In-depth immunophenotyping reveals significant alteration of lymphocytes in buffalo with brucellosis.

Water buffalo (Bubalus bubalis) has a prominent position in the livestock industry worldwide but still suffers from limited knowledge on the mechanisms regulating the immune against infections, including brucellosis (BRC), one of the most significant neglected zoonotic diseases of livestock. Seventy-three buffalo were recruited for the study. Thirty-five were naturally infected with Brucella spp. The aims of the study were to (i) verify the cross-reactivity of 16 monoclonal antibodies (mAbs) developed against human, bovine, and ovine antigens; (ii) evaluate lymphocyte subset alterations in BRC positive buffalo; (iii) evaluate the use of the canonical discriminant analysis (CDA), with flow cytometric data, to discriminate BRC positive from negative animals. A new set of eight mAbs (anti CD3e, CD16, CD18, CD45R0, CD79a; CD172a) were shown to cross-react with water buffalo orthologous molecules. BRC positive animals presented a significant (p < 0.0001) decrease in the percentage of PBMC (29.5 vs. 40.3), total, T and B lymphocytes (23.0 vs. 35.5, 19.2 vs. 28.9, 2.6 vs. 5.7, respectively). In contrast, they showed an increase in percentage of granulocytes (65.2 vs. 55.1; p < 0.0001) and B lymphocytes CD21neg (22.9 vs. 16.1; p = 0.0067), a higher T/B lymphocyte ratio (10.3 vs. 6.4; p = 0.0011) and CD3+ /CD21+ (14.7 vs. 8.3; p = 0.0005) ratio. The CDA, applied to 33 different flow cytometric traits, allowed the discrimination of all BRC positive from negative buffalo. Although this is a preliminary study, our results show that flow cytometry can be used in a wide range of applications in livestock diseases, including in support of uncertain BRC diagnoses.

Comprehensive phenotyping of peripheral blood monocytes in healthy bovine.

Monocytes are bone marrow derived innate myeloid cells that circulate in the blood and play important roles in infection and inflammation. As part of the mononuclear phagocytic system, monocytes provide innate effector functions, support the adaptive immune response, and play a role in the maintenance of tissue homeostasis. In addition to their role in sensing pathogen-associated molecular patterns using several pattern recognition receptors, monocytes are characterized by their ability to ingest and kill microbes, to produce cytokines and chemokines, and to present antigens to T cells. For a long time, monocytes have been considered as a homogenous cell population, characterized by the expression of CD14, the receptor of lipopolysaccharide. Studies in several species have shown that the monocyte population consists of phenotypically and functionally different cell subsets. In this review, we report a comprehensive phenotyping of monocyte subsets in cattle. In addition, the most characterizing cell markers and gating strategies for detailed immunophenotyping of bovine monocyte subsets are discussed.

Combined effects of CXCL8 (IL-8) and CXCR2 (IL-8R) gene polymorphisms on deregressed MACE EBV indexes of milk-related traits in Simmental bulls.

CXCL8 (also known as IL-8) is a member of the CXC subfamily of chemokines that binds two of the seven transmembrane G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, to mediate and regulate leucocyte accumulation and activation at sites of inflammation. They are known to play a critical role in both disease susceptibility and infection outcome. The aim of this study was to investigate the entire sequences of CXCL8 and CXCR2 genes in thirty-one Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, and for somatic cell score (SCS). Five new single nucleotide polymorphisms (SNPs) were found in CXCR2 gene. The analysis of association indicated that one SNP in CXCL8 and two in CXCR2 influenced the considered traits. To evaluate the existence of functional haplotypic effects, combinations among the three genomic variants (SNP 1 in CXCL8, SNP 6 and SNP 7 in CXCR2) were investigated. Four different haplotypic alleles were identified in the experimental population, one of which at a high frequency (61%). Bulls with Hap 4 (G-C-G at SNP 1, SNP 6, and SNP 7 respectively) had more favourable indexes for SCS (P < 0.05). These results suggest that the SNPs in CXCL8 and CXCR2 may be potential genetic markers to improve udder health in the Simmental breed.

Novel SNPs and haplotypes identified in the CD4 gene and their influence on deregressed MACE EBV indexes of milk-related traits in Simmental breed.

Cluster of differentiation 4 (CD4) is the accessory protein non-covalently bound to the T cell receptor that recognizes an invariant region of MHC class II on antigen presenting cells. Its cytoplasmic tail, physically associated with a protein tyrosine kinase, is important in the activation of helper/inducer T lymphocytes. In Bos taurus, CD4 gene is located on chromosome 5 from which two isoforms are transcribed, with a different number of amino acids due to splicing of exon 7 and variation in the reading frame. The aim of this study was to investigate the sequence of the entire CD4 gene in Simmental sires to evaluate the effects of genomic variants on the indexes of the bulls for milk, fat and protein yields, as well as somatic cell score. The associations among genomic variants and indexes were analysed using the Allele and GLM procedures of SAS 9.4. The analysis indicated that only four of the thirty-one identified SNPs influenced the considered traits. Identified variants insist on coding zones and intronic sequences, where we revealed the presence of sites for transcription factors. To evaluate the existence of haplotypic effects, combinations among the four genomic variants (SNP 3, SNP 8, SNP 11 and SNP 19) were investigated. Six different haplotypic alleles were identified, but only four of them were frequent enough to allow for an evaluation of any haplotypic effect (at least six copies in the examined sample): Hap1, Hap2, Hap3 and Hap6. The analysis of associations between the selected haplotypes in the CD4 gene with milk related indexes showed that bulls with Hap2 (T-A-C-C) had better indexes for milk and protein yields (P < 0.05), whereas the presence of the Hap1 haplotype (A-G-A-T) caused a significant decrease of the index for protein yield (P < 0.05). Frequencies of the two alleles Hap1 and Hap2 (9 and 36% respectively) make them of interest for their possible inclusion in breeding schemes and support the hypothesis of considering this gene as a candidate for the improvement of milk-related traits in the Simmental breed.

Comparison of metabolic, oxidative and inflammatory status of Simmental × Holstein crossbred with parental breeds during the peripartal and early lactation periods.

The aim of the research reported in this paper was to evaluate plasma concentrations of energy, oxidative and inflammatory biomarkers of Simmental (sire) × Holstein (dam) crossbred cows, in comparison with the two parental breeds during the peripartal and early lactation periods and to estimate the effects of heterosis for these traits. Thirty-three animals, managed under the same conditions, 8 Simmental (SI), 9 Holstein (HO) and 16 crossbred (CR) cows were enrolled in this study. Glucose, non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total protein, albumin, creatinine and urea were determined in blood sampled at six different time points (30 ± 3 and 15 ± 3 d before the expected calving date, at calving and 15, 30 and 60 d after calving). Furthermore, derived reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), interleukin-6 (IL-6), haptoglobin (Hp) and serum amyloid A protein (SAA) were determined to evaluate inflammatory and oxidative status. Results showed that the CR group had significantly lower average values of glucose and NEFA when compared to HO group; signifcantly lower values of urea than SI group and significantly higher values of creatinine than HO. Furthermore, CR cows showed the lowest average value of d-ROMs with respect to SI and HO parental breeds. Finally, the average value of haptoglobin was significantly lower in CR and HO groups, when compared to SI group. As for the heterosis we found the highest (positive) percentage for CK (98%) and BAP (47%) and the lowest (negative) percentage for OSi (-75%) and d-ROMs (-39%). A negative percentage was also found for the glucose (-11%) and NEFA (-20%) toward the Simmental parental breed. Our results suggest a different response among the three genetic groups during the peripartal and early lactation periods. In particular, CR and SI cows seem more adaptable regarding energy metabolism and oxidative status. Heterosis led to a positive effect on those parameters in Simmental (sire) × Holstein (dam) crossbred cows F1 population (50% Simmental and 50% Holstein).

Simmental × Holstein crossbred: comparison of immunological traits with parental breeds during peripartum and early lactation period.

The experiment described in this research communication aimed to compare the immunological traits of Simmental (sire) × Holstein (dam) crossbred cows with the two parental breeds in the peripartum and early lactation period and to estimate the effects of heterosis for these traits. Flow cytometric evaluation of leukocyte subpopulations was assessed in 16 Crossbred (CR), 8 Holstein (HO) and 8 Simmental (SI) cows. Estimated average values of innate and adaptive immune cells showed statistically significant differences between the crossbred cows and parental breeds. Interestingly, the most relevant differences between the three groups related to adaptive immune cells. In particular, the CR cows showed a lower percentage of CD3+ T lymphocytes compared with the SI group (P < 0.0001) and the highest proportions of CD21+ B lymphocytes among the three groups (P < 0.0001). Furthermore, we found the highest positive value of heterosis for the CD21+ B lymphocytes (7.0) and the lowest negative value for CD3+ T lymphocytes (-4.8) in F1 derived population. It seems reasonable to believe that these differences could affect immune function of crossbred cows.

Quality assessment of escaping silver eel (Anguilla anguilla L.) to support management and conservation strategies in Mediterranean coastal lagoons.

Silver eel samples, collected from the lagoons of Fogliano and Caprolace (Italy), were investigated for a broad range of contaminants (29 polychlorinated biphenyls, 9 polybrominated diphenyl ethers, 5 dichlorodiphenyltrichloroethane, 5 chlordanes, hexachlorobenzene, 3 hexachlorocyclohexane, and 5 metals). Concentrations of targeted compounds stand for a general low contamination pattern. Infestation by Anguillicola crassus and virus infections were also examined. No parasite infestation was found, while infected silver eels had a low prevalence for EVEX, and, for the first time in the Mediterranean area, for AngHV-1. Overall, a good quality status of escaping silver eels, for both lagoons, was highlighted by the use of integrative Indexes. A quality assessment of the ecological status of the two lagoons was carried out developing an expert judgment approach, in order to characterize the habitat of eel stocks. A Final Pressure Index was derived, whose values showed an overall limited global anthropogenic impact acting on both lagoons. Results stand for the suitability of an integrated approach to assess lagoon habitats and eel local stocks quality. This could be proposed as a tool to identify sites yielding high quality eel spawners in the Mediterranean region, in order to set up suitable management frameworks, providing elements to appraise and discuss the potential of coastal lagoons in the Mediterranean region towards the recovery of the eel global stock.

Systemic mastocytosis associated with myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis: Report of three cases.

The association of systemic mastocytosis with another hematologic neoplasia of myeloid or lymphoid origin is recognized as an advanced subvariant of mastocytosis. Here, we report the association of indolent or smoldering systemic mastocytosis with three cases of myelodysplastic/myeloproliferative neoplasms with ring sideroblasts and thrombocytosis, a recently recognized disease characterized by SF3B1 mutations. The hierarchical pattern of KIT, SF3B1, JAK2, and additional mutations was studied in whole and fractionated subpopulations of peripheral blood cells and whole bone marrow. In two cases, we could demonstrate a multilineage D816V KIT mutation, involving all myeloid lineages in one patient and also the lymphoid series in the other. Two patients displaying both SF3B1 and V617F JAK2 mutations had a very poor prognosis. Another patient bearing SF3B1, but not V617F JAK2 mutation, had a favorable response to erythropoietin treatment and long survival.

CAL2 monoclonal antibody is a rapid and sensitive assay for the detection of calreticulin mutations in essential thrombocythemia patients.

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.

Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload.

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available "first level" molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non-diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of "non-HFE" HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)-based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non-diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of "non-HFE" HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS-based "second level" genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations.

Clonal mast cell disorders in patients with severe Hymenoptera venom allergy and normal serum tryptase levels.

BACKGROUND: Systemic mastocytosis is a clonal mast cell (MC) disease that can lead to potentially fatal anaphylactic reactions caused by excessive MC mediator release. The prevalence of mastocytosis in patients with Hymenoptera venom allergy is high, and thus the disease should be suspected in patients with severe reactions caused by Hymenoptera stings and increased serum basal tryptase (SBT) levels. OBJECTIVE: We sought to evaluate the presence of clonal MC disorders in patients seen at our mastocytosis center with Hymenoptera sting-induced anaphylaxis, documented hypotension, absence of urticaria pigmentosa, and normal SBT levels. METHODS: Twenty-two patients with Hymenoptera sting-induced anaphylaxis, without skin lesions, and with tryptase levels of less than 11.4 ng/mL underwent bone marrow evaluation. Bone mineral density was assessed in those patients with ascertained mastocytosis. RESULTS: In 16 of 22 patients, a diagnosis of indolent mastocytosis could be established, and 1 patient had a monoclonal MC activation syndrome. Patients with mastocytosis had higher SBT levels (P = .03) but only rarely had angioedema/urticaria associated with hypotension (P = .004). CONCLUSIONS: The absence of urticaria or angioedema in severe reactions to Hymenoptera stings with hypotension might represent the most relevant factor in identifying patients with mastocytosis, regardless of their serum tryptase levels.

Reduced DNA methylation and hydroxymethylation in patients with systemic mastocytosis.

OBJECTIVE: As disruption of epigenetic control is a frequent event in solid tumors and leukemia, we investigated changes in DNA methylation (5mC) and hydroxymethylation (5hmC) in patients with systemic mastocytosis (SM), a rare myeloproliferative disease with a wide spectrum of severity, characterized by the accumulation of mast cells in various organs. METHODS: We measured overall genomic levels of 5hmC and 5mC in patients with SM by dot blot, as well as by quantitative immunofluorescence in samples of cutaneous mastocytosis. RESULTS: Overall 5hmC levels were reduced in all patients with SM, but to a greater extent in the presence of higher D816V mutational load in the KIT oncogene, which affects prognosis and therapeutic options in these patients. Loss of 5hmC was likely due to systemic effects of SM as it did not correlate with overall mast cell burden in these patients, nor it was due to inactivating mutations of TET2 or reduced TET2 expression. CONCLUSIONS: The correlation between SM diagnosis and significantly low 5hmC levels suggests that reduction of 5hmC represents a systemic effect of SM that may be useful for patient stratification and that measurements of 5hmC levels may serve as a better prognostic marker than TET2 mutations.

Role of JAK2 V617F mutation and aberrant expression of microRNA-143 in myeloproliferative neoplasms.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are clonal myeloid disorders characterized by the overproduction of mature blood cells. The pathogenetic hallmark of MPNs is the dysregulation of JAK-STAT signaling, usually associated with the JAK2 V617F mutation. Multiple additional genetic and epigenetic alterations that constitutively activate the JAK-STAT signaling pathway have been described, including the modulation of the microRNAs (miRs) expression levels. The aims of our study were to investigate JAK2 V617F mutation allele burden and miR-143 expression levels in MPNs patients and to investigate the correlation between these genetic signatures and hematological parameters. METHODS: In total 78 patients with a clinical diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM), made according to the WHO 2008 criteria, were included in the study. Twenty healthy subjects were checked as controls. Quantification of JAK2 V617F mutation and miR-143 expression levels were determined by real-time quantitative polymerase chain reaction. RESULTS: The miR-143 expression in MPNs patients was 2.97-fold higher than in controls. JAK2 V617F mutation allele burden and miR-143 expression level resulted higher in PV and IM respect to ET patients. Patients who had V617F allele burden >50% displayed a higher miRNA-143 expression level than patients with allele burden <50%. In MPNs patients, a statistically significant positive correlation was observed between JAK2 V617F mutation allele burden and hemoglobin and hematocrit values and between miR-143 expression levels and platelet count. CONCLUSIONS: Our findings of aberrant miR-143 expression support the concept that factors other than JAK2 V617F mutation may contribute to the pathogenesis and some clinical signs of MPNs.

Characterization of leptin receptor gene in Bubalus bubalis and association analysis with body measurement traits.

Leptin has a pleiotropic effect on regulating appetite, energy metabolism, growth, reproduction, body composition and immunity. This property supports leptin and its receptor as candidate genes for evaluating genetic polymorphisms to associate with growth, milk yield and other economic traits. The aim of this study is to characterize the leptin receptor gene in Bubalus bubalis, to identify single-nucleotide polymorphism (SNP) sites in different coding and non-coding regions and to analyse potential associations between SNPs identified and the body measurements traits of growing buffalo heifers. A group of 64 animals were genotyped by direct sequencing and twenty-eight SNPs were detected. A sequence analysis revealed the presence of nine interesting SNPs in gene sequence. The association analysis of polymorphisms with the body measurements traits of growing buffalo heifers shows significant statistical effects on chest depth and sacrum height. Therefore according to the results obtained from this study, the leptin receptor gene appears to have potential effects on the body measurement traits of Bubalus bubalis.

Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM.

BACKGROUND: Indolent systemic mastocytosis (ISM) without skin lesions (ISMs(-)) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs(+)). Interestingly, in almost one-half of ISMs(-) patients, MC-mediator release episodes are triggered exclusively by insects. OBJECTIVE: We aimed to determine the clinical and laboratory features of ISMs(-) associated with insect-induced anaphylaxis (insectISMs(-)) versus other patients with ISM. METHODS: A total of 335 patients presenting with MC activation syndrome, including 143 insectISMs(-), 72 ISMs(-) triggered by other factors (otherISMs(-)), 56 ISMs(+), and 64 nonclonal MC activation syndrome, were studied. RESULTS: Compared with otherISMs(-) and ISMs(+) patients, insectISMs(-) cases showed marked male predominance (78% vs 53% and 46%; P < .001), a distinct pattern of MC-related symptoms, and significantly lower median serum baseline tryptase levels (22.4 vs 28.7 and 45.8 µg/L; P ≤ .009). Moreover, insectISMs(-) less frequently presented BM MC aggregates (46% vs 70% and 81%; P ≤ .001), and they systematically showed MC-restricted KIT mutation. CONCLUSIONS: ISMs(-) patients with anaphylaxis triggered exclusively by insects display clinical and laboratory features that are significantly different from other ISM cases, including other ISMs(-) and ISMs(+) patients, suggesting that they represent a unique subgroup of ISM with a particularly low BM MC burden in the absence of adverse prognostic factors.

Effect of the Season on Blood Changes of Oxidative Stress Index in the Italian Mediterranean Buffalo (Bubalis bubalis).

Studies in cattle have shown that high temperatures increase the production of reactive oxygen species (ROS) causing an imbalance between ROS and the ability of antioxidant systems to detoxify and remove the reactive intermediates. As such studies remain limited in buffalo, the effect of temperature on oxidative stress was investigated through the oxidative stress index (OSi). Blood samples were collected from 40 buffaloes over 12 time points distributed over two years (2021, 2022). Samples were taken monthly during the hot and cold seasons. Plasma free oxygen radicals were determined using the d-ROMs test (Diacron, Italy), modified for a microplate procedure, and the results were expressed in arbitrary Carratelli Units (U.CARR). Plasma antioxidants were determined by using the BAP test (Diacron) in a dedicated spectrophotometer (Carpe Diem Free, Diacron). The OSi parameter was calculated as d-ROMs/BAP × 100. Temperature and humidity were recorded daily during the trial to calculate the Temperature Humidity Index (THI). For statistical analysis, year and season and their interactions were included in the model. The results of this study showed for the first time the effect of season on the oxidative stress in buffalo. The minimum and maximum THI values for the hot and cold season recorded during the experimental period were 79.27 ± 2.20 and 63.42 ± 3.20, respectively. Levels of d-ROMs and BAP were affected by the seasons (133.0 vs. 145.1 U.CARR, p = 0.0189, and 2489.19 vs. 2392.43 mml/L, p = 0.033, in the hot and cold season, respectively). A significant year × season interaction was found both for d-ROMs and BAP (p = 0.06 and p < 0.0001, respectively). Moreover, OSi was affected by season, showing a growing trend from hot to cold season (5.35 vs. 6.17, p < 0.0001), but, interestingly, it was unaffected by annual variation. Therefore, Osi could be considered a better and independent marker of oxidative status in buffalo, with respect to the evaluation of single determinations of d-ROMs and BAP. Lastly, there were no differences in the plasma 25OHD levels between seasons; concentrations were 12.24 and 10.26 ng/mL in the hot and cold season, respectively.

Interferon-Tau in Maternal Peripheral Blood and Its Relationship with Progesterone and Pregnancy-Associated Glycoproteins in the Early Phases of Gestation in Water Buffalo.

The aim of this study was to investigate the interferon tau (IFNt) concentration in the peripheral maternal blood during the early phase of pregnancy in buffalo cows and improve the knowledge on the physiological importance of circulating IFNt, evaluating the possible interaction with pregnancy-associated glycoproteins (PAGs) and progesterone (P4). Blood samples were taken from buffalo cows on day 0 (day of AI), 7, 14, 18, 28, and 40 post insemination for the IFNt, PAG, and P4 analysis and to determine the IFNt mRNA expression. The animals were categorized ex post into Pregnant, Non-pregnant and Embryo mortality groups. The interferon value was influenced by group (p = 0.003), being always higher in pregnant buffalo cows than in non-pregnant ones, while the embryo mortality group showed intermediate values between those for pregnant and non-pregnant animals. The mRNA expression of IFNt was not influenced by groups or any time points. The regression analysis that included IFNt as the independent variable showed that PAGs, from day 18 (p < 0.01), and P4, from day 28 (p < 0.05), were positively associated with IFNt values. The close associations among IFNt, PAGs and P4 demonstrate that all three molecules work together for fetal-placental well-being and pregnancy support. Unfortunately, the great individual variability in circulating IFNt makes this analysis unsuitable for early pregnancy diagnosis.

PAR level mediates the link between ROS and inflammatory response in patients with type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is characterized by disrupted glucose homeostasis and metabolic abnormalities, with oxidative stress and inflammation playing pivotal roles in its pathophysiology. Poly(ADP-ribosyl)ation (PARylation) is a post-translational process involving the addition of ADP-ribose polymers (PAR) to target proteins. While preclinical studies have implicated PARylation in the interplay between oxidative stress and inflammation in T2DM, direct clinical evidence in humans remains limited. This study investigates the relationship between oxidative stress, PARylation, and inflammatory response in T2DM patients. METHODS: This cross-sectional investigation involved 61 T2DM patients and 48 controls. PAR levels were determined in peripheral blood cells (PBMC) by ELISA-based methodologies. Oxidative stress was assessed in plasma and PBMC. In plasma, we monitored reactive oxygen metabolites (d-ROMs) and ferric-reducing antioxidant power. In PBMC, we measured the expression of antioxidant enzymes SOD1, GPX1 and CAT by qPCR. Further, we evaluated the expression of inflammatory mediators such as IL6, TNF-α, CD68 and MCP1 by qPCR in PBMC. RESULTS: T2DM patients exhibited elevated PAR levels in PBMC and increased d-ROMs in plasma. Positive associations were found between PAR levels and d-ROMs, suggesting a link between oxidative stress and altered PAR metabolism. Mediation analysis revealed that d-ROMs mediate the association between HbA1c levels and PAR, indicating oxidative stress as a potential driver of increased PARylation in T2DM. Furthermore, elevated PAR levels were found to be associated with increased expression of pro-inflammatory cytokines IL6 and TNF-α in the PBMC of T2DM patients. CONCLUSIONS: This study highlights that hyperactivation of PARylation is associated with poor glycemic control and the resultant oxidative stress in T2DM. The increase of PAR levels is correlated with the upregulation of key mediators of the inflammatory response. Further research is warranted to validate these findings and explore their clinical implications.

Vaccination of cattle with a virus vector vaccine against a major membrane protein of Mycobacterium avium subsp. paratuberculosis elicits CD8 cytotoxic T cells that kill intracellular bacteria.

Analysis of the recall response ex vivo in cattle vaccinated with a Mycobacterium avium subsp. paratuberculosis (Map) rel deletion mutant revealed the immune response was directed toward a 35 kD major membrane protein (MMP) of Map. Antigen presenting cells (APC) primed with MMP elicited expansion of CD8 cytotoxic memory T cells (CTL) with ability to kill intracellular bacteria. Development of CTL was MHC-restricted. The gene MAP2121c, encoding MMP, was modified for expression of MMP (tPA-MMP-2mut) in a mammalian cell line to explore the potential of developing MMP as a vaccine. Ex vivo stimulation of PBMC, from Map free cattle, with APC primed with tPA-MMP-2mut expressed p35 elicited a primary CD8 CTL response comparable to the recall response elicited with PBMC from cattle vaccinated with either the Maprel deletion mutant or MMP. In the present study, the modified gene for MMP, now referred to as p35NN, was placed into a bovine herpes virus-4 (BoHV4) vector to determine the potential use of BoHV-4AΔTK-p35NN as a peptide-based vaccine. Subcutaneous vaccination of healthy cattle with BoHV-4AΔTK-p35NN elicited a CTL recall response, as detected ex vivo. The results show use of a virus vector is an effective way for delivery of MMP as a vaccine. The immunogenic activity of MMP was not lost when modified for expression in mammalian cells. The next step is to conduct a field trial to determine if presence of an immune response to MMP prevents Map from establishing an infection.

Milk fatty acid variability: effect of some candidate genes involved in lipid synthesis.

In this work, the genetic variation of milk FA was investigated in three different bovine breeds, the Jersey, the Piedmontese and the Valdostana, and at different lactation stages. All animals were genotyped for 21 Single Nucleotide Polymorphisms located within nine candidate genes involved in lipid synthesis: diacylglycerol acyltransferase 1 and 2 (DGAT1, 2); stearoyl-CoA desaturase (SCD); growth hormone receptor (GHR); fatty acid synthase (FASN); acyl-CoA dehydrogenase (ACAD); fatty acid binding protein (FABP4); lipoprotein lipase (LPL); and leptin gene (LEP). The highest milk-fat Jersey breed also showed the highest content of saturated FA. Throughout lactation, the breeds showed a similar variation in the FA, with a decrease in the short-chain, this was accompanied by a general increase in the long chain FA at the end of lactation. The increase in long chain saturated FA was particularly evident in the case of the Jersey. The effect of SCD gene on the C14 desaturation index was confirmed; the DGAT1 gene was polymorphic only in the Jersey breed, but its effect was confirmed only on milk fat content; three further potential candidate genes were identified: first, the FABP4 gene, which was found to influence medium and long chain FA in all the breeds, but not the desaturation indices; second, the FASN gene, which was found to influence the amount of PUFA in the Piedmontese and the Valdostana, and third, the LPL gene, which was found to affect fat content in the Piedmontese.