In vivo NMR studies of regional cerebral energetics in MPTP model of Parkinson's disease: recovery of cerebral metabolism with acute levodopa treatment

In this study, we have evaluated cerebral atrophy, neurometabolite homeostasis, and neural energetics in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridin (MPTP) model of Parkinson's disease. In addition, the efficacy of acute l ‐DOPA treatment for the reversal of altered metabolic functions was also evaluated. Cerebral atrophy and neurochemical profile were monitored in vivo using MRI and ¹H MR Spectroscopy. Cerebral energetics was studied by ¹H‐[¹³C]‐NMR spectroscopy in conjunction with infusion of ¹³C labeled [1,6^‐13C₂]glucose or [2‐¹³C]acetate. MPTP treatment led to reduction in paw grip strength and increased level of GABA and myo‐inositol in striatum and olfactory bulb. ¹³C Labeling of glutamate‐C4 (1.93 ± 0.24 vs. 1.48 ± 0.06 μmol/g), GABA‐C2 (0.24 ± 0.04 vs. 0.18 ± 0.02 μmol/g) and glutamaine‐C4 (0.26 ± 0.04 vs. 0.20 ± 0.04 μmol/g) from [1,6‐¹³C₂]glucose was found to be decreased with MPTP exposure in striatum as well as in other brain regions. However, glutamine‐C4 labeling from [2‐¹³C]acetate was found to be increased in the striatum of the MPTP‐treated mice. Acute l ‐DOPA treatment failed to normalize the increased ventricular size and level of metabolites but recovered the paw grip strength and ¹³C labeling of amino acids from [1,6‐¹³C₂]glucose and [2‐¹³C]acetate in MPTP‐treated mice. These data indicate that brain energy metabolism is impaired in Parkinson's disease and acute l ‐DOPA therapy could temporarily recover the cerebral metabolism.

     

ProViDE: A software tool for accurate estimation of viral diversity in metagenomic samples

Given the absence of universal marker genes in the viral kingdom, researchers typically use BLAST (with stringent E-values) for taxonomic classification of viral metagenomic sequences. Since majority of metagenomic sequences originate from hitherto unknown viral groups, using stringent e-values results in most sequences remaining unclassified. Furthermore, using less stringent e-values results in a high number of incorrect taxonomic assignments. The SOrt-ITEMS algorithm provides an approach to address the above issues. Based on alignment parameters, SOrt-ITEMS follows an elaborate work-flow for assigning reads originating from hitherto unknown archaeal/bacterial genomes. In SOrt-ITEMS, alignment parameter thresholds were generated by observing patterns of sequence divergence within and across various taxonomic groups belonging to bacterial and archaeal kingdoms. However, many taxonomic groups within the viral kingdom lack a typical Linnean-like taxonomic hierarchy. In this paper, we present ProViDE (Program for Viral Diversity Estimation), an algorithm that uses a customized set of alignment parameter thresholds, specifically suited for viral metagenomic sequences. These thresholds capture the pattern of sequence divergence and the non-uniform taxonomic hierarchy observed within/across various taxonomic groups of the viral kingdom. Validation results indicate that the percentage of 'correct' assignments by ProViDE is around 1.7 to 3 times higher than that by the widely used similarity based method MEGAN. The misclassification rate of ProViDE is around 3 to 19% (as compared to 5 to 42% by MEGAN) indicating significantly better assignment accuracy. ProViDE software and a supplementary file (containing supplementary figures and tables referred to in this article) is available for download from http://metagenomics.atc.tcs.com/binning/ProViDE/     

AML-loaded DC generate Th1-type cellular immune responses in vitro

BACKGROUND: The generation of AML-specific T-lymphocyte responses by leukemia-derived DC has been documented by multiple investigators and is being pursued clinically. An obstacle to widespread use of this strategy is that it has not been possible to generate leukemic DC from all patients, and an alternative approach is needed if the majority of leukemia patients are to receive therapeutic vaccination in conjunction with other treatment protocols. METHODS: In the present study, we generated DC from CD14-selected monocytes isolated from healthy donor PBPC and loaded them with a total cell lysate from AML patient blasts. RESULTS: Immature in vitro-derived DC exhibited robust phagocytic activity, and mature DC demonstrated high expression of CD80, CD83, CD86 and the chemokine receptor CCR7, important for DC migration to local lymph nodes. Mature, Ag-loaded DC were used as APC for leukemia-specific cytotoxic T-lymphocyte (CTL) induction and demonstrated cytotoxic activity against leukemic targets. CTL lysis was Ag-specific, with killing of both allogeneic leukemic blasts and autologous DC loaded with allogeneic AML lysate. HLA-matched controls were not lysed in our system. DISCUSSION: These data support further research into the use of this strategy as an alternative approach to leukemia-derived DC vaccination.     

Ex vivo expanded umbilical cord blood T cells maintain naive phenotype and TCR diversity

BACKGROUND: Umbilical cord blood (CB) is a promising source of hematopoietic stem cells for allogeneic transplantation. However, delayed engraftment and impaired immune reconstitution remain major limitations. Enrichment of donor grafts with CB T cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We hypothesized that CB T cells could be expanded using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab. METHODS: CB units were divided into three fractions: (1) cells cultured without beads, (2) cells cultured with beads and (3) cells cultured with beads following CD3+ magnetic enrichment. All fractions were cultured for 14 days in the presence of IL-2 (200 IU/mL). RESULTS: A mean 100-fold expansion (range 49-154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. Following expansion, CB T cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity demonstrated by spectratyping. DISCUSSION: Our data provide evidence that naive and diverse CB T cells may be expanded ex vivo and warrant additional studies in the setting of human CB transplantation.     

Successful Treatment of Invasive Fungal Infection Due to Highly Resistant Aspergillus calidoustus in an Allogeneic Hematopoietic Cell Transplant Recipient

Invasive aspergillosis (IA) is the most common invasive fungal infection following a hematopoietic cell transplant, with emerging cryptic species exhibiting resistance to commonly used antifungals such as azoles. These species have been increasingly found after the introduction of anti-mold prophylaxis. We report a case of a 56-year-old female with primary myelofibrosis whose allogeneic hematopoietic cell transplant was complicated by disseminated fungal infection (skin, lung) due to Aspergillus calidoustus, a cryptic specie. Treatment of Aspergillus species remains challenging as these cryptic species are usually resistant to azoles including voriconazole which is the first line of treatment of IA. Infection was successfully treated with surgical excision and combination antifungal therapy based on in vitro susceptibility and synergy testing. Therapy included isavuconazole, a drug that has been shown to be non-inferior to voriconazole in the treatment of invasive mold infections.

     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

Eu-Detect: An algorithm for detecting eukaryotic sequences in metagenomic data sets

Physical partitioning techniques are routinely employed (during sample preparation stage) for segregating the prokaryotic and eukaryotic fractions of metagenomic samples. In spite of these efforts, several metagenomic studies focusing on bacterial and archaeal populations have reported the presence of contaminating eukaryotic sequences in metagenomic data sets. Contaminating sequences originate not only from genomes of micro-eukaryotic species but also from genomes of (higher) eukaryotic host cells. The latter scenario usually occurs in the case of host-associated metagenomes. Identification and removal of contaminating sequences is important, since these sequences not only impact estimates of microbial diversity but also affect the accuracy of several downstream analyses. Currently, the computational techniques used for identifying contaminating eukaryotic sequences, being alignment based, are slow, inefficient, and require huge computing resources. In this article, we present Eu-Detect, an alignment-free algorithm that can rapidly identify eukaryotic sequences contaminating metagenomic data sets. Validation results indicate that on a desktop with modest hardware specifications, the Eu-Detect algorithm is able to rapidly segregate DNA sequence fragments of prokaryotic and eukaryotic origin, with high sensitivity. A Web server for the Eu-Detect algorithm is available at http://metagenomics.atc.tcs.com/Eu-Detect/.     

Expression of mRNA for atrial natriuretic peptide receptor guanylate cyclase (ANPRA) in human retina

Summary 1. Guanylate cyclase plays an important role in the visual cycle. Here we report the mRNA expression for the atrial natriuretic peptide receptor type A form of guanylate cyclase (ANPRA) in human retina.2. Polymerase chain reaction using two sets of primers on the cDNAs reverse-transcribed from human retinal poly(A)⁺ RNA amplified two products under two different reaction conditions. The primers used in the reaction were designed from the reported sequence of human placental ANPRA cDNA.3. Sequencing of the amplified products showed 100% sequence homology to the human placental ANPRA gene. Northern blot analysis indicated the presence of a 4.4-kb ANPRA mRNA in human retina, similar to that present in human brain.     

Thymic function and allogeneic T-cell responses in stem-cell transplantation

The thymus is the primary site of T-cell production early in life, and has now been shown to continue to function in both healthy and immunocompromised individuals late into life. Positive and negative selection occurring in the thymus are two of the most important processes that govern the development and specificity of peripheral T cells, including their restriction to self HLA and their ability to respond in an alloreactive manner. In the chimeric state that follows successful allogeneic stem-cell transplants, the specificity of alloreactive cells may be governed by either host- or recipient-derived cellular elements, as well as maturing lymphoid cells, which are, in turn, derived from donor stem cells or host cells surviving transplant conditioning. The ability to measure recent thymic emigrants via the detection of T-cell receptor excision circles has facilitated studies of thymic function in immunodeficient individuals, including HIV-1 infected subjects and recipients of autologous or allogeneic stem-cell transplant (SCT). These studies have now demonstrated that thymic function is likely to play a beneficial role in immune reconstitution in these settings, but have yet to clearly demonstrate what clinical variables are the most important determinants of thymic persistence. It is also not yet clear how much the degree of thymic function following allogeneic SCT influences the alloreactive T-cell repertoire, although studies in animal models and early clinical studies suggest that GvHD results in thymic injury and dysfunction. Future studies will further clarify how thymic function shapes the repertoire of T cells that mediate alloreactivity, as well as protective pathogen-specific immune responses, following SCT. Finally, these studies will also demonstrate whether endogenous mediators of thymic function could be selectively applied to regulate post-SCT thymic function and alloreactivity.