Serum αKlotho as a Predictor of Graft Dysfunction After Kidney Transplantation.

OBJECTIVE: To improve the long-term survival rate after kidney transplantation (KTx), allograft injury should be identified as soon as possible. Regardless of aggressive immunosuppressive therapies, recipients of kidney transplants still have a significant risk of graft failure. No specific predictor for the progression of chronic kidney disease (CKD) after KTx has yet been found. Aberrant molecular mechanisms involving the αKlotho-fibroblast growth factor (FGF) 23 axis may be a useful determinant of renal impairment and graft failure over time. METHODS: Plasma and spot urine samples were collected from 47 patients 1 year after KTx. Evaluation of renal function after KTx was performed using levels of biomarkers including serum intact FGF23, soluble αKlotho, 25(OH) vitamin D (25(OH)D), and the difference in the estimated glomerular filtration rate (eGFR) between the first and third year after KTx (ΔeGFR). RESULTS: The median serum αKlotho, intact FGF23, and 25(OH)D were 516.3 pg/mL, 58.7 pg/mL, and 5.7 ng/mL, respectively. No marked changes in the standard biomarkers that regulate phosphate homeostasis were found. Serum αKlotho levels were associated with ΔeGFR. Multivariate regression analysis revealed that serum αKlotho levels significantly predicted a decrease in eGFR in the graft kidney 2 years after KTx, but serum 25(OH)D and FGF23 levels were not significant. Serum αKlotho levels showed an inverse correlation with fractional excretion of magnesium, which reflects tubular injury in the early stage of CKD. CONCLUSION: Measurement of serum αKlotho may serve as a useful predictor of KTx patients who require initiation of pre-emptive medication.

Efficacy of Eculizumab Therapy for Atypical Hemolytic Uremic Syndrome Recurrence and Antibody-Mediated Rejection Progress After Renal Transplantation With Preformed Donor-Specific Antibodies: Case Report.

Atypical hemolytic uremic syndrome (aHUS) develops as the result of unregulated complement progression and precipitates de novo thrombotic microangiopathy. Plasma therapy is used to control the progression of the complement cascade, but that therapy is not effective in all patients and is accompanied by risk of infection and/or allergy. Eculizumab has been reported as an efficient therapy for aHUS. We report the case of a 35-year old woman who underwent effective eculizumab therapy for aHUS recurrence and antibody-mediated rejection (AMR) progress after renal transplantation with preformed donor-specific antibodies (DSA). She developed end-stage renal disease due to suspicious IgA nephropathy at age 33 years. Kidney transplantation was performed at age 35 years, and aHUS recurred 2 weeks later, leading to the progressive hemolytic anemia and renal dysfunction. Therefore, she underwent plasma therapy several times. Because it was difficult to continue to plasma therapy for severe allergy, eculizumab was proposed as an alternate therapy. Treatment with eculizumab was initiated 36 days after renal transplantation. After 3 years of eculizumab treatment, and without plasma therapy, schistocytes decreased, haptoglobin increased to within normal limits, creatinine levels stabilized, and no further episodes of diarrhea were reported. At protocol biopsy 1 year after transplantation, she was diagnosed with C4d-negative subclinical AMR. However, her pathologic findings at follow-up biopsy 3 years after transplantation were recovered. We conclude that eculizumab alone, without plasma therapy, is sufficient to treat recurrence of aHUS and AMR due to DSA after renal transplantation and to maintain long-term graft function.

Encephalitis caused by human herpesvirus-6B in pancreas-after-kidney transplantation.

Human herpesvirus-6 (HHV-6) is a common pathogen among children, classically presenting with fever and rash that resolves without specific therapy. HHV-6 can be reactivated in the immunosuppressed patient. After bone marrow and solid organ transplantation, HHV-6 has been linked to various clinical syndromes, including undifferentiated febrile illness, encephalitis, myelitis, hepatitis, pneumonitis, and bone marrow suppression. However, HHV-6 encephalitis after pancreatic transplant has rarely been reported. Early diagnosis and treatment of HHV-6 encephalitis may be important for affected patients. We report the case of a 53-year-old pancreas-after-kidney transplant recipient who initially presented with high fever and confusion 3 weeks after operation. We managed to save the patient's life and preserve the pancreas graft function. We also review previously reported cases of HHV-6B encephalitis in solid organ transplant recipients.

Acute antibody-mediated rejection possibly due to anti-human leukocyte antigen DQB1 antibodies after renal transplantation - case report.

A 61-year-old Japanese woman, who had undergone hemodialysis because of chronic glomerulonephritis, received a living renal transplant from her ABO blood type-compatible spouse. HLA typing of A, B and DRB showed 3/6 mismatches. Complement-dependent cytotoxicity crossmatches, HLA antibody screening with the use of flow panel reactive antibody (PRA), and flow cytometry crossmatches (FCXM) were all negative. Tacrolimus, mycophenolate mofetil, methylprednisolone (MP), and basiliximab induction were used as the standard immunosuppressive therapy. After renal transplantation, her serum creatinine level favorably decreased, but urine output was not sufficiently obtained, contrary to our expectations. Doppler sonography revealed disappearance of diastolic arterial flow on postoperative day 2. The episode biopsy showed acute antibody-mediated rejection (AMR) based on the current Banff classification, although FCXM and flow PRA were still negative. To determine the cause of acute AMR, we expanded the HLA typing at high resolution levels to Cw, DQB1, and DPB1. Retrospective analysis of perioperative sera demonstrated the presence of low levels of donor-specific HLA IgG and moderate levels of IgM antibody against DQB1 before transplantation. There was an elevation of IgM antibody at the time of rejection, whereas IgG antibody showed no remarkable change. AMR was successfully treated with plasma exchange, low-dose intravenous immunoglobulin, high-dose intravenous MP pulse, and rituximab.

Clinical characteristics and outcomes of renal transplantation in elderly recipients.

BACKGROUND: Elderly renal transplant candidates constitute one the fastest-growing populations among end-stage renal disease patients. Since the impacts of advanced recipient age have not yet been fully defined, we evaluated the clinical characteristics and outcomes of elderly renal transplant recipients. METHODS: Among 564 adult renal transplant recipients, at our center between 2000 and 2009, 64 were at least 60 years of age (Elderly group), and 500 were younger than 60 years (Young group) at the time of the procedure. We compared their clinical features and surgical management. RESULTS: There were significant differences in mean donor age (55.6 years vs. 53.2 years, P = .030) and gender mismatch (77.0% vs. 63.4%, P = .035). However, there were no significant differences between the two groups in patient and graft survivals (P = .177 and P = .365, respectively). Malignancy after transplantation was a significant risk factor upon univariate evaluation but only ABO incompatibility upon multivariate analysis of patient and graft survival. The main cause of graft loss among the Elderly group was death with a functioning graft due to heart failure. CONCLUSIONS: Renal transplantation is a feasible, safe option for the elderly and should be actively implemented. However, screening for cancer and heart disease should be mandatory to improve outcomes.

Pathologic and immunologic profiles of a limited form of neuromyelitis optica with myelitis.

BACKGROUND: Neuromyelitis optica (NMO) is a demyelinating syndrome characterized by myelitis and optic neuritis. Detection of anti-NMO immunoglobulin G antibody that binds to aquaporin-4 (AQP4) water channels allows the diagnosis of a limited form of NMO in the early stage with myelitis, but not optic neuritis. However, the detailed clinicopathologic features and long-term course of this limited form remain elusive. METHODS: We investigated 8 patients with the limited form of NMO with myelitis in comparison with 9 patients with the definite form. RESULT: All patients with limited and definite form showed uniform relapsing-remitting courses, with no secondary progressive courses. Pathologic findings of biopsy specimens from the limited form were identical to those of autopsy from the definite form, demonstrating extremely active demyelination of plaques, extensive loss of AQP4 immunoreactivity in plaques, and diffuse infiltration by macrophages containing myelin basic proteins with thickened hyalinized blood vessels. Moreover, the definite form at the nadir of relapses displayed significantly higher amounts of the inflammatory cytokines interleukin (IL)-1beta and IL-6 in CSF than the limited form and multiple sclerosis. CONCLUSION: This consistency of pathologic findings and uniformity of courses indicates that aquaporin 4-specific autoantibodies as the initiator of the neuromyelitis optica (NMO) lesion consistently play an important common role in the pathogenicity through the entire course, consisting of both limited and definite forms, and NMO continuously displays homogeneity of pathogenic effector immune mechanisms through terminal stages, whereas multiple sclerosis should be recognized as the heterogeneous 2-stage disease that could switch from inflammatory to degenerative phase. This report is a significant description comparing the pathologic and immunologic data of limited NMO with those of definite NMO.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4 percent) or were fed a control diet (20 percent protein) ad libitum. When the experimental group had lost about 20 percent of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Identification of binding sites for anti-aquaporin 4 antibodies in patients with neuromyelitis optica.

OBJECTIVE: Anti-aquaporin 4 antibodies (AQP4-Ab) are specifically detected in patients with neuromyelitis optica. To investigate the role of AQP4-Ab, we examined the antibody binding epitope using human and mouse mutant AQP4. METHODS: We constructed human and mouse amino acid substitution AQP4 mutants and compared the reactivity with wild-form of human, mouse and rat AQP4. RESULTS: The decreased intensity of AQP4-Ab staining with mouse AQP4 was recovered to that of human AQP4 with the mouse mutant A228E for 9 of the 10 sera. CONCLUSIONS: The third extracellular loop of AQP4 is considered to be the major epitope for AQP4-Ab in NMO.

Protein-energy malnutrition halts hemopoietic progenitor cells in the G0/G1 cell cycle stage, thereby altering cell production rates.

Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4%) or were fed a control diet (20% protein) ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.

Anti-aquaporin 4 antibody in selected Japanese multiple sclerosis patients with long spinal cord lesions.

Multiple sclerosis (MS) in Asian populations is often characterized by the selective involvement of the optic nerve (ON) and spinal cord (SP) (OSMS) in contrast to classic MS (CMS), where frequent lesions are observed in the cerebrum, cerebellum or brainstem. In Western countries, inflammatory demyelinating disease preferentially involving the ON and SP is called neuromyelitis optica (NMO). Recently, Lennon et al. discovered that NMO-IgG, shown to bind to aquaporin 4 (AQP4), could be a specific marker of NMO and also of Japanese OSMS whose clinical features were identical to NMO having long spinal cord lesions extending over three vertebral segments (LCL). To examine this antibody in larger populations of Japanese OSMS patients in order to know its epidemiological and clinical spectra, we established an immunohistochemical detection system for the anti-AQP4 antibody (AQP4-Ab) using the AQP4-transfected human embryonic kidney cell line (HEK-293) and confirmed AQP4-Ab positivity together with the immunohistochemical staining pattern of NMO-IgG in approximately 60% of Japanese OSMS patients with LCL. Patients with OSMS without LCL and those with CMS were negative for this antibody. Our results accorded with those of Lennon et al. suggest that Japanese OSMS with LCL may have an underlying pathogenesis in common with NMO.

Roles of the glutamate receptor epsilon2 and delta2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex.

We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-D-aspartate (NMDA)- and delta-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRepsilon2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRepsilon subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRepsilon2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRepsilon2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRepsilon2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific delta2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

Effect of probucol in lecithin-cholesterol acyltransferase-deficient mice: inhibition of 2 independent cellular cholesterol-releasing pathways in vivo.

Cellular cholesterol release takes place by at least 2 distinct mechanisms: the lecithin-cholesterol acyltransferase (LCAT)-driven net efflux by cholesterol diffusion and the generation of high density lipoprotein (HDL) with cellular cholesterol and phospholipid on the cell-apolipoprotein interaction. Therefore, LCAT deficiency impairs the former pathway, and the latter can be inhibited by probucol, which interferes with the apolipoprotein-cell interaction. Hence, probucol was given to the LCAT-deficient mice in the attempt to suppress both of these pathways. The mice were fed low (0.2%) and high (1.2%) cholesterol diets containing 0.5% probucol for 2 weeks. LCAT deficiency and probucol markedly decreased plasma HDL, and the effects were synergistic. Tissue cholesterol content was lower in the adrenal glands and ovaries in the LCAT-deficient mice and in the probucol-treated mice, suggesting that HDL is a main cholesterol provider for these organs. It was also moderately decreased in the spleen of the low cholesterol-fed female mice and in the thyroid gland of the low cholesterol-fed male mice. On the other hand, the esterified cholesterol content in the liver was substantially increased by the probucol treatment with a high cholesterol diet in the LCAT-deficient mice but not in the wild-type mice. Among the groups, there was no significant difference in the tissue cholesterol levels in other organs, such as the liver, spleen, thymus, brain, erythrocytes, thyroid gland, testis, and aorta, resulting from either LCAT deficiency or probucol. Thus, the apolipoprotein-mediated mechanism plays a significant role in the export of cellular cholesterol in the liver, indicating that the liver is a major site of the HDL assembly. Otherwise, tissue cholesterol homeostasis can largely be maintained in mice even when the assembly of new HDL is inhibited by probucol in the absence of LCAT. Nonspecific diffusion of cholesterol perhaps adequately maintains the homeostasis in the experimental condition.

Purification and characterization of a monohalomethane-producing enzyme S-adenosyl-L-methionine: halide ion methyltransferase from a marine microalga, Pavlova pinguis.

A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 microM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I-, Br-, and Cl- to corresponding monohalomethanes and of bisulfide to methyl mercaptan.

catena-Poly[[diaqualithium(I)]-mu-[9H-purine-2,6(1H, 3H)-dionato-O2:N7]].

In the title compound, [Li(C(5)H(3)N(4)O(2))(H(2)O)(2)](n), the coordinate geometry about the Li(+) ion is distorted tetrahedral and the Li(+) ion is bonded to N and O atoms of adjacent ligand molecules forming an infinite polymeric chain with Li-O and Li-N bond lengths of 1.901 (5) and 2.043 (6) A, respectively. Tetrahedral coordination at the Li(+) ion is completed by two cis water molecules [Li-O 1.985 (6) and 1.946 (6) A]. The crystal structure is stabilized both by the polymeric structure and by a hydrogen-bond network involving N-H.O, O-H.O and O-H.N hydrogen bonds.

Discrimination between NK and LAK cytotoxic activities of murine spleen cells by MTT assay: differential inhibition by PGE(2) and EDTA.

In the present study we propose a mathematical approach to improve the analysis of NK and LAK activities measured by MTT assay adapted for murine cells. We found that to calculate NK activity, high E:T ratios should be used (up to 50:1) and the phenomenon fits to a linear least-squares analysis. However, 5-fold less effector cells (10:1, E:T) should be used to detect LAK activity and the phenomenon has a nonlinear exponential behavior. Using this approach, we showed that EDTA inhibits LAK but not NK activity whereas PGE(2) inhibits NK but not LAK activity. In conclusion, this analytical approach allowed the discrimination between NK and LAK activities and exposed differences between these two cytotoxic activities.

Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice.

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.

[Anesthetic management for distal aortic arch aneurysm stent grafting].

A 76-year-old woman with thoracic aortic aneurysm involving distal aortic arch was scheduled for graft replacement from ascending to proximal aortic arch with endovascular stent graft to descending aorta. Surgical procedures were performed under median sternotomy with hypothermic systemic circulation arrest and selective cerebral perfusion. The stent graft composed of 30 mm Gianturco Z stent and 27.5 mm woven Dacron graft was introduced into the descending aorta under the guidance of transesophageal echocardiography (TEE) and fluoroscopy. Ascending and proximal aortic arch replacement was then performed with four branched woven Dacron graft. The aortic pathology was confirmed by TEE and the extent of the aneurysmal lesion was defined. TEE was also useful to find the dislodgement of the stent graft after deployment. This surgical technique, being less invasive than conventional thoracotomy, would be indicated for elderly patients with distal aortic arch aneurysm. TEE is the vital imaging technique for placement of the stent graft, as well as for intraoperative cardiac monitoring.

Apolipoprotein AI efficiently binds to and mediates cholesterol and phospholipid efflux from human but not rat aortic smooth muscle cells.

Aortic smooth muscle cells (SMC) from several animal species have been reported to resist depletion of cellular cholesterol by the major apolipoprotein of HDL, apoAI. Resistance of SMC to this protective action of apoAI, if present in humans, could contribute to the overaccumulation of arterial wall cholesterol seen in atherosclerosis. We investigated the ability of human aortic medial SMC to bind and be depleted of cholesterol and phospholipids by apoAI. In contrast to rat aortic SMC, but similar to human fibroblasts, human SMC were readily depleted of cholesterol by apoAI, measured by a marked depletion of intracellular cholesterol available for esterification, and an increase in cholesterol efflux to the medium. Human SMC were also actively depleted of the phospholipids phosphatidylcholine and sphingomyelin by apoAI. In contrast, rat SMC released only a small fraction of these cellular phospholipids to apoAI-containing medium. (125)I-labeled apoAI bound with high affinity and specificity to human SMC, but failed to bind to rat SMC. Similar levels of expression of class B, type I scavenger receptor (SR-BI) and caveolin in human and rat SMC suggested these proteins do not account for the differences in apoAI binding or lipid efflux seen in these cells. An enhancer of apolipoprotein-mediated cholesterol efflux, tyrosyl radical-oxidized HDL, markedly amplified the depletion of cholesterol available for esterification in human SMC compared to HDL, but had no enhanced effect in rat SMC. These results show that human SMC bind and are readily depleted of cellular lipids by apoAI, and suggest that apoAI-mediated cholesterol efflux from arterial SMC may contribute significantly to the circulating pool of HDL cholesterol in vivo. The marked difference in apoAI binding to human and rat arterial SMC provides an excellent model to study the nature of the apoAI-cell binding interaction.

Cerebellar granule cell-specific and inducible expression of Cre recombinase in the mouse.

To develop a cell type-specific and temporal regulation system of gene targeting in the cerebellum, we used the NMDA-type glutamate receptor GluRepsilon3 subunit gene and Cre recombinase-progesterone receptor fusion (CrePR) gene in combination. Injection of the CrePR gene placed under the control of the 10 kb 5' region of the GluRepsilon3 gene into C57BL/6 eggs yielded the ECP25 line that strongly expressed the CrePR mRNA selectively in the granule cells of the cerebellum. Using a transgenic mouse carrying a reporter gene for Cre-mediated recombination, we showed that antiprogestins could induce the recombinase activity of CrePR protein in the cerebellar granule cells of the ECP25 line. Thus, the established mouse line will provide a valuable tool to investigate the mechanism of cerebellar function by manipulating molecules in the temporally regulated and granule cell-specific manner.

Selective down-regulation by protein kinase C inhibitors of apolipoprotein-mediated cellular cholesterol efflux in macrophages.

Extracellular apolipoprotein A-I removed cholesterol and phospholipid from cholesterol-loaded mouse peritoneal macrophage and thereby generated a prebeta high-density lipoprotein (HDL) particle having a weight ratio of cholesterol to phosphatidylcholine of approximately 1:1. Treatment of the cells with phorbol myristate slightly increased cholesterol efflux by this mechanism without influencing the nonspecific cholesterol efflux to the lipid microemulsion. When the cells were treated by protein kinase C (PKC) inhibitors, H7 and staurosporine, apolipoprotein-mediated cellular cholesterol efflux was substantially reduced without a significant change in phosphatidylcholine efflux, resulting in generation of cholesterol-poor prebeta-HDL particles having a weight ratio of cholesterol to phosphatidylcholine as low as 1:10. In spite of this change, specific binding of apoA-I to the cellular surface was unaffected. Cellular cholesterol available for acylCoA:cholesterol acyltransferase (ACAT) was rapidly depleted by adding apoA-I to the medium, and the PKC inhibitor treatment reversed this effect. In contrast, nonspecific cellular cholesterol efflux to the lipid microemulsion did not influence the ACAT-available cellular cholesterol pool, and it was not influenced by the PKC inhibitors. Thus, we concluded that apolipoprotein-mediated cellular cholesterol efflux is linked to mobilization of cholesterol from an intracellular pool used by ACAT to a specific pool for apolipoprotein-mediated prebeta-HDL generation, in response to apolipoprotein-cell interaction and subsequent intracellular signaling. Binding of apolipoprotein to the cell surface is required for assembly of the prebeta-HDL particle with cellular phospholipid, and the intracellular cholesterol mobilization is needed for enrichment with cholesterol of the prebeta-HDL. These reactions are largely independent of diffusion-mediated nonspecific cell cholesterol efflux.