A comparison study between the Siemens ADVIA 120 and manual method for the differential white blood cell count in goats.

BACKGROUND: Although widely used, the ADVIA 120 hematology analyzer has not been previously validated for determining the differential leukocyte count in goats. OBJECTIVES: The aim of this study was to compare the differential leukocyte counts provided by the ADVIA 120 (A-diff) and the manual method (M-Diff) in goats. METHODS: EDTA blood samples that were analyzed within 4 h of collection were used in the study. The following exclusion criteria were applied: inappropriately filled tubes or tubes containing clots, erroneous ADVIA peroxidase cytograms, and blood smears of poor quality. The A-Diff was compared with the M-Diff performed by two independent observers on 200 leukocytes. RESULTS: Forty samples were included after previously excluding eight samples. The correlation between the A-Diff and M-Diff was very strong for eosinophils (r = .870, p < .001) and strong for lymphocytes (r = .796, p < .001) and neutrophils (r = .730, p < .001), while no significant correlation was observed for monocytes (r = .026, p = .872). The Passing-Bablok regression analyses revealed statistically significant constant errors for neutrophils (5.83%; 95% confidence interval [CI]: 0.41%, 12.18%) and eosinophils (1.89%; 95% CI: 1.17%, 2.71%). Bland-Altman analyses showed a statistically significant negative bias for lymphocytes (-5.0%) and a statistically significant positive bias for eosinophils (2.2%). The very low basophil percentages precluded a meaningful method comparison. CONCLUSIONS: The ADVIA 120 overall demonstrated good performance for the differential WBC count in goats under the conditions of this study. Therefore, it can be considered suitable for routine hematologic screening in goats. Nonetheless, it should be emphasized that any abnormal result should be confirmed with a blood smear evaluation.

Omalizumab in the treatment of bullous pemphigoid resistant to first-line therapy: a French national multicentre retrospective study of 100 patients.

BACKGROUND: Interest in the use of omalizumab to treat bullous pemphigoid (BP) in the event of resistance or contraindication to conventional therapies is currently based on limited evidence. OBJECTIVES: To assess the effectiveness and safety of omalizumab in BP and to identify predictive factors in response to treatment. METHODS: We conducted a French national multicentre retrospective study including patients with a confirmed diagnosis of BP treated with omalizumab after failure of one or several treatment lines. We excluded patients with clinically atypical BP, as per Vaillant's criteria. The criteria for clinical response to omalizumab were defined according to the 2012 international consensus conference. Anti-BP180-NC16A IgE enzyme-linked immunosorbent assay was performed on sera collected before initiating omalizumab, when available. RESULTS: Between 2014 and 2021, 100 patients treated in 18 expert departments were included. Median age at diagnosis was 77 years (range 20-98). Complete remission (CR) was achieved in 77% of patients, and partial remission in an additional 9%. CR was maintained 'off therapy' in 11.7%, 'on minimal therapy' in 57.1%, and 'on non-minimal therapy' in 31.2%. Median time to CR was 3 months (range 2.2-24.5). Relapse rate was 14%, with a median follow-up time of 12 months (range 6-73). Adverse events occurred in four patients. CR was more frequently observed in patients with an increased serum baseline level of anti-BP180-NC16A IgE (75% vs. 41%; P = 0.011). Conversely, urticarial lesions, blood total IgE concentration or eosinophil count were not predictive of CR. Patients with an omalizumab dosage > 300 mg every 4 weeks showed a similar final outcome to those with a dosage ≤ 300 mg every 4 weeks, but control of disease activity [median 10 days (range 5-30) vs. 15 days (range 10-60); P < 0.001] and CR [median 2.4 months (range 2.2-8.2) vs. 3.9 months (range 2.3-24.5); P < 0.001] were achieved significantly faster. CONCLUSIONS: We report the largest series to date of BP treated by omalizumab and confirm its effectiveness and safety in this indication. Serum baseline level of anti-BP180-NC16A IgE may predict response to treatment.

Early-to-mid stage idiopathic Parkinson's disease shows enhanced cytotoxicity and differentiation in CD8 T-cells in females.

Neuroinflammation in the brain contributes to the pathogenesis of Parkinson's disease (PD), but the potential dysregulation of peripheral immunity has not been systematically investigated for idiopathic PD (iPD). Here we showed an elevated peripheral cytotoxic immune milieu, with more terminally-differentiated effector memory (TEMRA) CD8 T, CD8+ NKT cells and circulating cytotoxic molecules in fresh blood of patients with early-to-mid iPD, especially females, after analyzing > 700 innate and adaptive immune features. This profile, also reflected by fewer CD8+FOXP3+ T cells, was confirmed in another subcohort. Co-expression between cytotoxic molecules was selectively enhanced in CD8 TEMRA and effector memory (TEM) cells. Single-cell RNA-sequencing analysis demonstrated the accelerated differentiation within CD8 compartments, enhanced cytotoxic pathways in CD8 TEMRA and TEM cells, while CD8 central memory (TCM) and naïve cells were already more-active and transcriptionally-reprogrammed. Our work provides a comprehensive map of dysregulated peripheral immunity in iPD, proposing candidates for early diagnosis and treatments.

Anti-BP180 IgG antibody ELISA values correlate with adverse pregnancy outcomes in pemphigoid gestationis.

BACKGROUND: Adverse pregnancy outcomes (APO) occur in 35% of patients with pemphigoid gestationis (PG). No biological predictor of APO has been established yet. OBJECTIVES: To assess a potential relationship between the occurrence of APO and the serum value of anti-BP180 antibodies at the time of PG diagnosis. METHODS: Multicentre retrospective study conducted from January 2009 to December 2019 in 35 secondary and tertiary care centres. INCLUSION CRITERIA: (i) diagnosis of PG according to clinical, histological and immunological criteria, (ii) ELISA measurement of anti-BP180 IgG antibodies determined at the time of PG diagnosis with the same commercial kit and (iii) obstetrical data available. RESULTS: Of the 95 patients with PG included, 42 had one or more APO, which mainly corresponded to preterm birth (n = 26), intrauterine growth restriction (IUGR) (n = 18) and small weight for gestational age at birth (n = 16). From a ROC curve, we identified a threshold of 150 IU ELISA value as the most discriminating to differentiate between patients with or without IUGR, with 78% sensitivity, 55% specificity, 30% positive and 91% negative predictive value. The threshold >150 IU was confirmed using a cross-validation based on bootstrap resampling, which showed that the median threshold was 159 IU. Upon adjusting for oral corticosteroid intake and main clinical predictors of APO, an ELISA value of >150 IU was associated with the occurrence of IUGR (OR = 5.11; 95% CI: 1.48-22.30; p = 0.016) but not with any other APO. The combination of blisters and ELISA values higher than 150 IU led to a 2.4-fold higher risk of all-cause APO (OR: 10.90; 95% CI: 2.33-82.3) relative to patients with blisters but lower values of anti-BP180 antibodies (OR of 4.54; 95% CI 0.92-34.2). CONCLUSION: These findings suggest that anti-BP180 antibody ELISA value in combination with clinical markers is helpful in managing the risk of APO, in particular IUGR, in patients with PG.

Treatment of dissecting cellulitis of the scalp with tumour necrosis factor inhibitors: a retrospective multicentre study.

Although retinoids are considered as the most effective treatment, management of dissecting cellulitis of the scalp (DCS) is often challenging. A multicentre retrospective study was conducted to evaluate the efficacy of anti-tumour necrosis factor (TNF) agents in treating DCS after failure of other conventional treatments. Twenty-six patients were included. After a mean treatment duration of 19 months (SD 21), the median Physician's Global Assessment score decreased from 3 to 1. The median number of inflammatory nodules and abscesses decreased from 7 to 0.5 and from 1 to 0, respectively. The median Dermatology Life Quality Index and numerical rating scale score for pain severity decreased from 10 to 8 and 6 to 1, respectively. The median treatment satisfaction was 7 out of 10 on the Patient Satisfaction Index. This study confirms the efficacy of anti-TNF agents in treating patients with DCS that is resistant to conventional therapies.

High-dimensional immune profiles correlate with phenotypes of peanut allergy during food-allergic reactions.

BACKGROUND: Food challenges carry a burden of safety, effort and resources. Clinical reactivity and presentation, such as thresholds and symptoms, are considered challenging to predict ex vivo. AIMS: To identify changes of peripheral immune signatures during oral food challenges (OFC) that correlate with the clinical outcome in patients with peanut allergy (PA). METHODS: Children with a positive (OFC+ , n = 16) or a negative (OFC- , n = 10) OFC-outcome were included (controls, n = 7). Single-cell mass cytometry/unsupervised analysis allowed unbiased immunophenotyping during OFC. RESULTS: Peripheral immune profiles correlated with OFC outcome. OFC+ -profiles revealed mainly decreased Th2 cells, memory Treg and activated NK cells, which had an increased homing marker expression signifying immune cell migration into effector tissues along with symptom onset. OFC- -profiles had also signs of ongoing inflammation, but with a signature of a controlled response, lacking homing marker expression and featuring a concomitant increase of Th2-shifted CD4+ T cells and Treg cells. Low versus high threshold reactivity-groups had differential frequencies of intermediate monocytes and myeloid dendritic cells at baseline. Low threshold was associated with increased CD8+ T cells and reduced memory cells (central memory [CM] CD4+ [Th2] T cells, CM CD8+ T cells, Treg). Immune signatures also discriminated patients with preferential skin versus gastrointestinal symptoms, whereby skin signs correlated with increased expression of CCR4, a molecule enabling skin trafficking, on various immune cell types. CONCLUSION: We showed that peripheral immune signatures reflected dynamics of clinical outcome during OFC with peanut. Those immune alterations hold promise as a basis for predictive OFC biomarker discovery to monitor disease outcome and therapy of PA.

Abortion Related to Toxoplasma gondii Infection in a Bactrian Camel (Camelus bactrianus) in Greece: A Case Report.

A female, 5 yr old Bactrian camel was presented to the Exotic and Wildlife Medicine Unit, School of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece, with severe dehydration, depression, anorexia, mild dyspnea and diarrhea. Supportive treatment immediately initiated with fluids, electrolytes and broad-spectrum antibiotics. The general condition of the animal was stable for the next 3 days, but at 4th day became worse, since the camel remained in sternal recumbency, denied to drink water and abortion of a mummified fetus was noticed. The aborted fetus and fetal membranes were submitted for laboratory examinations (bacterial cultures, MZN, cytology, PCR) that revealed Toxoplasma gondii infection. Treatment with sulfadimidine improved the situation of the animal that returned to its farm 1 week later. This seems to be the first reported case in the literature of confirmed toxoplasmic abortion in camels.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells.

SCOPE: Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs. METHODS AND RESULTS: Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel. CONCLUSION: BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

Association of platelet indices with glycemic status in diabetic dogs.

We investigated the platelet count (PLT), mean platelet volume (MPV), and plateletcrit (PCT) in dogs with type 1 diabetes mellitus (DM) compared to healthy controls, and their association with the major fraction of glycated hemoglobin (HbA1c). Blood samples from 33 clinically healthy dogs and 14 newly diagnosed diabetic dogs were included. CBCs were performed with the Advia 120; HbA1c was determined using a validated assay (Capillarys 2 flex-piercing; Sebia). Median [range] PLT and PCT were significantly higher (p = 0.040 and p = 0.010, respectively) in diabetic dogs (434 [176-987] × 109/L and 0.60 [0.26-1.22]%, respectively) compared to healthy dogs (297 [223-671] × 109/L and 0.35 [0.24-0.87]%, respectively]. Thrombocytosis was observed in 6 of 14 (43%) diabetic dogs. The median MPV was not significantly different (p = 0.114) between the diabetic (13.6 fL, 10.1-22.6 fL) and healthy dogs (11.9 fL, 8.6-19.1 fL). A significant, albeit weak, correlation was detected between HbA1c and PLT (rho = 0.298, p = 0.042) and PCT (rho = 0.340, p = 0.019), but no significant correlation was found with MPV (rho = 0.199, p = 0.180). Canine DM was associated with increased PLT and PCT, which was correlated with glycemic status. Our findings suggest dysregulated megakaryopoiesis in diabetic dogs, but this should be confirmed by large-scale studies, and the clinical implications should be investigated.

Prevalence of antinuclear antibodies and rheumatoid factor titers in dogs with arthritis secondary to leishmaniosis (Leishmania infantum).

Dogs with infectious arthritis may occasionally exhibit positive serum antinuclear antibody (ANA) and rheumatoid factor (RF) titers; however, relevant data are sparse for arthritis secondary to canine leishmaniosis (CanL) caused by Leishmania infantum. We determined the prevalence of positive serum ANA and RF titers in dogs with arthritis secondary to CanL. Blood samples from adult, client-owned dogs with purulent arthritis secondary to CanL, without any comorbidities, were collected for diagnostic purposes. Serum ANA titers were measured by immunoperoxidase test and RF titers by the Rose-Waaler latex test. Twelve of 23 dogs enrolled prospectively in our study had clinical arthritis, and 11 of 23 had subclinical arthritis. Based on LeishVet clinical staging, 7 dogs had clinical stage II disease, 11 had clinical stage III disease, and 5 had stage IV. None of the 23 dogs was seropositive for ANA; 3 of 23 were positive for RF. ANA and/or RF seropositivity, in dogs with CanL-associated arthritis, appears to be weak, if present at all. Based on our results, positive serum ANA and RF titers should not be expected in dogs with arthritis secondary to CanL.

Combinatorial analysis reveals highly coordinated early-stage immune reactions that predict later antiviral immunity in mild COVID-19 patients.

While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-ß levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients.

Effect of major versus minor mastectomy on host immunity in canine mammary cancer.

Surgical procedures can affect host immunity proportionally to the extent of surgical trauma. In cancer cases, surgery-induced immunosuppression can potentially promote tumour metastasis. The aim of this study was to investigate, in bitches with malignant mammary tumours, whether major surgery (total unilateral mastectomy or bilateral regional mastectomy) has a more negative effect than minor surgery (unilateral regional mastectomy) on components of host immunity. Twenty bitches with mammary cancer of clinical stage II or III were allocated to group A (minor surgery) or group B (major surgery) of 10 animals each receiving the same anaesthetic protocol for mastectomy. Immune cell measurements in blood [number of leukocytes, neutrophils, lymphocytes and platelets, and relative percentages of T-lymphocytes (CD3+) and their CD4+, CD8+ and CD5low+ subpopulations] were performed before anaesthesia (day 0) and on days 3 and 10 post-mastectomy. On day 3, leukocytes, neutrophils and platelets numbers were higher (p = 0.016, 0.032 and 0.017, respectively) in group B than in group A. For all 20 bitches, T-lymphocytes and the CD4+, CD5low+ T-cells were significantly decreased on day 3, but no significant differences were noted between groups. Minor mastectomy seemed to preserve innate immunity better than major mastectomy, but cellular immunity was rather equally affected.

Bronchoscopy and Lung Fine-Needle Aspiration for Antemortem Evaluation of Pulmonary Involvement in Dogs with Naturally Occurring Canine Leishmaniosis.

Clinical manifestations from the lower respiratory tract are rare in canine leishmaniosis (CanL), making bronchoscopy and lung fine-needle aspiration (FNA) seldomly justified. The aim of this prospective study was to investigate the involvement of Leishmania infantum in the lungs of dogs with naturally occurring CanL by bronchoscopy and examination of bronchoalveolar lavage fluid (BALF), bronchial mucosa biopsies, and FNA, using immunodiagnostics. Dogs with relevant concurrent diseases and azotemia were excluded. Cough was detected in 5/31 (16.1%) dogs. Lesions (hyperemia, edema, mucosal granularity, secretions) were identified upon bronchoscopy in 19/31 (61.3%) dogs. The cytology of BALF revealed histiocytic inflammation in 14/31 (45.2%) dogs; the parasite was identified in one dog (3.2%). The immunofluorescence antibody test in BALF was positive in 15/31 (48.4%) dogs. Histopathology of bronchial mucosa and/or adjacent alveoli revealed lesions (mononuclear cell infiltration, fibrosis, edema, thickening of the inter-alveolar septa) in 24/31 (77.4%) dogs, with no Leishmania amastigotes. Positive antigen staining was observed within the cytoplasm of mononuclear cells in immunocytochemistry and immunohistochemistry. Μononuclear cells showed antigenic positivity in bronchial mucosa (27/31; 87.1%), BALF (30/31; 96.8%), and lung FNA (27/31; 87.1%). In conclusion, lungs seem to be affected from CanL more commonly than previously believed, and bronchoscopy allows obtaining valuable samples for antemortem diagnosis.

Effect of anaesthesia on cell-mediated immunity in dogs undergoing mastectomy for mammary cancer.

OBJECTIVE: To investigate if anaesthesia for canine cancer mastectomy further influences host cell-mediated immunity (CMI) promoting cancer progression. STUDY DESIGN: A randomized, controlled, blinded clinical study. ANIMALS: A total of 20 bitches with malignant mammary tumours of clinical stage II or III undergoing the same type of mastectomy (regional mastectomy). METHODS: Dogs were randomly allocated to one of two anaesthetic groups (10 per group). The anaesthetic protocol of group A used minimally immunosuppressive drugs (tramadol, robenacoxib, propofol), whereas that of group B (control) used more immunosuppressive drugs (morphine, fentanyl, thiopental, isoflurane). For each animal, measurements of white blood cells (WBCs), neutrophils and lymphocytes, and flow cytometric assessment of T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+) and CD5low+ T cells were performed prior to anaesthesia (day 0) and on days 3 and 10 postsurgery. Data were analysed using a General Linear Model for repeated measures and presented as mean ± standard deviation, p ≤ 0.05. RESULTS: In all animals, on day 3, WBCs and neutrophils were significantly increased (p < 0.0005), while flow cytometry revealed significantly decreased relative percentages of T cells (CD3+) (p = 0.003) and their subpopulations CD4+ (p = 0.006), CD8+ (p = 0.029) and CD5low+ (p = 0.031). Specifically, on day 3, the cytotoxic T cells (CD8+) were significantly decreased (p = 0.05) only in group B, whereas the CD4+ (p = 0.006) and CD5low+ (p = 0.008) T cells in group A. The only significant difference between groups was found preoperatively in the CD4+/CD8+ ratio, which was higher in group A (p = 0.006). CONCLUSIONS AND CLINICAL RELEVANCE: In dogs with mammary cancer undergoing regional mastectomy, a significant decrease in components of CMI was observed on day 3 postsurgery in both anaesthetic groups. Some indication, however, for better preserved cellular immunity by less immunosuppressive anaesthetic/analgesic drugs was detected, rendering their use advisable.

Hematology reference intervals during the prepartum period, first week after calving, and peak lactation in clinically healthy Holstein cows.

Dairy cows face precipitous metabolic adaptations during the first postpartum week. Increasing knowledge of their hematologic profiles during this period could benefit both veterinary clinical pathologists and bovine practitioners. The objectives of this study were: (a) to establish period-specific RIs for Holstein dairy cows and (b) to assess the potential simultaneous effect of several factors on hematology parameters. Blood samples from clinically healthy cows of nine herds were used to establish hematology RIs for these periods. There were 68 cows in the prepartum period (PP), 247 and 218 cows at 2 and 8 days after calving (2 and 8 days in milk [DIM 2 and DIM 8], respectively), and 87 cows in peak lactation (PL). A complete blood count evaluation was performed using the ADVIA 120 analyzer. Linear models estimated the analytes associated with parity. RIs were calculated with Reference Value Advisor. Parity had a significant effect on analytes at all stages. Marked differences were evident between DIM 2 and 8 regarding red blood cell (RBC) counts, hematocrits, hemoglobin concentrations, and white blood cell (WBC), monocyte, and eosinophil counts. Reference intervals of RBC counts, hematocrits, and hemoglobin concentrations at DIM 2 were higher than those at PP and PL. This study provides evidence that period-specific RIs should be used for Holstein cows when making clinical decisions, especially during the critical first week postpartum.