Freeze-drying of red blood cells at ultra-Low temperatures

The hemolysis of human red blood cells (RBCs) after freeze-drying and resuspension depends on the vacuum-drying temperature. In an experimental study, RBCs were first solidified based on a modified high-yield cryopreservation protocol in the presence of hydroxyethyl starch and maltose. Afterward, they were vacuum-dried in a special low-temperature freeze-drying device at selected shelf temperatures between -5 and -65 degrees C. Subsequently, the dried samples were resuspended in an isotonic, phosphate-buffered saline solution. The hemolysis was determined according to a modified saline stability test. It decreases with a decreasing shelf temperature until a minimum is reached at -35 degrees C. A further decrease of the shelf temperature has no beneficial effect; the hemolysis even increases. To interpret these results, we assume that the hemolysis depends on two contrary damaging effects: (1) the higher the shelf temperature, the higher the probability of structural damages occurring during drying; (2) the lower the shelf temperature, the lower the driving force for water transport; this may lead to an incomplete intracellular dehydration which means that the cells are not in a glassy state at ambient temperature.     

Case report: Cutaneous nocardiosis complicating management of Crohn's disease with infliximab and prednisone

INFLIXIMAB IS A CHIMERIC ANTI-TUMOUR NECROSIS FACTOR-α antibody that is efficacious in treating Crohn''s disease. However, its immunomodulatory properties increase susceptibility to opportunistic infections. We present a case of cutaneous Nocardia infection in a patient who was taking infliximab for Crohn''s disease. The case illustrates the challenges in the diagnosis and management of this disease and serves as a reminder of the complications associated with the use of immunomodulatory agents.A 45-year-old HIV-negative man with fistulous Crohn''s disease had a history of inadequate disease control despite ongoing prednisone therapy. He had previously taken budesonide, mesalamine, ciprofloxacin and metronidazole in attempts to induce remission of his inflammatory bowel disease. The patient was born in Canada and, other than a 1-week holiday to Mexico 10 years before presentation, had travelled only locally. He denied a family history of tuberculosis, and he had never worked in a health care facility. Infliximab was introduced, and the patient received 3 infusions of 5 mg/kg at baseline and 2 and 6 weeks later. After he received his third infusion, prednisone was tapered to 40 mg at a rate of 5 mg weekly. One month after the third infusion, in February 2000, he reported multiple erythematous papulopustular lesions on his right leg (Fig. 1). There was no associated lymphadenopathy, cough, shortness of breath, fever or constitutional symptoms. He denied a history of insect bites, but in November 1999 he had received cuts to his right leg from a metal blade when at work. He had not immersed the leg in a whirlpool or swimming pool around the time of the leg injury. The patient continued to receive further infliximab infusions (at weeks 12 and 18 after baseline), and the lesions were treated with cloxacillin for suspected Staphylococcus aureus infection. Since improvement was minimal, a skin biopsy was performed in July 2000. Granulomatous inflammation was present, and acid-fast bacilli were visualized (Fig. 2). Cultures sent for mycobacteriology and mycology were incubated at 35°C for 8 weeks, but the results from the mycobacteriology culture proved negative. A polymerase chain reaction assay for Mycobacterium tuberculosus was also negative. A clinical diagnosis of M. marinum infection was made, and the patient''s antibiotic regimen was changed to minocycline. A tuberculin skin test was not performed; since the patient was immunocompromised, a negative result would not have excluded the disease. A chest radiograph appeared normal.Open in a separate windowFig. 2: Acid-fast bacilli visualized in skin biopsy.Open in a separate windowFig. 1: Multiple erythematous papulopustular lesions on the patient''s leg 1 month after the third infusion of infliximab.The patient failed to respond to the minocycline therapy, and he was referred for infectious disease consultation in October 2000. The infliximab infusions were discontinued, and 2 more skin biopsies were performed, with acid-fast bacilli visualized in both specimens. The patient was given trimethoprim–sulfamethoxazole, and his lesions began to heal slowly but progressively.Acid-fast bacilli were recovered from the second set of biopsies, and specific instructions were given to incubate the cultures at 30°C and 35°C to ensure that M. marinum, if present, would be detected. Again, the cultures failed to recover organisms. The laboratory, using polymerase chain reaction restriction analysis of the 439-base pair segment of the gene encoding a 65-kDa heat shock protein,¹ identified the presence of Nocardia species. Nevertheless, Nocardia organisms still could not be recovered in culture, and therefore final speciation could not be performed.The patient resumed taking prednisone, and the dosage was increased in order to ameliorate the symptoms of his Crohn''s disease. The trimethoprim–sulfamethoxazole therapy was continued until late 2003, and the dosage was reduced over the subsequent months. Complete healing of the lesions was eventually achieved 4 years after therapy was initiated.     

24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to approximately 165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 108 active virus particles yielded homogeneous transfection of approximately 80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.     

Feeding habits of guanacos<Emphasis Type="Italic">Lama guanicoe</Emphasis> in the high Andes of north-central Chile

The guanacoLama guanicoe Muller, 1776 has a wide distribution along the Andes and Patagonia. We studied the feeding behaviour of a guanaco population that lives over 4100 m altitude in the Andes of north-central Chile. By contrasting the diet of guanacos during a dry year with that of a wet year and comparing it with the plant availabilities in the field, we tested the hypothesis that the guanaco is a generalist herbivore. We predicted that under such extreme habitat conditions guanacos should consume whatever plant species are available in the environment, especially in a dry year, when vegetation is scarcer. In addition, we compared its diet at three different age classes. We estimated the diet through the microhistological analysis of plant remains found in guanaco pellets collected during January of 1997 (ie after a dry year) and 1998 (ie after a wet year; 41 vs 495 mm, respectively). Then, we computed dietary preferences, food niche-breadth, and food-niche overlap between years and among age classes. Vegetation cover and plant species richness were higher during the wet than during the dry year. The most common plants in the environment were perennial graminoids and legumes. Contrary to our prediction, the guanaco preferred a few plant species, showing a relatively narrow diet breadth that changed little between years differing in plant abundances. The diet proportions differed among the three age classes, however. Our data indicate that at least in this high-elevation population, guanacos are selective and non-opportunistic herbivores. This specialized feeding behaviour is puzzling given the energetic demands of living in a harsh environment with low availabilities of resources. The hypothesis that this is due to the lower palatability of the plants not eaten, remains to be tested.     

Identification of quinoide redox mediators that are formed during the degradation of naphthalene-2-sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.     

Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins

In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.     

Oxygen-insensitive nitroreductases NfsA and NfsB of Escherichia coli function under anaerobic conditions as lawsone-dependent Azo reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Interaction of Xenorhabdus nematophilus (Enterobacteriaceae) with the antimicrobial defenses of the house cricket, Acheta domesticus

Fifth instar Acheta domesticus nymphs exhibited a decline in total hemocyte counts during the first hour of exposure to dead Xenorhabdus nematophilus; the bacterial level in the hemolymph also declined during this time. Thereafter bacterial numbers in the hemolymph increased as the level of damaged hemocytes increased. The bacteria lowered phenoloxidase activity in vivo by initially reducing the number of hemocytes containing prophenoloxidase and later by inhibiting enzyme activation. Preincubating X. nematophilus in hemolymph with active phenoloxidase in vitro accelerated the removal of the bacteria from the hemolymph in vivo which may be due to modification of the bacterial surface by serine proteases. Lysozyme activity increased in bacteria-injected insects in parallel with an increase in counts of damaged hemocytes; most of the enzyme was located in hemocytes. Lipopolysaccharides of X. nematophilus caused changes in hemocyte counts and phenoloxidase and lysozyme levels comparable to whole bacteria. Lipopolysaccharides also slowed the removal rate of the bacteria from, and accelerated bacterial emergence into, the hemolymph.