Cell imaging and morphology: application to studies of inherited purine metabolic disorders

A number of inherited or drug-induced metabolic disorders involving dysfunctions in purines and pyrimidines are strongly associated with neurological dysfunction, e.g., Lesch Nyhan syndrome. Such disorders have been studied extensively using biochemical and molecular techniques in order to examine how such defects occur, sometimes using in vitro models based upon cultured neuroblastoma cell lines. However, these metabolic dysfunctions may manifest their effects in other ways, such as impaired synaptic transmission and gross abnormalities in neuronal growth and differentiation. This review outlines the latter novel facet of purine research. It is proposed that by employing cell imaging techniques and cultured neuroblastoma cell lines, believed to model the nervous system, significant insights into how inherited disorders of purine metabolism affect neuronal development can be obtained. This review provides an example of the application of these techniques to understand the etiology of Lesch Nyhan syndrome, and encourages further study of the role of purines and pyrimidines in the development of the nervous system.     

移植微囊化嗅鞘细胞对神经病理性疼痛及L4-5段脊髓P2X7受体表达的影响

目的 探讨微囊化嗅鞘细胞移植对坐骨神经损伤引起病理性疼痛及L4-5段脊髓P2X7受体的表达影响。 方法 取1只健康SD大鼠,取其嗅球组织并运用差速贴壁方法,在体外进行嗅鞘细胞培养扩增。取48只健康SD大鼠,随机分成4组,即对照组（control）、慢性压迫性坐骨神经损伤组（CCI）、嗅鞘细胞移植组（OECs）及微囊化嗅鞘细胞移植组（MC-OECs）。术后7 d及14 d,运用行为学方法检测各组大鼠机械缩足反射阈值,运用原位杂交方法检测各组大鼠L4-5段脊髓P2X7受体阳性细胞百分比及平均吸光度表达情况。 结果 术后7 d及14 d,与对照组比较,CCI组大鼠机械缩足反射阈值明显减低,L4-5段脊髓P2X7受体阳性细胞百分比及平均吸光度明显增加（P<0.05）;与CCI组比,OECs组大鼠机械缩足反射阈值明显增高,P2X7受体阳性细胞百分比及平均吸光度明显减低（P<0.05）,与OECs组对比,MC-OECs组大鼠机械缩足反射更高。P2X7受体阳性细胞百分比及平均吸光度更低（P<0.05）。 结论 微囊化嗅鞘细胞移植能更好地缓解神经病理性疼痛,减低L4-5段脊髓内P2X7受体的表达水平,修复坐骨神经损伤。     

The First Purine Nucleoside Phosphorylase Deficiency Patient Resembling IgA Deficiency and a Review of the Literature

Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive primary immunodeficiency disorder characterized by decreased numbers of T-cells, variable B-cell abnormalities, decreased amount of serum uric acid and PNP enzyme activity. The affected patients usually present with recurrent infections, neurological dysfunction and autoimmune phenomena. In this study, whole-exome sequencing was used to detect mutation in the case suspected of having primary immunodeficiency. We found a homozygous mutation in PNP gene in a girl who is the third case from the national Iranian registry. She had combined immunodeficiency, autoimmune hemolytic anemia and a history of recurrent infections. She developed no neurological dysfunction. She died at the age of 11 after a severe chicken pox infection. PNP deficiency should be considered in late-onset children with recurrent infections, autoimmune disorders without typical neurologic impairment.     

Synthesis of C6-Substituted Purine Nucleoside Analogues via Late-Stage Photoredox/Nickel Dual Catalytic Cross-Coupling

Nucleoside analogues have been and continue to be extremely important compounds in drug discovery. Despite the significant effort dedicated to their synthesis, medicinal chemistry campaigns around these structures are often hampered by synthetic challenges. We describe a strategy for the functionalization of purine nucleosides via photoredox and nickel-catalyzed sp²–sp³ cross-coupling. The conditions described herein allow for coupling of unprotected nucleosides with readily available alkyl bromides, providing opportunities for their application to parallel medicinal chemistry.     

NAD metabolism in HPRT-deficient mice

The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT^- and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT^- mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.     

Recent progress in the management of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a clonal disease characterized by proliferation and accumulation of small CD5-positive B cells. More than 50% of patients are asymptomatic at diagnosis and usually require no treatment. However, treatment is needed in the advanced and progressive disease. Chlorambucil with or without steroids has been the drug of choice for many years in previously untreated patients with CLL. The purine nucleoside analogs (PNAs), fludarabine (FA), cladribine (2-CdA-chlorodeoxyadenosine) and pentostatin (DCF, 2'-deoxycoformycin) also have been introduced for treatment of CLL. Significantly higher overall response (OR) and complete response (CR) and longer progression free survival (PFS) in patients with CLL treated with FA or 2-CdA have been confirmed in randomized, multicenter trials and more recently in meta-analysis. However, the median survival time did not differ between patients treated with PNA and alkylating agents. Combination therapies with PNAs and cyclophosphamide and especially with cyclophosphamide and rituximab are more active than monotherapy in terms of OR, CR and PFS. Several reports have shown significant activity of alemtuzumab in previously untreated and pretreated patients even when refractory to FA. Alemtuzumab also can be used in CLL as a preparative regimen before stem cell transplantation (SCT) and to eliminate minimal residual disease (MRD). Recently, several new agents have shown promise in treating CLL, including new monoclonal antibodies, agents targeting bcl-2 family of proteins, antisense oligonucleotides and other agents. Moreover, autologous and allogenic hematopoietic cell transplantations are increasingly considered for treatment of patients with CLL. In this review current therapeutic strategies in CLL are presented.     

Older and new purine nucleoside analogs for patients with acute leukemias

Purine nucleoside analogs (PNAs) compose a class of cytotoxic drugs that have played an important role in the treatment of hematological neoplasms, especially lymphoid and myeloid malignancies. All PNA drugs have a chemical structure similar to adenosine or guanosine, and they have similar mechanisms of action. They have many intracellular targets: they act as antimetabolites, competing with natural nucleosides during DNA or RNA synthesis, and as inhibitors of key cell enzymes. In contrast to other antineoplastic drugs, PNAs act cytotoxically, both in the mitotic and quiescent cell cycle phases. In the last few years, three PNAs have been approved for the treatment of lymphoid malignancies and other hematological disorders: 2-chlorodeoxyadenosine (2-CdA), fludarabine and pentostatin. 2-CdA and fludarabine are also active in the treatment of acute myeloid leukemia (AML). These drugs, in combination with cytarabine and other agents, are commonly used as salvage regimens in relapsed or refractory AML. Moreover, the addition of 2-CdA to the standard induction regimen is associated with an increased rate of complete remission and improved survival of adult patients with AML. More recently three novel PNAs have been synthesized and introduced into clinical trials: clofarabine, nelarabine and forodesine. Clofarabine is the most promising PNA in current clinical trials in pediatric and adult patients with acute leukemias. Nelarabine is more cytotoxic in T-lineage than in B-lineage leukemias. Clofarabine and nelarabine have been approved for the treatment of refractory patients with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma. Clofarabine is also an active drug in AML treatment when administered either alone or in combination regimens as front-line treatment and in relapsed or refractory patients. Unlike other PNA, forodesine is not incorporated into DNA but displays a highly selective purine nucleoside phosphorylase inhibitory action. Forodesine is undergoing clinical trials for the treatment of T-cell malignancies, including T-cell ALL. This article summarizes recent achievements in the mechanism of action, pharmacological properties and clinical activity and toxicity of PNAs, as well as their emerging role in lymphoid and myeloid acute leukemias.     

Current Clinical Practice: Treatment of Hairy Cell Leukemia at the Close of the 20th Century

In the last half of this century, hairy cell leukemia was recognized as a distinct B-cell malignancy, accounting for 2% of all leukemias. Characteristics include splenomegaly, pancytopenia, a usually indolent course, and responsiveness to both interferon and purine analog therapy. Accurate diagnosis requires the demonstration of malignant cells in the bone marrow and peripheral blood which contain cytoplasmic projections and characteristic surface antigens. Splenectomy was identified early as a palliative therapy, and in 1984 systemic treatment with interferon alpha was first reported to induce complete remissions. Soon thereafter, the purine analog deoxycoformycin was found to induce more durable complete remissions in a higher percentage of patients. In 1990, 2-Chlorodeoxyadenosine, a new purine analog therapy, was reported to be capable of inducing long-term durable responses in most patients after a single cycle. Current challenges include identifying which purine analog is the least toxic since both appear similarly effective, and neither appear to add to the already increased rate of second malignancies occurring in these patients. Moreover, up to 25% of patients with hairy cell leukemia fail initially or eventually to respond to standard therapy, making the development of new approaches necessary. The characteristic bright expression of several B-cell antigens on the malignant cells, including CD20, CD22 and CD25, has led to the development of targeted biotherapeutic approaches. A recombinant immunotoxin targeting CD25 has recently been reported to induce major responses and it is likely that other successful targeted approaches will be reported early in the new century.     

Combined immunodeficiency due to purine nucleoside phosphorylase deficiency: Outcome of three patients

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway. PNP deficiency, caused by the autosomal recessive mutations in the PNP gene, can lead to severe combined immunodeficiency (SCID).PNP deficiency patients typically have profound T-cell deficiency with variable B and NK cell functions. They present clinically with recurrent infections, failure to thrive, various neurological disorders, malignancies, and autoimmune diseases. Hematopoietic stem cell transplantation (HSCT) is the only available cure for patients with PNP deficiency.We present three patients, two of whom were successfully treated with HSCT. One patient died prior to HSCT due to EBV-associated lymphoma. Over the course of post-HSCT, there was no further aggravation of the patients’ neurological symptoms. Although both of the patients still had mild developmental delay, new developmental milestones were achieved.