Immune and Metabolic Effects of Rumen-Protected Methionine During a Heat Stress Challenge in Lactating Holstein Cows

Multiparous, lactating Holstein cows (n = 32) were randomly assigned to 1 of 2 dietary treatments [TMR with rumen-protected Met (RPM) or TMR without RPM (CON)], and within each dietary treatment group cows were randomly assigned to 1 of 2 environmental treatment groups in a split-plot crossover design. In phase 1 (9 d) all cows were fed ad libitum and in thermoneutral conditions (TN). In phase 2 (9 d), group 1 (n = 16) was exposed to a heat stress (HS) challenge (HSC). Group 2 cows (n = 16) were pair-fed (PFTN) to HSC counterparts and remained in TN. After a 21-d washout period, the study was repeated (period 2) and the environmental treatments were inverted relative to treatments from phase 2 of period 1, while dietary treatments remained the same for each cow. During phase 1, cows in RPM had greater plasma Met concentration compared to cows in CON (59 µM and 30 µM, respectively; P < 0.001). Cows in PFTN had a greater decrease (P < 0.05) in plasma insulin than cows in HSC at 4 h (-2.7 µIU/mL vs. -0.7 µIU/mL) and 8 h (-7.7 µIU/mL vs. -0.4 µIU/mL) during phase 2. Compared to cows in PFTN, cows in HSC had an increase (P < 0.05) in plasma serum amyloid A (-59 µg/mL vs. +58 µg/mL), serum haptoglobin (-3 µg/mL vs. +33 µg/mL), plasma lipopolysaccharide binding protein (-0.27 µg/mL and +0.11 µg/mL), and plasma interleukin-1β (-1.9 pg/mL and +3.9 pg/mL) during phase 2. In conclusion, HSC elicited immunometabolic alterations; however, there were limited effects of RPM on cows in HSC.

Usefulness of Selected Acute-Phase Proteins in the Postsurgical Monitoring of Arthroscopy and Splint Bone Removal in Horses

Background: Arthroscopy and splint bone removal are the common orthopedic procedures in horses. Estimation of the dynamics of acute phase proteins in postoperative monitoring seems to be interesting diagnostic approach. The aim of the study was to investigate changes in the concentrations of plasma inflammatory markers—fibrinogen, haptoglobin, and protease inhibitors—following orthopedic surgery in horses. The study involved 114 horses, divided into two study groups undergoing: arthroscopy (41 horses) and splint bone removal (13 horses). The control group consisted of 60 healthy horses. The blood was collected before the surgery and 24, 48, 72 h, 5, 7, 10, 14 and 28 days after the surgery. Plasma fibrinogen, serum haptoglobin and proteinase inhibitors were measured. Results: In non-complicated cases of arthroscopy and splint bone removal, fibrinogen and haptoglobin increased stepwise from 24 h, achieved the maximum level at 72 h and returned to preoperative levels after 10–14 days. In one complicated case after arthroscopy surgery the marked increase in fibrinogen and haptoglobin concentrations was observed 24 h earlier than standard parameters of inflammation Conclusion: The study shows the evolution of APPs after arthroscopy and splint bone removal in 28 days postsurgery period and in the case of one complicated case of arthroscopy.

Purple Thrombocytopenic Thrombotic

Thrombotic thrombocytopenic purpura is a rare disease of immune origin, belonging to thrombotic microangiopathies. In most cases, it can present as microangiopathic hemolytic anemia accompanied by thrombocytopenia, neurological deficit and kidney abnormalities. The present clinical case belongs to a female patient with no significant personal or family history, who went to the doctor for a clinical picture of 2 days of evolution with hematuria, general malaise, asthenia and adynamia, on physical examination without alterations, with vital signs in ranges of normality. The diagnosis of thrombotic thrombocytopenic purpura was made due to hemolysis, elevated levels of lactate dehydrogenase and a reduction in serum haptoglobin accompanied by the presence of serum schistocytes> 6% in the peripheral blood smear. Thrombocytopenic purpura is a diagnostic challenge because its clinical picture is often nonspecific, making it more difficult to start its treatment in a timely manner. Another drawback is the high costs for ADAMTS13 activity tests. Despite the fact that the treatments for this disease have low success rates, in our clinical case our patient responded favorably to the treatment instituted

PRM-MS Quantitative Analysis of Isomeric N-Glycopeptides Derived from Human Serum Haptoglobin of Patients with Cirrhosis and Hepatocellular Carcinoma

Currently, surveillance strategies have inadequate performance for cirrhosis and early detection of hepatocellular carcinoma (HCC). The glycosylation of serum haptoglobin has shown to have significant differences between cirrhosis and HCC, thus can be used for diagnosis. We performed a comprehensive liquid chromatography—parallel reaction monitoring—mass spectrometry (LC-PRM-MS) approach, where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system, to study the site-specific isomerism of haptoglobin (Hp) extracted from cirrhosis and HCC patients. We found that our strategy was able to identify a large number of isomeric N-glycopeptides, mainly located in the Hp glycosylation site Asn207. Four N-glycopeptides were found to have significant changes in abundance between cirrhosis and HCC samples (p < 0.05). Strategic combinations of the significant N-glycopeptides, either with alpha-fetoprotein (AFP) or themselves, better estimate the areas under the curve (AUC) of their respective receiver operating characteristic (ROC) curves with respect to AFP. The combination of AFP with the isomeric sialylated fucosylated N-glycopeptides Asn207 + 5-6-1-2 and Asn207 + 5-6-1-3, resulted with an AUC value of 0.98, while the AUC value for AFP alone was 0.85. When comparing cirrhosis vs. early HCC, the isomeric N-glycopeptide Asn207 + 5-6-0-1 better estimated AUC with respect to AFP (AUCAFP = 0.81, and AUCAsn207 + 5-6-0-1 = 0.88, respectively).

Determining Serum Haptoglobin and Proinflammatory Cytokines Concentration in the Calves Clinically Diagnosed with Pneumonitis, Pneumoenteritis and Enteritis

Background: This study aims to determine the concentration of serum haptoglobin (Hp), interleukin 1 (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) in cases of Pneumonia, Pneumoenteritis and Enteritis. Methods: 60 calves were subjected to the study and they were divided into four groups. The study group consisted of the claves diagnosed with clinical pneumonia (Group P; n=15), pneumoenteritis (Group PE; n=15) and enteritis (Group E; n=15) while the control group included the healthy calves (Group C; n=15). The measurements of the concentration of serum haptoglobin (Hp), interleukin 1 (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α), Total protein (TP) and Albumin (ALB) were made by using commercial kits. Conclusion: In all infection groups (P, PE ve E), Haptoglobin concentration, serum cytokine (IL-1β, IL-6 and TNF-α) and Albumin values were found to have been higher than the control group (p≤0,005). However, there was no difference in total protein. In the light of these findings, it is suggested that routine controls for Haptoglobin and cytokine (IL-1β, IL-6 and TNF-α) concentrations would be rewarding to determine the severity of the infection, to choose the suitable treatment and to detect subclinical infections in veterinary medicine.

Detection of Aberrant Glycosylation of Serum Haptoglobin for Gastric Cancer Diagnosis Using a Middle-Up-Down Glycoproteome Platform

Gastric cancer is a frequently occurring cancer and is the leading cause of cancer-related deaths. Recent studies have shown that aberrant glycosylation of serum haptoglobin is closely related to gastric cancer and has enormous potential for use in diagnosis. However, there is no platform with high reliability and high reproducibility to comprehensively analyze haptoglobin glycosylation covering microheterogeneity to macroheterogeneity for clinical applications. In this study, we developed a middle-up-down glycoproteome platform for fast and accurate monitoring of haptoglobin glycosylation. This platform utilizes an online purification of LC for sample desalting, and an in silico haptoglobin glycopeptide library constructed by combining peptides and N-glycans to readily identify glycopeptides. In addition, site-specific glycosylation with glycan heterogeneity can be obtained through only a single MS analysis. Haptoglobin glycosylation in clinical samples consisting of healthy controls (n = 47) and gastric cancer patients (n = 43) was extensively investigated using three groups of tryptic glycopeptides: GP1 (including Asn184), GP2 (including Asn207 and Asn211), and GP3 (including Asn241). A total of 23 individual glycopeptides were determined as potential biomarkers (p < 0.00001). In addition, to improve diagnostic efficacy, we derived representative group biomarkers with high AUC values (0.929 to 0.977) through logistic regression analysis for each GP group. It has been found that glycosylation of haptoglobin is highly associated with gastric cancer, especially the glycosite Asn241. Our assay not only allows to quickly and easily obtain information on glycosylation heterogeneity of a target glycoprotein but also makes it an efficient tool for biomarker discovery and clinical diagnosis.

PRM-MS Quantitative Analysis of Isomeric N-Glycopeptides Derived From Human Serum Haptoglobin of Patients With Cirrhosis and Hepatocellular Carcinoma

Currently surveillance strategies have inadequate performance for the early detection of hepatocellular carcinoma (HCC). Protein glycosylation is a potential source of biomarkers to differentiate between cirrhosis and HCC. We performed a comprehensive LC-PRM-MS approach where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system to study the microheterogeneity of haptoglobin (Hp) extracted from 15 patients with cirrhosis and 15 with HCC. We found that our strategy was able to identify a large number of isomeric N-glycopeptides mainly located in the glycosylation site Asn207. Nine out of twelve N-glycopeptides, located in the Asn207 site, had significant differences in abundance between patients with cirrhosis and HCC (p < 0.05). The area under the curve (AUC) of alpha-fetoprotein (AFP) was alone 0.85, which improved to 0.95 (95% CI: 0.88, 1) when NLF_5613 Isomer 1 was combined with AFP. When comparing the early HCC vs. cirrhosis, four sialylated-fucosylated glycopeptides better estimated AUCs with respect to AFP (AUCAFP = 0.66, and AUCN−glycopeptides = 0.86, 0.84, 0.88, and 0.80, respectively). Further large scale validation of glycopeptides for the early detection of HCC is warranted.

Serum haptoglobin concentrations in feline inflammatory bowel disease and small-cell alimentary lymphoma: a potential biomarker for feline chronic enteropathies

Objectives The aim of this study was to evaluate serum haptoglobin as a biomarker to differentiate between small-cell alimentary lymphoma and inflammatory bowel disease in cats. Methods Client-owned domestic cats with and without chronic gastrointestinal signs were enrolled in the study. Serum was collected from each patient and serum haptoglobin levels were measured using ELISA. In cats with gastrointestinal signs, histopathologic evaluation of endoscopic biopsies harvested from the intestinal tract was used to separate them into inflammatory bowel disease and small-cell lymphoma cohorts. Serum haptoglobin levels were statistically analyzed and compared among the three groups: healthy cats; cats with inflammatory bowel disease; and cats with small-cell alimentary lymphoma. Results Sixty-two cats were enrolled in the study, including 20 clinically normal cats, 14 cats with small-cell alimentary lymphoma and 28 cats with inflammatory bowel disease. The mean ± SD serum haptoglobin was 73.2 ± 39.1 mg/dl in normal cats, 115.3 ± 72.8 mg/dl in cats with inflammatory bowel disease and 133.1 ± 86.1 mg/dl in cats with small-cell alimentary lymphoma. Cats with inflammatory bowel disease and lymphoma had significantly higher serum haptoglobin than controls, with P values of 0.0382 and 0.0138, respectively. There was no statistical difference between the inflammatory bowel disease and lymphoma cohorts ( P = 0.4235). For every one unit increase in serum haptoglobin, the odds of gastrointestinal inflammatory disease (inflammatory bowel disease or small-cell alimentary lymphoma) increased by 1.41% ( P = 0.0165). Conclusions and relevance Serum haptoglobin is a useful biomarker for distinguishing between normal cats and those with gastrointestinal inflammatory disease, but it could not significantly differentiate between inflammatory bowel disease and lymphoma. Additional studies may be beneficial in determining the prognostic significance of serum haptoglobin as it may relate to the severity of gastrointestinal inflammation.