Elevated Dickkopf‐2 levels contribute to the abnormal phenotype of human osteoarthritic osteoblasts

The Wnt signaling pathway is crucial for osteogenesis and regulates terminal osteoblast differentiation. Although osteoarthritic (OA) osteoblasts show an abnormal phenotype and poor in vitro mineralization, the mechanism leading to this situation still remains unknow. Recent evidence indicates that Wnt signaling may be altered in OA osteoblasts. In this study we determined whether an alteration of the Wnt/β‐catenin signaling pathway is responsible for the abnormal phenotype of OA osteoblasts. Expression of the Wnt signaling antagonist Dickkopf‐1 (DKK1) was similar in normal and OA osteoblasts, whereas DKK2 expression was higher in OA osteoblasts than in normal osteoblasts. OA osteoblasts showed a decrease of Wnt3a‐dependent Wnt/β‐catenin signaling, measured by the TOPflash reporter assay and by Western blot analysis, compared with normal osteoblasts. Correcting DKK2 levels in OA osteoblasts by siRNA techniques enhanced Wnt/β‐catenin signaling. Elevated DKK2 levels could be explained by elevated transforming growth factor β1 (TGF‐β1) in OA osteoblasts, and exogenous TGF‐β1 increased DKK2 expression in normal osteoblasts, whereas ablating TGF‐β1 expression in OA osteoblasts reduced DKK2 expression. Inhibiting TGF‐β1 or DKK2 expression corrected the abnormal phenotype of OA osteoblasts. In vitro mineralization of OA osteoblasts also was increased by DKK2 siRNA. We conclude that elevated TGF‐β1 levels in OA osteoblasts can stimulate DKK2 expression, which, in turn, is responsible, at least in part, for their abnormal phenotype. © 2011 American Society for Bone and Mineral Research.     

Long-term outcomes of cytomegalovirus infection and disease after lung or heart–lung transplantation with a delayed ganciclovir regimen

Abstract: Background: Information is limited on long‐term outcomes after preemptive use of ganciclovir to control cytomegalovirus (CMV) infection in lung transplantation. Methods: We studied 78 lung recipients who received antithymocyte globulin induction from 1994 to 2000. All patients received six months of oral acyclovir (800 mg TID). This was interrupted three wk post transplantation for a two‐wk course of IV ganciclovir. Additional courses of ganciclovir were administered based on serial virological monitoring. CMV‐mismatched patients (R?D+) also received four doses of CMV immunoglobulin between weeks 2 and 8. Results: The one yr cumulative risk of CMV disease was 2% (1/61) in CMV seropositive (R+) patients, but was 37% (6/17) in R?D+ patients (p < 0.0001). Over 4.3 yr of follow‐up, patients with CMV infection developed more chronic graft dysfunction caused by bronchiolitis obliterans or bronchiolitis obliterans syndrome than patients without CMV infection (p = 0.012). This effect was also apparent in the subgroup of R+ recipients (p = 0.043). Acute rejection and overall survival were not associated with CMV infection. Conclusions: The use of prophylactic acyclovir and short preemptive courses of ganciclovir effectively controlled CMV disease in R+ patients, but was a relative failure in R?D+ patients. CMV infection was significantly associated with chronic graft dysfunction, even in R+ recipients who had good control of CMV symptoms.     

Bone mass is inversely proportional to Dkk1 levels in mice

The Wnt/beta-catenin signaling pathway has emerged as a key regulator in bone development and bone homeostasis. Loss-of-function mutations in the Wnt co-receptor LRP5 result in osteoporosis and "activating" mutations in LRP5 result in high bone mass. Dickkopf-1 (DKK1) is a secreted Wnt inhibitor that binds LRP5 and LRP6 during embryonic development, therefore it is expected that a decrease in DKK1 will result in an increase in Wnt activity and a high bone mass phenotype. Dkk1-/- knockout mice are embryonic lethal, but mice with hypomorphic Dkk1d (doubleridge) alleles that express low amounts of Dkk1 are viable. In this study we generated an allelic series by crossing Dkk1+/- and Dkk1+/d mice resulting in the following genotypes with decreasing Dkk1 expression levels: +/+, +/d, +/- and d/-. Using muCT imaging we scanned dissected left femora and calvariae from 8-week-old mice (n=60). We analyzed the distal femur to represent trabecular bone and the femur diaphysis for cortical endochondral bone. A region of the parietal bones was used to analyze intramembranous bone of the calvaria. We found that trabecular bone volume is increased in Dkk1 mutant mice in a manner that is inversely proportional to the level of Dkk1 expression. Trabeculae number and thickness were significantly higher in the low Dkk1 expressing genotypes from both female and male mice. Similar results were found in cortical bone with an increase in cortical thickness and cross sectional area of the femur diaphysis that correlated with lower Dkk1 expression. No consistent differences were found in the calvaria measurements. Our results indicate that the progressive Dkk1 reduction increases trabecular and cortical bone mass and that even a 25% reduction in Dkk1 expression could produce significant increases in trabecular bone volume fraction. Thus DKK1 is a negative regulator of normal bone homeostasis in vivo. Our study suggests that manipulation of DKK1 function or expression may have therapeutic significance for the treatment of low bone mass disorders.     

Cytomegalovirus prevention strategies in seropositive kidney transplant recipients: an insight into current clinical practice

There is notable heterogeneity in the implementation of cytomegalovirus (CMV) prevention practices among CMV‐seropositive (R+) kidney transplant (KT) recipients. In this prospective observational study, we included 387 CMV R+ KT recipients from 25 Spanish centers. Prevention strategies (antiviral prophylaxis or preemptive therapy) were applied according to institutional protocols at each site. The impact on the 12‐month incidence of CMV disease was assessed by Cox regression. Asymptomatic CMV infection, acute rejection, graft function, non‐CMV infection, graft loss, and all‐cause mortality were also analyzed (secondary outcomes). Models were adjusted for a propensity score (PS) analysis for receiving antiviral prophylaxis. Overall, 190 patients (49.1%) received preemptive therapy, 185 (47.8%) antiviral prophylaxis, and 12 (3.1%) no specific intervention. Twelve‐month cumulative incidences of CMV disease and asymptomatic infection were 3.6% and 39.3%, respectively. Patients on prophylaxis had lower incidence of CMV disease [PS‐adjusted HR (aHR): 0.10; 95% confidence interval (CI): 0.01–0.79] and asymptomatic infection (aHR: 0.46; 95% CI: 0.29–0.72) than those managed preemptively, with no significant differences according to the duration of prophylaxis. All cases of CMV disease in the prophylaxis group occurred after prophylaxis discontinuation. There were no differences in any of the secondary outcomes. In conclusion, antiviral prophylaxis was associated with a lower occurrence of CMV disease in CMV R+ KT recipients, although such benefit should be balanced with the risk of late‐onset disease.     

人输卵管Wnt家族部分基因的表达及米非司酮对其表达的影响

目的 了解Wnt基因及其抑制因子在人输卵管上有无表达,并观察其在月经周期中的变化,探讨米非司酮对Wnt基因表达的调节作用.方法 选取育龄妇女的正常输卵管组织共18例,分为3组,即增殖期输卵管组织6例、分泌期输卵管组织6例,以及分泌期服用米非司酮50 mg并于24 h后手术的输卵管6例.应用实时定量聚合酶联反应（PCR）技术检测各组的Wnt-3、Wnt-5a、Wnt-7a、FrpHE和DKK1的mRNA表达;应用蛋白印迹方法检测FrpHE和DKK1的蛋白表达.结果 Wnt-3、Wnt-5a、Wnt-7a、FrpHE和DKK1在正常育龄妇女的增殖期和分泌期输卵管组织中均有表达.增殖期Wnt-3和FrpHE的mRNA表达要显著高于分泌期（P〈0.05）,而分泌期DKK1的mRNA表达则显著高于增殖期 （P〈0.05）,其它基因的表达无显著性差异.FrpHE和DKK1的蛋白表达与其mRNA表达相符.分泌期服用米非司酮的输卵管组织的DKK1的mRNA和蛋白表达均较分泌期未服用米非司酮的输卵管组织明显降低（P〈0.05）,而其余各基因的表达在两组间无显著性差异.结论 在正常育龄妇女的输卵管组织中存在Wnt部分家族成员的表达;它们在月经周期中的不同时期发生了相应改变,提示Wnt信号通路可能受到卵巢性激素的调节作用;孕激素可能是DKK1的重要调控因子.     

Circulating Sclerostin and Dickkopf-1 (DKK1) in Predialysis Chronic Kidney Disease (CKD): Relationship with Bone Density and Arterial Stiffness

Abnormalities of bone metabolism and increased vascular calcification are common in chronic kidney disease (CKD) and important causes of morbidity and mortality. The Wnt signaling pathway may play a role in the bone and vascular disturbances seen in CKD, termed collectively "CKD-MBD." The aim of the study was to investigate the possible association of circulating concentrations of the secreted Wnt signaling inhibitors DKK1 and sclerostin with BMD and arterial stiffness in predialysis CKD. Seventy-seven patients (48 M, 29 F), mean age 57 (SD = 14) years with CKD stages 3B (n = 32) and 4 (n = 45) were studied. Sclerostin, DKK1, PTH, and 1,25(OH)(2)D were analyzed. BMD was measured at the lumbar spine (LS), femoral neck (FN), total hip (TH), and forearm (FARM). Arterial stiffness index was determined by contour analysis of digital volume pulse (SI(DVP)). There was a positive correlation between sclerostin and age (r = 0.47, p < 0.000). Sclerostin was higher in men than women (p = 0.013). Following correction for age and gender, there was a negative association between GFR and sclerostin (p = 0.002). We observed a positive association between sclerostin and BMD at the LS (p = 0.0001), FN (p = 0.004), and TH (p = 0.002). In contrast, DKK1 was negatively associated with BMD at the FN (p = 0.038). A negative association was seen between DKK1 and SI(DVP) (p = 0.027). Our data suggest that the Wnt pathway may play a role in CKD-MBD. Prospective studies are required to establish the clinical relevance of sclerostin and DKK1 as serological markers in CKD.     

Altered Levels of CC Chemokines During Pulmonary CMV Predict BOS and Mortality Post-Lung Transplantation

Pulmonary CMV infection (CMVI) and disease (CMVD) is associated with reduced long-term survival post-lung transplantation, however, the specific biologic mechanisms remain unclear. We have demonstrated a role of CC chemokines during lung allograft dysfunction. Based on these findings, we hypothesized that pulmonary CMV upregulates the expression of multiple CC chemokines that leads to allograft dysfunction and decreased long-term survival.
We performed a nested case control study in lung transplant recipients to investigate alterations in CC chemokine biology during pulmonary CMV. Levels of CC chemokines were measured in bronchoalveolar lavage fluid ( BALF) from recipients with CMVI (n = 33), CMVD (n = 6), and in healthy lung transplant controls (n = 33). We found a trend toward increased levels of MIP-1α/CCL3 during pulmonary CMVI. Levels of MCP-1/CCL2 and RANTES/CCL5 were significantly elevated during pulmonary CMV. Interestingly, elevated levels of CCL3 in BALF were protective with regards to survival. Importantly, elevated levels of CCL2 in BALF predicted the development of BOS, while elevated levels of CCL5 in BALF predicted an increase in mortality post-lung transplant.
Altered levels of specific CC chemokines during pulmonary CMV are associated with future clinical outcomes. These results suggest a possible utility of BALF CC chemokines as biomarkers for guiding risk assessment during pulmonary CMV post-lung transplantation.     

Sclerostin and DKK1 in Primary Hyperparathyroidism

Bone formation is influenced by the Wnt pathway through effects on osteoblast functionality, and these actions are opposed by two antagonists: sclerostin and Dickkopf-1 (DKK1). Decreased levels of serum sclerostin were found after treatment with the PTH analogue teriparatide and in patients with primary hyperparathyroidism (PHPT), while treatment with teriparatide of postmenopausal osteoporosis is associated with increases in serum DKK1. We studied mineral metabolism and Wnt pathway in 21 postmenopausal women affected by PHPT and in 42 age-matched healthy women. Mean serum calcium and PTH were significantly higher and serum phosphates significantly lower in the PHPT group compared with the control group. Serum 25-OH-vitamin D (25OHD) was lower in PHPT patients and 1,25 dihydroxy-vitamin D [1,25(OH)₂D] was significantly higher. Patients with PHPT had significantly higher levels of bone alkaline phosphatase (BAP) and of serum C-terminal telopeptides of type I collagene (sCTX). Serum sclerostin in PHPT was significantly lower (?26 %) and serum DKK1 significantly higher (+57 %) than in healthy control subjects. Serum PTH was positively correlated with 1,25OH₂D (p < 0.001), BAP (p = 0.036), sCTX (p = 0.003), and DKK1 (p = 0.007) and negatively with 25OHD (p = 0.002) and sclerostin (p = 0.02). In PHPT patients, serum sclerostin was negatively correlated with BAP (p = 0.038) and sCTX (p = 0.07). Patients with PHPT have significantly lower sclerostin and higher DKK1 levels compared with healthy postmenopausal control subjects. Further studies are warranted in order to verify whether the balance between these two opposite effects on Wnt function might help explain the variable bone involvement among patients with PHPT.     

Natural Killer Cell Receptor Repertoire and Their Ligands,and the Risk of CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is the most common viral complication after solid organ transplantation (SOT). Whilst current immunosuppression is known to impair antiviral‐specific T‐cell immunity in SOT, a potential role for natural killer (NK) cells not affected by immunosuppressive therapy remains to be determined. To address this, we compared the genotype of the NK immunoglobulin‐like receptor (KIR) genes and their HLA cognate ligands to the rate of CMV infection in 196 kidney transplant recipients. We have shown that the absence of the HLA‐C ligand for inhibitory KIR and the presence of activating KIR genes in the recipients were both associated with a lower rate of CMV infection after transplantation. In a cohort of 17 recipients with acute CMV infection, NK cells were phenotyped over a period of time after diagnosis by their expression profile of C‐type lectin receptors and capacity to secrete IFN‐γ. The increased expression of the activating C‐type lectin receptors NKG2C and NKG2D was paralleled by the decreased IFN‐γ secretion during the early phase of CMV infection. In conclusion, our findings suggest that KIR/HLA genotype and expression of NKG2C and NKG2D might play a significant role in regulating NK cell function and anti‐CMV immunity after kidney transplantation.     

Sclerostin and DKK1 in postmenopausal osteoporosis treated with denosumab

The bone mass benefits of antiresorbers in postmenopausal osteoporosis are limited by the rapid coupling of decreasing bone resorption with bone formation. Wnt signaling is involved in this coupling process during treatment with bisphosphonates, whereas its role during treatment with the anti‐receptor activator of NF‐κB ligand (RANKL) antibody denosumab is unknown. The study population includes patients participating in a placebo‐controlled trial lasting 36 months: 19 women were on placebo and 24 on subcutaneous 60 mg denosumab every 6 months. All measured parameters (serum C‐terminal telopeptide of type I collagen [sCTX], serum bone alkaline phosphatase [bAP], Dickkopf‐1 [DKK1], and sclerostin) remained unchanged during the observation period in the placebo group. sCTX and bAP were significantly suppressed by denosumab treatment over the entire follow‐up. Denosumab treatment was associated with significant (p < 0.05) increases (28% to 32%) in serum sclerostin over the entire study follow‐up. Serum DKK1 significantly decreased within the first 6 months with a trend for further continuous decreases, which reached statistical significance (p < 0.05) versus placebo group from the 18th month onward. The changes in DKK1 were significantly and positively related with the changes in sCTX and bAP and negatively with hip bone mineral density (BMD) changes. The changes in sclerostin were significantly and negatively related only with those of bAP. The changes in bone turnover markers associated with denosumab treatment of postmenopausal osteoporosis is associated with significant increase in sclerostin similar to those seen after long‐term treatment with bisphosphonates and significant decrease in DKK1. This latter observation might explain the continuous increase over 5 years in BMD observed during treatment of postmenopausal osteoporosis with denosumab. © 2012 American Society for Bone and Mineral Research.     

Dickkopf‐related protein 3 promotes pathogenic stromal remodeling in benign prostatic hyperplasia and prostate cancer

BACKGROUND

Compartment‐specific epithelial and stromal expression of the secreted glycoprotein Dickkopf‐related protein (Dkk)‐3 is altered in age‐related proliferative disorders of the human prostate. This study aimed to determine the effect of Dkk‐3 on prostate stromal remodeling that is stromal proliferation, fibroblast‐to‐myofibroblast differentiation and expression of angiogenic factors in vitro.

METHODS

Lentiviral‐delivered overexpression and shRNA‐mediated knockdown of DKK3 were applied to primary human prostatic stromal cells (PrSCs). Cellular proliferation was analyzed by BrdU incorporation ELISA. Expression of Dkk‐3, apoptosis‐related genes, cyclin‐dependent kinase inhibitors and angiogenic factors were analyzed by qPCR, Western blot analysis or ELISA. Fibroblast‐to‐myofibroblast differentiation was monitored by smooth muscle cell actin and insulin‐like growth factor binding protein 3 mRNA and protein levels. The relevance of Wnt/β‐catenin and PI3K/AKT signaling pathways was assessed by cytoplasmic/nuclear β‐catenin levels and phosphorylation of AKT.

RESULTS

Knockdown of DKK3 significantly attenuated PrSC proliferation as well as fibroblast‐to‐myofibroblast differentiation and increased the expression of the vessel stabilizing factor angiopoietin‐1. DKK3 knockdown did not affect subcellular localization or levels of β‐catenin but attenuated AKT phosphorylation in PrSCs. Consistently the PI3K/AKT inhibitor LY294002 mimicked the effects of DKK3 knockdown.

CONCLUSIONS

Dkk‐3 promotes fibroblast proliferation and myofibroblast differentiation and regulates expression of angiopoietin‐1 in prostatic stroma potentially via enhancing PI3K/AKT signaling. Thus, elevated Dkk‐3 in the stroma of the diseased prostate presumably regulates stromal remodeling by enhancing proliferation and differentiation of stromal cells and contributing to the angiogenic switch observed in BPH and PCa. Therefore, Dkk‐3 represents a potential therapeutic target for stromal remodeling in BPH and PCa. Prostate 73: 1441–1452, 2013. © 2013 Wiley‐Liss, Inc. The Prostate published by Wiley Periodicals, Inc.     

Primary response against cytomegalovirus during antiviral prophylaxis with valganciclovir,in solid organ transplant recipients

Antiviral prophylaxis has proved successful for prevention of cytomegalovirus (CMV) disease in solid organ transplant (SOT) patients; though emerging data suggest that antiviral agents interfere with immunity, and may inhibit immune priming. In this context, we investigated levels and phenotype of primary CMV‐specific immune responses that developed during antiviral prophylaxis in a cohort of CMV seronegative recipients (R^?) of a SOT from a seropositive donor (D⁺). We longitudinally monitored CMV viral load, antibodies and levels of the negative immuno‐modulator IL‐10. PBMC were stimulated with CMV‐specific peptide libraries to measure CD137 activation marker on CMV‐specific T‐cells and levels of PD‐1 receptor, which is over expressed on exhausted T‐cells. Unexpectedly, the majority (13/18) of D⁺R^? patients who developed a primary CMV response showed early post‐transplant CMV‐specific responses, though levels of PD‐1 on CMV‐specific T‐cells remained elevated throughout prophylaxis. A strong inverse association was found between levels of plasma IL‐10 and CMV‐specific cellular immune responses. Our study suggests that during prophylaxis, subclinical CMV infection might have occurred in the D⁺R^? patients, and primary CMV‐specific responses were detected early post‐transplant when levels of plasma IL‐10 were low. Extended prophylaxis or antiviral treatment did not appear to suppress CMV‐specific antibodies or T‐cells, which, however, showed exhaustion phenotypes.     

Prophylactic CMV therapy does not improve three‐yr patient and graft survival compared to preemptive therapy

Despite increasing evidence in favor of prophylactic valganciclovir treatment in kidney transplant recipients for the prevention of cytomegalovirus (CMV) infection, the impact of preemptive vs. prophylactic treatment on long‐term clinical outcomes is unclear. In this retrospective study, 187 kidney transplant recipients with serologic intermediate‐risk constellation (recipient CMV IgG positive) received either preemptive or prophylactic treatment with valganciclovir. Patient survival (primary endpoint), graft survival, viremia rates, and other CMV‐related outcomes were analyzed. Prophylactic therapy reduced the rates for CMV viremia during the first year (hazard ratio: 0.48, 95% confidence interval [CI] 0.30–0.75; p < 0.001). There was a trend for higher three‐yr patient mortality in the prophylactic group (hazard ratio: 5.08, 95% CI 0.62–41.3; p = 0.091), and the rate of graft loss was not reduced (hazard ratio: 0.93, 95% CI 0.32–2.68; p = 0.894). Estimated glomerular filtration rate over three yr was on average 6.8 mL/min/1.73 m² lower in the prophylactic group (95% CI −11.68 to −1.81; p = 0.007) using a multivariate random effects model but showed more improvement over time. Prophylactic valganciclovir treatment reduced the rate of CMV infections during the first year post‐transplant but no effects of prophylactic treatment on patient and graft survival or kidney function over three yr were observed.     

Targeted inhibition of T-cell factor activity promotes syndecan-2 expression and sensitization to doxorubicin in osteosarcoma cells and bone tumors in mice

Alterations of Wnt signaling appear to be involved in the pathogenesis of osteosarcoma, presenting mutations of adenomatous polyposis coli (APC) and epigenetic downregulation of Wnt inhibitory factor 1. However, the precise role of Wnt effectors in the bone cancer progression remains unclear. We previously showed that Wnt/β‐catenin/T‐cell factor (TCF) activation are responsible for the repression of syndecan‐2, a key modulator of apoptosis and chemosensitivity in osteosarcoma cells, suggesting a role of Wnt signaling in chemoresistance. In this study, we investigated the functional relationship between syndecan‐2, Wnt/β‐catenin/TCF signaling and chemosensitivity in these cells. To this goal, we selected resistant osteosarcoma cells from sensitive human cell lines using repeated exposures to doxorubicin. In doxorubicin‐responsive but not in doxorubicin‐resistant‐derived cells syndecan‐2 expression was upregulated by doxorubicin treatment. Moreover, syndecan‐2 overexpression restored the sensitivity to doxorubicin in resistant‐derived cells. We found that syndecan‐2 induction by doxorubicin is forkhead box protein O3A (Foxo3a)‐dependent. Foxo3a overexpression resulted in increased syndecan‐2 expression in sensitive and resistant‐derived cells. Doxorubicin modulated Foxo3a binding on syndecan‐2 gene promoter and induced Foxo‐dependent inhibition of Wnt/TCF activity. Conversely, β‐catenin/TCF activation impaired syndecan‐2 induction by doxorubicin, indicating that Wnt signaling is competing with the action of the cytotoxic drug. However, β‐catenin was also found to be required for Foxo3a activity. Consistently, Dickkopf 1 (DKK1) and secreted frizzled‐related protein 1 (sFRP‐1) altered doxorubicin action in sensitive cells, whereas inhibition of TCF activity strongly decreased cell viability and increased sensitivity to doxorubicin in sensitive and resistant cells. TCF inhibition also increased the effect of doxorubicin treatment in an orthotopic bone tumor model in mice. Altogether, these data provide evidence that the repression of syndecan‐2 by Wnt/β‐catenin/TCF signaling contributes to the resistance of osteosarcoma cells to doxorubicin and suggest that TCF inhibition may represent a novel therapeutic strategy in osteosarcoma. © 2012 American Society for Bone and Mineral Research.     

The significance of cytomegalovirus viremia at day 100 or more following allogeneic hematopoietic stem cell transplantation

We conducted a single‐center retrospective review of patients who had received allogeneic hematopoietic stem cell transplantation (HSCT) between January 2003 and December 2007, to assess the incidence and risk factors for late CMV infection and evaluate its effects on outcomes. Twenty of 49 HSCT recipients (41%) developed CMV infection at day ≥100 after transplant. Univariable analysis showed that having a matched unrelated donor, having early CMV infection, having a diagnosis of lymphoma, and receipt of antithymocyte globulin were risks for developing late CMV. On multivariable analysis, the occurrence of CMV prior to day 100 and lymphoma conferred a significant risk for late CMV infection. Of the 20 patients with late CMV infection, two patients manifested CMV disease (10%). Despite the relatively low incidence of CMV disease, patients with late CMV infection had a 4.8‐fold increased risk of death compared to patients without late CMV. Identifying patients at increased risk for developing late CMV infection may be important for prompting more intensive monitoring of infection late after HSCT, particularly because this manifestation of CMV is associated with poorer outcomes.     

Improving our mechanistic understanding of the indirect effects of CMV infection in transplant recipients

Cytomegalovirus (CMV) is an immunomodulatory virus that indirectly increases the risk for bacterial, fungal, and viral infections. However, the pathogenesis of this phenomenon is poorly understood. We determined whether inflammatory responses to different Toll‐like receptor (TLR) ligands are blunted during CMV infection in solid‐organ transplant (SOT) patients. Peripheral blood mononuclear cells from 38 SOT patients with and without CMV were incubated in the presence of various viral, fungal, and bacterial TLR ligands. Cytokines were measured in the supernatant by multiplex enzyme‐linked immunosorbent assay. Patients had blunted cytokine responses to bacterial, fungal, and viral ligands during CMV infection when compared to the absence of CMV infection. This was independent of viral load, clinical presentation of CMV infection or immunosuppression, supporting the clinical observation in SOT recipients that CMV infection increases susceptibility to bacterial, fungal, and other viral infections. Moreover, in the absence of CMV infection, patients with subsequent CMV infection had lower cytokines in response to TLR ligands compared to those without subsequent CMV infection, suggesting that inherent differences in patients not directly related to CMV also contribute to this increased susceptibility. In summary, these data provide novel ex vivo evidence to support indirect effects of CMV.     

Demonstration of HHV‐6 antigens in biopsies of kidney transplant recipients with cytomegalovirus infection

The activation of human herpesvirus‐6 (HHV‐6) commonly coexists with that of cytomegalovirus (CMV) in organ transplant recipients. No data exist of HHV‐6 in renal allografts, whereas persistent CMV in the kidney associates with poor outcome and histopathologic changes. We examined HHV‐6 and CMV antigens from kidney transplants with previous CMV infection. HHV‐6 and CMV pp65 antigens were demonstrated by monoclonal antibodies and immunohistochemistry from 22 kidney transplants with previous CMV infection. CMV was diagnosed by antigenemia tests and/or viral cultures. HHV‐6 antigens were found in 7/22 biopsies 18–1330 days after CMV infection, in infiltrating leukocytes in six, and in tubular epithelial cells in two patients. CMV infections were treated, and no virus could be detected from urine or blood thereafter, or at the time of the biopsy. Only 1/7 of these biopsies demonstrated also CMV antigens, whereas CMV antigens were found in 6/15 of the biopsies with no HHV‐6. HHV‐6 in the graft was associated with previous acute rejections, but not with any histopathological changes or reduced renal function. In conclusion, HHV‐6 was a common finding in late renal allograft biopsies of patients with previous CMV infection, but its significance remains to be elucidated.     

A Six‐Year Follow‐Up After Interferon‐Alpha Monotherapy for Chronic Hepatitis C Infection in Hemodialysis Patients

Interferon‐alpha (IFN) has been accepted as an effective treatment for chronic hepatitis C virus (HCV) infection in hemodialysis (HD) patients. We prospectively assess the long‐term clinical, biochemical, and virological effects of interferon in the treatment of HD patients with chronic HCV infection. This study was performed in 20 HCV‐RNA‐positive HD patients with evidence of chronic hepatitis on liver biopsy. The patients received IFN administered after HD sessions in doses ranging from 3 to 6 million units for 6 to 12 months. The patients were followed up for a period of 6 years with determinations of serial alanine aminotransferase (ALT) levels and serum HCV‐RNA. At the time of the final follow‐up, the patients had no cirrhosis or hepatocellular carcinoma. Among the nonresponder group, only 1 patient died due to sudden cardiac death. Sustained normal serum ALT levels occurred in 9 (45%) of the patients. Nine patients had variable ALT levels, and 2 patients had persistently elevated ALT levels. Eight (40%) patients were continuously HCV‐RNA negative, whereas 12 patients (60%) had variable HCV‐RNA results at the end of the 6‐year follow‐up. These findings show that the long‐term clinical, biochemical, and virological response to interferon monotherapy is good in HD patients with HCV infection.     

Increased urinary excretion of transforming growth factor-beta(1) in renal transplant recipients during cytomegalovirus infection

AIMS: Cytomegalovirus (CMV) is a suggested risk factor for chronic allograft nephropathy, and transforming growth factor-beta (TGF-beta) is a key fibrogenic molecule in this process. CMV has been shown to induce the expression of TGF-beta and several cytokines. We analyzed the impact of CMV on urinary excretion of TGF-beta, ICAM-1, TNF-alpha and correlated findings with biopsy histology. MATERIAL: Urine samples from 46 renal transplant recipients were available for the study. Urine samples were taken when CMV infection was suspected, or for controlling of proteinuria or bacteriuria. METHOD: CMV was diagnosed by antigenemia and viral cultures. Patients with previous CMV infection were excluded from the analysis. Urine samples were analyzed by ELISA-method to detect the levels of TNF-alpha, ICAM-1 and TGF-beta(1). Banff '97 criteria were used for scoring of protocol biopsies taken 6 months after transplantation. RESULTS: At the time of the urine collection, 13/46 patients had CMV infection. Eight patients with no CMV infection were used as controls. TGF-beta(1) was significantly increased in the CMV group (samples taken mean 137+/-79 days post-transplantation) compared to controls (samples 139+/-64 days post-transplantation) (51.1+/-28.0 vs. 13.3+/-6.7 ng/mmol crea, p<0.001). No differences in the levels of other molecules were recorded. In the biopsies, interstitial fibrosis was significantly increased in the CMV group compared to controls. CONCLUSIONS: Urinary excretion of TGF-beta(1) was increased in patients during CMV infection. This was associated with increased fibrosis in the biopsies.