Release of pig leukocytes and reduced human NK cell recruitment during ex vivo perfusion of HLA‐E/human CD46 double‐transgenic pig limbs with human blood

Background

In pig‐to‐human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non‐classical human leukocyte antigen‐E (HLA‐E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood.

Methods

Six pig forelimbs per group, respectively, stemming from either wild‐type (wt) or HLA‐E/hCD46 double‐transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12 hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells.

Results

Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60 minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10 minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12 hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA‐E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells.

Conclusions

Ex vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell‐mediated rejection mechanisms of vascularized pig‐to‐human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA‐E/hCD46 in pig limbs provides partial protection from human NK cell‐mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.     

NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells

The phenotype of NFATc2(-/-) c3(-/-) (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4(+) CD25(+) T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and CD25. However, neither wild-type nor DKO CD4(+) CD25(+) regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4(+) CD25(-) T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4(+) CD25(+) T reg cells but renders conventional CD4(+) T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.     

Human Pharmacology of Cefotaxime (HR 756), a New Cephalosporin

Cefotaxime (HR 756) is a new semisynthetic parenteral cephalosporin with exceptional activity against gram-negative organisms and considerable stability against their beta-lactamases. To study its pharmacokinetic properties, 0.5-, 1-, and 2-g doses were administered to each of six volunteers intravenously over 15 min, followed by a sustaining infusions of 0.5, 1, and 2 g/h, respectively, for 3 consecutive hours. The loading doses produced mean peak levels of 41, 93, and 160 mug/ml, and mean steady-state serum concentrations were 27, 64, and 138 mug/ml, respectively. The mean terminal half-life was 75 +/- 7 min. The total volume of distribution averaged 0.22 +/- 0.03 liters/kg of body weight. Total body and renal clearances were 232 +/- 30 and 145 +/- 24 ml/min per 1.73 m(2), respectively; 63 +/- 9% of the administered dose was excreted through the kidneys in 24 h. To determine the effect of cefotaxime on the renal tubules, urinary alanine aminopeptidase excretion was measured before, during, and after the infusions. It remained within the normal range in all instances; however, 48 +/- 14% of the total daily alanine aminopeptidase output was recovered during the infusion period. Side effects were dose related and included fatigue, loose stools, and night sweats. No significant changes in hematology, serum chemistry, or urinalysis were recorded.     

Tartrate-resistant acid phosphatase 5b and C-terminal telopeptides of type I collagen as markers for diagnosis of aseptic loosening after total hip replacement

Introduction  

Plain radiography, bone scintigraphy, digital subtraction arthrography and various other techniques can be used to evaluate loosening of hip replacements. These methods are associated with radiation exposure and some of them have an increased morbidity. Furthermore, in some cases the results are not conclusive.     

Influences of heat seal lacquer thickness on the quality of blister packages

A sealability of aluminium lidding foils against formable polymer materials of blister packages is usually achieved by a coating of aluminium with certain grammages of heat seal lacquers. To investigate influences of their thickness on quality of blister packages, lidding foils with different grammages of two lacquer types were manufactured. Sealing experiments (variation of temperature, pressure and sealing time) were performed. Sealed seam strengths were determined with mechanical tensile tests, tightness of cold form blisters were analysed by means of helium leakage tests. Time-dependent moisture uptake of stored blisters was monitored with micro-gas chromatography. By means of a simple calculation model the permeability coefficients of the heat seal lacquers were determined. Lidding foils with higher lacquer grammages showed significantly greater sealed seam strengths. Helium leakage tests showed only slight effects of heat seal lacquer grammage on tightness of blisters. But cold form blisters with lidding foils of higher lacquer grammages showed a significantly greater moisture uptake. Since the heat seal lacquers and the rigid polyvinyl chloride of the formable aluminium compound foils had similar permeability coefficients, the contribution of the lacquers to the total permeability of the investigated cold form blisters was only slightly.     

Lp(a) lipoprotein and plasminogen activity in patients with different etiology of ischemic stroke

BACKGROUND AND PURPOSE: Lp(a) lipoprotein plays an important part in atherothrombogenesis and is considered an independent risk factor for coronary heart disease. However, its role in cerebrovascular disease remains unclear, in particular because of the heterogeneous nature of strokes. We investigated whether elevated Lp(a) is more frequent in ischemic stroke related to atherothrombosis than in other etiologies of stroke. Because of the close structural homology between Lp(a) and plasminogen, we also studied the role of plasminogen in different stroke subtypes and whether there is a dependency on Lp(a) plasma levels. METHODS: Lp(a) levels and plasminogen activity were measured in 253 consecutive patients with acute ischemic stroke and in 63 controls (CS). Subtypes of stroke were established according to the TOAST criteria. RESULTS: Median Lp(a) levels were found to be higher in the total cerebral infarction group and in patients with large artery atherosclerosis (LAA) when compared with CS (20.9 and 22.0 mg/dl, respectively, vs. 16.0 mg/dl; p < 0.05). In addition, elevated Lp(a) levels >30 mg/dl were more frequent among the LAA subgroup than among CS (39.4 vs. 11.1%; p < 0.001). Mean plasminogen activity was lower in the total cerebral infarction group (110.8 vs. 120.3%; p < 0.001) and in patients with cardioembolic stroke (109.8 vs. 120.3%; p < 0.05) when compared with CS. There was no correlation between Lp(a) levels and plasminogen activity. CONCLUSIONS: Our results support the hypothesis that elevated Lp(a) is a risk factor for ischemic stroke and especially for strokes caused by LAA. Low plasminogen activity may play a role in the pathogenesis of cerebrovascular disease, especially for the development of cardioembolic stroke.     

Mast cells are crucial for early inflammation, migration of Langerhans cells, and CTL responses following topical application of TLR7 ligand in mice

Until recently, IgE-activated mast cells have been regarded merely as effector cells of adaptive immune responses, involved in allergic reactions and mucosal immunity to parasites. Herein, we report that murine dermal mast cells, activated by local administration of a cream containing the synthetic TLR7 ligand imiquimod, are essential to initiate an early inflammatory reaction. The mast-cell-derived cytokines TNF-alpha and IL-1beta play an important role in this process. Furthermore, TLR7-activated mast cells are also able to promote the emigration of Langerhans cells, which partly depends on the expression of mast-cell-derived IL-1beta. We have previously shown that TLR7 ligation enhances transcutaneous immunization evoked by topical application of vaccine antigens to the skin, a procedure that directly targets skin-resident antigen-presenting cells. Consequently, we now demonstrate here that the capacity to mount a peptide-specific cytotoxic T-lymphocyte response following transcutaneous immunization using imiquimod as adjuvant is severely impaired in mast-cell-deficient mice. Thus, these findings demonstrate the potent versatility of alternatively activated mast cells at the interface of innate and adaptive immunity.     

Endorectal MRI assessment of local relapse after surgery for prostate cancer: A model to define treatment field guidelines for adjuvant radiotherapy in patients at high risk for local failure

PURPOSE: To assess the role of endorectal magnetic resonance imaging (MRI) in defining local relapse after radical prostatectomy for prostate cancer to help to reassess the clinical target volume (CTV) for adjuvant postprostatectomy radiotherapy. METHODS AND MATERIALS: Sixty patients undergoing an endorectal MRI before salvage radiotherapy were selected. Spatial coordinates of the relapses were assessed using two reference points: the inferior border of the pubic symphysis (point 1) and the urethro-vesical anastomosis (point 2). Every lesion on MRI was delineated on the planning computed tomography and center of mass coordinates were plotted in two separate diagrams (along the x, y, and z axes) with the urethro-vesical anastomosis as the coordinate origin. An "ideal" CTV was constructed, centered at a point defined by the mathematical means of each of the three coordinates with dimensions defined as twice 2 standard deviations in each of the three axes. The dosimetric impact of the new CTV definition was evaluated in six adjuvantly treated patients. RESULTS: The ideal CTV center of mass was located at coordinates 0 (x), -5 (y), and -3 (z) mm with SDs of 6 (x), 6 (y), and 9 (z) mm, respectively. The CTV size was 24 (x) x 24 (y) x 36 (z) mm. Significant rectal sparing was observed with the new CTV. CONCLUSIONS: A CTV with an approximately cylindrical shape (approximately 4 x 3 cm) centered 5 mm posterior and 3 mm inferior to the urethro-vesical anastomosis was defined. Such CTV may reduce the irradiation of normal nontarget tissue in the pelvis potentially improving treatment tolerance.