Downregulation of CX3CR1 ameliorates experimental colitis: evidence for CX3CL1-CX3CR1-mediated immune cell recruitment.

PURPOSE: Inflammatory conditions like inflammatory bowel diseases (IBD) are characterized by increased immune cell infiltration. The chemokine ligand CX3CL1 and its receptor CX3CR1 have been shown to be involved in leukocyte adhesion, transendothelial recruitment, and chemotaxis. Therefore, the objective of this study was to describe CX3CL1-CX3CR1-mediated signaling in the induction of immune cell recruitment during experimental murine colitis. METHODS: Acute colitis was induced by dextran sodium sulfate (DSS), and sepsis was induced by injection of lipopolysaccharide (LPS). Serum concentrations of CX3CR1 and CX3CL1 were measured by ELISA. Wild-type and CX3CR1-/- mice were challenged with DSS, and on day 6, intravital microscopy was performed to monitor colonic leukocyte and platelet recruitment. Intestinal inflammation was assessed by disease activity, histopathology, and neutrophil infiltration. RESULTS: CX3CR1 was upregulated in DSS colitis and LPS-induced sepsis. CX3CR1-/- mice were protected from disease severity and intestinal injury in DSS colitis, and CX3CR1 deficiency resulted in reduced rolling of leukocytes and platelets. CONCLUSIONS: In the present study, we provide evidence for a crucial role of CX3CL1-CX3CR1 in experimental colitis, in particular for intestinal leukocyte recruitment during murine colitis. Our findings suggest that CX3CR1 blockade represents a potential therapeutic strategy for treatment of IBD.

Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco.

Complex multimeric proteins such as dimeric and secretory immunoglobulin A (IgA) can be difficult to produce in heterologous systems, although this has been achieved using several platforms including plants. As well as topical mucosal applications, dimeric IgA (dIgA), and secretory IgA (sIgA) can be used in tumor and anti-viral therapy, where their more potent cell-killing properties may increase their efficacy compared to current drugs based on IgG. However, the development of therapeutic IgA formats is hampered by the need to co-express four different polypeptides, and the inability to purify such molecules using conventional protein A or protein G affinity chromatography. The light chain (LC)-specific affinity ligand protein L is a potential alternative, but it only recognizes certain kappa light chain (LC(κ)) subtypes. To overcome these limitations, we have adapted a framework-grafting approach to introduce LCs that bind protein L into any IgA. As a model, we used the chimeric anti-human chorionic gonadotropin (hCG) antibody cPIPP, since this contains a murine LC((κ)) subtype that does not bind protein L. Grafting was achieved by replacing selected framework region 1 (FR1) residues in the cPIPP LC(κ) variable domain with corresponding residues from LC(κ) subtypes that can bind protein L. The grafted antibody variants were successfully purified by protein L affinity chromatography. These modifications affected neither their antigen-binding properties nor the yields achieved by transient expression in tobacco plants. Our results therefore show that LC FR1 grafting can be used as generic strategy for the purification of IgA molecules.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Measuring recent thymic emigrants in blood of normal and HIV-1-infected individuals before and after effective therapy.

The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed alpha1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that alpha1 circle numbers in blood remain high for the first 10-15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, alpha1 circle numbers in HIV-1-infected adults were significantly reduced; however, there were many individuals with normal alpha1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on alpha1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in alpha1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as alpha1 circle numbers are normal in a substantial subset of HIV-1-infected individuals.

Hepatic ischaemia-reperfusion injury from bench to bedside.

BACKGROUND: Vascular occlusion to prevent haemorrhage during liver resection causes ischaemia-reperfusion (IR) injury. Insights into the mechanisms of IR injury gathered from experimental models have contributed to the development of therapeutic approaches, some of which have already been tested in randomized clinical trials. METHODS: The review was based on a PubMed search using the terms 'ischemia AND hepatectomy', 'ischemia AND liver', 'hepatectomy AND drug treatment', 'liver AND intermittent clamping' and 'liver AND ischemic preconditioning'; only randomized controlled trials (RCTs) were included. RESULTS: Twelve RCTs reported on ischaemic preconditioning and intermittent clamping. Both strategies seem to confer protection and allow extension of ischaemia time. Fourteen RCTs evaluating pharmacological interventions, including antioxidants, anti-inflammatory drugs, vasodilators, pharmacological preconditioning and glucose infusion, were identified. CONCLUSION: Several strategies to prevent hepatic IR have been developed, but few have been incorporated into clinical practice. Although some pharmacological strategies showed promising results with improved clinical outcome there is not sufficient evidence to recommend them.

Experimental models of temporary normothermic liver ischemia.

Hepatic ischemia/reperfusion injury has so far been investigated in various experimental models. A clinical transfer of experimental results is, however, problematic because of anatomical and physiological differences and also the inevitable simplification of experimental work. The choice of model must therefore be adapted to the clinical question to be answered. The simplest procedure for inducing normothermic ischemia is to clamp the hepatoduodenal ligament. Models that do not avert portal congestion are regarded as unsuitable. Our current understanding of the pathogenesis of ischemia/reperfusion injury depends mainly on studies whose authors have investigated either global liver ischemia with a portocaval shunt, spleen transposition and in the isolated perfused system, or partial ischemia. This review is a critical examination of various approaches to the study of normothermic hepatic ischemia in experimental animals.

Acute liver failure: from bench to bedside.

Acute liver failure constitutes a challenge to clinicians and scientists alike. The course of the disease, usually unpredictable and polarizing, is associated with a high mortality unless liver transplantation is feasible, but can end in a spontaneous restitution. It poses many scientific questions regarding the mechanisms of liver cell damage and regeneration and the possibility of new therapeutic approaches. However, the performance of clinical studies in patients in acute liver failure presents problems because of the varied etiology, the small number of cases, and furthermore due to ethical and logistical difficulties. For this reason experimental investigations have gained a special importance. Arising from the improved understanding of the mechanisms of liver cell damage in acute liver failure, which may be primarily due not to the initial noxious agent alone but may also be triggered secondarily by the release of proinflammatory mediators, there are numerous options for liver cell protection, some of which have already proved successful in experimental studies. New insights into the mechanisms of regulation of liver regeneration and the physiological liver mass, gathered in particular from experimental models of partial hepatectomy and by the use of gene-manipulated animals, have contributed to the development of new therapeutic approaches for the stimulation of liver cell regeneration. Temporary liver support systems have already been successfully employed in some cases under clinical conditions. Although the systematic experimental investigation of many of the questions of acute liver failure has significantly contributed to a better understanding of liver cell damage and regeneration, the application of this new knowledge to clinical practice is to some extent made difficult by the artificial simplification that experimental studies inevitably entail and needs to be validated by controlled clinical studies.

Therapy and prognosis of tumors of the genitourinary tract after kidney transplantation.

There is an increased incidence of tumors of the genitourinary tract among kidney graft recipients. From 1979 to 2001, all patients who received kidney transplants had records of both their underlying diseases and their initial immunosuppression. Patients who developed a genitourinary tract malignancy were evaluated for tumor type, location, stage, tumor therapy and clinical course. During this period, 1804 patients underwent 2068 kidney transplantations. Thirty-four patients had 39 tumors of genitourinary origin. One patient was lost to follow-up. There were 15 patients with 18 renal cell carcinomas (one of them multifocal): six had seven transitional cell carcinomas; six, prostatic carcinoma; six, tumor of the female genital tract (one also had a renal cell carcinoma); and two, a seminoma. Most tumors were diagnosed in their early stages (< or = pT3, N0, M0; n = 31 tumors) and thus accessible to curative therapy, achieving good long-term results: 1- and 5-year survival rates of 100% and 91%, which were better than those obtained in advanced stages (N+, M+; n = 7 tumors), namely both 1- and 5-year survival rates of 38% (P < .05). Death was caused by tumor growth in nine patients (27%) and by other causes in three patients (9%). With appropriate treatment genitourinary tumors at early stage show a good prognosis. New immunosuppressants with supposed antiproliferative effects may help to decrease the incidence of malignancies. The most important factor is risk-adapted screening to identify malignancies early and to initiate appropriate therapy.

Immunologic and virologic responses to HAART in severely immunocompromised HIV-1-infected children.

OBJECTIVE: To determine the long-term immunologic and virologic effects of highly active antiretroviral therapy (HAART) in children with AIDS. DESIGN: A prospective observational study. SETTING: Two pediatric HIV clinics. PARTICIPANTS: Twenty-five protease-inhibitor naive HIV-infected children (aged 2-18 years) with advanced disease (CD4 < or =6%). INTERVENTION: HAART (one protease inhibitor and one or more nucleoside analogs). Diphtheria and tetanus immunization in six patients after 18 months of therapy. MAIN OUTCOME MEASURES: Changes in percentage of CD4 cells and plasma HIV-1 RNA levels; post-treatment assays of lymphoproliferative responses to recall antigens; CD4 cell memory phenotype. RESULTS: Median duration of follow-up was 18.8 months (range, 7.5-28 months). At baseline the CD4 cell percentage was 2% (range, 0-6%), this increased significantly to 16% (range, 3-48%) above baseline at 12 months (P = 0.002). The mean maximum CD4 cell increase was 20.7% (range 4-48%) which corresponds to 657x10(6) cells/l (range, 30-2240x10(6) cells/l) above baseline. By contrast, the median viral load was not significantly lower at 12 months than at baseline (P = 0.34), and only 25% of the patients had sustained undetectable viral load. Of the reconstituted CD4 cells 70% were naive, and none of the subjects had lymphoproliferative responses to tetanus and diphtheria although 40% did develop responses to Candida, an environmental antigen. A single immunization with diphtheria and tetanus toxoid produced lymphoproliferative responses to tetanus in three out of six patients. CONCLUSIONS: HAART was associated with sustained increases in CD4 cell counts, despite a high incidence of 'virologic failure'. CD4 counts and the proportion of naive cells were higher than have been reported in adults, which may be a reflection of greater thymic activity in children. Memory cell clones for antigens encountered in the past which are not prevalent before therapy could not be expanded without additional antigenic exposure.

Interferon kinetics and adverse reactions after intravenous, intramuscular, and subcutaneous injection.

Three groups of six subjects each received a single 36 X 10(6) U dose of recombinant leukocyte A interferon (rIFN-alpha A) as a 40-min infusion, an intramuscular injection, or a subcutaneous injection. Blood samples were collected at specific times after dosing for analysis of rIFN-alpha A serum concentrations by an enzyme immunoassay method, ELISA. The rIFN-alpha A was rapidly distributed and moderately eliminated (t 1/2 = 5.1 hr) after intravenous infusion. The maximum concentrations at the end of intravenous infusion were tenfold the maximum concentrations after intramuscular and subcutaneous injections. Renal tubular secretion or extrarenal elimination was suggested by clearance values of 1.8 times the glomerular filtration rate. After intramuscular and subcutaneous injection, rIFN-alpha A was absorbed slowly (time to reach maximum concentration ranged from 4 to 8 hr), which resulted in prolonged serum concentrations. Estimated bioavailability was more than 80% for both intramuscular and subcutaneous injection shares qualitatively the same adverse reactions, the reactions differ in severity and duration. The adverse effects appear to be related to route of administration of herpes labialis were also noted. There were no significant clinical laboratory abnormalities of medical concern. Although rIFN-alpha A injected by intravenous infusion or intramuscular or subcutaneous injection shares qualitatively the same adverse reactions, the reactions differ in severity and duration. The adverse effects appear to be related to route of administration.

Legal principles in the psychiatric assessment of personal injury litigants.

The authors review the legal principles that a psychiatrist must understand in assessing the emotional and psychiatric sequelae of a personal injury leading to a litigation claim. The principles of the establishment of fault or liability, the assessment of pain and suffering, causal connection, the credibility of the plaintiff, the credibility of the expert witness, the determination of prognosis, the award of damages, and the adversarial system are discussed. An appreciation of these principles will enable psychiatrists to provide assessments that will be useful to the legal system in arriving at a fair and accurate determination of the compensation to which a victim is entitled.

Neurogenic and hysterical seizures in children and adolescents: differential diagnostic and therapeutic considerations.

For patients whose seizures are not controlled by medication alone, psychotherapeutic intervention can sometimes play a useful adjunctive role in both differential diagnosis and treatment. Diagnostic issues include the differentiation between neurogenic and hysterical seizures as well as the delineation of the possible role of psychogenic stress in triggering neurogenic seizures. Therapeutic considerations focus on the alleviation of precipitating stresses as well as the development of a sense of mastery in the patient.

Predictors of smoking abstinence following a single-session restructuring intervention with self-hypnosis.

OBJECTIVE: This study examined the relation of smoking and medical history, social support, and hypnotizability to outcome of a smoking cessation program. METHOD: A consecutive series of 226 smokers referred for the smoking cessation program were treated with a single-session habit restructuring intervention involving self-hypnosis. They were then followed up for 2 years. Total abstinence from smoking after the intervention was the criterion for successful outcome. RESULTS: Fifty-two percent of the study group achieved complete smoking abstinence 1 week after the intervention; 23% maintained their abstinence for 2 years. Hypnotizability and having been previously able to quit smoking for at least a month significantly predicted the initiation of abstinence. Hypnotizability and living with a significant other person predicted 2-year maintenance of treatment response. CONCLUSIONS: These results, while modest, are superior to those of spontaneous efforts to stop smoking. Furthermore, they suggest that it is possible to predict which patients are most likely and which are least likely to respond to such brief smoking cessation interventions.

Lack of antiscorbutic activity of ascorbate 2-sulfate in the rhesus monkey.

Oral administration of 10 mg per kilogram of body weight of ascorbic acid (AA) completely prevented development of scurvy in juvenile rhesus monkey (Mucaca mulata) fed an AA-free liquid diet. The same dose cured scurvy when injected intramuscularly. An equimolar dose of ascorbic acid 2-sulfate (AA-2-S) did not prevent or cure scurvy. Neither AA nor AA-2-S altered serum cholesterol. AA but not AA-2-S reduced serum triglyceride. A case of scurvy in an AA-2-S treated monkey is described in detail.

Age differences in vitamin B6 status of 617 men.

The effect of age on vitamin B6 metabolism was studied in 617 community-dwelling subjects, ages 18 to 90. These are, for the most part, clinically healthy, educated men whose intake of nutrients is not limited by economic factors. Plasma pyridoxal phosphate (PLP) was used as the primary criterion of vitamin B6 status. About one-third of the subjects were taking supplementary vitamins on their own initiative. The amount of pyridoxine-HCl varied from 0.1 to 105 mg/day. The average plasma PLP of the men not taking a supplement (N = 414) was 12.3 +/-0.3 ng/ml, with 25% of the values below 7.5 ng/ml and 7% below 5 ng/ml. There was a statistically significant decrease in plasma PLP with age of 0.9 ng/ml per decade. For those taking a supplement, the average plasma PLP was 20.5 +/- 1.0 ng/ml, with only 8% of the values below 7.5 ng/ml and none below 5 ng/ml. Glutamic-oxaloacetic transaminase activity in plasma (PGOT) and erythrocytes (EGOT) was determined on all subjects. The ratio of EGOT with in vitro stimulation by PLP to EGOT actual (alpha-EGOT) was also studied. These studies provide the most extensive normative data on vitamin B6 status available on men in the adult years of life.

Hypnosis and evidence: help or hindrance?

Clarifying misconceptions about hypnosis can reduce confusion about the place of hypnosis in forensic medicine. Hypnosis identifies a person's capacity for attentive, receptive concentration with parallel awareness. While in trance concentration, memory recall under interrogation should not only be subject to all the usual investigative safeguards with checks and balances, but even more so because the leverage effect of hypnotically enhanced memory is achieved at the risk of contamination by external and/or internal cues. This Janus-like feature enables incredibly accurate revivification and recall of perceived events but can also evoke false memories, false confessions, and the "honest liar syndrome." The internal and external factors that account for these contradictory possibilities--and the appropriate safeguards--are considered with case illustrations. In addition, a new use of trance capacity assessment contributes to clarifying diagnosis and the mental defect/disease issue. Knowledge of the uses and limits of hypnosis by the interrogating professionals enhance the judicious process of eliciting information and evidence.

Bactericidal effect of extracorporeal shock waves on Staphylococcus aureus.

Despite considerable knowledge about the effects of shock waves on eukaryotic soft tissues, no data are available concerning their effect on prokaryotic micro-organisms. In vitro studies on the bactericidal effect of extracorporeal shock waves on staphylococci were performed with energy levels that are standard for the disintegration of calculi. Suspensions containing 10(4)-10(5) cfu of Staphylococcus aureus/ml were sealed in plastic tubes and exposed to shock waves, resulting in a mean decrease of 3.1 log(10). Whereas impulse rates of > or =350 resulted in a decrease of cfu/ml equalling the detection limit, lower numbers of impulses did not result in an appreciable bactericidal effect. The bactericidal effect of extracorporeal shock waves might provide the basis for the development of novel therapeutic strategies for bacterial infections.

Callus formation and fixation rigidity: a fracture model in rats.

To produce different amounts of callus in rats, we devised a procedure to fix leg fractures using intramedullary nails that differ in bending rigidity. We inserted a silicone cannula into the intact diaphysis of the tibia of rats that had already been killed and then fractured the tibia by a three-point bending technique. The fracture was stabilised by insertion of either a stainless-steel or polypropylene nail into the silicone cannula. Biomechanical testing showed that the initial stiffness of the fractures differed between the two nail types by a factor of 16. In vivo, 16 Wistar rats were operated on by the same technique to study the formation of callus. Four weeks after fracture, the size of the callus differed significantly between the steel-nailed and polypropylene-nailed fractures. In conclusion, mechanically differing internal fixation devices led to different callus responses in rats when all other factors were kept equal.

Achilles tendon healing: long-term biomechanical effects of postoperative mobilization and immobilization in a new mouse model.

The aim of the study was to investigate the long-term effects of postoperative immobilization as opposed to mobilization on the biomechanical attributes of healing Achilles tendons in a new experimental mouse model. In 114 Balb-C-mice the left Achilles tendon was transected and sutured by the Kirchmayr-Kessler technique. The tendons healed either under postoperative immobilization effected by fixing the upper ankle joint in equinus position or under mobilization through a limited range of movement. The contralateral Achilles tendons served as internal control. All tendons were tested biomechanically at short intervals up to the 112th postoperative day in terms of load to failure [N], tendon deflection [mm] and tendon stiffness [N/mm], and were evaluated histologically after 8 and 112 days. Postoperative mobilization resulted in a continuous and significantly more rapid restoration of load to failure in comparison to the immobilization group. Tendon deflection was decreased by postoperative mobilization, whereas under immobilization it paradoxically increased still further in the later course. After 112 days the tendons of the mobilization group had regained their original tendon stiffness, whereas the tendons after immobilization reached only about half the values seen in the control tendons. Histologically, postoperative mobilization led to increased immigration of inflammatory cells in the early phase. In the late phase, as compared to immobilization, tendon structure was more mature, with fibre bundles arranged in parallel and interposed tendocytes. Tensile loading of the healing tendon by postoperative mobilization leads to fundamental changes in the biological process of tendon healing resulting in accelerated restoration of load to failure and reduced tendon deflection.

Alteration of fracture stability influences chondrogenesis, osteogenesis and immigration of macrophages.

Mechanical conditions at the fracture line determine the mode of fracture healing (osteonal versus non-osteonal bone union). The aim of this study was to investigate the influence of differing degrees of fracture stability on the time course of chondrogenesis, enchondral ossification and immigration of macrophages into the fracture callus. Using a fracture model of the rat's tibia, histological (Azan staining), immunohistological (antibodies directed against the macrophage-specific surface antigen ED2), and molecular biological techniques (expression of the mRNA of the cartilage-specific collagen IX, osteocalcin - a marker for mature osteoblasts - and the macrophage-specific macrosialin) were employed. In terms of histology and molecular biology (collagen IX mRNA expression) chondrogenesis in the fracture gap continued for longer in less stable fractures. In more stable fractures bone formation - identified by osteocalcin mRNA expression - increased from day 12 onwards. The expression of the macrophage-specific surface antigen ED2 and the mRNA of macrosialin was more pronounced but of shorter duration in the more stable fractures. This study shows that differing degrees of fracture stability not only influence the interplay between osteogenesis and chondrogenesis but also alter the kinetics of macrophage immigration into the fracture callus. These findings could aid in better understanding the cytobiologic mechanisms of callus formation and may suggest that macrophages are an important factor not only in soft tissue healing but also in bone healing.