Evaluation of leukocyte counts and neutrophil-to-lymphocyte ratio as predictors of local recurrence of feline injection site sarcoma after curative intent surgery.

Local recurrence (LR) is the major concern in the treatment of feline injection-site sarcoma (FISS). Pretreatment leukocyte counts and ratios have been reported as diagnostic and/or prognostic markers in human and canine oncology. The aim of this retrospective study was to explore the prognostic impact on LR and overall survival time (OST) of pretreatment neutrophil-to-lymphocyte ratio (NLR), white blood cell count (WBCC), neutrophil count (NC) and lymphocyte count (LC) in cats with surgically excised FISS. Eighty-two cats with histologically confirmed FISS at first presentation, without distant metastases, and with available pretreatment haematological analyses were retrospectively enrolled. The correlation of NLR, WBCC, NC, LC with tumour variables and patient variables was explored. NLR was correlated with tumour size (P = .004), histological pattern of tumour growth (P = .024) and histotype (P = .029), while WBCC and NC were associated with ulceration (P = .007, P = .011) and pattern of growth (P = .028, P = .004). No significant relationships emerged between LC and any of the considered variables. The impact of NLR, WBCC, NC, LC on LR and OST was then estimated in univariate and multivariate analysis. In univariate analysis, NLR, WBCC and NC were significant prognostic factors for both LR and OST. NLR, WBCC and NC remained prognostic in multivariate analysis for LR but not for OST. When NLR, WBCC and NC were jointly analysed, WBCC was the marker with the greater impact on LR. Preoperative NLR, WBCC and NC may aid in identifying cats at higher risk of LR.

Utility of flow cytometry in canine primary cutaneous and matched nodal mast cell tumor.

Mast cell tumors (MCT) are among the most frequent tumors in dogs, but studies regarding canine mast cell immunophenotype are lacking. The aim of this study was to assess the feasibility of flow cytometric analysis of MCTs, to describe canine MCTs immunophenotype(s), and to evaluate the ability of flow cytometry to detect mast cells in lymph node aspirates. Thirty-four primary canine MCTs and 12 draining lymph nodes were evaluated regarding the expression of CD117, IgE, CD11b, CD18, CD44, CD34, CD25 and CD45. Distinct populations attributable to mast cells and eosinophils were recognized based on light scatters and CD117 positivity. Common antigens (CD18, CD45, CD44) and CD117 were detected in all cases; positivity for IgE and CD11b was found in 28 (82%) and 23 (68%) cases respectively, while CD34 and CD25 were occasionally expressed. A single multicolor tube (IgE/CD117/CD11b/CD21/CD5) allowed the identification of mast cells in lymph nodes, showing a high correlation with cytology in quantifying mast cells infiltration. In conclusion, flow cytometric analysis can be applied to characterize canine MCTs and can be used to detect the presence of mast cells in lymph nodes. The immunophenotype abnormalities observed may be useful to confirm the neoplastic nature of such mast cells but the diagnostic usefulness of atypical antigen expression remains to be clarified.

Flow Cytometric Characterization of S-phase Fraction and Ploidy in Lymph Node Aspirates from Dogs with Lymphoma.

Canine lymphoma is a multifaceted disease encompassing numerous entities with different prognosis. Objective assessment of the proliferation rate is of importance from the pathological and clinical perspectives. Different methods have been described in the literature to assess proliferation rate, including evaluation of Ki67 expression in fresh lymph node (LN) aspirates measured by flow cytometry (FC). This test has a high accuracy in discriminating between low- and high-grade lymphomas, and provides prognostic information among high-grade B-cell lymphomas. DNA content analysis is less expensive and suitable for well-preserved samples. We describe DNA-content analysis using LN aspirates from 112 dogs with lymphoma. S-phase fraction (SPF) accurately discriminated between low- and high-grade lymphomas, with 3.15% being the best discriminating cut-off value. SPF values strongly correlated with Ki67 expression as assessed by FC. Survival analyses were restricted to 33 dogs with high-grade B-cell lymphoma receiving standardized multi-agent chemotherapy, but no significant result was obtained for SPF. We also describe a subset of aneuploid cases and their respective follow-up. We conclude that DNA content analysis may be combined with morphological examination of LN aspirates to improve the objectivity in lymphoma subtype classification in dogs. Further studies are needed to assess the possible prognostic role of SPF and ploidy status within specific lymphoma subtypes in dogs.

Severe Heinz body anemia and methemoglobinemia in a kitten with chronic diarrhea.

INTRODUCTION: A 2-month-old kitten was referred for depression and partial anorexia since 3 days and chronic diarrhea lasting for over 3 weeks. General physical examination showed pale and cyanotic mucous membranes. Blood sample was of brownish appearance. Venous blood gas analysis and complete blood count showed 16% methemoglobin level and severe regenerative anemia with Heinz bodies in about 40% of the erythrocytes, respectively. The kitten was transfused with fresh whole blood and treated with supportive care, antimicrobial and antioxidant agents. The kitten totally recovered. To the authors' knowledge, this represents the first case report of severe Heinz body hemolytic anemia and methemoglobinemia with concurrent chronic diarrhea in a young kitten. Diarrhea resolution coincided with Heinz bodies and methemoglobin disappearance. The possibility that diarrhea might have stimulated an inflammatory state causing release of oxygen radicals and prolonged erythrocytes oxidative damage has been discussed.

Altered Erythropoiesis in Mouse Models of Type 3 Hemochromatosis.

Type 3 haemochromatosis (HFE3) is a rare genetic iron overload disease which ultimately lead to compromised organs functioning. HFE3 is caused by mutations in transferrin receptor 2 (TFR2) gene that codes for two main isoforms (Tfr2α and Tfr2ß). Tfr2α is one of the hepatic regulators of iron inhibitor hepcidin. Tfr2ß is an intracellular isoform of the protein involved in the regulation of iron levels in reticuloendothelial cells. It has been recently demonstrated that Tfr2 is also involved in erythropoiesis. This study aims to further investigate Tfr2 erythropoietic role by evaluating the erythropoiesis of two Tfr2 murine models wherein either one or both of Tfr2 isoforms have been selectively silenced (Tfr2 KI and Tfr2 KO). The evaluations were performed in bone marrow and spleen, in 14 days' and 10 weeks' old mice, to assess erythropoiesis in young versus adult animals. The lack of Tfr2α leads to macrocytosis with low reticulocyte number and increased hemoglobin values, together with an anticipation of adult BM erythropoiesis and an increased splenic erythropoiesis. On the other hand, lack of Tfr2ß (Tfr2 KI mice) causes an increased and immature splenic erythropoiesis. Taken together, these data confirm the role of Tfr2α in modulation of erythropoiesis and of Tfr2ß in favoring iron availability for erythropoiesis.

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high-grade B-cell lymphoma.

Ki67 can discriminate between high- and low-grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67-positive cells), evaluated by flow cytometry, in 40 dogs with high-grade B-cell lymphoma, treated with a modified Wisconsin-Madison protocol (UW-25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1-40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high-grade B-cell lymphoma.

Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples.

Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses. Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order to assess the influence of breed, sex and age on the expression of CD4 in a larger case series. Based on univariate GLMs, none of these variables influenced CD4 RFI. Normalizing fluorescence data using lymphoid CD4 MFI as a reference would improve the comparison of results obtained by different laboratories, patients or times in diagnostic and research analyses of FI. Further studies are needed to confirm our results with different FC approaches.

Analysis of cerebrospinal fluid from cattle with central nervous system disorders after storage for 24 hours with autologous serum.

BACKGROUND: We compared the changes in cell morphology, total and differential cell counts between cerebrospinal fluid (CSF) samples analyzed within an hour of collection (fresh sample) and after the addition of autologous serum and storage for 24 h (stored sample) in 27 cattle with central nervous system disorders. RESULTS: There was a positive linear correlation between total and differential cell counts in the fresh and the stored samples. Cell morphology was preserved in all stored samples, except for increased vacuolization of mononuclear cells and cleaved nuclei of some small mononuclear cells. In the stored CSF samples, the total nucleated cell count and monocyte percentage were decreased (P = 0.01; P = 0.03), while the lymphocyte percentage was increased (P = 0.04). Mononuclear pleocytosis diagnosed in 20 fresh samples was cytologically confirmed in 12 of the 20 stored samples. In the remaining eight stored samples, the number of total nucleated cells was within the normal range. Neutrophilic pleocytosis was confirmed in all seven stored samples. The overall agreement rate between cytologic interpretation of the fresh and the stored CSF samples was 70 % (100 % for neutrophilic pleocytosis and 60 % for mononuclear pleocytosis). CONCLUSIONS: Adding 11 % of autologous serum to CSF samples might allow delayed analysis with a good agreement rate for CSF cytological interpretation. Caution is nonetheless warranted, as animal age, anamnesis, and neurological presentation need to be considered when interpreting stored CSF without pleocytosis.

Seizure disorders in 43 cattle.

BACKGROUND: Large animals have a relatively high seizure threshold, and in most cases seizures are acquired. No published case series have described this syndrome in cattle. OBJECTIVES: To describe clinical findings and outcomes in cattle referred to the Veterinary Teaching Hospital of the University of Turin (Italy) because of seizures. ANIMALS: Client-owned cattle with documented evidence of seizures. METHODS: Medical records of cattle with episodes of seizures reported between January 2002 and February 2014 were reviewed. Evidence of seizures was identified based on the evaluation of seizure episodes by the referring veterinarian or 1 of the authors. Animals were recruited if physical and neurologic examinations were performed and if diagnostic laboratory test results were available. RESULTS: Forty-three of 49 cases fulfilled the inclusion criteria. The mean age was 8 months. Thirty-one animals were male and 12 were female. Piedmontese breed accounted for 39/43 (91%) animals. Seizures were etiologically classified as reactive in 30 patients (70%) and secondary or structural in 13 (30%). Thirty-six animals survived, 2 died naturally, and 5 were euthanized for reasons of animal welfare. The definitive cause of reactive seizures was diagnosed as hypomagnesemia (n = 2), hypocalcemia (n = 12), and hypomagnesemia-hypocalcemia (n = 16). The cause of structural seizures was diagnosed as cerebrocortical necrosis (n = 8), inflammatory diseases (n = 4), and lead (Pb) intoxication (n = 1). CONCLUSION AND CLINICAL IMPORTANCE: The study results indicate that seizures largely are reported in beef cattle and that the cause can be identified and successfully treated in most cases.

Effects of competition on acute phase proteins and lymphocyte subpopulations - oxidative stress markers in eventing horses.

The aim of the study was to evaluate markers of the acute phase response (APR) in eventing horses by measuring acute phase proteins (APP) (haptoglobin, Hp, and serum amyloid A, SAA), lysozyme, protein adducts such as pentosidine-like adducts (PENT), malondialdehyde adducts (MDA), hydroxynonenal adducts (HNE) and total advanced glycation/glycoxidation end products (AGEs), complete blood count and lymphocyte subpopulations (CD4+, CD8+ and CD21+) both at rest and at the end of an eventing competition. Blood samples were collected from eight Warmblood horses (medium age 10 ± 3) during an official national 2-day event competition at rest (R) and 10 min after the arrival of the cross-country test on the second day. Exercise caused a significant increase in red blood cell number, haemoglobin, packed cell volume, neutrophils, white blood cell and lymphocyte number; however, these values remained within the normal range. The CD4+ and CD8+ cells significantly increased, whereas the CD21+ lymphocytes decreased; a significant increase in serum SAA, lysozyme and protein carbonyl derivates was also observed. Two-day event causes significant changes in APR markers such as lysozyme, protein carbonyl derivates (HNE, AGEs, PENT) and lymphocyte subpopulations. The data support the hypothesis that 2-day event may alter significantly APR markers. Limitations of the study were the relatively small sample size and sampling time conditioned by the official regulations of the event. Therefore, further studies are needed to investigate the time required for recovery to basal values in order to define the possible effects on the immune function of the athlete horse.

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma.

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non-Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B- and T-immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut-off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.

Acute undifferentiated leukaemia in a dog.

BACKGROUND: Acute undifferentiated leukaemia (AUL) is considered a separate entity in the context of acute leukaemias. AUL is extremely rare in both humans and dogs, has a rapid clinical course and does not respond to treatment. It is characterised by the presence of blast cells within the bone marrow and/or peripheral blood at levels ≥ 20% and even up to 100% of all nucleated cells. Blast cells are unable to be differentiated on morphological, cytochemical and phenotypic criteria into myeloid or lymphoid lineages because of their immaturity and/or atypia. CASE REPORT: An 8-year-old German Shepherd dog was referred for depression, asthenia, mild anaemia, thrombocytopenia and marked leucocytosis. Abdominal ultrasound showed hepatomegaly, splenomegaly, bilateral nephromegaly and enlargement of mesenteric lymph nodes. Echocardiography revealed biventricular hypertrophy with abnormal tissue density of the myocardium. Blood and bone marrow smears were composed of 95% unclassifiable and/or atypical blast cells and signs of dysplasia of the erythroid and thrombocytic/megakaryocytic lineages were present. Blast cells were negative for all cytochemical stains used and flow cytometry of peripheral blood revealed 85% of total leucocytes consisting of small-to-medium-sized cells, negative for all lymphoid and myeloid markers except CD45 and CD34. After necropsy, cytology and histology revealed that blast cells had diffusely infiltrated all tissues examined. Both erythroid and megakaryocytic extramedullary haemopoiesis was also detected in the spleen, lymph nodes and liver. All immunohistochemical stains used were negative. CONCLUSION: On the basis of all the results, a diagnosis of acute leukaemia involving a very primitive haematopoietic precursor was made.

Urinary tract infections in sows in Italy: accuracy of urinalysis and urine culture against histological findings.

In order to investigate the prevalence of urinary tract infections (UTI) in sow, lower urinary tract (LUT), kidney and urine samples were collected at slaughterhouse from 65 multiparous culled sows. Histopathology was performed on urethra, urinary bladder and -kidney sections. Urine collected by cystocentesis was analysed for physical and biochemical parameters, in addition to microscopic examination of the sediment and quantitative culture ( > 10(5) CFU/ml urine). The diagnostic accuracy of urinalysis and urine culture was calculated for the parameters that correlated with histological diagnosis: bilateral chronic lesions were found in 54 per cent of kidney samples and diffuse/multifocal lymphoplasmacytic infiltration of the submucosa in 53 per cent of the bladder and 68 per cent of the urethra samples. In 49 per cent of cases, the co-occurrence of bladder and urethra lesions was statistically significant (P < 0.009). Turbid urine (80 per cent sensitivity, 50 per cent specificity), > 5 white blood cells per high-power field (34 per cent sensitivity, 90 per cent specificity), intracellular or free bacteria (43 per cent sensitivity, 90 per cent specificity), and urine culture (49 per cent sensitivity, 97 per cent specificity) correlated with a finding of histopathological changes in the bladder. UTI appears to be common in culled sows in northern Italy. Compared with histopathology, urinalysis and urine culture showed low sensitivity but high specificity in detecting UTI.

Sepsis and bacterial suppurative meningitis-meningoencephalitis in critically ill neonatal Piedmontese calves: clinical approach and laboratory findings.

Sepsis (S) and bacterial suppurative meningitis-meningoencephalitis (M-ME) are common causes of death in bovine neonates. The aim of this prospective study was to evaluate the prevalence of S and M-ME in critically ill neonatal Piedmontese calves. Critically ill animals up to 15 days old referred by practitioners were registered according to their status and subsequently assigned to clinical standardized score. Calves with a clinical score > = 5 were further assessed under a clinical and clinical-pathological protocol to strengthen the suspicion of S and M-ME. Critically ill neonatal calves sent for necropsy were included in the study as well. Fifty-nine calves were investigated, 26 of which referred alive and 33 dead. Ten out of the 26 clinically evaluated calves were classified as suspicious of S on the basis of the clinical and clinical-pathological protocols. S was confirmed by positive bacteriologic culture in 7 cases and in 3 cases on the basis of necroptic lesions. Concomitant suppurative M-ME suspected in 6 of these 10 calves was subsequently confirmed by CSF analysis or histological findings. Of the 33 calves examined only post-mortem, 20 showed pathognomonic findings of S and 14 signs of M-ME. The prevalence of S and M-ME was 46 and 36 %, respectively. Clinical signs of S were confirmed to be vague and overlapping with other diseases. The developed protocol was highly accurate in predicting S in these neonatal calves.

Canine leishmaniosis: in vitro efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum.

Despite the availability of different therapeutic options, canine visceral leishmaniosis (CVL) remains a challenging disease to treat. Recently miltefosine has been registered for use in dogs, and different studies have demonstrated its leishmanicidal effect. Moreover, it has been suggested that fluoroquinolones, compared to standard chemotherapeutic agents, could be an effective and pragmatic alternative to treat CVL. The efficacy of miltefosine and marbofloxacin alone or in combination with allopurinol against clinical strains of Leishmania infantum was assessed in vitro by incubating increasing concentrations of the drugs with a standard parasite inoculum. Miltefosine was significantly more efficacious than marbofloxacin (P < 0.05) against the two strains of L. infantum either alone or in combination with allopurinol. Both drugs were significantly (P < 0.05) more efficacious when associated with allopurinol than alone.

Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy. OBJECTIVES: The aim of the study was to determine if the immunophenotype of neoplastic cells predicts survival in canine CLL. DESIGN: Retrospective study. ANIMALS: Forty-three dogs with CLL. PROCEDURES: Records of dogs with a final diagnosis of CLL were reviewed. For each included dog, a CBC, blood smear for microscopic reevaluation, and immunophenotyping data had to be available. Data on signalment, history, clinical findings, therapy, follow-up, as well as date and cause of death were retrieved. RESULTS: Seventeen dogs had B-CLL (CD21+), 19 had T-CLL (CD3+ CD8+), and 7 had atypical CLL (3 CD3- CD8+, 2 CD3+ CD4- CD8-, 1 CD3+ CD4+ CD8+, and 1 CD3+ CD21+). Among the variables considered, only immunophenotype was associated with survival. Dogs with T-CLL had approximately 3-fold and 19-fold higher probability of surviving than dogs with B-CLL and atypical CLL, respectively. Old dogs with B-CLL survived significantly longer than did young dogs, and anemic dogs with T-CLL survived a significantly shorter time than dogs without anemia. CONCLUSIONS: Although preliminary, results suggested that immunophenotype is useful to predict survival in dogs with CLL. Young age and anemia are associated with shorter survival in dogs with B-CLL and T-CLL, respectively.

Analysis of cerebrospinal fluid from 20 calves after storage for 24 hours.

Samples of CSF collected from 20 normal healthy calves were analysed either immediately or after having been stored for 24 hours at 4 degrees C in the presence of 11 per cent autologous serum. There were no significant differences between the total and differential cells counts of the fresh and stored samples, but there was a positive linear correlation between them. There were some morphological changes to the nuclei of the mononuclear cells in the stored samples.

Effects of anabolic and therapeutic doses of dexamethasone on thymus morphology and apoptosis in veal calves.

Three groups of 10 veal calves were treated, respectively, with 5 mg of dexamethasone-21-isonicotinate administered intramuscularly on days 0 and 7 (group A); 0.4 mg/day of dexamethasone-21-phosphate administered orally for 20 days (group B); or left untreated as controls (group C). Two animals from each group were slaughtered on day 3, 7, 14, 32 and 52. The size and weight of the thymus decreased progressively in both treated groups until day 32. On day 14, in comparison with the controls, there was a mean reduction of 76 per cent in the thymus weight of group A and 35 per cent in group B. On day 32, the reductions were 13 per cent in group A and 50 per cent in group B, but the thymus weight of both groups had recovered completely by day 52. Dexamethasone-induced changes in thymus weight associated with lymphoid depletion and fat replacement, and there were clear correlations between these changes and apoptosis of thymocytes.

A case of disseminated mycosis in a German shepherd dog due to Penicillium purpurogenum.

The genus Penicillium is among the most common contaminant fungi in the environment. Around 15 species are known to cause opportunistic human mycoses, in immunocompromised patients. Until now, Penicillium purpurogenum has been involved in only three human cases of pulmonary diseases but no infections in animals have been reported. Most disseminated mycoses in dogs are caused by members of the genus Aspergillus, with the predisposing factors in these cases being difficult to define. The case reported here involved a 4-year-old female German shepherd dog (GSD) with forelimb instability and back pain. Clinical examination showed hyperthermia, generalized lymphadenomegaly and kyphosis. Radiological findings of the spine revealed areas of discospondilitis involving thoracic and lumbar vertebrae. Microscopic observations of fine needle aspiration biopsies (FNAB) of lymph-nodes showed regular, septate, branching fungal hyphae. Itraconazole therapy was started but the subject died six days later. Disseminated necrotic areas were detected in enlarged lymph-nodes, liver and spleen. Vertebral granulomas within lytic areas in T10-T11 and L2-L3, were observed. Cultures inoculated with samples obtained from lymph-node FNAB and bioptic material from necropsied organs revealed the presence of pure cultures of Penicillium, subsequently identified as P. purpurogenum. Apart from female GSD's suspected predisposition to disseminated mycoses described in literature, no other predisposing factors were ascertained in this case.

Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes.

Triphenyltin acetate (TPTA), a triorganotin compound used in agriculture as a biocide, is immunotoxic in vivo and in vitro. The present study was undertaken to ascertain whether apoptosis might play a role in the TPTA toxicity in vitro. Mouse thymocyte primary cultures were exposed to 0, 4 and 8 micromol/L TPTA; methyl prednisolone (1 micromol/L) was used as a positive control. Cell aliquots were harvested after 0, 1, 2, 4, and 8 h and the presence of early or late apoptotic phenomena was checked by (a) morphological investigations; (b) spectrophotometric quantification of fragmented DNA and agarose gel electrophoresis; (c) cell flow cytofluorometry, using an annexin V-FITC kit; and (d) detection of in situ apoptosis by a colorimetric detection kit (Titer-Tacs). TPTA cytotoxicity was also evaluated using the trypan blue dye exclusion test. Morphological investigation indicated apoptosis and/or necrosis. After 8 h of incubation, cells exposed to 4 micromol/L TPTA showed an increase in DNA fragmentation (on electrophoresis), which was confirmed by spectrophotometry (p < 0.05). Flow cytofluorometry pointed out an early (p < 0.05) increase of annexin V-positive (apoptotic) cells in TPTA-exposed flasks, whereas at least partly contradictory, results were obtained with the Titer-Tacs kit. Overall, these results provide evidence that TPTA, at low concentrations (4 micromol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 micromol/L) are likely to undergo necrosis rather than apoptosis.