Human leukemia-associated antigens: detection on cells of established lymphoblastoid lines.

Primate and rabbit antisera to different morphologic classes of human leukemia cells, after appropriate absorptions, detected leukemia-associated antigens present on cultured lymphoblastoid cell lines derived from leukemia patients. The primate antisera distinguished antigens on cells derived from myeloid leukemia patients from those on cells derived from lymphocytic leukemia patients. Of particular interest was the fact that antigens of myeloid leukemia, but not of lymphatic leukemia, were detected on lymphoid cell lines established from blood of patients with myeloid leukemia. One of four lymphoblastoid cell lines derived from normal donors expressed antigens of lymphatic leukemia. Leukemia-associated antigens were not found on the HRIK lymphoblastoid line derived from a Burkitt's lymphoma patient on skin fibroblasts or HeLa cells. Expression of these antigens on cultured cells derived from leukemia patients could not be related to the presence of the EB virus or the EB virus genome. Rabbit antisera detected antigens common to cells from patients with myeloid and lymphocytic leukemia. Absorption experiments demonstrated that the antigens detected on cell lines derived from leukemia patients are similar to those detected by the primate and rabbit antisera on fresh peripheral blood leukemic cells. The serologic detection of leukemia-associated antigens on lymphoblastoid cell lines indicates that some of these cultures contain cells with antigenic properties similar to those of human peripheral blood leukemic cells.     

A mitochondrial basis for Huntington’s disease: therapeutic prospects

Huntington’s disease (HD) is an autosomal dominant disease, with overt movement dysfunctions. Despite focused research on the basis of neurodegeneration in HD for last few decades, the mechanism for the site-specific lesion of neurons in the brain is not clear. All the explanations that partially clarify the phenomenon of neurodegeneration leads to one organelle, mitochondrion, which is severely affected in HD at the level of electron transport chain, Ca²⁺ buffering efficiency and morphology. But, with the existing knowledge, it is not clear whether the cell death processes in HD initiate from mitochondria, though the Huntingtin (Htt) aggregates show close proximity to this organelle, or do some extracellular stimuli like TNFα or FasL trigger the process. Mainly because of the disparity in the different available experimental models, the results are quite confusing or at least inconsistent to a great extent. The fact remains that the mutant Htt protein was seen to be associated with mitochondria directly, and as the striatum is highly enriched with dopamine and glutamate, it may make the striatal mitochondria more vulnerable because of the presence of dopa-quinones, and due to an imbalance in Ca²⁺. The current therapeutic strategies are based on symptomatic relief, and, therefore, mainly target neurotransmitter(s) and their receptors to modulate behavioral outputs, but none of them targets mitochondria or try to address the basic molecular events that cause neurons to die in discrete regions of the brain, which could probably be resulting from grave mitochondrial dysfunctions. Therefore, targeting mitochondria for their protection, while addressing symptomatic recovery, holds a great potential to tone down the progression of the disease, and to provide better relief to the patients and caretakers.     

Apoptotic Mode of Cell Death in Substantia Nigra Following Intranigral Infusion of the Parkinsonian Neurotoxin,MPP<Superscript>+</Superscript> in Sprague-Dawley Rats: Cellular,Molecular and Ultrastructural Evidences

The potent parkinsonian neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is known to cause dopaminergic neurodegeneration in nigrostriatal system. In the present study we investigated the nuclear morphology of cells in the substantia nigra pars compacta (SNpc) region of rats following unilateral intranigral infusion of the active metabolite, 1-methyl-4-phenyl pyridinium ion (MPP⁺), which resulted in a dose-dependent and prolonged dopamine depletion in the ipsilateral striatum. There appeared a substantial loss of tyrosine hydroxylase immunoreactive neurons in the SNpc that received the neurotoxin. Specific nuclear staining with Hoechst 33342 or acridine orange revealed bright pyknotic, shrunken, distorted nuclei and condensed chromatin with perinuclear nucleolus respectively following visualization with the former and latter dyes in the ipsilateral SNpc, as compared to the round, intact nuclei and centrally positioned nucleolus in the contralateral side. Ultrastructural details of the nucleus under transmission electron microscope confirmed distorted nuclear organization with shrunken or condensed nuclei and disrupted nuclear membrane. These features are typical of nucleus undergoing apoptosis, and suggest that MPP⁺ causes dopaminergic neuronal death through an apoptotic mode. Typical laddering pattern of genomic DNA isolated from the ipsilateral SN in agarose gel electrophoresis conclusively established apoptosis following intranigral administration of MPP⁺ in rats. Rebecca Banerjee and Sen Sreetama contributed equally to this paper.     

Striatal dopamine level contributes to hydroxyl radical generation and subsequent neurodegeneration in the striatum in 3-nitropropionic acid-induced Huntington's disease in rats

We tested the hypothesis that dopamine contributes significantly to the hydroxyl radical (OH)-induced striatal neurotoxicity caused by 3-nitropropionic acid (3-NP) in a rat model of Huntington's disease. Dopamine (10–100 μM) or 3-NP (10–1000 μM) individually caused a significant increase in the generation of hydroxyl radical (OH) in the mitochondria, which was synergistically enhanced when the lowest dose of the neurotoxin (10 μM) and dopamine (100 μM) were present together. Similarly, systemic administration of l-DOPA (100–250 mg/kg) and a low dose of 3-NP (10 mg/kg) potentiated OH generation in the striatum, and the rats exhibited significant decrease in stride length, a direct indication of neuropathology. The pathology was also evident in striatal sections subjected to NeuN immunohistochemistry. The significant changes in stride length, the production of striatal OH and neuropathological features due to administration of a toxic dose of 3-NP (20 mg/kg) were significantly attenuated by treating the rats with tyrosine hydroxylase inhibitor α-methyl-p-tyrosine prior to 3-NP administration. These results strongly implicate a major contributory role of striatal dopamine in increased generation of OH, which leads to striatal neurodegeneration and accompanied behavioral changes, in 3-NP model of Huntington's disease.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

Mitochondrial functional alterations in relation to pathophysiology of Huntington’s disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease which is characterized by psychiatric symptoms, involuntary choreiform movements and dementia with maximum degeneration occurring in striatum and cerebral cortex. Several studies implicate mitochondrial dysfunction to the selective neurodegeneration happening in this disorder. Calcium buffering imbalance and oxidative stress in the mitochondria, critically impaired movement across axons and abnormal fission or fusion of this organelle in the cells are some of the salient features that results in the loss of mitochondrial electron transport chain (ETC) complex function in HD. Although several models involving mutant huntingtin, excitotoxins and mitochondrial complex-II inhibitors have been used to explore the disease, it is not clear how disturbances in mitochondrial functioning is associated with such selective neurodegeneration, or in the expression of huntingtonian phenotypes in animals or man. We have carefully assessed various mitochondrial abnormalities observed in human patient samples, postmortem HD brains, cellular, vertebrate and invertebrate models of the disease, to conclude that ETC dysfunction is an integral part of the disease and justify a causal role of mitochondrial ETC dysfunction for the genesis of this disorder     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Antibodies to MHC Class II Molecules Induce Autoimmunity: Critical Role for Macrophages in the Immunopathogenesis of Obliterative Airway Disease

Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- α1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naïve animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.     

HIF-1α signaling by airway epithelial cell K-α1-tubulin: role in fibrosis and chronic rejection of human lung allografts

Long term survival of the human lung allografts are hindered by chronic rejection, manifested clinically as bronchiolitis obliterans syndrome (BOS). We previously demonstrated significant correlation between the development of antibodies (Abs) to K-α1-tubulin (Kα1T) and BOS. In this study, we investigated the molecular basis for fibrinogenesis mediated by ligation of Kα1T expressed on airway epithelial cells by its specific Abs. Using RT-PCR we demonstrate that normal human bronchial epithelial (NHBE) cells upon ligation of Kα1T with specific Abs caused upregulation of pro-fibrotic growth factors. Western blot analysis of NHBE incubated with Kα1T Abs increased hypoxia inducible factor (HIF-1α). Kα1T Ab-mediated growth factor expression is dependent on HIF-1α as inhibition of HIF-1α returned fibrotic growth factor expression to basal levels. In conclusion, we propose that HIF-1α -mediated upregulation of fibrogenic growth factors induced by ligation of Kα1T Abs is critical for development of fibrosis leading to chronic rejection of lung allograft.