Closed Culture System Research Articles

GMP Scale up for a Clinical Gene Therapy Trial - High Efficiency Human T Cell Expansion and Transduction in a Closed Culture System Utilizing Serumfree Medium and Low IL-2 Concentrations.

Translational research is an ever increasing focus in the field of cellular and gene therapy. Here we demonstrate the scale-up process for a gene therapy clinical trial aimed at abrogation of Graft versus Host Disease (GvHD) using a suicide gene fused to a truncated CD34 receptor as a selectable marker in a Donor Lymphocyte Infusion setting to treat relapsed hematologic malignancies after allogeneic bone marrow transplantation. Transduced T cells can be purified to over 95% in a magnetic cell sorter (CliniMacs, Miltenyi, Auburn, CA) using the CD34 receptor, normally not present on T cells. After infusion, these T cells produce a Graft versus Leukemia effect, but can be eliminated by administration of Ganciclovir if GvHD develops. Donor T Cells were cultured in a closed system using 2 types of serumfree media: X-VIVO 15 (Cambrex, Walkersville, MD) and the newly developed GMP grade Stemline T Cell Expansion Medium (Sigma, St. Louis, MO). Stimulation was done using clinical grade magnetic beads coated with antibodies to CD3 and CD28 (Xcyte, Seattle, WA). High concentrations of IL-2 (500 U/ml) impair the in vivo functionality of cultured T cells, as shown in our NOD SCID/B2 M deficient mouse model of T cell expansion. To maintain in vivo functionality of transduced T cells, it was imperative to lower the IL-2 concentration to 50 U/ml during culture. At least a 2 fold T cell expansion with maintenance of a physiological CD4 and CD8 compartment is necessary, since transduction using a Moloney leukemia virus based retroviral vector depends on cell division. It is also important to transduce both CD4 and CD8 cells in the same ratio for normal function of the infused T cell population, and expression of the selectable surface marker for subsequent enrichment of the transduced cells in both fractions. Requirements stipulated by regulatory agencies demanded not to introduce more than 1–2 vector copies per cell to keep the risk for insertional mutagenesis to a minimum. We demonstrated that a transduction frequency of 20–30% obtained at an MOI of 1–2 is required to generate this copy number (Rettig et al., 2003). After 48 hours of pre-stimulation, the cells were transduced 2 times, medium was replaced by spinning the culture bags, removing 2/3 of the supernatant and replacing it with fresh vector containing medium. Cells were harvested on day 5 by disrupting bead/cell clumps and bead removal in a magnetic field. With X-VIVO 15, strong variability in expansion and viability at the end of culture could be seen using low concentrations of IL-2. However, the consistency of cell expansion increased dramatically when the concentration of IL-2 was increased to 1000U/ml. In contrast, the newly formulated Stemline serumfree medium consistently worked equally well in high and low concentrations of IL-2. At 50 U/ml of IL-2, a 3 fold expansion of T cells with a 30% transduction efficiency, equally well distributed in the CD4 and CD8 compartment, was observed. CD4/CD8 ratio was maintained at input ratio, with greater than 95% cell viability. Removal of magnetic beads after culture was consistently more than 2 logs. The transduced cells could be purified by magnetic cell sorting using the selectable marker, which was highly expressed. In summary, a new serumfree media formulation allows for reproducible, high efficiency expansion and transduction of T cells in low IL-2 concentrations to optimize in vivo T cell functionality for human gene therapy trials.

Dendritic cell culture: a simple closed culture system using ficoll, monocytes, and a table-top centrifuge.

Dendritic cells (DCs) are potent antigen-presenting cells involved in the induction of T cell-mediated immune responses and as such have emerged as important candidates for cellular-based therapies. Critical to safe clinical use is the easy manipulation of DCs and their precursors in a closed system. We have developed a serum-free, closed culture system applying a simple wash-Ficoll centrifugation method to reduce platelet and red blood cell (RBC) contamination. This procedure optimized adherence of monocytes (44 +/- 10.9% recovery, >85% expressed CD14(+)/CD163(+)) for the generation of DCs from mononuclear cell (MNC) apheresis units. Most RBCs and up to 98% of platelets were removed. Following density sedimentation, cell viability remained high (98 +/- 2%) with only minimal loss of monocytes (3 +/- 3%). Importantly, Ficoll-treated monocytes retained their ability to differentiate to mature DCs demonstrated by morphology, phenotype (MHC class II(+), CD1a(+), CD80(+), CD86(+), and CD83(+)), ability to stimulate mixed lymphocyte responses (MLR), present antigen, and produce interleukin-12 (IL-12). Nonadherent CD3(+) (80 +/- 4%) were also isolated for functional assays. Ficoll can be easily incorporated into a simple adherence-based closed system for collection of lymphocytes and adherent monocytes for DC culture. The procedure is relatively fast (effective working time 5-6 h), does not impair monocyte function or induce substantial cell activation, and can be performed economically using equipment found in a typical blood banking environment.

Large-scale monocyte enrichment coupled with a closed culture system for the generation of human dendritic cells

Conventional methods for generating monocyte-derived dendritic cells (DC) for clinical trials utilize the property of plastic adherence to select monocytes from leukapheresis samples. This method is labor-intensive and has the potential for contamination at various steps. We evaluated a large-scale monocyte enrichment procedure using a cell selector (Isolex 300i®) followed by culture in a sterile bag system (Stericell®) for generation of DC. DC generated in tissue culture flasks after monocyte selection by plastic adherence were compared to those generated in Stericell® bags after monocyte enrichment by negative selection with the Isolex® 300i. DC were matured with lipopolysaccharide and pulsed with a peptide derived from the melanoma antigen gp100. Peptide-pulsed DC cultured by the two techniques were evaluated for phenotype, viability, ability to induce allogeneic and peptide-specific autologous proliferative responses as well as peptide-specific cytotoxic T-cell responses. The mean monocyte yield from leukapheresis collections was 17±2.4%, which increased to 52±11% after Isolex® selection. The DC yield of plated mononuclear cells from flasks or bags was 2.7±0.96% and 4.84±2.65%, respectively. DC cultured by both methods expressed high levels of CD86, CD80, CD40, CD83, CD44, CD11c and CD58, and was comparable in their ability to induce allogeneic and peptide-specific autologous proliferative responses as well as gp100 peptide-specific cytotoxic T-cell responses. These results indicate that potent monocyte-derived DC can be generated in a closed culture bag system after monocyte enrichment by immunomagnetic negative selection. Due to the closed nature of the enrichment and culture systems, the potential for contamination is minimized. This protocol is well suited for culturing large numbers of DC for clinical immunotherapy trials.

Growth Characteristics of the Australian Redclaw Crayfish, Cherax quadricarinatus, Cultured in an Indoor Recirculating System

The redclaw crayfish has been cultured in ponds since about 1990. As interest in redclaw has expanded, some culturists have begun to ask about culture potential in areas where pond culture may not be feasible. This study was conducted to determine growth characteristics of redclaw crayfish in an indoor, closed-system culture unit. Redclaw crayfish of two initial sizes, small (0.3±0.1 g) and medium (6.9±2.9 g) were cultured in tanks (1 m2) for five months. Temperature was maintained at 29±1.0°C, and the photoperiod was set at a 12:12 light:dark cycle. Survivals ranged from 65% to 70%. Final mean weights for the two size groups were 5.0 g and 29.0 g, respectively. Redclaw crayfish growth was much lower in this study than has been routinely reported for redclaw crayfish growth in outdoor ponds but not much different from what others have reported for other crayfish species cultured in indoor tanks. Factors that might cause the reduced growth include inadequate diet, density effects, and pheromones.

Generation of large numbers of dendritic cells in a closed system using Cell Factories™

There is a growing interest in using dendritic cells (DC) for vaccine approaches in the treatment of cancer and infectious diseases. This requires a reproducible method for the generation of large numbers of DC in a closed culture system suitable for clinical use and conforming to the current guidelines of good manufacturing practices. We designed a system in which the DC were generated in a closed system from adherent monocytes using Cell Factories™ (DC-CF). Monocytes were enriched from apheresis products by adherence and then cultured in the presence of AB serum or autologous plasma and GM-CSF and IL-4 for 6 days. The DC generated in Cell Factories™ were extensively compared to research-grade DC generated in conventional tissue culture flasks (DC-TCF). At day 6, the immature DC were harvested and the yield, the viability, the immunophenotype and the functional characteristics of the DC were compared. DC-CF and DC-TCF showed similar viability and purity and scored equally when tested for stability, dextran and latex bead uptake, in MLR and in the activation of influenza-specific memory cells after electroporation with influenza matrix protein 1 (IMP1) mRNA. These data indicated that large numbers of functional clinical-grade DC could be generated from adherent cells in a closed system using Cell Factories™.

Cultivation of Paracentrotus lividus (Echinodermata: Echinoidea) on extruded feeds: digestive efficiency, somatic and gonadal growth

This study assessed the use of extruded feeds, in the form of pellets, for the growing of echinoid Paracentrotus lividus within a closed-culture system. Two feed types, one with soya-bean protein, the other with both soya-bean and fish protein were compared with dried Lessonia sp. and fresh Laminaria sp. as food sources. Pellets present a very high conversion efficiency (about 80%) against about 50% for Laminaria and 35% for Lessonia. However, as pellets are less absorbed, somatic growth (SG) is statistically equivalent for the sea urchins fed with pellets and Laminaria between 2 and 2.2% g soma day–1. Sea urchins fed with pellets produced significantly more gonadal tissue in a shorter time, resulting in a gonadal index (GI) twice higher (6.5%) than Laminaria (3%) in the second month of the experiment. Dry Lessonia does not promote gonadal growth (GG). This study shows that extruded feeds are well assimilated by P. lividus and promote both SG and production of gonadal tissue.

CELL DEATH IN TETRAHYMENA THERMOPHILA: NEW OBSERVATIONS ON CULTURE CONDITIONS

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain ‘physical’ conditions, in addition to the availability of oxygen within closed culture systems.

Efficient plant regeneration from multiple shoots formed in the leaf-derived callus of Lavandula vera, using the “open culture system”

To develop an efficient procedure for plant regeneration from leaf-derived callus of Lavandula vera DC, the production of multiple shoots and the formation of roots from these shoots were studied. When calli were cultured in a medium with 4.0×10 −7 M N-(2-chloro-4-pyridyl)- N′-phenylurea (CPPU), urea-type cytokinin, multiple shoots were obtained from the greenish surface of the callus efficiently at a rate of 52.2%. For multiple shoot development, the shoots were divided into 7–20 shoot clusters with 2–3 shoots and subcultured every 2 weeks. Two modified culture-systems were employed for subculture; a “closed culture system” and an “open culture system”. In the open-system, shoot clusters grew and elongated vigorously. In the closed-system, most of the clusters showed vitrification and did not elongate. In root induction culture, shoot clusters in the open-system were well rooted and grew vigorously, the highest rate of root formation being 74.0%, in the 1 2 MS medium supplemented with 1.0×10 −6 M indoleacetic acid (IAA). On the other hand, shoot clusters in the closed-system showed vitrification and did not root well. About seven times more regenerants were obtained from the multiple shoots than from normal shoots treated with 4.4×10 −6 M 6-benzylamino purine (BA), a purine-type cytokinin. These results indicate that efficient plant regeneration in L. vera from callus using multiple shoots is possible by means of an open-system.

Selective depletion of alloreactive t cells from allografts using the isolex® and closed system culture technology

Abstract Graft-Versus-Host Disease (GVHD) is a major problem with allogeneic stem cell transplantation. Depletion of donor T cells reduces GVHD, but increases the risk of graft rejection and infection. Selective depletion of donor T cells that react to the recipients' alloantigens might ameliorate GVHD while retaining the majority of the T cell repertoire. We developed large-scale procedures using Nexell Lifecell ® culture bags for mixed lymphocyte reaction culture (MLR) and experimental software on the Isolex ® 300i system to eliminate alloreactive T cells. Alloreactive donor T cells cultured with irradiated recipient cells can be identified by their expression of activation antigens such as CD25 and CD69. We evaluated the use of anti-CD25 and anti-CD69 antibodies to deplete alloreactive T cells. Peripheral blood mononuclear cells (PBMC) from normal donors were co-cultured with irradiated recipient cells in Lifecell R bags for 96–120 hours. Alloreactive activated cells sensitized with either murine anti-CD69 PE mAb or anti-CD25 FITC were removed using M-450 Dynabeads ® sheep anti-mouse IgG. Anti-CD25 mAb had higher purging efficiency (93 ± 5% for n=8) compared to anti-CD69 antibody (74 ± 12% for n=6) in a small-scale depletion procedure (30–50 million cells). We then performed large-scale depletion procedures using the Isolex ® 300i. A 77.3 ± 9% depletion (n = 3) with anti-CD69 antibody and a 98 ± 2% (n = 3) depletion with anti-CD25 mAb was achieved. Depleted donor T cells exhibited decreased cytotoxic activities against recipient target cells. This procedure provides a large-scale method for depleting alloreactive T cells from grafts.

Préparation de produits de thérapie cellulaire: apport des systèmes clos

A phase I clinical trial is being currently performed in our institution, aiming at evaluating the feasibility and toxicity related to the administration of Herpes Simplex-thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using sampling and transfer pack bags, culture bags and a sterile connection device which avoids opening the culture system. This closed system allows safe and reproducible ex vivo preparation of gene-modified primary T-lymphocytes for clinical use.

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.

Ex vivo generation of dendritic cells from CD34+ cells in gas-permeable containers under serum-free conditions.

Dendritic cells (DC) are efficient and potent APCs that can be generated ex vivo. For them to be used clinically, however, a closed culture system using serum-free medium should be used. Our goal was to differentiate DC from human blood CD34+ cells in serum-free media in a new gas-permeable culture container, PL2417. Apheresis products were collected from healthy G-CSF-mobilized donors, and CD34+ cells were selected using the Isolex immunomagnetic cell selection system. Cells were cultured in the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in various serum-free media and compared with serum-containing medium in 4-well plates. One of the serum-free media was then selected and used in PL2417 containers and compared with serum-containing medium in standard flasks. The cells were evaluated at days 0, 7, and 14 for the presence of DC, which were identified morphologically after Wright-Giemsa staining by cytoplasmic processes extending from the surface of the cell. The cultures were evaluated phenotypically by flow cytometry and immunohistochemistry. The stimulatory capacity was examined in MLR. Overall, results from serum-free media and PL2417 containers were comparable results obtained under the other conditions. These data indicate that culture-deriving DC from CD34+ cells in PL2417 closed system containers using serum-free media is as effective as using standard flasks and serum-supplemented media.

Corrigendum

Journal of HematotherapyVol. 7, No. 5 Corrigendumis erratum ofA Closed Culture System for the Ex Vivo Transduction and Expansion of Human T LymphocytesPublished Online:27 Mar 2009https://doi.org/10.1089/scd.1.1998.7.478AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail AbstractIn the article, "A Closed Culture System for the Ex Vivo Transduction and Expansion of Human T Lymphocytes," by E. Robinet et al., Journal of Hematotherapy 7:205-215, 1998, there was an error in Table 3 on page 214. We reprint the corrected table here.FiguresReferencesRelatedDetailsRelated articlesA Closed Culture System for the Ex Vivo Transduction and Expansion of Human T Lymphocytes27 Mar 2009Journal of Hematotherapy Volume 7Issue 5Oct 1998 To cite this article:Corrigendum.Journal of Hematotherapy.Oct 1998.478-478.http://doi.org/10.1089/scd.1.1998.7.478Published in Volume: 7 Issue 5: March 27, 2009PDF download

Performance of Pinus contorta inoculated with two strains of root endophytic fungus, Phialocephala fortinii: effects of synthesis system and glucose concentration

Melanized, root-colonizing fungi are ubiquitous. Their ecological role, however, is poorly understood, and results of studies of associations between these fungi and their potential host plants are controversial. The culture system under which the association is studied may also affect the host-fungus interaction. Two experiments on the association between Pinus contorta Dougl. ex Loud., and a root-inhabiting endophytic fungus, Phialocephala fortinii Wang & Wilcox, were conducted to study the host response to inoculation. First, Pinus contorta seedlings were inoculated with two strains of Phialocephala fortinii and grown under aseptic conditions with five levels of glucose in the medium. Multiple linear regression analysis was employed to study the effects of inoculation and glucose concentration. Second, the same two strains of Phialocephala fortinii were inoculated on Pinus contorta seedlings in open pot cultures. Inoculation resulted in substantial increase in all biomass components of the host plant in the aseptic culture system. Total biomass, for example, was increased approximately 60 and 90% when seedlings were inoculated with strain 1 and strain 2, respectively. No seedling mortality was observed following fungal inoculation after 6 months of incubation. Inoculation increased host biomass with increasing glucose concentration, while glucose concentration did not significantly affect host biomass when no inoculum was added. Inoculation lowered foliar nitrogen and phosphorus concentrations. The effect of glucose concentration on the foliar nutrient concentrations varied between the two strains. In the open pot cultures, inoculation did not affect biomass or foliar nutrient concentration. We hypothesise that the observed increases in host growth in the aseptic culture system are due to fungal respiration in a closed culture system, the carbohydrates made available to the host plant by the fungus, or, most likely, to a combination of both factors. The ecological role of Phialocephala fortinii and the validity of aseptic culture assays are discussed.Key words: dark-septate endophytes, deuteromycetes, fungi, host-fungus associations, root endophytes, symbiosis.

養液栽培における培養液濃度がイチジクの生長と果実品質に及ぼす影響

イチジク'桝井ドーフィン'を用い, ロックウール底面給液と潅水チューブによる給液を併用した非循環閉鎖型養液栽培システムにおいて, 挿し木1年目から年間に2作をする栽培体系の検討を行った.第1作は1月10日に挿し木し, 2月24日に定植した.培養液濃度はEC 1.8 dS/mと2.4 dS/mを与えたところ, 第1作の収穫を打ち切った6月30日まで貯溜水槽のECは両区ともしだいに上昇しそれぞれ9.5と10.1 dS/mとなった.培養液濃度がEC 2.4 dS/m区ではEC 1.8 dS/m区に比べ吸液量の増加速度が劣った.5月上旬からEC 2.4 dS/m区の給液濃度を0.6 dS/mに低下すると吸液速度も促進された.最上位の葉面積は吸液量の少ないEC 2.4 dS/m区で特に劣った.収穫初期の果実重は, EC 1.8 dS/m区およびECを0.6 dS/mに変化させた区において大きかった.糖度は逆にEC 2.4 dS/m区が他の2区よりも高かった.6月30日に切り戻しせん定すると, 12日後に再発芽した.しかし, 10節目まではほとんど着果せず, 11月16日が収穫開始日となり, 12月28日までには大部分の果実が収穫された.最上位の葉面積, 果実数, 果実重および糖度ともいずれも第1作には及ばなかった.また, 9月からECを0.3 dS/mに低下させると裂果が多発した.

Evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts.

Injury to articular cartilage predisposes that joint to further degeneration and eventually osteoarthritis. Recent studies have demonstrated the feasibility of using chondrocytes together with different biomaterial carriers as grafts for the repair of cartilage defects. The following study was undertaken to determine the effect of a variety of these materials on chondrocyte growth and extracellular matrix synthesis. We cultured chondrocytes on several commonly used materials and compared their rates of synthesis of proteoglycan and collagen. Additionally, we evaluated them in a closed culture recirculating system on these materials and compared them with standard culture techniques. This was done to see whether such a bioreactor-type system can be used to enhance the quality of in vitro reconstructed tissues. Our results demonstrated marked variability with respect to how chondrocytes responded to culture on the various materials. Bioabsorbable polymers such as polyglycolic acid (PGA)--enhanced proteoglycan synthesis, whereas collagen matrices stimulated synthesis of collagen. The use of the closed culture system, in general, improved the rates of synthesis of collagen and proteoglycan on the different material scaffolds. Exceptions were collagen synthesis on collagen matrices: use of the closed culture system did not enhance the rate of synthesis. Rates of proteoglycan synthesis on PGA scaffold initially was higher in the closed culture system but did not sustain a difference over the entire course of the 3-week culture period. This study demonstrates the importance of carrier material for the purpose of cartilage tissue reconstruction in vitro.

Design Procedure for Hooded Surface Oxygen Absorption Systems

AbstractA design procedure addressing effluent total dissolved gas pressure limits along with standard performance indicators such as oxygen absorption efficiency (kg absorbed/kg applied), and transfer efficiency (kg/kW.h), is presented for surface agitation equipment operating in an oxygen‐enriched atmosphere. Application of this contactor type in closed culture systems is attractive given its insensitivity to biological fouling and ability to operate without the need for a significant hydraulic gradient. Performance algorithms were developed through application of chemical reactor theory, Henry's Law and the Ideal Gas Law. In the analysis, gas and liquid phases were treated as being homogeneous. The design steps presented are unique in that required mass transfer coefficients (KLa), operating pressures (CP), and oxygen feed rates (G/L) are calculated for target changes in both dissolved oxygen and nitrogen without the use of iterative numerical procedures. A second calculation sequence establishes the sensitivity of system performance to changes in G/L when CP and KLa are known.

A thermochemical study of metabolic pathways in activated and triggered 2C11–12 mouse macrophage hybridoma cells

Abstract Hybridization with lymphosarcoma cells immortalized mouse macrophages for the study of phagocytosis and cytobicidal properties. In the study of macrophage physiology, it became necessary to know more of their cellular metabolism and the changes which occurred when the cells were triggered into the respiratory burst. It was found that, in common with many other types of growing cell, activated 2C11-12 macrophage hybridomas produced considerable lactate under fully aerobic conditions, judging from the highly exothermic CR ratio and subsequent spectrophotometric analysis. Most of both the substrates (glucose and glutamine) was converted to lactate, respectively, by glycolysis and glutaminolysis, in the demand for biosythetic precursors during growth. Glucose was the more important energy source. Approximately 60% of heat production was explained in terms of enthalpy changes in glucose and glutamine metabolism. It was suspected that fatty acid oxidation from contaminants in the bovine serum albumin needed for cell culture may be important in catabolism. The respiratory burst was triggered by phorbol-12-myristate-13-acetate and recorded by greatly (5-fold) increased heat production and enhanced chemiluminescence. Oxygen consumption was very rapid and soon led to anoxia in the closed culture system. The calorimetric-respirometric (CR) ratio was less negative and analysis confirmed that there was less lactate production. Radioisotope studies indicated that glycolysis and glutaminolysis were less intensive, with respiration of glucose accounting for over 90% of the heat production. The imperative for producing NADPH and cytotoxic oxygen metabolites heavily biased catabolism, reducing the supply of biosynthetic precursors. Known sources of heat production accounted for 87% enthalpy recovery and the remainder may well be caused by fatty acid oxidation.

Arthritis in the highlands of Papua New Guinea.

Acute polyarthritis is an important cause of morbidity in many tropical countries. Classification has often been difficult, with the term tropical polyarthritis used for those in whom a diagnosis could not be made. The implication that this is a distinct entity is probably incorrect, with likely causes being septic arthritis or post-infective reactive arthritis. This study aimed to determine the types of arthritis found in 43 patients (30 men) presenting consecutively to the Goroka Base Hospital in the Eastern Highlands of Papua New Guinea. Gonococcal arthritis was diagnosed in eight patients (six men) on the basis of isolation of Neisseria gonorrhoeae from the joint aspirate. In all cases the N gonorrhoeae was identified by the closed culture system on chocolate agar, but not always by routine plating. There were no specific clinical features that identified patients with a gonococcal septic arthritis. The remaining 34 patients had an undifferentiated oligoarthritis. The pattern of arthritis in men and women was of a lower limb pauciarticular arthritis with a predilection for the knee and ankle joints. A total of 30% of male patients had a history of urethral discharge and 44% of all patients had preceding diarrhoea. Arthritis was the only feature in 59% of patients and in 32% there was an associated enthesitis. In this study most patients had an oligoarthritis consistent with a reactive arthritis or a septic arthritis due to N gonorrhoeae. Broth inoculation of synovial fluid was the best method to isolate N gonorrhoeae, with standard methods for gonococcal isolation failing in some patients. It is recommended that the term 'tropical polyarthritis' is no longer used as it does not refer to a specific entity but consists of several known arthritides.

Direct measurement of nitric oxide in headspace gas produced by a chicken macrophage cell line in a closed culture system

A simple and rapid method was applied for direct measurement of nitric oxide (NO) gas produced by cultured macrophages using a modified chemiluminescence detector, the thermal energy analyzer (TEA). HD 11 chicken macrophages (1–3 × 106/ml) were cultured on microcarrier beads (100 mg/ml) in 140 ml air-tight glass jars (5 ml cell suspension per jar) containing 0.5 μg/ml of LPS and different concentrations ofl-arginine. Headspace gas was sampled at 24 hours of culture via a rubber septum and directly injected into a TEA with a liquid nitrogen trap set at −130 to −140°C. The concentration of NO in the gas sample was quantified using a standard gas mixture of NO (2 μl/L) in nitrogen. Gas samples froml-arginine-supplemented cultures contained NO (0.028 – 0.066 pl/μl), whereas NO was not detected in samples from controls. These results suggest that chicken macrophages synthesize NO gas in a dose-dependent manner relative tol-arginine concentration.