Epidemiology of hepatitis D virus (HDV) infection in an urban area of northern Italy.

OBJECTIVES: The introduction of vaccination against hepatitis B initially reduced the number of HBV (hepatitis B virus) and HDV (hepatitis delta virus) infections, but the decreasing trend of HDV infection seems to have stopped. The aim of this study was to assess the prevalence of HDV infection in the general population living in the catchment area of Legnano Hospital in northern Italy. METHODS: Of the 22,758 subjects tested in 2007-2008, the 488 who were HBsAg (hepatitis B surface antigen)-positive [including 107 (21.9%) of non-Italian origin] were subsequently tested for anti-HDV antibodies. RESULTS: Of the 488 subjects who tested positive for HBsAg, 24 (4.9%) were anti-HDV positive, all aged between 30 and 60 years. The difference in prevalence between males (7.1%) and females (1.9%) was statistically significant (p < 0.05), but not that between Italian (5.0%) and non-Italian patients (4.7%). The differences in anti-HDV seropositivity between the patients with acute (0%) and chronic infections (6.3%), and between the incident (2.5%) and prevalent cases (7.4%), were not statistically significant, but there was a significant difference (p < 0.01) between those with asymptomatic (2.1%) and clinically symptomatic infections (10.3%). Intravenous drug abuse was the main source of infection. CONCLUSIONS: In the catchment area of our hospital, the prevalence of HDV infection does not seem to be due to patients of non-Italian origin, but to Italian patients who are not vaccinated against HBV and who survived the HDV epidemic of the 1970s and 1980s. Nevertheless, the increase in the number of immigrants from non-EU countries in recent years is soon likely to lead to a change in the epidemiology of HDV.

Quantification of neutrophil and monocyte CD64 expression: a predictive biomarker for active tuberculosis.

SETTING: QuantiFERON TB assay (QFT) is used to screen tuberculosis (TB) infection, but it cannot distinguish active TB from latent TB infection (LTBI).OBJECTIVE: To evaluate the quantitative expression of the high-affinity FCgamma receptor I (CD64) on neutrophils (NE) and monocytes (MO) in peripheral blood using flow cytometry, measured in antibody binding capacity (ABC) units as a predictive biomarker of TB.DESIGN: Fifty-two patients were enrolled (45 QFT-positive and 7 QFT-indeterminate). Cultures and molecular analyses were performed.RESULTS: Of the 45 QFT-positive patients, 29 were culture-positive (active TB) and 16 were negative (LTBI). The median NE CD64 ABC and MO CD64 ABC expression was significantly higher (P < 0.001) in culture-positive patients. The NE CD64 and MO CD64 area under the receiver operating characteristic curve values were respectively 0.948 (95%CI 0.838-0.992) and 0.989 (0.901-1.000). By setting the cut-off NE CD64 value at >2400 ABC or MO CD64 value >25 800 the assay sensitivity increased to 95.5% with 100% specificity and 100% positive predictive value. In the QFT-indeterminate group, five culture-positive cases had NE CD64 >2400 ABC or MO CD64 value >25 800; two culture-negative cases had lower values.CONCLUSION: The CD64 quantitative expression on peripheral blood cells may be used as a predictive biomarker for active TB.

Hexamethylene bisacetamide inhibits malignant phenotype in T-ALL cell lines.

T acute lymphoblastic leukemia cell lines treated with hexamethylene bisacetamide (HMBA) undergo a delay in cell cycle progression and increase susceptibility to apoptosis, although they never overcome the differentiation block. In accordance with changes in cell cycle and apoptosis, transitory p53 pathway activation commonly occurs. Bcl-2 inhibition further favours the pro-apoptotic effect of HMBA. Notch1 expression is down regulated by reduction of its transcription level. Accordingly, Notch1 protein and transcriptional activity were affected. Even if HMBA generally reduces Notch1 level in T acute lymphoblastic leukemia (T-ALL) cell lines, this does not commonly influence the biological response; in fact all the analysed cell lines, except CEM cells, display no biological effect following DAPT-induced Notch inhibition.

Immunotoxin-mediated inhibition of chronic lymphocytic leukemia cell proliferation in humans.

We evaluated the cytotoxicity of the immunotoxin OKT1-SAP on fresh B-chronic lymphocytic leukemia (B-CLL) cells from 31 consecutive patients. OKT1-SAP comprised the OKT1 (CD5) monoclonal antibody disulfide linked to saporin-6 (SAP) ribosome-inactivating protein from the plant Saponaria officinalis. The effect of OKT1-SAP on target CD5-positive B-CLL cells was estimated using an in vitro proliferation inhibition assay in which control or OKT1-SAP-treated B-CLL cells were induced to proliferate by sequential stimulation with insolubilized anti-C3b receptor CB04 (CD35) antibody and low molecular weight B-cell growth factor. In 90% of patients, OKT1-SAP specifically suppressed B-CLL cell proliferation in a dose-related manner (50% inhibitory concentration, 4.0-6.8 nM). Taken together the findings reported in this article provide information relevant to the clinical development of immunotoxins because: (a) the in vitro conditions under which B-CLL cell proliferation is inhibited by OKT1-SAP are achievable in vivo without nonspecific toxicity according to our previous toxicology and pharmacokinetics studies in primates; and (b) the B-CLL cell proliferation inhibition assay described here provides a basis for future comparative studies.

Successful CD34+ cell mobilization by intermediate-dose Ara-C in chronic lymphocytic leukemia patients treated with sequential fludarabine and Campath-1H.

Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.

Panleucogating as an accurate and affordable flow cytometric protocol to analyse lymphocyte subsets among HIV-positive patients on HAART treatment in Mozambique.

In Africa tens of millions of people are HIV+. Prevention alone is not effective, and needs to be coupled with anti-retroviral treatment (HAART). Laboratory tests as CD4+ T cell count are fundamental tools in HIV disease monitoring, but they require costly equipment, reagents and specialised manpower. The goal of this study was to minimise and optimise the reagents needed for a reliable routine CD4+ cell count in a resource-poor setting (Mozambique). Panleucogating protocol (PLG), requires two antibodies only, CD45 and CD4, or three if CD8 is requested for special clinical reasons. PLG was compared with the current protocol used in two Mozambique hospitals, based on FSC/SSC gating and CD3/CD4/CD8 staining. 189 samples from HIV+ patients, included in the Community of Sant'Egidio's DREAM program and on HAART were processed with both protocols. The overall correlation of the lymphocyte subsets measurements was satisfactory, with r2 always >0.96. The Bland-Altman analysis of CD4+ cell count showed a negative bias when CD4+ cells were <15%, due to the imprecise FSC/SSC gating used previously. When CD4+ cells were >15% the negative bias tended to zero, further confirming the better quality of the PLG gating strategy. Two- or three color PLG protocol, in double platform, currently seems the most accurate and affordable method to monitor CD4+ lymphocytes and CD4/CD8 ratio by flow cytometry in resource-poor medical settings.

The presence of posttransplant HLA-specific IgG antibodies detected by enzyme-linked immunosorbent assay correlates with specific rejection pathologies.

Posttransplant monitoring of anti-HLA antibodies with routine techniques gives unsatisfactory results due to a variety of technical limitations. We investigated how a new alternative technique correlates with posttransplant clinical events. A total of 313 nonselected serum samples from 136 patients were screened by an ELISA utilizing captured soluble HLA class I antigens. We observed the absence of anti-HLA antibody production in acute rejection cases responding to standard antirejection therapy. On the other hand, we showed a clear presence of these antibodies in acute rejection episodes not responding to standard therapy (P<0.0001) and in chronic rejection (P<0.001). We conclude that routine posttransplant monitoring by ELISA offers early risk assessment that is crucial for proper immunosuppression and for antirejection therapy choice.

Effect of familial hypertension on glomerular hemodynamics and tubulo-glomerular feedback after uninephrectomy.

Familial hypertension, glomerular hemodynamic alterations, and dysregulation of tubulo-glomerular feedback (TGFB) have all been associated with the development of chronic renal failure. In the present study we evaluated renal and glomerular hemodynamics and TGFB responses in healthy kidney donors either with or without familial hypertension, before and after nephrectomy. Para-amino-hippurate plasma clearance (CPAH) and inulin plasma clearance (CInu) were measured in 15 kidney donors before and 1 year after nephrectomy. All subjects were normotensive and were kept in a sodium-replete state. Both clearances were measured after 40 min of constant infusion of PAH and Inu, as well as 20, 30, 50, and 60 min after the intravenous administration of acetazolamide (5 mg/kg). Glomerular hemodynamics were calculated by means of the Gomez formulae. Nephrectomy caused the expected decreases in CPAH and CInu and increase in the filtration fraction (all P < .0001). The decrease in renal resistances of the remaining kidney was greater at the afferent (-24%, P = .0075) than at the efferent arteriolar level (-17%, P < .0001). The TGFB activation was not altered by nephrectomy or by familial hypertension. Effective renal plasma flow (ERPF) decrease after TGFB activation appeared earlier than glomerular filtration rate (GFR) decrease before (P = .01), but not after, nephrectomy (P = .48). The presence of familial hypertension was associated with increased glomerular pressure (P = .0004). This study suggests that uninephrectomy in healthy human subjects causes a greater decrease in afferent arteriolar resistances, but that TGFB responses are not quantitatively altered. Familial hypertension is associated with a tendency toward higher glomerular pressures.

Diagnostic and prognostic value of flow cytometric immunophenotyping in malignant hematological diseases.

Flow cytometry is a diagnostic cell analysis technique with ever increasing applications in modern hematological practice. To date immunophenotyping of clonal hematological diseases represents one of the primary clinical applications of flow cytometry. Immunophenotyping of abnormal cells is now considered a fundamental tool to establish the cell lineage assignment and to obtain a more precise identification of the various cell subtypes. A number of observations have emerged showing strong association between specific immunophenotypes and genetic recurrent abnormalities underlying the malignant transformation, with prognostic value.

Immunotoxin mediated killing of clonable T-lymphocytes infiltrating an irreversibly rejected human renal allograft.

We isolated and treated in vitro with a novel CD5-specific saporin immunotoxin, referred to as OKT1-SAP, the cells infiltrating an irreversibly rejected renal allograft from a patient who rejected while on cyclosporine plus steroids and then failed to respond to multiple courses of high-dose steroids, intravenous OKT3 antibody, and local irradiation to the graft. We report here that under experimental conditions achievable in vivo the immunotoxin OKT1-SAP was capable of eliminating in vitro more than 95% of clonable T-lymphocytes infiltrating the rejected allograft of this patient despite their resistance to previous aggressive immunosuppression. To our knowledge, this is the first report of an immunotoxin-mediated suppression of the clonogenic growth of rejected renal allograft infiltrating T-lymphocytes.

Effect of n-3 polyunsaturated fatty acids on cyclosporine pharmacokinetics in kidney graft recipients: a randomized placebo-controlled study.

Highly concentrated marine polyunsaturated fatty acids (n-3 PUFA), affecting the lipids and lipophilic drugs metabolism, can interfere with cyclosporine (CyA) pharmacokinetics. This prospective, randomized and placebo-controlled, double-blind study involved 42 kidney graft recipients. From day +1, 21 pts (E) received 6 g n-3 PUFA (85% EPA + DHA, Esapent, Pharmacia) and 21 pts (P) received placebo (olive oil), both reduced to 3 g from day +30 on. A quadruple immunosuppressive regimen was employed. Plasma creatinine, lipids and CyA pharmacokinetics were investigated 1, 3, 6, 9 and 12 months after graft. The two groups were comparable for age, weight, M/F ratio, hypertension prevalence and baseline lipids. Active treatment did not affect total and HDL-cholesterol, but significantly lowered triglycerides (E:120 +/- 12 vs P:166 +/- 21 mg/dl, p < 0.0001). At one year, E pts had lower creatinine than P (1.26 +/- 0.06 vs. 1.88 +/- 0.2 mg/dl, p < 0.05), comparable CyA dosage, and a larger CyA area under the curve (AUC) (n.s.), with a higher blood peak level (Cmax) (p < 0.04) and less variance in time to peak (n.s.). The larger AUC in the E group at all intervals and the better pattern of plasma creatinine, with no rise in blood pressure, provided evidence of better CyA absorption and metabolism in n-3 PUFA supplemented kidney graft recipients.

Immunophenotyping of mononuclear cell infiltrates associated with renal disease.

Twenty-eight frozen renal biopsy specimens with a marked mononuclear cell interstitial infiltrate (MCI) were analyzed with monoclonal antibodies and a biotin-avidin peroxidase technique to define the surface phenotype distribution of the infiltrating cells. Twelve cases were diagnosed as tubulointerstitial nephritis of acute and chronic presentation, of unknown cause in 5 cases or secondary to multiple myeloma or drug reactions. Sixteen cases occurred in primary and secondary glomerulonephritis, 3 cases being associated with lymphoproliferative disorders. The results showed a remarkable heterogeneity of the MCI composition, even in cases with similar clinical and pathological findings. Namely, the T cells accounted for the majority of the infiltrating cells in most cases but a variable predominance of the T cell subsets Leu3 and Leu2 was observed. B cells and monocytes were also prominent in some cases. Such differences in the MCI composition may indicate the activation of different mechanisms of tissue damage, or a different phase of the renal disease. In the three cases of glomerulonephritis associated with lymphoproliferative disorders, the malignant origin of the MCI was demonstrated in one case, while in the remaining cases it was excluded.

Fluorocytometric analysis of anti-HLA class 2 autoantibodies in patients with lupus nephritis.

Systemic lupus erythematosus (SLE) patients are known to produce a variety of autoantibodies (AAb), some of which may be directed against immunocompetent cells. Anti-B cell autoimmunity may encompass reactivity against HLA-class 2 molecules, which are also expressed on kidney tissue. We studied 15 patients with moderate to severe renal involvement and 5 lupus patients with no clinical renal disease, in order to detect the presence of anti-HLA class 2 AAb. Flow cytometry was employed in an inhibitory assay using patient sera, autologous cells and two anti-class 2 monoclonals, to establish the specificity of anti-B cell AAb. Seven out of 15 nephritis patients had detectable anti-class 2 AAb with an epitopic heterogeneity, as demonstrated by different degrees of inhibition on the binding of non-overlapping monoclonals. The specificity of the reaction was confirmed by the lack of inhibition of non-class 2 antibody binding. The presence of such AAb was not correlated with disease activity but with the presence of a diffuse proliferative glomerulonephritis on renal biopsy. Anti-class 2 AAb may be a marker of SLE diffuse proliferative nephritis.

The role of donor and recipient factors in initial renal graft non-function.

ATN is a deleterious problem in the outcome of kidney transplantation. This complication is usually related to multiple factors including donor parameters, surgical technique, ischemic time, and recipient variables. In order to develop prophylactic measures, out of 430 kidney transplants performed in our Department, a series of 90 consecutive cadaveric renal allografts has been considered in this study. The overall incidence of IGNF was 23/90 (25.5%). Kidneys from MOD revealed a lower rate of IGNF (7/35 = 20%) when compared with organs from SOD (16/55 = 29%, P = NS). No difference was noted when kidneys were removed together with heart and/or liver and/or pancreas. Out of the donor factors, only CID was significant (17 +/- 9 hours in IGNF v 11 +/- 10 hours in patients with IGF, P = less than .05). Analysis of data concerning the fate of paired kidneys revealed two cases of IGNF in both kidneys from the same donor v 14 cases of IGNF in only one of the two paired grafts (P = NS). We conclude that: 1. Donor factors are clearly associated with a minority of IGNF. 2. The introduction of multiorgan procurement programs does not complicate early function. 3. Recipient factors (immunological events and intraoperative fluid management) provides important additive effects on initial graft nonfunction.

Clinical relevance of fractionation characteristics in cascade filtration.

Plasmapheresis performance is improved in the treatment of hyperviscosity syndromes with one of the several cascade filtration techniques (CF), intended for plasma fractionation and reinfusion of albumin-enriched plasma filtrate to the patients, avoiding the need for exogenous reinfusion solutions. A retrospective open analysis of 103 CF, performed by dead-end mode, in 14 patients with macroglobulinemia, cryoglobulinemia, multiple myeloma and other diseases, has been performed. Protein fractions removals have been calculated, normalized to the treatment of one plasma volume, compared in different macromolecular diseases and according to the different plasma fractionators employed. [table: see text] Protein removal is partially dependent of the surface area of the fractionator, but a wide variability has been reported, mainly depending on the underlying macromolecular disease.

Impaired efficacy of selective LDL-apheresis in primary biliary cirrhosis.

Low-density lipoprotein apheresis (LDL-apheresis) was done with either cascade filtration (DF) or dextran sulfate cellulose adsorption (DSC) in a patient with primary biliary cirrhosis who developed severe dyslipidemia associated with cholestasis and accumulation of lipoprotein-X (LP-X). The extracorporeal treatment was initially performed weekly, and resulted in a sharp drop in total cholesterol from 1038 to 430 mg/dl. During the next four months the patient was treated every 10-15 days, and pre-apheresis cholesterol levels were maintained between 438 and 505 mg/dl, until an orthotopic liver transplantation was successfully performed. With semi-selective DF a mean 47.1% of total cholesterol was removed per procedure compared to 30.0% with DSC, although the volume of treated plasma was 38.0 vs 49.9 ml/kg body weight. The changes in plasma cholesterol levels during DSC and DF showed that the kinetics of cholesterol removal were similar with both techniques, but the efficacy differed; DF removed both LDL and LP-X from plasma, whereas DSC selectively lowered the LDL content. Cascade filtration may therefore be considered as a first-choice treatment for patients with LP-X accumulation due to cholestasis.

Performance evaluation of cascade filtration with high flow rate recirculating plasma on the secondary filter.

Cascade filtration (CF) has been performed in 67 on-line procedures in 9 normolipidemic patients with paraproteinemic disorders. A modified dead-end technique has been employed, with high flow rate recirculating plasma on the plasma fractionation filter (QD recycled CF), and an albumin-rich, globulin-poor filtrate was reinfused into the patient. Postprocedure recoveries were 81 +/- 15% for albumin, 55 +/- 23% for IgM and 48% for cryocrit, with an increase in A/G ratio from 1.8 to 2.1. An improvement was observed also in antiatherogenic/atherogenic lipoproteins ratio, suggesting a possible use of this technique in the treatment of familial hypercholesterolemia. Plasma primary separation was obtained by centrifugation or by filtration, and no significant differences were observed on subsequent protein fractionation process. An albumin priming of the plasmafractionation circuit accounted for an additional 13% saving in postprocedure level. Different surface area secondary filters have been employed: with larger surfaces, larger volumes were processed without any increase in the waste volume and with reduced need for washouts, but with an additional loss of small molecules possibly due to entrapping onto the membrane.

Efficacy of a combined treatment with plasma exchange and cytostatics in macroglobulinemia.

Monthly plasma exchange (PE) sessions have been carried out in 3 patients with advanced Waldenström macroglobulinemia, in order to reduce electrophoretic M band under 2g/100 ml. When PE was combined to low doses of cytostatics (n = 18), 3 procedures per session were required to obtain a mean 57.4 +/- 12.3% IgM reduction, from 4.2 +/- 1.2 to 1.7 +/- 0.5 g/100 ml. A mean 61.5 +/- 13.1% IgM reduction, from 5.5 +/- 1.3 to 2.1 +/- 1 g/100 ml, was obtained in 64 procedures carried out as the only therapy in 12 sessions, with 5.3 procedures requirement per monthly session. IgM percent reduction 24 hours after PE was greater with combined treatment (45 +/- 9.7 vs. 28.9 +/- 15.4%; p = 0.001). The advantage of a combined treatment is therefore either a lowered PE requirement or a tapered maintenance cytostatic dosage.

Different cascade filtration operating modalities in clinical use.

Cascade filtration (CF) can be performed in either the single-pass or dead-end configuration. The distinction, as predicted by experimental models, is that solute removal is lower but constant in the former, and higher but variable with the quantity of filtrate in the latter. Moreover, unpredictable plugging reduces permeability during operation. It is therefore unclear which configuration is preferable in clinical use. In four cryo- and three macroglobulinemic patients, the data of 10 dead-end CF (QP) and 11 modified single-pass CF with high flow rate recycling plasma (QD) were compared. Both groups had similar starting values. Centrifugal primary plasma separation was performed. No exogenous reinfusions were used. Either 0.8 and 1.2 sq.m. surface secondary filters were employed. The ratio of plasma processed to patients' plasma volume was 1.1 +/- 0.3 in QP, and 0.94 +/- 0.09 in QD. The mean percentage removals of albumin, IgG and IgM respectively were 25.8 +/- 9, 32 +/- 17 and 47.5 +/- 26% in QP, 30.5 +/- 12, 40.5 +/- 19 and 48 +/- 17.5% in QD: albumin vs IgM p less than 0.025 in QP and p less than 0.02 in QD. A/G ratio increased from 1.6 +/- 0.5 to 2.06 +/- 0.6 in QP, and from 1.5 +/- 0.3 to 1.7 +/- 0.3 in QD. Plugging occurred in both groups, requiring 2.3 +/- 1.8 washouts/run in QP vs 1.5 +/- 1.2 in QD. IgM removal was comparable. Removal of albumin and IgG was slightly higher in QD. Similar performances can be obtained with either technique in clinical use, provided an adequate fractionation surface is available.

Renal abnormalities reverted by plasma exchange in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is usually accompanied by renal disfunction presumable due to diffuse thrombotic occlusions in the microcirculation. Two patients with TTP and slight renal failure with proteinuria and microscopic hematuria, were treated by repeated plasma exchanges with fresh frozen plasma, associated with prednisone and cyclophosphamide in one case, and prednisone alone in the other one. Platelet count, hematocrit and lactic dehydrogenase reverted to normal values within the fourth exchange; circulating immune complexes were never detected. Plasma factor stimulating prostacyclin activity lacked in only one patient and returned to normal levels after plasma exchange without being affected during a hematologic relapse. Renal function and urinary abnormalities reverted to normal by the end of plasma exchange and nine and six months renal and hematologic follow-up is still negative. Renal abnormalities in TTP seem to take advantage of early treatment by plasma exchange, which further to replacement of missing plasma factors, can account for the removal of toxic substances to be further investigated on.