Severe bleeding complications and multiple kidney transplants in a patient with tuberous sclerosis complex caused by a novel TSC2 missense variant.

We presented an extremely severe case of tuberous sclerosis complex (TSC) in a female patient with recurring, life-threatening bleeding complications related to renal angiomyolipomas. Massive intratumoral hemorrhage required surgical removal of both angiomyolipomatous kidneys and kidney transplantation. During the follow-up period, the patient developed severe metrorrhagia that eventually led to hysterectomy and salpingo-oophorectomy. Bleeding from the operative sites caused the loss of the first kidney transplant received from the mother, and immediate hemorrhagic shock led to the loss of the second, cadaveric kidney allograft. The third kidney transplant had a successful outcome. Pathological analysis of all tissue specimens showed TSC-associated lesions and deformed blood vessels in the surgically removed organs. Molecular genetic analysis of TSC1 and TSC2 in the DNA of peripheral leukocytes identified a novel TSC2 c.3599G>C (p.R1200P) variant. Functional assessment confirmed the likely pathogenicity of the TSC2 c.3599G>C (p.R1200P) variant. To the best of our knowledge, this is the first report of the c.3599G>C (p.R1200P) variant in exon 29 of the TSC2 gene related to a severe clinical course and multiple kidney transplants in a patient with TSC.

Different Ecological Niches of Poisonous Aristolochia clematitis in Central and Marginal Distribution Ranges-Another Contribution to a Better Understanding of Balkan Endemic Nephropathy.

Aristolochia clematitis L. is a perennial herbaceous plant distributed throughout Europe, Asia Minor and Caucasus. It has been used as a medicinal plant since antiquity but not in recent times because it contains poisonous aristolochic acid, causing progressive kidney failure. The aim of this work was to study Aristolochia clematitis ecology on the basis of vegetation plots from the European Vegetation Archive, and to investigate the differentiation of its ecological niche using a co-occurrence-based measure of ecological specialization (ESI). The ecological niche was studied on three spatial scales: on the entire distribution area, its differentiation across 200 × 200 km grid cells and the differences between three central and three marginal regions. Our results suggest that Aristolochia clematitis has a very broad ecological niche occurring in a range of different habitats and climatic conditions, with a trend of a niche width decrease with the distance from the geographical center. The plant prefers more stable communities with less anthropogenic influence moving towards the margin of the distribution area. Specialization towards the marginal area is a result of evolutionary history, which refers to the recent anthropogenically induced spread from its original home range. A high incidence of Aristolochia clematitis in the vegetation of arable lands and market gardens as well as anthropogenic herbaceous vegetation in the distribution center corresponds to the geographical incidence of Balkan Endemic Nephropathy.

Comparison between external locking plate fixation and conventional external fixation for extraarticular proximal tibial fractures: a finite element analysis.

BACKGROUND: Good clinical outcomes for locking plates as an external fixator to treat tibial fractures have been reported. However, external locking plate fixation is still generally rarely performed. This study aimed to compare the stability of an external locking plate fixator with that of a conventional external fixator for extraarticular proximal tibial fractures using finite element analysis. METHODS: Three models were constructed: (1) external locking plate fixation of proximal tibial fracture with lateral proximal tibial locking plate and 5-mm screws (ELP), (2) conventional external fixation of proximal tibial fracture with an 11-mm rod and 5-mm Schanz screws (EF-11), and (3) conventional external fixation of a proximal tibial fracture with a 7-mm rod and 5-mm Schanz screws (EF-7). The stress distribution, displacement at the fracture gap, and stiffness of the three finite element models at 30-, 40-, 50-, and 60-mm plate-rod offsets from the lateral surface of the lateral condyle of the tibia were determined. RESULTS: The conventional external fixator showed higher stiffness than the external locking plate fixator. In all models, the stiffness decreased as the distance of the plate-rod from the bone surface increased. The maximum stiffness was 121.06 N/mm in the EF-11 model with 30-mm tibia-rod offset. In the EF-7 model group, the maximum stiffness was 40.00 N/mm in the model with 30-mm tibia-rod offset. In the ELP model group, the maximum stiffness was 35.79 N/mm in the model with 30-mm tibia-plate offset. CONCLUSIONS: Finite element analysis indicated that external locking plate fixation is more flexible than conventional external fixation and can influence secondary bone healing. External locking plate fixation requires the placement of the plate as close as possible to the skin, which allows for a low-profile design because the increased distance from the plate to the bone can be too flexible for bone healing. Further experimental mechanical model tests are necessary to validate these finite element models, and further biological analysis is necessary to evaluate the effect of external locking plate fixation on fracture healing.

Kidney Transplants from Elderly Donors: The Experience of a Reference Center in Croatia.

OBJECTIVES: Our country Croatia is among the global leaders regarding deceased donation rates, yet we are facing organ shortage and concurrently a sharp decline in our acceptance rates for kidney offers. To reevaluate our organ acceptance policy, we retrospectively analyzed the factors that influenced the posttransplant outcomes of kidneys from elderly deceased donors at our center during a 20-year period and the changes to our organ acceptance criteria during Eurotransplant membership. MATERIALS AND METHODS: We studied all kidney transplants from donors ≥60 years old during the two 5-year episodes of Eurotransplant membership from 2007 to 2017 (period II and period III) and compared those data to data from the decade before Eurotransplant membership (period I, 1997-2007). Differences in acceptance rates and reasons for the decline of kidney offers between the two 5-year periods of Eurotransplant membership were analyzed. RESULTS: In period I, 14.1% of all kidney allografts were obtained from donors ≥60 years old; in period II and period III the rates were nearly 2-fold higher (27.0% and 25.7%, respectively; P = .007 and P = .008). During the first 5-year period of Eurotransplant membership (period II), we accepted significantly more grafts from marginal donors with a higher number of human leukocyte antigen mismatches compared with period I. Consequently, the 3-month survival rate of kidneys from donors ≥60 years old dropped from 91.1% to as low as 74.2% (P = .034). After application of morestringent human leukocyte antigen matching, especially in human leukocyte antigen DR, and morestringent donor acceptance criteria in period III, graft survival improved to 91.1%. CONCLUSIONS: Our experience indicates that careful selection of kidneys from elderly deceased donors and allocation to human leukocyte antigen-matched recipients is important to improve transplant outcomes.

The role of the spectral domain ocular coherence tomography in detection of age-related macular degeneration.

Age-related macular degeneration (ARMD) is one of the most common causes of the vision loss and blindness in developed countries. Among other harmful effects, exposure to the UV radiation is the most prominent factor for the development of the disorder. Using the method of SD OCT (Spectral Domain Ocular Coherence Tomography) we performed measurement of the neurosensory retinal thickness of 19 eyes of low vision patients from the population of Primorsko-Goranska County of Republic of Croatia, with dry form of the terminal macular degeneration. These results we compared with control measurements performed on 28 eyes of healthy, normal vision subjects from same County. We determined following parameters: central foveal thickness (CFT), macular volume (MV) and mean foveal thickness (MFT) in the both groups. Results showed statistically significant reduction of CFT in the group of normal vision female patients when compared to males, while any significant difference of CFT between total groups of normal vision individuals and low vision patients was not detected. Furthermore, we noticed statistically significant (p < 0.000001) decrease of the MV in the group of the low vision patients in comparison to healthy subjects and statistically significant (p < 0.000001) reduction of the MFT of the low vision patients when compared to normal vision individuals. In our study we detected the absence of any significant difference of the CFT between healthy and low vision population, what looks like controversial finding, because neurosensory retina in the ARMD is thin and atrophic, but on the other side it is known that fixation point in low vision patients is translocated from the damaged fovea to extrafoveal region, usually above the fovea, where neurosensory retina is of the normal thickness, but with the less sensitivity. Furthermore, our results suggest possible connection of higher incidence of ARMD with lower CFT in females. Owing to the thicker neurosensory retina in males and better protection, damaging effect of the UV irradiation, which is the proven factor of ARMD development, is smaller. From the evolutionary point of view it is possible that males in all vertebrates have more resistant macula because during the evolutionary process they have spent much more time outside in the sunlight than females.

F. novicida-Infected A. castellanii Does Not Enhance Bacterial Virulence in Mice.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

Thymic alterations induced by partial hepatectomy: Upregulation of glycoprotein 96, CD91 and TLR2 and generation of regulatory T cells.

Glycoprotein 96 (gp96) is an endoplasmic reticulum (ER)-resident heat shock protein. It controls the folding of nascent membrane-spanning and secretory proteins, participates in stress-induced unfolded protein response (UPR) and in pathways leading to proteolysis of damaged proteins through ER-associated degradation pathways and chaperone-mediated autophagy. In addition, gp96 controls the steroid biosynthesis and Ca²âº homeostasis and participates in insulin-IGF/signaling pathways. Besides, owing to its peptide chaperone capacity and ability to interact with antigen-presenting cells, gp96 has been implicated in priming of innate and adaptive immunity. In an attempt to visualize the intensity of ER-stress in thymus and possible participation of gp96 in generation of auto-reactive T cell clones that were detected in regenerating liver, in this study we investigated the dynamics of gp96 expression in partially hepatectomized (pHx) and sham Hx mice. Simultaneously, we detected the thymic expression of receptors responsible for endocytosis of gp96-chaperoned peptides (CD91) and intracellular activation of ER-stress pathways (TLR2), as well as the expression of TGF-ß and the distribution of CD4+CD25+FoxP3+ cells. The data have shown that both pHx and sham Hx induced an accelerated apoptosis and hypoplasia in thymus. Partial Hx induced, however, a higher expression of gp96, the translocation of the CD91, TLR2 and TGF-ß immunostaining from medulla to cortex and an appearance of Treg cells. The data show that pHx triggers in thymus the ER-stress and UPR response and suggest that gp96 participates in the generation of natural Treg cells, which might be involved in the control of liver regeneration in the periphery.

Immune reactivity of renal transplant recipients receiving interleukin-2 receptor antagonists during the early posttransplant period.

PURPOSE: There is a need for methods that would enable monitoring of the effects of immunosuppression on the recipient's immune system to avoid rejection, immunodeficiency-related complications and non-immune toxicities of the drugs used in therapy. METHODS: This prospective trial included thirty patients who underwent renal transplantation in our center. All patients received an interleukin-2 receptor (IL-2R) antagonist in combination with mycophenolate, corticosteroid and calcineurin inhibitor. During the first 6 weeks after transplantation, the anti-CD3-stimulated proliferative response of peripheral blood T lymphocytes (PBTL) was studied by cell cycle analysis. The proportion of PBTL in different phases of the cell cycle and expression of IL-2R were determined by flow cytometry. RESULTS: As an effect of quadruple immunosuppressive therapy including IL-2R antagonists, cell cycle analysis showed an incremental decrease in the proliferative response of PBTL during the first 6 weeks after renal transplantation. A sudden drop in the proportion of IL-2R-positive cells was observed immediately after the first dose of the IL-2R antagonist and a significant antiproliferative effect on PBTL after the second dose. In vitro, IL-2R antagonists showed a dose-dependent inhibition of the anti-CD3-stimulated proliferation of PBTL of healthy blood donors. CONCLUSIONS: Cell cycle analysis of the immune reactivity of renal allograft recipients may represent a valuable tool for the immunological posttransplant follow-up and optimization of the immunosuppressive therapy.

Gabapentin-induced changes of plasma cortisol level and immune status in hysterectomized women.

AIM: We have examined the effects of gabapentin (GBP) on stress-related changes of cortisol and catecholamines in patients who underwent hysterectomy because of uterine fibrinoids. Additionally, we have observed the effect of GBP on the immune status in the acute stress response to surgery. METHODS: Sixty patients scheduled for an abdominal hysterectomy were randomly assigned to the GBP administration 1h before surgery (n=30 pts), or to the placebo group (n=30 pts). Blood samples were collected before and 24h after the surgery. The intensity of pain was assessed by a visual analogue scale (VAS) every 8h at rest. Immunomodulatory effects of GBP were determined by flow cytometry. We followed the total proportion of CD3(+) lymphocytes, CD3(+)CD4(+), CD3(+)CD8(+), CD19(+) B lymphocytes, CD16(+)CD56(+)CD3(-)NK cells and CD16(+)CD56(+)CD3(+) NKT cells before and 24h after hysterectomy. The plasma cortisol and catecholamines concentration was used to estimate the level of the stress response. RESULTS: VAS pain score at rest was significantly lower in the GBP group than in the placebo group (P=0.003). Application of GBP significantly decreased the plasma cortisol level 24h after the operation in comparison to the placebo group (P<0,001). We found significant positive correlation between the VAS pain score and concentration of cortisol in all patients (P=0.025). GBP reduced the concentration of catecholamines (p<0.05). The proportion of CD3(+) (P=0.027) and CD3(+)CD4(+)cells (P=0.006) was significantly lower in the GBP group 24h after operation, while the contribution of CD19(+) (P=0.033) was significantly higher. CONCLUSION: Preoperative administration of GBP reduced the pain scores at rest in patients at 0, 16 and 24h after abdominal hysterectomy. Additionally, GBP reduced the stress response and changed immune parameters in the reaction to surgery.

Predicting carotid restenosis by comparison of plaque MCP-1 mRNA expression and serum levels.

As cardiovascular pathology grows in numbers, research into the discovery of new chemokine biomarkers should not be neglected, as they seem to be paramount in atherosclerosis prevention and its early detection. Chemokines attract and activate leukocytes and are well recognized in the environment of inflammatory response. MCP-1 is a valuable chemokine whose potential to become a new crucial atherosclerosis marker is surely worth investigating. Since quantities of MCP-1 found in lesions are as low as immeasurable, we propose the use of an immunohistochemical method for the quantification of MCP-1 levels in atherosclerotic lesions. Additionally, serum levels of MCP-1 can be measured by commercially available immunoassays. Proposed MCP-1 concentration increase may explain the acceleration in lesion's atherosclerosis progression as chemokine activation occurs once they bind to specific ligands. If proven, this hypothesis would indicate the need for further studies in order to objectively link the increased MCP-1 expression with carotid restenosis.

Heat shock protein Gp96 as potential regulator of morphostasis after partial hepatectomy in mice.

Gp96 (also known as glucose-regulated protein 94, endoplasmin) is the endoplasmic reticulum (ER)-resident protein, which belongs to the heat shock protein HSP90 family. It is upregulated in response to glucose starvation and other stressful stimuli that disrupt protein synthesis in the ER. There, it is acting as a molecular chaperon involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of protein translations that modulate the polypeptide traffic into the ER. In addition, it has been implicated in antigen presentation and MHC class I and II upregulation, in the activation and maturation of dendritic cells and proinflammatory cytokine secretion, as well as in chaperoning of integrins and Toll-like receptors, acting as a "danger signal" to the innate and adaptive immunity. Moreover, owing to its specific function in Ca2+ homeostasis and in the insulin- IGF/signaling pathways, it has been proposed that gp96 might participate in mechanisms that are critical for cell growth, differentiation, and responses to ER stress. Emphasizing that gp96, as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, may also be involved in the maintenance of morphostasis and self tolerance, in this survey we show that high levels of upregulation of gp96 in regenerating liver and thymus are followed by signs of transient autoimmunity, augmented apoptosis in thymus, and the presence of autoreactive NKT and regulatory T cells that might be involved in the control of rapid liver growth induced by partial hepatectomy.

Heat shock protein-GP96 as an innate sensor of damage and activator of autoreactive NKT and regulatory T cells during liver regeneration.

Tissue disintegration after injury leads, in the endoplasmic reticulum (ER), to activation of adaptive pathways known as the ER stress response. It is directed to the correction of unfolded proteins and to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, but induces also a rapid activation of natural and adaptive immunity, since a ER resident heat shock protein-gp96 acts not only as a molecular chaperone, but also as a strong adjuvant, able to cross-present the antigenic peptides onto MHC class I or MHC class II pathways. Analyzing its potential role in processes of normal growth, in mice subjected to 1/3 partial hepatectomy (pHx) we determined the tissue expression of gp96 protein and mRNA in regenerating liver, thymus and spleen, determining simultaneously the phenotypic profile and spontaneous cytotoxic activity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) against NKT- and NK-cells sensitive targets (syngeneic thymocytes and YAC-1) in wild, perforin and FasL deficient mice. The data have shown that pHx induces fast overexpression of gp96 protein and mRNA in hepatocytes, spleen and thymus, with accumulation of CD3intermediate/NK1.1+/CD69+ cells (liver) and Foxp3+CD4+CD25+ cells (liver and thymus). Simultaneously, intrahepatic MNLC acquired the FasL-dependent cytotoxic potential against NKT-sensitive targets and both, intrahepatic and splenic MNLC, acquired the perforin-dependent cytotoxic potential against NK-sensitive targets, implying that during the disturbance of morphostasis gp96 serves as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT and regulatory cells, as well as NK cells. Moreover, cell cycle analysis revealed that G2+M phase of regenerating hepatocytes in PKO mice was translocated from the 1st to the 7th p. o. day, as well as that hepatocytes from FasL deficient mice were arrested in G0/G1 phase.

Early events in the pathogenesis of a murine transfer colitis.

Inflammatory bowel disease (IBD) is a chronic, disabling disease. A dysregulated immune response seems to play a pivotal role in the pathogenesis of this disorder. Here we will review current concepts of the adoptive transfer model of IBD with particular emphasis on early events in disease development. In the adoptive transfer model, the reconstitution of immunoincompetent mice with CD4+ T cells from congenic donor animals leads to severe colitis. We will address the question as to which CD4+ T cell subsets might be involved in the induction, suppression, or regulation of disease, and review data concerning the specificity of their T cell receptor and its putative MHC restriction elements. We will also discuss whether and at what anatomical sites donor T cells could be primed in the recipient.

Dendritic cells pulsed with exogenous hepatitis B surface antigen particles efficiently present epitopes to MHC class I-restricted cytotoxic T cells.

L(d)- and K(b)-binding epitopes processed by murine dendritic cells (DC) pulsed with exogenous, particulate hepatitis B surface antigen (HBsAg) are presented to cytotoxic T lymphocytes (CTL). The specific and dose-dependent induction of IFN-gamma release and cytotoxicity in CTL by metabolically active DC did not depend on antigenic peptides contaminating the particles, was cytochalasin D resistant, independent of the maturation state of DC, and blocked by primaquine, amiloride and NH(4)Cl (indicating involvement of acid proteolysis). The specific immunostimulatory phenotype of pulsed DC was maintained for about 3 h after the end of the pulse but rapidly decayed thereafter. Processing of L(d)- and K(b)-binding epitopes from exogenous HBsAg particles by pulsed DC for presentation was TAP independent. Surface-associated 'empty' (presentation-deficient) 64(+) L(d) molecules (defined by the mAb 64-3-7), but not trimeric (presentation-competent) 30(+) L(d) molecules (defined by the mAb 30-5-7) had to be available during the pulse of DC with exogenous HBsAg particles to generate 30(+) L(d)molecules that present the antigenic S(28-39) peptide. Exogenous beta2-microglobulin present during the pulse of DC with HBsAg particles facilitated presentation of L(d)- and K(b)-restricted epitopes. DC generated from bone marrow progenitors in vitro, as well as splenic and liver DC (generated in vivo) presented epitopes to specific CTL. HBsAg particles thus efficiently enter an alternative processing pathway in DC that leads to presentation of epitopes to MHC class I-restricted CTL.

Colonic lamina propria dendritic cells in mice with CD4+ T cell-induced colitis.

CD11c(+) (F4/80(-) CD68(-)) dendritic cells (DC) in the colonic lamina propria (cLP) of normal and immunodeficient (RAG1(-/-)) C57BL/6 (B6) mice show high surface expression of MHC class I/II molecules and CD1d, and low surface expression of CD40, CD80, CD86 costimulator molecules. CD4(+) alpha beta T cells from normal or MHC class II-deficient B6 mice transferred into congenic RAG1(-/-) hosts induce a progressive, lethal colitis. Concomitant with colitis development, DC in the inflamed cLP increase in number and up-regulate surface expression of CD1d, MHC class II molecules and CD40, CD80, CD86 costimulator molecules. cLP DC from non-transplanted (healthy) and transplanted (diseased) mice produce similar amounts of IL-12 p70 and IL-10 in response to CD40 signaling, but the inducible IL-12 p40 release is 5-15-fold higher in mice with colitis than in non-transplanted mice. Binding of IL-12 p40 to p19 generates IL-23. Freshly isolated cLP lymphocytes (cLPL) from transplanted, diseased mice express 3-10-fold more p19 transcripts than cLPL from non-transplanted, healthy mice. p19 expression by cLPL is further up-regulated in response to CD40 ligation. Freshly isolated cLP DC from transplanted mice with colitis (but not from non-transplanted controls) stimulate IFN-gamma (but not IL-4 or IL-13) release by co-cultured NKT cells. Incolitis, DC accumulate in the cLP, show an activated surface phenotype, up-regulate IL-12 p40 and p19 expression, and 'spontaneously' stimulate NKT-like cells. cLP DC may be interesting targets for novel therapeutic approaches to modulate mucosal T cell responses in situ.

Activating immunity in the liver. II. IFN-beta attenuates NK cell-dependent liver injury triggered by liver NKT cell activation.

Dendritic cell (DC)-dependent activation of liver NKT cells triggered by a single i.v. injection of a low dose (10-100 ng/mouse) of alpha-galactosyl ceramide (alphaGalCer) into mice induces liver injury. This response is particularly evident in HBs-tg B6 mice that express a transgene-encoded hepatitis B surface Ag in the liver. Liver injury following alphaGalCer injection is suppressed in mice depleted of NK cells, indicating that NK cells play a role in NK T cell-initiated liver injury. In vitro, liver NKT cells provide a CD80/86-dependent signal to alphaGalCer-pulsed liver DC to release IL-12 p70 that stimulates the IFN-gamma response of NKT and NK cells. Adoptive transfer of NKT cell-activated liver DC into the liver of nontreated, normal (immunocompetent), or immunodeficient (RAG(-/-) or HBs-tg/RAG(-/-)) hosts via the portal vein elicited IFN-gamma responses of liver NK cells in situ. IFN-beta down-regulates the pathogenic IL-12/IFN-gamma cytokine cascade triggered by NKT cell/DC/NK cell interactions in the liver. Pretreating liver DC in vitro with IFN-beta suppressed their IL-12 (but not IL-10) release in response to CD40 ligation or specific (alphaGalCer-dependent) interaction with liver NKT cells and down-regulated the IFN-gamma response of the specifically activated liver NKT cells. In vivo, IFN-beta attenuated the NKT cell-triggered induction of liver immunopathology. This study identifies interacting subsets of the hepatic innate immune system (and cytokines that up- and down-regulate these interactions) activated early in immune-mediated liver pathology.