional weeks, and asymmetries in forelimb use were quantified at 3, 10, 17, and 24 days postoperatively. After killing, dopaminergic damage was assessed by both quantifying 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester ([(125)I]RTI-55) binding to striatal dopamine transporters and counting tyrosine hydroxylase-positive cells in the substantia nigra. Exercised 6-hydroxydopamine-infused rats showed improved motor outcomes relative to sedentary lesioned controls, effects that were most apparent at postoperative days 17 and 24. Despite this behavioral improvement, 6-hydroxydopamine-induced loss of striatal dopamine transporters and tyrosine hydroxylase-positive nigral cells in exercised and sedentary groups did not differ. Since prior studies suggested that forced limb use improves motor performance by sparing nigrostriatal dopaminergic neurons from 6-hydroxydopamine damage, experiment 2 used a combined regimen of forced plus voluntary wheel running. Again, we found that the motor performance of exercised rats improved more rapidly than that of sedentary controls, but that there were no differences between these groups in the damage produced by 6-hydroxydopamine. It appears that voluntary exercise can facilitate recovery from partial nigrostriatal injury, but it does so without evident sparing of dopamine nerve terminals.

beta(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter.

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that beta(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition beta(3)-integrin regulates u-PAR expression. Overexpression of beta(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (-1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when beta(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

Use of large-scale veterinary data for the investigation of antimicrobial prescribing practices in equine medicine.

BACKGROUND: As antimicrobial resistant bacterial strains continue to emerge and spread in human and animal populations, understanding prescription practices is key in benchmarking current performance and setting goals. Antimicrobial prescription (AP) in companion veterinary species is widespread, but is neither monitored nor restricted in the USA and Canada. The veterinary use of certain antimicrobial classes is discouraged in some countries, in the hope of preserving efficacy for serious human infections. OBJECTIVES: The aim of this study was to ascertain the rate of prescription of a number of 'reserved' antimicrobials in a first-opinion US and Canadian horse cohort, and identify trends in their empirical use. STUDY DESIGN: Retrospective cohort study. METHODS: A large convenience sample of electronic medical records (2006-2012) was interrogated using text mining to identify enrofloxacin, clarithromycin and ceftiofur prescriptions. Time series analysis and logistic regression were used to identify trends and risk factors for prescription. RESULTS: Prescription of these antimicrobials as a first-line intervention, without culture and sensitivity testing (CST), was common in this population. Enrofloxacin prescriptions were found to increase over the study period, and there was evidence of either a reducing, or static trend in the proportion of reserved APs informed by CST. MAIN LIMITATIONS: Dose adequacy could not be included due to the nature of the data used. CONCLUSIONS: Empirical use of reserved antimicrobials was common in this population, and further advice and guidance should be issued to first-opinion veterinarians to safeguard antimicrobial efficacy.

Tissue uptake, distribution, and potency of the photoactivatable dye chloroaluminum sulfonated phthalocyanine in mice bearing transplantable tumors.

The potency of chloroaluminum sulfonated phthalocyanine (ClAlSPc) as a photosensitizing agent for photodynamic therapy of cancer was evaluated in vivo by its ability to be taken up and retained by murine tumors of diverse histological origin. Antitumor effects following laser irradiation were evaluated by measurement of the tumor weights of dissected-out tumor masses. Three tumors (Colo 26, a colorectal carcinoma; M5076, a reticulum cell sarcoma; and UV-2237, a fibrosarcoma) growing s.c. in the flank region retained substantially greater quantities of ClAlSPc than did adjacent skin and muscle achieving peak values 24-48 h after the i.v. administration of ClAlSPc (10 mg/kg). The relative magnitude of ClAlSPc retention by these tumors was Colo 26 greater than M5076 greater than UV-2237. However, normal liver and spleen were organs which retained the greatest amounts of ClAlSPc even compared to the s.c. grown tumors and other normal tissues examined. Flow cytometric analysis of tumor cell suspensions obtained from collagenase-digested tumors showed that individual neoplastic cells were capable of taking up and retaining ClAlSPc. Photodynamic therapy, undertaken by i.v. administration of dye (5 mg/kg) followed 24 h later by local laser light irradiation (675 nm, 100 J), brought about significant (Colo 26, M5076, and 3LL tumors) and obvious but nonsignificant (UV-2237 tumor) reductions in tumor weights, as assessed 5 days later. Thus, selective tumor retention of ClAlSPc coupled with a significant response to red light produced dramatic alterations in cancer growth.

Identification by differential display of annexin-VI, a gene differentially expressed during melanoma progression.

To identify genes involved in the melanocyte to malignant melanoma conversion, we have applied differential display to the comparison of syngeneic murine B16F10 (metastatic melanoma) and Melan-a-immortalized melanocyte cell lines. Approximately 7000 bands were analyzed, revealing approximately 80 to be differentially displayed. Reverse Northern blotting and subsequent Northern blotting confirmed the reproducible differential expression of four transcripts. Three B16F10-specific bands encode novel genes or partially sequenced cDNAs of unknown function. One Melan-a-specific band was found to be identical to the 3' end region of the mouse Annexin VI mRNA and shown to have a reduced message expression in B16F10 relative to Melan-a. Differential expression was confirmed at the protein level with Western blotting using a rabbit polyclonal antiserum. Immunohistochemistry of human melanoma specimens with this antiserum revealed a decrease or loss of Annexin VI expression as melanomas progressed from a benign to a more malignant phenotype. Our results provide further evidence for a potential role of Annexin VI in tumor suppression.

Effect of sulfonation on the cell and tissue distribution of the photosensitizer aluminum phthalocyanine.

Aluminum sulfonated phthalocyanine has potential as a suitable photosensitizer for use in the photodynamic therapy of cancer. In the present study, cellular uptake and retention of the individual mono-, di-, tri-, and tetrasulfonated derivatives (AlS1-4Pc) were examined in tissue culture and in normal and neoplastic tissue of tumor-bearing mice. Uptake and retention of the various derivatives by cells in tissue culture correlated inversely with the degree of sulfonation. Accordingly, Colo 26 cells in monolayer culture, 24 h after addition of 10 microM of appropriate photosensitizer, had accumulated approximately 25-fold more AlS1Pc than AlS3Pc and retained this species longer than more sulfonated derivatives. In contrast to these in vitro results, it was found that Colo 26 growing s.c. in BALB/c mice accumulated photosensitizer to a greater extent when the degree of sulfonation increased, such that A1S4Pc greater than AlS3Pc greater than AlS2Pc greater than AlS1Pc. By 24-48 h after the i.v. injection of 0.1 ml 2.27 mM solution of individual photosensitizer, the relative ratios of tumor:adjacent tissue varied from greater than 10:1 to greater than 2:1, showing that selective tumor uptake may be affected profoundly by the composition of the phthalocyanine compound. The livers and spleens of both normal and tumor-bearing mice, unlike other normal tissue, took up the sulfonated derivatives in an order that provided a mirror image of that observed in neoplastic tissue. These complex in vivo distribution and retention characteristics appear to be a consequence of relative hydrophilicity/hydrophobicity properties of the sulfonated species and indicate the extent to which these characteristics may influence photosensitizer distribution and accumulation.

Influence of tumor-derived interleukin 1 on melanoma-endothelial cell interactions in vitro.

Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.

Suppression of TGFß and Angiogenesis by Type VII Collagen in Cutaneous SCC.

BACKGROUND: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGFß signaling and angiogenesis in vitro and in vivo. METHODS: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGFß pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. RESULTS: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between α2ß1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFß signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFß markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFß markers, n = 6; VEGF, n = 21). Inhibition of TGFß receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siTßRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFß signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.

Fos protein expression and cocaine-seeking behavior in rats after exposure to a cocaine self-administration environment.

To examine neuronal activation associated with incentive motivation for cocaine, cocaine-seeking behavior (operant responding without cocaine reinforcement) and Fos expression were examined in rats exposed to saline and cocaine priming injections and/or a self-administration environment. Rats were first trained to self-administer cocaine or received yoked saline administration ("control"). They then received 21 daily exposures to either the self-administration environment ("extinction") or a different environment ("no extinction") without cocaine available. Extinction training, used to decrease incentive motivation for cocaine elicited by the self-administration environment, decreased cocaine-seeking behavior elicited by both the environment and the cocaine priming injection. Exposure to the self-administration environment enhanced Fos expression in the no extinction group relative to control and extinction groups in the anterior cingulate, basolateral amygdala, hippocampal CA1 region, dentate gyrus, nucleus accumbens shell and core, and central gray area, regardless of whether or not priming injections were given. The priming injections enhanced Fos expression in the ventral tegmental area, caudate putamen, substantia nigra pars reticulata, entorhinal cortex, central amygdala, lateral amygdala, arcuate nucleus, and central gray area, regardless of group. Thus, these changes likely reflect an unconditioned effect from either cocaine or injection stress. The priming injections also enhanced Fos expression in the anterior cingulate, but only in cocaine-experienced groups, suggesting that this enhancement reflects an experience-dependent motivational effect of the priming injections. The results suggest that different neural circuits may be involved in the incentive motivational effects of cocaine-paired environmental stimuli versus priming injections and that the anterior cingulate may be part of a common pathway for both.

Regulation of apolipoprotein gene expression and plasma high-density lipoprotein composition in experimental nephrosis.

Hepatic and intestinal RNA levels were measured in rats made nephrotic by injection of puromycin aminonucleoside (PAN). The following increases in hepatic RNA levels, relative to controls, were measured: poly A+ (1.2), ribosomal (1.2), mRNA levels for transferrin (1.8), albumin (3.8) apolipoprotein (apo)E (2.3), apoB (2.5), apoA-II (1.9) and apoA-I (6.1). Increases of 1.5- to 2.2-fold in hepatic mRNA levels for albumin, apoA-II, apoB and apoE were measured in pre-nephrotic animals killed before the onset of proteinuria. Intestinal RNA levels in pre-nephrotic and nephrotic animals were not significantly different from control values. Transcription of the hepatic apoA-I gene increased 1.8-fold in nephrotic animals compared to controls. Immunological detection of apolipoproteins in high-density lipoproteins (HDL) separated by gradient gel electrophoresis indicated an increase in apoA-I and a decrease in apoA-IV and apoE containing HDL particles in nephrosis. To simulate the effects of increased apoA-I gene expression, human apoA-I was added to rat plasma in vivo and in vitro. ApoE was displaced from HDL by increased concentration of apoA-I. The results indicate that relatively small changes in apoA-I levels in the serum lead to significant changes in the apolipoprotein composition of HDL.