An improved method for generating human spinal cord neural stem cells.

Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.

Enhanced axonal transport: A novel form of "plasticity" after primate and rodent spinal cord injury.

Deficient axonal transport after injury is believed to contribute to the failure of CNS regeneration. To better elucidate neural mechanisms associated with CNS responses to injury, we transected the dominant voluntary motor system, the corticospinal tract (CST), in the dorsolateral T10 spinal cord of rhesus monkeys. Three months later, a 4.5-fold increase in the number of CST axons located in the spared ventral corticospinal tract at both the lesion site and, surprisingly, remotely in the cervical spinal cord was observed. Additional studies of increases in corticospinal axon numbers in rat and primate models demonstrated that increases were transient and attributable to enhanced axonal transport rather than axonal sprouting. Accordingly, increases in axonal transport occur after CNS injury even in the longest projecting pathways of the non-human primate, likely representing an attempted adaptive response to injury as observed in the PNS.

First Report of Phakopsora pachyrhizi, the Causal Agent of Asian Soybean Rust, on Florida Beggarweed in the United States.

Phakopsora pachyrhizi Syd. & P. Syd., which causes Asian soybean rust (SBR), was observed on Florida beggarweed, Desmodium tortuosum (Sw) DC., in Attapulgus, GA during late October and early November 2005. Tan to brown lesions (<1.0 mm in diameter) consistent with symptoms of SBR (2) were observed on older leaves of several plants collected near an SBR-infected soybean trial. Dissection (40 to 60×) and compound microscopy (×200 to 400) revealed conical pustules and ellipsoid, echinulate urediniospores (average size 15 × 20 µm) on the abaxial leaf surface. Polymerase chain reaction (PCR) (primers Ppm1 and Ppa2) (1) was conducted on four samples to confirm identification of P. pachyrhizi or P. meibomiae. Three were positive for P. pachyrhizi, and one was negative for both species. Using morphology and real-time PCR, SBR was confirmed as P. pachyrhizi by the USDA/APHIS in Beltsville, MD. Six noninfected Florida beggarweed plants were transplanted to pots during December 2005 and grown at 22 to 24°C in a greenhouse. On 11 January 2006, a water suspension of urediniospores collected from SBR-infected soybeans (1 × 105 spores per ml) was spray inoculated on all leaves to almost runoff and incubated for 48 h in a plastic humidity chamber. Lesions, pustules, and urediniospores consistent with SBR (2) were observed on 3 February 2006. A PCR assay was conducted on six samples from the infected greenhouse plants and all were positive for P. pachyrhizi. Florida beggarweed is widespread in the southern United States and may serve as an additional overwintering source for P. pachyrhizi and a potential inoculum source for the soybean crop. References: (1) R. D. Fredrick et al. Phytopathology 92:217, 2002. (2) J. B. Sinclair and G. L. Hartman. Soybean rust. Pages 25-26 in: Compendium of Soybean Diseases. 4th ed. G. L. Hartman et al., eds. The American Phytopathological Society, St. Paul, MN, 1999.

First Demonstration of Koch's Postulates for Lasiodiplodia theobromae Fruit Spot on Eggplant (Solanum melongena).

During October 2004, diseased eggplant fruit from a commercial farm in Colquitt County, Georgia, developed circular, tan, water-soaked lesions. Gray, septate mycelia quickly covered the fruit. Diseased fruit became shriveled, spongy, and mummified. Disease incidence in the field was approximately 1%. Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (synonym Botryodiplodia theobromae Pat.) (2) was isolated from the margins of lesions and cultured on acidified potato dextrose agar. The fungus produced grayish colonies with aerial hyphae and black ostiolate pycnidia massed into stroma. Mature elliptical conidia (25.8 × 15.6 µm) were brown, had a single septation, and longitudinal striations. Isolates obtained from peanut and pecan were included in the pathogenicity tests. Mature fruit cv. Nightshade were surface disinfested for 30 s in 70% ethanol, followed by 60 s in 0.5% sodium hypochlorite, rinsed twice in sterile distilled water, and allowed to dry. Inoculations were made by placing an agar plug containing L. theobromae mycelial side down on the surface of the fruit or wounding with a sterile toothpick containing mycelium of the fungus. Fruit similarly inoculated with agar plugs or sterile toothpicks served as controls. There were a total of three replicates. Fruit were placed in plastic containers lined with moistened paper towels. Containers were placed in a dew chamber and incubated (28°C, relative humidity >95%) for 3 days, and then evaluated. Symptoms identical to those observed on naturally infected fruit developed on inoculated fruit. Controls remained disease free. L. theobromae was reisolated from all symptomatic tissue, satisfying Koch's postulates. Disease damage on wounded fruit was twice that of nonwounded fruit. However, seven of nine inoculations with agar plugs containing L. theobromae resulted in infection. Lesion lengths from wound inoculations were 9.8, 7.3, and 5.2 cm for isolates from peanut, pecan, and eggplant, respectively. Generally, L. theobromae is considered a facultative wound pathogen or a secondary invader (3). However, this study suggests that direct infection can occur. Although fruit spot has been reported previously on eggplant (1), to our knowledge, this is the first report verifying L. theobromae as the causal agent. References: (1) S. A. Alfieri et al. Index of Plant Diseases in Florida. Fla. Dep. Agric. Consum. Serv. Bull. 11, 1984. (2) H. L. Barnett and B. B. Hunter. Illustrated Guide of Imperfect Fungi. 4th ed. The American Phytopathological Society St. Paul, MN, 1998. (3) P. M. Phipps and D. M. Porter. Plant Dis. 82:1205, 1998.

The immediate effects of Hurricane Iniki on intertidal fauna on the south shore of O'ahu.

When Hurricane Iniki struck the Hawaiian Islands in September 1992, it provided a rare opportunity to examine the immediate effects of a hurricane on two intertidal benthic communities off the reefs of O'ahu, Hawai'i. The Niu Beach site contained large, obvious aggregations of the tube building polychaete Diopatra dexiognatha, and the Wailupe Beach site was without obvious tubiculous fauna at the surface. Ten replicate sediment cores were taken before and after the hurricane with a 7.6 cm PVC corer and organisms were identified to family and enumerated. There were no substantial depletions or loss of taxa after the hurricane. Oligochaetes were the most dominant taxa pre-and post-hurricane. The abundance of all dominant polychaete families increased post-hurricane. The three most abundant polychaetes were capitellids and D. dexiognatha (Onuphidae) at Niu Beach and Pygospio muscularis (Spionidae) at Wailupe Beach. We suggest that D. dexiognatha and P. muscularis help stabilize the sediments since they both form dense tube mats while capitellids and oligochaetes are considered highly adaptive surface burrowers that can take advantage of newly disturbed sediments. Overall, there was no substantial effect observed on the intertidal fauna exposed to this severe disturbance. It is suggested here that invertebrate communities in this area are adapted to survive and thrive in high-energy environments and possibly benefit from dense aggregations of tube building polychaetes.

First Report of a Leaf Blight of Onion Caused by Xanthomonas spp. in Georgia.

In October of 2001 and 2002, a leaf blight was reported affecting Vidalia onion (Allium cepa) cvs. Pegasus and Sweet Vidalia, respectively, in one field each. Lesions on onion seedlings began as a water-soaked, tip dieback that gradually blighted the entire leaf. Symptoms on onion transplants appeared as elongated, water-soaked lesions that typically collapsed at the point of initial infection. In both cases, disease was very severe on seedlings, and disease incidence was 50% or more in both fields. Warm temperatures combined with overhead irrigation and above average rainfall likely enhanced the severity and spread of disease. Disease was not detected on more mature onions once cool, dry conditions occurred later in the season, and no significant economic loss occurred. Seed was tested from seed lots of the aforementioned cultivars and Xanthomonas spp. were not found. Diseased tissue was macerated in sterile, phosphate-buffered saline, and 10 µl of the resulting suspension was streaked on nutrient agar plates. Yellow-pigmented, gram-negative, rod-shaped bacteria were isolated routinely from diseased tissue. Bacteria were catalase-positive, cellulolytic, oxidase-negative, amylolytic, proteolytic, and utilized glucose in an oxidative manner. Analysis of whole cell, fatty acid methyl esters (FAME) using the Microbial Identification System (MIS, Sherlock version 3.1; MIDI, Inc., Newark, DE) identified four representative strains of the bacterium as a pathovar of Xanthomonas axonopodis (similarity indices 0.75 to 0.83). Known Xanthomonas spp. from onion from Colorado and Texas (1,2) had similar FAME profiles when analyzed by the MIDI system. Onion plants were grown under greenhouse conditions for 2 months and inoculated by injecting the base of a quill with 1.0 ml of bacterial suspensions (1 × 107 CFU ml-1) of the Xanthomonas sp. isolated from Georgia, and negative controls were inoculated with 1 ml of sterile water. Disease symptoms developed on plants inoculated with bacterial suspensions in 4 to 7 days and Xanthomonas sp. was isolated from the lesions produced. Disease symptoms occurred when the same suspension was sprayed on onion foliage. No symptoms occurred on plants inoculated with 1 ml of sterile water. To our knowledge, this is the first report of Xanthomonas spp. affecting Vidalia onions. References: (1) T. Isakeit et al. Plant Dis. 84:201, 2000. (2) H. F. Schwartz and K. Otto. Plant Dis. 84:922, 2000.

First Report of a Field Outbreak of a Bacterial Leaf Spot of Cantaloupe and Squash Caused by Pseudomonas syringae pv. syringae in Georgia.

In March 2000, a leaf spot was reported affecting yellow summer squash (Cucurbita pepo) and cantaloupe (Cucumis melo) in commercial fields in Colquitt, Echols, and Grady counties in Georgia. All of the crops affected were reported within a 10-day period, and average temperatures during that time were 8 to 22.5°C, which is very close to the 50-year normal temperatures for these areas located in southwest Georgia. Incidence in affected fields was 100%. Lesions on squash leaves appeared irregularly shaped, dark, water soaked, somewhat vein restricted, and were 5 to 10 mm in diameter. Lesions on cantaloupe were angular, light tan, and necrotic with a lesion diameter of 2 to 5 mm. A general chlorosis was observed around lesions of both crops. Leaf distortion was observed on squash. Four isolates collected were used in biochemical, pathogenicity, and physiological tests. Gram-negative, rod-shaped bacteria were isolated from diseased tissue from squash and cantaloupes. Bacteria were aerobic, catalase-positive, fluorescent on King's medium B (1), oxidase-negative, nonpectolytic on potato, arginine dihydrolase-negative, utilized sucrose as a carbon source, produced levan, and gave a hypersensitivity response on tobacco (HR). Analysis of fatty acid methyl ester (FAME) profiles using the Microbial identification system (Sherlock version 3.1, Microbial identification system, Newark, DE) characterized representative strains as Pseudomonas syringae (similarity indices 0.65 to 0.80). Upon further characterization, the strains were negative for l (+)-tartarate utilization but utilized l-lactate and betaine and also exhibited ice nucleation activity. These characteristics are consistent with those of Pseudomonas syringae pv. syringae. Squash and cantaloupes were grown in a greenhouse for 4 weeks. Bacteria were grown in nutrient broth, resuspended in sterile tap water, and standardized using a spectrophotometer. Plants were inoculated by infiltrating leaves with 1 ml of bacterial suspensions (1 × 107 CFU/ml) using sterile syringes. Sterile water was used as a negative control, and 1 ml was infiltrated into leaves of squash and cantaloupes. Water-soaked lesions developed in 4 to 6 days on squash and cantaloupes inoculated with bacterial suspensions, and Pseudomonas syringae pv. syringae was isolated from diseased tissue. No symptoms developed on squash and cantaloupes used as negative controls. This outbreak of Pseudomonas syringae pv. syringae did not cause significant economic damage to either crop as symptoms subsided once daily high temperatures reached 28 to 32°C. This disease has been isolated from several cucurbit transplants reared in greenhouses, but to our knowledge, this is the first report of this disease occurring in the field. Reference: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954.

Expression of ferritin, transferrin receptor, and non-specific resistance associated macrophage proteins 1 and 2 (Nramp1 and Nramp2) in the human rheumatoid synovium.

OBJECTIVE: To gain a better understanding of how iron accumulates in human rheumatoid synovium. METHODS: The distribution of ferritin, transferrin receptor, and non-specific resistance associated macrophage proteins 1 and 2 (Nramp1 and Nramp2) in the human rheumatoid synovium was investigated by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Both heavy and light ferritin subunit types were detected in the lining layer and the subinitimal zone of rheumatoid synovium, heavy ferritin generally being more abundant than light. Both heavy and light ferritin were detected in isolated synovial macrophages and fibroblasts. Transferrin receptor expression was largely confined to fibroblasts of the synovial lining layer. Nramp2 was detected by PCR in both isolated synovial macrophages and fibroblasts, whereas Nramp1 was detected by PCR and immunocytochemistry in macrophages and neutrophils in the lining and subinitimal zone, and in inflammatory infiltrates, but was absent from fibroblasts. CONCLUSION: A complex chain of events, perhaps initiated by proinflammatory cytokines, may culminate in a toxic build up of iron in the rheumatoid joint.

Bacteriostatic activity of human lactoferrin against Staphylococcus aureus is a function of its iron-binding properties and is not influenced by antibiotic resistance.

The in vitro antistaphylococcal activity of lactoferrin and the antibiotic resistance of clinical Staphylococcus aureus isolates obtained from three different sites of infection were examined. Antibiotic, but not lactoferrin resistance correlated with selective antibiotic pressure, and nosocomial and most community isolates were antibiotic resistant, whereas only a third of each group was resistant to lactoferrin. The antimicrobial activity of lactoferrin, both in defined medium and in normal human plasma serum, was dependent upon its ferrochelating properties. Therapeutic approaches based on the use of ferrochelating agents such as lactoferrin combined with antimicrobial drugs may help to counteract the reduced efficacy of current antibiotics.

Liposomes as possible carriers for lactoferrin in the local treatment of inflammatory diseases.

Liposomes prepared from naturally occurring biodegradable and nontoxic lipids are good candidates for local delivery of therapeutic agents. Treatment of arthritis by intra-articular administration of anti-inflammatory drugs encapsulated in liposomes prolongs the residence time of the drug in the joint. We have previously shown that intra-articular injection of human lactoferrin (hLf), a glycoprotein that possesses anti-inflammatory and antimicrobial activities, into mice with collagen-induced arthritis reduces inflammation. We have now investigated the possibility of using liposome-entrapped hLf as a delivery system to prolong hLf retention at sites of local inflammation such as the rheumatoid joint. Entrapment of hLf in negatively charged liposomes enhanced its accumulation in cultured human synovial fibroblasts from rheumatoid arthritis (RA) patients, compared with positively charged formulations or free protein. However, in the presence of synovial fluid, positively charged liposomes with entrapped hLf were more stable than the negatively charged formulations. In vivo experiments in mice with collagen-induced arthritis showed that the positive liposomes were more efficient in prolonging the residence time of hLf in the inflamed joint as compared with other liposomes. Thus, the amount of hLf retained in the joint after 2 hr was 60% of the injected dose in the case of positive liposomes and only 16% for negative pH-sensitive liposomes. The results suggest that entrapment of hLf in positively charged liposomes may modify its pharmacodynamic profile and be of therapeutic benefit in the treatment of RA and other local inflammatory conditions.

Growth inhibition of Bacteroides fragilis by hemopexin: proteolytic degradation of hemopexin to overcome heme limitation.

The stimulatory effect of heme on growth of Bacteroides fragilis, an anaerobic human pathogen, was strongly inhibited by hemopexin, an avid (Kd<1 pM) heme-binding plasma protein. Both rabbit and human hemopexins were bacteriostatic for a limited period of time, suggesting an adaptation by B. fragilis to heme-limited growth, and that hemopexin-bound heme can eventually be utilized by the bacteria. The inhibitory effect of hemopexin was lost when heme in the medium was replaced by protoporphyrin IX, which is bound less strongly by hemopexin (Kd approximately 1 microM). Protease activity was detected in the culture supernatant of B. fragilis grown in the presence of heme plus hemopexin but not in the presence of free heme, protoporphyrin IX or protoporphyrin IX plus hemopexin, suggesting that the enzyme(s) is induced by heme macrocycle limitation due to the scavenging effect of hemopexin. This protease activity was able to degrade rabbit hemopexin and human hemopexin, as well as human transferrin and ovalbumin, and may be a due to a serine protease since it was inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by EDTA, leupeptin, pepstatin A or aprotinin. Thus, B. fragilis may overcome hemopexin-mediated heme limitation by secreting inducible protease(s), shown here to make protein-bound heme available to the microorganism.

Staphylococcus aureus fibronectin binding proteins are essential for internalization by osteoblasts but do not account for differences in intracellular levels of bacteria.

Staphylococcus aureus is a major pathogen of bone that has been shown to be internalized by osteoblasts via a receptor-mediated pathway. Here we report that there are strain-dependent differences in the uptake of S. aureus by osteoblasts. An S. aureus septic arthritis isolate, LS-1, was internalized some 10-fold more than the laboratory strain 8325-4. Disruption of the genes for the fibronectin binding proteins in these two strains of S. aureus blocked their ability to be internalized by osteoblasts, thereby demonstrating the essentiality of these genes in this process. However, there were no differences in the capacity of these two strains to bind to fibronectin or osteoblasts. Analysis of the kinetics of internalization of the two strains by osteoblasts revealed that strain 8325-4 was internalized only over a short period of time (2 h) and to low numbers, while LS-1 was taken up by osteoblasts in large numbers for over 3 h. These differences in the kinetics of uptake explain the fact that the two strains of S. aureus are internalized by osteoblasts to different extents and suggest that in addition to the fibronectin binding proteins there are other, as yet undetermined virulence factors that play a role in the internalization process.

The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis.

OBJECTIVE: To determine whether lactoferrin can modify articular inflammation in murine models of autoimmune and septic arthritis. METHODS: Collagen arthritis was induced in DBA/1 mice and Staphylococcus aureus septic arthritis in Swiss mice. Joints with established inflammation were injected periarticularly with 0.5 mg or 1 mg of human lactoferrin, and arthritis was monitored for 3 days. RESULTS: DBA/1 mice injected with lactoferrin showed significantly suppressed local inflammation for up to 3 days, achieving up to 71% of the effect of corticosteroid. Periarticular injection of 125I-lactoferrin confirmed that 25% of lactoferrin was retained in paws after 6 hours. Serum levels of interleukin-6, however, were not significantly reduced, suggesting a predominantly local antiinflammatory effect. Similarly, local, periarticular administration of lactoferrin into S aureus-infected Swiss mice significantly suppressed paw inflammation and did not enhance bacterial survival. CONCLUSION: Lactoferrin may have clinical utility in reducing articular inflammation, particularly in septic arthritis, in which antiinflammatory effects may be achieved without promoting bacterial survival.

A novel endoproteolytic processing activity in mitochondria of erythroid cells and the role in heme synthesis.

The erythroid isoform of aminolevulinate synthase (eALAS) protein is a major control point in erythroid heme synthesis and hemoglobin formation. Erythroid cells were extracted from mouse blood and bone marrow and metabolically labeled with (35)S-methionine. This was followed by immunoprecipitation of eALAS protein products. The results show that the N-terminus of the expected full-length 59-kd form of the eALAS protein is truncated in bone marrow erythroid cells by approximately 7 kd. More differentiated erythroid cells in the peripheral blood exhibit very little of this protein truncation. Erythroid cells from the bone marrow were isolated using monoclonal antibody TER-119 and were shown to contain a unique endoprotease activity that could cleave the eALAS protein to the shorter form in vitro. With or without the mitochondrial signal sequence, the eALAS protein could serve as a substrate for the cleavage. This cleavage renders a functional eALAS protein and only removes a domain of unclear function, which has previously been reported to vary in size as a result of alternative RNA splicing. The protease activity was enriched from the membranes of mitochondria from bone marrow cells and was shown to be different from mitochondrial processing peptidase, medullasin, and other known proteases. Apart from the mitochondrial processing peptidase that cleaves the import signal sequence, this is the first description of a mitochondrially located site-specific processing protease activity. (Blood. 2000;96:740-746)

Inhibition of binding of lactoferrin to the human promonocyte cell line THP-1 by heparin: the role of cell surface sulphated molecules.

Lactoferrin, an iron-binding protein of the transferrin family, is a highly basic protein which interacts with many acidic molecules, including heparin proteoglycans. Such interactions may modify some of the biological properties of lactoferrin. In the present work we found that heparin caused a dose-dependent inhibition of specific binding of both human and bovine lactoferrin to human monocytic THP-1 cells. Low-affinity binding sites (Kd 500 nM) were more susceptible to inhibition by heparin than the high-affinity sites (Kd 100 nM). The effect was mediated by interaction between lactoferrin and heparin rather than by competition between heparin and lactoferrin for common binding sites on the cells. Pretreatment of cells with NaClO3 to prevent sulphation of surface glycosaminoglycans reduced lactoferrin binding, and de-N-sulphated heparin did not inhibit binding of lactoferrin to THP-1 cells. These results suggest that heparin binding and monocyte/macrophage binding by lactoferrin both involve interactions between basic regions in the N1 domain of lactoferrin and sulphate groups. The N-terminal Arg2-Arg5 sequence of human lactoferrin may be involved, but it does not seem to be the key element in these interactions.

Cellular and molecular aspects of iron and immune function.

Fe plays a critical role in the immune system and defence against infection. However, many aspects of the way in which Fe influences these processes at the molecular and cellular level are unclear. The present review summarizes the role of Fe in lymphocyte activation and proliferation, and discusses how Fe is handled by macrophages.

Regulation of iron metabolism in murine J774 macrophages: role of nitric oxide-dependent and -independent pathways following activation with gamma interferon and lipopolysaccharide.

To elucidate the pathways by which nitric oxide (NO) influences macrophage iron metabolism, the uptake, release, and intracellular distribution of iron in the murine macrophage cell line J774 has been investigated, together with transferrin receptor (TfR) expression and iron-regulatory protein (IRP1 and IRP2) activity. Stimulation of macrophages with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) decreased Fe uptake from transferrin (Tf), and there was a concomitant downregulation of TfR expression. These effects were mediated by NO-dependent and NO-independent mechanisms. Addition of the NO synthase (NOS) inhibitor N-monomethyl arginine (NMMA) partially restored Fe uptake but either had no effect on or downregulated TfR expression, which suggests that NO by itself is able to affect iron availability. Analysis of the intracellular distribution of incorporated iron revealed that in IFN-gamma/LPS-activated macrophages there was a decreased amount and proportion of ferritin-bound iron and a compensatory increase in insoluble iron, which probably consists mainly of iron bound to intracellular organelles. Finally, although NO released by IFN-gamma/LPS-activated macrophages increased the iron-responsive element (IRE)-binding activity of both IRP1 and IRP2, IFN-gamma treatment decreased IRP2 activity in an NO-independent manner. This study demonstrates that the effect of IFN-gamma and/or LPS on macrophage iron metabolism is complex, and is not entirely due to either NO-or to IRP-mediated mechanisms. The overall effect is to decrease iron uptake, but not its utilization.

Benefits and dangers of iron during infection.

Iron is essential for both human and microbial metabolism, and it is therefore important that iron status is maintained at a level that permits optimal immune responses by the host but does not facilitate availability of iron to microorganisms. This paper reviews the role of iron in resistance to infection, with particular reference to malaria and hepatitis C, and discusses the desirability of iron supplementation of populations at risk of infection.