Molecular markers and targeted therapy of skin rejection in composite tissue allotransplantation.

Skin rejection remains a major hurdle in reconstructive transplantation. We investigated molecular markers of skin rejection with particular attention to lymphocyte trafficking. Skin biopsies (n = 174) from five human hand transplant recipients were analyzed for rejection, characteristics of the infiltrate and lymphocytic adhesion markers. The cellular infiltrate predominantly comprised CD3+ T cells. CD68, Foxp3 and indoleamine 2, 3-dioxygenase expression and the CD4/CD8 increased with severity of rejection. Lymphocyte adhesion markers were upregulated upon rejection, intercellular adhesion molecule-1 and E-selectin correlated best with severity of rejection. Guided by the findings, a specific E- and P-selectin inhibitor was investigated for its effect on skin rejection in a rat hind limb allotransplant model. While efomycine M (weekly s.c. injection into the graft) alone had no effect, long-term allograft survival was achieved when combined with antithymocyte globulin and tacrolimus (control group without efomycine M rejected at postoperative day [POD] 61 +/- 1). Upregulation of lymphocyte trafficking markers correlates with severity of skin rejection and time after transplantation in human hand transplantation. Blocking E- and P-selectin in the skin holds potential to significantly prolong limb allograft survival.

Alemtuzumab: key for minimization of maintenance immunosuppression in reconstructive transplantation?

Minimization of immunosuppression has become the key effort in solid organ transplantation. Alemtuzumab, the humanized CD-52 monoclonal antibody, is an effective depleting agent increasingly used in transplantation trials. In this article, we summarize the current experience with alemtuzumab use in hand transplantation and discuss its role in current and future approaches toward minimization of maintenance immunosuppression in reconstructive transplantation.

The Banff 2007 working classification of skin-containing composite tissue allograft pathology.

Composite tissue allotransplantation (CTA) is a recently introduced option for limb replacement and reconstruction of tissue defects. As with other allografts, CTA can undergo immune-mediated rejection; therefore standardized criteria are required for characterizing and reporting severity and types of rejection. This article documents the conclusions of a symposium on CTA rejection held at the Ninth Banff Conference on Allograft Pathology in La-Coruna, Spain, on 26 June 2007, and proposes a working classification, the Banff CTA-07, for the categorization of CTA rejection. This classification was derived from a consensus discussion session attended by the first authors of three published classification systems, pathologists and researchers from international centers where clinical CTA has been performed. It was open to all attendees to the Banff conference. To the extent possible, the format followed the established National Institutes of Health (NIH) guidelines on Consensus Development Programs. By consensus, the defining features to diagnose acute skin rejection include inflammatory cell infiltration with involvement of epidermis and/or adnexal structures, epithelial apoptosis, dyskeratosis and necrosis. Five grades of severity of rejection are defined. This classification refines proposed schemas, represents international consensus on this topic, and establishes a working collective classification system for CTA reporting of rejection in skin-containing CTAs.

Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type 1 diabetes.

Immunoprecipitating IgG autoantibodies to glutamic acid decarboxylase, GAD65, and/or a tyrosine phosphatase, IA2, are present in the majority of individuals experiencing pancreatic beta cell destruction and development of type 1 diabetes. Here we identify a third islet cell autoantigen, a novel 38-kD protein, which is specifically immunoprecipitated with sera from a subset of prediabetic individuals and newly diagnosed type 1 diabetic patients. The 38-kD autoantigen, named glima 38, is an amphiphilic membrane glycoprotein, specifically expressed in islet and neuronal cell lines, and thus shares the neuroendocrine expression patterns of GAD65 and IA2. Removal of N-linked carbohydrates results in a protein of 22,000 Mr. Glima 38 autoantibodies were detected in 16/86 (19%) of newly diagnosed patients, including three very young children, who had a rapid onset of disease, and in 6/44 (14%) of prediabetic individuals up to several years before clinical onset. The cumulative incidence of GAD65 and glima 38 antibodies in these two groups was 83 and 80%, respectively, and the cumulative incidence of GAD65, glima 38, and IA2 antibodies in the same groups was 91 and 84%, respectively. GAD65, IA2, and glima 38 represent three distinct targets of immunoprecipitating IgG autoantibodies associated with beta cell destruction and type 1 diabetes.

The effect of soybean soapstock on the quality parameters and fatty acid composition of the hen egg yolk.

The effects of increasing proportions of soybean soapstock (SSS) in laying hen diets on egg quality parameters and on the fatty acid (FA) composition of the yolk were studied. One hundred sixty Babcock B-300 x laying hens, 20 wk of age, were allotted to 5 dietary treatments comprising a control diet (D5) with soybean oil at 100% or SSS in proportions of 25% (D1), 50% (D2), 75% (D3), and 100% (D4) replacing the oil source in commercial-type diets throughout the 15-wk laying period. Egg quality parameters were recorded weekly. Four pooled yolks for each treatment were collected at 0, 9, and 15 wk, and their FA profiles were determined. Egg weight, shell thickness, shape index, and Haugh unit were not influenced by dietary treatment. The concentrations of saturated FA (SFA) in yolks were generally kept at a constant level, regardless of the percentage of SSS in the hen diets. Total monounsaturated FA (MUFA) content did not vary over the course of the experiment, except for C14:1. After 15 wk of feeding, the total polyunsaturated FA (PUFA) n-3 increased in yolks of all diets. Among the long-chain PUFA, the percentage of C20:4 (arachidonic acid) was less affected. Inclusion of 100% SSS allowed production of eggs with similar PUFA/SFA and n-6/n-3 ratios when compared with eggs from hens fed D5. As SSS represents one-third of the oil cost, its inclusion in layer diets could represent an important economic benefit.

A syndrome of gonadotropin resistance possibly due to a luteinizing hormone receptor defect.

An 18-yr-old 46,XY man with primary hypogonadism and a microphallus is described whose Leydig cells appear to be partially insensitive to gonadotropin action. The external genitalia were well differentiated though abnormally small. The mean +/- SE baseline plasma testosterone (T) level was 62 +/- 3.9 ng/dl, and androstenedione was 34.5 +/- 7.3 ng/dl. Plasma levels of dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-hydroxyprogesterone, 17-hydroxypregnenolone, corticosterone, deoxycorticosterone, and 17 beta-estradiol were all normal. After the im administration of hCG, plasma T increased insignificantly from 71 to 78 ng/dl, and androstenedione increased from 22 to 47 ng/dl; there was no significant change in the levels of precursor steroids. The mean +/- SE serum FSH level was 17.4 +/- 3.6 mIU/ml, and LH was 15.4 +/- 1.1 mIU/ml (normal, 5-20); both responded briskly to iv GnRH. Exogenous T therapy resulted in normal virilization, whereas therapy with hCG was ineffectual. Testicular biopsy revealed Leydig cells in normal numbers, some spermatogenesis, and thickened tubular basement membranes. In vitro binding studies using [125I]hCG were performed with testicular homogenates from the patient and three normal subjects. With 7.4 fmol labeled hCG, the specific binding (mean +/- SD), expressed as femtomoles of hCG per mg protein, was 1.16 +/- 0.44 compared to 2.49 +/- 0.41 in normal subjects (P less than 0.05). These data demonstrate partial resistance to hCG and suggest that the defect in Leydig cell function may be at the LH receptor or postreceptor level.

Inhibition of nitric oxide synthesis improves the vasoconstrictive effect of noradrenaline in sepsis.

BACKGROUND: Septic shock is characterized by systemic vasodilation and an impaired reactivity to vasoconstrictor agents. It has been suggested that an excessive release of nitric oxide has a role in this hemodynamic derangement. OBJECTIVE: To investigate whether inhibition of nitric oxide synthesis by the administration of N omega-nitro-L-arginine (LNNA), improves the vasoconstrictor effects of catecholamines in sepsis. MATERIAL AND METHODS: Mechanically ventilated and pentobarbital-anesthetized sheep received either no treatment (n = 6) or LNNA (100 mg/kg IV bolus, n = 4). Other sheep (septic group) received live Escherichia coli (E coli) (1,5* 10(9) micro-organisms/kg over 30 min) followed 1 hour later by either no treatment (n = 5) or LNNA (100 mg/kg IV bolus, n = 7). After those interventions, all sheep were given noradrenaline in a continuous IV infusion at three different doses (0.5, 1.5, and 4.5 micrograms, kg-1, min-1). Cardiovascular parameters were recorded at maximal blood pressure response achieved with each dose. RESULTS: The administration of live E coli to the septic group resulted in systemic hypotension, high cardiac output, and hyperlactatemia. The LNNA caused a significant systemic and pulmonary vasoconstriction in both septic and nonseptic sheep. In nonseptic sheep, noradrenaline induced a significant increase in systemic vascular resistance (from 2,973 +/- 637 to 4,561 +/- 1,287 dyn/s/cm-5/m-2), whereas the increase caused in those that received LNNA was nonsignificant (5,562 +/- 3,489 to 6,693 +/- 2,871 dyn, s, cm-5, m-2). Septic sheep showed a nonsignificant vasoconstriction during the infusion of noradrenaline (from 1,438 +/- 1,132 to 2,244 +/- 1,391 dyn/s/cm-5/m-2). However, treatment with LNNA markedly improved the vasoconstrictor effect of noradrenaline (from 2,804 +/- 2,317 to 4,894 +/- 3,435 dyn/s/cm-5/m-2). The dose-response curve of systemic vascular resistance in these LNNA-pretreated septic sheep became very similar to the corresponding curve obtained in nonseptic animals. CONCLUSIONS: Inhibition of nitric oxide synthesis by the administration of LNNA significantly improves the vasoconstrictor effect of noradrenaline in septic sheep, allowing an increase in systemic vasomotor tone similar to that observed in nonseptic sheep. It is concluded that increased synthesis of nitric oxide contributes to the depressed vascular reactivity to vasoconstrictor agents characteristic of sepsis.

Time course of hemostatic abnormalities in sepsis and its relation to outcome.

OBJECTIVES: To investigate the time course and the relation to prognosis of coagulation and fibrinolytic abnormalities in patients with septic shock. PATIENTS AND METHODS: Forty-eight consecutive patients admitted to the medical ICU with the diagnosis of septic shock (diagnosed by defined criteria) were studied. Mortality was 25 of 48. Mean age was 57 +/- 7.3 years. Blood samples were obtained on days 1, 4, and 7 after hospital admission to measure tissue-type plasminogen activator antigen (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor antigen (PAI-1), plasminogen, alpha 2-antiplasmin, fibrinogen, antithrombin III, protein C, protein S, thrombin-antithrombin complexes (TAT), D-dimer, and von Willebrand factor-related antigen (vWF:Ag). RESULTS: All patients showed marked abnormalities in both the coagulation and fibrinolytic systems. There were signs of coagulation activation and elevation of both activators and inhibitors of fibrinolysis. Nonsurvivors showed lower levels of protein C and antithrombin III and higher concentration of TAT than survivors. While both t-PA and PAI-1 concentrations were high in survivors and nonsurvivors, only survivors showed a progressive normalization of both parameters during the study period. Low plasminogen levels and plasminogen/alpha 2-antiplasmin ratio were found in both groups, presenting a trend toward normalization only in survivors. The differences reported were not apparent at the time of hospital admission. CONCLUSIONS: Septic shock is characterized by coagulation activation and fibrinolysis activation and inhibition. Nonsurvivors present a particular hemostatic profile characterized by a more marked activation of coagulation and a more intense inhibition of fibrinolysis. None of the abnormalities studied was significantly different between survivors and nonsurvivors at the time of hospital admission. In the presence of fibrin formation, nonsurvivors present a maintained imbalance in the fibrinolytic response determined by higher PAI-1 plasma concentration, probably contributing to their poor outcome.

Hemodynamic changes during whole bronchoalveolar lavage in two cases of pulmonary alveolar proteinosis.

Five whole bronchoalveolar lavages were performed in 2 patients with pulmonary alveolar proteinosis with continuous monitoring of mixed venous and arterial oxygen saturation. Hemodynamic parameters and gas-exchange status were measured during the different phases of the lavage. In the phase of filled lung, a significant increase of arterial partial pressure (PaO2) and arterial saturation of oxygen were observed, secondary to a decrease in the intrapulmonary shunt. The mean pulmonary arterial pressure, pulmonary vascular resistances and cardiac index were higher during the filling of the lung as compared to the controls. During the empty lung phase, although PaO2 decreased (without reaching statistical significance), due to an increase in the intrapulmonary shunt, the increase in cardiac output during this phase left the oxygen delivery (DO2) unchanged.