Dual role of splenic mononuclear and polymorphonuclear cells in the outcome of ciprofloxacin treatment of Salmonella enterica infections.

OBJECTIVES: To determine the immune cell populations associated with Salmonella enterica serovar Typhimurium before and after ciprofloxacin treatment using a murine model of systemic infection. The effect of depletion of immune cells associating with Salmonella on treatment outcome was also determined. METHODS: We infected mice with a Salmonella enterica serovar Typhimurium strain expressing GFP and used multicolour flow cytometry to identify splenic immune cell populations associating with GFP-positive Salmonella before and after treatment with ciprofloxacin. This was followed by depletion of different immune cell populations using antibodies and liposomes. RESULTS: Our results identified CD11b+CD11chi/lo (dendritic cells/macrophages) and Ly6G+CD11b+ (neutrophils) leucocytes as the main host cell populations that are associated with Salmonella after ciprofloxacin treatment. We therefore proceeded to test the effects of depletion of such populations during treatment. We show that depletion of Ly6G+CD11b+ populations resulted in an increase in the number of viable bacterial cells in the spleen at the end of ciprofloxacin treatment. Conversely, treatment with clodronate liposomes during antimicrobial treatment, which depleted the CD11b+CD11chi/lo populations, resulted in lower numbers of viable bacteria in the tissues. CONCLUSIONS: Our study identified host cells where Salmonella bacteria persist during ciprofloxacin treatment and revealed a dual and opposing effect of removal of Ly6G+CD11b+ and CD11b+CD11chi/lo host cells on the efficacy of antimicrobial treatment. This suggests a dichotomy in the role of these populations in clearance/persistence of Salmonella during antimicrobial treatment.

Nintendo Wii Fit as an adjunct to physiotherapy following lower limb fractures: preliminary feasibility, safety and sample size considerations.

OBJECTIVE: The Nintendo Wii Fit integrates virtual gaming with body movement, and may be suitable as an adjunct to conventional physiotherapy following lower limb fractures. This study examined the feasibility and safety of using the Wii Fit as an adjunct to outpatient physiotherapy following lower limb fractures, and reports sample size considerations for an appropriately powered randomised trial. METHODOLOGY: Ambulatory patients receiving physiotherapy following a lower limb fracture participated in this study (n=18). All participants received usual care (individual physiotherapy). The first nine participants also used the Wii Fit under the supervision of their treating clinician as an adjunct to usual care. Adverse events, fracture malunion or exacerbation of symptoms were recorded. Pain, balance and patient-reported function were assessed at baseline and discharge from physiotherapy. RESULTS: No adverse events were attributed to either the usual care physiotherapy or Wii Fit intervention for any patient. Overall, 15 (83%) participants completed both assessments and interventions as scheduled. For 80% power in a clinical trial, the number of complete datasets required in each group to detect a small, medium or large effect of the Wii Fit at a post-intervention assessment was calculated at 175, 63 and 25, respectively. CONCLUSIONS: The Nintendo Wii Fit was safe and feasible as an adjunct to ambulatory physiotherapy in this sample. When considering a likely small effect size and the 17% dropout rate observed in this study, 211 participants would be required in each clinical trial group. A larger effect size or multiple repeated measures design would require fewer participants.

Effect of X- and Y-box deletions on the development of diabetes in H-2Ealpha-chain transgenic nonobese diabetic mice.

The development of type 1 diabetes in nonobese diabetic (NOD) mice is influenced by major histocompatibility complex (MHC) class II genes. The NOD-E transgenic mouse, which expresses H2-E as a result of the introduction of an Ead gene, is protected from development of type 1 diabetes. While the mechanism of protection remains unclear, the effect has been regarded as a model system for MHC protection from autoimmunity. We have investigated the effect of deletions of the Ea promoter region, which, in turn, affect H2-E expression patterns in transgenic NOD mice. We have constructed transgenic NOD mice where the X (DeltaX) and Y (DeltaY) boxes of the Ead gene have, respectively, been functionally deleted. Previous reports, using X- or Y-box-deleted H2-E transgenic mice, made by crossing the appropriate transgenes onto the NOD background from C57BL/6 transgenic mice, indicated that promoter mutation abrogated the H2-E-mediated protection seen in NOD-E. The NOD DeltaX and NOD DeltaY transgenic mice generated in the present study differ in susceptibility to diabetes from wild-type NOD mice. NOD DeltaY1 animals are protected from diabetes development, while DeltaX mice remain susceptible, albeit to a lesser extent than the parental NOD strain.

In vivo depletion of CD8+ cells does not affect primary maedi visna virus infection in sheep.

T-cells have been implicated both, in promoting and reducing viral replication during lentivirus infection. CD8+ lymphocytes are believed to be important in controlling viral load through direct killing of virus-infected cells and by secretion of inhibitory chemokines and cytokines. To evaluate the role of CD8+ T-cells in the induction and control of the primary phase of a lentivirus infection, we have used a non-T-cell tropic lentivirus, maedi-visna virus (MVV), to study the initial pathogenesis and subsequent immune responses in sheep depleted in vivo of CD8+ cells. Sheep were depleted of CD8+ cells in both blood and efferent lymph for up to 14 days. No difference in MVV replication was observed in either the draining efferent lymph or lymph node of these sheep. Surprisingly, these animals displayed a normal induction of pCTL whereas the virus-specific proliferative responses were reduced. This could reflect either that a proportion of functional CD8+ lymphocytes remained in these animals, as suggested by the appearance of pCTLs, or that CD8+ cells are not required for control of primary MVV infection.

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node.

Infection with Schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice.

The spontaneous development of insulin dependent diabetes mellitus in non-obese diabetic (NOD) mice has been shown to be mediated by a Th1 response against beta cell antigens. It is known that in murine models of Schistosoma mansoni infection, egg production is associated with a switch from a Th1 to Th2 response. This subsequent dominance of a Th2 response in S.mansoni infected mice has been shown to influence the response to other infectious agents or antigens. We therefore determined whether infection with S.mansoni could influence the spontaneous incidence of insulin dependent diabetes mellitus (IDDM) in NOD mice. Infection with this helminth significantly reduced the spontaneous incidence of IDDM. IDDM was also prevented by injecting parasite eggs alone. Because until relatively recently humans might expect to succumb to a variety of infectious agents, the current freedom from infection might permit the expression of a genetic predisposition to autoimmune pathology and be responsible for the increased incidence of IDDM.

Investigation of the role of delayed-type-hypersensitivity responses to myelin in the pathogenesis of Theiler's virus-induced demyelinating disease.

The contribution of autoimmune responses to the pathogenesis of Theiler's virus-induced demyelinating disease was investigated. Delayed-type hypersensitivity responses to myelin were examined in both symptomatic and asymptomatic mice at different times post-infection, in order to determine whether autoreactivity correlates with the development of demyelination. The results indicate that although autoimmune responses probably do not play a major role in the initiation of demyelination at early times post-infection, autoreactivity to myelin antigens dose eventually develop in symptomatic animals, perhaps through the mechanism of epitope spreading. Autoimmunity to myelin components is therefore an additional factor that may contribute to lesion progression in chronically diseased animals.

Team briefing--helping to rediscover the road to Utopia.

The BDC has implemented several team briefing schemes into various NHS organisations over the last seven years. Evaluation over the last three years has highlighted several problems with the team briefing system. The article explores these and suggests ways of overcoming the problems with clear, definable actions. It highlights that the basic team briefing model is sound but that organisations need to consider their commitment and whether the culture is compatible or not with some reinforcement of certain processes which includes training team briefers and having effective feedback as part of the process. The article concludes that team briefing is a useful tool, which can help organisations communicate effectively with their employees.

Effect of MHC transgene expression on spontaneous insulin autoantibody class switch in nonobese diabetic mice.

A study of spontaneous anti-insulin autoantibodies in nonobese diabetic (NOD) mice revealed that when first detected, the antibodies are immunoglobulin M (IgM), but by age 10 weeks, immunoglobulin G (IgG) autoantibodies have appeared in many of these animals. When NOD strains, partially or completely protected from IDDM by the insertion of transgenes in the class II region, were compared, it was found that the switch to IgG autoantibodies was inhibited and the autoantibodies remained IgM indefinitely. We speculate that the switch to IgG may be a marker of events leading to IDDM in NOD mice and an indication that T-cell help has been generated for responses to beta-cell antigens. Such help not only directs the development of IgG autoantibodies, but more importantly, allows the emergence of potentially pathogenic T-cell clones that are capable of infiltrating the pancreas and mediating beta-cell damage.

The PI capsid region of Theiler's virus controls replication in mouse glial cell cultures.

The GDVII strain of Theiler's virus is virulent. The DA strain is avirulent and can persist and initiate lesions of inflammatory demyelination in the CNS of susceptible strains of mice. Other, resistant strains of mice clear the infection. Replication of the GDVII and DA strains of Theiler's virus and their genetic recombinants R2, R3 and R4 were compared in mixed glial cell cultures derived from the mouse CNS. Differences were observed in the early rate of viral production. These mapped to the P1 capsid region of the viral genome. Viruses with GDVII P1 sequences produced virus and spread more rapidly than viruses with DA P1 sequences. GDVII virus infected greater numbers of cells than DA virus. Both strains of virus rapidly replicated at least to the level of translation in astrocytes (GFAP+), macrophage/microglial cells (F4/80+), oligodendrocytes (O4+) and bipotential precursor (A2B5+) cells. Early in infection many A2B5+ and GFAP+ cells were infected and destroyed. In contrast, O4+ cells were relatively resistant to cell-death. The cultures survived and produced virus over 14 days of study, at which time all 4 cell-type were present in the culture but < 1% of all the cells, the majority of which were O4+, expressed viral protein. Most of these infected O4+ cells retained a healthy morphology with extensive sheets of cytoplasm, suggesting that Theiler's virus infection of mature oligodendrocytes was non-destructive.

Lymphocyte recognition elements on the VP1 protein of Theiler's virus.

Theiler's virus is a murine picornavirus that persists in the central nervous system in susceptible mouse strains, and gives rise to immune mediated demyelinating disease. Antiviral CD4 T cells are necessary to protect from overwhelming virus replication in the acute phase of the disease, and are thought to act by stimulating the antibody response. The present study used overlapping synthetic peptides to map the location of epitopes recognized by CD4 T cells. One T-cell epitope was identified between amino acids 33-47 of VP1, which was recognized by virus-reactive T cells. 'Cryptic' epitopes were also present within VP1 at positions 153-167, 166-180, 225-239 and 233-247. A linear B-cell epitope was identified in the C-terminal region 225-276. Immunization of CBA mice with inactivated virus, but not peptides containing VP1 B- or T-cell epitopes, reduced the virus titre in the CNS in the acute phase of the disease.

Beyond the business plan.

Acknowledges that business planning in the NHS frequently disappoints. Reasons for this are found in the tendency for managers to view the production of a plan as an end rather than a means. A further difficulty resides in the perception managers have of their world. Argues that marketing is the most appropriate paradigm for understanding and structuring this world at present. However, an adaptive cognitive style is necessary to allow constant reframing within the dominant paradigm or even reframing of the paradigm itself. In adopting these approaches, the probability of achieving competitive advantage is heightened. If they are ignored, however, it is likely that training and development techniques, however sophisticated, will have little lasting impact.

Positioning the human resource business using service level agreements.

Explores the introduction and development of Service Level Agreements (SLAs) in relation to Human Resource Departments. Considers approaches to SLAs and highlights four dimensions necessary for the completion of an SLA. Stresses that Human Resource Specialists should have a thorough understanding of how directorates and other departments relate to one another to provide added value in terms of contribution to the organizational outcomes. Suggests the idea of adding value is an integral part of the SLA process which ensures that it operates as a means to an end and does not become an end in itself. Examines the degree of devolved freedom given to a department to seek work or sell its products outside its Trust/Unit. Scrutinizes the format of SLAs and concludes that the benefits of SLAs for users of Human Resource Departments and the benefits to the Human Resource Departments are similar.

The role of CD8+T cells in the acute and chronic phases of Theiler's murine encephalomyelitis virus-induced disease in mice.

The technique of in vivo depletion with T cell subset-specific monoclonal antibodies was used to study the involvement of CD8+T cells in protection/pathogenesis during the acute and chronic demyelinating phases of Theiler's murine encephalomyelitis virus (TMEV)-induced disease. Mice rendered CD8-deficient prior to infection with TMEV were less efficient at clearing virus from the central nervous system compared to intact animals and also suffered demyelinating disease of earlier onset and increased severity. This indicates that CD8+ cells have a protective role in virus clearance at early times post-infection, and may also be involved in downregulating the severity of the chronic demyelinating disease. How CD8+ T cells function to produce these effects is discussed.

Observations on demyelinating lesions induced by Theiler's virus in CBA mice.

We examined demyelinating lesions in the spinal cord of CBA mice infected with the BeAn strain of Theiler's virus to see if it was possible to document the sequence of changes which result in demyelination. It was found that the lesions which develop in the late stages of the disease were progressive. Therefore, by examining the different zones of a single lesion, it was possible to follow a sequence of changes which lead to demyelination. There was a clear progression from normal myelin, to vacuolated myelin, to myelin phagocytosis, to demyelinated axons, to remyelinated axons. Virus was detected in degenerating oligodendrocytes in the area showing myelin vacuolation by both electron microscopy and immunocytochemistry, a finding which indicated that virus infection precedes demyelination. The area of normal myelin which surrounded the zone of vacuolated myelin was infiltrated by lymphocytes, indicating that lymphocytic infiltration preceded viral replication and oligodendrocyte degeneration. Our observations indicate that cells of the immune system may play a role in the initiation of virus replication which appears to be a prerequisite for demyelination.

The effect of L3T4 T cell depletion on the pathogenesis of Theiler's murine encephalomyelitis virus infection in CBA mice.

Theiler's murine encephalomyelitis virus (TMEV) gives rise to a biphasic disease of the central nervous system (CNS) following intracranial inoculation of susceptible strains of mice. The early phase, during the first month, resembles poliomyelitis and in the late phase the mice suffer from inflammatory demyelination reminiscent of multiple sclerosis. In order to investigate the role of helper T cells in the acute and chronic phases of the disease we depleted mice of their L3T4 T cells in vivo with rat monoclonal antibodies, prior to infection and prior to the onset of clinical signs of demyelination. Mice depleted of their helper cells failed to produce antibodies to TMEV and consequently were unable to clear virus from the CNS and died within the first month of infection. Depletion of T cells before the demyelinating phase of the disease resulted in a marked decrease in the incidence of disease from 77% of the immunocompetent animals with clinical signs of paralysis to 36%. Immunocompetent TMEV-infected mice also developed antibodies against myelin suggesting that autoimmune mechanisms may play a role in TMEV-induced demyelination.