Inhibition of HIV-1 replication in primary human monocytes by the IκB-αS32/36A repressor of NF-κB

BACKGROUND: The identification of the molecular mechanisms of human immunodeficiency virus type 1, HIV-1, transcriptional regulation is required to develop novel inhibitors of viral replication. NF-κB transacting factors strongly enhance the HIV/SIV expression in both epithelial and lymphoid cells. Controversial results have been reported on the requirement of NF-κB factors in distinct cell reservoirs, such as CD4-positive T lymphocytes and monocytes. We have previously shown that IκB-αS32/36A, a proteolysis-resistant inhibitor of NF-κB, potently inhibits the growth of HIV-1 and SIVmac239 in cell cultures and in the SIV macaque model of AIDS. To further extend these observations, we have generated NL(AD8)IκB-αS32/36A, a macrophage-tropic HIV-1 recombinant strain endowed to express IκB-αS32/36A. RESULTS: In this work, we show that infection with NL(AD8)IκB-αS32/36A down-regulated the NF-κB DNA binding activity in cells. NL(AD8)IκB-αS32/36A was also highly attenuated for replication in cultures of human primary monocytes. CONCLUSIONS: These results point to a major requirement of NF-κB activation for the optimal replication of HIV-1 in monocytes and suggest that agents which interfere with NF-κB activity could counteract HIV-1 infection of monocytes-macrophages in vivo

Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis.

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R2 = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%

Evaluation of the capillary electrophoresis method for measurement of immunoglobulin concentration in ewe colostrum

ABSTRACT Capillary electrophoresis (CE) is a technique routinely used in clinical laboratories that allows the separation and quantification of blood serum proteins in a rapid, precise, accurate, and inexpensive manner. Recently, CE has been proposed to separate and measure colostral proteins, but an evaluation of the agreement between CE and radial immunodiffusion (RID) method, currently used to quantify IgG in colostrum, is still lacking. The purpose of this study was to test the ability of a CE instrument, normally used in blood serum protein analysis, to realize the correct quantification of total Ig concentration in ewe colostrum, using RID assay as reference. Colostrum samples (n = 68) were collected from 35 multiparous Sarda ewes at first milking (n = 33) and at 24 h postpartum (n = 35). The mean ± standard deviation of IgG concentration measured by RID and whey colostrum total Ig concentration measured by CE were 54.76 ± 41.82 g/L and 54.70 ± 41.43 g/L, respectively. Lin's concordance correlation coefficient (r = 0.993; 95% confidence interval=0.989 to 0.996) and linear regression analysis results (RID = 1.0022CE − 0.063; R 2 = 0.986) showed an excellent agreement between these 2 methods. Bland-Altman analysis confirmed that CE method can be a suitable alternative to RID: the mean of the differences between CE and RID was −0.055 ± 4.95 g/L (95% confidence interval=−1.25 to 1.14 g/L) and the agreement limits were −9.75 to 9.60 g/L (low limit 95% confidence interval=−11.82 to −7.68 g/L; high limit 95% confidence interval=7.57 to 11.72 g/L). In conclusion, the current study indicates that CE method may be a reliable tool for the quantification of the total Ig concentration in ewe colostrum

Technical note: Capillary electrophoresis as a rapid test for the quantification of immunoglobulin G in serum of newborn lambs

ABSTRACT Finding a rapid and simple method of serum IgG determination in lambs is essential for monitoring failure of passive transfer of immunity. The aim of this study was to assess the ability of capillary electrophoresis (CE), an instrument mainly used in blood serum protein analysis, to estimate IgG content in serum of newborn lambs through determination of only total Ig percentage by comparing the results with those obtained with radial immunodiffusion (RID), the reference method for serum IgG quantification. Serum samples were collected at 24 h after birth from 40 Sarda lambs. The IgG concentration measured by RID and serum total Ig concentration measured by CE were (mean ± standard deviation) 29.8 ± 16.1 g/L and 37.8 ± 15.0%, respectively. Data provided by RID and CE analysis showed a polynomial trend (RID = 0.02CE2 − 0.04CE + 4.13; coefficient of determination, R2 = 0.96), displaying a strong relationship between these 2 methods. Applying the polynomial equation, the IgG values were predicted. Predicted IgG values were highly correlated (r = 0.98) and related (R2 = 0.96) to IgG values obtained by RID assay. These data were subjected to Bland–Altman analysis, revealing a high level of agreement between CE and RID methods with a bias that was not different from 0 (−0.04 g/L) and agreement limits of −6.38 g/L (low) and +6.30 g/L (high). In addition, the linear regression analysis between differences (dependent variable) and average of IgG concentration by CE and RID (independent variable) did not show proportional bias (R2 = 0.01). In conclusion, CE is a reliable instrument for a lamb health monitoring program, where Bland–Altman analysis also confirmed that the CE method can be a suitable alternative to RID

Immunometabolic status and productive performance differences between periparturient Simmental and Holstein dairy cows in response to pegbovigrastim

In the present study, we aimed to investigate the side effects of pegbovigrastim, injected approximately 7 d before parturition and on the day of calving, on a panel of plasma biomarkers to evaluate energy, inflammatory, oxidative, and liver function status. We also addressed treatment responses in different breeds during the transition period. Holstein and Simmental cows were randomly assigned into 2 groups based on expected calving date and according to parity: the treated group (PEG; 14 Holstein and 12 Simmental cows) received pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN), and the control group (CTR; 14 Holstein and 14 Simmental cows) received saline solution. The PEG or CTR treatments were administered via subcutaneous injection in the scapular region at approximately 7 d (mean 7.80 ± 5.50 d) before expected parturition and within 24 h after calving. Blood samples were collected at -21, -7 (before injection), 1, 3, and 28 d relative to calving. Milk production was recorded at 7, 15, 21, 30, and 42 d. A mixed model with repeated measures was fitted to the normalized data using Proc MIXED of SAS (SAS Institute Inc., Cary, NC). Simmental PEG cows showed higher plasma protein concentrations at 1 and 3 d after calving compared with Simmental CTR and Holstein PEG cows, whereas no differences were detected between Holstein PEG and CTR cows. Albumin was greater at 1 d in Simmental PEG compared with Simmental CTR cows. In contrast, γ-glutamyl transferase was higher overall (across breed) in PEG than in CTR. The PEG group had higher values throughout the postcalving period compared with CTR. Cows treated with pegbovigrastim had also higher alkaline phosphatase (ALP) activity at 1 and 3 d after calving. The Holstein PEG group had higher ALP activity at 3 d compared with the Holstein CTR and Simmental PEG groups, and higher ALP at 1 d compared with the Simmental CTR group. The PEG group had higher levels of IL-6 at 3 and 28 d but higher IL-1β only at 28 d after calving compared with the CTR group. Overall, Holstein cows were characterized by a greater response in the production of inflammation biomarkers (cytokines, haptoglobin, and ceruloplasmin). In addition, PEG cows had higher values of zinc at 1 and 3 d after calving compared with CTR cows. The response observed in plasma biomarkers for energy metabolism and liver functionality after pegbovigrastim treatment in Simmental and Holstein cows was not different from that in control cows. However, our data shed light on the different metabolic adaptations during the transition period between Simmental and Holstein cows, characterized by different energy, inflammatory, and oxidative pattern responses. For the first time, we have highlighted the effect of pegbovigrastim in maintaining stable cytokine levels during the first month after parturition, reflecting greater regulation of neutrophil recruitment, trafficking, and maturation during the inflammatory response. These results provide evidence of the immunomodulatory action of pegbovigrastim around parturition, when dairy cows are highly immunosuppressed. At the same time, these data demonstrate that increasing release of cytokines after parturition is not linked to exacerbation of a systemic inflammation evaluated based on haptoglobin and ceruloplasmin levels

Effect of Pegbovigrastim on Hematological Profile of Simmental Dairy Cows during the Transition Period

Pegbovigrastim is a long-acting analog of recombinant bovine granulocyte colony-stimulating factor, that promotes and increases the count and functionality of polymorphonuclear cells in dairy cows. The present study aimed to explore, for the first time in Simmental cows, the clinical and hematological effect of pegbovigrastim during the transition period (TP). Cows were randomly assigned into two groups: treated group (PEG; n = 16) received pegbovigrastim at approximately 7 days before expected parturition and within 6 h after calving, and control group (CTR; n = 16) received saline solution. Blood samples were obtained at −7, 0, 1, 3, 7, 14, 21, and 30 days relative to calving. PEG group showed white blood cells (WBC) count consistently higher compared with CTR group (p < 0.001) until to 3 weeks after calving. Neutrophils remained higher in PEG group (p < 0.001) up to three weeks after calving, compared with CTR group, with slight increment of band cells. Moreover, PEG group displayed a lower index of myeloperoxidase at 1, 3, and 7 days after calving (p < 0.01) compared with CTR. Basophils and lymphocytes showed a similar trend to those observed for neutrophils at 1 day after calving in PEG group. Finally, monocytes remained markedly elevated until 3 days after calving in PEG compared to CTR group (p < 0.001), whereas in PEG group, eosinophils population showed lower percentage values at 1 and 3 days after calving but higher values at 30 days compared with CTR group. PEG group was characterized by lower red blood cells (RBCs) count compared with CTR group (p < 0.05) and higher % of red cell volume distribution width (RDW) from week 2 and mean corpuscular volume (MCV) at 30 days after calving. In addition, the mean platelet volume (MPV) was significantly higher in PEG group at calving, 1, 3, and 7 days after calving compared with CTR group (p < 0.05). For the first time, we described the effect of pegbovigrastim in a breed not specialized exclusively in milk production as Holstein, but with dual purpose (meat and milk), evaluating the complete hematological profile in cows during the transition period. These results provide evidence on the proliferative effect of pegbovigrastim on WBC in Simmental breed highlighting its possible side effect on RBCs

Evaluation of freezing point in milk from buffalos reared in Calabria, Italy

Evaluation of freezing point is one of the most common technique used to detect milk adulteration such as addition of external water to increase volume. The aim of this study was to evaluate the freezing point of buffalo milk using infrared spectroscopy and to assess how it is influenced by other milk components. A total of 361 individual buffalo milk samples were collected monthly from March to August of 2017 in a dairy farm in Catanzaro district, Italy. Samples were tested for freezing point, urea, acetone and beta-hydroxybutyrate, percent of fat, protein, lactose, casein, by Fourier Transformed Spectroscopy. The pH and daily milk production were also recorded. Freezing point ranged from -0.574°C to - 0.512°C and the mean values was -0.545°C ±0.010. According to lactation stage, freezing point decreased until 210 days post-partum reaching the minimum value of −0.550°C, then it slightly increased during lactation; according to sampling month the highest and lowest values were recorded in August and June, respectively. A positive correlation between freezing point and lactose content were evidenced (r=0.1806, P<0.05). Moreover, a faintly positive correlation was also found between freezing point and beta-idroxibutirrate (r=0.0869, P<0.05) and acetone (r=0.0096, P<0.05), whereas a negative correlation with fat (r=−0.2356, P<0.05), protein (r=-0.1855, P<0.05), casein (r=-0.2127, P<0.05) and urea (r=-0.1229, P<0.05) was evidenced

Detection and quantification of breast arterial calcifications on mammograms: a deep learning approach

ObjectiveBreast arterial calcifications (BAC) are a sex-specific cardiovascular disease biomarker that might improve cardiovascular risk stratification in women. We implemented a deep convolutional neural network for automatic BAC detection and quantification.MethodsIn this retrospective study, four readers labelled four-view mammograms as BAC positive (BAC+) or BAC negative (BAC-) at image level. Starting from a pretrained VGG16 model, we trained a convolutional neural network to discriminate BAC+ and BAC- mammograms. Accuracy, F1 score, and area under the receiver operating characteristic curve (AUC-ROC) were used to assess the diagnostic performance. Predictions of calcified areas were generated using the generalized gradient-weighted class activation mapping (Grad-CAM++) method, and their correlation with manual measurement of BAC length in a subset of cases was assessed using Spearman rho.ResultsA total 1493 women (198 BAC+) with a median age of 59 years (interquartile range 52-68) were included and partitioned in a training set of 410 cases (1640 views, 398 BAC+), validation set of 222 cases (888 views, 89 BAC+), and test set of 229 cases (916 views, 94 BAC+). The accuracy, F1 score, and AUC-ROC were 0.94, 0.86, and 0.98 in the training set; 0.96, 0.74, and 0.96 in the validation set; and 0.97, 0.80, and 0.95 in the test set, respectively. In 112 analyzed views, the Grad-CAM++ predictions displayed a strong correlation with BAC measured length (rho = 0.88, p &lt; 0.001).ConclusionOur model showed promising performances in BAC detection and in quantification of BAC burden, showing a strong correlation with manual measurements

Serum 25-hydroxyvitamin D measurement: Comparative evaluation of three automated immunoassays

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Thyroid papillary carcinoma arising in ectopic thyroid tissue within a neck branchial cyst

BACKGROUND: Thyroid gland derives from one median anlage at the base of the tongue, and from the two fourth branchial pouches. A number of anomalies may occur during their migration. These can be in form of ectopic tissues, which are frequently found along the course of thyroglossal duct and rarely in other sites, many of these may develop same diseases as the thyroid gland. CASE PRESENTATION: A 36-years-old female presented with a 3 month history of left side neck mass. The mass disappeared following aspiration of brown colored fluid, which on cytological examination showed cells with nuclear irregularities that warranted the resection of the lesion. The histology demonstrated a thyroid papillary carcinoma arising within the branchial cyst. Thereafter, the patient underwent a total thyroidectomy with central lymph nodes dissection. Histology showed a multifocal papillary carcinoma with central lymph nodes metastases. Only four cases of primary thyroid carcinomas in neck branchial cyst have been described so far. CONCLUSION: In a lateral cystic neck mass, although rare, occurrence of ectopic thyroid tissue and presence of a papillary thyroid carcinoma should be kept in mind