Use and adequacy of non-pregnancy diagnosis in cow. Which future?

In cattle, early detection of gestation is very important from an economic and management point of view in all types of farming. However, due to the poor efficiency of oestrous detection, it is essential to determine non-pregnant cows as early as possible, in order to minimize the inter-insemination interval, thus de facto, reducing herd open days. Direct and indirect gestation diagnostic methods have been developed with the aim of improving the reproductive performance of the herd. Today, the most accurate method for making an early diagnosis of gestation from 28 to 30 days post-insemination is B-mode ultrasound. In recent years, indirect methods have included techniques that allow non-pregnant cows to be identified with a minimum margin of error, the most widely utilized of which is the colour Doppler. This technique is rapidly becoming established for the diagnosis of non-pregnancy, which allows for the identification of non-pregnant animals earlier compared with the pregnancy diagnosis. Some limitations of this technique in dairy cow have been presented.

Challenges in Diagnosis and Clinical Management of COVID-19 in Patient with B-Cell Chronic Lymphocytic Leukemia (CLL): Report of One Case.

We report here a case of a patient affected by B-cell chronic lymphocytic leukemia (CLL) that developed COVID-19 during the actual SARS-CoV-2 outbreak. The coexistence of CLL and COVID-19 raises many questions regarding the possible increased risk of developing COVID-19 among patients with CLL, the problems in managing therapies for both diseases and, above all, the difficulties in diagnosing COVID-19 in patients affected by CLL. In our patient, an 84-year-old man, the recognition of COVID-19 was delayed because of its atypical clinical presentation and technical problems related to the methods used for the diagnosis. Based on the symptoms and the radiological aspect of the lung, the occurrence of COVID-19 was suspected. Repeated tests on oro/nasopharyngeal swabs gave negative results, causing a delay in the diagnosis. Moreover, different methods used to identify the SARS-CoV-2 antibodies in serum gave conflicting results, and only two tests were able to identify SARS-CoV-2 Abs of the IgG type. During the clinical course of unrecognized COVID-19, our patient developed severe complications and did not receive any specific treatment for the two diseases. Recognition of COVID-19 in patients with CLL is a challenging task and the most accurate methods are necessary to overcome the diagnostic difficulties encountered.

Medial Entorhinal Cortex Selectively Supports Temporal Coding by Hippocampal Neurons.

Recent studies have shown that hippocampal "time cells" code for sequential moments in temporally organized experiences. However, it is currently unknown whether these temporal firing patterns critically rely on upstream cortical input. Here we employ an optogenetic approach to explore the effect of large-scale inactivation of the medial entorhinal cortex on temporal, as well as spatial and object, coding by hippocampal CA1 neurons. Medial entorhinal inactivation produced a specific deficit in temporal coding in CA1 and resulted in significant impairment in memory across a temporal delay. In striking contrast, spatial and object coding remained intact. Further, we extended the scope of hippocampal phase precession to include object information relevant to memory and behavior. Overall, our work demonstrates that medial entorhinal activity plays an especially important role for CA1 in temporal coding and memory across time.

Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos).

The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1 ) and receptor 2 (OX2 ) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2 -IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2 . Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.

Presence and distribution of urocortin and its receptors in the epididymis of alpaca (Vicugna pacos).

Urocortin 1 (UCN) is a 40-amino acid peptide belonging to the corticotrophin-releasing hormone (CRH) family. The biological effects of this peptide are modulated by binding two G-coupled receptors named CRH receptor 1 (CRHR1) and CRH receptor 2 (CRHR2). CRHR2 has high affinity for UCN. The aim of the present study was to investigate the presence and distribution of UCN, CRHR1 and CRHR2 in the epididymis of the South America camelid Alpaca (Vicugna pacos) by Western blotting analysis and immunohistochemistry. Tissue extracts of the organ reacted with the anti-UCN, anti-CRHR1 and anti-CRHR2 antibodies, recognizing in all the cases a single specific protein band. UCN- and CRHR2-immunoreactivities (IRs) were found in the cytoplasm of the principal cells (PCs) of the caput epididymis. A prevalent supranuclear localization of granular-shaped positive material was observed. CRHR1-IR was observed in the fibromuscular stromal cells encircling the tubules and in the smooth musculature of the blood vessels throughout the three epididymal segments. In addition, in the cauda, CRHR1-IR was observed in some apical epithelial cells (ACs) which were morphologically similar to apical mitochondria-rich cells (AMRCs). These results suggest that UCN, CRHR1 and CRHR2 are expressed in the alpaca epididymis and that CRH-related peptides might play multiple roles in maturation and storage of spermatozoa.

Presence and distribution of urocortin and corticotrophin-releasing hormone receptors in the bovine thyroid gland.

Urocortin (UCN), a 40 amino acid peptide, is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated via two types of G-protein-coupled receptors, CRH type 1 (CRHR1) and CRH type 2 (CRHR2). The aim of this study was to investigate the expression of UCN, CRHR1 and CRHR2 by immunoprecipitation, Western blot, immunohistochemistry and RT-PCR in the bovine thyroid gland. Immunoprecipitation and Western blot analysis showed that tissue extracts reacted with the anti-UCN, anti-CRHR1 and anti-CRHR2 antibodies. RT-PCR experiments demonstrated that mRNAs of UCN, CRHR1 and CRHR2 were expressed. UCN immunoreactivity (IR) and CRHR2-IR were found in the thyroid follicular and parafollicular cells and CRHR1-IR in the smooth muscle of the blood vessels. These results suggest that a regulatory system exists in the bovine thyroid gland based on UCN, CRHR1 and CRHR2 and that UCN plays a role in the regulation of thyroid physiological functions through an autocrine/paracrine mechanism.

Expression of orexin A and its receptor 1 in the epididymis of the South American camelid alpaca (Vicugna pacos).

Orexins A (ox A) and B are two peptides originally discovered in neurons of rat hypothalamus, and later found in different cellular types of the gastrointestinal and genital tracts. They arise from the proteolytic cleavage of a common precursor molecule, prepro-orexin, and bind to two receptors, namely receptor 1 (ox1r) and receptor 2 for orexins, that show different binding affinity. The central role of the two peptides has been extensively studied, whereas their activity in the periphery is still poorly known. Here, we investigated the presence of ox A and ox1r in the epididymis of a South American camelid species, the alpaca, by immunohistochemistry, and we also assessed the expression of prepro-orexin and ox1r in tissue extracts by Western blotting analysis. Ox A- and ox1r-immunoreactivity was found in the cytoplasm of principal cells of the caput epididymis. A prevalent supranuclear localization of granular-shaped positive material was observed. No positivity was present in the other cytotypes of epididymis. The expression of two peptides with molecular weight corresponding to those of prepro-orexin and ox1r, respectively, was detected in the tissue extracts from the organ.

Outcome of patients with splanchnic venous thrombosis presenting without overt MPN: a role for the JAK2 V617F mutation re-evaluation.

INTRODUCTION: Although investigation for JAK2 V617F mutation is recommended in patients presenting with splanchnic venous thrombosis (SVT), no specific clinical advice is given to SVT patients presenting without myeloproliferative neoplasms (MPN) and JAK2 V617F mutation. In MPN-free SVT patients, to investigate the clinical outcome, the clinical impact of re-evaluation for the JAK2 V617F mutation, and relationships with the occurrence and time to diagnosis of MPN. MATERIALS AND METHODS: A cohort of non-cirrhotic SVT patients, enrolled at a single centre and prospectively analyzed. RESULTS: In 121 SVT patients prospectively followed from 1994 to 2012, a MPN was present in 28 (23.1%). Additional 13 patients (10.7%) showed only the JAK2 V617F mutation. During the follow-up, the JAK2 V617F mutation and/or MPN were identified in 8 patients (median time of development: 21 months, range 6-120), whereas 72 remained (MPN and JAK2 V617F)-free until the end of the observation. The mortality rate was higher among patients presenting with MPN and/or the JAK2 V617F mutation than in patients who developed later or remained disease-free (p=0.032). The thrombosis-free survival was lower in patients with (p=0.04) or developing later MPN and the JAK2 V617F mutation (p=0.005) than in patients (MPN and JAK2 V617F)-free. The incidence of bleeding was similar among groups. CONCLUSIONS: MPN with or without circulating positive clones for JAK2 V617F mutation can occur long after a SVT, identifying at risk patients for new thrombotic events. If confirmed in other studies, re-evaluation for JAK2 V617F mutation may be of help in early MPN detection and clinical management of SVT patients.

Maternal hemodynamics influence fetal hemodynamics in normal and hypertensive pregnancy.

OBJECTIVES: This observational case-control study aims to test whether there is a relationship between maternal systemic hemodynamics, maternal renin-angiotensin system and fetal hemodynamics in normal and hypertensive pregnancy. STUDY DESIGN: Four groups of non-pregnant women (n=18), pregnant controls (n=25), women with gestational hypertension (n=21) and preeclampsia (n=10) were included. MAIN OUTCOME MEASURES: Maternal echocardiography parameters, plasma renin and aldosterone were correlated with fetal Doppler parameters in third trimester pregnancy. RESULTS: Higher maternal mean arterial pressure and total peripheral vascular resistance were associated with lower fetal middle cerebral artery pulsatility index (PI) (r=-.51, p<0.01 and r=-.49, p<0.01, respectively); mean arterial pressure correlated negatively with ductus venosus PI (r=-.35, p=0.01); higher maternal plasma aldosterone levels were associated with lower maternal uterine artery resistance (r=-0.33, p=0.03). CONCLUSIONS: It seems that maternal hemodynamics influence fetal hemodynamics with protective adaptation in fetal cerebral and ductus venosus blood flow observed as maternal blood pressure and vascular resistance increase.

Immunocytochemical characterisation of cultures of human bladder mucosal cells.

BACKGROUND: The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation. METHODS: Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence. RESULTS: Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections. CONCLUSIONS: Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.

Antiphospholipid antibodies and acute-phase response in non-metastatic colorectal cancer patients.

AIM: To investigate the plasma levels and prevalence of the most common antiphospholipid antibodies, as well as their relationships with several plasma markers of inflammation, in order to characterize some aspects of cancer thrombophilia. MATERIALS AND METHODS: Eighty-three cancer patients with non-metastatic colorectal solid tumors and 94 control subjects were tested for the presence of IgG/IgM/IgA anti-cardiolipin and anti-Beta2-glycoprotein I antibodies and of several acute-phase reactants, i.e., fibrinogen, factor VIII:C and C4b-binding protein. RESULTS: In cancer patients the plasma levels of the acute-phase reactants and the IgA/IgG anti-cardiolipin and IgA anti-Beta2- glycoprotein I antibodies were significantly higher; the acute-phase reactants were significantly correlated with anti-cardiolipin antibodies; the prevalence of antiphospholipid antibodies was not significantly higher. CONCLUSIONS: In patients with non-metastatic colorectal cancer the acute-phase response is associated with antiphospholipid generation. This could represent a further pathogenetic mechanism for the short-term post-surgery thrombotic complications of patients with colorectal cancer.

Coagulation factor levels in non-metastatic colorectal cancer patients.

UNLABELLED: There is evidence that high plasma levels of factor (F) VIII, FIX, FXI and fibrinogen are independent risk factors for venous thromboembolism. AIM: To determine the plasma concentrations of several coagulation factors and C4b-binding protein (C4BP) in a group of patients with non-metastatic colorectal cancer in order to investigate some aspects of cancer-acquired thrombophilia. METHODS: Plasma fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI and FXII activity levels and C4BP concentrations were determined in 73 patients with non-metastatic colorectal cancer (48 colon and 25 rectum) and in 67 matched control subjects. No one in either group had had previous thrombotic events. RESULTS: Mean plasma concentrations of fibrinogen (functional and antigen), FVIII, FIX, FV and C4BP were significantly higher in colorectal cancer patients than in control subjects, while FVII and FXII levels were significantly decreased. Several correlations were found between the increased coagulation factors and C4BP concentrations, while FVII was highly correlated with FXII. CONCLUSIONS: In colorectal cancer patients high plasma fibrinogen, FVIII and FIX levels might represent further risk factors for venous thrombotic complications in the immediate post-surgery period, while decreased FVII and FXII concentrations may be an index of intravascular coagulation activation, still in a subclinical phase.

In-vitro activity of the synthetic protegrin IB-367 alone and in combination with antifungal agents against clinical isolates of Candida spp.

The in vitro activity of the peptide IB-367, alone or combined with either fluconazole (FLU) or amphotericin B (AMB), was investigated against 25 Candida isolates belonging to five species. IB-367 minimum inhibitory concentrations (MICs) ranged from 2.0 to 32 microg/ml and it was active against both FLU-susceptible and - resistant isolates. A rapid fungicidal activity was observed. Synergism was documented in 41.6% and 44% of IB-367/FLU and IB-367/AMB interactions, respectively. Antagonism was never observed. The broad antifungal activity and the positive interactions with AMB and FLU suggest that IB-367 represents a promising candidate against infections due to Candida spp.

Protein S in cancer patients with non-metastatic solid tumours.

AIMS: To study protein S, as an acute phase protein, for its relationships with C4b-BP (C4BP), fibrinogen and Factor VIII:C in a group of patients with solid tumours, without proven metastases. METHODS: Eighty-one consecutive patients with gastrointestinal or pelvic adenocarcinoma (TNM staging: T1-3, N0-2, M0) and 58 healthy subjects were evaluated for plasma free and total protein S antigen, protein S activity, C4BP, fibrinogen and Factor VIII:C. RESULTS: When compared to the control group, the total protein S, the C4BP, the fibrinogen and the Factor VIII:C mean levels were significantly higher in the cancer group, but there was no significant difference for the free and the functional protein S mean concentrations. In both groups the free protein S was correlated with the functional and the total protein S; moreover the latter was significantly correlated with the C4BP, whereas it was significantly correlated with the fibrinogen and the Factor VIII:C only in the cancer group. In addition, a high correlation was found among the C4BP, the fibrinogen and the Factor VIII:C. CONCLUSIONS: Our data show that in these patients there is an acute phase response and suggest that, in the thrombophilic early cancer screening, determination of free protein S is redundant.

Signal amplification by combining two advanced immunohistochemical techniques.

The immunohistochemical techniques known as EnVision trade mark + System (EVS) and Mirror Image Complementary Antibody (MICA) were recently introduced into laboratory practice because of their high sensitivity. In this paper these techniques were compared and their sequences combined to obtain a new method possibly more sensitive than the original ones. The immunohistochemical staining employing the avidin-biotin complex (ABC), largely used as routine, was adopted as a term of comparison. Samples from the small and large intestine of pigs and sheep were fixed in Bouin and embedded in Paraplast. The primary antibodies utilized were directed against the neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and chromogranin A (Cr A). Targets of these antibodies were nerve structures of the intestinal wall, as well as endocrine cells scattered in the mucosa of the bowel, defined neuroendocrine cells or paraneurons. The EVS method appeared as slightly superior to the MICA method regarding sensitivity of detection. The EVS/MICA (combined) method resulted four/eight times more effective than the original techniques regarding sensitivity of detection and staining intensity, both at low and high dilutions of the primary antibodies. Of these latter, immunopositive structures were still clearly identifiable, at a dilution of 1:256,000. Such efficiency could be explained by the high number of revealing molecules of peroxidase contained in the new sequence. The application of the combined method is recommended when a small quantity of tissue antigens needs to be detected immunohistochemically.

NADPH-d positive neurons of the lizard Podarcis s. sicula oviduct and their relationship to 17beta-estradiol hormone.

The distribution of nicotinamide adenine dinucleotide phosphate reduced diaphorase (NADPH-d) containing neurons was examined in the oviduct of the lizard Podarcis s. sicula and the relationship between these neurons and 17beta-estradiol hormone was studied. NADPH-d-histochemistry and indirect immunofluorescence method were applied to cryostat sections. NADPH-d-nerve structures were found throughout the oviduct. Positive neurons were primarily located in the reproductive oviduct, and were more numerous in the intermuscular and circular muscle layers than in the mucosa. The vagina revealed a reactive nerve population denser than elsewhere. The NADPH-d-positive neurons densities and the 17beta-estradiol plasma levels coincided throughout the lizard sexual cycle. In addition, after 17beta-estradiol treatments, non-reproductive lizards showed an increase of NADPH-d neurons. We suppose that nitric oxide (NO) neurons play an estrogen-dependent role in the oviduct muscle motility.

Nitric oxide-containing neurons in the bovine gut, with special reference to their relationship with VIP and galanin.

The presence and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d)-containing neurons have been studied by means of NADPH-d histochemistry in different regions of the adult cow gut, from the esophagus to the rectum. NADPH-d and nitric oxide synthase (NOS) were constantly recognized to be colocalized in the same neuron. The colocalization of vasoactive intestinal polypeptide (VIP) and galanin in such nitrergic neurons was also studied by means of combined histochemical and immunofluorescence techniques. NADPH-d-positive neurons were present along the myenteric plexus of the entire gut, and in the submucous plexus from the abomasum to the rectum. Notably, they formed two types of nerve networks in the submucous connective tissue of the jejunum-ileum. NADPH-d-positive innervation of the muscle layers occurred throughout the tract, and sometimes a clear correspondence was noted between the number of reactive fibres and the thickness of the muscle. Nitrergic fibres also occurred in the mucosa and often were in relation to glands and blood vessels. The nitrergic neurons varied in size, shape, and intensity of staining, and often their terminals were seen to surround unstained perikarya. Various types of neurons were recognized on the basis of their chemical content; one of them contained galanin, VIP and NOS simultaneously. The present results suggest that the nitrergic neurons of the bovine gastrointestinal tract play roles presumably for controlling the motility of the gut and the conduction of interneuronal impulses.

Behavior of circulating endothelin-1 in a group of patients with acute myocardial infarction.

The aim of this study was to investigate the behavior of plasma endothelin-1 (ET-1) in 23 patients with acute myocardial infarction, complicated and uncomplicated by left ventricular failure, and treated with and without thrombolytic agents. ET-1 was measured on admission; on days 2, 3, and 5; and again on discharge. In addition, on discharge, ET-1 was correlated with left ventricular systolic function. Left ventricular failure was present, on admission, in 14 patients, whereas the other nine did not have any hemodynamic impairment. On discharge, no patients had left ventricular failure, but 11 had moderate to severe left ventricular systolic dysfunction, defined as left ventricular ejection fraction (LVEF) < 40%. Fourteen subjects, matched for age and sex, served as a control group. Compared with the control range, ET-1 was highly elevated on the first day, in both uncomplicated (p < 0.01) and complicated patients (p < 0.001). Then it decreased rapidly in the uncomplicated group, reaching the control range within day 5, whereas in the complicated group it remained significantly elevated in comparison with both the control subjects and the uncomplicated patients, until discharge. ET-1 was not correlated with the peak of creatine-kinase MB isoenzyme in any group. In seven patients submitted to thrombolytic treatment ET-1 was always significantly lower than in the nonthrombolyzed patients (p < 0.05), but the pattern of variation across time was no different. On discharge, the difference in plasma ET-1 between patients with LVEF < 40% and the control group was significant (p < 0.001), as was the difference between patients with and without moderate to severe systolic dysfunction (p < 0.01). ET-1 was closely and inversely correlated with LVEF when patients were considered as a whole (p < 0.001). These results suggest that the ET-1 increase in the early phase of myocardial infarction could be due to an ischemic process, to stress reaction, and to cardiac hemodynamic impairment, and therefore, ET-1 may be a good marker of disease. In the following phase the ET-1, being correlated with LVEF, could be a reliable index of systolic function.