Thymus-independent T-cell differentiation in vitro

The generation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRalphabeta+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor beta-chain rearrangements were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.     

Glucose may induce cell death through a free radical-mediated mechanism

It has been reported that glucose may autooxidize generating free radicals which have been hypothesized to induce important cellular abnormalities. To investigate the cell damage induced by glucose-dependent oxidative stress, the FRTL5 cell strain was incubated in 10 or 20 mM glucose, either alone or in the presence of buthionine-sulfoximine, a transition state inhibitor that blocks glutathione synthesis. We found indeed that buthionine-sulfoximine greatly inhibited glutathione production and increased malondialdehyde (a marker of oxidative cell damage) levels, especially in 20mM glucose. We also found that, when glutathione production was inhibited, 10mM glucose induced apoptosis and 20 mM glucose induced necrosis. These data show that the glucose-dependent cell damage is a function of glutathione production. They also show that such glucose-dependent free radical production may be critical for determining cell damage, even for small variations as the ones we tested (from 10 to 20 mM glucose).     

Klebsiella bacteraemia: community versus nosocomial infection

In the period 1988-1993, 241 patients had Klebsiella bacteraemia at our medical centre. The annual number of patients with positive blood cultures increased from 306 in 1988 to 622 in 1993, representing a 4.5-6% positivity rate of drawn cultures. After E. coli, Klebsiella was the leading cause of Gram-negative bacteraemia. During this period, the absolute number of Klebsiella bacteraemia increased from 25 in 1988 to 84 in 1993; this represents a true increase in Klebsiellaa bacteraemia, from 6-7% of positive cultures in the late 1980s to 12-13% in more recent years. There were 210 cases with K. pneumoniae and 31 with K. oxytoca. A representative sample of 80 records was retrieved and subdivided into two groups: community-acquired Klebsiella bacteraemia (CAKB) vs. hospital-acquired Klebsiella bacteraemia (HAKB). Urinary tract infection was the underlying source of 58% of CAKB vs 28% of HAKB (p < 0.01); pneumonia occurred significantly more often in HAKB (25%) than in CAKB (7%) (p < 0.01). In HAKB, as compared to CAKB, serious manifestations of illness were more common, e.g. shock (65% vs. 37%, p < 0.046) and respiratory failure (45% vs. 20%, p < 0.046). Overall mortality was 32%, 22% of patients with CAKB died vs. 42% of those with HAKB (p < 0.05). Multiple drug resistance was very common: only 57% of all Klebsiella strains were susceptible to gentamicin, 66% to ceftriaxone, 70% to ciprofloxacin, and 83% to amikacin. The susceptibility rates of Klebsiella spp isolated from patients with HAKB were significantly lower (p < 0.001). Sepsis due to multiple-drug-resistant Klebsiellaa has become frequent, carrying significant morbidity and mortality. Restriction of broad-spectrum antimicrobials in the hospital and the community as well as implementation of infection control measures are needed to contain this problem.     

Increased response to antigen and histamine release in smaller sensitized canine bronchi

Presynaptic alpha 2-autoreceptors in mouse atria were characterized in terms of the alpha 2A, alpha 2B, alpha 2C and alpha 2D subtypes. Segments of the atria were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC30% values. i.e. concentrations that increased previously autoinhibited release of 3H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pKd values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s). The pKd values correlated well with the pEC30% values (r = 0.98; P < 0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native alpha 2 radioligand binding sites, (2) radioligand binding sites in COS cells transfected with alpha 2 subtype genes, and (3) previously classified presynaptic alpha 2-autoreceptors-all taken from the literature-indicated that the mouse atrial autoreceptors corresponded to the alpha 2D subtype. For example, the pKd values at mouse atrial auto-receptors correlated closely with pKd values at native alpha 2D binding sites in the bovine pineal gland (r = 0.96; P < 0.001); with pKd values at alpha 2D binding sites in COS cells transfected with the rat alpha 2D gene (r = 0.85; P < 0.01); and with pKd values at guinea-pig cerebral and atrial and mouse cerebral alpha 2D-autoreceptors (r = 0.96-0.98; P < 0.001). The antagonist pKd values at mouse atrial autoreceptors correlated less with pKd values at alpha 2A, alpha 2B and alpha 2C sites. It is concluded that the presynaptic alpha 2-autoreceptors in mouse atria are alpha 2D. This identification supports the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D pair of orthologous alpha 2-adrenoceptors.     

Dietary fish oil affects monoaminergic neurotransmission and behavior in rats

We studied the effects of a fish oil enriched diet on fatty acid composition of cerebral membranes and on several neurochemical and behavioral variables of monoaminergic function in rats. The frontal cortex, striatum, hippocampus and cerebellum were studied in rats fed fish oil (FPO, 50% salmon oil + 50% palm oil), which provided an (n-6)/(n-3) polyunsaturated fatty acid (PUFA) ratio of 0.14 versus 6. 19 in controls fed a diet containing a mixture of African peanut oil and rapeseed oil. In the FPO group compared to the control group, the major modifications in fatty acid composition of cerebral membranes included the following: higher levels in 22:6(n-3), lower levels in 20:4(n-6) and a significantly greater proportion of phosphatidylserine. Dopamine levels were 40% greater in the frontal cortex of rats fed FPO than from those fed the control diet. In this cerebral region there was also a reduction in monoamine oxidase B (MAO-B) activity and greater binding to dopamine D2 receptors. By contrast, a lower binding to dopamine D2 receptors (-7%) was observed in the striatum. Ambulatory activity was also reduced in FPO-fed rats, possibly related to observed changes in striatal dopaminergic receptors. This suggested that the level of (n-6) PUFA, which was considerably lower in the FPO diet than in the control diet, could act on locomotion through an effect on striatal dopaminergic function, whereas the high level of (n-3) PUFA could act on cortical dopaminergic function.     

Effects of a metalloproteinase that truncates P-selectin glycoprotein ligand on neutrophil-induced cardiac dysfunction in ischemia/reperfusion

This study was designed to test the effects of polymorphonuclear leukocytes (PMNs) in the presence and absence of a P-selectin blocker, mocarhagin, in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Control rat hearts not subjected to ischemia were perfused without blood cells for 80 min. Additional control rat hearts were perfused with 100 x 10(6) PMNs in the presence and absence of 0.2 microgram/ml mocarhagin over a 5-min perfusion followed by a 45-min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 min of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of PMNs. I/R hearts perfused with PMNs exhibited decreases of 50-60% in all measurements of cardiac function (P < 0.001). These PMN perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selection expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were attenuated markedly by the metalloproteinase, mocarhagin, which inhibits P-selectin-mediated cell adhesion by cleaving its high-affinity receptor, PSGL-1, present on neutrophils. These results provide evidence that neutrophils provoke post-reperfusion cardiac dysfunction, and that this may be largely due to P-selectin-induced adherence of neutrophils to the endothelium.     

Three sibs with mild variety of osteopetrosis

We report three sibs with mild autosomal recessive variety of osteopetrosis. The prominent clinical features were short stature, malocclusion of teeth, hepatosplenomegaly and a typical facial appearance. The only atypical features were microcephaly, a normal upper segment to lower segment ratio and a normal arm span.     

Detection of infectious disease agents in tissue by immunocytochemistry

1. Immunocytochemical procedures have played an increasingly larger role in the identification of infectious disease agents in tissue sections owing to the increased availability and specificity of antibody reagents, the great sensitivity of the methods, and the relative facility with which the studies are performed. 2. Immunocytochemical methods can be applied to routine formalin-fixed tissue for the detection of infectious agents such as viruses, bacteria, fungi, and protozoa among other microorganisms for diagnostic and research purposes.     

Developmental expression of G proteins in a migratory population of embryonic neurons

Directed neuronal migration contributes to the formation of many developing systems, but the molecular mechanisms that control the migratory process are still poorly understood. We have examined the role of heterotrimeric G proteins (guanyl nucleotide binding proteins) in regulating the migratory behavior of embryonic neurons in the enteric nervous system of the moth, Manduca sexta. During the formation of the enteric nervous system, a group of approx. 300 enteric neurons (the EP cells) participate in a precise migratory sequence, during which the undifferentiated cells populate a branching nerve plexus that lies superficially on the visceral musculature. Once migration is complete, the cells then acquire a variety of position-specific neuronal phenotypes. Using affinity-purified antisera against different G protein subtypes, we found no apparent staining for any G protein in the EP cells prior to their migration. Coincident with the onset of migration, however, the EP cells commenced the expression of one particular G protein, Go alpha. The intensity of immunostaining continued to increase as migration progressed, with Go alpha immunoreactivity being detectable in the leading processes of the neurons as well as their somata. The identity of the Go alpha-related proteins was confirmed by protein immunoblot analysis and by comparison with previously described forms of Go alpha from Drosophila. When cultured embryos were treated briefly with aluminium fluoride, a compound known to stimulate the activity of heterotrimeric G proteins, both EP cell migration and process outgrowth were inhibited. The effects of aluminium fluoride were potentiated by alpha toxin, a pore-forming compound that by itself caused no significant perturbations of migration. In preliminary experiments, intracellular injections of the non-hydrolyzable nucleotide GTP gamma-S also inhibited the migration of individual EP cells, supporting the hypothesis that G proteins play a key role in the control of neuronal motility in this system. In addition, once migration was complete, the expression of Go alpha-related proteins in the EP cells underwent a subsequent phase of regulation, so that only certain phenotypic classes among the differentiated EP cells retained detectable levels of Go alpha immunoreactivity. Thus Go may perform multiple functions within the same population of migratory neurons in the course of embryonic development.