GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth.

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.

Rapid, effective, and long-lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves.

Behavioral function lost in mammals (including humans) after peripheral nerve severance is slowly (weeks to years) and often poorly restored by 1-2-mm/day, nonspecifically directed outgrowths from proximal axonal stumps. To survive, proximal stumps must quickly repair (seal) plasmalemmal damage. We report that, after complete cut- or crush-severance of rat sciatic nerves, morphological continuity, action potential conduction, and behavioral functions can be consistently (>98% of trials), rapidly (minutes to days), dramatically (70-85% recovery), and chronically restored and some Wallerian degeneration prevented. We assess axoplasmic and axolemmal continuity by intra-axonal dye diffusion and action potential conduction across the lesion site and amount of behavioral recovery by Sciatic Functional Index and Foot Fault tests. We apply well-specified sequences of solutions containing FDA-approved chemicals. First, severed axonal ends are opened and resealing is prevented by hypotonic Ca²âº-free saline containing antioxidants (especially methylene blue) that inhibit plasmalemmal sealing in sciatic nerves in vivo, ex vivo, and in rat B104 hippocampal cells in vitro. Second, a hypotonic solution of polyethylene glycol (PEG) is applied to open closely apposed (by microsutures, if cut) axonal ends to induce their membranes to flow rapidly into each other (PEG-fusion), consistent with data showing that PEG rapidly seals (PEG-seals) transected neurites of B104 cells, independently of any known endogenous sealing mechanism. Third, Ca²âº-containing isotonic saline is applied to induce sealing of any remaining plasmalemmal holes by Ca²âº-induced accumulation and fusion of vesicles. These and other data suggest that PEG-sealing is neuroprotective, and our PEG-fusion protocols that repair cut- and crush-severed rat nerves might rapidly translate to clinical procedures.

Striatal neuroprotection with methylene blue.

Recent literature indicates that low-dose Methylene Blue (MB), an autoxidizable dye with powerful antioxidant and metabolic enhancing properties, might prevent neurotoxin-induced neural damage and associated functional deficits. This study evaluated whether local MB may counteract the anatomical and functional effects of the intrastriatal infusion of the neurotoxin rotenone (Rot) in the rat. To this end, stereological analyses of striatal lesion volumes were performed and changes in oxidative energy metabolism in the striatum and related motor regions were mapped using cytochrome oxidase histochemistry. The influence of MB on striatal levels of oxidative stress induced by Rot was determined, and behavioral tests were used to investigate the effect of unilateral MB coadministration on motor asymmetry. Rot induced large anatomical lesions resembling "metabolic strokes," whose size was greatly reduced in MB-treated rats. Moreover, MB prevented the decrease in cytochrome oxidase activity and the perilesional increase in oxidative stress associated with Rot infusion in the striatum. MB also prevented the indirect effects of the Rot-induced lesion on cytochrome oxidase activity in related motor regions, such as the striatal regions rostral and caudal to the lesion, the substantia nigra compacta and reticulata, and the pedunculopontine nucleus. At a network level, MB maintained a global strengthening of functional connectivity in basal ganglia-thalamocortical motor circuits, as opposed to the functional decoupling observed in Rot-alone subjects. Finally, MB partially prevented the behavioral sensorimotor asymmetries elicited by Rot. These results are consistent with protective effects of MB against neurotoxic damage in the brain parenchyma. This study provides the first demonstration of the anatomical, metabolic and behavioral neuroprotective effects of MB in the striatum in vivo, and supports the notion that MB could be a valuable intervention against neural damage associated with oxidative stress and energy hypometabolism.

Alleviating the suffering of seriously ill children.

Modern medicine has largely focused on the physical aspects of disease, aggressively attacking the illness, often at the expense of caring for pain and suffering. Medical interventions based solely on the diagnosis and treatment of disease limit the medical care of the severely ill child. Such an approach is particularly detrimental when caring for the terminally ill. Successful care of children with chronic, life-threatening, or terminal illnesses requires a comprehensive assessment of their physical, psychological, and spiritual needs as well as a process of collaboration between members of the multiple disciplines involved in the care of the patient and the family unit as a whole. Supportive/palliative care serves as a bridge between a scientific (disease-oriented) and humanistic (person-oriented) approach to patient care. Bridging this gap early in the course of life-threatening illness is essential for successful palliative intervention to relieve suffering and improve the quality of life for the child and his or her family. A model that introduces supportive, palliative, and hospice services into the mainstream of medical therapy is emphasized as a standard for the care of all children with significant chronic, life-threatening, or terminal illness. This article expands on a previous paper published in the American Journal of Hospice & Palliative Care (Kane JR, Barber RG, Jordan M, et al.: Supportive/palliative care of children suffering from life-threatening and terminal illness. May/June 2000; 17(3): 165-172).

A multiplex RT-PCR assay for the detection of chimeric transcripts encoded by the risk-stratifying translocations of pediatric acute lymphoblastic leukemia.

Modern therapy for pediatric acute lymphoblastic leukemia (ALL) is based on the principle of risk stratification. One of the most important laboratory features used to accurately risk stratify patients is the presence of specific chromosomal translocation within the leukemic blasts. In this paper, we describe a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the accurate, sensitive, and rapid identification of chimeric transcripts encoded by the major risk-stratifying translocations of pediatric ALL. This assay will identify both the CML- and ALL-type BCR-ABL transcripts encoded by the t(9;22), all described variants of the E2A-PBX1 transcripts encoded by the t(1;19), the MLL-AF4 transcripts encoded by the t(4;11), and all variants of TEL-AML1 encoded by the t(12;21). In addition, we have developed a reverse dot-blot detection system as an alternative to traditional post-PCR Southern blot analysis. Application of this combined assay to the analysis of 70 leukemic samples and five cell lines resulted in a complete concordance between this multiplex assay and individual PCR reactions. The characteristics of the multiplex assay suggest that its application to routine clinical screening will significantly improve the ability of clinical laboratories to accurate risk stratify pediatric ALL patients.

Molecular analysis of the PML/RAR alpha chimeric gene in pediatric acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized cytogenetically by the t(15;17)(q22;q11-21) translocation. To compare molecular events among pediatric and adult APL cases, we designed two sets of oligonucleotide primers using published cDNA sequence for PML/RAR alpha fusion transcripts, and undertook reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of 22 US pediatric cases of APL. PML/RAR alpha fusion transcripts were detected in all APL cases, including two cases lacking cytogenetic evidence of t(15;17). Breakpoint usage in PML was determined using a combination of PCR amplification with differing 5' primers, junction-specific probes, and sequence analysis in selected cases. Consistent with previously published data, case analysis demonstrated fusion products resulting from three breakpoint cluster regions (bcr) in PML, and a single breakpoint region in intron 2 of RAR alpha. Transcripts resulting from breakpoints in bcr1 were detected in 59 percent of cases, bcr2 in 27 percent and bcr3 in 14 percent. This distribution is dissimilar to that observed in adults, where bcr2 comprises a lesser and bcr3 a greater portion of cases. These results suggest that the pathogenesis of the t(15;17) in APL may differ among patient sets. RT-PCR with these primer sets is a reliable method for detecting PML/RAR alpha chimeric transcript in t(15; 17)-containing APL.

Biology and treatment of infant leukemias.

The leukemias of infancy, characterized by an equal distribution of lymphoid and myeloid subtypes, account for 2.5-5% of the acute lymphoblastic leukemias (ALL) and 6-14% of the acute myeloid leukemias (AML) of childhood. Rearrangements of the MLL gene on chromosome 11q23 are the most common genetic abnormalities in both ALL and AML, occurring in 70-80% and approximately 60% of cases, respectively. Infants with ALL and a rearrangement of MLL typically present with hyperleukocytosis, massive organomegaly, CNS involvement, CD10- B-lineage phenotype and myeloid-associated antigen (CD15) expression. Prognosis in these cases is uniformly poor, whereas in similar cases without the genetic defect, it is good to intermediate. The presenting features of infant AML include monoblastic or myelomonoblastic morphology, hyperleukocytosis and extramedullary involvement. Expected outcome approximates that for ALL (approximately 30% long-term survival rate). Rare congenital forms of lymphoid or myeloid leukemia, manifested at birth or during the first month of life, carry a dismal prognosis, especially when a MLL/11q23 rearrangement is present; such cases should be carefully distinguished by chromosomal/molecular analysis and cell culture techniques from transient myeloproliferative disorders which require only supportive care but close follow-up for subsequent development of leukemia. Juvenile chronic myeloid leukemia also can occur in infants and may be responsive to chemotherapy alone. Rapid progress has been made over the past decade in understanding the biology of infant leukemias. The biggest challenge now is to develop more effective treatment, especially for patients with MLL rearrangements.

Localization of the murine homolog of the anaplastic lymphoma kinase (AlK) gene on mouse chromosome 17.

The murine homolog of the human anaplastic lymphoma kinase gene, which encodes a membrane-spanning receptor tyrosine kinase in the insulin receptor kinase subfamily, was assigned to mouse Chromosome 17 by interspecific backcross analysis. This assignment further confirms the homology between a portion of the distal Chromosome 17 and the short arm of human chromosome 2 and extends this region in the mouse by an additional 3 cM.

Diffuse alveolar hemorrhage associated with Mycoplasma hominis respiratory tract infection in a bone marrow transplant recipient.

An 18-year-old woman developed respiratory distress and diffuse pulmonary infiltrates after allogeneic bone marrow transplantation. Bronchoalveolar lavage findings indicated diffuse alveolar hemorrhage. Cultures of the lavage fluid and the pharynx grew Mycoplasma species; the pharyngeal isolate was identified as Mycoplasma hominis. Mycoplasma hominis infection may have an etiologic role in diffuse alveolar hemorrhage.