Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

Mild hypothermia reduces the inflammatory response and hepatic ischemia/reperfusion injury in rats.

BACKGROUND/AIMS: Hypothermia is known to protect against ischemia/reperfusion (I/R) injury. The mechanisms of protection are incompletely understood and a temperature threshold for protection has not been established. METHODS: In anesthetized Wistar rats, partial (70%) hepatic ischemia was applied for 45 min. Three study groups were used. Group T31 (n = 6) spontaneously cooled to 31.3 +/- 0.8 degrees C, while group T34 (n = 6) spontaneously cooled to 34 degrees C and was then maintained at 34.0 +/- 0.1 degrees C using a heat lamp. The normothermic group (T37, n = 6) was maintained at 37.1 +/- 0.3 degrees C. Hepatic injury, inflammation, lipid peroxidation and metabolic function (using quantitative 1H-NMR) were assessed 24 h after reperfusion. RESULTS: At 24 h following reperfusion, alanine aminotransferase and aspartate aminotransferase increased to 5101 +/- 2378 and 6409 +/- 4202 U/l in the normothermic T37 group (P < 0.05 vs. T34 and T31), whereas transaminases in hypothermic groups (T31 and T34) were significantly lower. Severe liver necrosis was only noted with T37. Myeloperoxidase activity was increased in the T37 group when compared with hypothermic groups (223 +/- 161 (T37) vs. 16 +/- 10 (T31) and 8 +/- 5 (T34) mU/min/mg of tissue, P<0.05 vs. T31 and T34). 1H-NMR analysis of the blood of normothermic animals revealed metabolic changes consistent with increased ischemic injury, which was almost completely ameliorated in T34 and T31 groups. CONCLUSIONS: Mild hypothermia of 34 degrees C is sufficient to reduce I/R injury by inhibiting the inflammatory response. Further spontaneous cooling to 31 degrees C did not demonstrate any additional protective effect.

Liver-specific loss of beta-catenin blocks glutamine synthesis pathway activity and cytochrome p450 expression in mice.

There is accumulating evidence that Wnt/beta-catenin signaling is involved in the regulation of liver development and physiology. The presence of genetic alterations resulting in constitutive beta-catenin stabilization in human and murine liver tumors also implicates this pathway in hepatocyte proliferation. In the present study, we generated hepatocyte-specific beta-catenin knockout mice to explore the role of beta-catenin in liver function. Conditional knockout mice were born at the expected Mendelian ratio and developed normally to adulthood, indicating beta-catenin is dispensable for essential liver function under normal breeding conditions. However, the liver mass of knockout mice was 20% less than those of mice in the control groups. Expression analysis revealed loss of genes required for glutamine synthesis in knockout mice. Loss of the liver glutamine synthesis pathway did not affect the blood ammonia level in mice fed a standard diet, yet, knockout mice showed significantly elevated blood ammonia levels with high-protein dietary feeding. Furthermore, the expression of two cytochrome P450 enzymes, CYP1A2 and CYP2E1, was almost completely abolished in livers from hepatocyte-specific beta-catenin knockout mice. Consequently, these mice were resistant to acetaminophen challenge, confirming the requirement of these cytochrome P450 enzymes for metabolism of xenobiotic substances. In conclusion, in addition to regulating hepatocyte proliferation, beta-catenin may also control multiple aspects of normal liver function.

Glutamine does not protect against hepatic warm ischemia/reperfusion injury in rats.

The administration of glutamine before experimental ischemia/reperfusion (I/R) has been shown to protect intestinal, pulmonary, and myocardial tissue by inducing heat shock proteins (HSP). However, it is not known whether glutamine is protective for all organs. We therefore tested whether pretreatment with glutamine reduces injury following hepatic I/R in rats. Male lean Zucker rats were pretreated with either glutamine (0.75 g/kg intraperitoneally, n = 6) or saline (n = 6), 24 and 6 hours before ischemia. Seventy percent of the liver was exposed to 75 minutes of warm ischemia followed by 24 hours reperfusion. Liver enzymes, histology, neutrophil accumulation, survival, and heat shock protein (HSP) 70 induction were examined. Glutamine administration did not reduce liver injury. In both groups, 5 of 6 animals survived 24 hours of reperfusion. There was no difference in serum transaminase levels with AST 15113 +/- 4336 U/L (glutamine) vs. 17695 +/- 8531 U/L (control, P > 0.05), and ALT 7763 +/- 2524 (glutamine) U/L vs. 5884 +/- 2063 U/L (control, P > 0.05). The degree of neutrophil accumulation and necrosis was not different between groups at 24 hours of reperfusion. Pretreatment did not result in HSP70 upregulation in any of the groups. Pretreatment with glutamine did not reduce hepatic ischemia/reperfusion injury. The lack of protection was associated with an absence of HSP70 upregulation prior to ischemia.

Mild hypothermia provides significant protection against ischemia/reperfusion injury in livers of obese and lean rats.

OBJECTIVE: To evaluate the effect of anesthesia induced mild systemic hypothermia on hepatic injury in lean and obese rats during warm ischemia. SUMMARY BACKGROUND DATA: Hepatic warm ischemia during surgery remains a significant problem, particularly in organs with presumed baseline dysfunction. METHODS: The left and median lobes of male lean and obese Zucker rats were exposed to 75 minutes of ischemia under either mild hypothermic or normothermic conditions. After 75 minutes of ischemia, the organs were reperfused and animals were observed for 24 hours. Surviving animals were killed and blood and tissue was harvested to determine liver enzymes and examine the histology. RESULTS: Mild hypothermia significantly decreased hepatocellular injury in both lean and obese rats. Biochemical markers of hepatic injury were significantly reduced in hypothermic groups (P < 0.01). Survival in normo- and hypothermic lean groups were not different at 24 hours of reperfusion. However, hypothermia profoundly increased survival in obese rats when compared with normothermic obese rats (100% versus 20%, P < 0.01). Necrosis was more pronounced in both normothermic lean and obese animals who experienced more than >75% necrosis when compared with hypothermic animals. In contrast, mild hypothermia reduced necrosis in lean rats to less than 25% and in obese rats to less than 50%. CONCLUSIONS: We demonstrated in a clinically relevant model that mild hypothermia significantly reduces hepatic injury in a warm ischemia model in lean and obese rats and significantly improved 24-hour survival in obese rats.

Antitumor immunity induced by dendritic cell-based vaccination is dependent on interferon-gamma and interleukin-12.

BACKGROUND: This study was conducted to determine whether dendritic cells (DCs) pulsed with a tumor cell lysate can effectively vaccinate against tumor cells and to establish which cytokines are necessary. MATERIALS AND METHODS: Each wild-type mouse received two subcutaneous immunizations (days 14 and 7) with either saline, tumor lysate, DCs, or tumor-lysate-pulsed DCs. Gamma-interferon (gamma-IFN), knock-out (KO), and interleukin-12 (IL-12) KO mice were also used in immunizations. A tumor challenge was given at day 0. Splenocytes were assayed for gamma-IFN production. RESULTS: All saline-injected mice (n = 19) and all mice injected with tumor lysate (n = 9) developed tumors. Six of nine mice immunized with DCs alone and 6/24 mice treated with lysate-pulsed DCs developed a tumor. Splenocytes from both the saline- and lysate-immunized groups produced undetectable levels of gamma-IFN, while those from mice immunized with either DCs or pulsed DCs produced high levels of gamma-IFN. Four of five gamma-IFN KO mice developed tumors after immunization with tumor-lysate-pulsed DCs. None of four IL-12 KO mice developed a tumor after immunization with wild-type pulsed DCs and 1/10 wild-type mice developed tumor after immunization with IL-12 KO pulsed DCs. Three of four IL-12 KO mice developed tumors after immunization with IL-12 KO pulsed DCs. CONCLUSIONS: Tumor-lysate-pulsed DCs can initiate an effective antitumor immune response. The presence of gamma-IFN in the host is essential for antitumor protection. In contrast, tumor protection is observed if IL-12 is present in either the host or the DCs.

CD69 expression on peripheral CD8 T cells correlates with acute rejection in renal transplant recipients.

BACKGROUND: The development of a noninvasive method to diagnose renal allograft rejection could prevent the complications associated with graft biopsy and allow more accurate surveillance of allograft function. The present study determines whether expression of CD69 on peripheral T lymphocytes of renal allograft recipients correlates with the presence of acute graft rejection. METHODS: Peripheral blood T lymphocytes from healthy volunteers, renal allograft recipients with elevated creatinine but no evidence of rejection on biopsy, and renal allograft recipients with biopsy-proven rejection were analyzed by flow cytometry for the expression of CD69 and various intracellular cytokines (interleukin-2, interferon-gamma). Results were then compared with the degree of rejection on biopsy. RESULTS: CD69 expression on CD3+, CD4+, and CD8+ T-cell subsets was low in controls and transplant recipients without allograft rejection. In contrast, patients with renal allograft rejection showed significantly elevated percentages of CD69+ cells in the CD3+ (P<0.01) and CD8+ subsets (P<0.01). The fraction of CD69+ and CD8+ T cells was found to be a more clinically useful test based on receiver-operator characteristics. CD69 expression on CD4+ T cells did not correlate with rejection. Significant intracellular cytokine levels were not detected in unstimulated T cells from any of the groups; stimulation with mitogens increased expression equally among the three groups. CONCLUSIONS: We demonstrate that expression of CD69 on CD3+ and CD8+ peripheral blood T cells correlates closely with the presence of acute graft rejection in renal allograft recipients. Measurement of this surface marker may provide a rapid, noninvasive, and accurate means by which graft rejection can be identified.

Targeted gene therapy with CD40Ig to induce long-term acceptance of liver allografts.

BACKGROUND: The purpose of this study was to modulate the immune response of rat liver transplant recipients by adenovirus-mediated gene transfer of CD40Ig, a secretable fusion protein designed to block the CD40-CD154 T-cell costimulation pathway. METHODS: CD40Ig complementary DNA was created by joining the reverse transcriptase-polymerase chain reaction complementary DNA products for the extracellular domain of murine CD40 to the Fc portion of murine IgG2a. AdCD40Ig and AdSIg (IgG2a-Fc control) recombinant adenoviruses were used to transduce donor liver grafts before nonarterialized orthotopic rat liver transplantation. Donor specific unresponsiveness was examined with skin transplants. RESULTS: All rats (n = 6) that received liver allografts transduced with AdCD40Ig survived >100 days with normal liver histology. Serum levels of CD40Ig at 10, 30, 60, and 100 days after transplantation ranged from 100 to 500, 100 to 250, 5 to 40, and 2 to 10 microg/mL, respectively. Mean survival of rats (n = 4) that received liver allografts transduced with AdSIg control adenovirus was 9.25 +/- 2.9 days. Long-term survivors were rechallenged with skin grafts 100 days after liver transplantations. Survival was 72, >100 (x4) days for donor specific allogeneic skin grafts and 14, 14, 18, 19, and 21 days for third-party allogeneic skin grafts. CONCLUSIONS: Adenovirus-mediated gene transfer of CD40Ig into cold-preserved liver allografts before transplantation results in high levels of transgene expression with resultant long-term survival of hepatic allografts and donor specific unresponsiveness.