Tracking the progeny of adoptively transferred virus-specific T cells in patients posttransplant using TCR sequencing.

Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirus-specific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor ß-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation.

Genomewide Approach Validates Thiopurine Methyltransferase Activity Is a Monogenic Pharmacogenomic Trait.

We performed a genomewide association study (GWAS) of primary erythrocyte thiopurine S-methyltransferase (TPMT) activity in children with leukemia (n = 1,026). Adjusting for age and ancestry, TPMT was the only gene that reached genomewide significance (top hit rs1142345 or 719A>G; P = 8.6 × 10-61 ). Additional genetic variants (in addition to the three single-nucleotide polymorphisms [SNPs], rs1800462, rs1800460, and rs1142345, defining TPMT clinical genotype) did not significantly improve classification accuracy for TPMT phenotype. Clinical mercaptopurine tolerability in 839 patients was related to TPMT clinical genotype (P = 2.4 × 10-11 ). Using 177 lymphoblastoid cell lines (LCLs), there were 251 SNPs ranked higher than the top TPMT SNP (rs1142345; P = 6.8 × 10-5 ), revealing a limitation of LCLs for pharmacogenomic discovery. In a GWAS, TPMT activity in patients behaves as a monogenic trait, further bolstering the utility of TPMT genetic testing in the clinic.

Transport of D-glucose and 2-fluorodeoxyglucose across the blood-brain barrier in humans.

The deoxyglucose method for calculation of regional cerebral glucose metabolism by PET using 18F-2-fluoro-2-deoxy-d-glucose (FDG) requires knowledge of the lumped constant, which corrects for differences in the blood-brain barrier (BBB) transport and phosphorylation of FDG and glucose. The BBB transport rates of FDG and glucose have not previously been determined in humans. In the present study these transport rates were measured with the intravenous double-indicator method in 24 healthy subjects during normoglycemia (5.2 +/- 0.7 mM). Nine subjects were restudied during moderate hypoglycemia (3.4 +/- 0.4 mM) and five subjects were studied once during hyperglycemia (15.0 +/- 0.7 mM). The global ratio between the unidirectional clearances of FDG and glucose (K1*/K1) was similar in normoglycemia (1.48 +/- 0.22), moderate hypoglycemia (1.41 +/- 0.23), and hyperglycemia (1.44 +/- 0.20). This ratio is comparable to what has been obtained in rats. We argue that the global ratio is constant throughout the brain and may be applied for the regional determination of LC. We also determined the transport parameters of the two hexoses from brain back to blood and, assuming symmetrical transport across the BBB, we found evidence of a larger initial distribution volume of FDG in brain (0.329 +/- 0.236) as compared with that of glucose (0.162 +/- 0.098, p < 0.005). The difference can be explained by the very short experimental time, in which FDG may distribute both intra- and extracellularly, whereas glucose remains in a volume comparable to the interstitial fluid of the brain.

Changes in cerebral blood flow and carbohydrate metabolism during acute hyperketonemia.

During starvation, brain energy metabolism in humans changes toward oxidation of ketone bodies. To investigate if this shift is directly coupled to circulating blood concentrations of ketone bodies, we measured global cerebral blood flow (CBF) and global cerebral carbohydrate metabolism with the Kety-Schmidt technique before and during intravenous infusion with ketone bodies. During acute hyperketonemia (mean beta-hydroxybutyrate blood concentration 2.16 mM), cerebral uptake of ketones increased from 1.11 to 5.60 mumol.100 g-1.min-1, counterbalanced by an equivalent reduction of the cerebral glucose metabolism from 25.8 to 17.2 mumol.100 g-1.min-1, with the net result being an unchanged cerebral uptake of carbohydrates. In accordance with this, global cerebral oxygen metabolism was not significantly altered (144 vs. 135 mumol.100 g-1.min-1). The unchanged global cerebral metabolic activity was accompanied by a 39% increase in CBF from 51.0 to 70.9 ml.100 g-1.min-1. Regional analysis of the glucose metabolism by positron emission tomography-[18F]fluoro-2-deoxy-D-glucose indicated that mesencephalon does not oxidize ketone bodies to the same extent as the rest of the brain. It was concluded that the immediate oxidation of ketone bodies induced a decrease in cerebral glucose uptake in spite of an adequate glucose supply to the brain. Furthermore, acute hyperketonemia caused a resetting of the coupling between CBF and metabolism that could not be explained by alterations in arterial CO2 tension or pH.

Blood-brain barrier permeability of glucose and ketone bodies during short-term starvation in humans.

The blood-brain barrier (BBB) permeability for glucose and beta-hydroxybutyrate (beta-OHB) was studied by the intravenous double-indicator method in nine healthy subjects before and after 3.5 days of starvation. In fasting, mean arterial plasma glucose decreased and arterial concentration of beta-OHB increased, whereas cerebral blood flow remained unchanged. The permeability-surface area product for BBB glucose transport from blood to brain (PS1) increased by 55 +/- 31%, whereas no significant change in the permeability from brain back to blood (PS2) was found. PS1 for beta-OHB remained constant during starvation. The expected increase in PS1 due to the lower plasma glucose concentration was calculated to be 22% using previous estimates of maximal transport velocity and Michaelis-Menten affinity constant for glucose transport. The determined increase was thus 33% higher than the expected increase and can only be partially explained by the decrease in plasma glucose. It is concluded that a modest upregulation of glucose transport across the BBB takes place after starvation. Brain transport of beta-OHB did not decrease as expected from the largely increased beta-OHB arterial level. This might be interpreted as an increase in brain transport of beta-OHB, which could be caused by induction mechanisms, but the large nonsaturable component of beta-OHB transport makes such a conclusion difficult. However, beta-OHB blood concentration and beta-OHB influx into the brain increased by > 10 times. This implies that the influx of ketone bodies into the brain is largely determined by the amount of ketones present in the blood, and any condition in which ketonemia occurs will lead to an increased ketone influx.

Brain metabolism during short-term starvation in humans.

During prolonged starvation, brain energy requirements are covered in part by the metabolism of ketone bodies. It is unknown whether short-term starvation of a few days' duration may lead to reduced brain glucose metabolism due to the change toward ketone body consumption. In the present study we measured the cerebral metabolism of glucose and ketone bodies in nine healthy volunteers before and after 3.5 days of starvation. Regional glucose metabolism was measured by dynamic positron emission tomography using [18F]2-fluoro-2-deoxy-D-glucose. The mean value of K1* in gray and white matter increased by 12% (p < 0.05), whereas k2* and k3* were unchanged compared with control values. Regional glucose metabolism in cortical gray matter was reduced by 26% from 0.294 +/- 0.054 to 0.217 +/- 0.040 mumol g-1 min-1 (p < 0.001). White matter glucose metabolism decreased by 27% (p < 0.02). The decrease was uniform in gray and white matter with regional decreases ranging from 24 to 30%. A determination using Fick's principle confirmed the reduction in glucose metabolism yielding a decrease of 24% from 0.307 +/- 0.050 to 0.233 +/- 0.073 mumol g-1 min-1 (p < 0.05), whereas CBF did not change (0.57 +/- 0.07 vs. 0.57 +/- 0.06 ml g-1 min-1). The global net uptake of beta-hydroxybutyrate increased 13-fold from 0.012 +/- 0.024 to 0.155 +/- 0.140 mumol g-1 min-1 (p < 0.05). Net uptake of acetoacetate and net efflux of lactate and pyruvate did not change significantly during starvation. The present study shows that the human brain adapts to the changes in energy supply as early as 3 days following initiation of starvation, at which time ketone bodies account for approximately one-fourth of the cerebral energy requirements.

Outcome in patients with subarachnoid haemorrhage and negative angiography according to pattern of haemorrhage on computed tomography.

15% of patients with spontaneous subarachnoid haemorrhage have normal cerebral angiograms; they fare better than patients with demonstrated aneurysms, though rebleeding and cerebral ischaemia can still occur. In patients with a normal angiogram and accumulation of blood in the cisterns around the midbrain--"perimesencephalic nonaneurysmal haemorrhage"--outcome is excellent. To test the hypothesis that rebleeding and disability in angiogram-negative subarachnoid haemorrhage might be limited to those with other patterns of haemorrhage on initial computed tomography (CT), complications and long-term outcome were studied in 113 patients with angiogram-negative subarachnoid haemorrhage, admitted between January, 1983, and July, 1990. All patients were investigated with third-generation CT scans within 72 h of the event, and with cerebral angiography. The mean follow-up period was 45 (range 6-96) months. None of 77 patients with a perimesencephalic pattern of haemorrhage on CT died or was left disabled as a result of the haemorrhage (0% [95% confidence interval 0-5%]). Among the other 36 patients, who had a blood distribution on CT indistinguishable from that in proven aneurysmal bleeds, 4 had rebleeds and 9 died or were left disabled as result of the haemorrhage (25% [14-43%]). Thus, two distinct subsets of patients with angiogram-negative subarachnoid haemorrhage should be recognised. Patients with a perimesencephalic pattern of haemorrhage have an excellent prognosis. Rebleeding, cerebral ischaemia, and residual disability occur exclusively in patients with aneurysmal patterns of haemorrhage on initial CT. Repeated angiography in search of an occult aneurysm is justified only in the patients with aneurysmal patterns.

Outcome in perimesencephalic (nonaneurysmal) subarachnoid hemorrhage: a follow-up study in 37 patients.

We interviewed 37 patients with perimesencephalic hemorrhage, 18 months to 7 years after the bleed. None rebled or had persisting neurologic deficits. These findings are remarkably good compared with recent series of patients with subarachnoid hemorrhage and normal angiogram. When blood is confined to the mesencephalic cisterns in patients with normal angiogram, repeat angiography may not be indicated.

Circumstances surrounding aneurysmal subarachnoid hemorrhage.

The circumstances surrounding aneurysmal subarachnoid hemorrhage were investigated in a group of 500 consecutive patients admitted to a neurosurgical center. Subarachnoid hemorrhage occurred during stressful events in 42.8% of the patients, during nonstrenuous activities in 34.4%, and during rest or sleep in 11.8%. The activities or events preceding subarachnoid hemorrhage were not known in the remaining 11.0%. Men were more likely to have suffered their hemorrhage during stressful events than women (54.1% versus 36.6%; p less than 0.00025). Only 30.1% of aneurysms arising from the internal carotid artery ruptured during stressful events compared with 48.1% of aneurysms at other locations (p less than 0.005). Physiological responses to the various activities and events are discussed as they may relate to mechanisms underlying aneurysmal subarachnoid hemorrhage. Two important factors in the precipitation of aneurysmal rupture are increased arterial blood pressure and decreased cerebrospinal fluid pressure around the aneurysm. These factors result in a high transmural pressure and concomitant wall stress. Physical exertion, activities involving the Valsalva maneuver, and emotional strain are associated with an increase in blood pressure and frequently preceded subarachnoid hemorrhage. Fluctuations in cerebrospinal fluid pressure around the aneurysm mainly occur during Valsalva's maneuver and postural changes.

Referral pattern of patients with aneurysmal subarachnoid hemorrhage.

The referral pattern of 334 patients admitted to a neurosurgical clinic with aneurysmal subarachnoid hemorrhage (SAH) was analyzed. Forty-nine percent of the patients were admitted after the day following the SAH. Failure of patients to seek prompt medical care was a cause of delay in 29 patients and of physician diagnostic errors in 95 patients. Common misdiagnoses included migraine, mental exhaustion, sinusitis, and influenza. A delay at the referring hospital was observed in 97 patients. Early intervention is important for the optimal management of patients with SAH. Educating the public, medical students, and physicians about the signs and symptoms of SAH and the importance of prompt therapy is likely to improve overall outcome after aneurysmal rupture.

Relationship between blood viscosity and cerebral ischemia after surgical treatment of ruptured intracranial aneurysms.

To determine the role of blood viscosity after surgical treatment of ruptured intracranial aneurysms, the relationship between blood viscosity and clinical condition was examined in 17 patients. A total of 213 blood samples were analyzed. An inverse correlation was found between blood viscosity and level of consciousness; in addition, blood viscosity was higher when focal neurologic deficit was observed. Hematocrit was similarly related to clinical condition, although the correlations observed were less strong. Postoperative plasma viscosity was higher in patients with focal neurologic deficit. Regular blood viscosity measurements are of value in patients at risk for developing cerebral ischemia.

Maturation of a primitive neuroectodermal brain tumor? A case study with some remarks on the classification and nomenclature of 'primitive' CNS tumors.

A brain tumor in a 4-year-old child is described. The neoplasm was partly cystic and showed an a-typical multi-differentiated aspect. Microscopically the neoplasm had a clear-cut 'malignant' morphology. This tumor represents possibly a partly maturated primitive neuroectodermal brain tumor. The term PNET is briefly discussed in relation to the clinical implications.