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The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

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Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.

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One of the most useful tools for investigating the aetiopathology of cancer is the mutation spectrum, which comprises the type and distribution of mutations within a gene sequence. Many studies have generated mutagen-induced spectra using in vitro or in vivo model systems in an attempt to find correlations with those observed in cancer-associated genes such as the TP53 tumour suppressor gene. Consequently, meaningful similarities in the types of mutation found in induced and human spectra have been demonstrated. However, it is more difficult to draw such conclusions about the distribution or sequence context of mutations when they arise in different target sequences. We have developed an analytical approach for base substitution spectra that capture information for both sequence context and mutation type simultaneously. The resulting mutation signature is a fixed set of data points that allows comparison of multiple mutation spectra regardless of sequence. We have applied this method to a mixed set of mutation spectra observed in exons 5, 7 and 8 of TP53 from cancers of brain, breast, skin, colon, oesophagus, liver, head and neck, stomach and lung (smokers and non-smokers) and spectra induced by benzo[a]pyrene diol epoxide, ultraviolet (UV) B, UVC, simulated sunlight and hydroxyl radicals in the cII, supF and yeast p53 model systems. We demonstrate that this approach allows human cancer and mutagen-induced signatures to be grouped together according to similarity. Specifically, the analysis reveals key differences between smoking- and non-smoking-related lung cancer for TP53 mutations and the mutability of CpG sites between exons in skin cancer.

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Here we describe the development of a novel software tool entitled Loader Lite that generates plate records or sample sheetsfor the ABI PRISMs 3700 DNA sequencer. The major advantage of this program is that it enables the ongoing operation of sequencing instruments without reference to external network(s). The autonomous operation of sequencing instruments is critical if sample throughput is to be maintained during periods of network outage. Loader Lite employs a deliberate strategy of inputting anonymous tray barcodes at run time. After sequencing, the barcodes are reconciled with relevant project details by reference to a database. This software takes advantage of barcode scanning technology by creating plate records directly on the local computer, serving an individual sequencer, immediately before importing and linking. This real-time synthesis of the plate records at the point of loading all but eliminates loading errors. Loader Lite is user-friendly, fully configurable, and permits the running of partial or full 384-well sample trays, using any standard combinations of run modules, dye sets, mobility files, analysis modules, etc. The 96-well format is not supported; however, this capability will appear in subsequent versions that are currently under development. This application is designed as an added value, adjunct program to the regular ABI PRISM 3700 Data Collection software. We have successfully used Loader Lite over the past six months to load approximately 7 million sequencing reactions and believe its utility and functionality will prove to be attractive to the wider sequencing community.

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DNA sequences and three distinct in vitro functions of Nef were evaluated in a group of seven perinatally infected children. nef gene sequences obtained before and after virus culture showed that one of the five non-/slow progressors harbored a virus with large deletions. nef genes from the remaining four children were full length but contained discrete changes at a higher frequency than the rapid progressors. In functional studies, 40 of 44 Nef proteins derived from the whole study group were capable of binding the cellular serine kinase p62, indicating that this function is well conserved among naturally occurring viruses. In contrast, representative Nef proteins derived from the long-term non-/slow progressors were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells. On reversion of highly prevalent point mutations in the defective proteins, viral replication could be restored to wild-type levels. Our results suggest that nef genes derived from pediatric long-term nonprogressors have gross deletions in isolated cases but a higher prevalence of discrete changes that may impair Nef function in primary T cell assays, but not all functions reported for Nef.

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OBJECTIVE: To determine the influence of prenatal zidovudine (ZDV) prophylaxis on the course of HIV- 1 infection in children by comparing the clinical outcome of infants born to HIV- 1-seropositive mothers who did versus those who did not receive ZDV during pregnancy. METHODS: Medical records of HIV-1-seropositive mothers and their infants were reviewed retrospectively. Participants were divided according to maternal ZDV use: no ZDV (n = 152); ZDV (n = 139). The main outcome measure was rapid disease progression (RPD) in the infant, defined as occurrence of a category C disease or AIDS-related death before 18 months of age. RESULTS: HIV vertical transmission rates were significantly different (no ZDV versus ZDV: 22.3% versus 12.2%; p = .034). Among infected infants, the RPD rate was 29.4% in the no ZDV group compared with 70.6% in the ZDV group (p = .012), and prematurity was significantly associated with a higher risk of RPD (p = .027). CONCLUSIONS: The rate of RPD was significantly higher among perinatally infected infants born to HIV-infected mothers treated with ZDV than among infected infants born to untreated mothers. The decreased proportion of infected infants with nonrapid disease progression in the former group might be related to the ability of ZDV to block intrapartum transmission preferentially and also to nonrapid disease progression resulting from intrapartum transmission.

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OBJECTIVES: This study sought to determine the number, characteristics, and living situations of children of HIV-infected adults. METHODS: Interviews were conducted in 1996 and early 1997 with a nationally representative probability sample of 2864 adults receiving health care for HIV within the contiguous United States. RESULTS: Twenty-eight percent of infected adults in care had children. Women were more likely than men to have children (60% vs 18%) and to live with them (76% vs 34%). Twenty-one percent of parents had been hospitalized during the previous 6 months, and 10% had probably been drug dependent in the previous year. Parents continued to have children after being diagnosed with HIV: 12% of all women conceived and bore their youngest child after diagnosis, and another 10% conceived before but gave birth after diagnosis. CONCLUSIONS: Clinical and support services for people affected by the HIV epidemic should have a family focus.

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Significant advances have been made in the understanding of the pathophysiology of HIV infection since the beginning of the epidemic. This knowledge has translated into the development of new therapies for HIV and opportunistic infections, laboratory advances in monitoring viral and immune status, and a better understanding of factors affecting patient outcome. Concomitantly, significant progress has been made in the medical management of children with HIV infection in the past 5 years. The number of children reported with AIDS in the United States is decreasing, and efforts are shifting from caring for children with advanced immunosuppression and severe opportunistic infections to early HAART, maintenance of the immune system, and prevention of opportunistic infections. Primary care physicians are now more involved and informed in the care of HIV-infected patients. Although published data are limited, physicians who have been working with this population have observed a dramatic improvement in the quality of life and length of survival of these patients. Unfortunately, this progress is not shared by developing countries where resources are minimal and antiretroviral agents are commonly unavailable. Although efforts to develop a vaccine to prevent HIV infection are ongoing, progress has been slow. Education and awareness continue to be the most powerful weapons against HIV.

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BACKGROUND: HIV-infected children are particularly susceptible to serious bacterial infections including Gram-negative bacillary bacteremia (GNB). However, the information available on GNB in these children is limited. METHODS: Retrospective review of hospital charts of HIV-infected children with GNB diagnosed between 1980 and 1997. The association between bacteremic episodes, degree of immunosuppression, HIV severity, medical treatment and clinical outcome was assessed. RESULTS: Of 680 HIV-infected children, 72 (10.6%) had 95 episodes of GNB. Statistical analyses were restricted to data from the first episode. The mean age (+/-SD) at diagnosis of GNB was 2.5 +/- 2.7 years (median, 1.6). The predominant organisms were Pseudomonas aeruginosa (26.4%), nontyphoidal Salmonella (15.3%), Escherichia coli (15.3%) and Haemophilus influenzae (12.5%). The relative frequency, per 5-year interval, of P. aeruginosa bacteremia steadily increased from 13% during 1980 through 1984 to 56% during 1995 through 1997. There were no cases of H. influenzae bacteremia after January 1, 1990. Eighty percent of GNB developed in children with AIDS and 72.2% developed in those with severe immunosuppression. Hypogamma-globulinemia and neutropenia were present in only 4.9 and 10.4% of first episodes, respectively. The overall case-fatality rate of GNB was 43.0%, and in children younger than 12 months it was 54.2%. CONCLUSIONS: A diagnosis of AIDS and/or severe immunosuppression was associated with increased risk of GNB, especially among younger children. Because of the high mortality of GNB, a broad spectrum antimicrobial therapy that effectively covers these organisms should be promptly instituted when bacteremia is suspected in HIV-infected children.

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1. This study investigated the ability and the behaviour of laying hens jumping between perches 2 different distances apart using hungry and satiated birds to determine whether behaviour and success were related to motivation. 2. Four groups of 10 birds (Lohmann brown) trained to jump from one perch to another were subjected 4 times to each of the experimental treatments (2 perches with a distance of either 50 or 150 cm between them) whilst either hungry or satiated, controlling for time of day and order of presentation. 3. The behaviour of each bird on the 1st perch was observed for a maximum of 2 min or until the bird jumped from the perch. The outcome of each jump, number of head movements and steps, incidence and duration of calling, and activity score were recorded. 4. The probability of birds jumping was less at the greater distance and was not affected significantly by other experimental factors. 5. Head movements were more frequent in birds which jumped, at the shorter distance, in motivated (hungry) birds and in the morning rather than the afternoon. 6. More stepping was performed by birds which jumped and at the shorter distance; there was no significant effect of motivation or time of day on stepping rate. 7. Birds called more at the greater distance and this was interpreted as indicative of frustration. 8. Subjective scores for activity were lower when perches were separated by the greater distance and when birds were satiated (less motivated) rather than hungry. 9. Agitated head movements and stepping activity thus occurred mainly when birds were motivated and on the point of jumping whereas calling was associated with an apparent inability or unwillingness to jump.

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OBJECTIVE: To determine the independent contribution of specific nutritional factors on disease progression and survival in HIV-1-infected children. POPULATION: HIV-infected children (N = 24), who were perinatally exposed to the virus and symptomatic, were recruited between October and December of 1990 from the Jackson Memorial Pediatric Immunology Clinic, Miami, Florida, and observed for 5 years. METHODS: Immune status was measured by CD4 cell count; nutritional status was determined using serum albumin and plasma trace elements including iron, zinc, and selenium. Cox proportional hazards regression models were used to evaluate the relationship of these parameters to survival. Use of antiretroviral treatment was considered in the statistical model, and age at death was considered a parameter of disease progression. RESULTS: Over the course of the study, 12 children died of HIV-related causes. The final Cox multivariate analysis indicated that, of the variables evaluated, only CD4 cell count below 200 (risk ratio [RR] = 7.05; 95% confidence interval [CI], 1.87-26.5); p = .004], and low levels of plasma selenium (RR = 5.96; 95% CI, 1.32-26.81; p = .02) were significantly and independently related to mortality. Among the children who died, those with low selenium levels (< or =85 microg/L), died at a younger age, suggesting more rapid disease progression. CONCLUSIONS: In pediatric HIV-infection, low plasma level of selenium is an independent predictor of mortality, and appears to be associated with faster disease progression.

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Although the character of acquired immunodeficiency syndrome is changing into a chronic illness, it is estimated that by the end of this century, 80 000 children and adolescents in the United States will be orphaned by parental death caused by human immunodeficiency virus infection. Plans for these children need to be made to ensure not only a stable, consistent environment that provides love and nurturing, but also the medical and social interventions necessary to cope with the tragic loss. Pediatricians should become aware of local laws and community resources and initiate discussion early in the course of parental illness to facilitate planning for the future care and custody of the children. States need to adopt laws and regulations that provide flexible approaches to guardianship and placement of children orphaned by acquired immunodeficiency syndrome.

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The association between antibody reactivity to the neutralizing epitope ELDKWA in the transmembrane glycoprotein gp41 and disease progression was investigated in 29 children perinatally infected with HIV-1. Levels of antibody reactivity to this epitope, measured over time, were associated with absolute CD4+ lymphocyte numbers and disease status, and inversely associated with the levels of acid-dissociated p24 antigen in the plasma. Early virus isolates from 10 of 12 children with no detectable antibody reactivity to this epitope were sequenced. Only three contained sequences that differed from the consensus, indicating that this epitope is well conserved in this population. None of these three children developed antibodies to the autologous sequences, indicating that at least 80% of children with negative antibody reactivity to this epitope were true nonresponders. Together, these results indicate that the ELDKWA determinant could be an important component in the formulation of a vaccine or for immunotherapeutic approaches to HIV-1 infection.

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All end-of-lay hens must be transported from the production site to the site for slaughter, usually by road. The acts of being crated and transported are likely to be novel and potentially frightening. Frightened birds can be put into tonic immobility (TI), an unlearned, catatonic state, the duration of which is positively related to the fear level of the birds. Ninety ISA Brown free-range birds (in three groups of 30) and 90 caged ISA Brown birds (in similar groups) were transported (one group from cages and free-range per day) on a journey of 68 km (74 min approximately). On their return, each of the birds was subjected to TI. Although no significant differences were recorded between free-range and caged birds after they were transported (P = 0.087), in the comparison between the same birds after 7 wk, without transport, a significant difference in fear levels was observed (P < 0.001). The free-range birds were less frightened than the caged birds. No significant difference was ever found in fear levels for caged birds from different tiers. Handling and transport on this relatively short journey was apparently equally frightening for caged and free-range birds. It cannot be assumed that just because fear levels differ between free-range and caged birds, this difference will be apparent for all novel stimuli. If all birds, at all tiers, receive the same amount of human contact during the production period, there is no reason to expect differences to occur in fear levels of birds from different tiers.

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Observers named visually presented words as quickly as possible when the words were presented to the centre of a viewing screen (Experiments 1, 5, and 6) or were flashed to either the left visual field/right hemisphere (LVF/RH) or right visual field/left hemisphere (RVF/LH) on each trial (Experiments, 2, 3, and 4). Words varied in frequency of occurrence in the language and in regularity of pronunciation. On lateralised trials, there was a RVF/LH advantage for both error rate and reaction time, with this hemispheric asymmetry for word naming being independent of both frequency and regularity. The RVF/LH advantage was also independent of whether the letters within a word were arranged horizontally or vertically. Error rates and reaction times were lower for high-than for low-frequency words and lower for words with a regular pronunciation than for exception words. In addition, on centre trials the effects of regularity were larger for low-frequency words than for high-frequency words. However, when words were presented in the visual periphery, the effects of regularity were of the same magnitude for high-and low-frequency words. In view of the theoretical importance of the Frequency by Regularity interaction, this latter result suggests that word processing is qualitatively different in the visual periphery than in the centre of vision; indicating that perceptual asymmetries in a typical visual half-field experiment may be limited in what they can tell us about the relative contributions of the two hemispheres to processing words in the centre of vision.

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Observers identified consonant-vowel-consonant trigrams with the letters arranged vertically by pronouncing the stimulus (treating the bottom letter as the first letter) and spelling it from bottom to top. On each trial, the trigram was presented to the left visual field/right hemisphere (LVF/RH), to the right visual field/left hemisphere (RVF/LH), or to both visual fields simultaneously (BILATERAL trials). Quantitative and qualitative visual field differences were identical to those found when observers used a more natural response output order, treating the top letter of the trigram as the first letter. The results suggest that, regardless of output order, attention is distributed across the three letters in a relatively slow, top-to-bottom fashion on LVF/RH and BILATERAL trials, whereas attention is distributed more rapidly and evenly across the three letters on RVF/LH trials.

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OBJECTIVE: To identify the population of human immunodeficiency virus-infected pediatric long- term survivors (LTS) followed in major medical institutions in California, Florida and New Jersey. METHODS: A cross-sectional survey was performed with data collection forms sent to all investigators. Demographic, clinical, and laboratory data were obtained on all living patients >/=8 years infected in the perinatal period with human immunodeficiency virus. RESULTS: A total of 143 perinatally infected and 54 children infected by neonatal transfusion were identified. Fifty-four children (27%) had absolute CD4 counts >/=500 cells/mm (group 1: mean age 9.8 years), 54 children (27%) had CD4 counts between 200 and 500 cells/mm (group 2: mean age 10.1 years), and 89 children (45%) had CD4 counts <200 cells/mm (group 3: mean age 10.4 years). Ninety-five (48%) patients had developed AIDS defining conditions; 14 (26%) in group 1, 26 (48%) in group 2, and 55 (62%) in group 3. Ninety-two percent of patients had received antiretrovirals. Perinatally human immunodeficiency virus-infected children tended to be younger (mean age 9.8 years) than children infected via a blood transfusion (mean age 11 years). Generalized lymphadenopathy was the most prevalent clinical finding. Lymphoid interstitial pneumonia and recurrent bacterial infections were the most prevalent acquired immune deficiency syndrome-defining conditions. Twenty percent of LTS had CD4 counts >/=500 cells/mm and no immune deficiency syndrome-defining conditions. CONCLUSIONS: Pediatric LTS were in variable stages of disease progression. The proportion of children within each CD4 strata did not differ by mode of acquisition of infection. Increased CD4 counts were inversely proportional to age. Only 20% of pediatric LTS had minimal to no disease progression.

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1. This paper describes how birds move between horizontal perches at different heights with different angles separating them, using the time to complete the task and the number of birds failing to indicate ability. 2. For the first experiment, 4 groups of 15 birds (ISA Brown) were subjected to each of the experimental treatments (using three perches with angles of 0 degree (horizontal control), 30 degrees, 45 degrees or 60 degrees between them). The birds were required to move upward between the perches for each treatment and then to move downward between the perches, as a second part of the experiment. 3. Birds moving up between the perches took significantly longer at 60 degrees than at any other angle. No birds failed to complete the task. 4. For birds moving downward between the perches, the median time to complete the task increased significantly with angle. Furthermore, the number of birds failing to complete the task increased with angle. 5. A second experiment was conducted to test whether the vertical or horizontal component of distance affected birds' ability to negotiate perches separated by different angles. 6. For the second experiment 4 groups of 10 Lohmann Brown laying hens were subjected to each of the following treatments: two horizontal perches separated by 30 degrees or 60 degrees and either directly, horizontally or vertically separated by a set distance of 50 cm. Birds were required to move both up and down between perches. 7. Birds negotiated horizontally-separated perches more successfully at 30 degrees than at 60 degrees. However, when the vertical distance between the perches was 50 cm there was no significant difference in the ability of birds to move downward at 30 degrees or 60 degrees. For birds moving upward, 60 degrees was easier to negotiate than 30 degrees. 8. The vertical and horizontal separations, as well as the different angles affected the ability of birds to move between perches. There was a general decreasing trend in ability with increasing vertical separation between perches. 9. The findings are important in terms of arrangements of perches to improve bird welfare. To minimise the risk of injury, the angle between perches at different heights should be no more than 45 degrees, and the horizontal and vertical distances between these perches minimised, to allow the birds to be able to move downwards more easily.

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Stockworkers should be able to easily manage alternative systems for commercial egg production. Such production systems can be analysed in terms of human welfare, based on ergonomic criteria. Work-related postures can cause discomfort and strain to workers (Stoffert, 1985). The OVAKO Working posture Analysing System (OWAS) developed in 1974 (Karhu et al, 1977) defines body positions during working practices and scores them according to the strain caused. This technique has so far not been used to assist in the design of working systems for the poultry industry. This pilot study was carried out to determine if such a technique could be easily applied to a perchery system and whether it could lead to an improved design. The stockworker was video recorded performing normal duties within the perchery and the positions of the body were assessed, using the OWAS system. It was found that manual collection of floor eggs and reaching into the middle of the perches from the litter side put the most strain on the stockworker. Modification of the perchery, in light of these results, will establish an improved perchery system for the stockworkers.