James X. Hartmann

Using Hyaluronic Acid to Create a Fetal-like Environment in vitro

The fetal wound healing matrix is exceptionally rich in hyaluronic acid (HA). Fetal wounds heal without scarring or contraction. Noting these observations, we cultured adult dermal explants in the presence of various concentrations of medical-grade HA in vitro. In the presence of HA, fibroblasts migrated from the dermal explant and multiplied more rapidly than control explants. Subsequently, sterile toothpicks were used to disrupt (wound) fibroblast monolayers mechanically and the rate of closure was monitored. Cells cultivated in the presence of 5 mg/ml of exogenous HA changed in morphology and closed the wound more quickly than control cultures. Cells surrounding the wound extended numerous podalic processes and showed increased interdigitation. The effect of HA on cell proliferation is usually discussed in terms of the mechanical effects HA exerts on cells and the extracellular matrix. The physiological effect of HA may lie in its ability to act as an accessory receptor in cooperative ligand-binding pathways. For example, HA may bind growth and/or other factors, and thereby increase the effective concentration of these factors at the cell surface. Shepard S, Becker H, Hartmann JX. Using hyaluronic acid to create a fetal-like environment in vitro. Ann Plast Surg 1996;36:65-69

Pathogenic and Epiphenomenal Anti-DNA Antibodies in SLE

The discoveries of natural and the development of manufactured highly efficient catalytic antibodies (abzymes) opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy and prevention (vaccination), of cancer, AIDS, autoimmune diseases. A special entity of naturally occurring DNA hydrolytic anti-DNA antibodies is emerging within past decades linked to autoimmune and lymphoproliferative disorders, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjogren Syndrome (SS), B - Chronic lymphocytic leucosis (B-CLL), and Multiple Myeloma (MM). The origin of the antibodies is unknown. The underlying mechanisms of these activities are suggested to be penetration into the living cells and translocation in the nucleus, with recognition of the specific binding sites at particular (ss or ds) DNA. There are controversies in the literature whether hydrolysis is a sequence-specific event. The interplay between anti-DNA antibodies and DNA is not yet elucidated. This molecular “twist” also suggests that anti-DNA antibodies with DNA hydrolytic capacity could be the organisms immune response to a microbial attack, with microbial DNA, or specific genes within microbial DNA sequence, as a target for neutralization. The catalytic antibody-based approach can become a key tool in selective chemotherapeutic strategies.

Effect of vitamin D on T-helper type 9 polarized human memory cells in chronic persistent asthma

BACKGROUND Vitamin D suppresses inflammation and vitamin D deficiency is linked to the severity of asthma symptoms. T-helper type 9 (TH9) cells are important in the pathogenesis, yet the effects of vitamin D on this subset of inflammatory T-helper cells from patients with chronic asthma is unknown. OBJECTIVE To determine the effects of vitamin D and dexamethasone on TH9 memory cells from adults with chronic persistent asthma and on a recall response to dust mite allergen. METHODS T-helper memory cells were cultured with cytokines that drive TH9 polarization with vitamin D and/or dexamethasone. Peripheral blood mononuclear cells (PBMCs) from patients with radioallergosorbent test results for house dust mite were stimulated with allergen in the presence or absence of vitamin D. Intracellular cytokines, transcription factors, and identification of cell surface phenotypic markers were determined by flow cytometry. RESULTS Vitamin D decreased interleukin (IL)-9, IL-5, and IL-8 but increased IL-13(+) cells in TH9 cultures. Transcription factors PU.1 and interferon regulatory factor 4 were downregulated by vitamin D but not GATA3 and c-MAF. When PBMCs from patients with positive radioallergosorbent test results were stimulated with dust mite allergen, vitamin D decreased IL-9, IL-5, and IL-13 in T-helper cells (CD4(+)). TH9 cells present in a recall response were classically TH2 (CD294(+)), and polarization by transforming growth factor-β and IL-4 altered that phenotype. CONCLUSION Vitamin D decreased inflammatory cytokine profiles in TH9 memory cells and CD4(+) cells stimulated with dust mite allergen. Vitamin D is additive with dexamethasone in decreasing inflammatory cytokine production from T-cell subsets implicated in asthma.

Electron microscopic study of plant-animal cell fusion.

SummaryAvian erythrocytes and protoplasts isolated from mesophyll cells of tobacco plants were suspended in 1% protease, agglutinated with polyethylene glycol (PEG) and subsequently fused upon elution of the PEG. The fusion reaction was monitored by scanning (SEM) and transmission (TEM) electron microscopy. SEM studies showed a marked difference in the topography of agglutinated cells. During, and subsequent to fusion, the markedly different surfaces of the two cell types became homogeneous and lines of demarcation between the cells were no longer visible. TEM revealed that adhesion occurred over the entire membrane area between agglutinated cells. Incipient fusion was evidenced by the appearance of vacuoles at the intermembrane surfaces. During initial elution of the PEG, cytoplasmic channels between erythrocytes and protoplasts were evident. With continued elution of the PEG, starch-containing plant chloroplasts and starch grains were seen within erythrocytes and homogenous erythrocyte cytoplasm was present inside plant protoplasts. Cytoplasmic mixing between the two cell types occurred within 3 hours of elution. The frequency of interkingdom fusion was estimated to be 0.5–1%.

Highly Specific Novel Method for Isolation and Purification of Lupus Anti‐DNA Antibody via Oligo‐(dT) Magnetic Beads

Abstract:  A novel method for isolation and purification of anti‐ssDNA antibodies from human sera is developed. The process involves: antibody purification based on their affinity for single‐stranded sequence of thymidines and removal of remaining components via protein G coated magnetic beads, with high affinity for only IgG subclass. The high degree of purity and molecular weights of healthy versus lupus anti‐ssDNA antibodies were confirmed by SDS‐PAGE and silver staining. Western blot confirmed IgG isotype. This novel technique allows for diagnostic purposes, structural and functional analysis of anti‐DNA antibodies, and studies of their role in autoimmune diseases.

Production of functional native human interleukin-2 in tobacco chloroplasts.

Interleukin-2 (IL-2) is an important T lymphocyte-derived cytokine in the mammalian immune system. Non-native, recombinant IL-2 derived from Escherichiacoli is used widely in both medical research and treatment of diseases. Recombinant human IL-2 gene has been expressed in plant nuclear genomes, therefore it can be spread to the environment through pollen. Furthermore, all the plant-produced IL-2 reported thus far had been attached with artificial tags or fusion proteins, which may trigger unintended immunological responses and therefore compromise its full utility as a medicine. To expand the potential of using plant chloroplasts to produce functional native human therapeutic proteins, we inserted an engineered human interleukin-2 (hIL-2)-coding gene, without any tags, into the chloroplast genome of tobacco (Nicotiana tabacum L.). Partially purified hIL-2 protein from the leaves of the transplastomic plants induced in vitro proliferation of IL-2-dependent murine T lymphocytes. Our study demonstrates that plant chloroplasts can serve as a bio-factory for production of an active native human interleukin in a self-contained and therefore environmentally safe manner.

A Novel Method for Real-Time, Continuous, Fluorescence-Based Analysis of Anti-DNA Abzyme Activity in Systemic Lupus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of antibodies against a variety of self-antigens including nucleic acids. These antibodies are cytotoxic, catalytic (hydrolyzing DNA, RNA, and protein), and nephritogenic. Current methods for investigating catalytic activities of natural abzymes produced by individuals suffering from autoimmunity are mostly discontinuous and often employ hazardous reagents. Here we demonstrate the utility of dual-labeled, fluorogenic DNA hydrolysis probes in highly specific, sensitive, continuous, fluorescence-based measurement of DNA hydrolytic activity of anti-ssDNA abzymes purified from the serum of patients suffering from SLE. An assay for the presence and levels of antibodies exhibiting hydrolytic activity could facilitate disease diagnosis, prediction of flares, monitoring of disease state, and response to therapy. The assay may allow indirect identification of additional targets of anti-DNA antibodies and the discovery of molecules that inhibit their activity. Combined, these approaches may provide new insights into molecular mechanisms of lupus pathogenesis.

Are filtration rates for the rough tunicate Styela plicata independent of weight or size

The filtration rate of the rough tunicate Styela plicata was determined as an aid for potential use as a bioremediator of algae and bacteria contamination in estuarine waters. Filtration rates were calculated hourly over a period of six hours for tunicates (16.8 to 57.8 grams) exposed to two targeted concentrations (105 and 106 cells mL−1) of the microalgae Nannochloropsis sp. (n = 7 per treatment) and the bacteria Escherichia coli (n = 6 per treatment). Filtration rates for individual tunicates exposed to microalgae differed as much as 3520 mL hr−1 within an hour and 2349 mL hr−1 with bacteria. However, the average filtration rate of tunicates exposed to microalgae at 105 cells mL−1 was 3065 mL hr−1 animal−1(± 1284 mL hr−1 s.d.), 3252 mL hr−1 animal−1 (± 1039 mL hr−1 s.d.) at 106 cells mL−1 and 3158 mL hr−1 animal−1 when combined. The average filtration rate with bacteria at 105 cells mL−1 was 4654 mL hr−1 animal−1 (± 810 mL hr−1 s.d.), 2296 mL hr−1 animal−1 (± 1460 mL hr−1 s.d.) at 106 cells mL−1 and 3475 mL hr−1 animal−1 when combined. There was no relationship between average hourly filtration rate and whole animal weight (r2 = 0.0001) or dry organ weight (r2 = 0.0067) indicating that filtration rate should not be reported on a live or dry weight basis. It is suggested that averaging the filtration rate of a population of animals over time would yield a more accurate value, especially for use in modeling of bioremediation effects.

Routine establishment of primary elasmobranch cell cultures

Dear Editor: Members of the class Elasmobranchiomorphi (Elasmobranchii/ Chondrichthyes of some authors), such as sharks, skates, and rays are interesting organisms for a number of reasons. Unlike most marine mammals and marine bony fishes, elasmobranchs have intracellular osmolarities nearly equal to that of seawater (11). This high osmolarity is maintained by concentrating urea to an average of 0.4 M. In order to counteract the destabilizing effects of the urea on proteins and other macromolecules, they utilize methylamine compounds and amino acids (10,17). Shark cartilage is a source of compounds that prevent the vascularization of animal and human tumors (8). The mechanism whereby sharks exhibit resistance to the development of malignant neoplasias (4) remains unknown, even though sharks possess oncogenes (12). It would be advantageous to employ cultivated elasmobrancb cells to address the areas of research described above on the cellular level and provide a means to isolate elasmobranch viruses. Garner (2) obtained limited growth in brain cells from a sharpnose shark (Rhizoprionodon porosus) after one month of culture. Our Laboratory (6) reported limited success in primary culture using a medium supplemented with urea and TMAO. Grogan and Lund (3) used urea and TMAO in the short term culture of elasmobranch immunocytes. In this paper, we describe a medium and procedures for the routine and rapid establishment of shark and sting ray primary cultures. Heart, kidney, brain, liver, testes, spleen, and fin epidermis tissues were aseptically resected from 20 nurse sharks (Ginglymostoma cirratum), 4 horn sharks (Heterodontus francisc 0, 1 silky shark (Carcharhinusfalciformis), and 8 yellow stingrays (Urolophus jamaicensis). The tissues were placed in phosphate buffered saline modified for use with shark cells (SPBS), which is Dulbeccos phosphate buffered saline to which 333 mM urea, 240 mM sodium chloride (NaCI) and 54 mM trimethylamine N-oxide (TMAO)/(Sigma Chemical Co., St. Louis, MO) are added. Samples from all tissues examined were minced and divided evenly. Half were placed directly onto the surface of 25 cm 2 cell culture flasks (Coming Works, Coming, NY) (14). The other half of each sample was treated for 15 min with 0.25% trypsin (DIFCO, Detroit, MI). The trypsin was removed by pipeting and the tissues were then placed into flasks (15). Culture medium was gently added after the tissues were allowed to adhere to the surface of the flask for 1 h (9). Eagles minimum essential medium, Medium 199, RPMI164o, Leibovitzs L-15, Hams F-12 and Opti-MEM I (Grand Island Biological Co., Grand Island, NY) were modified by the addition of 333 mM urea, 188 mM NaCI and 54 mM TMAO, with the pH adjusted to 7.1 Fetal bovine serum (FBS), shark serum, or skate serum, all heat inactivated at 56 ° C for 30 min, were added to a final concentration of 10% to the modified medium. Gentamicin at 50 #g/ml was added to all media and the final osmolarity of the media measured 9 3 0 1 0 0 0 raPs. The flasks were placed at 26 ° C in either ambient air or 5% CO2. Half the conditioned medium was changed with fresh medium weekly. Of the eight tissue types tested for growth in vitro, heart atrium, kidney, testis, and brain cells could be routinely cultivated from all species examined. No marked differences in the extent and type of cell growth were noted between species. Growth was evident within one week following the plating of tissues into flasks, and extensive growth with mitotic activity was obtained after three weeks of cultivation (Fig. 1}. The morphology of kidney cells from the shark were seen as fibroblast-like (Fig. I a), epithelial-like (Fig. 1 b), and large epithelial-like (Fig. 1 c). The shark heart, kidney, and brain grew as epithelial-like cells (Fig. 1 d). Spleen, liver, and fin epidermis did not grow. Heart atrium and brain tissues could be grown by plating these tissues directly into medium following mincing. These two tissue types began to adhere to a flask surface within 10 minutes, and more than 90% of the tissue pieces adhered within 1 h of incubation. Kidney and testicular tissues required exposure to trypsin for successful cultivation, and only 50% of the tissue pieces remained adherent after 1 h of incubation. If the minced tissues were treated with trypsin for periods longer than 15 min they became gelatinous, the trypsin was difficult to remove, and the ceils subsequently died. Eagles minimum essential medium, RPMI1640, and Medium 199 modified as described above, did not support the growth of primary cultures. The modified Opti-MEM I was superior to that of Hams F-12 and Leibovitzs in both initiating and maintaining growth. Cultures initiated on Opti-MEM I and changed to Leibovitzs or Hams F-12 media became granular and ceased growing (Table 1).

Ascorbate, cyclic nucleotides, citrus and a model for preventing large bowel cancer

Abstract There is evidence that the risk for developing large bowel cancer is dependent on dietary practice. The “western” diet, high in meats, proteins, fats and refined carbohydrates, and low in fiber, has been causally related to this disease. Diet is known to affect microflora, bile acids, neutral sterols and bacterial enzyme activities in the bowel. These changes may result in the production of carcinogens/ cocarcinogens within the large bowel (in situ). Current evidence suggests that nitrosamines and secondary bile acids and/or cholesterol metabolites may be the carcinogens and cocarcinogens respectively. Although the population of the United States is considered at high risk for developing large bowel cancer, the South-East as well as southern California, Arizona and Florida residents are interesting exceptions. The incidence of large bowel cancer is two-fold lower in these regions, in spite of many residents having previously lived in the higher risk north-eastern and mid-western states. Preliminary studies of dietary practices (meat consumption and dietary fiber) did not find differences which would account for the low risk regions. Our hypothesis is that citrus fruit consumption is the prime factor which can account for these low risk regions. Consumption of citrus fruits may prevent in situ production of fecal nitrosamines, secondary bile acids, and cholesterol metabolites. It has also been reported that ascorbate induces regression of preneoplastic bowel polyps. We suggest that polyp regression is the result of a cellular response in which ascorbate increases cyclic adenosine monophosphate, and the result is biochemical differentiation with an enhanced extrusion of aberrant polyp cells. Florida, which has a large immigrating population from the high risk north-eastern United States, provides an excellent model for metabolic-epidemiological studies of an area at low risk for colon cancer.