Matthias Krams

Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection

BackgroundConstitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract.MethodsWe investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria.ResultsConstitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level.ConclusionsUpregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.

Pediatric follicular lymphoma – a clinico-pathological study of a population-based series of patients treated within the Non-Hodgkin’s Lymphoma - Berlin-Frankfurt-Münster (NHL-BFM) multicenter trials

Background Pediatric follicular lymphoma has recently been recognized as a novel variant of follicular lymphoma in the World Health Organization classification of lymphomas. Given the rarity of the disease, histopathological and genetic data on this type of lymphoma are still scarce. Design and Methods We analyzed 25 cases of pediatric follicular lymphoma (patients aged ≤18 years) by morphology, immunohistochemistry and interphase fluorescence in situ hybridization. All patients analyzed were treated within Non-Hodgkin’s Lymphoma - Berlin-Frankfurt-Münster (NHL-BFM) multicenter trials, and the cohort was representative of the German population. Results The genetic hallmark of adult follicular lymphoma, t(14;18)(q32;q21), was not detectable in any of the pediatric cases, although BCL2 protein was expressed in 55% of the latter cases. No correlation was found between BCL2 protein expression and outcome. Chromosomal breaks in the immunoglobulin heavy chain gene (IGH) and the BCL6 locus were detected in 5 of 17 and 1 of 18 cases, respectively. Patients with pediatric follicular lymphoma had long event-free survival and, in contrast to adult follicular lymphoma, the clinical course was not dominated by relapses. A simultaneous diffuse large B-cell lymphoma was frequently detected at initial diagnosis in children but did not indicate an aggressive clinical course. Conclusions Our data suggest that pediatric follicular lymphoma is a disease that differs from its adult counterpart both genetically and clinically.

Impact of Latent Epstein-Barr Virus Infection on Outcome in Children and Adolescents With Hodgkin's Lymphoma

PURPOSE The prognostic significance of latent Epstein-Barr virus (EBV) infection in Hodgkins lymphoma (HL) is debated controversially. Especially in the pediatric age group, no conclusive data are available. PATIENTS AND METHODS Eight hundred forty-two children and adolescents (median age, 13.7 years) from pediatric multicenter treatment studies HD-90 and HD-95 were studied for latent EBV infection in Hodgkins and Reed-Sternberg cells by immunostaining against latent membrane protein 1 (LMP-1). Results were compared with established risk factors. RESULTS Two hundred sixty-three patients (31%) were LMP positive. EBV infection correlated with sex (39% male v 23% female; P < .001), histologic subtype (69% mixed cellularity v 22% nodular sclerosis v 6% lymphocyte predominance; P < .001) and young age. With a median follow-up of 4.9 years, 820 patients (97%) are alive. Probability of overall survival at 10 years (+/- standard deviation) for EBV-negative and -positive patients was 98.1% +/- 0.6% and 95.1% +/- 1.4%, respectively (P = .017 by log-rank test). A negative effect of EBV infection became evident for patients with nodular sclerosis subtype Bennett II (P = .02), and those treated for advanced stages (P = .003). In multivariate analysis, LMP positivity was an independent factor for adverse outcome (RR = 3.08). Probability of failure-free survival (FFS) in LMP positive and negative patients was 89.1% +/- 2.3% and 84.1% +/- 3.9%, respectively (P = .86). CONCLUSION With effective combined treatment modalities in pediatric HL, latent EBV infection has no influence on FFS but is associated with an inferior overall survival in crucial subgroups.

Full-length telomerase reverse transcriptase messenger RNA is an independent prognostic factor in neuroblastoma.

Telomerase activity (TA) is the most recently recognized prognostic factor in neuroblastoma, and its outstanding predictive power was documented by several studies. However, TA measurements require fresh tumor tissue that is not always available in daily clinical practice. We previously described a reverse transcriptase-polymerase chain reaction assay that we used to investigate the possible prognostic relevance of the telomerase catalytic subunit, hTERT, at the mRNA level. Because hTERT mRNA undergoes alternative splicing as a regulatory mechanism of TA, we discriminated between truncated and full-length hTERT transcripts. In a retrospective study on 124 neuroblastomas, 56 (45.2%) tumors showed spliced hTERT transcripts, whereas 30 (24.2%) contained full-length hTERT transcripts. The presence of both spliced and full-length hTERT transcripts was significantly associated with MYCN amplification. hTERT in general showed no correlation to other prognostic factors, ie, International Neuroblastoma Staging System stage, International Neuroblastoma Pathology classification grade, or age at diagnosis, whereas the presence of full-length transcripts was significantly associated with higher stages. The presence of any hTERT transcripts carried no significant prognostic information, yet full-length hTERT transcripts were highly predictive of poor outcome (P < 0.0001). In a multivariate analysis, full-length hTERT transcripts and International Neuroblastoma Pathology classification grade emerged as the sole independent predictors of event-free survival, with relative risks of 10.0 and 3.9, respectively. The strong statistical correlation of full-length hTERT transcripts with clinical outcome in neuroblastoma suggests that the reverse transcriptase-polymerase chain reaction analysis of hTERT transcripts may be equatable to TA measurements. Because this assay is well suited for archival material, it could become a useful adjunct in evaluating the prognosis of individual neuroblastoma cases.

Expression of the c-kit receptor characterizes a subset of neuroblastomas with favorable prognosis.

Expression of the c-kit proto-oncogene product in neuroblastomas has been reported, but its clinical relevance is unclear. We determined the expression of c-kit by immunohistochemistry in a series of 155 neuroblastomas with long-term follow-up. The specificity of the reaction was verified by Western blot analysis and quantitative RT–PCR, and exon 11 of the kit gene was screened for mutations by PCR and capillary electrophoresis. No mutations were detected, and transcription of the kit gene correlated with protein expression. c-kit expression was associated with lower tumor stages and a low rate of MYCN amplification. More importantly, it coincided with tumor differentiation (P<0.0001), and portended a favorable outcome with a relative risk of 0.18 (P<0.0001). In a multivariate analysis of event-free survival, loss of c-kit (relative risk 4.25, P<0.0001) was an independent prognostic factor next to INSS stage 4 and before MYCN amplification. It is concluded that c-kit is transcriptionally regulated in neuroblastomas. Its expression likely identifies a subset of neuroblastomas with conserved capacity for differentiation, which may represent the embryonal variety of the disease. Assessment of c-kit may improve prognostic models for neuroblastoma and provide a basis for new therapy concepts.

Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas

It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases.

The superior prognostic value of humoral factors compared with molecular proliferation markers in renal cell carcinoma

The American Joint Committee on Cancer and the Union Internationale Contre le Cancer have acknowledged routine laboratory parameters, such as serum calcium, alkaline phosphatase, hemoglobin, and the erythrocyte sedimentation rate (ESR), as predictors of survival in patients with renal cell carcinoma. The predictive value of these parameters compared with proliferation markers, such as Ki‐67, proliferating cell nuclear antigen (PCNA), topoisomerase II‐α, and p100, has not been determined.

Loss of expression of LyGDI (ARHGDIB), a rho GDP‐dissociation inhibitor, in Hodgkin lymphoma*

The guanosine triphosphatase (GTPase) inhibitor LyGDI (ARHGDIB, Ly/D4‐GDI, RhoGDIb or RhoGDI 2) is abundantly expressed in haematopoetic cells and possibly plays a role in the onset of apoptosis. Gene expression profiling of Hodgkin cell lines revealed that LyGDI expression was downregulated in these cell lines. The present study evaluated the expression of LyGDI in Hodgkin cells in vivo and studied the function of LyGDI in Hodgkin cell lines in vitro. Our results showed that virtually all Hodgkin and Reed‐Sternberg cells in classical Hodgkin lymphoma lacked LyGDI protein expression. On the other hand, almost all non‐Hodgkin lymphomas, except for anaplastic large cell lymphomas, expressed LyGDI protein. Transfection of the classical Hodgkin cell line L428 with a vector containing full‐length LyGDI‐induced apoptosis in a subset of cells. However, the majority of Hodgkin cells with transgenic expression of LyGDI escaped apoptosis. Our data show that lack of LyGDI expression is a frequent feature of cHL but that it is not of vital importance for the growth and survival of these cells.

repp86 Expression and Outcome in Patients With Neuroblastoma

PURPOSE Given the well-known challenges of neuroblastoma prognosis, we investigated whether the expression of restrictedly expressed proliferation-associated protein of 86 kDa theoretical molecular mass (repp86), a proliferation-associated protein expressed in S, G2, and M phases of the cell cycle, correlates with the clinical outcome in patients with neuroblastoma. PATIENTS AND METHODS 161 children with different stages of neuroblastoma were studied; the median follow-up time was 72.8 months. The patients were staged according to the International Neuroblastoma Staging System, and histologic grading of the tumors was performed according to the criteria of Hughes and those of the International Neuroblastoma Pathology Classification. The MYCN gene copy number was determined by Southern blot analysis or fluorescence in situ-hybridization, and repp86 expression was assessed immunohistochemically by means of monoclonal antibody Ki-S2 on paraffin sections from archival tumor samples. RESULTS A repp86 labeling index (RI) of more than 10% positive tumor cells significantly predicted a shortened disease-free interval and an increased tumor mortality (both P <.0001). Moreover, the RI allowed the identification of patients with favorable and adverse prognosis in subsets defined by stage, grade, age, and MYCN status. In a multivariate analysis, the RI emerged as the most important predictor of event-free and disease-specific survival with hazard ratios of 11.7 and 10.5, respectively (both P <.0001). CONCLUSION It seems that repp86 expression is closely associated with the biologic behavior of neuroblastoma. Assessment of the RI might, therefore, considerably refine prognostic models.

Topical inhibition of nuclear factor-κB enhances reduction in lung edema by surfactant in a piglet model of airway lavage

Objective:Acute respiratory distress syndrome is occasionally seen in newborn infants due to a severe inflammatory process in the lungs that affects capillary–alveolar permeability, epithelial integrity, and type I and II pneumocyte function. The aim of this study was to investigate the effect of a topically applied nuclear factor-&kgr;B inhibitor (I&kgr;B kinase–NF-&kgr;B essential modulator binding domain [IKK-NBD] peptide) on gas exchange, lung function, lung fluids, and inflammation in a piglet model of repeated airway lavage that is characterized by surfactant deficiency, lung edema, and an inflammatory response. Design:Prospective, randomized, controlled animal study. Setting:Research laboratory of a university childrens hospital. Subjects:A total of 24 anesthetized, mechanically ventilated newborn piglets. Interventions:Repeated airway lavage was carried out until both the Pao2 decreased to approximately 40 mm Hg, while ventilating the piglets with an Fio2 of 0.6, and a peak inspiratory pressure of ≥18 cm H2O was needed to maintain tidal volume at 6 mL/kg. One group of piglets served as a control (n = 8), a second group (S, n = 8) received a porcine surfactant preparation (Curosurf), and a third group received IKK-NBD peptide admixed to surfactant (S+IN, n = 8). Measurements and Main Results:After 6 hrs of mechanical ventilation after intervention, S+IN group piglets showed decreased extravascular lung water (S+IN vs. S, 20 ± 3 vs. 28 ± 10 mL/kg; p < .05) and a lesser protein content in the epithelial lining fluid (S+IN vs. S, 38 ± 5 vs. 50 ± 5 mg/L; p < .05). Functional residual capacity (S+IN vs. S, 16.7 ± 6.3 vs. 12.2 ± 4.3 mL/kg; p < .05), alveolar volume (S+IN vs. S, 5.4 ± 1.8 vs. 4.6 ± 1.5 mL/kg; p < .05), and lung mechanics were improved. Bronchoalveolar lavage showed a lesser percentage of polymorphonuclear leukocytes (S+IN vs. S, 70% ± 6% vs. 82% ± 3%; p < .01) and a reduction in the chemokine leukotriene B4 (S+IN vs. S, 2.0 ± 0.6 vs. 3.5 ± 1.4 pg/mL; p < .01). Conclusions:A topically applied nuclear factor-&kgr;B inhibitor improves lung edema and lung volumes and reduces inflammation in this newborn piglet model of airway lavage.