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A key predictor for the success of gene-modified T cell therapies for cancer is the persistence of transferred cells in the patient. The propensity of less differentiated memory T cells to expand and survive efficiently has therefore made them attractive candidates for clinical application. We hypothesized that redirecting T cells to specialized niches in the BM that support memory differentiation would confer increased therapeutic efficacy. We show that overexpression of chemokine receptor CXCR4 in CD8+ T cells (TCXCR4) enhanced their migration toward vascular-associated CXCL12+ cells in the BM and increased their local engraftment. Increased access of TCXCR4 to the BM microenvironment induced IL-15-dependent homeostatic expansion and promoted the differentiation of memory precursor-like cells with low expression of programmed death-1, resistance to apoptosis, and a heightened capacity to generate polyfunctional cytokine-producing effector cells. Following transfer to lymphoma-bearing mice, TCXCR4 showed a greater capacity for effector expansion and better tumor protection, the latter being independent of changes in trafficking to the tumor bed or local out-competition of regulatory T cells. Thus, redirected homing of T cells to the BM confers increased memory differentiation and antitumor immunity, suggesting an innovative solution to increase the persistence and functions of therapeutic T cells.

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.

Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are associated, respectively, with either increased risk or apparent protective effects for atherothrombosis. The ability of purified LDL and HDL to downregulate thrombin formation, a contributor to atherothrombotic processes, was assessed. Purified HDL, but not LDL, significantly enhanced inactivation of coagulation factor Va by activated protein C (APC) and protein S, and HDL stimulated protein S-dependent proteolytic inactivation of Va by APC, apparently due to cleavage at Arg306 in Va. In normal plasma, added HDL enhanced APC/protein S anticoagulant activity in modified prothrombin-time clotting assays. When the anticoagulant potency of HDL was compared with phospholipid (PL) vesicles of well-defined composition using this assay, HDL appeared qualitatively different from PL vesicles because HDL showed only good anticoagulant activity, whereas PL vesicles were rather procoagulant. When 20 normal plasmas were tested using this clotting assay, apoA-I levels correlated with anticoagulant response to APC/protein S (r = 0.47, P = 0.035), but not with activated partial thromboplastin time-based APC resistance ratios. Because HDL enhances the anticoagulant protein C pathway in vitro, we speculate that HDL may help downregulate thrombin generation in vivo and that this anticoagulant action is one of HDL's beneficial activities.

Evidence for a qualitative abnormality in the androgen receptor was obtained by studies of temperature sensitivity. The binding of [(3)H]dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) was studied in monolayers of cultured genital skin fibroblasts from genetic males with abnormal sexual differentiation resulting from androgen resistance. Binding in cells from eight patients with a female phenotype (complete and incomplete testicular feminization) fell from half-normal levels at the usual assay temperature of 37 degrees C to levels <20% of normal when cells were incubated at 42 degrees C. This thermal inactivation was rapidly reversed when the assay temperature was lowered to 37 degrees C, was not associated with altered dihydrotestosterone metabolism, and was also demonstrable with [(3)H]methyltrienolone as the binding ligand. Binding increased to overlap the normal range when the assay temperature was lowered to 26 degrees C. The patients with receptor-deficient testicular feminization include three pairs of siblings; the pedigrees in two of these families are compatible with X-linkage. Only minor changes in the amount of binding at elevated temperatures were observed in cells from 10 control subjects and from 2 male pseudohermaphrodites with normal levels of androgen receptors. In 10 patients with androgen resistance and partial receptor deficiency associated with a predominantly male phenotype (Reifenstein syndrome and infertile men), dihydrotestosterone binding also did not change consistently with elevated temperature. Binding was approximately half-normal at 37 degrees C and either increased or decreased slightly at 42 degrees C. The thermal instability in receptor-deficient testicular feminization represents a new molecular defect associated with hereditary male pseudohermaphroditism that appears to be caused by an alteration in the tertiary structure of the androgen receptor protein.

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

The properties of 5alpha-reductase have been compared in genital skin fibroblasts cultured from five patients from three families (Los Angeles, Dallas, and Dominican Republic) in which hereditary male pseudohermaphroditism has been established to result from deficient conversion of testosterone to dihydrotestosterone. Despite the fact that 5alpha-reductase was immeasurable in a homogenate of epididymis removed from one of the Los Angeles patients, 5alpha-reductase activity was normal in intact fibroblasts and fibroblast extracts from both patients from the Los Angeles family. Although the apparent K(m) for testosterone was also near normal, the apparent K(m) for NADPH in these mutants is elevated some 40-fold above normal. Furthermore, the enzyme is not protected against denaturation at 45 degrees C by concentrations of NADPH that stabilize normal 5alpha-reductase, and in intact fibroblasts from these patients (but not from controls), enzyme activity decreases promptly when protein synthesis is inhibited. We conclude that the mutation in this family results in an unstable enzyme. In contrast 5alpha-reductase activity in fibroblast extracts from a patient from the Dominican Republic family is similar to that previously described in two members of the Dallas family, namely total enzyme activity is low at the optimal pH for the normal reaction, and the apparent K(m) for testosterone is some 20-fold higher than that of the controls. We conclude that the mutations in the Dallas and Dominican Republic families are similar and result in low activity of the enzyme as the result of a decreased affinity for testosterone.Thus, two distinct types of mutations can produce male pseudohermaphroditism due to deficient dihydrotestosterone formation.

We have studied 17 elderly and 27 non-elderly, nonobese subjects (mean age 69+/-1 and 37+/-2 yr, respectively) to assess the mechanisms responsible for the abnormal carbohydrate tolerance associated with aging. Serum glucose and insulin levels were significantly elevated in the elderly subjects compared with the nonelderly subjects during a 75-g oral glucose tolerance test, suggesting an insulin resistant state. Peripheral insulin sensitivity was assessed in both groups using the euglycemic glucose clamp technique during an insulin infusion rate of 40 mU/m(2) per min. Similar steady-state serum insulin levels led to a peripheral glucose disposal rate of 151+/-17 mg/m(2) per min in the elderly compared with a value of 247+/-12 mg/m(2) per min in the nonelderly, thus documenting the presence of insulin resistance in the elderly subjects. Insulin binding to isolated adipocytes and monocytes was similar in the elderly and nonelderly groups (2.34+/-0.33 vs. 2.62+/-0.24% and 5.04+/-1.10 vs. 5.12+/-1.07%), respectively. Thus, insulin resistance in the presence of normal insulin binding suggests the presence of a postreceptor defect in insulin action. This was confirmed by performing additional euglycemic clamp studies using infusion rates of 15 and 1,200 mU/m(2) per min to assess the contours of the dose-response relationship. These studies revealed a 39 and 25% decrease in the glucose disposal rate in the elderly subjects, respectively. The results confirm the presence of a postreceptor defect as well as a rightward shift in the dose-response curve. Insulin's ability to suppress hepatic glucose output was less in the elderly subjects during the 15 mU/m(2) per min insulin infusion (77+/-5 vs. 89+/-4% suppression), but hepatic glucose output was fully and equally suppressed in both groups during the 40 and 1,200 mU/m(2) per min infusion. Finally, a significant inverse relationship was observed between the degree of glucose intolerance in the individual elderly subjects, as reflected by the 2-h serum glucose level during the oral glucose tolerance test, and the degree of peripheral insulin resistance as assessed by the glucose disposal rate during the 40 mU/m(2) per min insulin infusion (r = 0.59, P < 0.01).We conclude that carbohydrate intolerance develops as part of the aging process. This carbohydrate intolerance appears to be the consequence of peripheral insulin resistance caused by a postreceptor defect in target tissue insulin action, which causes both a decrease in the maximal rate of peripheral glucose disposal and a rightward shift in the insulin action dose-response curve. In elderly subjects, the severity of the abnormality in carbohydrate tolerance is directly correlated to the degree of peripheral insulin resistance.

The ability of human Hageman factor (coagulation factor XII) to bind to a glass surface and its susceptibility to limited proteolytic cleavage during the contact activation of plasma have been studied using normal human plasma and plasmas genetically deficient in factor XI, prekallikrein, or high molecular weight kininogen (HMWK). When diluted normal plasma containing (125)I-Hageman factor was exposed to a glass surface for varying times, the Hageman factor was found to bind to the surface, and within 5 min became maximally cleaved from its native 80,000 mol wt to yield fragments of 52,000 and 28,000 mol wt. Hageman factor in factor XI-deficient plasma behaved similarly. In prekallikrein-deficient plasma, the binding of Hageman factor to the glass surface occurred at the same rate as in normal plasma but the cleavage was significantly slower, and did not reach maximum until 60 min of incubation. Cleavage of Hageman factor in HMWK-deficient plasma occurred at an even slower rate, with greater than 110 min of incubation required for maximal cleavage, although the rate of binding to the glass was again the same as in normal plasma. Normal rates of cleavage of Hageman factor were observed for the deficient plasmas after reconstitution with purified human prekallikrein or HMWK, respectively. These observations suggest that normal contact activation in plasma is associated with proteolytic activation of surfacebound Hageman factor. The cleavage of the surface-bound Hageman factor molecule responsible for the formation of the 52,000-and 28,000-mol wt fragments occurred at two closely situated sites, one of which was within a disulfide loop. Cleavage at the site external to the disulfide bond resulted in the release from the surface of the 28,000-mol wt fragment. Cleavage at the site within the disulfide loop resulted in the formation of a 28,000-mol wt fragment which remained surface bound, presumably by virtue of the disulfide linkage to the larger fragment.

Factor VII can be activated, to a molecule giving shorter clotting times with tissue factor, by incubating plasma with kaolin or by clotting plasma. The mechanisms of activation differ. With kaolin, activated Factor XII (XII(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-deficient plasma, was partially activated in prekallikrein and high-molecular weight kininogen (HMW kininogen)-deficient plasmas, but was activated in other deficient plasmas. After clotting, activated Factor IX (IX(a)) was the apparent principal activator. Thus, Factor VII was not activated in Factor XII-,HMW kininogen-, XI-, and IX-deficient plasmas, but was activated in Factor VIII-, X-, and V-deficient plasmas. In further studies, purified small-fragment Factor XII(a) (beta-XII(a)), kallikrein, and Factor IX(a) were added to partially purified Factor VII and to plasma. High concentrations of beta-XII(a) activated Factor VII in a purified system; much lower concentrations of beta-XII(a) activated Factor VII in normal plasma but not in prekallikrein or HWM kininogen-deficient plasmas. Kallikrein alone failed to activate partially purified Factor VII but did so when purified Factor IX was added. Kallikrein also activated Factor VII in normal, Factor XII-, and Factor IX-deficient plasmas. Purified Factor IX(a) activated partially purified Factor VII and had no additional indirect activating effect in the presence of plasma. These results demonstrate that both Factor XII(a) and Factor IX(a) directly activate human Factor VII, whereas kallikrein, through generation of Factor XII(a) and Factor IX(a), functions as an indirect activator of Factor VII.