Cytological, Bacteriological and Antibiogram Studies for the Management of Uterine Infection in Repeat Breeder Bovines

Background: Repeat breeding syndrome (RBS) associated with sub-clinical uterine infection (UI) remains to be a major reproductive problem faced by Indian farmers. Present study documents its diagnosis, prevalent etiological agents, antibiogram pattern and efficacy of the treatment at field level. Methods: Seventy-eight RBS affected bovines were selected. The cervical mucous (CM) was collected for study of its characteristics, white-side test, endometrial cytology, microbial examination and antibiogram. The treatment protocol was developed and the animals’ response to the treatment was assessed. Result: The overall incidence of RBS was found to be 12.9% and the cases associated with uterine infection (RBS/UI+ve) and without uterine infection (RBS/UI-ve) were 44.87% and 55.13%, respectively. The mean scores of CM character, odour, pH and number of polymorphonuclear cells in RBS/UI +ve were 2.09±1.39, 1.14±0.12, 8.49±0.08 and 12.46±0.96, respectively, and differed significantly (P less than 0.05) from RBS/UI-ve cases. The microbial examination revealed the presence of gram negative bacilli, Trueperella spp., Pseudomonas spp., Staphylococcus spp., Escherichia coli and yeast infection. Antibiogram studies recorded the response of Tetracyclin as best (48.57%) followed by Cephalexin (22.86%), Chloramphenicol (20.0%) and Streptomycin (8.57%). The RBS/UI+ve cases were treated individually, on one-to-one basis, obeying antibiogram. The infection appeared to be considerably controlled and overall success rate was observed in the form of confirmed pregnancy in 71.43% cases. Thus, it was concluded that prompt diagnosis using endometrial cytology and antibiogram guided therapeutic approach may aid for effective management of RBS/UI +ve cases, under field conditions.

Osteochondral necrosis of the femoral condyles in Thoroughbred foals: eight cases (2008–2018)

OBJECTIVE To describe clinical, imaging, gross, and histopathological abnormalities associated with osteochondral necrosis of the femoral condyles in foals and identify features suggestive of a common pathogenesis. ANIMALS 8 Thoroughbred foals euthanized with a presumptive diagnosis of necrosis of the femoral condyles. PROCEDURES Postmortem CT was performed on all distal femoral epiphyseal samples. The articular epiphyseal cartilage complex (AECC) of affected distal femurs was examined grossly and histologically, focusing on lesions of interest identified on CT images. RESULTS 7 foals were between 9 and 23 days old at the time of euthanasia; 1 foal was 85 days old. Concurrent illness (neonatal maladjustment syndrome, neonatal isoerythrolysis, or infection such as enteritis and omphalitis) was diagnosed in 7 foals. The characteristic antemortem radiographic and postmortem CT finding was a crescent-shaped osteochondral flap displaced from the affected medial femoral condyle. Synovial fluid cytology from affected joints was either within normal limits or consistent with mild inflammation. Histologically, all lesions were characterized by osteochondral necrosis and detachment of the AECC. In 6 foals, polymorphonuclear cells were found within growth cartilage canals, representing septic cartilage canals. CLINICAL RELEVANCE Osteochondral necrosis was interpreted to be secondary to bacterial colonization of the distal femoral AECC, evidenced by septic cartilage canals identified in 6 of 8 foals. This uncommon condition was previously thought to arise from an ischemic event, but the disease process was not well understood. An improved understanding of the pathogenesis of osteochondral necrosis is the first step in formulating more successful preventative and treatment strategies.

Neutrophils—From Bone Marrow to First-Line Defense of the Innate Immune System

Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue.

Inflammation in rats born to mothers treated with dexamethasone 15cH during pregnancy: an immunohistochemical study

In previous studies, we observed that rats born to mothers treated with dexamethasone 15CH (10-33M) had a higher level of mast cell degranulation and greater arteriolar dilation after the exposure of an inflammatory stimulus, suggesting the possibility of vertical transmission of the effects of ultra-diluted substances between mother and offspring. In this study, a more detailed assessment of the cellular events in acute inflammation was made using techniques of immunohistochemistry. The identification of adhesion molecules expression was made by the markers: anti-CD54 (ICAM-1) and anti-CD18 (β2-Integrin). The identification of inflammatory cells was performed by the markers anti-MAC387 (mononuclear cells) and anti-CD163 (active macrophages). Polymorphonuclear cells were identified by hematoxylin-eosin staining. The number of labeled cells per field was recorded, except for the anti-CD54 marker, whose intensity of staining on the endothelial cells was defined by scores assigned by two independent observers. The results point toward to an up regulation of the whole inflammatory process in rats born to mothers treated with dexamethasone 15CH during pregnancy. This conclusion is justified by the following statistically significant (p≤0.05) findings: a) bigger mast cell degranulation and increased of arteriolar diameter; b) increased migration of polymorphonuclear cells in relation to the mononuclear cells; c) earlier expression of CD163 in monocytes, d) higher level of adhesion molecules expression.

Polymorphonuclear Cells Show Features of Dysfunctional Activation During Fatal Sepsis

Sepsis and septic shock remain leading causes of morbidity and mortality for patients in the intensive care unit. During the early phase, immune cells produce various cytokines leading to prompt activation of the immune system. Polymorphonuclear leukocytes (PMNs) respond to different signals producing inflammatory factors and executing their antimicrobial mechanisms, resulting in the engulfment and elimination of invading pathogens. However, excessive activation caused by various inflammatory signals produced during sepsis progression can lead to the alteration of PMN signaling and subsequent defects in their functionality. Here, we analyzed samples from 34 patients in septic shock, focusing on PMNs gene expression and proteome changes associated with septic shock. We revealed that, compared to those patients who survived longer than five days, PMNs from patients who had fulminant sepsis were characterized by a dysfunctional hyper-activation, show altered metabolism, and recent exit from the cell cycle and signs of cellular lifespan. We believe that this multi-omics approach, although limited, pinpoints the alterations in PMNs’ functionality, which may be rescued by targeted treatments.

What are the best diagnostic tests for diagnosing bacterial arthritis of a native joint?

Aims This study aimed to answer two questions: what are the best diagnostic methods for diagnosing bacterial arthritis of a native joint?; and what are the most commonly used definitions for bacterial arthritis of a native joint? Methods We performed a search of PubMed, Embase, and Cochrane libraries for relevant studies published between January 1980 and April 2020. Of 3,209 identified studies, we included 27 after full screening. Sensitivity, specificity, area under the curve, and Youden index of diagnostic tests were extracted from included studies. We grouped test characteristics per diagnostic modality. We extracted the definitions used to establish a definitive diagnosis of bacterial arthritis of a native joint per study. Results Overall, 28 unique diagnostic tests for diagnosing bacterial arthritis of a native joint were identified. The following five tests were deemed most useful: serum ESR (sensitivity: 34% to 100%, specificity: 23% to 93%), serum CRP (sensitivity: 58% to 100%, specificity: 0% to 96%), serum procalcitonin (sensitivity: 0% to 100%, specificity: 68% to 100%), the proportion of synovial polymorphonuclear cells (sensitivity: 42% to 100%, specificity: 54% to 94%), and the gram stain of synovial fluid (sensitivity: 27% to 81%, specificity: 99% to 100%). Conclusion Diagnostic methods with relatively high sensitivities, such as serum CRP, ESR, and synovial polymorphonuclear cells, are useful for screening. Diagnostic methods with a relatively high specificity, such as serum procalcitonin and synovial fluid gram stain, are useful for establishing a diagnosis of bacterial arthritis. This review helps to interpret the value of various diagnostic tests for diagnosing bacterial arthritis of a native joint in clinical practice. Cite this article: Bone Joint J 2021;103-B(12):1745–1753.

Milk Somatic Cell Count and Polymorphonuclear Cells in Healthy Quarters of Cows That Underwent Blanket and Selective Dry Therapy: An Italian Case Study

The incidence of mastitis increases with parity in dairy cattle and multiparous cows are often treated at drying off to limit the risk of udder health issues and support mammary gland tissues recovery. Milk somatic cells count (SCC, cells/mL) comprises different white blood cells fractions and is worldwide used to monitor and genetically improve udder health. Nevertheless, only certain SCC fractions increase when an udder inflammation occurs. Considering that antibiotic use for preventive purposes will be forbidden in 2022, we compared two different dry therapy protocols, blanket (BDCT) and selective (SDCT), on different SCC fractions in healthy quarters milk. Multiparous Holstein cows were enrolled in a randomized controlled trial and SCC, neutrophils, macrophages, lymphocytes, polymorphonuclear cells (PMN) and differential somatic cell count (DSCC) recorded after the experimental drying off were available. Significant differences were observed between the two protocols, with more favorable parameters in BDCT than SDCT cows. Results showed that moving from BDCT to SDCT is expected to significantly increase some SCC fractions, such as PMN, in healthy quarters. The baseline SCC level at the onset of lactation was greater in cows under SDCT than BDCT. Although not significant, clinical mastitis prevalence was numerically lower in BDCT (7.32%) than SDCT (8.62%). In this study we referred to a limited number of cows, but still findings will be useful to improve the knowledge on the impact of SDCT on milk SCC fractions in healthy quarters.

Regulation of T-Cell Immune Responses by Pro-Resolving Lipid Mediators

Both the initiation and the resolution of inflammatory responses are governed by the sequential activation, migration, and control/suppression of immune cells at the site of injury. Bioactive lipids play a major role in the fine-tuning of this dynamic process in a timely manner. During inflammation and its resolution, polymorphonuclear cells (PMNs) and macrophages switch from producing pro-inflammatory prostaglandins and leukotrienes to specialized pro-resolving lipid mediators (SPMs), namely, lipoxins, resolvins, protectins, and maresins, which are operative at the local level to limit further inflammation and tissue injury and restore homeostasis. Accumulating evidences expand now the role and actions of these lipid mediators from innate to adaptive immunity. In particular, SPMs have been shown to contribute to the control of chronic inflammation, and alterations in their production and/or function have been associated with the persistence of several pathological conditions, including autoimmunity, in human and experimental models. In this review, we focus on the impact of pro-resolving lipids on T cells through their ability to modulate T-cell responses. In particular, the effects of the different families of SPMs to restrain effector T-cell functions while promoting regulatory T cells will be reviewed, along with the underlying mechanisms. Furthermore, the emerging concept of SPMs as new biological markers for disease diagnostic and progression and as putative therapeutic tools to regulate the development and magnitude of inflammatory and autoimmune diseases is discussed.

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A). The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date. Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members. Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C). In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. Figure 1 Figure 1. Disclosures Voorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

The Roles of Transient Receptor Potential Vanilloid 1 and 4 in Pneumococcal Nasal Colonization and Subsequent Development of Invasive Disease

Transient receptor potential (TRP) channels, neuronal stimulations widely known to be associated with thermal responses, pain induction, and osmoregulation, have been shown in recent studies to have underlying mechanisms associated with inflammatory responses. The role of TRP channels on inflammatory milieu during bacterial infections has been widely demonstrated. It may vary among types of channels/pathogens, however, and it is not known how TRP channels function during pneumococcal infections. Streptococcus pneumoniae can cause severe infections such as pneumonia, bacteremia, and meningitis, with systemic inflammatory responses. This study examines the role of TRP channels (TRPV1 and TRPV4) for pneumococcal nasal colonization and subsequent development of invasive pneumococcal disease in a mouse model. Both TRPV1 and TRPV4 channels were shown to be related to regulation of the development of pneumococcal diseases. In particular, the influx of neutrophils (polymorphonuclear cells) in the nasal cavity and the bactericidal activity were significantly suppressed among TRPV4 knockout mice. This may lead to severe pneumococcal pneumonia, resulting in dissemination of the bacteria to various organs and causing high mortality during influenza virus coinfection. Regulating host immune responses by TRP channels could be a novel strategy against pathogenic microorganisms causing strong local/systemic inflammation.