Development of Immune Response to Experimental Bovine Trichophyton vervucosum Infection

Abstract— Cell mediated and humoral immune responses to experimental Trichophyton verrucosum infection were assessed by sequential cutaneous biopsies, antibody assessments and microscopic monitoring of fungal presence. Histopathologic examination showed the accrual of lymphocytes and other inflammatory cells in the dermis of infected sites. Immunoperoxidase staining of frozen sections with monoclonal antibody preparations revealed an influx of macrophages, BoCD4+ and B0CD8+ lymphocytes and γδ T cells from the 5th day to the 33rd day of infection. A moderate influx of B cells was observed. Protein G-colloidal gold staining revealed the presence of immunoglobulins in the dermis and superficial epidermal layers. Trichophyton specific serum antibodies appeared between days 33 and 55. Microscopic assessment of infected tissues revealed an increase in T, verrucosum elements (mycelium and ectothrix spores) from days 19 to 55. Fungal elements in infected areas did not decrease until after both humoral and cell mediated elements of the immune response were established. These responses imply a combination of cell mediated and humoral events were associated with T. verrucosum immunity and clearance in the calf. Résumé— Le réponse immunitaire humorale et cellulaire a une infection expérimentale àT. verrucosum a été appréciée par des biospsies cutanées successives, des dosages d'anticorps et la recherche microscopique de champignons. L'examen histopathologique a montré un afflux de lymphocytes et d'autres cellules inflammatoires dane le derme des sites infectés. Les marquages en immunopéroxydase par un anticorps monoclonal de coupes congelées a montré un influx de macrophages, lymphocytes BoCD4+ et BoCD8+ et des cellules T γδ, du 5e au 33e jour de l'infection. Un marquage par une protéine G—or colloidal a révélé d'immunogolglobulines dans le derme et les couches supéerficielles de l'épiderme. Les anticorps spécifiques de Trichophyton sont apparus entre 33 et 55 jours. L'examen microscopique des tissus infectés a révélé une augmentation du nombre d'éléments de T. verrucosum (mycelium et spores ectothrix) du 19e au 55e jours. Les éléments fongiques dans les zones infectées n'ont pas diminué avant que les réponses humorales et cellulaires ne solent établies. Ces résponses impliquent qu'une coopération des réponses humorales et cellulaires étaient associées dans l'immunité et la défense contre T. verrucosum. Zusammenfassung— Die zellvermittelten und humoralen Immunantworten auf die experimentelle Infektion mit T. verrucosum wurden durch eine Serie von Hautbiopsien, Antikörperuntersuchungen und mikroskopischer Untersuchung auf das Vorhandensein von Pilzen ausgewertet. Die histopathologische Untersuchung zeigte eine Ansammlung von Lymphozyten und anderen Entzündungszellen in der Dermis der infizierten Stellen. Die Immunperoxidasefärbung der Gefrierschnitte mit monoklonalen Antikörperzubereitungen zeigte einen Influx von Makrophagen, BoCd4-und BoCD8-Lymphozyten und gamma-delta-T-Zellen vom 5. bis zum 33. Tag der Infektion. Es wurde auch ein mäßiger Influx von B-Zellen beobachtet. Die Protein-G-kolloidale Goldfärbung zeigte die Anwesenheit von Immunglobulinen in der Dermis und den oberflächlichen epidermalen Schichten. Trichophyton-spezifische Serumantikörper traten zwischen Tag 33 und 55 auf. Die mikroskopische Untersuchung infizierter Gewebe zeigte eine Zunahme von T. verrucosum-Bestandteilen (Myzel und exktothrixe Sporen) vom Tag 19 bis 55. Pilzteile in infizierten Bereichen verminderten sich weder, nachdem humorale, noch nachdem zellvermittelte Elemente der Immunantwort auftraten. Diese Reaktionen deuten an, daß eine Kombination von zellvermittelten und humoralen Vorgängen im Zusammenhang mit T. verrucosum-Immumtät und Abheilung beim Kalb vorliegt. Resumen Por medio de biposias cutáneas secuenciales, medida de anticuerpos y exámen microscópico de presencia de hongos, se estudió la respuesta inmunitaria de tipo humoral y celular producida por la infección experimental con T. verrucosum. El exámen histopatológico reveló la presencia de agregación de linfocitos y otras células inflamatorias procedentes de la dermis de los puntos infectados. La tintura por medio de inmunoperoxidasa de las secciones congeladas, con preparaciones monoclonales de anticuerpos, demostró un aflujo de macrófagos BoCD4 + y BoCD8 + linfocitos y linfocitos, Tαδ, desde el quinto hasta el día 33 la infección. También se observó un aflugo moderado de linfocitos B. La tintura aúrica de proteina coloidal G reveló la presencia de inmunoglobulinas en al dermis y capas superficiales de la epidermis. Los anticuerpos específicos para la especie Trichophyton aparecieron entre los días 33 y 55. El exámen microscópico de los tejidos afectados demostró un incremento, de los elementos füngicos T. verrucosum (micelio y esporas exótricas) desde los días 19 al 55. Los elementos fúngicos en áreas infectadas no disminuyeron hasta después del establecimiento de ambos tipos de respuesta inmunitaria, humoral y celular. Estas respuestas implican que la combinación de ciertos fenómenos de inmunidad celular y humoral, están relacionados con la desaparición y la inmunidad de la infección producia por T. verrucosum en el ternero.     

Identification of feline blood B and T lymphocytes by cell electrophoresis.

The electrokinetic properties of feline blood lymphocytes isolated by centrifugation over Ficoll-Isopaque gradient were investigated. A biphasic electrophoretic mobility (EPM) distribution was regularly observed with a low-mobility (LM) population (mean EPM: 0.82) accounting for 32% of blood lymphocytes and a high-mobility (HM) population (mean EPM: 1.09) representing 68% of blood lymphocytes. Following fractionation on nylon-wool columns, lymphocytes with B-cell properties (64% sIg⁺; 9% guinea pig erythrocytes (GPE)-rosette⁺, PHA and Con A unresponsive) were enriched in the adherent fraction and belonged mainly (78%) to the LM population. In contrast, lymphocytes with T properties (5% sIg⁺, 42% GPE-rosette⁺, PHA and Con A responsive) were recovered in the effluent fraction and comprised 84% of HM elements.Thus, in cat blood, LM lymphocytes are likely to represent in majority B cells and HM lymphocytes T cells. This indicates that cell electrophoresis provides an interesting mean for differentiating B and T cells in the cat.     

雏鸡感染传染性法氏囊病毒后血液的T细胞变化

本项研究对雏鸡感染传染性法氏囊病毒(IBDV)后,1～8周外周血液的 ANAE~+淋巴细胞(T 细胞)、淋巴细胞和白细胞数量的动态变化进行了检测。结果表明,1日龄雏鸡感染 IBDV 后,其血液中 ANAE~+,淋巴细胞数量明显低于对照雏鸡,表明其细胞免疫机能降低;4周龄雏鸡感染 IBDV 后,其 ANAE~+淋巴细胞数量呈一过性降低,变化不如1日龄感染雏鸡明显,说明 IBDV 对雏鸡存在感染年龄差异;对 ANAE~+淋巴细胞绝对数的检测较百分比能更真实地反映 IBD 鸡的细胞免疫功能变化的实质。     

Disseminated lymphoma with large granular lymphocyte morphology diagnosed in a horse via abdominal fluid and transtracheal wash cytology

A 22‐year‐old Tennessee Walking Horse mare was presented to the Auburn University Large Animal Teaching Hospital with a 3‐day history of lethargy, anorexia, and mild signs of colic. The mare had a several‐month history of weight loss and refractory cough. Physical examination revealed an increased respiratory rate, and crackles and wheezes were heard on thoracic auscultation. Thoracic ultrasonographic examination showed disseminated, minor, bilateral comet tail‐like lesions on the parietal pleural surfaces. Abdominal ultrasonographic examination was unremarkable. Trans‐rectal palpation revealed a firm small colon impaction with concomitant diarrhea. Laboratory data were characterized by a very pronounced acute inflammatory leukogram with severe neutropenia and significant left shift, evidence of hepatocellular damage/necrosis, cholestasis, and possibly mixed metabolic alkalosis and acidosis. On cytologic evaluation of a peritoneal fluid sample, there were many large granular lymphocytes (LGL). Large numbers of LGL were also observed on cytologic examination of a subsequent transtracheal wash. The final cytologic interpretation was disseminated lymphoma with LGL morphology. Due to worsening of the clinical signs and poor prognosis, the mare was euthanized. On necropsy and in histopathologic examination, disseminated lymphoma with LGL morphology was noted in a mesenteric lymph node, lungs, liver, spleen, kidneys, and right dorsal colon. Lymphoma with LGL morphology is rarely diagnosed in the horse. This report provides unique cytologic findings of a case of disseminated lymphoma with LGL morphology in a horse, confirmed with histopathologic evaluation.     

Effects on adhesion molecule expression and lymphocytes in the bovine mammary gland following intra-mammary immunisation

Changes to adhesion molecule expression and lymphocyte populations were evaluated in alveolar mammary tissue collected from cows following an immunisation protocol that involved intra-mammary inoculation to induce an IgA response in mammary secretions. The right quarters of the udder were immunised; the left side acted as a control. Antibody titres in secretions showed that at least two animals responded with antigen-specific IgA. Numbers of T-lymphocytes were 4-fold higher in immunised glands compared with controls (P < 0.05). IgA-, IgM- and IgG-positive cell numbers were significantly higher (P < 0.01) in immunised glands compared with controls in three of the four cows. No mucosal addressin molecule-1 (MAdCAM-1), vascular cell-adhesion molecule-1 (VCAM-1) or peripheral node addressin (PNAd) protein expression was detected on smaller venules that stained positively for von Willebrand factor in alveolar mammary tissues, from either immunised or control glands. Both VCAM-1 and PNAd were detected on smaller venules in supramammary lymph nodes, however, there was no significant difference between immunised and control glands. Quantification of MAdCAM-1 mRNA showed very low expression in both immunised and control alveolar tissue compared with Peyer's patch positive-control tissue. These findings suggest that the bovine mammary gland is capable of a mucosal antibody response; however, MAdCAM-1 is not involved with lymphocyte homing to the mammary gland in this species.     

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.     

Clinical and pharmacokinetic characteristics of intracavitary administration of pegylated liposomal encapsulated doxorubicin in dogs with splenic hemangiosarcoma

BACKGROUND: Canine splenic hemangiosarcoma (HSA) is a fatal malignancy, and most affected dogs die within a few months of diagnosis. Most dogs present with signs from tumor rupture, resulting in hemoabdomen and intra-abdominal dissemination. The abdomen is also the main site of disease recurrence. HYPOTHESIS: Intraperitoneal (IP) administration of doxorubicin will delay or prevent intra-abdominal tumor recurrence and prolong survival in dogs with HSA. ANIMALS: Fourteen dogs with splenic HSA. METHODS: A prospective, unmasked, uncontrolled clinical trial. After staging of disease status and splenectomy, pegylated liposomal encapsulated doxorubicin was administered intraperitoneally (1 mg/kg body weight) every 3 weeks for 4 cycles. All dogs were monitored for recurrence of HSA. Samples of plasma and abdominal fluid were collected for measurement of doxorubicin concentration and pharmacokinetic analysis. Nonlinear mixed-effect modeling was used to describe the pharmacokinetics of liposomal doxorubicin administered IP. RESULTS: All 14 dogs died, 12 because of HSA and 2 from other causes. Postmortem examination was performed on 12 dogs. All 12 dogs died because of HSA-related causes and had hepatic metastases and hemoabdomen. The IP-treated dogs had fewer serosal, mesenteric, and omental metastases than historical controls treated with systemic doxorubicin. Results of the postmortem examination and pharmacokinetic analysis confirmed that IP delivery of doxorubicin resulted in an effective drug concentration with a clearance comparable with that after i.v. delivery. CONCLUSIONS AND CLINICAL IMPORTANCE: IP pegylated liposomal encapsulated doxorubicin administration did not prevent intraabdominal recurrence of HSA in dogs.     

The Effects of Salivary Gland Extracts from <Emphasis Type="Italic">Boophilus microplus</Emphasis> Ticks on Mitogen-stimulated Bovine Lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.     

Association of tumor budding with tumor-infiltrating lymphocytes and prognosis in breast cancer patients

AIM: To investigate the relationship of tumor budding with clinicopathologic parameters, tumor-infiltrating lymphocytes (TILs) of tumor microenvironment and the prognosis in breast cancer patients.METHODS: A total of 178 HE section samples were collected from the breast cancer patients treated with surgery in the First Affilated Hospital of Jinan University during Jan. 2012 to Dec. 2016. The tumor budding and stromal tumor-infiltrating lymphocytes were observed under light microscope. The correlation of tumor budding with the clinicopathologic status and TILs were analyzed by χ² test. Kaplan-Meier survival analysis and Log-rank test were used to estimate the disease-free survival (DFS) and overall survival (OS).RESULTS: High tumor budding level was associated with more positive lymph nodes, higher grade, and more lymphovascular invasion. In addition, the patients with higher tumor budding level showed fewer TILs, while the patients with lower tumor budding level had more TILs. Furthermore, the patients with higher tumor budding level had a worse disease-free survival and overall survival than those with lower tumor budding level.CONCLUSION: Tumor budding is significantly associated with adverse clinicopathological characteristics of breast cancer and negatively correlated with TILs. Therefore, tumor budding may serve as a potential biomarker to predict the prognosis of breast cancer.     

天门冬多糖对猪脾淋巴细胞体外增殖的影响

试验旨在研究不同浓度的天门冬多糖(ASP),在ConA或LPS的协同刺激下对猪脾淋巴细胞体外增殖的影响。猪脾淋巴细胞体外培养体系中加入不同浓度的天门冬多糖使其终浓度为400、200、100、50、25、12.5μg/ml,在ConA(5μg/ml)或者LPS(10μg/ml)的协同刺激作用下,细胞培养24、48、72 h时,观察猪脾淋巴细胞增殖情况。结果表明,天门冬多糖及其协同ConA或LPS能显著或极显著的促进猪脾淋巴细胞体外增殖(P<0.05或P<0.01)。