The hEag1 K+ Channel Inhibitor Astemizole Stimulates Ca2+ Deposition in SaOS-2 and MG-63 Osteosarcoma Cultures.

The hEag1 (Kv10.1) K+ channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC50 = 0.135 µM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K+ channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 µM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

Prevalence of WU and KI polyomaviruses in plasma, urine, and respiratory samples from renal transplant patients.

WU and KI polyomaviruses (WUPyV, KIPyV) have been detected in respiratory, blood, stool, and lymphoid tissue, but not in urine samples. PCR based detection revealed higher frequency in immunocompromised individuals. In this study the prevalence of WUPyV and KIPyV was analyzed in respiratory, urine, and blood samples from renal transplant patients compared with healthy individuals. WUPyV and KIPyV were detected by nested PCR. The PCR products were sequenced and viral DNA loads were determined by quantitative real-time PCR. WUPyV and KIPyV were found in plasma (3.6%; 7/195), urine (14%; 7/50), and respiratory samples (10%; 9/90) of renal transplant patients, but not in plasma (0/200) and urine (0/36) specimens from healthy blood donors. WUPyV and KIPyV were detected mainly early after renal transplantation and the viral loads were low. A higher prevalence of WUPyV was found in plasma and urine samples, KIPyV was found more frequently in respiratory samples from renal transplant patients. It is hypothesized that immunosuppression due to the transplantation may result in reactivation of these viruses or may establish greater susceptibility to infection with KIPyV and WUPyV.

Dominance of variant A in human herpesvirus 6 viraemia after renal transplantation.

BACKGROUND: Human herpesvirus 6 (HHV-6), mostly variant B reactivation in renal transplant patients has been published by other authors, but the pathogenetic role of HHV-6 variant A has not been clarified. Our aims were to examine the prevalence of HHV-6, to determine the variants, and to investigate the interaction between HHV-6 viraemia, human cytomegalovirus (HCMV) infection and clinical symptoms. METHODS: Variant-specific HHV-6 nested PCR and quantitative real-time PCR were used to examine blood samples from renal transplant patients and healthy blood donors for the presence and load of HHV-6 DNA and to determine the variants. Active HHV-6 infection was proved by RT-PCR, and active HCMV infection was diagnosed by pp65 antigenaemia test. RESULTS: HHV-6 viraemia was significantly more frequent in renal transplant patients compared to healthy blood donors (9/200 vs. 0/200; p = 0.004), while prevalence of HHV-6 latency was not significantly different (13/200 vs. 19/200; p > 0.05). Dominance of variant A was revealed in viraemias (8/9), and the frequency of HHV-6A was significantly higher in active infections compared with latency in renal transplant patients (8/9 vs. 2/13; p = 0.0015). Latency was established predominantly by HHV-6B both in renal transplant patients and in healthy blood donors (11/13 and 18/19). There was no statistical significant difference in occurrence of HCMV and HHV-6 viraemia in renal transplant patients (7/200 vs. 9/200). Statistical analysis did not reveal interaction between HHV-6 viraemia and clinical symptoms in our study. CONCLUSIONS: Contrary to previous publications HHV-6A viraemia was found to be predominant in renal transplant patients. Frequency of variant A was significantly higher in cases of active infection then in latency.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells.

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic H+extrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA). Inhibition of hHv1 by ClGBI significantly decreases mineral matrix production of cMSCs induced by conditions mimicking physiological or pathological (inorganic phosphate, Pi) induction of osteogenesis. Wound healing assay and single cell motility analysis show that ClGBI significantly inhibits the migration of cMSCs. Thus, seminal functions of cMSCs are modulated by hHv1 which makes this channel as an attractive target for controlling advantages/disadvantages of MSCs therapy.

WU and KI polyomaviruses in respiratory, blood and urine samples from renal transplant patients.

BACKGROUND: It is suggested that immunosuppression due to transplantation might be a risk for human polyomavirus KI (KIPyV) and WU (WUPyV) infection. Most of the publications report data about stem cell transplant patients, little is known about these virus infections in renal transplant patients. OBJECTIVES: To study the presence of KIPyV and WUPyV in upper respiratory, plasma and urine samples from renal transplant patients. To analyse clinical and personal data. STUDY DESIGN: 532 respiratory, 503 plasma and 464 urine samples were collected from 77 renal transplant patients. KIPyV and WUPyV were detected by nested and quantitative real-time PCR. Patient and clinical data from medical records were analyzed. RESULTS: KIPyV was detected in respiratory, plasma and urine samples from 14.3%, 3.9% and 4.1% of renal transplant patients. WUPyV was found in respiratory and plasma specimens from 9.1% and 5.3% of the patients. Significant association was revealed between the detection of KIPyV and WUPyV and the time of samples collection and the age of the patients. KIPyV was presented in respiratory and plasma sample at the same time. KIPyV was detected in plasma samples from two patients and in urine samples of three other patients providing also KIPyV positive respiratory samples at the same time. No clinical consequences of KIPyV or WUPyV infection were found. CONCLUSION: Although no clinical consequences of KIPyV and WUPyV infections were found in renal transplant patients, it is suggested that renal transplantation might result in higher susceptibility or reactivation of these infection.

Prevalence of human herpesvirus 6A and 6B during pregnancy.

The aim of the present study was to assess the frequency of human herpesvirus 6A (HHV-6A) and human herpesvirus 6B (HHV-6B) infection during pregnancy. 100-100 blood samples were collected from pregnant and non-pregnant women, then nucleic acid was isolated from both plasma and leukocytes fraction. Nested and real-time PCR were used to detect and differentiate HHV-6A and HHV-6B DNA and to determine viral loads. Reverse transcription PCR (RT-PCR) for HHV-6 U79/80 mRNA was performed in order to reveal active HHV-6 replication.HHV-6A and HHV-6B active infections were not detected in blood samples neither from pregnant nor from non-pregnant women. Frequency of HHV-6B and HHV-6A latency did not show difference between the studied groups (15% vs. 16%). HHV-6B latency was dominant in both studied groups (14/15 and 15/16). Beside these results, in leukocyte samples of one pregnant and three non-pregnant women high HHV-6A viral loads (1.28 × 105 - 5.07 × 105 GEq / 1.5 × 106 leukocytes) were detected, and viral DNA was also found in plasma samples. Although RT-PCR did not confirm virus replication, but chromosomal integration was also not proved unequivocally, the number of 0.08-0.33 HHV-6 copy / 1 leukocyte refers more to postnatal infection.

Elevated tumor necrosis factor-alpha expression in periapical lesions infected by Epstein-Barr virus.

INTRODUCTION: In apical periodontitis, there is an intense inflammatory response to endodontopathogenic bacteria, an essential component of the pathogenic microbiota. The inflammation can be aggravated by herpesviruses acting as nonessential pathogens in periapical lesions. This study aimed to determine the levels of tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-ß) in periapical lesions in relation to local occurrence of Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV-6), and human herpesvirus 8 (HHV-8). METHODS: Fifty-eight samples with apical periodontitis and 20 clinically healthy gingival control tissues were collected. Viral DNA was determined with nested polymerase chain reaction, and cytokine mRNA expression was detected with real-time polymerase chain reaction assays. RESULTS: Periapical lesions harbored EBV (75.9%) and HHV-6 (22.4%) at significantly higher frequencies compared with controls (P < .000001 and P < .05, respectively), whereas HCMV (12%) and HHV-8 (0%) occurred rarely. The median TNF-α expression was 13 times higher (P < .001) and TGF-ß expression was 5 times higher in periapical lesions than in controls (P < .001). TNF-α expression was significantly higher in EBV-positive lesions than in EBV-negative lesions (P = .032). Presence of symptoms, lesion size, and infection by HCMV or HHV-6 had no significant association with either TNF-α or TGF-ß expression. CONCLUSIONS: The herpesviral component of the endodontic microbiota did not correlate with TGF-ß expression, whereas EBV infection was associated with a median 1.5 times further elevation of the high TNF-α expression characteristic for periapical lesions.

Novel human polyomaviruses in pregnancy: higher prevalence of BKPyV, but no WUPyV, KIPyV and HPyV9.

BACKGROUND: Immunosuppression due to pregnancy may lead to higher susceptibility to infections and reactivation of latent infections, such as BK polyomavirus (BKPyV). There is lack of information about the prevalence of novel human polyomavirus 9 (HPyV9), WU (WUPyV) and KI (KIPyV) during pregnancy. OBJECTIVES: To study whether pregnancy results in higher prevalence of HPyV9, WUPyV, KIPyV and their correlation with BKPyV. STUDY DESIGN: Plasma, urine and throat swab samples from 100 pregnant and 100 non pregnant women were screened for the presence of WUPyV, KIPyV, HPyV9 and BKPyV by PCR. RESULTS: No WUPyV DNA was detected in plasma, urine and respiratory samples from pregnant and non pregnant women. KIPyV DNA was found in two plasma samples from non pregnant women (2%) and not detected in other samples from neither pregnant nor non pregnant women. HPyV9 DNA was determined in all sample types of pregnant and non pregnant women, respectively. There were no significant differences between pregnant and non pregnant women in HPyV9 DNA frequencies for plasma (2% vs. 6%), urine (3% vs. 2%) and respiratory samples (2% vs. 2%). Prevalence of BKPyV in urine samples was significantly higher (p=0.039) in pregnant women (13%) then in non pregnant women (4%); co infection with KIPyV and/or HPyV9 was not detected. CONCLUSIONS: In contrast with BKPyV, infection with WUPyV, KIPyV and HPyV9 was not detected more frequently during pregnancy. To the best of our knowledge HPyV9 was detected first in respiratory samples in our study.