The immunopathology of experimental allergic encephalomyelitis. I. Quantitative analysis of inflammatory cells in situ

Acute experimental allergic encephalomyelitis (EAE) is a T cell-mediated, neurologic disease that is under immunogenetic control. We systematically analyzed the quantity and distribution of T cells, B cells, and macrophages in the central nervous system (CNS) of susceptible and resistant guinea (GP) with a panel of seven monoclonal antibodies by using the avidin-biotin complex (ABC) immunoperoxidase technique and alpha-naphthyl-butyrate esterase (ANBE) staining. Adult EAE-susceptible strain 13 GP immunized with isogeneic spinal cord homogenate (SC) or with myelin basic protein (MBP) developed clinical signs (paralysis, weight loss, etc.) in 2 to 3 wk. T cells were present in all CNS inflammatory foci and comprised 44% of the perivascular mononuclear cells. T cells diffusely infiltrated the neuropil away from inflammatory cell aggregates. These T cells were judged to be extravascular by the lack of an associated identifiable vessel in counter-stained sections, and by their persistence following exhaustive perfusion of the brains. In routine sections, mononuclear cells could be detected only in perivascular aggregates. IgM+ B cells comprised 9% of the perivascular infiltrates and did not diffusely infiltrate the parenchyma. ANBE+ macrophages comprised the remaining 47% of the identified perivascular cells. SC- and MBP-immunized GP showed equivalent numbers of inflammatory foci, T cells, and macrophages, but SC-immunized GP had more IgM+ cells in the meninges and choroid plexus (p less than 0.001, p less than 0.02, respectively). Virtually all cells in perivascular locations were Ia+. Ia+ mononuclear cells were also present in the neuropil. EAE-resistant strain 2 GP immunized with SC developed no clinical signs. These GP had fewer perivascular foci than strain 13 GP but, when present, the cellular composition, including the density of diffuse parenchymal T cell infiltrates, was indistinguishable. Significantly fewer parenchymal mononuclear cells in the strain 2 GP, however, displayed Ia, both in perivascular and diffuse infiltrates (p less than 0.001). We conclude that T cell migration into the CNS parenchyma is a characteristic feature of acute EAE in the GP, but that T cells can occur in this pattern without clinical signs of disease. The two features that distinguish susceptible and resistant strains were the frequency of perivascular infiltrates and the expression of Ia on parenchymal mononuclear cells, which probably reflects their enhanced immunologic activation in situ.     

Phosphatase of Regenerating Liver 2 (PRL2) Is Essential for Placental Development by Down-regulating PTEN (Phosphatase and Tensin Homologue Deleted on Chromosome 10) and Activating Akt Protein

The PRL (phosphatase of regenerating liver) phosphatases are implicated in the control of cell proliferation and invasion. Aberrant PRL expression is associated with progression and metastasis of multiple cancers. However, the specific in vivo function of the PRLs remains elusive. Here we show that deletion of PRL2, the most ubiquitously expressed PRL family member, leads to impaired placental development and retarded growth at both embryonic and adult stages. Ablation of PRL2 inactivates Akt and blocks glycogen cell proliferation, resulting in reduced spongiotrophoblast and decidual layers in the placenta. These structural defects cause placental hypotrophy and insufficiency, leading to fetal growth retardation. We demonstrate that the tumor suppressor PTEN is elevated in PRL2-deficient placenta. Biochemical analyses indicate that PRL2 promotes Akt activation by down-regulating PTEN through the proteasome pathway. This study provides the first evidence that PRL2 is required for extra-embryonic development and associates the oncogenic properties of PRL2 with its ability to negatively regulate PTEN, thereby activating the PI3K-Akt pathway.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

THE SIZE OF THE CELLULOSE MICROFIBRIL

Recently the lateral width of the cellulose microfibril has been estimated as 30 A rather than about 150 to 200 A, by extrapolation of data from model shadowing experiments. The difference was attributed to a layer of metal deposited during shadowing. However, direct photographs of the same microfibrils parallel and perpendicular to the direction of shadowing, of unshadowed portions of microfibrils compared with shadowed portions of the same microfibrils, of silver-stained unshadowed microfibrils, and of unshadowed, unstained segments of microfibrils give no evidence of a layer of metal of this thickness in material shadowed under normal conditions. Furthermore, the evidence for microfibril strands of about 35 A in width from negative-staining experiments is subject to a bias from the form of the filaments and from variable positive adsorption of phosphotungstic acid by cellulose. Consequently, the conclusion that the true lateral width of native cellulose microfibrils is about one-fifth of the presently accepted value is not yet justified by unequivocal direct experimental evidence.     

Evidence for pH dependent Zn2+influx in K562 erythroleukemia cells: studies using ZnAF-2F fluorescence and 65Zn2+ uptake

Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.     

Optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228

Metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM-1) have spread world-wide and present a serious threat. Expression of MBLs confers resistance in Gram-negative bacteria to all classes of β-lactam antibiotics, with the exception of monobactams, which are intrinsically stable to MBLs. However, existing first generation monobactam drugs like aztreonam have limited clinical utility against MBL-expressing strains because they are impacted by serine β-lactamases (SBLs), which are often co-expressed in clinical isolates. Here, we optimized novel monobactams for stability against SBLs, which led to the identification of LYS228 (compound 31). LYS228 is potent in the presence of all classes of β-lactamases and shows potent activity against carbapenem-resistant isolates of Enterobacteriaceae (CRE).     

A cryptic invasion within an invasion and widespread introgression in the European water frog complex: consequences of uncontrolled commercial trade and weak international legislation

In Western Europe, many pond owners introduce amphibians for ornamental purposes. Although indigenous amphibians are legally protected in most European countries, retailers are circumventing national and international legislation by selling exotic nonprotected sibling species. We investigated to what extent non‐native species of the European water frog complex (genus Pelophylax) have become established in Belgium, using morphological, mitochondrial and nuclear genetic markers. A survey of 87 sampling sites showed the presence of non‐native water frogs at 47 locations, mostly Marsh frogs (Pelophylax ridibundus). Surprisingly, at least 19% of all these locations also harboured individuals with mitochondrial haplotypes characteristic of Anatolian water frogs (Pelophylax cf. bedriagae). Nuclear genotyping indicated widespread hybridization and introgression between P. ridibundus and P. cf. bedriagae. In addition, water frogs of Turkish origin obtained through a licensed retailer, also contained P. ridibundus and P. cf. bedriagae, with identical haplotypes to the wild Belgian populations. Although P. ridibundus might have invaded Belgium by natural range expansion from neighbouring countries, our results suggest that its invasion was at least partly enhanced by commercial trade, with origins as far as the Middle East. Also the invasion and rapid spread of Anatolian lineages, masked by their high morphological similarity to P. ridibundus, is likely the result of unregulated commercial trade. We expect that Anatolian frogs will further invade the exotic as well as the native range of P. ridibundus and other Pelophylax species elsewhere in Western and Central Europe, with risks of large‐scale hybridization and introgression.     

Water-soluble quantum dots for biomedical applications

Semiconductor nanocrystals are 1-10nm inorganic particles with unique size-dependent optical and electrical properties due to quantum confinement (so they are also called quantum dots). Quantum dots are new types of fluorescent materials for biological labeling with high quantum efficiency, long-term photostability, narrow emission, and continuous absorption spectra. Here, we discuss the recent development in making water-soluble quantum dots and related cytotoxicity for biomedical applications.     

Design of High-Specificity Nanocarriers by Exploiting Non-Equilibrium Effects in Cancer Cell Targeting

Although targeting of cancer cells using drug-delivering nanocarriers holds promise for improving therapeutic agent specificity, the strategy of maximizing ligand affinity for receptors overexpressed on cancer cells is suboptimal. To determine design principles that maximize nanocarrier specificity for cancer cells, we studied a generalized kinetics-based theoretical model of nanocarriers with one or more ligands that specifically bind these overexpressed receptors. We show that kinetics inherent to the system play an important role in determining specificity and can in fact be exploited to attain orders of magnitude improvement in specificity. In contrast to the current trend of therapeutic design, we show that these specificity increases can generally be achieved by a combination of low rates of endocytosis and nanocarriers with multiple low-affinity ligands. These results are broadly robust across endocytosis mechanisms and drug-delivery protocols, suggesting the need for a paradigm shift in receptor-targeted drug-delivery design.