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A thoracic suspensory jacket has been used in four patients with recurring ischial pressure sores to transfer the site of skin pressure from the insensate ischial area to the sensory area over the rib cage. This modality is not an alternative to surgery, but rather an adjunct to prevent an early recurrence after an ischial pressure sore has been repaired with a proper surgical procedure.
The standard mixed lymphocyte culture assay, which measures the incorporation of [3H]thymidine into DNA, usually requires 5 days. We describe a more rapid assay based on changes in the activity of ornithine decarboxylase. An increase in the activity of ornithine decarboxylase was observed in mixed lymphocyte cultures from genetically defined, major histocompatibility complex (MHC)-nonidentical miniature swine as early as 18 hr after plating. No increase was found in mixed cultures from inbred MHC-identical animals. Similar results were obtained with the enzyme S-adenosylmethionine decarboxylase with the increase in activity starting at about 32 hr. There was a good correlation between the ornithine decarboxylase values at 18 hr and the results of the [3H]thymidine incorporation assay on day 5. Preliminary experiments with human lymphocytes revealed similar results.
Cytologic analysis was performed on 128 bile specimens collected by schedule from 12 liver transplant recipients over a 4-month period. Clinical diagnoses at the time of specimen collection were determined retrospectively, as follows: clinically stable, 75; acute rejection, 15; CMV hepatitis, 1; systemic infection, 8; ischemic injury, 24 (all within the first 4 days postop); nonclassifiable, 5. Bile analysis was done by a blinded investigator. Specimens contained ductal epithelial cells (EC) and inflammatory cells (IC), which were counted using Cytospin slide preparations. Greater than 10 cells/slide were seen in 93.3% of rejections, 91.7% of ischemic injuries, 100% of systemic infections, and 14.6% of stable patients. In samples collected after POD 4, IC were seen in 86.7% of rejections, yielding a specificity of 94.4% (P less than 0.001). If lymphoblastic cells were also seen, the specificity increased to 96.6%. Five specimens were obtained the day before the clinical diagnosis of rejection; all demonstrated IC. Seven specimens were obtained 3 days after beginning therapy for rejection. In 5 the bile contained no IC, and clinical improvement occurred; in the 2 in whom IC were found, further therapy was subsequently required. IC were seen in 5 of 8 specimens taken when systemic infection was present; the clinical setting allowed differentiation from rejection. Only 1 case of CMV hepatitis was included, thus no conclusions can be drawn for this entity. Cytoplasmic vacuolization of EC was observed in 30% of cases, in these, cyclosporine levels were significantly higher (989.9 +/- 356.9 vs. 672.8 +/- 421.2, P = 0.02). In summary, bile cytology analysis aides in the monitoring of the onset and duration of rejection. It may be an indicator of persistent rejection, and it may help prevent overimmunosuppression in those cases with normal cytological findings.
Donor-recipient microchimerism has recently been suggested to play a critical role in the induction and maintenance of allograft tolerance. In this study we sought evidence for this hypothesis using the LEW-to-ACI cardiac allograft as a model system. Donor-specific tolerance to cardiac allografts was induced by intravenous or intraportal injection of graft recipients with donor peripheral blood, T cells, or B cells 7 days before transplantation. All the graft recipients injected with donor antigens accepted donor heart grafts indefinitely when compared with control recipients that rejected donor allografts in 12 days. Long-term graft survivors rejected third-party BN heart allografts in 14 days without an adverse effect on the survival of the first LEW heart allografts, demonstrating the specificity of the tolerance. Tissue lysates prepared from heart, kidney, liver, bone marrow, thymus, lymph nodes, and spleen of tolerant (>120 days) graft recipients were analyzed for the presence of donor DNA using LEW T cell receptor C beta gene-specific primers for polymerase chain reaction that detects donor DNA at > or = 1:10,000 dilution. Donor DNA was detected in 77% of tolerant graft recipients. Chimeric recipients showed variations in the levels and presence of donor DNA in different tissues. The status of donor microchimerism, with respect to its presence and tissue distribution, was dependent upon the donor cell type and route of injection used for the induction of tolerance. Intraportal injection of the graft recipients with donor peripheral blood resulted in the highest degree of chimerism, whereas intravenous injection with donor B cells did not induce detectable microchimerism in this group of recipients. These data clearly demonstrate that the presence of microchimerism is common following administration of donor cells, but that its presence is not an absolute requirement for the long-term survival of allografts.
Bone marrow cells may be a useful vehicle for pretransplant delivery of alloantigen to induce tolerance in vivo. However, infusion of fully allogeneic bone marrow cells carries the risk of graft-versus-host disease. In order to reduce this risk while retaining the tolerogenic potential of the bone marrow infusion, we have investigated the ability of recipient bone marrow cells expressing a single donor MHC class I antigen to induce specific unresponsiveness in vivo. We show that 5x10(7) and 5x10(6) bone marrow cells from a transgenic strain of CBA mice, CBK, that express a single donor class I MHC gene, H2Kb (H2k + H2Kb), were able to induce long term survival of a fully allogeneic C57BL/10 (H2b) cardiac allograft in 80% and 20% of unmanipulated CBA (H2k) recipients, respectively, when administered intravenousely on the day of transplantation. In contrast, the same doses of fully allogeneic C57BL/10 donor bone marrow were completely ineffective at prolonging graft survival. When the interval between bone marrow infusion and transplantation was increased to 14 days, CBK bone marrow at either dose (5x10(6) and 5x10(7)) induced long term survival of C57BL/10 cardiac allografts in all recipients (MST>100 days) while fully allogeneic donor bone marrow was ineffective (MST=7, 5x10(6) cells; MST=6, 5x10(7)). Only when 27 or 42 days had elapsed between bone marrow infusion and transplantation did fully allogeneic bone marrow exert a beneficial effect on graft survival. Administration of 5x10(6) C57BL/10 bone marrow cells 27 and 42 days before transplantation resulted in long term survival of C57BL/10 hearts in 67% and 75% of CBA recipients. Next, we investigated whether manipulating the periphery of the recipient with a depleting anti-Cd4(4) monoclonal antibody before bone marrow infusion would facilitate the induction of unresponsiveness. When pretreatment with bone marrow cells was combined with anti-Cd4 monoclonal antibody 28 days before transplantation, a 10-fold reduction in the number of either C57BL/10 or CBK bone marrow cells required to induce tolerance was observed. These data confirm that bone marrow is a suitable vehicle for alloantigen delivery at the time of, or before, transplantation, on its own or in combination with anti-Cd4. The use of recipient type bone marrow cells expressing one or more donor MHC genes may be more effective than fully allogeneic, donor bone marrow cells in inducing tolerance in vivo. This difference may have important clinical implications for the current trials of donor bone marrow given at the time of transplantation in order to augment chimerism and to prolong graft survival.
The motoneuron has a strong ability to regenerate after injury. However, the problems of nerve cell survival after a proximal axotomy, difficulty in axonal elongation after intraspinal lesion and the lack of target specificity during nerve fibre regrowth interfere with a good functional restitution. Current research has addressed these impediments. New approaches for the management of complicated as well as intraspinal lesions and peripheral nerve trunk injuries are described.
Analysis of genetic changes is often hampered by insufficient starting DNA from limited clinical tissue specimens. We employed ligation-mediated PCR (LM-PCR) for global amplification of the genome to overcome this limitation, generating up to 5 microg of representative amplicons of genomic DNA from as little as one cell. We demonstrate successful global genome amplification in high-quality starting DNA source like laser-captured cultured cells, as well as partially degraded starting DNA from old formalin-fixed paraffin-embedded tissue sections. This process generates adaptor-tailed templates that can be repeatedly amplified almost ad infinitum. We have further modified this technique such that, instead of a single endonuclease digest, we can achieve higher amplicon coverage by combining 3 endonuclease digests prior to LM-PCR. As tested by examining amplification of STS sequences scattered genome-wide, the coverage was improved from the published 70% to 96%. The faithful representation of global losses and gains in the amplified genomic DNA was confirmed by array-comparative genomic hybridization. Further, we exemplify the utility of this technique for finer p53 point mutation analysis by PCR-SSCP. This technique is thus a clinically useful tool for globally amplifying and archiving DNA from finite sources like paraffin tissue sections, providing a potentially unlimited resource for genetic analyses.
STUDY DESIGN: An animal study in immune competent rabbits and athymic rats was conducted. OBJECTIVES: To develop an animal model for simulation of previous human Type 5 adenovirus (Ad5) exposure, to determine the impact of adenoviral pre-exposure on spine fusion induced with ex vivo Ad5-LMP-1, and to test strategies for overcoming any potential immune response. SUMMARY OF BACKGROUND DATA: Cells transduced with adenovirus containing the osteoinductive LMP-1 cDNA (Ad5-LMP-1) can induce spine fusion in rabbits. Because up to 80% of the human population has been exposed to adenovirus, immune responses to the vector may limit this strategy in humans. Few studies have modeled previous adenoviral exposure and tested strategies to circumvent it. METHODS: Adult New Zealand white rabbits were injected with 10 or 10 viral particles of Ad5-LacZ. At 4 or 16 weeks after Ad5 injection, autologous buffy coats were prepared from peripheral blood, and 4 million cells per side were infected ex vivo for 10 minutes with Ad5-LMP-1 (multiplicity of infection = 4). Cells were implanted on a collagen matrix instead of an autograft for posterolateral lumbar arthrodesis. Unimmunized rabbits served as control subjects. Additional immunized rabbits underwent arthrodesis at 4 weeks with increased cell number (10 million) and viral dose (multiplicity of infection = 10), or with both parameters increased. The rabbits were killed at 4 weeks, and the spines were assessed by palpation and radiograph. A parallel study was performed in athymic rats using immunized rabbits for the donor cells. RESULTS: All the unimmunized rabbits had solid spine fusions. None of the rabbits arthrodesed 4 weeks after Ad5 pre-exposure achieved fusion. At 4 weeks after Ad5 exposure, increasing the multiplicity of infection to 10 did not overcome the immune response (0/3 fused), but increasing the cell number to 10 million (2/3 fused) or increasing both cell number and multiplicity of infection (3/3 fused) did overcome the immune effects. Delaying arthrodesis until 16 weeks after Ad5 pre-exposure also overcame the immune response (3/3 fused). Similar results were seen in the athymic rat ectopic implant model, suggesting that the immune effect was mediated by humoral antibodies rather than a T-cell response. CONCLUSIONS: Two model systems were developed that simulate previous exposure to human Ad5 and could separate the cellular and humoral components of the response. There was a dose-dependent inhibition of ex vivo Ad5-LMP-1 gene transfer to cells from animals previously exposed to human Ad5. Data suggested that the inhibition of Ad5 infection was caused by humoral antibodies rather than a T-cell-based response. Minor modifications in the gene transfer protocol, such as doubling the viral dose or number of cells infected, or increasing the infection time, could overcome the immune response for an ex vivo approach.
Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.
BACKGROUND: Chronic allograft nephropathy (CAN) is the most common cause of long-term renal-allograft dysfunction and the second most common cause of transplant loss. There are concerns that prolonged patient exposure to calcineurin inhibitors (CNIs) exacerbates or promotes the process of CAN. METHODS: In our unit, we carried out an observational study over the period 2000 to 2003 using low-dose mycophenolate mofetil (MMF) to facilitate a phased reduction in the dose of CNI in a group of patients with CAN. Low doses of MMF were chosen to minimize adverse effects and to reduce levels of CNIs without the attendant risks of under-immunosuppression. RESULTS: A step-wise reduction in mean reciprocalised creatinine was evident over the run-in period (mean 1/creatinine before MMF=0.005739-0.000083*month; R=0.77) with a step-wise monthly improvement postintroduction of MMF and dose reduction of CNI (mean 1/creatinine after MMF=0.004609+0.000049*month; R=0.74) (P<0.0001). The mean Cockroft-Gault estimated creatinine clearances were 47.1+/-24.2, 37.2+/-16.3, and 41.6+/-21.1 mL/min at time [t]=-12, t=0 and t=+12 months, respectively. Low-dose MMF therapy was well tolerated (only 7/89 patients stopped MMF because of side-effects in the first 12 months), and acute rejection was noted in only one patient. At latest follow-up, only 17 transplant losses had occurred, of which 6 patients had died with a functioning graft. CONCLUSIONS: Low-dose MMF was well tolerated and resulted in prolonged graft survival.
BACKGROUND: Bronchiolitis obliterans (BO) is a common complication and is associated with high mortality after allogeneic stem-cell transplantation (SCT). Early diagnosis of BO may improve outcome. Low levels of Clara cell secretory protein (CC16) have previously been associated with BO in lung transplant recipients. METHODS: Serum samples were collected from eight patients with BO, eight patients with chronic graft-versus-host disease (GVHD), and eight control patients with neither BO nor chronic GVHD in a matched patient analysis. Patients were matched for diagnosis, conditioning, donor match, and GVHD prophylaxis. Another seven patients with BO were also analyzed separately. CC16 was measured with an enzyme-linked immunosorbent assay method. RESULTS: In the matched analysis, eight patients were diagnosed with BO at a median of 11.5 months (range, 4-13 months) after SCT and in non-matched BO patients at a median of 12 months (range, 9-36 months). In the matched patient analysis, patients with BO had significantly lower (P=0.03) or decreasing (P=0.02) levels of CC16 compared with patients with only chronic GVHD or controls. In the matched patient analysis, measurement of CC16 showed a sensitivity of 88% and a specificity of 81%. With the criteria of low levels of CC16 or a decrease of more than 40% compared with the previous sample, BO was detected with analysis of CC16 in 13 of 15 patients. In 11 of the 13 patients, low or decreasing values of CC16 were detected at a median of 10 months (range, 1-30 months) before BO was diagnosed clinically. CONCLUSIONS: Low levels of CC16 are associated with BO after allogeneic SCT. Monitoring of CC16 in serum after SCT may have potential as an early marker for BO.
Over the past 10 years considerable insight into intracellular interactions leading to long-term memory formation have been gleaned from various neural circuits within invertebrate and vertebrate species. This review suggests that, while certain intracellular signaling pathways are commonly involved across species, it is important to analyze specific neural systems because critical differences among systems appear to exist. The olfactory bulb has been used by our group to estimate the influence of neuromodulatory systems (serotonin and norepinephrine) on intracellular processes leading to learning. We describe here how activation of noradrenergic input to mitral cells increases cAMP leading to CREB phosphorylation when paired with a conditioning stimulus, odor. CREB phosphorylation is causal in odor preference learning leading to long-term memory for the odor. However, the relationship between cAMP activation and CREB phosphorylation is not straight forward; overstimulation of cAMP pathways impedes learning and prevents CREB phosphorylation. Excessive CREB phosphorylation also interferes with learning.
Population sex ratios in monogamous birds are often male biased. One factor that can affect population sex ratios is sex-biased predation. However, most estimates of sex-biased predation in birds have focused on species with obvious sexual colour dimorphism or body size dimorphism. Data on sexually monomorphic birds are generally lacking. In the present study, we adopt a PCR-based sexing procedure to help test for sex-biased predation in a wintering population of western sandpipers (Calidris mauri), a shorebird that shows only subtle sexual size dimorphism. Specifically, by comparing the a priori determined sex ratio of live birds wintering at a site in western Mexico to the molecular estimate obtained from depredated birds at this same site, we were able to perform a population-specific test for sex bias in predator-induced mortality. The proportion of females estimated from living (ca. 25%) versus dead (ca. 24%) individuals was in fact not significantly different, indicating that the strong male bias in this population is not due to differential predation. However, molecular sexing of prey remains is a hitherto unexploited test of sex-biased predation in birds, and is potentially applicable to any species for which prey remains can be gathered. We discuss our results in the context of alternate ecological hypotheses for population sex biases.
It has recently been established that infection with a virus is the most common cause of transient arrest of erythrocyte production in the bone marrow, leading to aplastic crisis, in persons suffering chronic haemolytic anaemias. The physical characteristics of this human virus have suggested that it may be a member of the Parvoviridae. We report here that extraction of nucleic acid from this virus under annealing conditions yielded a single species of double-stranded DNA 5.5 kb in length. Treatment with heat or alkali converted this DNA into a rapidly migrating form sensitive to the single-strand-specific nuclease S1. Extraction of the virion DNA under conditions of low ionic strength where annealing would not be expected to occur yielded DNA which comigrated with the 5.5 kb single-stranded molecule. The results indicate that this virus packages equal numbers of complementary DNA strands into separate virions. It is suggested that this virus can be classified as a member of the genus parvovirus.
A phage-resistant mutant of Lactobacillus casei (strain YIT 9021) was capable of adsorbing both PL-1 and J-1 phages, but did not yield phage-infected cells. The mutant and wild-type strains were identical in morphology and sugar composition of the cell walls. Attempts to induce prophages from strain YIT 9021 were unsuccessful. Electron microscopic examination of negatively stained mixtures of phage PL-1 and YIT 9021 bacteria revealed that the phages were adsorbed to the cells in a tail-first orientation. All the absorbed phages had DNA-filled heads. It was concluded that PL-1 adsorbed normally but was blocked in the injection of the phage genome into the cell.
An acute graft versus host disease (GvHD) murine model was developed to study the pathogenic and protective mechanisms against viruses that replicate in cells of the human immune system. The model allowed efficient replication of lymphotropic, macrophage and amphitropic strains of human immunodeficiency virus type 1 (HIV-1) and measles virus (MV). Cytopathic lymphotropic strains of HIV-1 and a wild-type MV strain replicated in a 'burst'-like manner, whereas a non-cytopathic lymphotropic HIV-1 strain and all macrophage-tropic HIV-1 strains caused persistent infection of the graft. The replication kinetics of infection with these viruses were highly reproducible and were very similar to those observed in natural infection of humans. Infection with these viruses, with the exception of HIV-1SF2, led to a delay [corrected] and abrogation of the GvHD, indicating a direct immunosuppressive effect. Interestingly, infection with the lymphotropic HIV-1SF2 strain was rapidly and spontaneously abrogated. The model was also shown to be suitable for the evaluation of passive immunization strategies. Administration of a combination of antibodies against the HIV-1 V3 loop and the HIV-1 CD4 binding sites prevented subsequent infection with HIV-1IIIB. In contrast, administration of CD4 binding site specific human monoclonal antibody at a concentration that would neutralize the virus in vitro enhanced in vivo infection with HIV-1IIIB. The model also allowed evaluation of in vivo immunization studies. Immunization with a live attenuated measles vaccine resulted in protection from a wild-type MV challenge, whereas immunization with a subunit candidate vaccine appeared to give partial protection.
Monoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full-length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.
Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which are thought to locate to the envelope of virus particles. These proteins form two complexes in tissue culture systems, a high molecular mass aggregate that contains intermolecular covalent bonds and a native complex in which E1 and E2 associate by non-covalent interactions. The contribution of either complex to the structures of the proteins on virus particles is not known. Using dithiothreitol to reduce inter- and intramolecular disulphide bonds in situ, we have studied the nature of the interactions within the aggregate and the role of covalent bonds in the early stages of E1-E2 association. Results with two HCV type 1a strains, Glasgow and H77, showed that the aggregate contains not only covalent interactions but also non-covalent associations between E1 and E2. These non-covalent associations are complex since deletion mutant analysis failed to identify any single region which was required for non-covalent interaction. Complex formation by de novo synthesized proteins was not arrested under reducing conditions which prevented the production of inter- and intramolecular disulphide bonds. Moreover, a conformation-specific antibody continued to recognize the E2 protein in reduced complexes, indicating that covalent bonds do not stabilize certain structures of E2 that can interact with E1. These data suggest that disulphide bonds are not required either to allow association between the proteins or to stabilize E1-E2 complexes.