Rapid memory T-cell reconstitution recapitulating CD45RA-depleted haploidentical transplant graft content in patients with hematologic malignancies.

T-cell depletion of an HLA-haploidentical graft is often used to prevent GVHD, but the procedure may lead to increased graft failure, relapse and infections due to delayed immune recovery. We hypothesized that selective depletion of the CD45RA+ subset can effectively reduce GVHD through removal of naive T cells, while providing improved donor immune reconstitution through adoptive transfer of CD45RA- memory T cells. Herein, we present results from the first 17 patients with poor-prognosis hematologic malignancy, who received haploidentical donor transplantation with CD45RA-depleted progenitor cell grafts following a novel reduced intensity conditioning regimen without TBI or serotherapy. Extensive depletion of CD45RA+ T cells and B cells, with preservation of abundant memory T cells, was consistently achieved in all 17 products. Neutrophil engraftment (median day +10) and full donor chimerism (median day +11) was rapidly achieved post transplantation. Early T-cell reconstitution directly correlated with the CD45RA-depleted graft content. T-cell function recovered rapidly with broad TCR Vß spectra. There was no infection-related mortality in this heavily pretreated population, and no patient developed acute GVHD despite infusion of a median of >100 million per kilogram haploidentical T cells.

Normal table of embryonic development in the four-toed salamander, Hemidactylium scutatum.

We present a complete staging table of normal development for the lungless salamander, Hemidactylium scutatum (Caudata: Plethodontidae). Terrestrial egg clutches from naturally ovipositing females were collected and maintained at 15 °C in the laboratory. Observations, photographs, and time-lapse movies of embryos were taken throughout the 45-day embryonic period. The complete normal table of development for H. scutatum is divided into 28 stages and extends previous analyses of H. scutatum embryonic development (Bishop, 1920; Humphrey, 1928). Early embryonic stage classifications through neurulation reflect criteria described for Xenopus laevis, Ambystoma maculatum and other salamanders. Later embryonic stage assignments are based on unique features of H. scutatum embryos. Additionally, we provide morphological analysis of gastrulation and neurulation, as well as details on external aspects of eye, gill, limb, pigmentation, and tail development to support future research related to phylogeny, comparative embryology, and molecular mechanisms of development.

Early infections after autologous hematopoietic stem cell transplantation in children and adolescents: the St. Jude experience.

INTRODUCTION: Advances in autologous hematopoietic stem cell transplantation (HSCT) over the past 20 years may have had an impact on the morbidity and mortality associated with infections post transplant. PATIENTS AND METHODS: We sought to retrospectively analyze the epidemiology of the first episode of bacterial, fungal, viral, or parasitic infections 0-30 days post transplant in a cohort of 320 children and adolescents who underwent autologous HSCT in a single institution, between 1990 and 2009 for solid tumors or lymphoma, and in 65 children transplanted for acute leukemia during the same period. RESULTS: Infections occurred in 66 (21%) patients with solid tumors or lymphoma. Bacterial infections occurred in 33 (10%) including bacteremia in 23 (7%), and viral infections in 34 (11%) patients. Gram-positive bacterial infections were more prevalent than gram-negative bacterial infections (P = 0.03). Infections caused by fungal or parasitic pathogens were uncommon. The decade when transplant was performed (1990-1999 vs. 2000-2009) had no impact on the incidence of bacterial (P = 0.41) or viral (P = 0.47) infection. Between 1990 and 1999, a total of 60 (92%) children were transplanted for leukemia, and 5 (8%) in the 2000-2009 period (P < 0.0001). Infections occurred in 32 (49%) patients. Bacterial (P = 0.004), candidal (P = 0.003), and herpes simplex viral (P = 0.03) infections were more common in patients transplanted for leukemia. In patients transplanted for leukemia, 3 deaths occurred attributed to infection, all before 2000. CONCLUSION: Changes in epidemiology of infection are likely a result of decline in autologous transplantation for childhood leukemia in the recent era. Autologous transplantation for solid tumors or lymphoma was not associated with mortality from early infections at our institution.

Natural killer cell engineering for cellular therapy of cancer.

Natural killer (NK) cells can kill transformed cells and represent a promising tool for the treatment of cancer. Their function is governed by a balance of stimulatory and inhibitory signals triggered by surface receptors. Advances in NK cell therapy require the development of dependable methods for obtaining an adequate number of effector cells; additional activation or genetic modification may further increase their anticancer capacity. A method for NK cell expansion used in our laboratory relies on a genetically modified form of the K562 myeloid leukemia cell line, engineered to express a membrane-bound form of interleukin-15 and the ligand for the costimulatory molecule 4-1BB (CD137). Expanded NK cells can be transduced with genes encoding chimeric antigen receptors that stimulate tumor cell-specific cytotoxicity. These methods for NK cell expansion and genetic modification have been adapted to large-scale, clinical-grade, Current Good Manufacturing Practice conditions and support two active clinical trials. Summarized are current efforts for NK cell immunotherapy for cancer and future perspectives.

Quantitative trait loci affecting survival and fertility-related traits in Caenorhabditis elegans show genotype-environment interactions, pleiotropy and epistasis.

We have identified, using composite interval mapping, quantitative trait loci (QTL) affecting a variety of life history traits (LHTs) in the nematode Caenorhabditis elegans. Using recombinant inbred strains assayed on the surface of agar plates, we found QTL for survival, early fertility, age of onset of sexual maturity, and population growth rate. There was no overall correlation between survival on solid media and previous measures of survival in liquid media. Of the four survival QTL found in these two environments, two have genotype-environment interactions (GEIs). Epistatic interactions between markers were detected for four traits. A multiple regression approach was used to determine which single markers and epistatic interactions best explained the phenotypic variance for each trait. The amount of phenotypic variance accounted for by genetic effects ranged from 13% (for internal hatching) to 46% (for population growth). Epistatic effects accounted for 9-11% of the phenotypic variance for three traits. Two regions containing QTL that affected more than one fertility-related trait were found. This study serves as an example of the power of QTL mapping for dissecting the genetic architecture of a suite of LHTs and indicates the potential importance of environment and GEIs in the evolution of this architecture.

Quantitative trait loci controlling halothane sensitivity in Caenorhabditis elegans.

Genetic analysis is an essential tool for defining the molecular mechanisms whereby volatile anesthetics (VA) disrupt nervous system function. However, the degree of natural variation of the genetic determinants of VA sensitivity has not been determined nor have mutagenesis approaches been very successful at isolating significantly resistant mutant strains. Thus, a quantitative genetic approach was taken toward these goals. Recombinant-inbred strains derived from two evolutionarily distinct lineages of the nematode Caenorhabditis elegans were tested for sensitivity to clinically relevant concentrations (0.3-0.5 mM) of the VA halothane. The halothane sensitivities of coordinated movement and male mating behavior were highly variant among the recombinant-inbred strains with a range of EC50 values of 13- and 4-fold, respectively. Both traits were highly heritable (H2 = 0.82, 0.87, respectively). Several strains were found to be significantly resistant to halothane when compared with the wild-type strain N2. A major locus or loci mapping to the middle of chromosome V accounted for more than 40% of the phenotypic variance for both traits. Five weaker loci, four of which interact, explained most of the remaining variance. None of the halothane-sensitivity quantitative trait loci significantly affected behavior in the absence of halothane or halothane's potency for C. elegans immobilization, which requires 5-fold higher drug concentrations. Thus, the quantitative trait loci are unlikely to result from differences in halothane-independent (native) behavior or differences in halothane metabolism or permeability. Rather, these loci may code for targets and/or downstream effectors of halothane in the C. elegans nervous system or for modifiers of such gene products.

Optical and digital techniques for enhancing radiographic anatomy for identification of human remains.

Out of a total of more than 300 radiographic identifications made by one of us (JJF), there were 11 cases in which radiologic adjuncts were used because the antemortem radiographs were either miniaturized or because anatomical landmarks could not be clearly discerned. The techniques used included slide projection (two cases), photographic enlargement and enhancement (two cases), digitization (three cases), and digitization with computer enhancement (three cases), commercial digitization (one case). In a 12th case, where identification was made by comparison of antemortem and postmortem film X-rays, the films were digitized as a further evaluation of a commercial system. This is the first reported use of these techniques.

Mapping quantitative trait loci affecting life history traits in the nematode Caenorhabditis elegans.

We have identified chromosomal regions containing quantitative trait loci (QTLs) specifying life history traits in recombinant-inbred strains of the nematode Caenorhabditis elegans. This approach also allows us to examine epistatic interactions between loci and pleiotropic effects on different traits at specific loci. QTLs for mean life span were identified on chromosomes II (near stP101), IV (stP5) and the X (stP61), and QTLs for fertility were identified on II (maPI), III (stP19) and IV (stP51). The QTLs for mean life span accounted for 90% of the genetic component of variance. The loci for mean fertility accounted for 88% of the genetic component of variance. Additional QTLs for temperature-sensitive fertility [II (stP36) and V (stP6)] and internal hatching [IV (stP5)] were also mapped in these crosses. We found evidence for epistatic effects on mean life span between maP1 and bP1 (V), and for epistatic effects on mean fertility between stP36 and stP6, between stP98 (II) and stP192 (V), between maP1 and stP127 (III), between maP1 and stP103 (X), and between stP5 and stP6. Negatively correlated, pleiotropic effects on mean life span and internal hatching were found linked to stP5.

Evidence that the deep keratin filament systems of the Xenopus embryo act to ensure normal gastrulation.

To study the role of keratin filaments in Xenopus development, fertilized eggs were injected with anti-keratin monoclonal antibodies. The anti-keratin monoclonal antibodies AE1 and AE3 induce abnormal gastrulation; in the most severely affected embryos gastrulation fails completely. In contrast, embryos injected with the anti-keratin antibody 1h5 develop normally. Immunocytochemical data indicate that injected 1h5 binds to the dense superficial keratin filament system of the embryo but not to the deeper keratin filament networks of ectodermal and subectodermal cells. Injected AE1 and AE3 do not bind to the superficial keratin system but appear to interact preferentially with the deep keratin filament systems of the embryo. We conclude that the superficial keratin filament system is not involved in the process of gastrulation per se but may protect the embryo from mechanical damage. On the other hand, our results suggest that the integrity of the deeper keratin filament systems is required for the mechanical integration of the morphogenetic movements that underlie gastrulation in Xenopus.

Carotid artery mixing with diastole-phased pulsed drug infusion.

Focal injury to the brain or retina is a frequent complication of drug delivery to the internal carotid artery (ICA) and may be due to poor mixing of the drug with blood at the infusion site. Rhesus monkeys were studied to determine whether phased drug delivery during diastole from a modified pulsatile angiographic injector would improve drug mixing in vivo. A radiolabeled flow tracer, carbon-14-iodoantipyrine (14C-IAP), was injected into the ICA of three monkeys in 80-msec pulses, each ending at least 50 msec before the end of local diastole. Local isotope concentration in the brain was determined by quantitative autoradiography. The ratio of highest to lowest concentration was 1.86 +/- 0.26 (mean +/- standard deviation) in the frontoparietal cortex, 1.65 +/- 0.42 in the frontoparietal white matter, 1.89 +/- 0.28 in the temporal cortex, and 1.39 +/- 0.17 in the basal ganglia. These results were similar to recordings in three control animals that received intravenous 14C-IAP to demonstrate complete drug mixing (1.37 +/- 0.12, 1.41 +/- 0.11, 1.70 +/- 0.08, 1.22 +/- 0.24, respectively), and contrasted to findings in five animals which received continuous intracarotid infusions to demonstrate standard ICA drug delivery (4.54 +/- 2.07, 2.94 +/- 1.45, 5.43 +/- 3.57, 3.60 +/- 2.90, respectively). Pulsed intra-arterial infusion during diastole provides a technically simple method for improving intravascular drug mixing, and results in drug delivery to tissue capillaries that is proportional to blood flow.

Uniformity of intracarotid drug distribution with diastole-phased pulsed infusion.

Drug streaming has been implicated in the development of focal necrotic lesions in perfused tissues following intracarotid chemotherapy of brain tumors at low infusion rates. The narrow infusate path characteristic of streaming within laminar blood flow is not observed at high infusion rates such as are typical in contrast injection for angiography. By periodically pulsing the infusate at a high rate, the mechanisms of rapid mixing can be exploited while retaining the practicality of low average infusion rates. This in vitro study demonstrates the effects of the pulse-controlling parameters and the catheter characteristics and placement on mixing effectiveness. An internal carotid artery model including eight cerebral branches was infused with dye through various indwelling catheters, and individual branch effluents were collected and analyzed spectrophotometrically for dye concentration. While catheter placement dominates the factors that control infusate distribution, judicious selection of the pulse parameters can alleviate that dependence. A primary advantage is gained by phasing the pulse to occur during that period of the cardiac cycle when the blood flow is lowest at the injection site. The data clearly showed that diastole-phased pulsed infusions are highly effective in producing a uniform infusate distribution at low average infusion rates.

Everting (toposcopic) catheter for broad clinical application.

The advanced development of the clinical everting (toposcopic) catheter is described. A detailed discussion of the design and outline of the fabrication techniques are followed by a thorough performance evaluation and summary of the first two clinical applications. The everting element is a low-durometer thermoplastic polyurethane elastomer. Surface treatments include the bonding of a hydrophilic polymeric coating, optimized for lubricity, to the sliding internal surfaces of the catheter. Eversion pressures and infusion/aspiration flow rates have been measured under various conditions and the infusate-in-blood mixing potential investigated. A preliminary assessment is given of the clinical performance of the catheter in the vascular delivery of chemotherapy and standard endoscopic retrograde cholangiopancreatography.

Glioblastoma: catheter techniques for isolated chemotherapy perfusion.

Techniques have been developed for isolated perfusion of chemotherapeutic agents in patients with glioblastoma. Three catheters that facilitate crossing the carotid siphon have been developed; two are based on an everting or toposcopic principle, and one uses microjets for deflectability and improved mixing. Blood from the ipsilateral jugular vein is aspirated at high volumes (300 ml/min) for extracorporeal circulation through an adsorption column (for recovery of carmustine) or dialysers (for recovery of cisplatin). Preliminary experience in 10 patients suggests that high doses of chemotherapeutic agent can be administered using these catheters, with reduced retinal and systemic toxicity.

Intrathoracic meningocele and vertebral anomalies in a case of neurofibromatosis.

A case of an intrathoracic meningocele associated with vertebral scalloping, enlarged intervertebral foramina and scoliosis in neurofibromatosis without spinal or nerve root rumors is described. A markedly attenuated dura mater anteriorly was present at autopsy and may have predisposed to meningeal herniation and scalloping.

Pulmonary venous air embolism in hyaline membrane disease.

A newborn infant with the respiratory distress syndrome in whom pulmonary venous air embolism (PVAE) developed as a complication of positive pressure therapy is reported. The underlying pathophysiology in this disorder is probably the development of alveolar-capillary fistulae secondary to unduly high intrabronchial pressures. An increased awareness by radiologists and clinicians of PVAE as a potential complication of aggressive respiratory therapy will result in more frequent recognition of this uncommon but lethal disorder.

Increased uptake of 99mTc-sulfur colloid in vertebral compression fractures.

Increased uptake of 99mTc-sulfur colloid in vertebral bodies of two patients with vertebral compression fractures was noted on a liver-spleen scan. This finding has not previously been reported in the literature. The mechanism of localization of 99mTc-sulfur colloid in bone marrow depends on regional blood flow and increased reticuloendothelial and phagocytic cell activity. Both mechanisms are felt to be involved in these cases. In fractures, hyperemia and phagocytic activity are transient phenomena and therefore the above observations may be useful in determining fracture age.