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Infection of Immunodeficient Horses with Sarcocystis neurona Does Not Result in Neurologic Disease

ABSTRACT Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 × 105 sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 × 108 merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.

Equine protozoal myeloencephalitis (EPM) is one of the most common neurologic disorders of horses in the Americas. Although there are occasional reports of neurologic disease in horses due to Neospora hughesii, EPM is most frequently associated with infection with the apicomplexan parasite Sarcocystis neurona (9). Despite recent advances in our understanding of the life cycle of S. neurona (1,2,10), very little is known about the pathogenesis of infection in horses (9). S. neurona can parasitize all regions of the equine central nervous system (CNS). The route of migration of the parasite from the time of ingestion of sporocysts to neuroinvasion is unknown. Studies of gamma interferon (IFN-␥) knockout (GKO) mice, which develop fulminant neurologic disease after infection, suggest that S. neurona may initially multiply in visceral tissues (6,8).
The major goal of our laboratory is to investigate the pathogenesis of S. neurona infection in horses by determining steps in the progression of infection and the role of innate and adaptive immunity in the control of infection and neuropathology. GKO mice develop profound neurologic disease when they are infected with S. neurona (6,8), suggesting that IFN-␥ is crucial in the elimination of that infection in mice. IFN-␥ is produced by lymphocytes during an adaptive Th1 immune response, and adaptive immune responses are presumed to be important in the protection of horses from S. neurona. However, in the absence of adap-tive immune responses, mice are protected from Toxoplasma gondii infection, presumably through the production of IFN-␥ by cells of the innate immune system (5,13). Similarly, two mice with severe combined immune deficiency (SCID) experimentally infected with S. neurona did not develop neurologic disease (18). In a preliminary experiment, we infected an Arabian horse with SCID (19,28) with sporocysts of S. neurona. The infected horse became parasitemic, and S. neurona merozoites were isolated from the blood on postinfection day (PID) 21 (16). This isolate was designated the WSU-1 strain of S. neurona. Although this SCID horse did not exhibit convincing neurologic deficits, S. neurona DNA was detectable by PCR after long-term culture of brain tissue obtained at necropsy.
In the present study, we further investigate the pathogenesis of S. neurona infection in SCID and immunocompetent (IC) horses. Horses were infected orally with sporocysts isolated from the feces of opossums or intravenously (i.v.) with merozoites of the WSU-1 strain of S. neurona. Parasitemia was monitored after infection by collection of blood samples for culture and PCR. Horses were monitored for the development of neurologic abnormalities. At the time of necropsy, tissues were collected for culture, PCR, immunohistochemistry (IHC), and histopathology to determine the presence of S. neurona and associated inflammatory lesions in peripheral tissues or the CNS.

MATERIALS AND METHODS
Experimental animals. All horses were obtained from the Arabian herd at Washington State College of Veterinary Medicine and were the offspring of horses that were heterozygous for SCID (28). All mares were seronegative for antibodies to S. neurona, and foals were allowed to suckle colostrum immediately after birth. A complete blood count was performed on each foal 24 to 48 h after birth to identify immune status. Foals with blood lymphocyte counts of Ͻ200/l were identified as probable SCID horses. Their immune status was subsequently confirmed by PCR for detection of the specific mutation in the DNA-dependent protein kinase gene. SCID foals were removed from their dams and placed in isolation stalls as soon as their status was confirmed. IC foals were weaned from their dams a few days prior to scheduled infection. All horses were anesthetized with xylazine (1.1 mg/kg of body weight i.v.) and ketamine (2.2 mg/kg i.v.) prior to infection for collection of cerebrospinal fluid (CSF) from the atlanto-occipital space. Serum and CSF from each horse were analyzed by Western blotting to confirm the absence of specific antibodies to S. neurona (Equine Biodiagnostics, Inc., Lexington, Ky.). SCID horses were maintained using procedures previously described (19), except that they did not receive trimethoprim-sulfa antibiotics. Horses were infected with S. neurona at 2 to 4 weeks of age.
Infection with S. neurona. Sporocysts of S. neurona were obtained from the intestines and feces of wild opossums or opossums experimentally infected with S. neurona as previously described (1,2,4). Prior to infection, sporocysts were identified as S. neurona by a PCR protocol which differentiates S. neurona from the closely related organism Sarcocystis falcatula (27). Horses were infected orally with 5 ϫ 10 5 sporocysts of S. neurona as previously described (16). For i.v. infections, merozoites of the previously described WSU-1 strain of S. neurona were harvested from culture (16). Merozoites were washed and resuspended in medium, and 5 ϫ 10 8 were injected i.v. via the jugular vein. IC horses were anesthetized as described above after infection to obtain CSF for Western blotting to detect an antibody response to S. neurona. To assess parasitemia and seroconversion, blood for culture and Western blotting, respectively, was obtained by jugular venipuncture (16). Horses were monitored for development of neurologic abnormalities. All SCID horses and selected IC horses were euthanized by pentobarbital overdose, and a complete necropsy was performed to collect tissues (cerebral cortex, brain stem, spinal cord, spleen, liver, kidney, cardiac muscle, skeletal muscle, tongue, diaphragm, and adrenal gland) for culture, PCR, and histopathology. A CSF sample was obtained from the atlantooccipital space of each horse immediately after euthanasia.
Identification of parasites in blood and tissue samples. In order to assess the presence and duration of parasitemia and determine the tissue distribution of parasites, blood and tissue samples were obtained for PCR and culture as previously described (16). Samples were frozen at Ϫ70°C until processed for extraction of DNA, and PCR was performed to identify S. neurona as previously described (27). Purified DNA samples were amplified, and reaction products were analyzed by electrophoresis through an agarose gel stained with ethidium bromide. Controls consisted of a no-DNA control and either uninfected equine tissues or noninoculated cell cultures. Positive controls included DNA derived from cultured bovine turbinate cells infected with S. neurona. As an additional positive control, samples were reacted with primers that amplify a segment of the equine tumor necrosis factor alpha gene. Aliquots of blood or tissue were cultured on bovine turbinate cells as previously described (16). Cultures were maintained for 3 months or until cells were losing viability because of senescence or infection. At that time, cells were harvested, DNA was extracted, and the presence or absence of S. neurona was determined by PCR as previously described (16,27).
Samples of liver, lung, spleen, brain, spinal cord, cardiac muscle, skeletal muscle, and tongue were obtained at necropsy, fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin for routine histopathological evaluation. Slides were examined for evidence of inflammatory changes or parasites. Formalin-fixed, paraffin-embedded samples of CNS were also examined for the presence of S. neurona antigen by IHC using a polyclonal rabbit antiserum against the parasite. Control samples demonstrated that this antibody did not cross-react with related protozoal organisms, such as Neospora caninum (data not shown). Slides stained for IHC were examined by light microscopy for positive staining, indicative of parasite antigen in tissues.

Infection of IC horses. (i) Clinical outcome.
The clinical observations after oral and i.v. infection of IC horses are shown in Table 1. After oral infection, both IC horses remained systemically healthy and afebrile and their serum and CSF were antibody positive by PID 30. Horse IC1 exhibited ataxia and proprioceptive deficits of all four limbs beginning on PID 42. Clinical signs were improved from PID 48 through PID 66.
This horse was not euthanized. At approximately 17 months postinfection, neurological examination revealed continuing mild proprioceptive deficits. However, the antibody to S. neurona was slightly positive in the serum and negative in the CSF at that time. Horse IC2 did not exhibit abnormal neurologic signs and was euthanized on PID 90.
Horses IC3, IC4, and IC5 had a mild to moderate febrile response for 2 to 3 days after i.v. infection. Otherwise, they remained systemically healthy and afebrile throughout the infection period and became serum and CSF antibody positive after infection. Horses IC5 and IC6 developed neurologic signs at PIDs 28 and 50, respectively. They exhibited ataxia and proprioceptive deficits in all four limbs and remained neurologically abnormal until they were euthanized on PIDs 41 and 73, respectively. Horse IC3 did not develop signs of neurologic disease after infection. That horse was treated with dexamethasone at 15 mg intramuscularly once daily for 8 days (approximately 0.1 mg/kg) beginning on PID 72 and euthanized on PID 80.
Horse IC4 did not develop neurological abnormalities during the 73-day observation period after infection and was returned to the Washington State University research herd. At that time, the horse had strongly positive serum and CSF antibody responses to S. neurona as detected by Western blotting. At PID 289, the horse was observed to have moderate ataxia and proprioceptive deficits in all four limbs but was otherwise clinically healthy. Serum and CSF exhibited low positivity and weak positivity (as reported by the lab running the assay), respectively, for antibody to S. neurona. Standing lateral radiographs of the cervical spine were considered to be within normal limits. The horse was treated conservatively with stall and paddock rest. Neurological abnormalities persisted, and the horse was euthanized on PID 471. At the time of euthanasia, serum and CSF were weakly positive for antibodies to S. neurona. No cause for the neurological abnormalities was apparent on gross or histopathological examination of tissues obtained at necropsy. Culture of neural tissues was negative for DNA of S. neurona, but a sample of the spinal cord obtained at the level of the first thoracic vertebra was weakly positive by PCR.
(ii) Parasitemia. Blood was collected from all horses for parasite culture at frequent intervals throughout the infection period ( Fig. 1 and 2). After oral infection, parasites were not detected in the blood of orally infected IC horses (IC1 and  Table 1. Oral infection of horses SCID1 and SCID2 resulted in no obvious neurologic signs; these horses were euthanized on PIDs 32 and 21, respectively. Horse SCID1 had a serum immunoglobulin G concentration of Ͻ400 mg/dl at 24 h of age and was treated i.v. with 1.5 liters of fresh plasma from his dam and intramuscularly with amikacin and sodium ceftiofur. A patent urachus and umbilical abscess developed. Antimicrobial therapy was continued until the horse was euthanized on PID 32 due to severe pneumonia. Horse SCID2 exhibited a dry cough and intermittent fever throughout the infection period. He was treated with sodium ceftiofur and/or gentamicin intermittently beginning on PID 14. However, the horse's condition continued to deteriorate, and he was euthanized on PID 21 due to severe pneumonia. Equine adenovirus, a common opportunistic pathogen of SCID horses, was isolated from samples of lung collected at necropsy (19). The sera and CSF of both SCID horses were antibody negative to S. neurona by Western blotting at the time of euthanasia.
Horse SCID3 was systemically healthy at the time of infection and did not exhibit a febrile reaction after i.v. injection of merozoites. This horse remained healthy throughout most of the infection period and was not treated with antimicrobial agents. The horse became persistently febrile at approximately PID 84, at which time he exhibited signs of respiratory tract disease that worsened until euthanasia on PID 95. Lung tissue obtained at necropsy was histologically normal. The horse remained neurologically normal on clinical examination and serum and CSF antibody negative to S. neurona by Western blotting throughout the experimental period.
(ii) Parasitemia. Blood was collected from all horses for parasite culture at frequent intervals throughout the infection period ( Fig. 1 and 2). Although direct PCR of DNA extracted from blood samples of SCID horses was negative at all time points, parasites were isolated from horses SCID1 and SCID2 by culture of blood samples on bovine turbinate cells on multiple days postinfection, beginning at PIDs 7 and 8, respectively. These parasites were morphologically indistinguishable from known isolates of S. neurona at the light microscopy level (data not shown). Amplification of DNA segments by use of primer pairs specific for S. neurona (27) confirmed the identity of all isolates (data not shown). Although the blood sample obtained from horse SCID3 at 3 h after i.v. infection was culture positive for parasites, confirming the viability of the inoculum administered to that horse, S. neurona was not isolated from any subsequent blood samples until PID 95, the day of euthanasia.
(iii) Postmortem evaluation. Culture of peripheral tissues, but not CNS tissues, from horses SCID1 and SCID2 yielded S. neurona. Liver, spleen, and skeletal muscle from horse SCID1 and skeletal and cardiac muscle from horse SCID2 were culture positive. Horse SCID1 was PCR positive for S. neurona in lung, cardiac muscle, spleen, liver, skeletal muscle, kidney, and tongue. Only the lung tissue of horse SCID2 was PCR positive. Peripheral tissues of horse SCID3 (heart, lung, liver, spleen, and skeletal muscle) were PCR and/or culture positive, but CNS tissues from this horse were consistently negative. Histopathologic evaluation of hematoxylin-and eosin-stained tissue sections revealed bronchopneumonia in horses SCID1 and SCID2 and mild multifocal hepatic necrosis in horse SCID3. Horse SCID1 also had evidence of moderate follicular lymph node necrosis. The results of IHC of CNS tissues from all SCID horses, with a polyclonal rabbit anti-S. neurona antiserum, were negative.

DISCUSSION
The present work demonstrates that infected SCID horses are unable to terminate parasitemia or clear S. neurona from visceral tissues, but the infection does not result in clinical signs of neurologic disease; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but experience neuroinvasion and clinical signs of neurologic disease. This finding suggests that adaptive immune responses are important for the termination of parasitemia and control of parasite reproduction in peripheral tissues but are not necessary for the prevention of signs of neurologic disease after infection.
Arabian horses with SCID lack specific B-and T-cell responses because of a mutation in the DNA-dependent kinase catalytic subunit (23). These horses have been used experimentally to determine the role of adaptive immune responses in the pathogenesis of equine infectious anemia and equine piroplasmosis (babesiosis) (14,20), and conditions for their maintenance have been described previously (19). The use of potentiated sulfonamide antimicrobials is recommended in these foals to prevent Pneumocystis carinii infection (19). However, because of the parasiticidal effect of these antimicrobials, they could not be used for the present study. Instead, two of the three SCID horses used in this study were treated with gentamicin or sodium ceftiofur when they showed signs of pneumonia. These drugs do not have significant parasiticidal activity   1, 2, 10), very little is known about the pathogenesis of infection in horses (9). S. neurona can parasitize all regions of the equine CNS and may be found in neurons, mononuclear cells, and glial cells. Studies of GKO mice, which develop fulminant neurologic disease after infection, suggest that S. neurona strains initially multiply in intestinal tissues, with parasitemia occurring 1 to 8 days after infection (6,7). We have previously reported the isolation of merozoites of S. neurona from the blood of a SCID horse after oral infection with sporocysts (16). The present report confirms the presence of persistent parasitemia in SCID horses after oral infection. The absence of detectable parasitemia in similarly infected IC horses suggests that parasitemia is controlled by specific immune responses.
After oral infection, SCID horses lived 21 to 98 days. At the time of necropsy, parasites were widely disseminated throughout the peripheral tissues of these horses, but there was no evidence of neuroinvasion or neuropathology. In contrast, S. neurona was detected in the CNS of IC horses with abnormal neurologic examination by PCR and/or culture. There was no evidence of pathology attributable to S. neurona infection (inflammation or parasites) in the CNS of any foal, and IHC was negative for S. neurona in all foals. It has been difficult for investigators to demonstrate pathology or parasites in the CNS after experimental infection of horses even when neurologic signs are evident. Saville et al. reported the presence of minimal numbers of or no inflammatory CNS lesions and no evidence of parasites in horses infected with S. neurona after transport stress (21). Fenger et al. reported mild to moderate inflammatory lesions consistent with S. neurona infection in three of five experimentally infected horses but no evidence of parasites (11). The present report provides the first evidence of parasites in the CNS of horses that develop neurologic disease after experimental infection.
Very little is known about innate or adaptive immune responses to S. neurona in horses. In murine models of infection, neurologic disease has been demonstrated only for GKO and nude mice (8,18). IC BALB/c mice apparently clear infection without neural invasion (6,8,16). In nude and GKO mice, neuropathology is first evident at approximately 14 to 21 days postinfection (6,7,18), which is consistent with our conclusion that parasitemia precedes neural invasion. After oral infection, SCID horses demonstrated parasitemia as early as 7 to 8 days postinfection.
The lack of neuropathology in SCID horses may have been the result of low virulence of the inoculum or insufficient time between infection and euthanasia, but these possibilities were considered unlikely. Four of six IC horses in the present study developed neurologic abnormalities after infection with sporocysts or merozoites of S. neurona, suggesting that a lack of neurovirulence was not a significant factor in these experiments. In addition, merozoites of the WSU-1 strain of S. neurona used for the i.v. infections in the present study have previously been shown to be neurovirulent in GKO mice (16). SCID horses lived only 21 and 30 days after oral infection; however, this length of time is sufficient for neuropathology in susceptible mice and these horses were persistently parasitemic from PIDs 7 and 8. In addition, horse SCID3 lived for Ͼ90 days after i.v. infection with no neurological abnormalities or evidence of parasite invasion into the CNS, despite widespread dissemination of S. neurona through peripheral organs. Despite these considerations, we cannot eliminate the possibility that neurologic signs might have developed in SCID foals if they had survived longer after infection.
IC horses that developed neurologic disease first showed clinical signs at 28 to 289 days postinfection. Horse IC4 was first observed to demonstrate neurologic abnormalities at PID 289. However, this horse was not closely evaluated for neurologic disease after PID 73, and subtle abnormalities could have been present quite some time prior to presentation at PID 289. This horse had strong blood and CSF Western blot antibody responses to S. neurona at PID 68. At the time clinical signs were noticed, the CSF and serum antibody responses were much weaker and continued to decline throughout the observation period. The declining antibody response is inconsistent with current clinical guidelines for the diagnosis of EPM due to S. neurona infection (3,9,17). However, there was no other cause for neurologic disease evident at necropsy, and the spinal cord of this horse was positive for S. neurona by PCR.
It is possible that the lack of neurovirulence in SCID horses is a result of either protective innate immune responses or a requirement for adaptive immune responses in the pathogenesis of neurologic disease. GKO mice rapidly succumb to infection with S. neurona, suggesting that IFN-␥ is crucial in elimination of this infection in mice (8,22,26). IFN-␥ plays the main role in host resistance to toxoplasmic encephalitis (24,25) and cryptoporidiosis (12) in mice and is produced by a variety of cell types, including CD8-positive T cells, Th1 cells activated during adaptive immune responses, ␥␦ T cells, natural killer (NK) cells, and a population of non-T, non-NK cells in the brain. Mice with SCID and a BALB/c genetic background do not develop neurologic disease after infection with S. neurona unless they are treated with an antibody to block the effects of IFN-␥ (22a). Similarly, doubly immunodeficient C57BL/6 SCID IFN-␥ knockout mice are more likely to become moribund or die after infection with C. parvum than are mice carrying only the SCID mutation or only the IFN-␥ knockout mutation (12).
Although SCID horses lack adaptive B-and T-cell responses, they should have functional innate immunity, including phagocytic and NK cell function, and it is possible that these innate responses are sufficient to control neural invasion by S. neurona. It is also possible that local production of IFN-␥ by nonlymphoid cells in the CNS is the determining factor in preventing neurologic disease.
An alternative hypothesis is that B and/or T cells are required for neural invasion and/or neurovirulence. The method by which S. neurona gains entrance to the CNS of horses or mice is undetermined. SCID horses develop persistent parasitemia, consistent with the hypothesis that S. neurona gains entrance to the CNS across the blood-brain barrier. In support of this hypothesis, a model of experimental infection of horses in which the parasite was intracellular in host lymphocytes has recently been described (S. Ellison, T. Kennedy, and K. Brown, abstract presented at the American Association of Veterinary Parasitologists, Nashville, Tenn., 2002). It is possible that SCID horses and SCID mice fail to develop neurologic disease because parasites cannot gain access to the CNS in the absence of lymphocytes. However, we have shown that administration of anti-IFN-␥ to SCID mice results in fulminant neurologic disease in the absence of T or B lymphocytes (our unpublished data).
Stress, especially transport stress, has been suggested to play a major role in the pathogenesis of infection with S. neurona in horses (21). The horses in the present study were stressed by weaning and isolation immediately prior to infection. In addition, horses SCID1 and SCID2 experienced continuous concurrent illness throughout the infection period. Horse SCID3 remained systemically healthy throughout most of the infection period, and parasitemia was demonstrable only at the time of euthanasia when the horse was suffering from a respiratory tract infection. It is possible that the stress of concurrent illness in horses SCID1 and SCID2 contributed to the ease with which parasites were isolated from blood samples obtained throughout the infection period.
Horse IC3 failed to develop evidence of neurologic disease after i.v. infection. Prior to euthanasia, this horse was treated for 8 days with dexamethasone at a dose that is sufficient to suppress immune responses and induce a relapse of equine infectious anemia virus in carrier horses (15), but there was no evidence of exacerbation of parasite infection in this horse on the basis of clinical signs or evaluation of tissues obtained at necropsy. The severe hepatic lipidosis observed in this horse was attributed to the effects of corticosteroids.
In the absence of specific adaptive immune responses, SCID horses infected with S. neurona develop persistent parasitemia and widespread dissemination of parasites throughout peripheral (non-CNS) tissues. However, these horses do not develop fulminant CNS infection, neuropathology, or clinical neurological abnormalities. This may be the result of enhanced innate immune responses controlling parasite replication in neuronal tissues or because of a requirement for adaptive responses for neuroinvasion or neurovirulence. The pathogenesis of S. neurona infection in SCID horses appears to be very similar to that of infection in SCID mice. Further investigations with SCID mouse and horse models are important to help define the role of the immune response in the pathogenesis of CNS disease in horses.


extfieldsofstudy: Biology Medicine

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Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina) Lymphocytes

Protein kinases (PKs) play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs), successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine) kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK) and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

INTRODUCTION
Protein kinases (PKs) play critical roles in cellular functions including signal transduction, cell cycle regulation, cell division and cell differentiation (Hunter et al., 1985;Edelman et al., 1987). Signal transduction controls many critical and complex cellular functions, including activation of lymphocytes in the adaptive immune response. Signal transduction through the B-and T-cell receptors (BCR and TCR, respectively) and cytokine receptors on the surface of lymphocytes occurs largely via tyrosine phosphorylation of intracellular substrates by protein tyrosine kinases (PTKs). Tyrosine kinases (TKs) play a major role in many disorders of cell proliferation, differentiation, survival and migration, which are fundamental to many diseases and abnormalities.
Although research in this area is limited and relatively recent, there is already experimental evidence that certain halogenated aromatic hydrocarbons [HAHs such as polychlorinated biphenyls (PCBs) and 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD)] and polycyclic aromatic hydrocarbons (PAHs)-affect tyrosine kinases.
For example, in vitro exposure of murine B lymphocytes to TCDD increased membrane protein phosphorylation and, in particular, stimulated tyrosine-specific protein phosphorylation (Clark et al., 1991). Similarly, certain PAHs have been shown to activate PTKs in human cell lines-specifically, FYN and LCK in T-cells and LYN and SYK in B cells (Archuleta et al., 1993;Mounho and Burchiel, 1998). These authors also reported that mobilization of intracellular calcium was coupled to increased tyrosine phosphorylation, and suggested that PAH-induced PTK activation and increased cellular Ca 2þ may alter antigen receptor signaling in human B cells.
Disruption of immune function associated with tissue accumulation of halogenated aromatic hydrocarbons in the marine environment has been suggested as a factor altering health in several marine mammal species (Addison, 1989;Tanabe et al., 1994). For example, it has been speculated that contaminant-induced immunosuppression may have contributed to the high mortality observed in several marine mammal populations during recent morbillivirus epizootics (Hall et al., 1992;Aguilar and Borrell, 1994). HAHs and PAHs are known to elicit a broad spectrum of immunotoxic effects in laboratory animals (Kerkvliet and Burleson, 1994;White et al., 1994). In marine mammals, PAHs and organochlorines (such as PCBs and DDT) also have been associated with impaired immunological function (Martineau et al., 1994;Lahvis et al., 1995;Ross et al., 1996;Beckmen et al., 2003). Recently, certain PCB and PAH compounds were shown to suppress harbor seal T-cell mitogenesis in vitro (Neale et al., 2002).
PKs provide the machinery for the differentiation and activation of lymphocytes, processes critical to cellmediated immunity and host resistance to pathogens. As part of a larger investigation of potential contaminantinduced immune alterations in the harbor seal, we wished to identify key signal transduction molecules in seal lymphocytes homologous to human and rodent PKs, for use as molecular biomarkers of contaminant-induced changes in kinase gene expression. We describe here the first nucleotide (partial) sequences of protein kinase genes in the harbor seal.

Sample Collection
Free-ranging harbor seals were captured as described previously (Neale et al., 2002) and blood drawn from the extradural vein into sterile evacuated blood collection tubes containing acid citrate dextrose (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA; NMFS Scientific Research Permit Nos. 555 -1565, 373 -1575). Material used for this study was collected from three clinically healthy seals caught in central California, including an adult female from San Francisco Bay on 7/16/01, a yearling male from San Francisco Bay on 1/24/02, and a male subadult from Monterey Bay on 2/7/02.

RNA Isolation
Within 12 h of collection, PBMC were isolated from approximately 8.5 ml whole blood by centrifugation of blood for 20 min at 300g. Buffy coats were diluted 1:2 in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA). Mononuclear cells were further purified by density gradient centrifugation (30 min at 700g) using 5 ml Histopaque-1077 (Sigma-Aldrich). The resulting white blood cell layer was removed and washed twice in Hank's Balanced Salt Solution (JRH Biosciences, Lenexa, KS, USA) with 10 min centrifugations at 200g. The white blood cell pellet was transferred to a 15-ml conical tube and TRIzol reagent (GibcoBrl/Life Technologies, Gaithersburg, MD, USA) was added in increments of 200 ml, with repetitive pipetting to ensure complete lysis of cells, for a total of 1 ml. After 10 min incubation, the mixture was transferred to a 1-ml microcentrifuge tube. RNA was isolated (phenol -chloroform separation) and precipitated from the aqueous phase with isopropanol, according to the manufacturer's instructions. The RNA pellet (approximately 20 ug) was then redissolved in 20 ml diethyl-pyrocarbonate (DEPC)-treated RNase-free water (hereafter, "water") and incubated at 608 C for 10 min.

RT-PCR
For reverse transcription, approximately 5 ug RNA (in 5 ml water) was added to a mixture of 1 ml (0.5 ug) oligo(dT) primers, 1 ml dNTP Mix (10 mM), and 5 ml water. This mixture was incubated at 708C for 10 min then cooled on ice. Next, 4 ml of 5 £ First Strand Buffer, 2 ml of DTT (0.1 M) and 1 ml (10 units) RNase inhibitor were added to the first mixture and incubated at 428C for 2 min. Lastly, 1 ml (200 units) Superscript II reverse transcriptase was added for a total volume of 20 ml. This was incubated for 428C for 50 min. The reverse transcriptase enzyme was heat-killed at 708C for 15 min. All reagents supplied from Invitrogen (Carlsbad, CA, USA).
Various protein kinase transcripts were PCR-amplified using degenerate primers derived from the conserved motifs DFG (5 0 ) and DVW (3 0 ) within the catalytic domains of (human) PTK. These have been described in detail previously (Robinson et al., 1996;Kung et al., 1998;Lin et al., 1998). Briefly, DFG is present in virtually all kinases, whereas DVW is primarily encoded by tyrosine kinases, although a small number of serine kinases can also be primed. These two motifs bound a conserved area of approx. 50 aa which, in phosphorylated tyrosine kinases, interact only with target molecules that contain SH2 domains. The 5 0 primer used to encode the amino acid sequence K[V/I][S/C/G]DFG is represented by: 5 0 -AAR RTT DCN GAY TTY GG. The 3 0 primer used to encode the amino acid sequence DVW[S/A][F/Y] is represented by: 5 0 -RHA IGM CCA IAC RTC. The mixed bases were defined as follows: PCR reactions (25 ml) contained 1 ml cDNA, 2.5 ml 10 £ buffer, 0.5 ml each of 3 0 , 5 0 , and dNTP Mix (10 mM), 1.5 ml MgCl 2 (25 mM), 0.5 ml (2.5 units) Taq polymerase, and 18 ml water (PCR profile: 948C, 11 min; 528C, 1 min; 728C, 1.5 min; 31 cycles; 728C, 10 min). PCR products were electrophoresced in an agarose gel. Due to the roughly even spacing between DFG and DVW in all kinases, we expected and obtained a relatively homogeneous PCR product of , 170 bp. This band was excised and DNA purified using the QIAEX II gel extraction kit (Qiagen, Valencia, CA, USA).

Cloning, Transformation, and Sequencing
Purified fragments were cloned into a plasmid vector using the TOPO TA Cloning Kit for Sequencing (Version H, Invitrogen) and the resulting recombinants were used to electro-transform E. coli, according to the manufacturer's instructions. Plasmid DNA was isolated using the Qiagen Plasmid Mini Kit (Qiagen, Valencia, CA, USA). PCR and FIGURE 2 Deduced amino acid sequences corresponding to nucleotide sequences of nine harbor seal PK genes. DFG and DVW motifs underlined. Reading frame starting base and total length (amino acids) given. gel electrophoresis were used at several steps to screen and select positive clones. In addition, because half (8/16) of the clones initially analyzed were identified as ITK, colonies were also screened using restriction endonuclease digestion, in this way we were able to selectively analyze non-ITK clones. Diversity of selection was further enhanced by visualization of slight size variations among positive clones using acrylamide gel electrophoresis. Inserts were sequenced at the DNA sequencing facility (University of California, Davis) using the M13 Reverse universal primer. We used the BLAST program (Altschul et al., 1997) to search protein and DNA databases for sequence similarities.

RESULTS
Clones positive for PK transcripts were generated from all three seals, and multiple clones were identified for most kinases. In all cases, replicate sequences were identical between the DFG and DVW domains. Because the greatest volume of blood (and thus lymphocyte RNA) was obtained from the yearling male, the majority (and greatest diversity) of PK sequences came from this individual.
We obtained nucleotide sequences encoding nine distinct protein kinases, including eight PTK-FYN, LYN and LYNþ , ITK, SYK, v-FGR, DDR2 and RETand one dual kinase (i.e. having both tyrosine and serine/ threonine kinase activities), MAPKK3 (Fig. 1). LYNþ , a variant of LYN tyrosine kinase, contained an insert of 29 bases beginning at 72, but was otherwise identical in nucleotide sequence to LYN. All other sequences were of the expected length (165 -171 nucleotides).
Deduced protein sequences corresponding to the nine harbor seal PK nucleotide sequences are presented in Fig. 2. The 29-base insert of Lynþ causes a shift in reading frame; consequently, the DVW motif is not encoded, but an open reading frame is maintained.
Identification was based on homology with published cDNA sequences. Nucleotide and deduced amino acid sequences were highly similar to human and rodent orthologs (Table I). Additional information based on the corresponding human proteins is provided in Table II.

DISCUSSION
Here, we demonstrated the ability to prime phocid PK cDNA using a human-based degenerate PCR primer mix and we identified, for the first time, protein kinases in an organism within the order Carnivora. Among the kinases identified were several key players involved in signal transduction through the BCR/TCR and activation of B-and T-cells-namely, FYN, LYN, SYK and MAPKK3.
The Src-family kinases FYN and LYN (together with BLK in B cells and LCK in T cells) are responsible for early events in BCR and TCR signaling (Qian and Weiss, 1997;Tsubata and Wienands, 2001). BCR proximal signaling occurs within "lipid rafts" and depends on the tyrosine kinase activity of LYN  (Tsubata and Wienands, 2001;Chakravarty et al., 2002). Likewise, FYN is critical for initiating TCR signaling and plays an important role in T-cell development as well (Howe and Weiss, 1995). Specifically, phosphorylation of the tyrosines in immunoreceptor tyrosine-based activation motifs (ITAMs) by Src-family PTKs serves as the initial intracellular signal indicating that the lymphocyte has detected its specific antigen (Janeway et al., 1999). SYK is a critical PTK in B-cell antigen receptor signaling and B-cell development (Cheng et al., 1995;Turner et al., 1995). Once the ITAMs in the receptor cytoplasmic tails have been phosphorylated, ITAMs in B cells bind SYK (ZAP-70 in T cells). Until SYK has been bound, it is inactive enzymatically. To be activated, it also must be phosphorylated; for this reason, SYK is thought to be important in the negative regulation of receptor TKcoupled signaling processes as well. Once activated, SYK phosphorylates target proteins to initiate a cascade of intracellular signaling molecules (Janeway et al., 1999).
MAPKK3 (mitogen-activated protein kinase kinase 3) is integral to the MAP kinase signaling cascade, one of several pathways leading to activation of transcription factors in the nucleus. The MAP kinase pathway is of great importance in both BCR/TCR signaling and T-cell development (Alberola-Ila et al., 1995;Li et al., 1996;Abbas and Lichtman, 2003). Activated kinase SYK (or ZAP-70 in T cells) activates phopholipase C-g (leading to the diacylglycerol and inositol trisphosphate pathways) and guanine-nucleotide exchange factors (GEFs). GEFs activate small G proteins, which in turn activate MAP kinase cascades. The role of the dual kinase MAPKK3 is to activate, via phosphorylation of a threonine and a tyrosine residue, particular MAP kinases, which then activate transcription factors to induce specific gene transcription leading to cell proliferation and differentiation.
ITK (murine IL-2 inducible T-cell kinase) is expressed in T lymphocytes (and NK cells) and is required for normal T-cell development (Janeway et al., 1999). There is strong evidence that ITK regulates TCR signaling, but the mechanism underlying this role has not been determined (Liao and Littman, 1995). The Tec family PTK preferentially expressed in T cells is ITK (Qian and Weiss, 1997). ITK was disproportionately represented (50%) in our clones, suggesting that ITK may be highly expressed in harbor seal lymphocytes, although the small scale of this preliminary study prevents quantitative assessment of gene expression.
The relevance to signal transduction and lymphocyte activation of the lesser-known TKs DDR2, v-FGR and RET is not clear, although all three appear to be involved in various disease processes. The recently identified receptor tyrosine kinase DDR2 (discoidin domain receptor family, member 2) is unusual in that it is activated by fibrillar collagens (types I and III) rather than a growth factor-like peptide (Vogel et al., 1997;Leitinger, 2003). In addition to its role as a DDR2 ligand, fibrillar collagen matrix can sequester and provide binding sites for immune mediators such as cytokines and chemokines which may lead to local tissue damage (Somasundaram et al., 2002). The activation of DDR2 also mediates the over-expression of matrix metalloproteinase 1 in cells, which is thought to be involved in the metastasis of some tumors (Wang et al., 2001).
V-FGR and RET were identifed as (proto-) oncogenes. V-FGR (feline sarcoma viral oncogene for fibroblast growth factor) arises from a recombination of a cellular structural gene (gamma actin) with a tyrosine kinase gene (c-fgr) (Baker et al., 1998). The transforming activity of v-FGR appears to lie in its TK, which may activate substrates not normally exposed to tyrosine phosphorylation (Sugita et al., 1989). Certain gene rearrangements of the RET receptor TK kinase proto-oncogene are responsible for cancer pathogenesis (Bongarzone et al., 2003). Activating mutations lead to the expression of deregulated products characterized by ligand-independent activation of the intrinsic tyrosine kinase of RET (Lanzi et al., 2003).
Because of their critical role in adaptive immunity, protein kinases (especially PTK) may represent important targets of immunomodulation by xenobiotics in marine mammals. Harbor seal sequences for PTK could serve as molecular biomarkers in semi-quantitative profiling of kinase gene expression. For example, differential expression of specific PTK, following in vitro exposures of seal lymphocytes to model compounds, could be identified via RT-PCR and restriction enzyme digest analysis. This technique has been applied previously to human samples to characterize kinase expression in disease (Robinson et al., 1996;Kung et al., 1998;Mao et al., 2002). In this approach, subsamples of labeled DNA (i.e. radioactive or fluorescent tags) are digested with multiple restriction enzymes, after which digested products are resolved via acrylamide gel electrophoresis. Nucleotide sequences of PK and known restriction sites of endonucleases are utilized to identify kinases and assess differential expression, e.g. for treated vs. control samples. Table III shows an optimized digest for the nine harbor seal PK presented here.


s2fieldsofstudy: Biology Medicine

text:
Ongoing angiogenesis in blood vessels of the abdominal aortic aneurysm

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the αvβ3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of αv mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the αvβ3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the αvβ3 protein. No staining was evident in control aortas for the αvβ3 antigen, tenascin or αv mRNA. The upregulation of av mRNA and the αvβ3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.

Introduction
The pathophysiology of the abdominal aortic aneurysm (AAA), which carries an annual mortality in the USA of ~15,000 (Brophy et al., 1991) is not well understood; and its treatment is restricted to surgical repair with its attendant risks of morbidity and mortality. The pathophysiological understanding of the aneurysm is based largely on biochemical and immunohistochemical studies that indicate aortic wall remodelling and proteolysis of matrix proteins (Tilson et al., 1990). A multicellular inflammatory infiltrate and increased expression of tissue-type and urokinasetype plasminogen activators have also been demonstrated in the aneurysm wall (Koch et al., 1990;Brophy et al., 1991;Schneiderman et al., 1995).
A conspicuous feature of the aneurysm is neovascularization of the aortic wall, which, in contrast to normal, becomes enriched with microvessels (Koch et al., 1990;Tilson et al., 1990;Brophy et al., 1991;Holmes et al., 1995;Schneiderman et al., 1995). Although the significance of the neovascularization remains unclear, Herron et al. (1991) speculated that the new vessels play a sustaining or even a causal role in the pathophysiology of the aneurysm by secreting proteinases that destabilize the aortic matrix. Supportive evidence for this hypothesis comes from immunohistochemical studies in which proteinases such as gelatinase (matrix metalloproteinase 2, MMP-2) (Herron et al., 1991) and collagenase (MMP-1) (Irizarry et al., 1993) have been localized to the neovascular endothelium.
A highly relevant but poorly understood question is whether the neovascularization is an ongoing process in well-developed aneurysms. If neovascularization were ongoing it would signify the presence of active disease in the aneurysm wall and would support the hypothesis that the new vessels are critical in the progression of the aneurysm. We address this question through detection of the αvβ3 integrin in adventitial vessels of aneurysms. The integrin αvβ3 has been proposed to play a critical role in angiogenesis. Brooks et al. have reported that this adhesion molecule is a marker for active angiogenic vessels in man and the chick, and that blocking its activity with a monoclonal antibody suppresses angiogenesis (Brooks et al., 1994).
During angiogenesis, the αvβ3 integrin uses the RGD sequence in the tenth fibronectin (III)-like domain of tenascin to bind to extracellular matrix (ECM) (Joshi et al., 1993;Sriramarao et al., 1993), and tenascin in turn has additional binding sites for matrix proteoglycans and heparin. Expression of αvβ3 is ubiquitous in the embryology and development of the vascular system, and expression of members of the tenascin superfamily is similarly widespread in fetal tissue. In adult tissues, although the αvβ3 integrin may be detectable in low amounts (Suzuki et al., 1993), expression of tenascin isoforms is markedly downmodulated within one month of birth (Saga et al., 1991). Under abnormal circumstances, such as in tumorigenesis and wound healing, αvβ3 expression increases significantly (Suzuki et al., 1987;Felding-Habermann and Cheresh, 1993;Juhasz et al., 1993). Thus, detection of these proteins associated with the neovascularization, as we report in the present work, indicates the presence of active angiogenesis in aneurysms.

Im m unohistochem istry
Aortas were obtained from 17 consecutive patients undergoing elective resection of AAA. All patients presented with abdominal aortic aneurysms of 5 cm in diameter size or greater. None of the patients suffered from rupture of AAA at the time of surgery. The sections of the AAA wall were reviewed macroscopically. Normal control aortic tissues were from 6 organ donors and 1 aorto-occlusive patient (ages average 38 yr; 4 male; 3 female). All tissues were fixed in buffered formalin (pH 7.4) for 16-24 h.
Histological sectioning and immunohistochemistry were processed as in previous work (Fu et al., 1994). Briefly, aortic paraffin embedded tissue sections (4 µm thick) were dewaxed in xylene and absolute ethanol, inhibited for endogenous peroxidase activity in 0.5% hydrogen peroxide in absolute methanol (20 min), and blocked with 0.2% BSA to prevent nonspecific binding. The anti-αvβ3 antibody R838 (1:100) and the anti-tenascin antibodies, EH5B01 (1:50) and TN2 (1:200), were applied to the sections overnight at 4 o C. The anti-eNOS antibodies (1:2000) were applied to slides following a microwaving step used to enhance antigenicity. Following PBS washes, the sections were exposed to the appropriate secondary antibody for 1 h at room temperature. After PBS washes the sections were incubated for 10 min at room temperature with either DAB solution in PBS containing 0.01% hydrogen peroxide, or NBT/BCIP solution, depending on whether the secondary antibody was respectively, HRP or alkaline phosphatase linked. The sections were finally washed in running tap water, counterstained with hematoxylin for DAB or Nuclear Fast Red for NBT/BCIP, dehydrated in a reversed alcohol-xylene series, and mounted for semi-quantitative morphometric analysis, microscopy and photography.

Sem i-quantitative m orphom etric analysis of m icrovessels
Quantitation of microvessel staining by anti-eNOS was carried out by an observer blinded to the source of each specimen, using three criteria: (1) overall impression of staining at 100X magnification, graded on 1 to 4 scale (1 = minimal, 2 = easily detectable, 3 = moderate, 4 = extensive), (2) the number of capillaries identified in the most densely positive 100 × field, and (3) the number of endothelial cells identified in the most densely positive 400 × field.

Reverse transcription (RT) in situ PCR
For in situ PCR, we modified Nuovo's method (Nouvo, 1994) and used the EZ rTth RNA PCR kit as follows: after dewaxing and air drying, all sections were digested in 2 mg/ml pepsin in 0.01 N HCl (45 min, room temperature). The pepsin was inactivated by 1 min washes in ultrapure water and absolute ethanol, then the sections were air dried. As a positive control, amplification of tissue genomic DNA was performed by carrying slides directly on PCR cycles without digestion by DNAse I. For detection, the sections were digested by RNase-free DNase I (400 U/ml in digestive buffer) overnight at 37 o C. To control for nonspecific binding and background staining, after digestion of DNAse I, the slides were further digested by RNase H (50 U/ml in the digestive buffer) for 2 hours at room temperature.
For RT and in situ PCR processes, 50 µl of the reagent mixure (1 × EZ buffer, 200 µM of dNTP, 15 µM of -11-dUTP, 2.5 mM of Mn(AOc)2 solution, 0.45 µM/each of αv or β3 5' and 3' primers, 2.5 U/50 µl of rTth DNA Polymerase, and 60 U RNasin) was added to each section and sealed by the In Situ PCR assembly tool (Perkin Elmer Corp., Newark. CT). After incubation for 25 min at 62 o C for reverse transcription, the PCR cycles were instituted in an in situ PCR Cycler (Ampligen PCR 1000, Perkin Elmer Corp., Newark, CT) under modified conditions (10 cycles: 94 C for 60 s, 65 C for 60 s and 72 C for 120 s).
To determine the specificity of αv primers, we sampled aliquots of the supernatant on tissue sections immediately following the RT in situ PCR procedure, with or without treating the sections with DNase I. The aliquots were loaded on to 1% agarose gel and Southern blotted by αv or β3 probes. These probes (kindly provided by Dr. D. Shinar) were synthesized and labeled with biotin-11-dUTP by PCR amplification of rat αv (RAV 611) or β3 (RIB 494/3) cDN A, using oligonucleotide prim ers (show n as above) (Shinar et al., 1993). The membrane was developed using Streptavidine-biotin-alkalinephosphatase and NBT/BCIP. The sections were chilled at 4 o C for 20 min, and blocked by 0.2% BSA in 0.1 SSC solution at 45 o C for 10 min. Then the slides were incubated in goat anti-digoxigenin antibody conjugated with alkaline phosphatase for 30 min at room temperature. After 3 washes of Tris buffer (0.1 M, pH 7.5), the sections were treated for 10 min at room temperature with NBT (2.5 µl/ml)/BCIP (2.5 µl/ml) in Tris-buffered CaCl2 solution (pH 9.5). The development was stopped by washing in running tap water. The sections were counterstained with Nuclear Fast Red, dehydrated and mounted for viewing.

R esults M icrovessels detected by H&E and anti-eNO S
Histological sections of aortic aneurysms by H&E staining showed the presence of several hemorrhagic regions, large numbers of inflammatory cells and numerous microvessels ( Figure 1). The inflammatory cells consisted mostly of macrophages and lymphocytes that were associated largely with the microvessels in the adventitia. In addition, some sub-intimal and medial regions showed the presence of neutrophils. By contrast, no inflammatory infiltrates or matrix abnormalities were evident in control aortas ( Figure 1D). Anti-eNOS immunohistochemical staining was employed for two reasons. First, detection of the intracellular eNOS enzyme in AAA and control tissue sections confirmed antigenic integrity of the preserved specimens. Second, anti-eNOS staining served as a marker for microvessel quantitation since the antibody detects the endothelial layer of both small and large microvessels.
In normal aortic wall, positive eNOS staining was seen in the endothelial cell layer of microvessels located in the adventitial vasa vasorum. The number of vessels seen per 100 × field was few, varying from none to 6. Intact medial smooth muscle cell nuclei were noted in most sections and medial elastin lamellae, as expected, were preserved in all control sections ( Figure 2B). In AAA sections positive staining was again seen in the endothelial lining of microvessels. These vessels, however, were numerous compared to controls. Of the 10-30 vessels present per high power field (100 ×), more than half were thin-walled capillaries of diameter 10 µm and the remainders were thick-walled microvessels of diameter 25 µm and wall thickness 10 µm (Figures 2A and C). The capillaries were localized to both the subintimal region and the adventitia, while the thick-walled vessels occurred almost exclusively in the adventitia. Also noted in many sections were dense inflammatory infiltrates surrounding both thick-walled microvessels as well as capillaries. These cells, based on their morphology on H&E staining, were mostly macrophages and lymphocytes. Lastly, elastic lamellae, in contrast to controls, were markedly disrupted and depleted. The results of the anti-eNOS morphometric analysis are shown in Figure 3. The differences between AAA and controls were striking. Mean capillaries were increased 15-fold per 100 × field (P 0.001), and mean endothelial cells 8-fold per 400 × field (P 0.001) in 17 AAA specimens by comparison to 7 controls. The general increase in staining for eNOS was highly remarkable in the AAA, although endothelial cells from control patients had approximately equivalent staining reactions on a per cell basis.

Anti-α v β 3 , anti-tenascin, and m RNA α v studies
To determine the specificity of αv primers, we sampled aliquots of the supernatant on tissue sections immediately following the RT in situ PCR procedure, with or without treating the sections with DNase I. Then we blotted the aliquots using our αv probe. As shown in Figure 4, in non-aneurysm tissues a band was detectable only in the absence of DNase I treatment, which is expected because the probe can The aortic wall distribution of the αvβ3 antigen is shown in Figure 5. In aneurysmal aortas, an overwhelming majority of microvessels, including capillaries, stained positively for the αvβ3 antigen. However, no staining occurred over the aortic endothelium, the medial and adventitial stroma, and in most large microvessels. A large number of macrophages, particularly those associated with microvessels, also stained positively. No staining was evident in either control aortas or in aneurysms in which R838 was replaced with non-immune rabbit serum as the primary antibody (negative control).
RT in situ PCR of tissue sections of aneurysms revealed co-localization of the αv mRNA with the αvβ3 antigen detected by immunohistochemistry ( Figure 5). mRNA staining was much more extensive than antigen staining. Almost all vessels, irrespective of diameter or location, most macrophages, and some lymphocytes, stained positively for αv mRNA. We studied β3 mRNA in one aneurysm specimen and found a distribution for β3 mRNA that closely resembled that of the αv mRNA (not shown). PCR products of αvβ3 integrin were not evident in either non-vascularized regions of aneurysms or in nonaneurysmal control aortas ( Figure 5). These results prove that both the transcription and the expression of the αvβ3 integrin are upregulated in aneurysm microvessels. The distribution of tenascin staining around microvessels was closely linked to that of αvβ3. Tenascin staining typically was seen circumscribing thick-walled adventitial microvessels ( Figure  6). Tenascin was also evident in subintimal regions and in areas of medial fibrosis.

D iscussion
Although neovascularization of the aneurysm wall has been previously reported (Koch et al., 1990;Tilson Figure 2. Anti-eNOS immunohistochemical staining (A) AAA wall at 40 × magnification. Most apparent are numerous eNOS-staining microvessels (arrow) outlined in brown stain. They are pictured infiltrating the AAA wall. Greater than 20 distinct lumens are identifiable. There is disarray of the normal regular elastic lamellar architecture (in comparison with B). Although the photomicrograph depicts the expected location for the adventitia/media interface, it is undiscernable. The arrow marks a single capillary, whose endothelial cell is better seen under higher magnification. (see panel C) (B) Non-aneurysmal aortic wall at 40 × magnification. Intact aortic media is free of microvessel invasion and hence dark brown eNOS staining is absent. The adventitia/media interface is clearly visible to the near left (arrow) and loose adventitial tissue (A) is seen to the far left. Long continuous strands of elastic tissue appear translucent, forming a regular orderly pattern within the media (M). Mutiple smooth muscle cells with blue staining nuclei are seen within the aortic media interspersed between elastic lamellae. (C) 100 × magnification of boxed portion of panel A. An endothelial cell (large arrow) outlines a microvessel, staining dark brown with anti-eNOS antibody. Intraluminal translucent red and white blood cells are easily seen at this magnification. Cell nuclei are stained blue but are poorly visualized in endothelial cells that stain positive for eNOS. The tissue stroma contains a single mononuclear inflammatory cell (small arrow). Brophy et al., 1991;Holmes et al., 1995;Schneiderman et al., 1995) the literature does not clarify whether new vessels appear early or late in the disease, or whether the vasculature undergoes continuous development and remodelling as the aneurysm enlarges. Our findings using an anti-eNOS Ab indicate that all layers of the aneurysm wall are vascularized and display characteristics typical of angiogenesis. Most extensive were thick-walled microvessels that displayed outer-medial to adventitial distribution, and thin-walled capillaries that invested the sub-intima. It is possible therefore, that vessels proliferating in the outer wall also developed transwall capillarization as part of a global neovascularization of the aneurysm. Two angiogenesis markers, namely the αvβ3 integrin and tenascin, were widely detectable in these blood vessels. We conclude from these findings, that vascularization of the aneurysm is extensive and that new blood vessels form in the aneurysm wall as a result of ongoing angiogenesis.
Evidence for angiogenesis was particularly conspicuous in the immunohistochemistry and the RT in situ PCR studies. The αvβ3 integrin, a vascular integrin of the cytoadhesin family (Albelda and Buck, 1990), is located in both the luminal and abluminal surfaces of endothelial cells as well in vascular smooth muscle cells, and has wide-ranging functions including cell adhesion and cell spreading (Albelda and Buck, 1990;Damjanovich et al., 1992;Joshi et al., 1993) and vascular permeability regulation (Tsukada et al., 1995). We confirmed that in thick-walled microvessels, both the endothelial lining as well as the smooth muscle layers stained positively for the integrin. Recently, integrin αvβ3 has been shown to be essential for angiogenesis and a mAb that blocks binding of ligands to αvβ3, promoted tumor regression of angiogenic blood vessels in chick embryos (Brooks et al., 1994). The authors suggest that inhibition of ligand binding to αvβ3 suppresses neovascularization and selectively promotes apoptosis of vascular cells. Interestingly, αvβ3 expressing blood vessels in aneurysms of the present study, were typically associated with macrophages that also expressed both the αvβ3 protein and αv mRNA. The αvβ3 integrin mediates removal of apoptotic cells by macrophages (Brooks et al., 1994;Flora and Gregory, 1994). Since apoptosis co-exists with growing tissue (Brooks et al., 1994), the presence of αvβ3 expressing macrophages in the vicinity of αvβ3 expressing vessels, further signifies that these locations were focal areas of active tissue growth and cell death.
From the experiments, two important results were, first, αv mRNA was undetectable in control aortas but strongly detectable in vessels of the aneurysm wall; second, some aneurysm microvessels failed to stain for αvβ3 although they stained positively for αv mRNA. These findings indicate that increased transcription of the αv gene is a significant feature of aneurysm microvessels. Increased transcription of the αv and β3 genes are co-associated (Shinar et al., 1993), hence, transcription of β3 mRNA and β3 expression also increased in these aneurysms. This is indirectly indicated in these experiments, in the increased staining with a polyclonal antibody that recognizes the αvβ3  dimer but not the individual subunits of the integrin (Suzuki et al., 1986). In one case, we determined the distribution of β3 mRNA by Rt in situ PCR and found this distribution to be similar to that of αv mRNA (data not shown). The mismatch between negative avb3 integrin staining and positive av mRNA expression in some areas indicates that increased αvβ3 expression did not always follow increased gene transcription. This may reconcile the apparent inconsistency between our findings and those recently reported by Cheuk and Cheng (2004) in which no significant difference in RNA transcripts for αv and β3 were ob-served in AAA vs. control homogenates. Hence, it is possible that increased αvβ3 expression is restricted to specific regions of active angiogenesis. The immunohistochemical evidence for blood vessel-associated tenascin expression also supports our proposal that angiogenesis was active in these aneurysms. Tenascin is a large oligomeric glycoprotein that appears transiently in the extracellular matrix during tissue modelling and tissue repair (Tremle et al., 1994). Two domains of tenascin, both of which contain the integrin-recognizing arg-gly-asp (RGD) tripeptide sequence, support endothelial cell adhesion  (Joshi et al., 1993). One of these, the third fibronectin-III domain, also supports cell spreading (Sriramarao et al., 1993). The involvement of endothelial integrins and in particular, the αvβ3 integrin is indicated in that endothelial adhesion to tenascin is RGD dependent (Joshi et al., 1993) and is partly inhibited by anti-αvβ3 antibodies (Sriramarao et al., 1993). The ability of endothelial cells to adhere to tenascin (Joshi et al., 1993) suggests that endothelial tenascin receptors play a supportive role for establishing blood vessel structure during angiogenesis. The circumscribed perivascular tenascin expression seen here, is consistent with this notion and suggests that that tenascin provides a cylindrical template for vessel tube formation. Addition of tenascin to a fibronectin-based substratum upregulates the synthesis of three important matrix metalloproteinases in fibroblasts: collagenase (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9) (Newman et al., 1994;Newman et al., 1994). We have reported that these three matrix metalloproteinases are present in significantly higher levels in aneurysms than in control aortic specimens (Newman et al., 1994). Tenascin may promote aneurysm progression by inducing these metalloproteinases in endothelial cells of new blood vessels as well as in mesenchymal cells.
Using anti-eNOS Ab directed against endothelial cells, we observed marked increases in neo-vessel number in the aneurysm wall. Although no conclusions can be made concerning the possible role of eNOS or nitric oxide in aneurysm formation from our study, Kuhlencordt et al. have suggested that alterations in eNOS expression play a role in the formation of AAA (2001). Furthermore, we have shown that the nitric oxide by-product, nitrite, is capable of invoking deleterious changes in connective tissue proteins such as collagen and elastin (Paik et al., 2001). Since levels of nitrogen oxide gases are high in tobacco smoke and cigarette smoking is a strong environmental risk factor for AAA disease (Lederle et al., 2003), future studies will be directed at clarifying the role of nitric oxide and its by-products in the formation of AAA.
Finally, our finding that angiogenesis is active in the mature aortic aneurysm, suggests that a potential intervention site exists for pharmacologically reducing the rate of aneurysm enlargement or preventing the disease in genetically susceptible individuals. The observation by Brooks et al. (1994) that the angiogenesis in tumorigenesis can be arrested by antibody to αvβ3 suggests that anti-angiogenic therapy might be considered among other novel interventions that are presently under investigation, based on recent developments in our understanding of aneurysm pathogenesis. Roosevelt Hospital Center, with NIH grant R01 HL64334-4 to MDT.