Assessment of longitudinal serum neutrophil extracellular trap-inducing activity in anti-neutrophil cytoplasmic antibody-associated vasculitis and glomerulonephritis in a prospective cohort using a novel bio-impedance technique.

Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation.

Antitumor activity of bovine lactoferrin and its derived peptides against HepG2 liver cancer cells and Jurkat leukemia cells.

Liver cancer and leukemia are the fourth and first causes, respectively, of cancer death in children and adults worldwide. Moreover, cancer treatments, although beneficial, remain expensive, invasive, toxic, and affect the patient's quality of life. Therefore, new anticancer agents are needed to improve existing agents. Because bovine lactoferrin (bLF) and its derived peptides have antitumor properties, we investigated the anticancer effect of bLF and LF peptides (LFcin17-30, LFampin265-284 and LFchimera) on liver cancer HepG2 cells and leukemia Jurkat cells. HepG2 and Jurkat cells were incubated with bLF and LF peptides. Cell proliferation was quantified by an MTT assay, and cell morphology and damage were visualized by light microscopy or by phalloidin-TRITC/DAPI staining. The discrimination between apoptosis/necrosis was performed by staining with Annexin V-Alexa Fluor 488 and propidium iodide, and the expression of genes related to apoptosis was analyzed in Jurkat cells. Finally, the synergistic interaction of bLF and LF peptides with cisplatin or etoposide was assessed by an MTT assay and the combination index. The present study demonstrated that bLF and LF peptides inhibited the viability of HepG2 and Jurkat cells, inducing damage to the cell monolayer of HepG2 cells and morphological changes in both cell lines. bLF, LFcin17-30, and LFampin265-284 triggered apoptosis in both cell lines, whereas LFchimera induced necrosis. These results suggested that bLF and LF peptides activate apoptosis by increasing the expression of genes of the intrinsic pathway. Additionally, bLF and LF peptides synergistically interacted with cisplatin and etoposide. In conclusion, bLF and LF peptides display anticancer activity against liver cancer and leukemia cells, representing an alternative or improvement in cancer treatment.

CD163 and CD206 expression define distinct macrophage subsets involved in active ANCA-associated glomerulonephritis.

INTRODUCTION: Macrophages are key players in the immunopathology of anti-neutrophil cytoplasmic antibody (ANCA) mediated-vasculitis (AAV) with glomerulonephritis (ANCA GN). Different macrophage phenotypes are expected to play distinct roles in ANCA GN. Macrophages expressing CD163 and CD206 are found in lesions associated with ANCA GN. Hence, we aimed to investigate the clinicopathological significance of CD206 and CD163 in ANCA GN in a multicenter retrospective cohort study. MATERIAL AND METHODS: Patients with ANCA-associated vasculitis, with clinical data, serum and urine samples were included from three cohorts. Serum soluble CD206 (ssCD206) and urinary soluble CD163 (usCD163) levels were measured. Human kidney tissue samples (n = 53) were stained for CD206 and CD163 using immunohistochemistry and immunofluorescence, and findings were correlated with clinical and pathological data. RESULTS: In total, 210 patients were included (i.e., ANCA GN, n = 134; AAV without GN, n = 24; AAV in remission n = 52). Increased levels of both ssCD206 and usCD163 were seen in ANCA GN. High levels of ssCD206 declined after reaching remission, however, ssCD206 did not improve the accuracy of usCD163 to detect ANCA GN. Soluble markers correlated with histopathological findings. CD163+CD206- macrophages were found in the glomerulus and may play pivotal roles in glomerulonephritis, whereas CD206+CD163- and CD206+CD163+ macrophages were located tubulointerstitially and likely play a more prominent role in ANCA-associated tubulointerstitial inflammation. In ANCA GN patients increasing levels of ssCD206 increased the risk for end-stage renal disease and mortality. CONCLUSIONS: Our results confirm and extend the notion that CD206+ and CD163+ macrophages are prominent components of the cellular infiltrate in ANCA GN. We found distinct macrophage phenotypes that may play distinct roles in the immunopathology of ANCA GN and elaborate on a potential mechanism underlying the findings of this study. usCD163 remains an excellent marker to detect active ANCA GN, whereas ssCD206 seems a more prominent marker for risk prediction.

Bovine lactoferrin and lactoferrin peptides affect endometrial and cervical cancer cell lines.

Cervical, uterine, and ovarian cancers are the most common malignancies of the female genital tract worldwide. Despite advances in prevention, early diagnosis, effective screening, and treatment programs, mortality remains high. Consequently, it is important to search for new treatments. The activity of bovine lactoferrin (bLF) and LF peptides against several types of cancer has been studied; however, only a few studies report the effect of bLF and LF peptides against cervical and endometrial cancers. In this study, we explored the effect of bLF as well as LF chimera and its constituent peptides LFcin17-30 and LFampin265-284 on the viability of cervical (HeLa, SiHa) and endometrial (KLE, HEC-1A) cancer cell lines. Cell proliferation was quantified with an MTT assay, cell morphological changes and damage were determined by Giemsa and phalloidin-TRITC and DAPI staining, and apoptotic and necrotic cells were identified by Alexa Fluor® 488 Annexin V and propidium iodide staining. Additionally, the effect of combinations of bLF and LF peptides with cisplatin was assessed. bLF and LF peptides inhibited the proliferation of uterine cancer cells and caused cellular morphological changes and damage to cell monolayers. bLF induced apoptosis, LFcin17-30 and LFampin265-284 induced apoptosis and necrosis, and LF chimera induced necrosis. Additionally, bLF and LF chimera showed an additive interaction with cisplatin against uterine cancer cells.

The natural course of pregnancies in women with primary atypical haemolytic uraemic syndrome and asymptomatic relatives.

Pregnancy has been linked to various microangiopathies, including primary atypical haemolytic uraemic syndrome (aHUS). Complement dysregulation, often linked to rare variants in complement genes, is key for primary aHUS to manifest and may play a role in pregnancy complications of the mother and fetus. The burden of such complications is unknown, making counselling of women with primary aHUS and asymptomatic relatives difficult. We analyzed the maternal and fetal outcomes of 39 pregnancies from 17 women with primary aHUS and two asymptomatic relatives. Seven out of 39 pregnancies were complicated by pregnancy-associated aHUS. Five out of 32 pregnancies not linked to pregnancy-associated aHUS were complicated by pre-eclampsia or HELLP. Rare genetic variants were identified in 10 women (asymptomatic relatives, n = 2) who had a total of 14 pregnancies, including 10 uncomplicated pregnancies. Thirty-five out of 39 pregnancies resulted in live birth. Eight out of 19 women had progressed to end-stage kidney disease, with an incidence of 2·95 (95% confidence interval, 1·37-5·61) per 100 person-years after the first pregnancy. Thus, we emphasized the frequency of successful pregnancies in women with primary aHUS and asymptomatic relatives. Pregnancies should be monitored closely. Rare genetic variants cannot predict the risk of a given pregnancy.

DNase-mediated eDNA removal enhances D-LL-31 activity against biofilms of bacteria isolated from chronic rhinosinusitis patients.

Chronic rhinosinusitis (CRS) is a chronic infection of the nasal cavity and paranasal sinuses associated with the presence of a microbial biofilm. Extracellular DNA (eDNA) is an important component of the biofilm matrix. Antimicrobial peptides (AMPs) are natural peptides with the ability to kill microorganisms. D-LL-31 is a synthetic variant of the AMP cathelicidin with increased resistance to proteolytic breakdown. In this study it is shown for 3 clinical CRS isolates that treatment of 24 h biofilms with DNase I enhanced the antimicrobial activity of D-LL-31. Conversely, co-incubation of D-LL-31 at the IC50 value with exogenous DNA resulted in reduced antimicrobial activity. DNase I alone did not show antimicrobial activity against the isolates tested but caused dispersal of an established biofilm. Hence, the presence of eDNA in the biofilm matrix reduced AMP-mediated killing. These results suggest that combination therapy with proteolysis resistant AMP D-LL-31 and DNase could be considered for effective treatment of CRS.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei.

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

C5b9 Formation on Endothelial Cells Reflects Complement Defects among Patients with Renal Thrombotic Microangiopathy and Severe Hypertension.

Background Severe hypertension can induce thrombotic microangiopathy (TMA) in the renal vasculature, the occurrence of which has been linked to mechanical stress to the endothelium. Complement defects may be the culprit of disease in patients who present with severe renal disease and often progress to ESRD, despite BP control.Methods We studied a well defined cohort of 17 patients with hypertension-associated TMA to define the prevalence of complement defects by a specific ex vivo serum-based microvascular endothelial cell assay.Results Compared with normal human serum and samples from patients with hypertensive arterionephrosclerosis, 14 of 16 (87.5%) serum samples collected at presentation from 16 patients with hypertension-associated TMA induced abnormal C5b9 formation on microvascular endothelial cells. We detected rare variants in complement genes in eight of 17 (47%) patients. ESRD occurred in 14 of 17 (82%) patients, and recurrent TMA after transplant occurred in seven of 11 (64%) donor kidneys. Eculizumab improved the renal function in three patients and prevented TMA recurrence in an allograft recipient.Conclusions These observations point to complement defects as the key causative factor of ESRD and recurrent TMA after transplant in patients presenting with severe hypertension. Complement defects can be identified by measurements of complement activation on microvascular endothelial cells, which should substantially influence treatment and prognosis.

Salivary peptide histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies.

Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of histatin 1, which are discussed here. Furthermore, we put forward a possible role of histatin 1 in various pathologies and its potential function in clinical applications.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Anti-GAD antibodies in a cohort of neuropsychiatric patients.

OBJECTIVE: Antiglutamate decarboxylase (anti-GAD) antibodies are associated with several neurological manifestations, like epilepsy and movement disorders. However, in daily neurological practice, it remains hard to define when to test for anti-GAD antibodies in patients with neurologic and/or psychiatric symptoms. Therefore, here, we report the patient characteristics of a large retrospective cohort of patients tested for anti-GAD antibodies in clinical practice and compare the characteristics of anti-GAD positive and anti-GAD negative patients. METHODS: We blindly assessed relevant clinical symptoms and comorbidities and functional outcome with the modified Rankin Scale (mRS) in a retrospective observational cohort of all patients in which the decision to assess anti-GAD levels had been made based solely on the presence of possible associated neurological and/or psychiatric symptoms (N=119). RESULTS: Out of 119 patients, 17 (14.3%) were anti-GAD positive. The anti-GAD positive patients had a median age of 30years (range: 3-64; 2 children). They all had epilepsy, with 8 (47%) patients reporting cognitive complaints. Psychiatric symptoms were less prevalent in anti-GAD positive patients, only 1 anti-GAD positive patient (6%) versus 34 anti-GAD negative patients (33%) reported psychiatric symptoms (p=0.021). The most frequent comorbidity of anti-GAD positive patients was diabetes mellitus type 1 (n=8). Twelve (71%) and 13 (78%) of the anti-GAD positive patients were functionally independent at the time of diagnosis and after one year, respectively (mRS score: 0 to 2). There was no significant difference in functional status at any time during follow-up compared with the anti-GAD negative group. CONCLUSION: Antiglutamate decarboxylase (anti-GAD) antibodies relate to epilepsy with or without cognitive complaints. However, psychiatric symptoms were almost absent in anti-GAD positive patients, and the presence of anti-GAD antibodies contributed little to the prognosis in our cohort.

LFchimera protects HeLa cells from invasion by Yersinia spp. in vitro.

Yersinia pestis is the causative agent of plague. As adequate antibiotic treatment falls short and currently no effective vaccine is available, alternative therapeutic strategies are needed. In order to contribute to solving this problem we investigated the therapeutic potential of the peptide construct LFchimera against the safer-to-handle Y. pestis simulants Yersinia enterocolitica and Yersinia pseudotuberculosis in vitro. LFchimera is a heterodimeric peptide construct mimicking two antimicrobial domains of bovine lactoferrin, i.e. lactoferrampin and lactoferricin. LFchimera has been shown to be a potent antimicrobial peptide against a variety of bacteria in vitro and in vivo. Also Y. enterocolitica and Y. pseudotuberculosis have been shown to be susceptible for LFchimera in vitro. As Yersiniae spp. adhere to and invade host cells upon infection, we here investigated the effects of LFchimera on these processes. It was found that LFchimera has the capacity to inhibit host-cell invasion by Yersiniae spp. in vitro. This effect appeared to be host-cell mediated, not bacteria-mediated. Furthermore it was found that exposure of human HeLa epithelial cells to both LFchimera and the bacterial strains evoked a pro-inflammatory cytokine release from the cells in vitro.

Patients with hypertension-associated thrombotic microangiopathy may present with complement abnormalities.

Thrombotic microangiopathy (TMA) is a pattern of endothelial damage that can be found in association with diverse clinical conditions such as malignant hypertension. Although the pathophysiological mechanisms differ, accumulating evidence links complement dysregulation to various TMA syndromes and in particular the atypical hemolytic uremic syndrome. Here, we evaluated the role of complement in nine consecutive patients with biopsy-proven renal TMA attributed to severe hypertension. Profound hematologic symptoms of TMA were uncommon. In six out of nine patients, we found mutations C3 in three, CFI in one, CD46 in one, and/or CFH in two patients either with or without the risk CFH-H3 haplotype in four patients. Elevated levels of the soluble C5b-9 and renal deposits of C3c and C5b-9 along the vasculature and/or glomerular capillary wall, confirmed complement activation in vivo. In contrast to patients without genetic defects, patients with complement defects invariably progressed to end-stage renal disease, and disease recurrence after kidney transplantation seems common. Thus, a subset of patients with hypertension-associated TMA falls within the spectrum of complement-mediated TMA, the prognosis of which is poor. Hence, testing for genetic complement abnormalities is warranted in patients with severe hypertension and TMA on renal biopsy to adopt suitable treatment options and prophylactic measures.

Anticancer activities of bovine and human lactoferricin-derived peptides.

Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukemia and breast-cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell-penetrating peptide; when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short cell-penetrating peptides and antimicrobial peptides can be combined to create new adducts with increased potency.

Antibacterial and cell penetrating effects of LFcin17-30, LFampin265-284, and LF chimera on enteroaggregative Escherichia coli.

Lactoferrin (LF) is a protein with antimicrobial activity, which is conferred in part by 2 regions contained in its N-terminal lobe. These regions have been used to develop the following synthetic peptides: lactoferricin17-30, lactoferrampin265-284, and LF chimera (a fusion of lactoferricin17-30 and lactoferrampin265-284). We have reported that these LF peptides have antibacterial activity against several pathogenic bacteria; however, the exact mechanism of action has not been established. Here, we report the effects of LF peptides on the viability of enteroaggregative Escherichia coli (EAEC) and the ability of these peptides to penetrate into the bacteria cytoplasm. The viability of EAEC treated with LF peptides was determined via enumeration of colony-forming units, and the binding and internalization of the LF peptides was followed via immunogold labeling and electron microscopy. Treatment of EAEC with 20 and 40 µmol/L LF peptides reduced bacterial growth compared with untreated bacteria. Initially the peptides associated with the plasma membrane, but after 5 to 30 min of incubation, the peptides were found in the cytoplasm. Remarkably, bacteria treated with LF chimera developed cytosolic electron-dense structures that contained the antimicrobial peptide. Our results suggest that the antibacterial mechanism of LF peptides on EAEC involves their interaction with and penetration into the bacteria.

Parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite Giardia intestinalis.

Giardia intestinalis is the most common infectious protozoan parasite in children. Despite the effectiveness of some drugs, the disease remains a major worldwide problem. Consequently, the search for new treatments is important for disease eradication. Biological molecules with antimicrobial properties represent a promising alternative to combat pathogens. Bovine lactoferrin (bLF) is a key component of the innate host defense system, and its peptides have exhibited strong antimicrobial activity. Based on these properties, we evaluated the parasiticidal activity of these peptides on G. intestinalis. Trophozoites were incubated with different peptide concentrations for different periods of time, and the growth or viability was determined by carboxyfluorescein-succinimidyl-diacetate-ester (CFDA) and propidium iodide (PI) staining. Endocytosis of peptides was investigated by confocal microscopy, damage was analyzed by transmission and scanning electron microscopy, and the type of programmed cell death was analyzed by flow cytometry. Our results showed that the LF peptides had giardicidal activity. The LF peptides interacted with G. intestinalis and exposure to LF peptides correlated with an increase in the granularity and vacuolization of the cytoplasm. Additionally, the formation of pores, extensive membrane disruption, and programmed cell death was observed in trophozoites treated with LF peptides. Our results demonstrate that LF peptides exhibit potent in vitro antigiardial activity.

Molybdenum cofactor deficiency: Identification of a patient with homozygote mutation in the MOCS3 gene.

We describe the clinical presentation and 17 years follow up of a boy, born to consanguineous parents and presenting with intellectual disability (ID), autism, "marfanoid" dysmorphic features, and moderate abnormalities of sulfite metabolism compatible with molybdenum cofactor deficiency, but normal sulfite oxidase activity in cultured skin fibroblasts. Genomic exome analysis revealed a homozygous MOCS3 missense mutation, leading to a p.Ala257Thr substitution in the highly conserved ubiquitin-like-domain of the protein. MOCS3 is the third protein, besides MOCS1 and MOCS2, involved in the biosynthesis of the molybdenum cofactor and has a dual ubiquitin-like function in tRNA thiolation. It is plausible that the phenotype results from deficiency of this dual function, not only from defective synthesis of molybdenum cofactor, which would explain similarities and differences from the MOCS1 and MOCS2-related disorders. This observation should encourage testing of additional ID patients with mild abnormalities of sulfite metabolism for MOCS3 mutations.