HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects.

BACKGROUND: The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus-associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients. OBJECTIVE: The presence of HPyV6, HPyV7 and TSPyV DNA in skin biopsies from patients affected by different skin diseases (cancers and inflammatory disorders) has been evaluated to confirm their skin tropism and the possible pathological association. METHODS: DNA extracted was amplified with HPyV6, HPyV7 and TSPyV specific PCR real time on Taqman platform with standard profile. RESULTS: HPyV7 and TSPyV sequences were not found in any skin specimen analysed. HPyV6, on the other hand, was detected in 30% of samples from healthy subjects vs. 14.3% of skin cancer patients and 2.9% of inflammatory disorders. HPyV6 sequences have been detected in primary cutaneous T-cell lymphoma (CTCL) patients (in 18.6% out of Mycosis Fungoides (MF) patients and in 16.7% out of CTCL not MF/SS(Sèzary syndrome) but have not been detected in primary cutaneous B-cell lymphoma (CBCL) patients. CONCLUSION: Our preliminary data suggest that these three novel human polyomaviruses seem not to play a significant role neither in the pathogenesis of cutaneous malignancies nor in that of inflammatory disorders but, according to literature, can inhabit the skin. On the basis of our data regarding the HPyV6 DNA presence with decreasing percentages in healthy subjects, skin cancer and inflammatory disorders patients, it could be an intriguing matter to study if the activated innate immune response in inflammatory disorders can suppress the virus. Further investigations are needed to better understand their relationship with the human host and its innate immune system.

Clinical impact of HSV-1 detection in the lower respiratory tract from hospitalized adult patients.

The occurrence and clinical impact of herpes simplex virus (HSV) were evaluated in 342 bronchoalveolar lavage specimens from 237 patients. HSV-1 and HSV-2 were detected in 32.1% and <1% of patients, respectively. A significant difference of HSV-1 prevalence and load was found in relation to admission to intensive care unit, mechanical ventilation and mortality within 28 days; in particular, a viral load ≥10(5) copies/mL bronchoalveolar lavage fluid was significantly associated with critical features. No association was found with immune status or other characteristics. Nine of 21 (42.9%) cases of ventilator-associated pneumonia were positive for HSV-1, with poor outcome in six.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. OBJECTIVES: To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). METHODS: We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. RESULTS: Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. CONCLUSIONS: These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Human polyomavirus BK in patients with lupus nephritis: clinical and histological correlations.

Polyomavirus BK (BKV) reactivation can occur in immunodeficient patients. Few studies on BKV infection in patients with systemic lupus erytematosus (SLE) nephritis are available. Aim of this study was to analyse the prevalence of BKV infection by quantifying viral load and to investigate the association with clinical and histological parameters indicating duration, type and activity of SLE.BKV-DNA was evaluated by polymerase chain reaction in serum (sBKV) and urine (uBKV) specimens from 40 patients with SLE nephritis and 29 healthy controls. Renal function, urinary activity, clinical index of SLE activity [SLE Disease Activity Index (SLEDAI) score], CD4+/CD8+ ratio, histological classes and duration of SLE nephritis were compared according to the BKV-DNA-positivity.sBKV was present in 15% of SLE patients and in 13.8% of controls; uBKV in 32% of SLE patients and in 17.2% of controls. There was no significant difference in terms of kidney function, urinary activity, SLEDAI score, presence of anti-dsDNA antibodies, CD4+/CD8+ ratio and BKV viremia and/viruria, as well as there was no significant correlation between SLEDAI score, anti-dsDNA antibodies titers and median viral load. Duration of nephropathy tended to be shorter in patients with BKV viremia and/or viruria; proteinuria/creatininuria ratio tended to be higher in patients with positive sBKV and uBKV. BKV-DNA-positivity tended to be more frequent in patients treated with an immunosuppressive agent versus those on steroid treatment. Reactivation of BKV infection can occur in patients with SLE, although prevalence data do not significantly differ from those obtained in the control group. The trend toward an association between BKV infection and degree of proteinuria and less duration of SLE nephritis could indicate a major susceptibility to develop BKV infection in more active phases of the disease. The role of BKV reactivation in terms of clinical parameters and histological pattern, as well as the role of therapeutic protocols in the onset of BKV reactivation and, conversely, the therapeutic implication of BKV reactivation in SLE patients remain to be defined and should be addressed in further studies on a larger number of patients.

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Development and utilization of a quantitative polymerase chain reaction assay to evaluate human polyomavirus JC DNA load.

AIM: Quantitative polymerase chain reaction (PCR) analysis to evaluate virus load in comparison with the patient's base-line virus levels would be an optimal diagnostic approach to monitoring human polyomavirus infections and to investigate their possible involvement in the onset of nephropathy in this patient group. Studies on the correlation between viral burden and renal disease have pointed to the incidence of JC virus (JCV) related progressive multifocal leukoencephalopathy (PML) occurring in renal and haematopoietic stem cell transplant recipients. METHODS: We developed a reliable internally-controlled quantitative PCR assay to measure JCV-DNA in fluid samples of urine, serum and cerebrospinal fluid (CSF) by densitometric analysis of the amplification products. The assay was also used to evaluate the JCV load in CFS samples from patients with suspected demyelinating syndrome and in urine and serum samples from healthy subjects and renal transplant recipients. RESULTS: All CSF samples from the 51 patients with suspected demyelinating syndrome tested JCV-DNA negative: none of them had a diagnosed PML. Analysis of the prevalence of JCV-viruria and JCV-viraemia confirmed our previous data. JCV-viruria was detected in 17% of renal transplant recipients and 26.6% of healthy controls; JCV-viraemia was found in 3.4% of transplant patients and 0% in controls. Noteworthy was a lower prevalence of JCV-viraemia in the 116 (3.4%) renal transplant patients than the prevalence previously reported for the 51 (11.8%) patients with suspected demyelinating syndrome. The mean viral load of viruria was much higher in the healthy controls than in the transplant recipients [104020 DNA copies/mL (DS+/-62284) vs 4136 DNA copies/mL (DS+/-77371)]. CONCLUSIONS: The quantitative PCR assay developed in our lab offers in 2 h time a reliable true quantification of viral DNA by densitometric analysis of the amplification product. To check for the possible presence of potential Taq polymerase inhibitors an internal control (the homemade pJCV-C plasmid) is used. The relation between polyomavirus infections and their possible involvement in post-transplant pathologies need further investigation. It would be useful to monitor the JCV-DNA load in urine and serum from more renal transplant recipients, including patients with nephropathy or active graft rejection over a longer period of time.

Co-detection and discrimination of JCV and BKV DNA by duplex nested-PCR in renal transplant recipients.

AIM: Several studies have disclosed a correlation between human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients. It has recently been hypothesized that some cases of nephropathy may be associated with human polyomavirus JC (JCV). METHODS: In this paper we describe the development of duplex nested-PCR assay which allows the simultaneous detection and discrimination of genomic sequences of JCV and BKV ''large T antigen'', resulting in amplicons of 150 bp and 278 bp, respectively. Thus, the presence of JCV and BKV DNA in urine and serum samples from 51 renal transplant recipients and 29 healthy controls was investigated and related to immunosuppressive regimens and renal function. RESULTS: The comparison between the incidence of the of BKV and/or JCV infections (detected by viruria and/or viraemia) in renal transplant recipients and the control group revealed a highly significant increase of the incidence of BKV infection in immunosuppressed patients vs healthy subjects (62.7% vs 27.6%; p=0.005). In particular, we found a significant increase of BKV-DNA viruria in renal transplant recipients vs healthy subjects (49% vs 17.2%; p=0.01), in agreement with the BKV urinary shedding in renal transplant recipients of the literature (5-45%). CONCLUSION: The nested-PCR technique is a valid diagnostic tool to detect viral presence in urine and its systemic diffusion. Our assay links the high sensitivity of nested amplification with the simultaneous detection and discrimination of genomic sequences of JC and BK polyomaviruses and thus provides a handy, rapid and sensitive means for DNA analysis of large numbers of samples.

Epstein Barr viral load monitoring by quantitative PCR in renal transplant patients.

Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are a severe complication arising in solid organ transplant patients. Their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and the age of recipients. A strong correlation between Epstein Barr virus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monitored EBV viral load monthly in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory that employes a previous screening of samples containing a significant number of viral DNA copies (> or =1000 copies/10(5) PBMC or 100 microl serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR. Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /10(5) PBMC (roughly corresponding to 10.000 copies/microg PBMC DNA), and 1/14 (7.1%) reached 5000 EBV copies /10(5) PBMC (roughly corresponding to 50.000 copies/microg PBMC DNA), at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/10(5) PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 microl serum), supporting the literature data. With regard to immunosuppressive treatment, 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/10(5) PBMC, were on FK506 whereas only 33.3% of them were on CyA. In conclusion, further investigations are needed to better understand the role of EBV infection in the pathogenesis of PTLD in immunosuppressed patients. Given the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk to develop PTLD.

[Epstein Barr viral load monitoring in mononuclear lymphocytes and serum of renal transplant recipients using a quantitative PCR protocol]. / Monitoraggio della carica virale dell'EBV nei linfomonociti e nel siero di trapiantati renali mediante un protocollo di PCR quantitativa.

BACKGROUND: Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are severe complications arising in solid organ transplant patients; their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and age of recipients. A strong correlation between Epstein-Barrvirus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monthly monitored EBV viral load in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory. METHODS: Our EBV-DNA quantification protocol employs a previous screening of samples containing a significant number of viral DNA copies (>=1000 copies/105 PBMC or 100 mL serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR. RESULTS: Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in the literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /105 PBMC, and 1/14 (7.1%) reached 5000 EBV copies /105 PBMC at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/105 PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 mL serum). With regard to the immunosuppressive treatment, it should be noted that 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/105 PBMC, were on FK506 whereas only 33.3% of them were on CyA. CONCLUSIONS: Since the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several Authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk of developing PTLD.

[Polyomavirus BK nephropathy in renal transplant: 2 cases with different clinical expressions and review of the literature]. / Nefropatia da Poliomavirus BK nel trapianto renale: 2 casi con espressione clinica diversa e revisione della letteratura.

INTRODUCTION: Polyomavirus BK nephropathy is emerging as a significant cause of interstitial nephritis and allograft dysfunction (1-2). CASE REPORT: Two patients with renal transplants from cadaveric kidneys were treated with Tacrolimus plus Mycophenolate Mofetil (MMF) and Cyclosporine plus MMF, respectively. Their renal function gradually deteriorated eight to twelve months after the transplant. The renal biopsy of the first patient showed signs of significant interstitial tubulite, which necessitated the anti-rejection therapy with intravenous steroid pulses. After the pulses there was an additional dramatic increase in plasmatic creatinine, which suggested a revaluation of the kidney biopsy because of suspected Polyomavirus BK (BKV) nephropathy. In fact, after a more careful review, the suspicion of BKV infection was confirmed by the presence of intranuclear inclusions of tubular epithelium cells and marked denudation of the tubular basal membrane. The subsequent screening in both cases confirmed the presence of decoy cells in the urine, while the immunohistochemical analysis of the renal biopsy was strongly positive for the SV40 antigen. Our diagnosis was that of interstitial nephritis due to Polyomavirus BK that, in the first patient, was expressed by more aggressive clinical progress, probably due to enhanced immunosuppression from incorrect diagnosis of the interstitial rejection. The pre-transplant clinical outcome of the first patient was characterised by proteinuric nephropathy without any histological confirmation. Furthermore, we observed abundant pre-transplant residual diuresis and glucose intolerance. All these elements led us to hypothesise that native kidneys could have a fundamental role as viral reservoirs. CONCLUSION: Even though we reconfirm the decisive role of the immunosuppressive therapy and of the donor s kidney as the fundamental causes of Polyomavirus reactivation, we believe that it cannot be the result of a possible active role by the native kidney. In fact, as already noted, the SV40 genome is important in the pathogenesis of focal gomerulosclerosis. Furthermore, reports of polyoma nephropathy in not-yet-transplanted patients could accredit the role of the native kidneys as important viral reservoirs capable of inducing nephropathy in renal transplant patients.

B19 virus infection in renal transplant recipients.

BACKGROUND: B19 virus infection with persistent anaemia has been reported in organ transplant recipients. Detection of B19 virus DNA in serum is the best direct marker of active infection. OBJECTIVE: The present study evaluated the incidence and clinical role of active B19 virus infection in renal transplant recipients presenting with anaemia. STUDY DESIGN: Forty-eight such recipients were investigated by nested PCR on serum samples. The controls were 21 recipients without anaemia. Active HCMV infection was also investigated as a marker of high immunosuppression. RESULTS AND CONCLUSIONS: In 11/48 (23%) patients B19 virus DNA was demonstrated in serum versus only 1/21 (5%) of the controls. Ten of these 11 patients had already been seropositive at transplantation and active infection occurred in eight of them during the first 3 months after transplantation. The remaining patient experienced a primary infection 9 months after transplantation. Eight (73%) of these 11 patients displayed a concomitant HCMV infection and four (36%) showed increasing serum creatinine levels but none developed glomerulopathy; 3/11 (27%) recovered spontaneously from anaemia whereas 8/11 (73%) needed therapy. In conclusion, the relatively high occurrence (23%) of B19 virus infection in patients presenting with anaemia, suggests that it should be considered in the differential diagnosis of persistent anaemia in renal transplant recipients. Presence of the viral DNA should be assessed early from transplantation and the viral load should be monitored to follow persistent infection and better understand the relation between active infection and occurrence of anaemia, and to assess the efficacy of IVIG therapy and/or immunosuppression reduction in clearing the virus.

Human polyoma virus BK DNA detection by nested PCR in renal transplant recipients.

Several studies report a correlation between the human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients in whom immunosuppressive treatment is thought to allow or induce reactivation of the virus. Furthermore, it is described that nephropathy may result from the use of newly introduced immunosuppressive drugs. In the present study, we evaluated the presence of BKV DNA by nested-PCR (n-PCR) in urine and serum samples from 35 renal transplant patients related to the immunosuppressive regimens and renal function.

HCMV pp67-mRNA detection by nucleic acid sequence-based amplification in renal transplant patients.

CMV infection is a major cause of morbidity and mortality following renal transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. The determination of the presence of viral transcripts is considered a direct marker of HCMV replication in vivo. In particular, it seems that the expression of late transcripts might better reflect active HCMV replication, dissemination and disease, and should cease upon effective blockage of viral polymerase by antiviral agents, such as gancyclovir. The unspliced pp67-mRNA can be specifically amplified using nucleic acid sequence-based amplification (NASBA) in a background of DNA. In the present study blood samples of forty-two renal transplant patients with active HCMV infection, demonstrated by pp65-antigenaemia, were investigated to detect pp67-mRNA using the NASBA technique. Thirty-one pp65-antigenaemia positive patients resulted NASBA positive (73.8%) also in case of very low levels of antigenaemia; in 9/42 (21.4%) pp67-m-RNA was detected between 6 and 15 days before antigenaemia. Our results indicate that pp67-mRNA NASBA is a useful tool for the early diagnosis of active HCMV infection and for starting and modulating antiviral therapy, in addition to quantitative techniques such as antigenaemia, in renal transplant patients.

Interaction of Bartonella henselae with the murine macrophage cell line J774: infection and proinflammatory response.

Bartonella henselae is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with B. henselae display typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of B. henselae with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-gamma) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis N(G)-monomethyl L-arginine. These findings suggest that IFN-gamma-mediated activation of macrophages may be important for the clearing of B. henselae infection and that anti-B. henselae microbicidal activity of IFN-gamma-activated macrophages is mediated to a large extent by NO production.

Quantitative PCR in EBV-infected renal transplant patients.

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.

Impact of co-amoxiclav on polymorphonuclear granulocytes from chronic hemodialysis patients.

Phagocyte-dependent host defenses are frequently impaired in maintenance hemodialysis patients who show an increased susceptibility to infections. In these individuals, the course of infections can be more aggressive than in normal hosts, and the antibiotic of choice should have a high antimicrobial effect without impairing host defenses. Hence, in uremic patients, the antibiotic enhancement of phagocyte functions may be of potential clinical importance in the outcome of bacterial infections. Because we demonstrated previously that co-amoxiclav had beneficial properties that result in enhancement of the microbicidal functions of human polymorphonuclear cells (PMNs) from healthy subjects, we investigated the influence of this combination on the activities of PMNs from chronic hemodialysis patients against Klebsiella pneumoniae, a human pathogen that can pose severe problems in patients whose immunity is impaired. PMNs from chronic dialysis patients showed a diminished in vitro phagocytic efficiency with a reduced phagocytosis and bactericidal activity towards intracellular K. pneumoniae compared with that seen in PMNs from healthy subjects. When co-amoxiclav was added to PMNs from chronic hemodialysis patients, it was able to restore the depressed primary functions of PMNs, resulting in a significant high increase in both phagocytosis or killing activity. A similar pattern was detected with PMNs collected from hemodialysis patients treated with co-amoxiclav. The results of the present study provide evidence that co-amoxiclav is able to induce stimulation of depressed phagocytic response of PMNs from patients on chronic hemodialysis, restoring their primary functions both in vitro and in vivo.

Cefonicid 'restores' the depressed activities of polymorphonuclear cells from chronic haemodialysis patients and renal transplant recipients in vitro.

BACKGROUND: Chronic haemodialysis patients and renal transplant recipients are highly susceptible to infection characterized by high morbidity and mortality and related to an impairment of the phagocytic response. SUBJECTS AND METHODS: In order to elucidate how cefonicid, a cephalosporin with a broad spectrum of activity and once-daily dosage, influences this phagocytic response, the effects of the drug upon the functions of human PMNs from both healthy individuals and immunocompromised patients were investigated. RESULTS: In vitro, PMNs from haemodialysed patients and renal transplant recipients showed a diminished phagocytic efficiency with reduced phagocytosis and bactericidal activity towards intracellular Klebsiella pneumoniae when compared with that seen in PMNs from healthy subjects. Cefonicid significantly affected the activity of PMNs from healthy volunteers, resulting in either an increased percentage of ingested klebsiellae or a reduced intracellular bacterial load when compared with the control, drug-free system. When cefonicid was added to PMNs from uraemic patients a pattern similar to that observed with phagocytes from healthy subjects was detected: the antibiotic was able to 'restore' the depressed primary functions of PMNs, resulting in a significant increase in both phagocytosis and killing activity. CONCLUSIONS: Cefonicid, with its several immunoproperties observed in this study, possesses interesting beneficial properties which make it suitable for the treatment of infections in patients with impaired components of the immune system.

Interleukin-15 activates proinflammatory and antimicrobial functions in polymorphonuclear cells.

Interleukin-15 (IL-15) is a recently discovered cytokine produced by a wide range of different cell types including fibroblasts, keratinocytes, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-15 may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-15, evaluated as enhancement of PMN anti-Candida activity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-15 depends on binding to the IL-15 receptor. Our experiments show that binding of a biotinylated human IL-15-immunoglobulin G2b IgG2b fusion protein was competed by the addition of human recombinant IL-15 (rIL-15) or of human rIL-2, suggesting that IL-15 binding to PMN might involve the IL-2Rbeta and IL-2Rgamma chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-15Ralpha chain monoclonal antibody that PMN express the IL-15Ralpha chain at the mRNA and protein levels. Incubation with IL-15 activated PMN to secrete the chemotactic factor IL-8, and the amount secreted was increased by costimulation with heat-inactivated Candida albicans. In addition, IL-15 primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed to C. albicans. IL-15 also increased the ability of PMN to phagocytose heat-killed C. albicans organisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-15 was as effective as gamma interferon (IFN-gamma) and IL-2 in increasing the C. albicans growth-inhibitory activity of PMN. Taken together, these results suggest that IL-15 is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response against C. albicans.