Immunotherapy by intralesional injection of BCG cell walls or live BCG in bovine ocular squamous cell carcinoma: a preliminary report.

Cows of the Dutch Frisian and Maas-Rijn-IJssel breed with histologically confirmed ocular squamous cell carcinoma showed complete regression of the primary tumor in 70 or 60% of the cases after intralesional injection of a BCG cell wall or live BCG vaccine, respectively. Recurrence of the tumor was observed in 57% of the animals treated with BCG cell walls and in 25% of the animals treated with live BCG vaccine. Spontaneous regression was seen in 20% of the untreated cows. In a second control group, radical surgery, the most successful treatment for primary stage I tumors in humans, resulted in a 90% cure. Influence of immunotherapy on metastases could not yet be fully evaluated. White blood cell counts were not changed after therapy. It was not possible to link a favorable response to BCG therapy with the intensity of the delayed type hypersensitivity (DTH) reaction to purified protein derivative of mycobacteriae (PPD) or the formation of antibodies to BCG as determined by a micro-enzyme-linked immunosorbent assay. However, in animals that showed tumor regression, the DTH reaction to PPD had a tendency to persist for a longer period of time. It was concluded that 1) block resection was the best method of treatment for this tumor, 2) a single intralesional injection of a BCG cell wall vaccine was as effective as live BCG vaccine in the induction of complete regression of the primary tumor, 3) in this preliminary study BCG cell wall vaccine was less effective than live BCG vaccine in the prevention of recurrence, and 4) this naturally occurring tumor model is well suited for the study of the influence of BCG immunotherapy in a primary stage I tumor.

Treatment of acute rejection of cadaveric renal allografts with rabbit antithymocyte globulin.

In a prospective randomized single-blind trial, we compared the effectiveness of rabbit antithymocyte globulin (RATG) in the treatment of acute renal graft rejection with the results of treatment by high oral doses of prednisone. Twenty recipients of cadaveric kidneys were included in each group. In the RATG group, the prednisone dose was not increased and a dose-by-rosette protocol was used to keep T cell levels between 50 and 150/mm3. In this group 15 of the 20 patients responded to the treatment. One of these patients lost her kidney afterward because of a technical failure. In five patients rejection was irreversible despite a subsequent course of high-dose prednisone orally. In the prednisone group, 13 patients showed a good response, but 3 of them only after a subsequent course of RATG. The remaining seven patients underwent nephrectomy before a course of RATG could be given. One patient in this group died of septicemia. In either group there were six second rejection episodes, but they developed 2.2 months later in the RATG group. All second rejection episodes were treated with the alternative regimen and all patients responded to this treatment. Renal function after 6 months was similar in both groups. Less infections occurred in the RATG group. Prior to rejection, there were no differences in concentrations of peripheral T cells between both groups. Treatment of acute rejections with RATG is an effective and safe procedure which is steroid sparing.

Absence of a correlation between humoral and cellular responses to a vaccinia virus and products of the major histocompatibility complex in rhesus monkeys.

Sixty-two Rhesus monkeys were tested at different times after vaccinia virus infection for virus-specific induction of lymphocyte proliferation in vitro or antibody production in vivo. No association was found between identifiable RhLA-controlled antigens and the strength of the cellular proliferative and/of humoral response.

Hexachlorobenzene-induced stimulation of the humoral immune response in rats.

Rats were fed diets containing 0, 500, 1000, and 2000 mg HCB/kg during a 3-week period. Marked weight increases of spleen, popliteal and mesenteric lymph nodes and of the liver were found. Histologically, the white pulp in the spleen was enlarged because of an increase in size of marginal zones and follicles. In addition, there was an increase of extramedullary hemopoiesis. In the lymph nodes, the number of high endothelial venules was increased at all dose levels. The number of neutrophils, basophils and monocytes in the peripheral blood was significantly increased, whereas peripheral lymphocyte counts were slightly higher. Total serum IgM levels were markedly increased, but IgG concentrations were unaltered. On the basis of this experiment, the 1000 mg HCB/kg diet level was chosen for the different function studies that were carried out after a 3-weeks dietary regimen. Regarding the humoral immunity, IgM antibodies to LPS were unaltered, whereas primary and secondary IgM and IgG antibody titers to tetanus toxoid were increased approximately three-fold. HCB did not significantly alter the cell-mediated immunity, as shown by the following parameters: resistance to Listeria monocytogenes infection, rejection of skin transplants, and delayed-type hypersensitivity to tuberculin. The phagocytizing capacity of macrophages was studied by measuring the blood clearance of carbon particles. HCB did slightly depress the phagocytic index, but the difference with control animals was statistically not significant. The in vitro responsiveness of thymus cells to the mitogens PHA, Con A, and PWM was not changed by in vivo HCB-treatment. On a cell-for-cell basis, the responsiveness of spleen cells was increased when cultured in the presence of LPS. On a whole organ basis, the response to PHA, Con A, PWM, and LPS was markedly enhanced because of an increase in the number of nucleated spleen cells. Regarding peripheral lymphocytes, only the response to the mitogen Con A was higher. On the basis of these studies it is concluded that HCB stimulates the humoral immune response in the rat, enhances the in vitro responsiveness of spleen cells to the different mitogens mainly as a result of an increase in the number of splenic lymphocytes, but does not alter the cell-mediated immunity as shown with in vivo tests. This result contrasts with data in the literature that show that HCB suppresses the humoral and cell-mediated immunity in mice. Finally, HCB pretreatment only marginally increased the susceptibility of rats to endotoxin, whereas mice have been shown to be 20-fold more susceptible to the lethal effects of bacterial endotoxin.

Vascular beta-adrenoceptor blocking activity of endotoxin and pertussis toxin from Bordetella pertussis in rats.

Isolated and purified leucocytosis promoting factor (LPF), alternatively described as pertussis toxin, reduced the hypotension after beta 2-adrenoceptor stimulation with salbutamol as well as the negative chronotropic activity induced by the muscarinic receptor stimulant arecoline 4 days after its injection into rats. These inhibitory effects of LPF were accompanied by a reduction in basal blood pressure. No effect on autonomic responsiveness or blood pressure was observed 5 h after injection of LPF. Sublethal doses of purified B. pertussis endotoxin (LPS) elicited neither vascular beta 2-adrenergic nor cardiac cholinergic blockade 4 days following injection. Only a distinct vascular beta 2-adrenolytic effect was measured 5 h after pretreatment with the same doses of LPS. This beta 2-adrenoceptor hyporesponsiveness was accompanied by neither an anticholinergic nor a hypotensive effect, but rather by a slight but significant elevation of the blood pressure. In conclusion, both components of B. pertussis (LPS and LPF) give rise to vascular beta 2-adrenergic hyporesponsiveness irrespective of blood pressure effects. There is an important difference between both components with respect to their various kinetic profiles for this phenomenon: an early occurring and short-lasting beta 2-adrenergic blockade for LPS and a late occurring LPF-mediated beta 2-adrenergic blockade.

Effects of dexamethasone and indomethacin on the vascular beta 2-adrenolytic action of pertussis toxin in rats; a prostaglandin-mediated phenomenon.

Lymphocytosis promoting factor (LPF), alternatively described as pertussis toxin, inhibits the vasodilation after beta 2-adrenoceptor stimulation with salbutamol as well as the negative chronotropic activity induced by the muscarinic receptor stimulant arecoline 4 days after vaccination of rats. To analyse whether arachidonic acid metabolites contributed to these phenomena the cyclo-oxygenase inhibitor indomethacin and the phospholipase A2 inhibitor dexamethasone were administered over a period of 4 days. Pretreatment with either drug restored beta 2-adrenoceptor responsiveness. The cardiac anticholinergic effect, however, was not changed. Interestingly, neither of the inhibitors prevented the blood pressure lowering effect of LPF. The reversing effect on vascular beta 2-hyporesponsiveness of indomethacin and dexamethasone therefore appears to be rather specific. It is concluded that endogenous prostaglandins may participate in the vascular beta 2-adrenergic impairment caused by LPF. Furthermore, the results are considered in view of desensitization theories and underlying mechanisms of LPF-induced autonomic impairment.

Effect of triphenyltin hydroxide on the immune system of the rat.

To evaluate the functional significance of triphenyltin hydroxide (TPTH)-induced lymphopenia and lymphocyte depletion in thymus-dependent areas of spleen and lymph nodes, various immune function studies were carried out after 3 or 4 weeks TPTH exposure. Weaned male rats were fed a diet containing 25 mg TPTH/kg, a concentration that did not influence food intake and weight gain. TBTO exposure was continued during the course of the function tests. As parameters of the cell-mediated immunity in 2 experiments the delayed-type hypersensitivity reactions to ovalbumin and tuberculin were significantly suppressed. No effect was observed on allograft rejection, splenic clearance of Listeria monocytogenes at days 5 and 6 after infection, and responsiveness of thymocytes to different T-cell mitogens. In contrast, the response of splenic lymphocytes to the T-cell mitogen phytohaemagglutinin was significantly suppressed. As TPTH treatment reduced the number of spleen cells, mitogenic response calculated per whole spleen was significantly depressed. Regarding the humoral immunity, no effect was observed on serum IgM and IgG levels, on the thymus-independent IgM response to E. coli lipopolysaccharide (LPS), and on the primary and secondary IgM and IgG response to the thymus-dependent antigen tetanus toxoid. Also, no effect was found on phagocytic and killing capacity of macrophages as demonstrated by unaltered splenic clearance of L. monocytogenes at days 1 and 2 after infection. Slightly enhanced mortality of TPTH-treated animals was observed in a L. monocytogenes mortality assay. Finally, TPTH did not increase the susceptibility of rats to endotoxin (LPS).

Consistency testing of diphtheria and tetanus to replace potency testing for lot release.

Diphtheria and tetanus vaccines are among the most effective and safe vaccines in the EPI programme. Their mechanism of toxicity and clinical protection is well documented and toxin neutralising antibodies induced by the vaccines are generally accepted as correlates of protection. Despite these positive aspects there are still no generally accepted methods to estimate their potency for routine lot release. Some of these tests use large numbers of animals and rely on lethal challenge tests. Consequently there are ethical and financial barriers to perform these tests. Test results expressed in IU, depend on the animal species or strain used and there is limited information about their predictive value for clinical protection. WHO, supported by the ECBS, has made a proposal to harmonise the current methods into simplified consistency tests, after clinical safety and efficacy, as well as consistency in manufacturing has been established to the satisfaction of the National Regulatory Authority. In principle these tests aim for a proof of consistency in biochemical and immunological characteristics in comparison with the lots shown to be clinically safe and effective. Given that many manufacturers have recently made, or are planning to make clinical trials with new combination vaccines in the near future, recent clinical data are already available, or will be soon, on the clinical safety and efficacy of the D and T components present in these combination vaccines. This will create a unique opportunity to compare the biochemical and immunological characteristics of routinely produced vaccine lots with the clinical lots and to use the consistency approach as suggested by WHO for lot release purpose. Background information and an update will be given about the proposed consistency approach of WHO for routine lot release of the D and T components in vaccines.

Report of an informal meeting about alternative methods for the potency control of the diphtheria and tetanus components in vaccines.

On the 10th of December 1985 an informal meeting was organized at the National Institute of Public Health and Environmental Hygiene (RIVM) in Bilthoven to discuss the reduction of animals used in the potency control of the diphtheria and tetanus components in vaccines. The objectives of the meeting were to discuss alternative potency tests with some participants of the IABS-symposium on "Use and standardization of combined vaccines" and to make proposals for modifications in the WHO requirements. It has to be mentioned that the meeting was quite informal. The participants (Table I) continued the discussions which took place among a few of them in London 1985. Based on the discussions in London and Bilthoven there was an agreement on the following items.

Standardization of acellular pertussis vaccines.

In comparison with the current whole cell pertussis vaccine, the new generation of acellular pertussis vaccines opens new opportunities to improve the standardization of the product, because well defined and characterized components are used in these new products.However, different compositions, purification and inactivation methods are used by different manufacturers. Consequently the various acellular pertussis vaccines in the world are difficult to compare in a meaningful manner using simple laboratory tests. In addition, the absence of a reliable animal model and serological correlates with protection in children are other complicating factors. For that reason it seems that the consistency in manufacturing based on a clinically validated production process is the best way to ensure the safety and efficacy of routinely produced acellular pertussis vaccines. Laboratory tests to monitor the antigen content, purity, safety and immunogenicity seem to be the best approach to standardize this new generation of pertussis vaccines against homologous standard vaccines with known clinical efficacy and safety and to support the consistency in manufacture.

Developments in pertussis vaccines: memorandum from a WHO meeting.

PIP: The WHO memorandum outlines the present situation regarding pertussis vaccines, discusses ways to evaluate candidate vaccines, and identifies future research needs. Most existing whooping cough vaccines are whole-cell vaccines, combined with diphtheria and tetanus toxoid adsorbed on an aluminum or calcium carrier. As whole bacterial cells, they contain a complex array of at least 7 toxins and antigens, and display a narrow margin between potency and toxicity. The Japanese introduced an acellular vaccine, admittedly sometimes less potent, called the Precipitated Purified Pertussis Vaccine, in 1981. This material contains far less bacterial mass, notably less endotoxin, and consequently produces less fever, erythema and induration. WHO has not yet established minimum requirements for standardization; even the mouse potency assay may not be suitable. There are techniques, however, which will measure amounts of component antigens and toxicity. Conflicting results on assays of potency and immunogenicity will have to be resolved. Besides the obvious need for large clinical trials of defined vaccines, a whole range of research needs were suggested, from genetic studies of the organism to specific details of the host response. It is generally agreed that a less reactogenic and more effective pertussis vaccine is needed and feasible.^ieng

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid.

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.

Antibody response of pregnant women to two different absorbed tetanus toxoids.

The antibody response in pregnant women vaccinated with either of two different adsorbed tetanus toxoids has been studied. One vaccine (A), prepared by toxoiding purified tetanus toxin followed by its adsorption onto calcium phosphate, exhibited a low titre expressed as international immunizing units, 69 IIU/0.5 ml. The other vaccine (B), prepared by purifying formalinized crude tetanus toxin and adsorbing it onto aluminium phosphate showed a high titre, 212 IIU/0.5 ml. No significant differences between titres of circulating antibodies were obtained after the first injection of either vaccine, but titres after the second injection were much higher for vaccine A as compared with those obtained using vaccine B. The results showed that the immune response in human beings is not correlated to titres expressed in IIU. These results confirm that other methods should be adopted for evaluating the potency of vaccines. A simplified technique based on the comparison of circulating antitoxin levels after vaccination of mice has recently been proposed.

B- and T-cell markers on human lymphoblasts after stimulation with mitogen or antigens.

Receptors for sheep erythrocytes and for the third component of complement were demonstrated on human lymphoblasts after stimulation with various antigens and mitogens. With these markers B- and T-cell stimulation could be differentiated. Phytohaemagglutinin (PHA) and purified protein derivative (PPD) proved to activate predominantly T cells, whereas foetal calf serum (FCS) was shown to be a B-cell stimulator. Candida albicans and allogeneic cells, on the other hand, stimulated both B and T cells.

Proposals for quality control methods of bacterial vaccines for immunostimulation. II. In vivo and in vitro stimulation of lymphocytes by BCG.

An in vitro test for measuring the reaction of the draining lymph node after BCG vaccination and an in vitro test based on the stimulation of lymphocytes by concanavalin A were used to demonstrate immunostimulating properties of different BCG vaccines. A marked difference was seen between vaccines grown as a surface pellicle and ground in a ball mill, and those which were prepared from dispersed homogeneous cultures. In both systems the ball mill preparations were the less active.