Rhesus macaque brain microvessel endothelial cells behave in a manner phenotypically distinct from umbilical vein endothelial cells.

Activation of endothelium is a critical step in leukocyte recruitment to the CNS and in development of neurological diseases, such as HIV-associated dementia. Due to limited availability of early disease course data, it is important to develop in vitro models of the blood-brain barrier (BBB) that can be used to address these early events. No such model of the BBB has been established for the macaque. Here, we characterize rhesus microvascular brain endothelial cells (MBEC), comparing them with rhesus umbilical vein endothelial cells (RUVEC), and discuss their suitability for future use in developing in vitro models of simian immunodeficiency virus (SIV) neuropathogenesis. We conclude that MBEC are distinct from RUVEC with respect to growth characteristics, culture requirements, morphology and expression of surface molecules important for leukocyte adhesion and immune activation.

Macaques with rapid disease progression and simian immunodeficiency virus encephalitis have a unique cytokine profile in peripheral lymphoid tissues.

The influence of host cytokine response on viral load, disease progression, and neurologic lesions was investigated in the simian immunodeficiency virus (SIV)-infected macaque model of AIDS. Cytokine gene expression (interleukin-1beta [IL-1beta], IL-2, IL-6, IL-10, gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and viral loads were evaluated by semiquantitative reverse transcription-PCR in lymph nodes of 5 control animals and 28 animals infected with SIVmac251 at the terminal stages of AIDS. Infected animals showed higher expression of IFN-gamma, IL-6, and IL-10 mRNAs compared with controls. Levels of all cytokines were comparable between animals with rapid (survival, <200 days) or slow/normal (survival, >200 days) disease progression. However, among rapid progressors, the eight animals with SIV encephalitis had a unique cytokine profile (increased IL-2, IL-6, and IFN-gamma) that was associated with higher viral loads. These observations provide evidence that host cytokine responses may influence SIV neuropathogenesis independent of disease progression.

CD8+ thymic lymphocytes express reduced levels of CD8beta and increased interferon gamma in cats perinatally infected with the JSY3 molecular clone of feline immunodeficiency virus.

Biological isolates of feline immunodeficiency virus (FIV) cause a relative expansion of activated single-positive CD8(+) (SP CD8(+)) lymphocytes within the thymus of infected cats. In this study, thymic SP CD8(+) lymphocytes were analyzed from cats inoculated as neonates with a pathogenic molecular clone of FIV, JSY3, which was previously derived from the wild-type biological isolate FIV(NCSU-1) (NCSU-1). Four cats were inoculated intraperitoneally with NCSU-1 and compared with 11 cats inoculated with JSY3. Five control cats matched in litter and age were administered an intraperitoneal sham inoculum. Between 12 and 16 weeks postinoculation, interferon-gamma (IFN-gamma) mRNA was quantified by RT-PCR in freshly isolated thymocytes and peripheral blood mononuclear cells (PBMCs). The quantity of IFN-gamma mRNA was increased more than 10-fold in thymocytes and PBMCs of 13 of 13 FIV-inoculated cats as compared with the sham-inoculated controls. IFN-gamma mRNA coenriched with magnetically sorted CD8(+) PBMCs and single-positive (SP) CD8(+) thymocytes. Cells expressing IFN-gamma mRNA were located within the thymic perivascular zone, along the corticomedullary junction, and adjacent to lymphoid follicles. The expansion of thymic SP CD8(+) cells was associated with an increase in CD8alpha(+)/beta(neg) and CD8alpha(+)/beta(lo) phenotypes, the latter population resembling a previously reported memory/effector peripheral blood cell with FIV suppressor activity. From these data we conclude that JSY3 and NCSU-1 induce similar phenotypic changes in thymic and peripheral blood CD8(+) cells. Thus, JSY3 is pathogenic for the thymus in vivo and will be useful for defining determinants of the CD8(+) cell response in this pediatric AIDS model.

Lymphocyte subsets in neonatal and juvenile cats: comparison of blood and lymphoid tissues.

OBJECTIVE: To compare lymphocyte subpopulations in the blood and lymphoid tissues of normal kittens between 1 and 90 days of age. METHODS: Lymphocyte subsets within the blood, thymus, and lymph node of 24 normal kittens were quantified by use of two-color fluorescence flow cytometry and were compared at 1, 23, 46, or 90 days after birth. RESULTS: Blood B and T lymphocytes increased over the 90-day postnatal period. The CD4+ and CD8+ sub-populations of T lymphocytes increased. However, CD8+ lymphocytes increased more than did CD4+ lymphocytes, resulting in reduced CD4-to-CD8 ratio. By 23 days of age, similar but more abrupt changes in the CD4-to-CD8 ratio occurred in the thymus and lymph nodes, coinciding with the highest thymus-to-body weight ratio and gradual increase in mature thymocytes expressing a pan-T lymphocyte marker. CONCLUSIONS: Postnatal thymopoiesis in the domestic cat favors production of mature CD8+ T lymphocytes over CD4+ T lymphocytes. This coincides with the emergence of CD8+ lymphocytes in the lymph node and precedes a more gradual increase in CD8+ cells in the blood. Therefore, the ontogeny of these effectors of cell-mediated immunity could be interrupted by infective agents that target lymphoid tissues of the neonate.

Biphasic thymus response by kittens inoculated with feline immunodeficiency virus during fetal development.

The objective of this study was to assess the response of the feline thymus to fetal infection with feline immunodeficiency virus (FIV), an animal model for human immunodeficiency virus infection. Thirteen feline embryos from four litters were directly inoculated with FIV during the sixth week postbreeding, a period corresponding to the late second trimester of pregnancy. Thymus tissue was collected and analyzed from randomly selected kittens at 2, 4, and 16 weeks postinoculation (PI) and compared to age-matched control kittens that did not receive fetal inoculations. Of three kittens evaluated at 2 weeks PI (week 8 of gestation), neither thymus:body weight ratio nor histologic structure differed from five age-matched control animals. However, analysis of thymocyte subpopulations by flow cytometry revealed a significant (P = 0.011) reduction in the percentage of cluster of differentiation (CD)4+/CD8+ cells from an average of 66% in control fetuses to 45% in infected fetuses. FIV RNA transcription, assessed by in situ hybridization using an FIVgag RNA probe, was widely distributed throughout the thymus in patterns suggestive of both stromal and parenchymal infection. By 4 weeks PI (week 1 postpartum), the thymus:body weight ratio was significantly reduced (P = 0.007) from 0.36% in five control kittens to 0.13% in four fetal inoculates. Severely atrophied thymus lobules supported minimal virus transcription and mean CD4+/CD8+ thymocyte percentages were lower (P = 0.021) in infected kittens (15%) compared to age-matched controls (66%). By 16 weeks PI (week 12 postpartum), thymus:body weight ratios of six inoculated kittens were not significantly different from six age-matched controls, suggesting that partial postnatal thymus regeneration had occurred. However, despite similar size, the regenerative thymus contained reduced percentages of CD4+/CD8+ thymocytes (infected: 40% versus control: 76%; P = 0.009) and increased percentages of CD4+/CD8- (11% versus 5%; P = 0.002) and CD4-/CD8+ (16% versus 9%; P = 0.035) lymphocytes. These changes were associated with widespread FIV transcription within thymic lymphocytes. Thus, the thymus of kittens infected with FIV during late fetal development is characterized by two distinct changes: neonatal atrophy and postnatal regeneration. Despite a recovery in thymus weight, thymus regeneration ineffectively restores the normal phenotypic distribution of thymocytes and supports FIV transcription.

Selective thymocyte depletion and immunoglobulin coating in the thymus of cats infected with feline immunodeficiency virus.

Thymus alterations associated with feline immunodeficiency virus (FIV), an AIDS animal model, were investigated by measuring phenotypic composition of thymocytes, structure of thymic epithelial cells, and transcription of viral RNA in the thymus of FIV-infected juvenile kittens. These kittens either acquired infection by natural vertical transmission or were experimentally inoculated with the virus at defined times of fetal or neonatal life. Thymocytes from FIV-infected cats were analyzed by flow cytometry for the differential expression of CD4, CD8, Pan T, and IgG and subpopulation percentages were compared to values from uninfected littermates. Infected cats demonstrated a decrease in the percentage of CD4+/CD8+ lymphocytes and a concurrent increase in the percentage of CD4-/CD8-, CD4-/CD8+, and IgG+ lymphocytes. Absolute numbers of IgG+ cells were increased with FIV infection. On bivariate distribution scatter plots generated by two-color flow cytometry, this population of IgG+ cells overlapped extensively with cells having low to minimally detectable levels of a pan-T lymphocyte marker, suggesting that thymocytes were coated with IgG. Immunohistochemical detection of feline IgG defined a broad zone of IgG+ cells within the residual cortex but outside lymphoid follicles. However, cells stained with B5, a feline B lymphocyte marker, localized almost exclusively to the centers of lymphoid follicles that were also characterized by a lack of internal cytokeratin staining. FIV RNA transcripts detected by in situ hybridization using an FIVgag RNA probe were evenly distributed throughout the thymic parenchyma except in lymphoid follicles, which were generally devoid of FIV expression. Despite these phenotypic and structural changes, thymus weight, expressed as a percentage of body weight, was not significantly reduced. From these data, we conclude that the clinically asymptomatic stage of FIV infection is associated with two distinct B cell-related phenomena within the thymus-the formation of germinal centers and the coating of thymocytes with IgG. These changes accompany a distorted thymocyte distribution characterized by a reduced percentage of CD4+/CD8+ lymphocytes and a relative increase in CD4-/CD8+ and CD4-/CD8- lymphocytes. Together, these findings suggest that degenerative thymic changes after lentivirus infection may involve humoral immune mechanisms.

Meal-stimulated canine jejunal ionic absorption. Effect of direct jejunal meal delivery and premeal intravenous hydration.

The ingestion of a meal stimulates water and ion absorption from the small intestine. The administration of nutrient substances directly to the small bowel can cause dumping symptoms, with intraluminal fluid accumulation and relative systemic hypovolemia. This study compared the effect of oral versus direct jejunal meal delivery on jejunal water and ion absorption, with and without premeal intravenous saline infusion. Jejunal absorption studies (N = 40) were performed on dogs with 25 cm proximal jejunal Thiry-Vella fistulas and feeding jejunostomies. Luminal perfusion with [14C]PEG was used to calculate fluxes of water and electrolytes. Five groups were randomly studied: (1) intravenous 0.9% saline alone, (2) oral meal alone, (3) intravenous 0.9% saline plus oral meal, (4) jejunal meal alone, and (5) intravenous 0.9% saline plus jejunal meal. Hydration status was assessed hourly by measurement of hematocrit. Water and electrolyte absorption was significantly stimulated by both oral and jejunal meal delivery (P less than 0.01). Intravenous saline hydration significantly reduced the hematocrit (P less than 0.05) but did not alter the proabsorptive response to an oral or jejunal meal. In conclusion, a postprandial signal for proximal jejunal water and electrolyte absorption was stimulated equally by orally or jejunally administered nutrients and was not affected by premeal hydration. These data support the hypothesis that the proabsorptive signal that stimulates water and ion absorption is an enteroenteric phenomenon originating from the small intestine, without implicating pathophysiologic events such as hypovolemia or dumping.

Influence of meal composition on canine jejunal water and electrolyte absorption.

The absorption of water and electrolytes from the proximal jejunal lumen increases immediately after a meal. This meal-induced jejunal absorption occurs in jejunal segments out of normal gastrointestinal continuity. This study was designed to characterize the jejunal absorptive response to a series of isovolumetric gavage-delivered stimuli. Twenty-five-centimeter canine proximal jejunal Thiry-Vella fistulas were constructed, and jejunal absorption studies (n = 66) were performed by luminal perfusion of the jejunal segments with an isotonic buffer containing 14C-labeled polyethylene glycol. Each study consisted of a 1-hour basal period, followed by a 3-hour experimental period. Nine groups were studied, each receiving one of the following isovolumetric stimuli delivered via the gavage route: water, 0.9% saline, mixed meal, protein, lipid, carbohydrate, and mannitol (150 mmol/L, 300 mmol/L, and 600 mmol/L). The water and 0.9% saline gavage groups showed no significant changes in integrated postprandial water and electrolyte absorption above basal. The isocaloric mixed meal, protein, lipid, carbohydrate, and mannitol groups all had significantly increased integrated postprandial jejunal water and electrolyte absorption above basal (P less than 0.05). These results indicate that a proabsorptive signal for meal-induced jejunal absorption originates from or distal to the stomach. Meal-induced jejunal absorption occurs in response to nutrients of diverse composition and is also responsive to nonnutritive solutes such as mannitol. These findings support a new role for gastric or intestinal chemo- or osmo-receptors in stimulating the neurohumoral mechanisms that mediate meal-induced jejunal absorption.

Meal-induced jejunal absorption requires intact neural pathways.

A signal for meal-induced absorption originates from the small intestine and is transmitted to a luminally excluded segment of the proximal jejunum (Thiry-Vella [TV] fistula). Using intraluminal topical anesthesia with oxethazaine, this study assessed the role of intestinal neural pathways in basal and postprandial jejunal water and electrolyte absorption. Studies (n = 45) were performed on dogs with 25-cm proximal jejunal TV fistulae and feeding jejunostomies, using luminal perfusion with 14C-polyethylene glycol. The animals were randomized into five study groups: (1) jejunostomy oxethazaine alone, (2) jejunostomy water and jejunal meal, (3) jejunostomy oxethazaine and jejunal meal, (4) TV fistula water and jejunal meal, and (5) TV fistula oxethazaine and jejunal meal. The jejunal meal significantly increased TV fistula absorption, whereas oxethazaine significantly reduced basal absorption when administered via the TV fistula and postprandial absorption when administered via the jejunostomy (p less than 0.05). TV fistula oxethazaine did not diminish the magnitude of postprandial absorption. We conclude that intact intestinal neurotransmission is necessary for maintenance of the normal basal absorptive state of the proximal jejunum and for the generation of a normal meal-stimulated proabsorptive signal from the small intestine. A nonneural mechanism appears to be of predominant importance in transmitting the proabsorptive signal from the intact gastrointestinal tract to the TV fistula.

Neuropeptide Y-induced intestinal absorption: mediation by alpha 2-adrenergic receptors.

In the intestine neuropeptide Y (NPY) is contained in sympathetic nerves, in neuroendocrine cells of the mucosa, and in neurons of the enteric plexuses. After a meal is ingested the concentration of NPY in the blood rises, and intestinal absorption of water and ions increases. We have recently demonstrated a proabsorptive effect of NPY on water and ion transport in the small intestine. The current experiments tested the hypothesis that the alpha 2-adrenergic receptor mediates NPY-induced intestinal absorption. Rabbit ileal segments (n = 35) were harvested and arterially perfused ex vivo. The intestinal lumen was perfused with an isotonic solution containing carbon 14-labeled polyethylene glycol. Net fluxes of H2O, Na+, and Cl- were calculated for three 20-minute periods: basal, drug infusion, and recovery. Five groups were randomly studied: (1) NPY (500 pmol/min); (2) terazosin (1 microgram/min, alpha 1-adrenergic receptor antagonist); (3) NPY + terazosin; (4) yohimbine (1 microgram/min, alpha 2-adrenergic receptor antagonist); and (5) NPY + yohimbine. The infusion of NPY alone caused a significant (p less than 0.05) proabsorptive response for H2O, Na+, and Cl-. Neither terazosin nor yohimbine alone had a significant effect on the transport state of the intestine. Yohimbine, but not terazosin, completely prevented the NPY-induced proabsorptive response. These data support the hypothesis that the proabsorptive effect of NPY is mediated by the alpha 2-adrenergic receptor system.

Neuropeptide Y is a proabsorptive agent in the small intestine.

In the mammalian intestine neuropeptide Y (NPY) is contained in sympathetic nerves and in enteric neurons originating from the myenteric and submucosal plexuses. This study investigated the role of NPY on small intestinal ionic transport using an isolated intestinal preparation. Rabbit ileal segments (n = 12) were harvested and arterially perfused with a nonrecirculating oxygenated sanguinous solution. The intestinal lumen was perfused with an isotonic solution containing [14C]PEG. Net fluxes of H2O, Na+, and Cl- were calculated for three 20-min periods: basal, drug infusion, and recovery. Two groups were studied: (1) NPY 50 pM/min (n = 6) and (2) NPY 500 pM/min (n = 6). NPY at 50 pM/min caused modest absorption and at 500 pM/min yielded a significant proabsorptive effect (P less than 0.05) for H2O, Na+, and Cl- during the drug infusion period. There were no significant changes in vascular perfusion pressure in either group. These data demonstrate a significant proabsorptive effect of NPY on water and electrolyte transport in the isolated perfused ileum. This proabsorptive effect occurs at a constant arterial blood flow and without alteration in perfusion pressure, supporting a direct effect of NPY on intestinal ionic transport.

Direct proabsorptive effect of octreotide on ionic transport in the small intestine.

Somatostatin is widely distributed within the nervous system and the gastrointestinal tract. Gastrointestinal actions of somatostatin include inhibition of hormone release, reduction of pancreatic secretion, inhibition of motility, and reduction of blood flow. The purpose of this study was to investigate the role of somatostatin and its analogue octreotide on water and electrolyte transport in the small intestine. Rabbit ileal segments (n = 17) were harvested and arterially perfused ex vivo with a nonrecirculating oxygenated sanguineous solution. The lumen was perfused with an isotonic solution containing carbon 14-labeled polyethylene glycol. Net fluxes of water, Na+, and Cl- were calculated for three 20-minute periods designated basal, drug infusion, and recovery. Three groups were studied: somatostatin at 10(-6) mol/L (n = 5), somatostatin at 10(-5) mol/L (n = 5), and octreotide at 10(-5) mol/L (n = 7). Somatostatin at 10(-5) mol/L yielded a proabsorptive effect on the flux of water and electrolytes. Octreotide at 10(-5) mol/L caused a significant (p less than 0.05) proabsorptive response in the fluxes of water, sodium, and chloride during the period of drug infusion, which returned to basal secretory levels during the recovery period. This proabsorptive effect occurred without alterations in vascular resistance and necessarily was independent of systemic hormone interaction, supporting a direct effect of octreotide on intestinal ionic transport.

Small-bowel origin of the signal for meal-induced jejunal absorption.

A meal stimulates the absorption of water and electrolytes from the proximal jejunal lumen. Neither sham feeding nor gastric distention alters this meal-induced jejunal absorption, implying no role for the cephalic or gastric phases of digestion. This study tested the hypothesis that the small bowel is the origin of the proabsorptive signal for meal-induced jejunal absorption. Twenty-five-centimeter canine proximal jejunal Thiry-Vella fistulas were constructed, and chronic duodenal catheters were placed. Jejunal absorption studies (n = 72) were performed by luminal perfusion of the jejunal segments with an isotonic buffer containing radioactive carbon-labeled polyethylene glycol. Each study consisted of a 1-hour basal period followed by a 3-hour experimental period. Ten groups were studied: control, orally ingested mixed meal, and 600 ml duodenal infusions of either water, saline solution, protein, lipid, carbohydrate, 150 mmol/L mannitol, 300 mmol/L mannitol, or 600 mmol/L mannitol, each delivered at 10 ml/min over 60 minutes. The control, water, and saline solution groups showed no significant changes in integrated 3-hour jejunal absorption above basal. The ingested mixed meal significantly increased water and electrolyte absorption (p less than 0.0001). The isovolumetric, isocaloric duodenal nutrient infusions of protein, lipid, and carbohydrate all significantly increased jejunal water and electrolyte absorption (p less than 0.0001). The poorly absorbed solute mannitol significantly increased absorption (p less than 0.0001) in a dose-dependent fashion. These results indicate that the proabsorptive signal for meal-induced jejunal absorption originates from or distal to the duodenum. This newly defined enteroenteric response occurs independently of nutrient composition and responds to increasing osmolarity of poorly absorbed solutes such as mannitol.

Neural blockade in basal and postreceptor-stimulated intestinal transport.

Postreceptor protein stimulation significantly alters the transport state of the ex vivo small intestine. This study investigated the effects of neural blockade on basal and stimulated ionic transport. Rabbit ileal segments (n = 46) were arterially perfused with an oxygenated sanguinous buffered electrolyte solution. The lumen was perfused with an isotonic solution containing [14C]polyethylene glycol as a nonabsorbable marker. Net fluxes of H2O, Na+, and Cl- were calculated. Tetrodotoxin (TTX) was used to block enteric neural transmission. Forskolin (FOR) was used to activate adenylate cyclase, and phorbol 12,13-dibutyrate (PDB) served to activate protein kinase C. Two groups were studied. Group A preparations had no TTX pretreatment, while group B preparations were pretreated with TTX. In the Group A preparations, TTX at 10(-6) M and PDB at 10(-5) M caused significant proabsorptive effects with a delta FH2O of +20 +/- 7 and +15 +/- 2 microliters/min, respectively (P less than 0.05), while FOR stimulated significant secretion with a delta FH2O of -14 +/- 3 microliter/min (P less than 0.05). In the Group B TTX-pretreated preparations, FOR did not cause secretion and PDB maintained an absorptive state. These results indicate that neural blockade with TTX reverses basal secretion in the ex vivo intestine, suggesting that an intact enteric nervous system maintains the secretory status of the intestine. FOR-induced adenylate cyclase-activated secretion does not occur in the presence of TTX, implying that intact neural transmission is required for the FOR effect. PDB-induced protein kinase C-activated absorption occurs despite neural blockade, suggesting that the PDB-induced proabsorptive effect is mediated without neural intermediaries.