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Introduction: The impact of COVID-19 pandemic represents a serious challenge for 'frail' patients' populations with inflammatory autoimmune systemic diseases such as systemic sclerosis (SSc). We investigated the prevalence and severity of COVID-19, as well the effects of COVID-19 vaccination campaign in a large series of SSc patients followed for the entire period (first 38 months) of pandemic., Patients and Method: This prospective survey study included 1755 unselected SSc patients (186 M, 1,569F; mean age 58.7 ± 13.4SD years, mean disease duration 8.8 ± 7.3SD years) recruited in part by telephone survey at 37 referral centers from February 2020 to April 2023. The following parameters were carefully evaluated: i. demographic, clinical, serological, and therapeutical features; ii. prevalence and severity of COVID-19; and iii. safety, immunogenicity, and efficacy of COVID-19 vaccines., Results: The prevalence of COVID-19 recorded during the whole pandemic was significantly higher compared to Italian general population (47.3 % vs 43.3 %, p < 0.000), as well the COVID-19-related mortality (1.91 % vs 0.72 %, p < 0.001). As regards the putative prognostic factors of worse outcome, COVID-19 positive patients with SSc-related interstitial lung involvement showed significantly higher percentage of COVID-19-related hospitalization compared to those without (5.85 % vs 1.73 %; p < 0.0001), as well as of mortality rate (2.01 % vs 0.4 %; p = 0.002). Over half of patients (56.3 %) received the first two plus one booster dose of vaccine; while a fourth dose was administered to 35.6 %, and only few of them (1.99 %) had five or more doses of vaccine. Of note, an impaired seroconversion was recorded in 25.6 % of individuals after the first 2 doses of vaccine, and in 8.4 % of patients also after the booster dose. Furthermore, the absence of T-cell immunoreactivity was observed in 3/7 patients tested by QuantiFERON® SARSCoV-2 Starter Set (Qiagen). The efficacy of vaccines, evaluated by comparing the COVID-19-related death rate recorded during pre- and post-vaccination pandemic periods, revealed a quite stable outcome in SSc patients ( death rate from 2.54 % to 1.76 %; p = ns), despite the significant drop of mortality observed in the Italian general population (from 2.95 % to 0.29 %; p < 0.0001)., Conclusions: An increased COVID-19 prevalence and mortality rate was recorded in SSc patients; moreover, the efficacy of vaccines in term of improved outcomes was less evident in SSc compared to Italian general population. This discrepancy might be explained by concomitant adverse prognostic factors: increased rate of non-responders to vaccine in SSc series, low percentage of individuals with four or more doses of vaccine, ongoing immunomodulating treatments, disease-related interstitial lung disease, and/or reduced preventive measures in the second half of pandemic. A careful monitoring of response to COVID-19 vaccines together with adequate preventive/therapeutical strategies are highly recommendable in the near course of pandemic in this frail patients' population., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Alessandro Antonelli reports financial support was provided by Italian 10.13039/100009647Ministry of Health, Ricerca Finalizzata (RF-2021-12374986) Destinatario istituzionale: Regione Toscana. Unità Operative:U.O.1: Azienda Ospedaliero-Universitaria Pisana; U.O.2: Azienda Ospedaliero-Universitaria Aldo Moro Bari; U.O.3: Azienda Ospedaliero-Universitaria Modena, CUP Master: D55E22000670001., (© 2023 Published by Elsevier B.V.)

Purpose: Mixed cryoglobulinemia syndrome (MCs) is a rare immunoproliferative systemic disorder with cutaneous and multiple organ involvement. Our multicenter survey study aimed to investigate the prevalence and outcome of COVID-19 and the safety and immunogenicity of COVID-19 vaccines in a large MCs series., Methods: The survey included 430 unselected MCs patients (130 M, 300 F; mean age 70 ± 10.96 years) consecutively collected at 11 Italian referral centers. Disease classification, clinico-serological assessment, COVID-19 tests, and vaccination immunogenicity were carried out according to current methodologies., Results: A significantly higher prevalence of COVID-19 was found in MCs patients compared to Italian general population (11.9% vs 8.0%, p < 0.005), and the use of immunomodulators was associated to a higher risk to get infected (p = 0.0166). Moreover, higher mortality rate was recorded in MCs with COVID-19 compared to those without (p < 0.01). Patients' older age (≥ 60 years) correlated with worse COVID-19 outcomes. The 87% of patients underwent vaccination and 50% a booster dose. Of note, vaccine-related disease flares/worsening were significantly less frequent than those associated to COVID-19 (p = 0.0012). Impaired vaccination immunogenicity was observed in MCs patients compared to controls either after the first vaccination (p = 0.0039) and also after the booster dose (p = 0.05). Finally, some immunomodulators, namely, rituximab and glucocorticoids, hampered the vaccine-induced immunogenicity (p = 0.029)., Conclusions: The present survey revealed an increased prevalence and morbidity of COVID-19 in MCs patients, as well an impaired immunogenicity even after booster vaccination with high rate of no response. Therefore, MCs can be included among frail populations at high risk of infection and severe COVID-19 manifestations, suggesting the need of a close monitoring and specific preventive/therapeutical measures during the ongoing pandemic., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

New-generation mRNA and adenovirus vectored vaccines against SARS-CoV-2 spike protein are endowed with immunogenic, inflammatory and immunomodulatory properties. Recently, BioNTech developed a noninflammatory tolerogenic mRNA vaccine (MOGm1Ψ) that induces in mice robust expansion of antigen-specific regulatory T (Treg) cells. The Pfizer/BioNTech BNT162b2 mRNA vaccine against SARS-CoV-2 is identical to MOGm1Ψ except for the lipid carrier, which differs for containing lipid nanoparticles rather than lipoplex. Here we report that vaccination with BNT162b2 led to an increase in the frequency and absolute count of CD4 pos CD25 high CD127 low putative Treg cells; in sharp contrast, vaccination with the adenovirus-vectored ChAdOx1 nCoV-19 vaccine led to a significant decrease of CD4 pos CD25 high cells. This pilot study is very preliminary, suffers from important limitations and, frustratingly, very hardly can be refined in Italy because of the >90% vaccination coverage. Thus, the provocative perspective that BNT162b2 and MOGm1Ψ may share the capacity to promote expansion of Treg cells deserves confirmatory studies in other settings.

This study monitored the anti-spike-receptor-binding domain (RBD) and neutralizing antibodies induced by the Pfizer/BioNTech mRNA BNT162b2 vaccine in a cohort of 163 healthcare workers aged ≤60 years. We have taken advantage of two study groups, both of whom received the first two doses in the same time window, but Group 1 (54 HCWs) received the third dose 2 months before Group 2 (68 HCWs) did. The cohorts were monitored from the 12th day after the first vaccine dose up to 1 month after the third vaccine dose for a total of eight time points and about 1 year of surveillance (T1 = 12 days after the first dose; T2 = 10 days after the second dose; T3 = 1 month after the second dose; T4 = 3 months after the second dose; T5 = 4 months after the second dose; T6 = 5 months after the second dose; T7 = 7 months after the second dose; T8 = 1 month after the third dose for Group 1; T8* = 9 months after the second dose for Group 2; T9 = 1 month after the third dose for Group 2). The mean value of anti-spike antibodies decreased faster over time, but at T7, its decline was significantly slowed (T7 vs. T8*). After the third dose, the anti-spike titer rose about 34-fold (T7 vs. T8 and T8* vs. T9) and the booster improved the anti-spike titer by about three times compared with that of the second dose (T3 vs. T8 and T3 vs. T9), and no difference was noted between the two groups. The neutralizing titer was evaluated at T3, T7, T8, and T9. Anti-spike and neutralizing antibodies were found to be strongly correlated (r 2 = 0.980; p < 0.001). At T3, 70% of the participants had a neutralizing antibody titer >91% of total anti-spike antibodies that increased to 90% after the third dose (T8 and T9). However, when the anti-spike titer reached its lowest value (T7), the neutralizing antibody levels decreased even further, representing only 44% of total anti-spike antibodies (p < 0.0001). Our findings show that the third vaccine dose improves the humoral response, but the wane of the anti-spike and neutralizing antibody titers over time is more marked in the neutralizing antibodies., Competing Interests: Author SS is employed by Synlab Lazio Srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Franzese, Coppola, Silva, Santini, Cinquanta, Ottomano, Salvatore and Incoronato.)

Autoimmune systemic diseases (ASD) show impaired immunogenicity to COVID-19 vaccines. Our prospective observational multicenter study aimed at evaluating the seroconversion elicited by COVID-19 vaccine over the entire vaccination cycle including the booster dose. Among 478 unselected ASD patients originally evaluated at the end of the first vaccination cycle (time 1), 344 individuals were re-evaluated after a 6-month period (time 2), and 244 after the booster vaccine dose (time 3). The immunogenicity of mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273) was assessed by measuring serum IgG-neutralizing antibody (NAb) on samples obtained at the three time points in both patients and 502 age-matched controls. In the 244 ASD group that received booster vaccine and monitored over the entire follow-up, the mean serum NAb levels (time 1, 2, and 3: 696.8 ± 52.68, 370.8 ± 41.92, and 1527 ± 74.16SD BAU/mL, respectively; p < 0.0001) were constantly lower compared to controls (p < 0.0001), but they significantly increased after the booster dose compared to the first two measurements (p < 0.0001). The percentage of patients with absent/suboptimal response to vaccine significantly decreased after the booster dose compared to the first and second evaluations (time 1, 2, and 3: from 28.2% to 46.3%, and to 7.8%, respectively; p < 0.0001). Of note, the percentage of patients with absent/suboptimal response after the booster dose was significantly higher compared to controls (19/244, 7.8% vs 1/502, 0.2%; p < 0.0001). Similarly, treatment with immune-modifiers increased the percentage of patients exhibiting absent/suboptimal response (16/122, 13.1% vs 3/122, 2.46%; p = 0.0031). Overall, the above findings indicate the usefulness of booster vaccine administration in ASD patients. Moreover, the persistence of a significantly higher percentage of individuals without effective seroconversion (7.8%), even after the booster dose, warrants for careful monitoring of NAb levels in all ASD patients to identify those with increased risk of infection. In this particularly frail patients' setting, tailored vaccination and/or therapeutic strategy are highly advisable., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Patients with autoimmune systemic diseases (ASDs) represent a frail population during the ongoing COVID-19 pandemic. The vaccination is the major preventive measure; however, a significant number of ASD patients show an impaired production of anti-COVID-19 neutralizing antibodies (NAb), possibly counterbalanced by adequate T-cell response. The present study aimed at evaluating both humoral and cellular response to COVID-19 vaccine booster dose in this particular setting., Patients and Methods: Serum NAb titer and T-cell response (measuring interferon gamma -IFN-γ- release) were evaluated 3 weeks after the COVID-19 vaccine booster dose, in 17 patients (12 F, mean age 68.8 ± 15.3 SD yrs) with different ASDs, compared to 17 healthy controls (HCs)., Results: The analysis excluded one patient reporting symptoms of COVID-19 only after the immunogenicity tests had been performed.The NAb levels were significantly lower in ASD compared to HCs (p < 0.0001); moreover, patients showed a higher percentage of negative/sub-optimal humoral response (31% vs 0% of HCs; p = 0.0184).The study of cellular response showed lower levels of IFN-γ for both Ag1 ( p  = 0.0032) and Ag2 (p = 0.0136) in ASD patients compared to HCs, as well lower rate of adequate T-cell response compared to HCs (50% vs 94%; p  = 0.0066).Disease modifying therapies (DMT) were administered in all patients with deficient NAb production (5/5, 100%), but in only 3/11 (27%) of responders ( p  = 0.025).Worthy to note, 3/16 (19%) ASD patients developed neither humoral nor cellular responses, all treated with DMT., Conclusions: The impaired immunogenicity to COVID-19 vaccine booster and even more the concomitant lack of both humoral and cellular response might represent a high risk for severe COVID-19, particularly in ASD patients undergoing DMT.These frail subjects should be tightly monitored for their immune protection and prioritized for the fourth dose of COVID-19 vaccine. Moreover, in the occurrence of SARS-CoV2 infection, treatments with specific monoclonal antibodies and/or antivirals may be highly recommendable., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Autoimmune systemic diseases (ASD) may show impaired immunogenicity to COVID-19 vaccines. Our prospective observational multicenter study aimed to evaluate the seroconversion after the vaccination cycle and at 6-12-month follow-up, as well the safety and efficacy of vaccines in preventing COVID-19. The study included 478 unselected ASD patients (mean age 59 ± 15 years), namely 101 rheumatoid arthritis (RA), 38 systemic lupus erythematosus (SLE), 265 systemic sclerosis (SSc), 61 cryoglobulinemic vasculitis (CV), and a miscellanea of 13 systemic vasculitis. The control group included 502 individuals from the general population (mean age 59 ± 14SD years). The immunogenicity of mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273) was evaluated by measuring serum IgG-neutralizing antibody (NAb) (SARS-CoV-2 IgG II Quant antibody test kit; Abbott Laboratories, Chicago, IL) on samples obtained within 3 weeks after vaccination cycle. The short-term results of our prospective study revealed significantly lower NAb levels in ASD series compared to controls [286 (53-1203) vs 825 (451-1542) BAU/mL, p < 0.0001], as well as between single ASD subgroups and controls. More interestingly, higher percentage of non-responders to vaccine was recorded in ASD patients compared to controls [13.2% (63/478), vs 2.8% (14/502); p < 0.0001]. Increased prevalence of non-response to vaccine was also observed in different ASD subgroups, in patients with ASD-related interstitial lung disease (p = 0.009), and in those treated with glucocorticoids (p = 0.002), mycophenolate-mofetil (p < 0.0001), or rituximab (p < 0.0001). Comparable percentages of vaccine-related adverse effects were recorded among responder and non-responder ASD patients. Patients with weak/absent seroconversion, believed to be immune to SARS-CoV-2 infection, are at high risk to develop COVID-19. Early determination of serum NAb after vaccination cycle may allow to identify three main groups of ASD patients: responders, subjects with suboptimal response, non-responders. Patients with suboptimal response should be prioritized for a booster-dose of vaccine, while a different type of vaccine could be administered to non-responder individuals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identified in China, in December 2019 determines COronaVIrus Disease 19 (COVID-19). Whether or not the virus was present in Italy earlier the first autochthonous COVID-19 case was diagnosed is still uncertain. We aimed to identify anti-SARS-CoV-2 antibodies in sera collected from 4th November 2019 to 9th March 2020, in order to assess the possible spread of the virus in Italy earlier than the first official national diagnosis., Patients and Methods: Anti-SARS-CoV-2 antibodies were evaluated in retrospective serum samples from 234 patients with liver diseases (Hep-patients) and from 56 blood donors (BDs). We used two rapid serologic tests which were confirmed by a validated chemoluminescence assay., Results: Via rapid tests, we found 10/234 (4.3%) IgG-positive and 1/234 (0.4%) IgM-positive cases in the Hep-patient group. Two/56 (3.6%) IgG-positive and 2/56 (3.6%) IgM-positive cases were detected in BD group. Chemoluminescence confirmed IgG-positivity in 3 Hep-patients and 1 BD and IgM-positivity in 1 Hep-patient. RNAemia was not detected in any of the subjects, rendering the risk of transfusion transmission negligible., Conclusions: Our results suggest an early circulation of SARS-CoV-2 in Italy, before the first COVID-19 cases were described in China. Rapid tests have multiple benefits; however, a confirmation assay is required to avoid false positive results.

Background: Infections, autoimmunity and cancer play a role as determinants of etiology in Hepatitis C virus (HCV) related mixed cryoglobulinemia (MC). Several factors of risk have been suggested as markers of pathogenesis and progression of HCV-related MC into B cell Non-Hodgkin's Lymphoma (B-NHL). Here, we evaluated IgG subclass distribution, free light chains (FLCs) and vascular endothelial growth factor (VEGF) as a new combination of biomarkers., Methods: We measured IgG1-4 subclasses, FLCs and VEGF levels in sera 53 from HCV-related MC, in comparison with 40 sera from HCV negative patients with rheumatoid arthritis (RA) and 30 from healthy blood donors (HBD)., Results: IgG3 levels were significantly higher in HCV-MC patients with a decrement of IgG2 and IgG4; FLC levels significantly increased in both MC and RA patients' groups; serological VEGF was higher in HCV-MC patients than in HBD in correlation with k and λ levels., Conclusion: Our results suggest that a specific IgG subclasses pattern together with raised levels of FLCs and VEGF could represent the biomarker "signature" of an inflammation multistage of acquired immune system., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: A still uncertain association between vitamin D levels and HCV chronic liver diseases has been reported. Increased levels of serum-free light chains (FLCs) and an altered k/λ FLC ratio correlate with Mixed Cryoglobulinemia (MC) vasculitis and/or B-cell non-Hodgkin's lymphoma in HCV-positive patients. We aimed to investigate the possible role of vitamin D, vitamin D Binding Protein (DBP), and FLCs levels as a tool for discriminating different stages of HCV- related MC and chronic liver diseases., Patients and Methods: Sixty-five untreated patients were retrospectively enrolled and 21 healthy blood donors (HBD) were used as controls. Vitamin D, DBP, FLCs, and cryoglobulins levels were measured. Based on cryoglobulins, patients were divided in three subgroups (without cryoglobulins, type II, and type III)., Results: We didn't find any significant differences in vitamin D and DBP levels between HCV patients' main groups and HBD. Serum FLCs levels were significantly higher in HCV patients than in HBD. FLCs ratio among patients' subgroups did not reveal differences., Conclusions: Our results confirm the presence of an increased serum level of FLCs in HCV patients and suggest that nor vitamin D and DBP or FLC levels can be considered reliable biomarkers for discriminating different stages of HCV-associated chronic liver diseases and/or HCV-associated extrahepatic manifestation. We confirm that serological FLCs levels are significantly higher in patients than in HBD as a signature of B cell activation in course of HCV infection.

Polycystic ovary syndrome (PCOS), as systemic disease, is accompanied by different indexes of inflammation. Free light chains of immunoglobulins (FLCs), produced by plasmacells, are released in slight excess for the immune requests, with still poorly defined physiological role but surely they represent a marker of inflammation. In order to evaluate their levels and correlate them with hyperandrogenism, we have studied a group of PCOS patients, age range 18-37 yrs, mean ± SEM body mass index (BMI) 24.1 ± 0.9 kg/m 2 ), compared with age- and BMI-matched controls, with assay of k and λ FLCs, by turbidimetric method, and their ratio in blood plasma. PCOs exhibited higher levels vs. controls: (mean ± SEM λ : 10.0 ± 0.85 mg/L vs. 8.41 ± 0.45 mg/L; k : 12.45 ± 0.72 mg/L vs. 6.41 ± 0.34 mg/L; k / λ : 1.31 ± 0.07 vs. 0.78 ± 0.04). A significant direct correlation was observed between λ -FLCs and testosterone levels, no correlation was indeed found with HOMA-IR index. These data confirm high levels of FLCs in PCOS, suggesting systemic inflammatory state and a possible role in the pathophysiology of such complex syndrome.

Clinical spectrum of hepatitis C virus (HCV)-related cryoglobulinemia varies from an asymptomatic presentation to severe vasculitis and lymphoma. A recent study in HCV-negative patients suggests that low cryoglobulins (CGs) levels are responsible for severe renal and neurological complications. The aim of this study was to identify a panel of serological biomarkers associated with low levels of CGs in HCV-positive patients. We studied a population of 79 untreated patients with chronic HCV infection: 13 naïve patients without CGs; 28 patients with asymptomatic mixed cryoglobulinemia (MC) and low levels of CGs (16/28 with polyclonal type III and 12/28 with microheterogeneous type III CGs); 38 patients with symptomatic MC and high levels of type II CGs. Serum samples were collected and examined for rheumatoid factor (RF) IgG and IgM, free light chains (FLCs) and C3 and C4 complement components. We found that RF-IgG and IgM, free k chains and k+λ were increased while C4 component was reduced, both in symptomatic and asymptomatic patients. Our results suggest that, even in absence of MC symptoms, the low levels of CGs may represent a trigger of activation for immune system in course of HCV infection. The identification of a correlated biomarkers panel could improve the clinical management of these patients and pave the way for target treatment strategies. © 2018 BioFactors, 45(3):318-325, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

Objective: Monoclonal plasma cell proliferative disorders comprise a wide spectrum of diseases associated to clonal B-cell expansion. Serum protein electrophoretic profile (SPEP) and circulating free light chains (FLCs) levels are the mainstay of diseases management. Recently, soluble (s) Syndecan-1 (SDC1, CD138) produced by myeloma plasma cells has been suggested in the monitoring and follow-up of patients with myeloma. The aim of our study is to evaluate sCD138 in addition with FLCs and SPEP for the screening of patients with different evolutive disease pathways., Patients and Methods: Sera from 73 patients with monoclonal gammopathy of undetermined significance (MGUS), 120 smoldering and 42 multiple myeloma (SMM and MM, respectively), 70 HCV-related mixed cryoglobulinemia (MC), 35 B-cell non-Hodgkin's lymphoma (B-NHL) and sera from 50 healthy donors (HD), were tested for sCD138, FLCs (assessed by means of ELISA and turbidimetric assay, respectively) and electrophoresis pattern (performed on Capillarys system) for the generation of a novel biomarker score (BS)., Results: Our results were grouped according to the two main lines of disease progression (vs. MM or B-NHL): in one group we found BS mean values of 0.2, 3.4, 5.3, 7.1 for HD, MGUS, SMM and MM, respectively; in the other group of 0.2, 4.4, 6.7 for HD, MC and B-NHL., Conclusions: We showed that BS mean values follow the ingravescence disease status towards the two main lines of progression to cancerous conditions; it could represent an additional useful tool in the management of screening and/or follow-up.

A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for identification and estimation of 18- β -glycyrrhetinic acid (GA) in HepG2 cell line. The analysis was carried out using a JASCO HPLC system with a C-18 (3  μ m) Supelco reversed phase column (150 x 4.7 mm) using a mobile phase of 80% CH 3 OH and 20% of CH 3 CN: tetrahydrofuran: water (10:80:10, v/v/v). The method was linear in the concentration range of 1.5-120  μ g /mL (n = 5). The LOD and LOQ were determined based on standard deviation of the y-intercept and the slope of the calibration curve. The LOD and LOQ values were found to be 11.46  μ g/mL and 34.72  μ g/mL, respectively. The mean percentage recovery by standard addition experiments of GA is 92.4 % ± 5.2%. The intracellular GA concentration value, obtained as mean of five different determinations, was 45.8 ± 7.45  μ g/mL. We have developed a HPLC-UV method for quantitative determination of GA inside cells, with advantages in the cost reduction and economy of the analytical process.

Objective: Mixed Cryoglobulinemia is the most well-known Hepatitis C Virus (HCV)-associated extrahepatic manifestation. MC is both an autoimmune and B-lymphoproliferative disorder. Cryoglobulins (CGs) are classified into three groups according to immunoglobulin (Ig) composition: type I is composed of one isotype or Ig class. Type II and type III mixed CGs are immune complexes composed of polyclonal IgGs acting as autoantigens and mono, polyclonal or oligoclonal IgM with rheumatoid factor activity. IgG1 and IgG3 are the predominant subclasses involved. This study shows the simultaneous presence of IgG-RF and IgG3, supporting the hypothesis of an involvement of this subclass in the initiation of early stages of CGs., Patients and Methods: We describe a case series of six HCV-positive patients, all of whom had peripheral neuropathy and transient ischemic attacks, presenting cryoprecipitates formed by IgG3 and IgG1. Cryoprecipitate IgG subclass research was carried out by immunofixation electrophoresis by using antisera against IgG1, IgG2, IgG3, and IgG4., Results: Our six patients presented with an immunochemical pattern characterized by the mere presence of IgG1 and IgG3 subclasses with probable RF activity and one of these six patients exhibited monoclonal IgG3 in his cerebrospinal fluid., Conclusions: We can hypothesize that the IgG passage through the blood-brain barrier could have contributed to the cause of TIAs, through a mechanism involving the precipitation of circulating immune complexes formed by the two subclasses in the intrathecal vessels.

Biological functions of immunoglobulin-free light chains (FLCs), other than in chronic inflammatory diseases, are still poorly defined; the field of insulin resistance (IR) has not been investigated, despite the strict relationships with oxidative stress (OS) and inflammation. Therefore, we evaluated FLCs levels and their relationships with metabolic parameters in adult growth hormone deficiency (GHD) and metabolic syndrome (MetS), both characterized by IR. One hundred subjects were enrolled: group A, patients with GHD [n =31, 24-69 years, mean ± SEM body mass index (BMI) 26.8 ± 1.5 kg/m 2 ]; group B, patients with MetS (n = 29, 21-70 years, BMI 31.9 ± 1.3); group C, controls (N = 40, 21-62 years, BMI 21.6 ± 1.1). Groups were matched by age range and, for patients, by BMI. Morning blood sample was collected for metabolic parameters and FLCs, assessed by turbidimetric assay. GHD patients show levels of FLCs significantly higher than MetS and controls (mean ± SEM κ 37.21 ± 6.97, 15.27 ± 0.86, 12.34 ± 0.85 mg/l; λ 19.44 ± 2.61, 11.78 ± 0.72 and 11.67 ± 0.77 mg/l; κ/λ ratio 1.77 ± 0.13, 1.38 ± 0.09; and 1.10 ± 0.06, respectively); only κ were higher in MetS versus controls. Therefore, the ratio showed progressive declining values in GHD versus MetS versus controls. Our data show increased FLCs levels in GHD and MetS, with the highest values in the former. Both conditions show OS, but with different molecular patterns. FLCs may contribute to chronic inflammation, leading to OS, and cardiovascular complications of GHD. Prognostic and therapeutic implications require further investigation. © 2018 BioFactors, 44(5):480-484, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Background: Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree., Methods: Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns., Results: After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate., Conclusion: Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: In order to establish a diagnosis of monoclonal gammopathy, it is necessary to detect and identify monoclonal components. To confirm the immunological nature of the proteins, the next step is to define their composition in heavy and light chains using immunofixation. The purpose of this study was to compare two different instruments, one semiautomated and the other fully automated for serum and urine immunofixation., Methods: We selected 150 sera and 100 urines from patients admitted for routine analysis, which were analyzed by immunofixation to characterize monoclonal components., Results and Conclusion: Comparison study showed a difference in the identification of small monoclonal components and hypogammaglobulinemia, in serum and urine, between the two analyzers. We also observed a difference in the length of the electrophoretic pattern that is of considerable importance as it leads to a better resolution of the gamma region, allowing to identify even the smallest monoclonal component that can be easily hide in an oligoclonal pattern. For this reason, there is need to ameliorate commercial immunofixation assays. It is essential to improve data harmonization and standardize measurement procedures in order to guarantee a correct diagnosis for the right patient care., (© 2016 Wiley Periodicals, Inc.)

Cryoglobulins are immunoglobulins that precipitate in serum at temperatures below 37°C and resolubilize upon warming. The clinical syndrome of cryoglobulinemia usually includes purpura, weakness, and arthralgia, but the underlying disease may also contribute other symptoms. Blood samples for cryoglobulin are collected, transported, clotted and spun at 37°C, before the precipitate is allowed to form when serum is stored at 4°C in a Wintrobe tube for at least seven days. The most critical and confounding factor affecting the cryoglobulin test is when the preanalytical phase is not fully completed at 37°C. The easiest way to quantify cryoglobulins is the cryocrit estimate. However, this approach has low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed of cryoglobulins. The characterization of cryoglobulins requires the precipitate is several times washed, before performing immunofixation, a technique by which cryoglobulins can be classified depending on the characteristics of the detected immunoglobulins. These features imply a pathogenic role of these molecules which are consequently associated with a wide range of symptoms and manifestations. According to the Brouet classification, Cryoglobulins are grouped into three types by the immunochemical properties of immunoglobulins in the cryoprecipitate. The aim of this paper is to review the major aspects of cryoglobulinemia and the laboratory techniques used to detect and characterize cryoglobulins, taking into consideration the presence and consequences of cryoglobulinemia in Hepatitis C Virus (HCV) infection.

Primary hyperparathyroidism (PHPT) is associated with hypovitaminosis D as assessed by serum total 25-hydroxyvitamin D (TotalD) levels. The aim of this study is to evaluate whether this is also the case for the calculated bioavailable 25-hydroxyvitamin D (BioD) or free 25-hydroxyvitamin D (FreeD), and whether the vitamin D status is influenced by genetic background. We compared vitamin D status of 88 PHPT patients each with a matched healthy family member sharing genetic background, i.e., first-degree relative (FDR), or not, namely an in-law relative (ILR). We compared TotalD and vitamin D-binding protein (DBP), using the latter to calculate BioD and FreeD. We also genotyped two common DBP polymorphisms (rs7041 and rs4588) likely to affect the affinity for and levels of vitamin D metabolites. TotalD was lower (p < 0.001) in PHPT (12.3 ± 6.6 ng/mL) than either family member group (FDR: 19.4 ± 12.1 and ILR: 23.2 ± 14.1), whether adjusted for DBP or not. DBP levels were also significantly lower (p < 0.001) in PHPT (323 ± 73 mg/L) versus FDR (377 ± 98) or ILR (382 ± 101). The differences between PHPT and control groups for TotalD, BioD, and FreeD were maintained after adjustment for season, gender, and serum creatinine. 25-hydroxyvitamin D, evaluated as total, free, or bioavailable fractions, is decreased in PHPT. No difference was seen between first-degree relative and in-law controls, suggesting that neither genetic nor non-genetic background greatly influences the genesis of the hypovitaminosis D seen in PHPT.

Objective: To investigate the role of CYP2D6 phenotype in the outcome of postoperative (PO) pain (POP) treatment., Design: Longitudinal cohort study. Open-label trial with post hoc analysis., Setting: General Hospital Surgery and Recovery Units., Patients: Ninety unrelated Caucasians submitted to abdominal/thoracic surgery., Interventions: Standard multimodal POP treatment including opioids (tramadol) and nonsteroidal anti-inflammatory drugs (ketoprofen) at different dosages and infusion rates according to the predicted mild, moderate, or severe POP., Outcome Measures: Pain (Numeric Rating Scale-NRS) and sedation (Ramsay Sedation Scale-RSS) up to 24 hours after surgery. By genotyping 16 CYP2D6 alleles, the four CYP2D6 phenotypes poor metabolizer (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM) were predicted., Results: As compared with the CYP2D6-EM phenotype, in the early PO time (30 min) a higher RSS mean score in IM was observed (P = 0.035). A suggestion towards higher mean score in PM (P = 0.091) and a minor mean score in UM (P = 0.091) was also detected. No difference in the outcome of pain across the CYP2D6 phenotypes was observed., Conclusions: In respect to the normal CYP2D6 phenotype, our results suggested that slowly metabolizers (IMs and PMs) might have a major sedation, whereas more rapid metabolizers (UM) a minor sedation, in the early time after surgery. A minor role of CYP2D6 phenotype in PO analgesia may be suggested., (Wiley Periodicals, Inc.)

Background and Aims: Several studies have reported that the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) K121Q polymorphism (rs1044498) interacts with increased adiposity in affecting glucose homeostasis and insulin sensitivity. Conversely, one would expect that the amelioration of glucose homeostasis observed after weight loss is modulated by the ENPP1 K121Q polymorphism. The aim of our study was to test such hypothesis, in non-diabetic overweight-obese individuals., Methods and Results: Two hundred eleven non-diabetic overweight-obese individuals were studied. Body mass index (BMI), fasting glucose, homeostasis model assessment of insulin resistance (HOMA-IR index) and lipid levels were obtained before and after 6-week lifestyle intervention (LI; diet and exercise) and their changes calculated as baseline minus 6-week values. LI decreased BMI, glucose, HOMA-IR and triglyceride levels (p < 0.001 for all). No difference across genotype groups (160 KK and 51 KQ or QQ - named as XQ - individuals) was observed in these changes. In a multivariate model, BMI changes predicted fasting glucose changes (β = 0.139 mmol/L (2.50 mg/dl) for 1 unit BMI change, p = 0.005). This correlation was not significant among KK individuals (β = 0.082; p = 0.15), while much steeper and highly significant among XQ individuals (β = 0.336; p = 0.00008) (p-value for Q121-by-weight loss interaction = 0.047)., Conclusion: Individuals carrying the ENPP1 Q121 variant are highly responsive to the effect of weight loss on fasting glucose. This reinforces the previously suggested hypothesis that the Q121 variant interacts with adiposity in modulating glucose homeostasis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aim: We investigated inpatients with and without Type 2 diabetes mellitus, aged over 60 yr, to compare their vitamin D status and calcium homeostatic parameters., Materials and Methods: We studied 140 patients consecutively admitted to our Internal Medicine Unit during the year 2010 (61 from November to April, 79 from May to October). The sample encompassed 70 patients with and 70 without diabetes. At admission we measured serum calcium (Ca), phosphate (P), sodium (Na), potassium (K), creatinine (Cr), alkaline phosphatase total activity (AP), albumin adjusted serum calcium (Caalb adj), 25 hydroxy-vitamin D (25OHD), PTH, and 24-h urinary Na/Cr (uNa/Cr), K/Cr (uK/Cr), Ca/Cr (uCa/Cr), P/Cr (uP/Cr) ratios, and calcium excretion (Ca ex)., Results: 25OHD levels of patients with and without diabetes did not significantly differ. In patients without diabetes recruited from November to April, 25OHD levels were significantly lower than those from May to October, whilst patients with diabetes did not show a significant seasonal variation. PTH had opposite non-significant seasonal variations, and negatively correlated with 25OHD in both groups of patients. This correlation was lost after adjusting for age and body mass index in patients with diabetes. These inpatients had higher serum P and lower uP/Cr, according to lower PTH. Their serum glucose negatively correlated with uCa/Cr and Ca ex, contrary to inpatients with other diseases. Instead, uCa/Cr and Ca ex correlated with uNa/Cr only in patients without diabetes., Conclusions: Inpatients with diabetes did differ from those with other disorders for vitamin D status and calcium-phosphate homeostatic mechanism.

The prevalence of therapeutic failures (TFs) and adverse drug reactions (ADRs) markedly increased in older subjects. However, both TFs and ADRs did not always appear related to the presence of multiple pharmacologic treatments, a common status in subjects aged 65 and over. Instead, they seem more related to variations in the genes encoding protein metabolizing and transporting drugs. Thus, variations in these proteins may account for the inter-individual differences observed in drug efficacy, including the most severe clinical consequences TFs and ADRs. The genetics of drug metabolizing enzymes (DMEs) and drug transporters (DTs) is a very active area of multidisciplinary research, overlapping the fields of medicine, biology, pharmacology, and genetics. These proteins are virtually responsible for metabolism and disposition, and thus the efficacy, of a number of drugs currently used in clinical practice. This article explored some basic concepts of the pharmacogenetics of DMEs and DTs. We also focused current knowledge of the genetic basis of TFs and ADRs of the most common drugs currently used in geriatric clinics. The knowledge of what we know and what we need to know is needed to advance the application of pharmacogenetics in clinical practice, in order to introduce personalized treatments for elderly people.

Objective: Cytochrome P450 (CYP) 2D6 enzyme is the major responsible for the metabolism of donepezil, an inhibitor of acetyl cholinesterase currently used for the symptomatic treatment of mild-to-moderate Alzheimer's disease (AD). Functional polymorphisms in the CYP2D6 gene may affect enzyme activity and thus, the metabolism of donepezil. The aim of this study was to evaluate the effect of 16 functional polymorphisms in the CYP2D6 gene on the clinical response to donepezil treatment in patients with mild-to-moderate AD., Methods: In this multicenter prospective cohort study we evaluated 57 unrelated Caucasians clinically diagnosed as AD according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group criteria. Patients were treated with donepezil (5-10 mg/daily) for 6 months. The response to donepezil treatment was evaluated at 6-month follow-up according to the National Institute for Health and Clinical Excellence requirements. The identification of 16 clinically relevant CYP2D6 gene variants was performed by a high-throughput genetic analysis., Results: Thirty-eight of 57 patients (67%) were responders and 19 patients (33%) were nonresponders to donepezil treatment. A significantly higher frequency of gene variants conferring decreased or absent enzyme activity was observed in responder than in nonresponder patients (73.68% vs. 36.84%; P=0.005). The presence of gene variants conferring decreased or absent activity of the CYP2D6 enzyme was significantly associated with a clinical response to donepezil treatment (odds ratio=6.286; 95% confidence interval=1.828-21.667)., Conclusions: Functional polymorphisms in the CYP2D6 gene can influence the clinical efficacy of donepezil. The analysis of CYP2D6 genotypes may be useful in identifying subgroups of AD patients with different clinical response to donepezil treatment.

Background: Recent studies investigating the single cytochrome P450 (CYP) 2D6 allele *2A reported an association with the response to drug treatments. More genetic data can be obtained, however, by high-throughput based-technologies. Aim of this study is the high-throughput analysis of the CYP2D6 polymorphisms to evaluate its effectiveness in the identification of patient responders/non-responders to CYP2D6-metabolized drugs., Methods: An attempt to compare our results with those previously obtained with the standard analysis of CYP2D6 allele *2A was also made. Sixty blood samples from patients treated with CYP2D6-metabolized drugs previously genotyped for the allele CYP2D6*2A, were analyzed for the CYP2D6 polymorphisms with the AutoGenomics INFINITI CYP4502D6-I assay on the AutoGenomics INFINITI analyzer., Results: A higher frequency of mutated alleles in responder than in non-responder patients (75.38 % vs 43.48 %; p = 0.015) was observed. Thus, the presence of a mutated allele of CYP2D6 was associated with a response to CYP2D6-metabolized drugs (OR = 4.044 (1.348 - 12.154). No difference was observed in the distribution of allele *2A (p = 0.320)., Conclusions: The high-throughput genetic analysis of the CYP2D6 polymorphisms better discriminate responders/non-responders with respect to the standard analysis of the CYP2D6 allele *2A. A high-throughput genetic assay of the CYP2D6 may be useful to identify patients with different clinical responses to CYP2D6-metabolized drugs.

Introduction: Previous papers investigating vitamin D status have often outlined the significant relationships between serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD), but the influence of ionized calcium levels has not been concomitantly considered., Design: Cross-sectional., Materials and Methods: In 1050 healthy men (547) and women (503), serum ionized calcium (iCa), creatinine (Cr), albumin, 25OHD, and PTH were measured. After conventional analysis, a regression tree was fitted on the data set., Results: 25OHD and PTH values showed significant opposite seasonal changes. 25OHD levels negatively correlated with PTH, which in turn negatively correlated with iCa. A regression tree was fitted to the whole data set using PTH as the response variable and 25OHD and iCa as covariates. PTH concentration depended on that of iCa only in subjects with 25OHD levels>16.35 ng/mL, while for 25OHD<16.35 ng/mL it depended on 25OHD values., Conclusions: Our results indicated that PTH levels were highly conditioned by those of 25OHD in subjects with 25OHD values lower than 16.35 ng/mL and by those of iCa only for higher 25OHD concentration., (Copyright 2010 Elsevier Inc. All rights reserved.)

In the recent decades, oxidative stress has become focus of interest in most biomedical disciplines and many types of clinical research. Increasing evidence from research on several diseases show that oxidative stress is associated with the pathogenesis of diabetes, obesity, cancer, ageing, inflammation, neurodegenerative disorders, hypertension, apoptosis, cardiovascular diseases, and heart failure. Based on this research, the emerging concept is that oxidative stress is the "final common pathway", through which risk factors of several diseases exert their deleterious effects. Oxidative stress causes a complex dysregulation of cell metabolism and cell-cell homeostasis. In this review, we discuss the role of oxidative stress in the pathogenesis of insulin resistance and beta-cell dysfunction. These are the two most relevant mechanisms in the pathophysiology of type 2 diabetes, and in the pathogenesis of diabetic vascular complications, the leading cause of death in diabetic patients.

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia (CAH), an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease- causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. In this report, we report the characterisation of two novel CYP21A2 missense mutations (p.H119R and p.I194N) found in two unrelated Italian patients with nonclassic (NC) CAH clinical diagnosis. Functional in vitro assays for mutagenized CYP21 enzymes were performed in transiently transfected mammalian cells to test the residual enzyme activity and the apparent kinetic values. The residual activities obtained for p.H119R and p.I194N mutants allowed to classify them as NC-CAH associated mutations. These results correlate with the rate of severity of the patients' disease. Finally, the new p.H119R and p.I194N mutations should be included in the panel of those already listed for association with the NC form of 21-hydroxylase deficiency., ((c) 2009 IUBMB IUBMB Life 61(3): 229-235, 2009.)

Background: The development of non-invasive procedure to determine HER2 status may represent a powerful method for monitoring disease progression and response to the treatment., Methods: Serum samples and RNA from peripheral blood were evaluated in 85 breast cancer patients (49 HER2 positive and 36 HER2 negative) and 22 healthy controls. HER2 mRNA levels were measured by real-time quantitative PCR (QPCR) and serum HER2 protein by immunoenzimatic assay (EIA). ROC curve analyses were used to determine the optimal cut off values., Results: A statistically significant difference was detected for both QPCR and EIA in HER2 positive patients as compared with both healthy controls and HER2 negative tumours. QPCR showed a 91% (CI95%: 84%-98%) specificity and a 78% (CI95%: 68%-88%) sensitivity for an optimal cut off value of 4.74. The optimal cut off value for EIA was 22 ng/ml yielding a 95% (CI95%: 90%-100%) specificity and a 59% (CI95%: 48%-70%) sensitivity. The QPCR assay was slightly less specific than EIA in discriminating HER2 positive breast cancers from HER2 negative tumours (78% CI95%: 69%-87% versus 86% CI95%: 79%-93%), but it was more sensitive (76% CI95%: 67%-85% versus 55% CI95%: 44%-66%)., Conclusions: Our results indicate that QPCR performs better than EIA in the determination of HER2 status of breast cancer patients and could be useful in monitoring the disease during follow up.

Objective: The aim of this study is to verify whether, early in the course of type 1 diabetes and assuming hyperglycemia as the only risk factor, women demonstrate a change in oxidative status due to an interaction between nitric oxide (NO) and uric acid production., Methods: Thirty-eight women with type 1 diabetes of less than 10 years' duration and with no diabetic complications were compared with 25 matched healthy female controls. Insulin, C-peptide, NO, HbA(1c) and oxidative stress metabolites were determined from venous blood samples taken from all patients after a 12 h overnight fast. Urine samples were used for urinary uric acid determination., Results: Most oxidative stress metabolites were significantly increased (p < 0.0001), while plasmatic and urinary uric acid levels were significantly lower (p < 0.0001) in patients with type 1 diabetes compared with controls. Mean NO levels were inversely related to uricemia. Bivariate regression analysis showed a significant correlation between plasmatic uric acid and NO (p = 0.004), ascorbic acid (p = 0.042), triglycerides (p = 0.014) and HbA(1c) (p < 0.0001). Linear multivariate regression analysis showed a significant relationship between HbA(1c) and plasmatic uric acid (beta = - 0.465, p = 0.0004)., Conclusions: Oxidative stress is already present in the early stages of type 1 diabetes. We conclude that the initial increase in oxidative stress could be linked to a reduction in plasmatic levels of uric acid, which is probably directly caused by an overproduction of NO., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Objectives: To report a first case of 21-hydroxylase deficiency associated with a new genotype determined by V281+I172N/V281L mutations of the CYP21A2 gene., Design and Methods: Direct genetic sequencing of CYP21A2 gene was performed., Results: Both siblings had the same genotype, namely V281+I172N/V281L, while their parents resulted as V281L and V281+I172N carriers, respectively., Conclusions: V281+I172N/V281L genotype should be included in the panel of mutations associated with the non-classical forms of 21-hydroxylase deficiency.

Background: Hypercortisolism is known to cause osteoporosis., Objective: To evaluate the prevalence of subclinical hypercortisolism in participants referred for evaluation of osteoporosis., Design: Cross-sectional study., Setting: Two community hospitals and research institutes in Italy., Patients: 219 patients without clinically overt hypercortisolism or other secondary causes of osteoporosis who were referred for evaluation of osteoporosis between January 2005 and December 2005., Measurements: Bone mineral density was measured by using dual-energy x-ray absorptiometry, and hypercortisolism was assessed with serum cortisol levels after a dexamethasone suppression test. Also measured were 24-hour urinary free cortisol levels and midnight plasma cortisol levels., Results: Seven of 65 patients with T-scores of 2.5 or less and vertebral fractures had subclinical hypercortisolism (prevalence, 10.8% [95% CI, 3.23% to 18.31%]). This prevalence was 4.8% (CI, 1.32% to 8.20%) among patients with osteoporosis. In multivariable analyses adjusted for age, sex, and body mass index, a positive dexamethasone suppression test result was associated with the presence of osteoporosis (odds ratio, 3.37 [CI, 1.78 to 6.43]; P < 0.001) and vertebral fractures (odds ratio, 1.70 [CI, 1.04 to 2.79]; P = 0.035)., Limitations: The study was conducted in a referral setting; its findings may not apply to the general population., Conclusions: Subclinical hypercortisolism may be more common than is generally recognized in patients with osteoporosis in whom secondary causes of osteoporosis have been excluded.

Background: Amplification of the Her2neu oncogene is a well known indicator of poor prognosis in breast cancer patients. The implementation into the clinical setting of therapeutical strategies directly targeting the Her2neu gene product, has create the need for the development of non-invasive analytical techniques in order to monitoring minimal residual disease and response to the treatment., Methods: To detect the expression of Her2neu mRNA in peripheral blood, we developed a specific real-time quantitative reverse transcription-PCR (QPCR) assay. The analyses were performed on blood samples obtained from 30 breast cancer patients positive for Her2neu overexpression by immunohistochemical analysis (IHC), 10 breast cancer patients negative for Her2neu overexpression, and 24 healthy controls., Results: Her2neu positive tumors showed a significant increase in mRNA transcripts as compared with both healthy controls (n=24) and Her2neu negative patients (n=10). After establishing a cut-off value, 18 out of the 30 Her2neu positive patient scored positive for Her2neu expression, whereas only 1 out of the 10 Her2neu negative patients was weakly positive., Conclusions: Her2neu QPCR is suitable method for Her2neu overexpression detection in peripheral blood from clinical samples of breast cancer patients. QPCR could be used to identify breast cancer patients with poor prognosis and for monitoring response to the therapy.

Mass spectrometry-based approaches are the reference techniques for the determination of nitrite and nitrate in plasma and serum. However, due to their simplicity and rapidity, assays based on the Griess reaction or HPLC are generally used in clinical studies, but they generate diverging values for nitrite/nitrate concentration. In this study, particular attention is paid to the optimization of the deproteinization procedure for plasma and serum samples prior to nitrite/nitrate analysis by an enzymatic batch Griess assay, HPLC and GC-MS. A method is reported to verify completeness of deproteinization and to correct for nonspecific contribution to the absorbance of the diazo dye at 540 nm. With the application of such optimized procedures, we were able to significantly improve the correlation between Griess and HPLC method or the GC-MS technique for nitrite+nitrate concentrations in human serum and plasma. Despite remaining potentially interfering pre-analytical and analytical factors, the procedures reported in the present study may be helpful in a critical evaluation of limits and possibilities of the enzymatic batch Griess assay as a large-scale method for nitrite/nitrate determination in human serum in clinical studies.

Background: Patients with type 1 diabetes (T1DM) present lower serum free IGF and IGFBP-3 values than healthy people. T1DM patients often present with associated autoimmune diseases such as thyroiditis or coeliac disease, and over time they frequently develop proliferative retinopathy, neuropathy or nephropathy in different combinations., Objective: The aim of this study was to evaluate the effect of two associated autoimmune diseases or three diabetic complications on the serum free IGF-I or IGFBP-3 levels in T1DM patients, who also have a family history of T1DM. Design. 246 T1DM patients were enrolled, and then subdivided into groups according to diabetic complications or associated autoimmune diseases. Demographic and clinical data were recorded. Serum free IGF-I and IGFBP-3 levels were determined by IRMA., Results: IGF-I and IGFBP-3 generally present correlated serum values as confirmed in this study. Those patients with autoimmune thyroiditis and coeliac disease presented with significantly lower serum values of IGFBP-3, whereas free IGF-I was significantly lower in patients with the different diabetic complications. Retinopathy presented a slightly significant reduction in serum free IGF-I, while neuropathy and nephropathy showed a more pronounced fall. The number of complications was related to progressively decreasing free IGF-I levels. T1DM family history was associated with lower serum free IGF-I concentrations. These findings were confirmed after correction for age, glycosylated haemoglobin levels, insulin treatment protocol, body mass index, serum creatinine and sex., Conclusion: Despite a direct correlation between serum free IGF-I and IGFBP-3, the correlation between the two molecules in patients with associated autoimmune diseases was lost, possibly due to different mechanisms of metabolic regulation.

Unlabelled: Insulin-like growth factor-1 (IGF-1) is involved in regulating the Th-1/Th-2 balance, favoring the development of the Th-2 compartment which enhances fibrosis, one of the main characteristics of Chronic Lung Disease (CLD) in premature newborns. Limited data is available concerning a possible association between early epithelial lining fluid (ELF) concentrations of IGF-1 (total and free forms), IGF-binding protein-3 (IGFBP-3), beta2-microglobulin and subsequent development of CLD in preterm neonates. If neutropenic, preterm neonates are frequently treated with recombinant human granulocyte colony stimulating factor (rhG-CSF). The objective of the study was to correlate ELF concentrations of IGF-1 and beta2 microglobulin during the first week of life both in non-neutropenic and in rhGCSF-treated neutropenic preterm neonates, with subsequent development in CLD. Thirty preterm neonates with Respiratory Distress Syndrome (6 with neutropenia) were studied. Eleven out of 24 non-neutropenic preterm infants (46%) and all of the six neutropenic subjects (100%) developed CLD. With the exception of first day values, there was a clear similarity in the behaviors of assayed molecules between non-neutropenic and neutropenic patients developing CLD. Non-neutropenic patients without CLD showed significantly lower values of free IGF-1 and beta2M both on days 1 and 3. Total IGF-I and cell counts were different only on the 3rd day., Conclusions: 1) the mechanisms leading to CLD might be mediated by high levels of IGF-family molecules soon after birth 2) beta2M could be a marker of increased bronchoalveolar lavage fluid cellularity with potential inflammatory properties 3) G-CSF treatment induces an increased synthesis of IGF-1 molecules by cells recruited in the lung, with possible enhancement of the fibrogenic mechanisms.

Oxidative stress is implicated in the pathogenesis of diabetes mellitus. Taurine and vitamin E+selenium supplementation has some benefits in experimental models of diabetes mellitus. This study evaluates whether taurine and vitamin E+selenium supplementations reduce a hard end-point such as mortality due to diabetes. Streptozotocin-induced diabetic rats were fed with standard diet or taurine (5%, w/w) or vitamin E (500 UI/Kg)+selenium (8 mg/Kg) enriched diets. Taurine significantly decreased mortality rate (p < 0.04), while vitamin E failed to increase survival. In the late phase of the disease, taurine significantly decreased glycaemia, being vitamin E ineffective. No correlation between glycaemia and survival was found. None of supplementations modified body weight. Thus, only taurine decreases the mortality rate and glycaemia. These results encourage new research in the field, since classical hypoglycaemic agents are unable to decrease mortality in diabetic patients.