Recombinant modified vaccinia virus Ankara-simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans.     

A neuronal network for computing population vectors in the leech

The correlation of neuronal activity with sensory input and behavioural output has revealed that information is often encoded in the activity of many neurons across a population, that is, a neural population code is used. The possible algorithms that downstream networks use to read out this population code have been studied by manipulating the activity of a few neurons in a population. We have used this approach to study population coding in a small network underlying the leech local bend, a body bend directed away from a touch stimulus. Because of the small size of this network we are able to monitor and manipulate the complete set of sensory inputs to the network. We show here that the population vector formed by the spike counts of the active mechanosensory neurons is well correlated with bend direction. A model based on the known connectivity of the identified neurons in the local bend network can account for our experimental results, and is suitable for reading out the neural population vector. Thus, for the first time to our knowledge, it is possible to link a proposed algorithm for neural population coding with synaptic and network mechanisms in an experimental system.     

Coenzyme A disulfide reductase, the primary low molecular weight disulfide reductase from Staphylococcus aureus. Purification and characterization of the native enzyme

The human pathogen Staphylococcus aureus does not utilize the glutathione thiol/disulfide redox system employed by eukaryotes and many bacteria. Instead, this organism produces CoA as its major low molecular weight thiol. We report the identification and purification of the disulfide reductase component of this thiol/disulfide redox system. Coenzyme A disulfide reductase (CoADR) catalyzes the specific reduction of CoA disulfide by NADPH. CoADR has a pH optimum of 7.5-8.0 and is a dimer of identical subunits of Mr 49,000 each. The visible absorbance spectrum is indicative of a flavoprotein with a lambdamax = 452 nm. The liberated flavin from thermally denatured enzyme was identified as flavin adenine dinucleotide. Steady-state kinetic analysis revealed that CoADR catalyzes the reduction of CoA disulfide by NADPH at pH 7.8 with a Km for NADPH of 2 muM and for CoA disulfide of 11 muM. In addition to CoA disulfide CoADR reduces 4,4'-diphosphopantethine but has no measurable ability to reduce oxidized glutathione, cystine, pantethine, or H2O2. CoADR demonstrates a sequential kinetic mechanism and employs a single active site cysteine residue that forms a stable mixed disulfide with CoA during catalysis. These data suggest that S. aureus employs a thiol/disulfide redox system based on CoA/CoA-disulfide and CoADR, an unorthodox new member of the pyridine nucleotide-disulfide reductase superfamily.     

The influence of antibodies to TNF-alpha and IL-1beta on haemodynamic responses to the cytokines, and to lipopolysaccharide, in conscious rats

Male, Long Evans rats (350-450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-alpha and human recombinant interleukin-1 (IL-1)beta, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-alpha and IL-1beta on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-alpha and IL-1beta had any influence on the responses to lipopolysaccharide (LPS). TNF-alpha (10, 100 and 250 microg kg(-1), in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum delta, +101+/-9 beats min(-1)) and modest hypotension (maximum delta, -10+/-2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum delta, +6+/-6, +27+/-9, and +61+/-12% at 10, 100 and 250 microg kg(-1), respectively). IL-1beta (1, 10, and 100 microg kg(-1) in separate groups, n = 8, 8 and 9, respectively) evoked changes similar to those of TNF-alpha (maximum delta heart rate, +69+/-15 beats min(-1); maximum delta mean blood pressure, -14+/-2 mmHg; maximum delta hindquarters vascular conductance, +49+/-17%), but with no clear dose-dependency. TNF-alpha (250 microg kg(-1)) and IL-1beta (10 microg kg(-1)) together caused tachycardia (maximum delta, +76+/-15 beats min(-1)) and hypotension (maximum A, -24+/-2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52+/-6%, +23+/-8%, and +52+/-11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum delta, -50+/-3% and -58+/-3%, respectively). Although bolus injection of LPS (3.5 mg kg(-1)) caused an initial hypotension (maximum delta, -27+/-11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-alpha and IL-1beta or after bolus injection of LPS, the secondary reduction in blood pressure was similar (-16+/-2 and -13+/-3 mmHg, respectively), but in the former group the tachycardia (+117+/-14 beats min(-1)) and increase in hindquarters vascular conductance (+99+/-21%) were greater than after bolus injection of LPS (+54+/-16 beats min ' and +439%, respectively). Pretreatment with antibodies to TNF-alpha and IL-1beta (300 mg kg(-1)) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h. Pretreatment with antibodies to TNF-alpha and IL-1beta before infusion of LPS (150 microg kg(-1) h(-1) for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion. the results indicate that although co-administration of TNF-alpha and IL-1beta can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.     

Neuroblastoma in Europe: differences in the pattern of disease in the UK. SENSE. Study group for the Evaluation of Neuroblastoma Screening in Europe

BACKGROUND: Neuroblastoma is a major contributor to childhood cancer mortality, but its prognosis varies with age and stage of disease, and some tumours regress spontaneously. Urinary screening programmes or clinical examination may detect the disease before symptoms appear, but the benefit of early diagnosis is uncertain. We examined the incidence, pattern, and presentation of neuroblastoma in four European countries. METHOD: Population-based incidence rates were derived for France, Austria, Germany, and the UK. Age, sex, and stage distribution were analysed by Mantel-Haenszel techniques and Poisson regression. The proportion of incidental diagnoses (cases without symptoms found at routine health checks or during investigation of other disorders) and mortality rates were also compared. FINDINGS: Between 1987 and 1991, 1672 cases of neuroblastoma were diagnosed in children under 15 years old (France, 624; Austria, 69; Germany, 493; UK, 486). Age-standardised annual incidence was significantly lower in the UK (10.1/million) than in France (12.5) and Germany (11.4). In the UK a deficit of low-stage disease in infants was accompanied by an excess of stage IV in older children. The UK had significantly fewer incidental diagnoses (8%) than Austria (27%) and Germany (34%). UK mortality rates were significantly higher than German or French rates. INTERPRETATION: In the UK, neuroblastoma diagnosis is delayed, possibly because of a less rigorous system of health checks for children. Although some overdiagnosis occurs in mainland Europe, our data suggest that in the UK some low-stage cases, undetected in infancy, may later present as advanced disease. This finding has implications for screening programmes and organisation of routine surveillance of infant health in the UK.     

The load applied to the foot in a patellar ligament-bearing cast

The purpose of this study was to determine whether a patellar ligament-bearing cast reduces the load applied to a foot in a cast. In a study of ten people who had no history of gait abnormalities, disease involving the motor system, or deformities of the lower extremities, we compared the load applied to the plantar aspect of a foot in a cast (as detected with F-Scan computer-monitored pedobarographic sensors) with the total load that an extremity in a cast receives relative to the ground (as detected with force-plates). Six trials were completed three times by each person. The trials consisted of walking (1) while wearing regular shoes; (2) with a patellar ligament-bearing cast on one leg; (3) with a patellar ligament-bearing cast and an overlying soft knee brace, locked in full extension, on the leg; (4) with only a below-the-knee cast on the leg; (5) with a below-the-knee cast and an overlying knee brace, locked in full extension, on the leg; and (6) with only a knee brace, locked in full extension, on the leg. The loads at peak heel-strike for all three trials were averaged and normalized to body weight. The load on the plantar aspect of the foot, as compared with the total load, was reduced a mean of 11 percent when the patellar ligament-bearing cast was worn alone, and it was reduced a mean of 26 percent when the patellar ligament-bearing cast was used with an overlying knee brace locked in full extension. This difference was significant (p = 0.007). With the numbers available, we could not detect a significant difference between the reduction in load when a patellar ligament-bearing cast was worn alone compared with that when a below-the-knee cast was worn alone or between the reduction when a below-the-knee cast was worn alone compared with that when a below-the-knee cast was used with a knee brace (p = 0.3). In conclusion, we could not demonstrate a significant reduction in the load on the foot when a patellar ligament-bearing cast was used in a traditional fashion; however, a significant (p = 0.007) reduction in load was found when a knee brace locked in full extension was worn in addition to the patellar ligament-bearing cast.     

Chemokine synthesis in the HSV-1-infected cornea and its suppression by interleukin-10

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea results in a tissue-destructive inflammatory response. In this study we show that virus infection induces the synthesis of macrophage inflammatory protein-2 (MIP-2), MIP-1alpha, and monocyte chemoattractant protein-1 (MCP-1). However, only the production of MIP-2 and MIP-1alpha coincided with the influx of leukocytes into the cornea. IL-10 treatment markedly suppressed chemokine message and protein synthesis in vivo. Local administration of IL-10 also dramatically reduced the number of T cells and neutrophils migrating into the cornea and suppressed the severity of corneal disease. The inflammatory response could also be suppressed by the passive transfer of neutralizing antibody to MIP-1alpha but not MCP-1. We conclude that local IL-10 administration can suppress chemokine synthesis, thereby ameliorating corneal disease. Furthermore, our results indicate that MIP-1alpha plays a major role in herpes stromal keratitis development, whereas MCP-1 does not.     

Somatic and dendritic mosaics formed by large ganglion cells in the retina of the common house gecko (Hemidactylus frenatus)

Recent studies of large ganglion cells in fishes and frogs have identified a shared inventory of three basic types, with characteristic forms and spatially independent mosaic distributions. These anamniote types and mosaics are hard to match to the large ganglion cell types and mosaics of mammals, implying that the underlying developmental programmes have diverged during evolution. Reptiles and mammals both belong to the amniote lineage, so the point of divergence can be investigated by comparing the large ganglion cells of reptiles with those of mammals, taking fishes and frogs as outgroups. With this aim, ganglion cells of the common house gecko, Hemidactylus frenatus, were labelled with horse-radish peroxidase by an in vitro method and studied in retinal flatmounts. Two prominent, regular, spatially independent mosaics were consistently present. One (alpha a) was characterized by somata displaced into the inner nuclear layer and dendrites forming planar trees in sublamina a; the other (alpha ab) comprised large orthotopic somata and distinctive, bistratified dendrites that formed discrete planar subtrees in sublaminae a and b. These subtrees were joined by up to 40 vertical link segments, whose distribution was found to correlate with the underlying photoreceptor mosaic. Some specimens also contained patches of a third mosaic (alpha c), characterized by large orthotopic somata and very large flat trees in sublamina c, but the labelling of this type was inconsistent. These reptilian mosaics share several distinctive characters with anamniote alpha-cell mosaics but differ markedly from the ganglion cell mosaics of any known mammal. The most parsimonious conclusion is that those mosaic features that are shared by the ganglion cells of all nonmammals are homologous and primitive (symplesiomorphic), while those that are shared by all therian mammals are homologous and derived (synapomorphic). This is consistent with other differences between mammalian and nonmammalian eyes. Mosaic formation itself, however, seems to be a universal characteristic of large ganglion cells.     

Determination of somatostatin receptor subtype 2 in carcinoid tumors by immunohistochemical investigation with somatostatin receptor subtype 2 antibodies

We have shown previously that expression of mRNA for somatostatin receptor subtype 2 (sst2) detected by in situ hybridization correlates to therapeutic outcome in patients with carcinoid tumors treated with somatostatin analogues. However, in situ hybridization is laborious and not practical in clinical routine work. We have, therefore, developed polyclonal antibodies directed against sst2 that may be used for immunohistochemistry on tissue specimens. The staining is specific and is highly correlated to expression of mRNA for sst2 (P < 0.01) as well as to tracer uptake at somatostatin receptor scintigraphy (P < 0.01). There is also a good correlation to the therapeutic response in carcinoid patients treated with somatostatin analogues (P < 0.05). Of 35 patients with carcinoid tumors included in this investigation, 25 stained positive with the antibodies. Twenty-two of these were investigated by somatostatin receptor scintigraphy and showed tracer uptake in metastases. An additional two patients that did not stain with the antibodies showed pathological uptake of the tracer in metastases, which might indicate binding to somatostatin receptor subtype 5. None of the 10 patients without positive immunostaining responded to somatostatin analogue treatment, whereas patients with a positive stain had a biochemical response or remained stable during treatment. Thus, these antibodies may be used to determine the presence of sst2 in carcinoid tumors and to select patients suitable for somatostatin analogue treatment. The method is easily applicable in clinical practice.