Poikilocytosis of Angora goats is associated with erythrocyte density and reticulocytosis.

Angora goats in South Africa experience several syndromes that result in notable morbidity and mortality in juveniles and adults, but not kids. Insight into their causes is hampered by the lack of normal reference values for this breed, and the present study therefore aimed to characterise (1) differences in the haematology of healthy kids at birth and weaning, and (2) the haematology of apparently healthy yearlings. Selected variables were measured by blood smear analysis, and complete blood counts were performed using an ADVIA 2120i. Variables at 1, 11, and 20 weeks of age were compared using the Friedman test and associations between variables of yearlings were determined by correlation analysis. In kids, red blood cell count, mean corpuscular haemoglobin concentration (MCHC), and poikilocytosis increased over time, while mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) decreased. Yearlings displayed a lower MCHC, and higher haemoglobin distribution width than previously reported for goats, and these were positively correlated with poikilocytosis, as were reticulocyte counts. White cell counts of yearlings exceeded normal values previously reported for goats, with some individuals displaying remarkably high mature neutrophil counts. Changes in haemoglobin variant expression or cation and water fluxes are possible explanations for the findings in kids, while in yearlings, the associations between MCHC, HDW, poikilocytosis, and reticulocytosis suggest alterations in red cell hydration in adulthood that are associated with increased red cell turnover. These findings may prove informative in the further investigation of various clinical syndromes in this population.

Temporally specific adrenocorticotropic hormone reference intervals for horses in South Africa.

An endogenous adrenocorticotropic hormone (ACTH) concentration above the reference interval (RI) is commonly used as means for diagnosing equine pituitary pars intermedia dysfunction (PPID). Basal ACTH concentrations are highly dependent on photoperiod and RIs should be month- and location-specific. To date, no ACTH RIs have been specifically established for South Africa. This study aimed to determine geographically and seasonally relevant RIs for equine ACTH in the Gauteng province of South Africa. A longitudinal prospective study was conducted over twelve months to determine ACTH RIs for a representative population of healthy South African horses in the Gauteng province. Eighty clinically healthy horses under 12 years of age were recruited for monthly venous blood sample collection, from July 2019 to June 2020. ACTH was measured using a chemiluminescent assay. RIs were constructed for each month of the year. This South African population showed similar temporal changes in ACTH concentrations to those previously observed in other locations. Upper reference limits were at their lowest in early summer (21.4 pg/ml, 90% CI 20.8-21.7) with a pronounced increase in autumn (60.6 pg/ml, 90% CI 53.1-62.7), and tapered off in winter (22.3 pg/ml, 90% CI 19.9-23.2). The month-specific ACTH RIs generated in this study will improve the accuracy of diagnosis and monitoring of PPID in the local equine population. These results highlighted the previously recommended need for seasonal and location-specific RIs.

The effects of feeding and transport length on the welfare of white rhinoceroses (Ceratotherium simum simum) during long-distance translocations: a preliminary study.

Translocation is a valuable conservation tool, but poses significant risks for the transported rhinoceroses. Interventions reducing these risks are required to ensure positive welfare during transportation. The aim of this study was to evaluate the effect of journey duration and feeding during the transport of white rhinoceroses (Ceratotherium simum simum). A total of 32 animals were transported by road during two events, five days apart. Fifteen rhinoceroses in the first transport event (37.0 ± 2.4 hr duration) were not fed, while 17 rhinoceroses in the second event (32.2 ± 1.5 hr duration) were offered lucerne. Blood samples were collected at capture and after transport for the evaluation of changes in serum clinical chemistry analytes. The Wilcoxon rank-sum test was used to compare differences between the groups. In all rhinoceroses, transport resulted in changes in serum electrolyte, metabolite and enzyme concentrations, indicating a loss in total body water, nutritional shifts, stress and fatigue. Fed rhinoceroses, transported over a shorter time, displayed greater changes in osmolality (p < 0.006), serum sodium and chloride concentrations (p = 0.005 and = 0.001, respectively) indicating a greater degree of total body water loss than non-fed rhinoceroses. Feeding and a shorter transport duration reduced, but did not prevent, nutritional challenges. A greater increase in the muscle enzymes CK and AST (p = 0.027 and = 0.001, respectively), indicated greater fatigue in non-fed rhinoceroses transported over a longer time. Further work to distinguish the effects of feeding and journey duration is required to better understand the role feeding may play in mitigating welfare challenges during rhinoceros translocation.

Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers.

Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear whether it confers worse survival in this setting. The prognostic value of KRAS mutations in both MSS and MSI CRC remains unclear. We examined the effect of BRAF and KRAS mutations on survival in stage II and III MSI colon cancer patients. BRAF exon 15 and KRAS exon 2-3 mutation status was assessed in 143 stage II (n = 85) and III (n = 58) MSI colon cancers by high resolution melting analysis and sequencing. The relation between mutation status and cancer-specific (CSS) and overall survival (OS) was analyzed using Kaplan-Meier and Cox regression analysis. BRAF V600E mutations were observed in 51% (n = 73) and KRAS mutations in 16% of cases (n = 23). Patients with double wild-type cancers (dWT; i.e., BRAF and KRAS wild-type) had a highly favourable survival with 5-year CSS of 93% (95% CI 84-100%), while patients with cancers harbouring mutations in either BRAF or KRAS, had 5-year CSS of 76% (95% CI 67-85%). In the subgroup of stage II patients with dWT cancers no cancer-specific deaths were observed. On multivariate analysis, mutation in either BRAF or KRAS vs. dWT remained significantly prognostic. Mutations in BRAF as well as KRAS should be analyzed when considering these genes as prognostic markers in MSI colon cancers.

Hereditary xanthinuria and urolithiasis in a domestic shorthair cat.

A 2-year-old domestic shorthair cat was presented with a history of hematuria, stranguria and intermittent urethral obstruction. Urine sediment showed hematuria, pyuria, and yellow-brown, amorphous and spherical crystals. Upon surgical correction of the obstructed urethra by perineal urethrostomy, many dark yellow to grey, irregular, gravel-like to millet grain-sized uroliths, consisting of 100% xanthine by crystallography were found. The urinary xanthine concentration was high. The cat subsequently developed bilateral nephroliths, recurrent urinary tract infection, and chronic kidney failure. Dietary management with a low-purine diet failed in part due to poor compliance, and the cat was euthanized at 6 years of age. Xanthinuria is rare inborn error of metabolism in cats and other species but should be considered as a differential diagnosis in cases of feline urolithiasis. No associated molecular genetic defect has been elucidated, and management of these cases is difficult. In the absence of calculi for analysis, measuring urinary xanthine concentration can help in diagnosing this metabolic defect.

Transient renal Fanconi syndrome in a Chihuahua exposed to Chinese chicken jerky treats.

Transient Fanconi syndrome without azotemia was diagnosed in a dog and was associated with ingestion of Chinese chicken jerky treats. Fanconi syndrome is a proximal renal tubular defect and a diagnosis was made based upon severe glucosuria with normoglycemia, and severe generalized aminoaciduria. The clinical signs of polyuria and polydipsia as well as the massive urinary metabolic abnormalities resolved after jerky treat withdrawal. While frequently seen in North America and Australia, this is the first report of jerky treat induced Fanconi syndrome in continental Europe. Clinicians should be aware of this potential intoxication and be vigilant for a history of jerky treat consumption in a dog with glucosuria.

Breed related odds ratio and anatomic distribution of canine mast cell tumours in Austria. Retrospective study of cases in the years 2000-2010.

OBJECTIVE: An increased risk of mast cell tumours (MCT) in certain breeds has been described repeatedly in the literature. The incidence of MCTs for registered breeds in Austria, an estimate of the risk by means of the odds ratios based on breed as well as the anatomic localisation of MCTs were examined. MATERIAL AND METHODS: In the first part of the study, the ranking of breeds in Austria based on 147,802 dogs with known breed (including mixed breed) was determined, based on those dogs included in the laboratory data base from 2000 to 2010. In the second part of the study, 476 dogs were identified with MCTs and analysed by age, sex, Patnaik grade of MCT and breed distribution. The odds ratios with confidence intervals were calculated for all breeds with skin tumours. RESULTS: The age distribution showed a peak in the age group from 6.1 to 8.0 years; 70% of MCTs were localised to the head and trunk. No significant difference was found based on gender. The evaluation of the odds ratios showed that only four of the 20 of the most popular in Austria breeds (Boxer, Bernese Mountain Dog, Golden Retriever, Spaniel) had an increased risk; on the other hand, some breeds which have not been previously identified in the literature were indicated to have a significantly increased risk for MCT (e.g., Dogo Argentino, Tibetan Spaniel, Pyrenean Mountain Dog, Beauceron, and Austrian Smooth-haired Hound). CONCLUSION AND CLINICAL RELEVANCE: Because disease risk may influence the popularity of some currently rare breeds, consultation with breeders and owners regarding the identification of the breeds newly identified in this study as an increased risk for development of mast cell tumours is indicated.

A retrospective study of 1,098 blood samples with anemia from adult cats: frequency, classification, and association with serum creatinine concentration.

BACKGROUND: Frequency and classification of anemia in terms of regeneration status and erythrocyte indices are not well described in cats. OBJECTIVE: To determine frequency and regenerative status of anemia in samples from adult cats, to assess the sensitivity and specificity of macrocytosis and hypochromasia for detecting regenerative anemia (RA), and to evaluate the association of anemia with increased serum creatinine concentration (SC). STUDY POPULATION: Laboratory records from 30,503 blood samples from cats (2003-2011). METHODS: Clinicopathologic data reviewed retrospectively. Anemia defined as hematocrit (Ht) ≤27%, red blood cell count (RBC) ≤5.5 × 10(6)/µL and hemoglobin (Hb) ≤9.0 g/dL. RA defined by manual absolute reticulocyte count >50 × 10(3)/µL. Macrocytosis was defined as mean corpuscular volume (MCV) >55 fL and hypochromasia as mean corpuscular hemoglobin concentration (MCHC) <31 g/dL. Cutoff for increased serum creatinine concentration was 1.6 mg/dL. RESULTS: Overall, 1,098 of 30,503 blood samples (3.6%) from cats fulfilled criteria for anemia, 633 of 1,098 (57.7%) classified as nonregenerative (NRA) and 465 of 1,098 (42.3%) as regenerative. RBC, Ht, and Hb were significantly lower in the RA compared to NRA group (P < .05). Sensitivity and specificity of the combined high MCV and low MCHC to detect samples with RA were 19.5 and 90.7%. SC was increased in 572 of the 1,098 anemic samples (52.1%) and in 11,121 of 29,405 of nonanemic samples (37.8%). CONCLUSIONS AND CLINICAL IMPORTANCE: Majority of anemic samples were classified as NRA. Anemia was more severe in cats with RA. Erythrocyte indices were not sensitive indicators of RA.

Diagnostic and predictive capability of routine laboratory tests for the diagnosis and staging of equine inflammatory disease.

BACKGROUND: A wide spectrum of laboratory tests is available to aid diagnosis and classification of equine inflammatory disease. OBJECTIVES: To compare diagnostic efficacy and combined predictive capability of the myeloperoxidase index (MPXI), and plasma fibrinogen, iron and serum amyloid A (SAA) concentrations for the diagnosis of inflammation. ANIMALS: Twenty-six hospitalized horses with systemic inflammation (SI), 114 with local inflammation (LI) and 61 healthy horses or those with noninflammatory disease (NI) were included. METHODS: A retrospective study was performed; clinicopathologic data from horses were compared between groups. Receiver-operator characteristic (ROC) curves were used to evaluate diagnostic efficacy; classification and regression tree analysis (CART) and logistic regression analysis were used to generate diagnostic algorithms. RESULTS: Horses with SI had significantly higher SAA than horses with LI (P = .007) and NI (P < .001) and lower iron concentrations than horses with LI (P < .001) and NI (P < .001). Fibrinogen concentration was higher in horses with inflammation than in those without inflammation (P = .002). There was no difference between the SI and LI groups. White blood cell count, neutrophil count and MPXI were similar between groups. SAA had the highest accuracy for diagnosing inflammation (area under ROC curve [AUC], 0.83 ± 0.06) and iron and SAA concentration had the highest accuracy for differentiating SI from LI (AUC, 0.80 ± 0.09 and 0.73 ± 0.10 respectively). Predictive modeling failed to generate useful algorithms and classification of cases was moderate. CONCLUSIONS AND CLINICAL IMPORTANCE: Very high SAA and low iron concentrations may reflect SI, but diagnostic guidelines based on quantitative results of inflammatory markers could not be formulated.

Increased cytotoxic capacity of tumor antigen specific human T cells after in vitro stimulation with IL21 producing dendritic cells.

Monocyte derived dendritic cells (moDC) electroporated with tumor associated antigen derived mRNA can elicit specific T cells against tumor cells in vivo. IL21 has been shown to enhance activation and cytotoxicity in CD8+ T cells. We therefore investigated in vitro effects on human CD8+ T-cells after stimulation with IL21 mRNA electroporated moDC. Codon modification of the IL21 gene significantly enhanced IL21 production upon electroporation of moDC. Tumor associated antigen specific CTL induction efficiency was significantly enhanced when codon modified IL21 mRNA was co-electroporated with tumor associated antigen mRNA. Tumor associated antigen specific T cells induced by codon modified IL21-DC demonstrated increased cytotoxic capacity and killing compared to control cultures. In conclusion, ectopic expression of codon modified IL21 by moDC enhances the priming efficiency of the DC as well as the cytotoxic potential of the induced CTL.

Effector memory T-cell frequencies in relation to tumour stage, location and HPV status in HNSCC patients.

BACKGROUND: The immune system plays an important role in tumour immune surveillance. Head and neck squamous cell carcinoma patients are often immune compromised. OBJECTIVE: To chart the baseline levels of T-cell subpopulation frequencies in patients with cancer prior to treatment. SUBJECTS AND METHODS: Blood samples of patients were taken at the time of diagnosis, analysed with flowcytometry and compared with blood samples of healthy donors. RESULTS: Compared to healthy donors, a significant shift from naive to effector memory T cells was observed. This effect was most prominent in stage II patients. A similar shift from naive to effector memory T cells was noted in patients with oropharynx or larynx squamous cell carcinomas. Furthermore, the percentage of effector memory and effector T cells was higher in the group of patients with human papillomavirus-positive oropharyngeal squamous cell carcinomas, compared with patients with human papillomavirus-negative tumours, suggestive of virus-induced T-cell activation. CONCLUSION: Here, we provide a simple and easily implementable tool to document T lymphocyte subsets in the peripheral blood of head and neck cancer patients, which might be useful for prognosis and/or therapy response prediction.

Preclinical safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7.

Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.

In-clinic laboratory diagnosis of canine babesiosis (Babesia canis canis) for veterinary practitioners in Central Europe.

OBJECTIVE: Haematological changes in dogs and climatic conditions favourable for the vector may assist in the quick in-house diagnosis of canine babesiosis. MATERIAL AND METHODS: Blood samples from 358 dogs suspected to have canine babesiosis were evaluated. The diagnosis was confirmed in 113 dogs by detection of Babesia canis by microscopic examination of a stained blood smear using the concentration line technique. RESULTS: Thrombocytopenia was present in all 113 dogs. Red blood cell count, packed cell volume and haemoglobin values were below the reference range in 62.8%, 61.1% and 46.0% of affected dogs, respectively. An increased reticulocyte count was apparent in five Babesia canis -positive dogs. Leukopenia, lymphopenia, neutropenia and monocytosis were present in 54.9%, 47.8%, 30.4% and 6.5% of the dogs, respectively. Evaluating haematological parameters by CART-analysis revealed a predictive model (accuracy= 93.5%) for canine babesiosis, when using the leucocyte, thrombocyte, and reticulocyte count. Climatic conditions present at the most probable time of Babesia canis- infection accounted for biseasonal occurrence. Changes of climatic factors during the year influence the vector activity and in conclusion should highlight babesiosis in the ranking of differentials for veterinarians. CONCLUSION: The results demonstrate that a tentative diagnosis of canine babesiosis can be made based on typical haematological changes. The results recorded match well with the seasonality of the tick vector and were confirmed here by the month of sample submission.

Absence of Granzyme B positive tumour-infiltrating lymphocytes in primary melanoma excisional biopsies is strongly associated with the presence of sentinel lymph node metastasis.

BACKGROUND: Sentinel Lymph Node (SLN) status is strongly related to clinical outcome in melanoma patients. In this study we investigated the possible association between the presence of activated and/or suppressive Tumour Infiltrating Lymphocytes (TILs) and SLN status in clinically stage I/II melanoma patients. METHODS: Diagnostic primary melanoma samples from 20 patients with a sentinel lymph node metastasis were compared to melanoma samples from 20 patients with a negative sentinel lymph node, who were matched for gender, age and Breslow thickness. Presence of activated Granzyme B positive (GrB+) TILs, presence of suppressive (FoxP3+) TILs and MHC class I antigen expression on tumour cells were analysed by immunohistochemistry. RESULTS: FoxP3 and MHC-I expression had no direct bearing on the presence of melanoma metastases in the SLN. Whereas the presence of activated GrB+ TILs in the primary melanoma had no predictive value for SLN status either, their absence was strongly associated with the presence of metastasis in the SLN (p=0.001). While both GrB+ and FoxP3+ TILs could be detected in SLN metastases, a majority did not display MHC-I expression. CONCLUSION: These data support a role for cytotoxic T cells in the prevention of early metastasis of melanoma to the draining lymph nodes.

Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation.

AIMS: Inhibition of apoptosis is important in the pathogenesis of lymphomas. c-FLIP, a regulator of caspase 8-mediated apoptosis, plays an important role in protecting normal B and T cells from apoptosis and possibly also in lymphomas. Because of contradictory reports about immunohistochemical detection of c-FLIP expression, the aim was to test the specificity of four antibodies in c-FLIP-transfected cells and subsequently to investigate expression of c-FLIP in different types of lymphoma. METHODS AND RESULTS: Two of four antibodies were specific. In primary lymphomas c-FLIP expression was restricted to Hodgkin's lymphomas (> 90%) and diffuse large B-cell lymphomas (44%). Burkitt lymphomas and indolent B-cell lymphomas were negative in all cases. No expression was detected in primary T-cell lymphomas, although expression was observed in one relapsed ALK+ anaplastic large cell lymphoma. Expression of c-FLIP was inversely correlated with caspase 8 activation. CONCLUSIONS: c-FLIP is important in escape of B cells from apoptosis during normal follicle centre cell reaction and may thus be an important early event in the development of B-cell-derived lymphomas. Moreover, non-specific staining of frequently used antibodies might explain discrepancies in different reports of c-FLIP expression.

Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells.

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

Differential effects of Notch ligands Delta-1 and Jagged-1 in human lymphoid differentiation.

Notch signaling is known to differentially affect the development of lymphoid B and T cell lineages, but it remains unclear whether such effects are specifically dependent on distinct Notch ligands. Using a cell coculture assay we observed that the Notch ligand Delta-1 completely inhibits the differentiation of human hematopoietic progenitors into the B cell lineage while promoting the emergence of cells with a phenotype of T cell/natural killer (NK) precursors. In contrast, Jagged-1 did not disturb either B or T cell/NK development. Furthermore, cells cultured in the presence of either Delta-1 or Jagged-1 can acquire a phenotype of NK cells, and Delta-1, but not Jagged-1, permits the emergence of a de novo cell population coexpressing CD4 and CD8. Our results thus indicate that distinct Notch ligands can mediate differential effects of Notch signaling and provide a useful system to further address cell-fate decision processes in lymphopoiesis.

Changing T cell specificity by retroviral T cell receptor display.

The diversity of the T cell receptor (TCR) repertoire is limited, because of the processes of positive and negative T cell selection. To obtain T cells with specificities beyond the immune system's capacity, we have developed a strategy for retroviral TCR display. In this approach, a library of T cell variants is generated in vitro and introduced into a TCR-negative murine T cell line by retroviral transfer. We document the value of TCR display by the creation of a library of an influenza A-specific TCR and the subsequent in vitro selection of TCRs that either recognize the parental influenza epitope or that have acquired a specificity for a different influenza A strain. The resulting in vitro selected TCRs induce efficient T cell activation after ligand recognition and are of equal or higher potency than the in vivo generated parent receptor. TCR display should prove a useful strategy for the generation of high-affinity tumor-specific TCRs for gene transfer purposes.

Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase.

Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8(+) T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.