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The fatty acid profile, vitamins A and E, cholesterol, antioxidant power colour and the phenols profile of Sarda sheep milk from 11 commercial sheep flocks managed under permanent grassland were investigated. In each farm, the structural and managerial data and milk samples were collected during four periods (sampling dates, SD): January, March, May, and July. Data from the milk composition (fat, protein, casein, lactose, and somatic cell count), 68 fatty acids, 7 phenols, 1 total gallocatechin equivalent, ferric reducing antioxidant power, vitamins A and E, cholesterol, degree of antioxidant protection, and the colour (b *, a * and L *) were analyzed by multivariate factorial analysis using a principal component analysis approach. A proc mixed model for repeated measurement to point out the studied factors affecting significant macro and micro milk composition was also used. Only the first five components were detailed in this paper, with approximately 70% of the explained variance detected. PC1 presented the highest positive loadings for milk lactose, de novo FA synthesis and the BH intermediate, whereas OBCFA had negative loadings values. The PC2, LCFA, UFA, MUFA, vitamins E, and DAP showed positive loadings values, while SFA had a negative value. The PC3 showed a high positive loading for total phenols and non-flavonoids. PC4 presented a high positive loading for the milk macro-composition and negative values for n-3 FAs. The PC5 is characterized by high positive loadings for the a * and L * colour parameters whereas negative loadings were detected for the milk flavonoids content. These preliminary results could help to establish future threshold values for the biomarkers in milk sourced from grazing dairy sheep in natural, permanent pasture-based diets.

Keywords: light-chain amyloidosis; T-cell receptor repertoire; regulatory T-cells; flow cytometry; spectratyping; CDR3

Although a number of studies suggest that different immune pathways may play a role in the pathogenesis of non-Hodgkin's lymphomas (NHL), the shape of the T-cell compartment has been only superficially explored in these patients. In our study, we analyzed the peripheral T-cell receptor (TCR) repertoire and the distribution of different T-cell subsets - including regulatory T cells (Treg) - in 30 patients with NHL, by combining flow cytometry and spectratyping. We first demonstrated by flow cytometry an increased frequency of expanded T-cell subpopulations expressing the same TCR beta variable (BV) subfamilies in CD8+ cells from NHL patients when compared with healthy controls, beside a higher frequency of Treg. Moreover, NHL patients were characterized by a higher percentage of BVs showing a skewed CDR3 profile both in CD4+ and CD8+ cells when analyzed by spectratyping. Our data suggest that the T-cell branch of the immune system of patients with B-cell NHL is deeply deranged, as witnessed by the increased degree of activation and skewing of their TCR repertoire along with the higher frequency of Treg.

Because different findings suggest that an immune dysregulation plays a role in the pathogenesis of myelodysplastic syndrome (MDS), we analyzed a large cohort of patients from a homogeneous Sardinian population using ImmunoChip, a genotyping array exploring 147,954 single-nucleotide polymorphisms (SNPs) localized in genomic regions displaying some degree of association with immune-mediated diseases or pathways. The population studied included 133 cases and 3,894 controls, and a total of 153,978 autosomal markers and 971 non-autosomal markers were genotyped. After association analysis, only one variant passed the genome-wide significance threshold: rs71325459 ( p = 1.16 × 10 −12 ), which is situated on chromosome 20. The variant is in high linkage disequilibrium with rs35640778, an untested missense variant situated in the RTEL1 gene, an interesting candidate that encodes for an ATP-dependent DNA helicase implicated in telomere-length regulation, DNA repair, and maintenance of genomic stability. The second most associated signal is composed of five variants that fall slightly below the genome-wide significance threshold but point out another interesting gene candidate. These SNPs, with p values between 2.53 × 10 −6 and 3.34 × 10 −6 , are situated in the methylene tetrahydrofolate reductase ( MTHFR ) gene. The most associated of these variants, rs1537514, presents an increased frequency of the derived C allele in cases, with 11.4% versus 4.4% in controls. MTHFR is the rate-limiting enzyme in the methyl cycle and genetic variations in this gene have been strongly associated with the risk of neoplastic diseases. The current understanding of the MDS biology, which is based on the hypothesis of the sequential development of multiple subclonal molecular lesions, fits very well with the demonstration of a possible role for RTEL1 and MTHFR gene polymorphisms, both of which are related to a variable risk of genomic instability.

Regulatory T cells (Treg) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed Treg frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25high+CD127low Treg than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal disorders.

Single nucleotide polymorphisms (SNPs) in the T-cell receptor (TCR) gene segments might play a role in shaping the TCR repertoire. Three polymorphisms have been described for the TCRBV20S1 gene segment, one of which is responsible for a nucleotide substitution at position 524, resulting in the introduction of a stop codon. Individuals homozygous for this inactivating polymorphism ("null allele") are unable to express TCRBV20 gene products. Using DNA restriction digestion analysis, we investigated the frequency of this polymorphism in 111 healthy Sardinian subjects. Inhabitants of the Mediterranean island of Sardinia are considered to represent a genetically isolated population. Our analyses revealed an incidence of 19.8% of homozygosity for the null allele, corresponding to an allele frequency of 0.45. Such an incidence, significantly higher than the one detected in 83 non-Sardinian Caucasians (6%), is the most elevated so far reported in the literature. BV20 is a single member subfamily and the null allele produces a gap in the potential TCR repertoire. Therefore, it is possible that an undetermined selective pressure could have played a role in determining the high frequency of this inactivating polymorphism in Sardinians. Alternatively, this finding could be related to a founder effect in this ancient island population.

The T-cell receptor (TCR) is the key player within the so called immunological synapse and the analysis of its repertoire offers a picture of both versatility and wideness of the whole immune T-cell compartment. Among the different approaches applied to its study the so-called spectratyping identifies the pattern of the third complementarity determining region (CDR3) length distribution in each one of the beta variable (TRBV) subfamilies encoded by the corresponding genes. This technique consists in a CDR3 fragment analysis through capillary electrophoresis, performed after cell separation, RNA extraction and reverse transcriptase PCR. This review will run through the most relevant studies which have tried to dissect the TCR repertoire usage in patients with different immune-mediated and infective diseases as well as solid or haematologic malignancies.

Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia show several immunological abnormalities. In this clinical setting, by combining flow cytometry and CDR3 spectratyping we monitored the kinetic of the T-cell repertoire during Azacitidine treatment, in order to explore its potential ability to reverse the immune derangement typical of these disorders. We firstly demonstrated by flow cytometry an increase in both CD4+ and CD8+ T-cell frequencies after starting treatment. Moreover, when monitored by spectratyping our patients showed significant changes in their T-cell receptor (TCR) CDR3 profiles, which were much more evident in helper T-cells. In fact, the frequency of BV (beta variable) subfamilies showing a skewed CDR3 profile significantly decreased from baseline to the following evaluations in CD4+ T-cells (81% vs. 70%). This pattern was even more pronounced in patients responding to Azacitidine (90% vs. 61%). Our data show that the overall derangement of the T-cell repertoire detectable in patients with MDS and AML with multilineage dysplasia gradually improves during Azacitidine treatment. These findings therefore suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these hematologic disorders.

Patient: Male, 64 Final Diagnosis: Acute myeloid leukemia (AML) Symptoms: — Medication: — Clinical Procedure: — Specialty: — Objective: Unusual clinical course Background: Central nervous system (CNS) involvement is a sporadic presenting finding in patients with acute myeloid leukemia (AML) both at diagnosis and at relapse. Moreover patients with CNS localization are often asymptomatic, while sometimes show meningeal signs and symptoms or, extremely rarely, signs of cranial nerve impairment. Case Report: Here we report on a patient with refractory AML who suddenly developed strabismus and diplopia. Both neurological and ophtalmologic examinations were suggestive of a bilateral VI cranial nerve palsy. Noteworthy, both a cranial CT and MRI were substantially normal, while a rachicentesis was performed and cerebrospinal fluid examination was clearly suggestive of a meningeal involvement by AML. Conclusions: This is to our knowledge the first reported case in which the clinical picture of meningeal localization in an AML patient was dominated by an isolated abducens cranial nerve impairment. Moreover it highlights as unexplained strabismus and diplopia can be considered as a potential sign of CNS involvement, even if conventional imaging is negative.

Regulatory T cells (T(reg)) have often been ascribed a role in the pathophysiology of several neoplastic diseases considering their potential ability to suppress anti-tumor immunity. This is particularly the case in myelodysplastic syndromes (MDS), which are clonal hematologic disorders characterized by marked immune dysregulation. We analyzed T(reg) frequencies in a cohort of 36 patients with early-stage MDS using a flow-cytometric approach based on the concomitant expression of CD25 and CD127. MDS patients showed a higher frequency of CD4+CD25(high)+CD127(low) T(reg) than healthy controls (1.51 vs. 1.14%), with no specific effect of patient- and disease-related factors. Our data point to impaired anti-tumor immunity in patients with MDS, even in the early stage, which has already been noted in other clonal disorders.

Among the different T-cell receptor (TCR) BV20S1 polymorphisms, nucleotide substitution at position 524 results in the introduction of a stop codon, whose potential functional relevance is still unknown. We have recently showed in Sardinian subjects the most elevated allele frequency ever reported worldwide for this "null allele" (0.44). As this variant generates a gap in the TCR repertoire, this preliminary finding prompted us to further analyze the role of this polymorphism in the susceptibility to type 1 diabetes (T1D) and multiple sclerosis (MS), which are extremely common in this population. With this aim, we evaluated the influence of the TCRBV20S1 polymorphism by assessing it with the transmission disequilibirum test (TDT) in 652 T1D and 616 MS families, without detecting any significant difference. We conclude that the high frequency of this null allele in Sardinia is not directly related to the high incidence of these autoimmune diseases observed in this founder population.

Several data suggest that stochastic rearrangements of the TCR could play a pathogenic role in both disease predisposition and protection in type 1 diabetes (T1D). As twin sets offer an enormous potential in evaluating the role of genetic and environmental factors in susceptibility to disease, the main goal of this study was to assess whether the degree of sharing of the expressed TCR repertoire of twin pairs discordant for T1D differs from that of disease concordant pairs. We performed our analysis in 5 pairs of monozygotic twins, 3 of which were concordant and 2 discordant for T1D, by combining flow cytometry and CDR3 spectratyping on both CD4+ and CD8+ T-cells. Our data show that TCR repertoires show increased level of concordance within each twin pair, especially in CD8+ cells, in terms of mean BV expression levels on flow cytometry as well as of CDR3 patterns and frequencies of skewed or oligoclonal BV subfamilies on spectratyping. It is worth noting that the degree of similarity among twins seems to be independent of concordance or discordance for T1D. Our findings seem to suggest that in monozygotic twins with T1D the TCR repertoire is influenced by genetic factors more than by the presence of the autoimmune disorder itself.

Objective Immune dysregulation plays a role in the pathophysiology of myelodysplastic syndromes (MDS), as T-cell clones seem to be involved in the inhibition of hematopoietic precursors. The purpose of this study was to analyze the T-cell receptor (TCR) repertoire of MDS patients, focusing on the third complementarity determining region (CDR3) pattern of their CD4 + and CD8 + lymphocyte expansions. Materials and Methods The study involved 30 patients and 15 age-matched controls. The β -variable ( β V) subfamily flow-cytometry analysis was performed on peripheral CD4 + and CD8 + T-cells. Spectratyping TCR-CDR3 analysis was carried out on isolated helper and cytotoxic T lymphocytes after immunomagnetic separation and reverse-transcriptase polymerase chain reaction. Results We first identified by flow cytometry an increased frequency of expanded βVs in both CD4 + and CD8 + T-cells in MDS patients. We then showed, by spectratyping, that the CDR3 profile was mostly Gaussian in their CD4 + T cells, whereas CD8 + T cells usually showed skewed or oligoclonal CDR3 regions. When we compared spectratyping and flow-cytometry findings in each patient, we showed that most CD4 + lymphocyte expansions detected by flow cytometry had Gaussian CDR3 profiles, whereas most CD8 + expansions were oligoclonal. Conclusion We confirm that in MDS patients the TCR- β V repertoire is overall extremely contracted, especially in cytotoxic T cells. This pattern is mainly determined by selective proliferations of both helper and cytotoxic T cells, which are, however, mostly polyclonal in the former and oligoclonal in the latter. Such a difference, possibly related to the different human leukocyte antigen restriction, could reflect the selective involvement of cytotoxic T cells either in the anti-tumor immune surveillance or in an autoreactive aggression toward hematopoietic precursors.

Even though the expression of CD4 and CD8 on thymocytes is considered mutually exclusive, CD4 + CD8 + double-positive T cells (DP) represent a small subset of T lymphocytes that have been described in the peripheral blood of normal individuals, as well as in some pathological conditions. In particular, an agedependent accumulation of monoclonal DP has been shown in elderly healthy subjects. From the functional point of view, DP are able to act as differentiated effector memory cells with specific antiviral functions.

Introduction and aims: Patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia are known to display several immunological abnormalities. Azacitidine represents a therapeutic options for these disorders and, beside the well known effects on bone marrow precursors, has been demonstrated to potentially influence T-cell polarization. The aim of this study is to monitor the kinetic of the T-cell receptor (TCR) repertoire during Azacitidine treatment in order to explore its potential ability, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these patients. Materials and methods: Our study consisted in a flow cytometric and spectratyping analysis performed on the peripheral blood of 11 patients (5 with MDS and 6 with AML with multilineage dysplasia) and 30 normal controls. Each patient was evaluated at baseline and then every 3 cycles of Azacitidine. The flow cytometry analysis was based on a panel of 24 beta variable (BV) family-specific antibodies. A BV expansion was defined as any value of BV family expression higher than the mean + 3 standard deviations calculated in controls. The profile of the third complementarity-determining-region (CDR3) in separated helper and cytotoxic T-cells was then analyzed by spectratyping. After immunomagnetic CD4+/CD8+ cell separation, RNA extraction and reverse transcriptase PCR, CDR3 fragment analysis was performed through capillary electrophoresis. Spectratyping evaluation was carried out by determining the percentage of Gaussian, skewed and oligoclonal BV subfamilies. Results: Our patients had a median of 3 assessments during their treatment with Azacitidine. On flow cytometry at baseline, in CD4+ cells 5 patients did not show any lymphocyte expansion while 5 of them showed a single BV expansion. One of these expansions disappeared after 3 cycles while 4 were stable during treatment. When reassessed during therapy, 2 of the patients showed each the appearance of 3 new BV expansions, some of which however disappeared at the following evaluations. Overall CD4+ expansions during the all period of study were 11 (5 at baseline and 6 emerged during treatment) and their size ranged from 5.1% in BV 13.6 to 23.9% in BV 11. Within the CD8+ subset, at baseline 7 out of 10 patients showed at least one T-cell expansion. In details 3 patients showed a single expansion while 4 of them displayed 2 different BV expansions. Of these 11 baseline expansions 7 were stable during treatment, while 4 of them quickly disappeared. Remarkably, one of these expansions which had disappeared in a patient in remission reappeared at disease relapse. Six patients showed the appearance of a single BV expansion during treatment, which once again was usually transient. Overall CD8+ expansions during the all period of evaluation were 17 (11 at baseline and 6 emerged later) and their size ranged from 2.5% in BV 5.3 to 50.1% in BV 3. Noteworthy, when analyzed by spectratyping during their treatment our patients showed significant changes in their CDR3 profiles, which were much more evident in helper T lymphocytes. In fact, the frequency of BV showing a skewed CDR3 profile was significantly decreased from baseline to the following evaluations in the CD4+ subset (mean 81.45% vs. 70.17%, p= 0.004). This pattern was even more pronounced in patients responding to Azacitidine (mean 89.60% vs. 61.47%, p= 0.002). Also in the CD8+ subset a trend towards a reduction in the frequency of skewed CDR3 profiles (mean 99.27% vs. 98.74%, p= 0.01) was demonstrated. In the patient with the longest follow up it was possible to observe a dramatic decrease in the degree of CD4+ CDR3 skewing from 91% at baseline to 22% at 18 months, when he was still responding to Azacitidine. Conclusions: Our findings firstly confirmed in our patients an overall derangement of the TCR repertoire. However this pattern seems to gradually improve during Azacitidine treatment, as witnessed by the disappearance of some BV expansions observed on flow cytometry but much more by the progressive restoration of the CDR3 diversity detected by spectratyping, especially within the CD4+ subset. Therefore our data suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of patients with MDS and AML with multilineage dysplasia. Disclosures No relevant conflicts of interest to declare.

Autoimmune manifestations including autoimmune hemolytic anemia (AIHA) are often described in association with splenic marginal zone lymphoma (SMZL) [1]. Moreover, AIHA has been recently reported as a potential risk factor of poor outcome for SMZL patients [2]. Among the different available therapeutic options, rituximab monotherapy has been recently shown to be extremely effective in SMZL [3]. On the other hand, this monoclonal antibody plays a key role in the treatment of steroid-refractory AIHA [4]. More specifically, although rituximab has been recently included among the possible strategies to treat AIHA secondary to chronic lymphocytic leukemia [5], very few cases describing its use in the course of SMZL-related AIHA have been described so far [6].

Very recently the JAK2V617F mutation has been described in BCR⁄ABL+ patients for the first time. Even though characterized by very different clinical patterns, these cases raise some questions about the biological meaning of the coexistence between these disease-specific molecular markers. The present report confirms that the unexpected coexistence between these two disease-specific molecular markers - the JAK2V617F mutation and the BCR⁄ABL translocation - is possible.

Abstract 4954 Introduction: Patients with myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) with multilineage dysplasia are known to display several immunological abnormalities. Azacitidine represents a therapeutic options for these disorders and, beside the well known effects on bone marrow precursors, has been demonstrated to potentially influence T-cell polarization. The aim of this study is to monitor the kinetic of the T-cell receptor (TCR) repertoire during Azacitidine treatment in order to explore its potential ability, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of these patients. Methods: Our study consists in a flow cytometric and spectratyping analysis performed on the peripheral blood of 9 patients (4 with MDS and 5 with AML with multilineage dysplasia) and 30 normal controls. Each patient is evaluated at baseline and then every 3 cycles of Azacitidine. The flow cytometry analysis is based on a panel of 24 beta variable (BV) family-specific antibodies. A BV expansion is defined as any value of BV family expression higher than the mean + 3 standard deviations calculated in controls. We then analysed the profile of the third complementarity-determining-region (CDR3) in separated helper and cytotoxic T-cells by spectratyping. After immunomagnetic CD4+/CD8+ cell separation, RNA extraction and reverse transcriptase PCR, CDR3 fragment analysis was performed through capillary electrophoresis. Spectratyping evaluation was carried out by determining the percentage of Gaussian, missing, skewed and oligoclonal beta-variable (BV) subfamilies. Results: Our patients had a median of 3 assessments during their treatment with Azacitidine. At baseline, in CD4+ cells 5 patients did not show any lymphocyte expansion while 4 of them showed a single BV expansion. One of these expansions disappeared after 3 cycles while 3 were stable during treatment. When reassessed during therapy, 2 of the patients showed each the appearance of 3 new BV expansions, some of which however disappeared at the following evaluations. Overall CD4+ expansions during the all period of study were 10 (4 at baseline and 6 emerged during treatment) and their size ranged from 7. 1% in BV 13. 6 to 23. 9% in BV 11. Within the CD8+ subset, at baseline 6 out of 9 patients showed at least one T-cell expansion. In details 2 patients showed a single expansion while 4 of them displayed 2 different BV expansions. Of these 10 baseline expansions 6 were stable during treatment, while 4 of them quickly disappeared. Noteworthy, one of these expansions which had disappeared in a patient in remission reappeared at disease relapse. Six patients showed the appearance of a single BV expansion during treatment, which once again was usually transient. Overall CD8+ expansions during the all period of evaluation were 16 (10 at baseline and 6 emerged later) and their size ranged from 2. 5% in BV 5. 3 to 50. 1% in BV 3. Preliminary data on the spectratyping analysis showed a gradual improvement in the TCR repertoire diversity, due to the progressive reduction in the frequency of non Gaussian CDR3 profiles, especially remarkable as regards the number of missing BVs. Conclusions: Our findings firstly confirmed in our patients an overall derangement of the TCR repertoire. However this pattern seems to gradually improve during Azacitidine treatment, as witnessed by the disappearance of some BV expansions observed on flow cytometry -particularly within the CD8+ subset-, as well as by the progressive restoration of the CDR3 diversity detected by spectratyping. These preliminary data seem to suggest that Azacitidine could be potentially able, not only to restore the hematopoietic function, but also to reverse the immune derangement typical of patients with MDS and AML with multilineage dysplasia. Disclosures: No relevant conflicts of interest to declare.