The immune reactivity of the regional and distant lymph nodes to autochthonous ovine squamous cell carcinomata

Anti-tumor immune reactivity of lymphocytes derived from lymph nodes regional to and distant from tumor growth, as well as that of peripheral blood leucocytes, against autochthonous tumor cells, was investigated. Experiments were carried out in vitro using a 51Cr cytotoxic assay and in vivo by cannulating the afferent and efferent lymphatics of regional and distant lymph nodes and challenging via the afferent lymphatics with 10(7) cultivated autochthonous tumor cells. No anti-tumor cytotoxic reactivity was detected in vitro using lymphocytes derived from any of the sources studied. In vivo, while challenge with autochthonous tumor cells produced no response in the regional lymph node, significant blast cell response was obtained in the distant node. The response at the distant node was associated with the production of antibodies that could bind to tumor cells without causing their demise. The anergy observed at the regional lymph node, and the possibility of a relation between the events occurring at that node and those observed at the distant node, are discussed.     

Successful isolation,cultivation and partial characterization of naturally occurring ovine squamous cell carcinomata

Seventeen epithelial cell lines have been successfully established from naturally occurring ovine squamous cell carcinomata. Culture establishment was most successful when tumor tissue was directly explanted rather than treated enzymatically. Success in establishing cultures also appeared to be related to the site on the body from which the tumor biopsy was taken, with tumors derived from the nose being most readily cultured. Several of the cell lines were successfully transplanted to nude mice where the growth patterns observed in the original host, i.e. expansive or invasive, were maintained. All cell lines assumed one of two distinct morphological types; however, no association could be seen between morphology and pattern of in vivo growth.     

In vitro response of lymphocytes of normal and ovine squamous cell carcinoma-bearing sheep to phytomitogens and tumour extracts

A comparison was made of the blastogenic responses of peripheral lymphocytes (BRPL) of normal sheep with those of sheep bearing ovine squamous cell carcinoma (OSCC). In normal tumour-free sheep, BRPL to phytomitogens were found to vary in different age groups, and BRPL to OSCC extracts were found to become significantly elevated in sheep over one year old. In tumour-bearing mature sheep, BRPL to phytomitogens and to OSCC extracts decreased significantly with increasing maturity of tumours. The results are interpreted to show that increasing tumour volume, due to natural growth or enhancement, associates with increasing suppression of cell-mediated responses, and that such suppression may be a cause and a result of increased tumour volume.     

Effect of cyclophosphamide on tumour growth and cell-mediated immunity in sheep with ovine squamous cell carcinoma

Cyclophosphamide (CY) is shown to associate with partial or complete regression of ovine squamous cell carcinoma (OSCC), the rate of regression varying directly with CY dosage and inversely with maturity of tumour at the start of therapy. CY treatment given coincidental with injection of tumour extracts (TA) completely neutralised the tumour enhancement effects regularly associated with TA injections, and caused tumour regression comparable to that seen in sheep treated with CY alone. In discussion of the results, which incorporate parallel studies on cell-mediated responses, it is suggested that immunosuppression during tumour growth is due to formation of B suppressor lymphocytes and that CY treatment of OSCC may exert beneficial effects through its immunosuppressive action on such lymphocytes as much as its direct cytotoxic action on tumour cells.     

Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue γδ TCR⁺ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R⁺) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by γδ T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.     

Effect of removal of ovine squamous cell carcinoma on peripheral lymphocyte stimulation by tumour extracts and phytohaemagglutinin

Mock-operation of normal tumour-free sheep was not associated with significant changes in blastogenic responses of peripheral lymphocytes (BRPL) to phytohaemagglutinin (PHA-P) or tumour extracts (TA). In age-matched tumour-bearing sheep, tumour removal was associated with a significant prolonged increase in BRPL to PHA-P and TA about three weeks after operation. This increase was significantly greater in sheep with mature than with early tumours. No increase in BRPL occurred in sheep with secondary lesions. Challenge of tumour-removed sheep with TA 15 weeks after operation was associated with a significant anamnestic-type short-term surge in BRPL to PHA-P and TA, which was significantly greater in sheep which had previously borne advanced tumours. Mock-operated normal mature sheep challenged with TA also showed a lowgrade anamnestic-type response of BRPL to PHA-P and TA. The results are discussed in terms of the mechanisms of BRPL suppression, and of specific reactions in normal sheep.     

Host response to bovine ocular squamous cell carcinoma

Specific humoral and cellular immunologic responses to autologous and heterologous tumors were evaluated in 35 Hereford cows with ocular squamous cell tumors and in 6 healthy cows. Sera from 5 healthy cows and 23 ocular tumor-bearing cows were evaluated for antibody to tumor, using radioimmunoassay, passive hemagglutination, agglutination, and microagglutination assays with various soluble tumor antigen and whole tumor cell preparations. Antibody to tumor was detected in only 2 cows. Using the microagglutination assay, antibody to autologous and heterologous tumor cells was found in the sera of 2 cows inoculated intraocularly with purified, viable autologous tumor cells. Twenty-eight tumor-bearing cows and 3 healthy cows were evaluated for delayed-type hypersensitivity responses, using 5 tumor preparations; positive skin test responses were not observed.     

Ki67 reactivity in nasal and periocular squamous cell carcinomas in cats treated with electron beam radiation therapy

Squamous cell carcinomas of sparsely haired skin are relatively common tumors in cats, and these tumors likely exhibit a rapid growth rate. Thus, we evaluated response and duration of response in relation to the Ki67 proliferative reactivity in such tumors. Seventeen cats with confirmed squamous cell carcinomas and treated with an accelerated, hypofractionated electron beam radiation protocol were included in the study. For all of them histologic grading, Ki67 reactivity, response, and disease-free interval (DFI) were evaluated. Response to therapy was excellent (94% complete response rate) with a median DFI of 414 days. Only moderate acute and few long-term adverse effects were seen. Cats with tumors with a low Ki67 reactivity had markedly shorter DFIs than cats with tumors with high Ki67 reactivity. We concluded that an accelerated, hypofractionated electron beam radiation therapy protocol is well suited for feline squamous cell carcinomas. The protocol appears especially efficacious in tumors with a high Ki67 reactivity.     

Immune exclusion, a mechanism of protection against the ovine nematode Haemonchus contortus

The fate of exsheathed Haemonchus contortus larvae injected directly into the abomasal lumen was studied in groups of naive and hyperimmune sheep. Within 48 hours of challenge, more than 90 per cent of the larvae were lost from hyperimmune sheep. The larvae had a propensity to associate with the superficial mucus in both groups and, in naive sheep, approximately 50 per cent of the larvae entered glandular tissue within four hours of challenge. By contrast, although initially associated with the mucosal surface, larvae in immune sheep were excluded from the glandular tissues. It is postulated that the rapid loss of worms from the hyperimmune abomasum is a consequence of 'immune exclusion' and the possible mechanisms underlying this process are discussed.     

The phagocytic cell response of the ovine lung to Pasteurella haemolytica

Conventionally-reared sheep were inoculated with (3.0 ± 0.6 × 10⁷) viable Pasteurella haemolytica type A1 by the intratracheal route and were killed immediately (0-time) or 2, 4, 12, 16, 24, 48 or 72 h later. Lung-wash cells and free bacteria were recovered by pulmonary layage.The number of recoverable bacteria tended to increase between 0-time and 4 h post-in-oculation (p.i.) then decline rapidly over the next 8 h. However, the rate of clearance was extremely variable and viable bacteria were recovered from $\text{3}\text{5}$ animals at 48 h p.i. and from $\text{1}\text{5}$ at 72 h p.i.In parallel with the clearance of the majority of the bacteria, total neutrophil numbers in the lung-wash rose to a peak of (36 ± 6) × 10⁸ cells/lung, which was, on average, 70-fold higher than 0-time levels. Their numbers remained constant from 12 to 24 h p.i. then fell to be 5-fold above 0-time levels at 72 h p.i. Macrophage numbers rose slowly throughout the experiment but most of the increase occurred between 24 and 48 h p.i. They reached a peak of (17 ± 11) × 10⁸ cells/lung at 48 h i.p. which was 3-fold higher than 0-time levels.     

Effects on ovine fetuses of exposure to ovine progressive pneumonia virus

Fetuses of 20 pregnant ewes at 4 gestational periods (45, 55, 85, and 100 days) were inoculated with ovine progressive pneumonia virus. Fourteen of 16 fetuses exposed to virus before gestational day 80 were either resorbed or expelled, whereas 10 of 15 fetuses exposed to virus after day 80 were normal at birth. Three of the 9 expelled fetuses and 1 of 2 newborn lambs had accumulations of lymphoid cells in the lungs. Virus was readily isolated from the tissues of expelled fetuses and newborn lambs. Lambs did not have precipitating antibody to the virus at birth, but 3 to 5 lambs had specific antiviral antibody at 18 months of age.     

Production of ovine chimeras by inner cell mass transplantation

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.     

Establishment and characterisation of ovine blood monocyte-derived cell lines

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.     

Immune reactions and immunopathologic changes induced in ovine fetuses and lambs by maedi-visna virus.

Seventy- to 80-day fetuses of Merino ewes were inoculated intramuscularly in utero and 2-week-old lambs of the same breed intratracheally with 10(6.3) TCID50/0.1 ml of maedi-visna virus strain K1512 isolated in Iceland. While no precipitins appeared in the serum of fetuses, such antibodies were demonstrable in the lambs from postinoculation (PI) day 30. Indirect immunofluorescence (IIF) revealed the presence of antibodies in samples from both fetuses and lambs; the detectability of these antibodies, however, varied even within a given animal during the experiment. The serologic results were inversely proportional to the kinetics of circulating immune complex (cIC) production. By the lymphocyte stimulation test (LST), the blastogenic transformation of lymphocytes as measured by 3H-TdR incorporation was 6-8% and 6-14% in the fetuses and lambs, respectively. By antibody-dependent cell-mediated cytotoxicity (ADCC) test, cytotoxic capacity (10-14%) was only demonstrable in lambs inoculated at 2 weeks of age, in the 2nd month of life. Histologic examination showed that in the lungs of both fetuses and lambs lympho-histiocytic infiltration developed from PI week 4. This was later joined by lymphoid hyperplasia in the peribronchial lymph nodes. T lymphocyte proliferation was dominant in these lesions as shown by a histochemical procedure (alpha-naphthyl-acetate-esterase, ANAE). By immunofluorescence (IF), deposited immune complexes (IC) were demonstrable in various organs (wall of cerebral ventricles, endothelium of blood vessels of the brain stem, cerebellum, lungs, kidneys). These IC may play an important role in the pathogenesis of maedi-visna.     

Ocular squamous cell carcinoma: immunological responses to tumour tissue and phytomitogens

Cows with histologically confirmed ocular squamous cell carcinoma were injected with autochthonous tumour brei in adjuvant. Lymphoproliferative responses of isolated peripheral blood lymphocytes exposed to phytomitogens and inhibition of cell migration indicated that afflicted animals were immunocompetent. Similar but lesser responses were evident when autochthonous tumour homogenates were used to stimulate lymphocytes.