NK cell intrinsic regulation of MIP-1α by granzyme M.

Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.

Lupus vasculopathy: Diagnostic, pathogenetic and therapeutic considerations.

A rare form of vascular disease in systemic lupus erythematosus (SLE), lupus vasculopathy is characterized by necrosis and accumulation of immunoglobulins (IGs) and complements in the wall of arterioles and small arteries resulting in luminal narrowing. Lupus vasculopathy often accompanies lupus nephritis and portends a poor prognosis. Although there is general agreement on the treatment of lupus nephritis, effective treatment strategies for lupus vasculopathy remain to be defined. We report a 20-year-old woman with SLE who presented with generalized tonic-clonic seizure. Her immunosuppressive regimen consisted of mycophenolate mofetil, prednisone and hydroxychloroquine. On physical examination, she was Cushingoid in appearance and hypertensive. Laboratory tests indicated renal disease. Coagulation studies disclosed de novo lupus anticoagulant. Magnetic resonance imaging of the brain demonstrated acute focal cerebral hemorrhage. Echocardiography revealed reduced ejection fraction and severe mitral regurgitation. Despite high-dose glucocorticoids and mycophenolate mofetil, renal function remained poor. Kidney biopsy demonstrated lupus vasculopathy and glomerulonephritis. Plasma exchange therapy and intravenous cyclophosphamide were administered. Over the ensuing four weeks, renal function improved, complement levels increased, autoantibody titers decreased and lupus anticoagulant disappeared. In conclusion, lupus vasculopathy can occur in SLE despite a heavy immunosuppressive regimen. Antiphospholipid antibodies might be involved in the pathogenesis of lupus vasculopathy. Plasma exchange therapy in conjunction with intravenous cyclophosphamide may represent an effective treatment strategy for lupus vasculopathy.

Determining the minimum sampling rate needed to accurately quantify cumulative spine loading from digitized video.

Cumulative low back loads have been linked to the reporting of low back pain. Traditional video-based methods used to estimate these loads are time intensive for data collection and analysis. Sampling less frequently would help to reduce the associated time and cost of this type of approach. The purpose of this study was to determine how the error in estimated cumulative low back loads is affected by reducing video sampling rate. Ten healthy male university students performed three laboratory, sagittal plane lifts of varying mass (2.3, 8.8, and 15.9 kg), speed (0.2, 0.4, 0.8 m/s), and postural demand (lift from floor to table; lower from shelf to table; lift from floor over barrier and lower to floor) while being videotaped (60 frames/s). Digitized body coordinates and anthropometrics were input into a static biomechanical model, resulting in estimates of low back compression and shear forces, and moment. Load-time histories for each condition underwent rectangular integration at 60 (gold standard), 30, 20, 15, 12, 10, 6, 5, 4, 3, 2 and 1 frames/s, resulting in estimates of low back cumulative loads. Mean relative errors with respect to 60 frames/s for all cumulative loads and all conditions were found to be below 8% at 1 frame/s, and less than 3% at 2 frames/s. In addition, analyses at sampling rates above 3 frames/s were not significantly different than the cumulative loads determined at 60 frames/s, for all conditions. The accuracy of cumulative loads exhibited even at low sampling rates can be attributed, in part, to the fact that overestimations and underestimations of the integrated loads tend to cancel out over the length of the tasks considered.

Function of CMV-encoded MHC class I homologues.

Homologues of MHC class I proteins have been identified in the genomes of human, murine and rat cytomegaloviruses (CMVs). Given the pivotal role of the MHC class I protein in cellular immunity, it has been postulated that the viral homologues subvert the normal antiviral immune response of the host, thus promoting virus replication and dissemination in an otherwise hostile environment. This review focuses on recent studies of the CMV MHC class I homologues at the molecular, cellular and whole animal level and presents current hypotheses for their roles in the CMV life cycle.

Infection of dendritic cells by murine cytomegalovirus induces functional paralysis.

Cytomegalovirus (CMV), measles and HIV are the main human pathogens known to induce immunosuppression. Unlike measles and HIV, and despite the availability of a well studied animal model, little is known about the mechanisms that control CMV-induced immunosuppression. We hypothesized that dendritic cells (DCs), which are crucial in generating and maintaining immune responses, represent a target for CMV and that the transient, but profound, immunosuppression that accompanies CMV infection results from viral interference with DC functions. Here we show that DCs were permissive to murine CMV infection. In addition, DC infection prevented delivery of the signals required for T cell activation. Thus, CMV-mediated impairment of DC function may be crucial for virally induced immunosuppression and interleukin 2 is implicated as a key factor.

An evaluation of predictive methods for estimating cumulative spinal loading.

The focus of this study was to assess the amount of error present in several approaches that have been commonly used to estimate the cumulative spinal loading during manual materials handling tasks. Three male subjects performed three sagittal plane lifting tasks of varying loads and postural requirements. Video recordings of the tasks were digitized and a biomechanical model was used to calculate the spinal loading (compression, joint shear, reaction shear, and flexion/extension moment) at L4/L5 for each frame of data. The 'gold standard' for cumulative loading experienced by the subjects was obtained by integrating the resultant biomechanical model outputs for the entire lifting cycle. Five approaches that quantify cumulative spinal loading, four that use discrete measures and one that reduces the number of frames used (5 Hz), were used and compared with the gold standard. The four methods using discrete measures to quantify the cumulative demands of a task resulted in substantial errors (average error across task and subjects was 27-69%). Reducing the number of frames of data processed to 5 frames/s preserved the time varying information and was the only approach examined that did not induce significant error into the cumulative loading estimates. This study indicates that errors in cumulative spinal loading estimates can be large depending upon the approach used, which will hinder any progress in developing a dose-response link between cumulative exposure and an increased risk of low-back pain or injury.

A novel autocrine pathway of tumor escape from immune recognition: melanoma cell lines produce a soluble protein that diminishes expression of the gene encoding the melanocyte lineage melan-A/MART-1 antigen through down-modulation of its promoter.

We have observed that malignant melanoma cells produce a soluble protein factor(s), which down-regulates melanocyte lineage Melan-A/MART-1 Ag expression by melanoma cells with concomitant loss of recognition by Melan-A/MART-1-specific T cells. This down-modulation of Melan-A/MART-1 expression, which we refer to as "Ag silencing," is mediated via its minimal promoter, whereas the promoter for the restricting Ag-presenting HLA-A2 molecule is not affected. Significantly, this Ag silencing is reversible, as removal of factor-containing supernatants from Melan-A/MART-1-expressing cells results in up-regulation of the promoter for the gene encoding this Ag, and renewed expression of the protein. We have evaluated over 20 known factors, none of which accounts for the Ag-silencing activity of the melanoma cell culture supernatants. The existence of this autocrine pathway provides an additional novel explanation for melanoma tumor progression in vivo in the presence of CTL specific for this melanocyte lineage Ag. These observations may have important implications for Melan-A/MART-1-specific CTL-mediated immunotherapy of melanoma tumors.

NK1.1+ cells and murine cytomegalovirus infection: what happens in situ?

NK cells mediate early host defense against viral infection. In murine CMV (MCMV) infection NK cells play a critical role in controlling viral replication in target organs, such as spleen and liver. Until now it has not been possible to directly examine the role of NK cells in MCMV-induced inflammation in situ due to the inability to stain specifically for NK cells in infected tissues. In this study, we describe a method of in vivo fixation, resulting in the first identification of NK cells in situ using NK1.1 as the marker. Using this method, we characterize the NK1.1(+) cellular component of the inflammatory response to wild-type MCMV in the spleen, liver, and lung of genetically susceptible and resistant mice following i.p. infection. This study provides the first in situ description of the cellular response mediated specifically by NK cells following MCMV infection.

Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum.

Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

Melanoma antigen recognition by tumour-infiltrating T lymphocytes (TIL): effect of differential expression of melan-A/MART-1.

We have isolated, from an individual patient with metastatic melanoma, a series of eight TIL clones capable of lysing autologous melanoma cell targets. Six of the eight clones expressed TCRAV2S1 and lysed targets expressing HLA-A2 and the Melan-A/MART-1 peptide: AAGIGILTV. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis showed that the Melan-A/MART-1-specific clones were predominant in the bulk culture prior to cloning. However, the tumour progressed in vivo even in the presence of these tumour cell-lytic clones. Using the anti-Melan-A/MART-1 MoAb (A-103), we noted that Melan-A/MART-1 expression on three melanoma cell lines varied considerably during in vitro culture, in the absence of T cell immunoselection, relative to cell density. Tumour cells which spontaneously decreased Melan-A/MART-1 expression were less susceptible to specific TIL lysis. Melan-A/MART-1 expression and susceptibility to lysis increased in cells cultured at lower density. These data suggest that modulation of tumour antigen may account for tumour progression in the presence of tumour cell-lytic T lymphocytes. The observations suggest a possible explanation for the common finding of Melan-A/MART-1-specific lytic TIL in clinically progressing melanomas, as well as a possible pathway for therapeutic intervention.

Comparison of four peak spinal loading exposure measurement methods and their association with low-back pain.

OBJECTIVES: This paper examines the performance of 4 different methods of estimating peak spinal loading and their relationship with the reporting of low-back pain. METHODS: The data used for this comparison was a subset of subjects from a case-referent study of low-back-pain reporting in the automotive industry, in which 130 random referents and 105 cases (or job-matched proxies) were studied. The peak load on the lumbar spine was determined using a biomechanical model with model inputs coming from a detailed self-report questionnaire, a task-based check list, a video digitization method, and a posture and load sampling technique. RESULTS: The methods were directly comparable through a common metric of newtons or newton meters of spinal loading in compression, shear, or moment modes. All the methods showed significant and substantial associations with low-back pain in all modes (odds ratios 1.6-2.3). The intraclass correlation coefficients (ICC) showed strong similarities between the checklist and video digitized techniques (ICC 0.84-0.91), moderate similarities between these techniques and the work sampling method (ICC 0.49-0.52), and poor correlations (ICC 0.16-0.40) between the self-report questionnaire and the observer recorded measures. CONCLUSIONS: While all the methods detected significant odds ratios, they cannot all be used interchangeably for risk assessment at the individual level. Peak spinal compression, moment, and shear are important risk factors for low-back pain reporting, no matter which measurement method is used. Questionnaires can be used for large-scale studies. At the individual level a task-based checklist provides biomechanical model inputs at lower cost and equal performance compared with the criterion video digitization system.

The severity of murray valley encephalitis in mice is linked to neutrophil infiltration and inducible nitric oxide synthase activity in the central nervous system.

A study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414-423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.

Mixed lymphohaemopoietic chimerism and graft-versus-lymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation.

BACKGROUND: HLA-mismatched donor bone-marrow transplantation after standard myeloablative conditioning therapy for haematological malignant disorders has been limited by severe graft-versus-host disease (GVHD) and graft failure. We tested a new approach to find out whether lymphohaemopoietic graft-versus-host reactions could occur without excessive GVHD in mixed haemopoietic chimeras produced across HLA barriers with non-myeloablative conditioning. METHODS: Five patients with refractory non-Hodgkin lymphoma underwent bone-marrow transplantation from haploidentical related donors sharing at least one HLA A, B, or DR allele on the mismatched haplotype. Conditioning included cyclophosphamide and thymic irradiation before transplantation, and antithymocyte globulin before and after transplantation. The only other GVHD prophylaxis was cyclosporin. FINDINGS: Four of five patients were evaluable and showed engraftment. Mixed haemopoietic chimerism was established, with a predominance of donor lymphoid tissue and varying degrees of myeloid chimerism. Two patients were in GVHD-free states of complete and partial clinical remission at 460 and 103 days after bone-marrow transplantation. INTERPRETATION: Mixed chimerism can be induced in adult recipients of HLA-mismatched bone-marrow transplantation by a non-myeloablative conditioning regimen. The antilymphoma responses seen in two patients suggest that allogeneic bone-marrow transplantation without myeloablative conditioning might have potent immunotherapeutic benefits.

Synthesis and influenza virus sialidase inhibitory activity of analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) modified in the glycerol side-chain.

Analogues of 4-Guanidino-Neu5Ac2en (Zanamivir) have been prepared containing carbamate substituents at the 7-hydroxy position. (4S,5R,6R)-5-Acetylamino-6-[1R-[(6-aminohexyl)carbamoyloxy]-2R,3-dihydroxypropyl]-4-guanidino-5,6-dihydro-4H-pyran-2carboxylic acid and (4S,5R,6R)-5-Acetylamino-6-[1R-[heptylcarbamoyloxy]-2R,3-dihydroxypropyl]-4-guanidino-5,6-dihydro4H-pyran2-carboxylic acid were the two analogues possessing activity comparable to Zanamivir, showing potent inhibition of influenza virus sialidases and good antiviral activity in vitro.

Oligoclonality of Vdelta1 and Vdelta2 cells in human peripheral blood mononuclear cells: TCR selection is not altered by stimulation with gram-negative bacteria.

Despite the enormous potential repertoire of gammadelta T cells, there are several observations which suggest that the expressed gammadelta repertoire in the periphery of normal individuals is often quite restricted. To assess selective expansions among gammadelta T cells from both adult and newborn blood samples, PBMC from 12 normal adults and cord blood from 15 normal newborns were analyzed for TCRDV1 and TCRDV2 junctional diversity by CDR3 size spectratyping and single-strand conformational polymorphism. Although TCRBV usage showed extensive heterogeneity in adults and newborns, both populations often showed CDR3 region restriction for TCRDV1 and TCRDV2. Analysis of the CDR3 spectratype patterns of newborn twins suggested that clonal selection for TCRDV is independent of genetic background. The possible role of Gram-negative bacteria in driving selective responsiveness of gammadelta T cells in PBMCs from adults was examined by in vitro stimulation with Escherichia coli and Pseudomonas aeruginosa. Donors whose TCRDV repertoire was highly clonal in the unstimulated blood cells showed the same predominant clones among the bacteria-stimulated cultures. In individuals whose gammadelta T cells were less restricted, in vitro stimulation did not select for clonality; rather, the TCRDV repertoires were similar before and after bacterial stimulation. Together, these data indicate that gammadelta T cells are often clonally restricted in adults as well as in newborns and suggest that the prominent stimulatory activity of Gram-negative bacteria does not by itself account for the restriction or diversity of the gammadelta T cell repertoire.

Malate dehydrogenase and glucose-6-phosphate dehydrogenase, key markers for studying the genetic diversity of Actinobacillus actinomycetemcomitans.

Cell-free extracts of strains belonging to the 5 serotypes of A. actinomycetemcomitans were screened for several enzymes. Enzymes representative of the pentose phosphate pathway/hexose monophosphate shunt and the TCA cycle were present. Of these glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH) were the most readily detected and stable. MDH and G6PDH retained more than 50% of their activities at alkaline pHs (10-11) for up to 6 h and 3 h at 25 degrees C, respectively, while at pH 6.5, 50% of their activities were lost within 2-3 h. The Km for malate oxidation catalysed by MDH was 5.8 x 10(-4) M while that for glucose-6-phosphate oxidation was 2.0 x 10(-4) M. The pH optima for MDH and G6PDH oxidation activities were 10 and 9.5, respectively. Among the 5 designated serotypes of A. actinomycetemcomitans three groups were delineated by multilocus enzyme electrophoresis using MDH and G6PDH.

Are humans able to voluntarily elicit maximum muscle force?

Previous investigators have used electrical stimulation superimposed on voluntary efforts to show that humans are capable of maximum muscle activation. In the present study this notion was tested using the interpolated twitch technique enhanced by triggered averaging and doublet stimulation of the human biceps brachii. It was found that the decline in extra torque evoked by percutaneous stimulation with increasing levels of voluntary effort was nonlinear and that none of the twelve subjects was able to elicit a voluntary effort large enough to prevent extra torque of elbow flexion. The exponential nature of the declining extra torque indicated that an extrapolated maximum muscle force could be considerably larger than that to which subjects were able to elicit voluntarily.