Tumor growth inhibition and potentiation of immunotherapy by indomethacin in mice.

Indomethacin was continuously administered in the drinking water of inbred C3H mice given grafts of syngeneic 3-methylcholanthrene-induced fibrosarcomas. A minor proportion of these animals died at the same time as the untreated controls, and others completely rejected their tumors; however, in most cases, the tumor growth rate was significantly slowed, and growth recommenced rapidly after drug withdrawal. This was the pattern for tumors either in their 10th to 14th transplant generation or only their third in vivo passage. Indomethacin exerted little prophylactic effect, in that it neither increased the minimal cell number required to initiate tumor growth nor significantly decreased the proportion of tumors established in drug-treated animals recieving tumor grafts. The injection of killed Corynebacterium parvum organisms into small, growing McC3-I tumors [intratumor (IT) route] caused the regression of most of these. In contrast, IT injection of BCG, ip injection of C. parvum, or IT injection of C. parvum into larger tumors had no effect. Oral administration of indomethacin enhanced BCG treatment and augmented the activity of C. parvum injected either systemically into animals with small tumors or IT into those with substantial tumor burdens. The duration of these effects was, however, often dependent on the continued administration of the drug.

Passive local anaphylaxis: demonstration of antitumor activity and complementation of intratumor BCG.

When an extracellular dye, Lissamine green, or 51Cr-labeled spleen cells were injected iv into C3H mice bearing small, partially necrotic 3-methylcholanthrene-induced transplantable fibrosarcomas (McC3), the tumor content of these circulating elements per unit weight was substantially lower than that of other selected organs. The level of these blood-borne materials was, however, significantly augmented by the intratumor induction of passive local anaphylaxis (PLA). The PLA-induced augmentation was inhibited by administration of the histamine and serotonin antagonist cyproheptadine; comparable increases were also induced by the intratumor injection of a histamine and serotonin mixture or BCG. The weekly intratumor induction of PLA in McC3 tumors resulted in the complete regression of a significant number of the tumors, and this therapeutic effect was eliminated by cyproheptadine treatment. The intratumor injection of BCG induced the regression of approximately 50% of injected tumors, and the combination of this immunostimulant treatment with the generation of PLA was more therapeutically effective than either treatment alone. PLA in the vicinity of solid tumors may, by increasing vascular permeability, potentiate antitumor effector mechanisms, particularly when these are BCG-stimulated. Despite this demonstration of a possible role of anaphylactic reactions in tumor immunity, no definitive evidence was found that active reagin-mediated local anaphylaxis occurred in C3H mice bearing the McC3 tumor, whether or not they were treated with immunostimulants.

Relationship between the degree of poverty and the IgE response to Ascaris infection in slum children.

We examined a group of urban slum children in Caracas, Venezuela, and demonstrated the relationship that exists between poverty, conditions of hygiene and the prevalence of helminth infection. Concordant with the high prevalence of helminth infection in these children, the total serum levels of immunoglobulin E (IgE) and blood eosinophilia were elevated and were directly related to the degree of poverty and lack of sanitary facilities. However, in contrast, the reverse pattern was observed for the specific allergic response to these parasites. Thus, the immediate hypersensitivity skin test reactivity to Ascaris antigens, and the serum levels of specific anti-Ascaris IgE antibody, were lowest in the poorest children, who had the highest prevalence of helminth infection and the highest total IgE levels. As allergic-type reactions may participate in protective mechanisms against helminths, these results suggest that poverty and poor sanitary conditions may, by influencing the IgE response, compromise the resistance of such children to parasitic infections.

In situ detection of amastigotes in American cutaneous leishmaniasis, using monoclonal antibodies.

Using an immunoperoxidase technique we have applied monoclonal antibodies against American Leishmania for the detection of amastigotes in biopsies from cutaneous leishmaniasis patients. The immunocytochemical procedure was notably superior to conventional histological staining in terms of the visualization and definition of the amastigotes. This technique could eventually prove to be of value in epidemiological studies, and possibly have prognostic importance, by allowing the in situ characterization of the species of infecting organism.

Seroprevalence of Toxocara canis infection in tropical Venezuela.

An enzyme-linked immunosorbent assay (ELISA) was used to determine the seroprevalence of Toxocara canis infection in different socio-economic groups of the tropical population of Venezuela. The lack of definitive independent diagnostic criteria for toxocariasis required the establishment of operational upper limits of normality for Toxocara ELISA values, based upon their log-normalized distribution in a presumptive "non-toxocariasis" sub-population. Only 1.8% of urban subjects of medium-high socio-economic level were considered to be clearly positive in Toxocara ELISA, compared to 20.0% of urban slum dwellers, 25.6% rural subsistence farmers and 34.9% Amazon Indians. As the test was performed using excretory-secretory antigen, and under conditions of competitive inhibition by soluble extracts of non-homologous parasites, co-infection by common intestinal helminths, protozoa or other organisms did not give rise to false positive results. However, strong cross-reactivity with Onchocerca volvulus may have influenced the prevalence figure obtained for the Amazon Indians. These results indicate that T. canis is yet another parasite that is widely distributed in economically underprivileged tropical populations.

Nutritional status and the IgE response against Ascaris lumbricoides in children from a tropical slum.

Helminthic infection can stimulate the interleukin-4 (IL-4)-dependent polyclonal synthesis of immunoglobulin E (IgE) in children endemically exposed to these parasites. As such children are also frequently at nutritional risk, in this study we considered the possible influence of malnutrition on serum IL-4 levels and the IgE response in helminthic infection. We evaluated 85 Ascaris-infected children living in an urban slum area of Caracas, Venezuela, and found that the serum levels of IL-4 and total IgE were significantly higher in malnourished children than in their well nourished counterparts. In contrast, the specific anti-Ascaris IgE antibody response was significantly lower in the malnourished group. After anthelmintic treatment of the children, the total serum IgE and IL-4 levels decreased significantly in the well nourished group, while the specific anti-Ascaris IgE antibody response increased. No significant change was detected, however, in the malnourished group. Our results suggested that malnutrition potentiates the polyclonal stimulation of IgE synthesis induced by helminths. As specific IgE antibody has been implicated in the resistance to helminthic infection, and the polyclonal stimulus diminishes this response, these factors may increase the susceptibility of malnourished children to such parasites.

Nonspecific changes in immunotherapy with house dust extract.

Despite the possible complications and inadequacies of the immunotherapy of allergic patients with house dust extracts (HDE), this is widely applied in Latin America and is commonly considered to be a cost-effective treatment for allergic diseases. Doubts about its efficacy and mechanism of action prompted us to evaluate the immunological changes that occurred in a group of 99 patients selected for their confirmed allergic sensitivity to house dust, and who showed significant clinical improvement after a minimum of 12 months of immunotherapy with HDE. The favorable clinical response of these patients was associated with some of the mechanisms reported for immunotherapy with pollen or venom allergens, such as increased levels of IgG or IgG4 "blocking" antibodies, and the increased affinity of these antibodies for house dust antigens, or diminished basophil sensitivity to HDE. However, the magnitude of these changes was relatively small, and their biological significance questionable. In contrast, particularly evident was a reduction in the cutaneous reactivity not only to the specific allergen, but also to the nonspecific mast cell degranulator codeine and to histamine. These results suggest that as yet unidentified "nonspecific" mechanisms might contribute to the effect of immunotherapy with extracts as heterogeneous and complex as HDE.

Bronchial challenge of tropical asthmatics with Ascaris lumbricoides.

Despite the fact that helminthic parasites can stimulate strong immediate hypersensitivity reactions, it is uncertain whether these are relevant to the development of allergic disease in infected patients. In order to examine this possibility, we tested 20 informed chronic asthmatic patients from an Ascaris-endemic area by bronchial challenge with a partially purified extract of this parasite. Sequential measurements were made of both the forced expiratory volume in the first second (FEV1) and the peak expiratory flow rate (PEFR) up to 6 h postchallenge, then of PEFR from 6 to 14 h and at 24 h. These were compared to the effect of control inhalations of saline. Extremely low doses of Ascaris antigen that did not exceed 10 PNU (6 x 10(-7) g of protein) induced significant reductions (> 20%) in FEV1 within 30 min in 3 (15%) patients, and in PEFR in 5 cases (25%). By 6 h postchallenge, 5 (25%) subjects showed significant alterations in FEV1, and 10 (50%) in PEFR. Significant changes in PEFR were recorded between 6 and 24 h in 12 (60%) patients. The challenge of nonasthmatic subjects from the same Ascaris-endemic area did not produce notable changes in pulmonary function, and although asthmatics with no evidence of prior contact with the parasite showed a certain degree of immediate bronchial reactivity to the parasite extract, the late responses were significantly less frequent than in the infected patients. No correlations were detected between the bronchial responses and skin test reactivities to the Ascaris extract, or serum levels of specific IgE or IgG antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of phagocytosis of Leishmania spp. by human polymorphonuclear leukocytes using anti-Leishmania monoclonal antibodies.

Phagocytosis of Leishmania is an early event in the capacity of human polymorphonuclear cells to limit the spread of this infectious agent. We compared two methods to assess the phagocytosis of Leishmania by PMN cells; the first using histochemical staining with Wright or Giemsa, and the second using the immunoperoxidase technique with anti-Leishmania monoclonal antibodies. The quantitative results obtained with either of the cytochemical methods were comparable with the immunoperoxidase technique, but the latter offered the advantage of an easier identification of the intracellular parasites. This improvement greatly reduced the time required to quantify phagocytosis compared to the conventional staining techniques.

Effect of age and helminthic infection on IgE levels in slum children.

Helminthic infections can cause an IL-4-dependent polyclonal synthesis of IgE, which is the major cause of the elevated serum levels of this immunoglobulin in tropical populations. In the present study, we measured the IgE levels in the serum of children of different ages in slum areas of Caracas, Venezuela, where intestinal helminths are endemic. As would be expected, in children over 5 years of age, the IgE levels were highly elevated, and in addition, the cord blood serum levels were higher than those reported for populations where parasitic infections are uncommon. However, the IgE levels measured in the sera of children under 5 years of age were relatively low, and within the generally accepted normal ranges. This was surprising because the latter children were infected by parasites and had high IgG antibody levels against helminths. We found, however, that whereas older children had detectable IL-4 levels in their sera, the younger children did not. These results suggest that, despite infection of these slum children by common intestinal helminths, a polyclonal stimulation of IgE synthesis did not occur, possibly due to a lack of IL-4 production by an immature immune system.

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis.

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.

Humoral and cellular immune responses of atopic individuals in a tropical environment.

In this study we compared immediate, intermediate and delayed skin test reactivity, total and specific serum IgE, IgG, A, M antibody and lymphocyte proliferative responses, between atopic and normal individuals in the tropical environment of Caracas, Venezuela (Lat. 10 degrees N). The allergenic extracts tested were prepared from house dust, mixed moulds and Aspergillus fumigatus. In lymphocyte stimulation the mitogen Concanavalin A was also employed, the cultures being supplemented with either autologous plasma, or a gamma globulin-depleted pool. The results revealed the association of immediate skin reactions with IgE antibody against house dust, and intermediate reactions with IgG, A, M antibody against moulds. No relation was, however, detected between delayed skin reactivity and in vitro lymphocyte transformation; skin reactions occurred at the highest frequency with moulds, while house dust provoked the strongest in vitro stimulation. Also, although the areas of positive delayed reactions were greatest in the atopic individuals, the lymphocyte proliferative responses were clearly highest in the normal subjects. The significance of the positivity of lymphocyte transformation tests in all of the study group, but lower reactivity in the atopics, is considered.

[Host-tumour relationships and immediate hypersensitivity reactions]. / Rapports hote-tumeurs et réactions d'hypersensibilité immédiate

When radiolabelled lymphoid cells and the extracellular dye Lissamine green were injected IV into C3H mice carrying non-necrotic methylcholanthrene-induced tumours, the tumour content of these agents was significantly lower than in other organs. The tumour content of these circulating blood components was significantly increased by the intratumoral (IT) induction of local passive anaphylactic reactions, and such reactions exerted an anti-tumour effect, and enhanced the therapeutic activity of IT BCG. No anti-tumour IgE antibody was detected in this model, but the tumour inhibited passive and active anaphylactic reactions in its host.

Histamine in the tissues of the marsupial Setonix brachyurus (the quokka).

Passive cutaneous anaphylactic (PCA) reactions in the marsupial Setonix brachyurus (the quokka) were completely inhibited by the histamine antagonist mepyramine maleate, but were unaffected by disodium cromoglycate or the serotonin antagonist, methysergide. Histological examination of quokka skin indicated that mast cell degranulation occurred during the PCA reaction in this marsupial and animals whose skin was relatively deficient in mast cells were poor PCA recipients. In contrast to many eutherian (placental) species, this marsupial was found to lack histamine in blood leukocytes and platelets. Also, while the peritoneal mast cells of rats and mice contain large quantities of histamine, this amine was not detected to quokka peritoneal washings, even after the induction of a peritoneal exudate or the regular intraperitoneal injection of antigen. Immunologic challenge of quokka blood or peritoneal cells did not induce the synthesis of histamine, but histamine release was elicited from sensitized quokka lung by antigenic challenge.

Tumour-associated inhibition of immediate hypersensitivity reactions in mice.

The intensity of anaphylactic shock was lower in C3H mice carrying a methylcholanthrene-induced tumour (McC3) than in their normal counterparts when immunized with ovalbumin and challenged i.v. after 14 days. This tumour-associated inhibitory effect on active systemic anaphylaxis was exerted mainly on events occurring after homocytotropic antibody synthesis because the serum titres of these antibodies were comparable in normal and tumour-bearing animals. In addition, passive systemic anaphylactic reactions were suppressed in animals carrying the tumour and the sensitivity of these animals to challenge with histamine and serotonin mixtures was also reduced. The presence of a growing McC3 tumour did not, however, diminish the amine-sensitizing effect of treatment with Bordetella pertussis vaccine. The McC3 tumour inhibited the generation of passive cutaneous anaphylactic reactions, an effect that was also exerted by a tumour extract, particularly when administered to the recipients shortly before antigen challenge. Thus immediate hypersensitivity reactions, like a variety of other immunological processes, can be inhibited by tumour products which by compromising the immune status of the host might permit tumour growth. The nature of the inhibiting factor is unknown, except that it is probably not the amine-degrading enzyme histaminase. In addition, which it is uncertain whether the inhibitory effect is exerted directly or indirectly, the possible importance of prostaglandins in the phenomenon is discussed.

Evolutionary adaptation of inflammatory immune responses in human beings.

Modern man's ancestors lived in an environment where infectious, tropical diseases would have been endemic. We postulate that this relatively hostile environment would have caused genetic selection for increased proinflammatory immune responses. On migrating to temperate regions, pronounced proinflammatory responses would have been less important and selected against due to increased mortality from overly vigorous responses to harmless environmental agents. This hypothesis is supported by the observation that proinflammatory alleles in several genes involved in inflammation are more prevalent in populations with long-term tropical ancestry than those with long-term residence in temperate regions. In addition, when the former populations relocate from a tropical to a temperate region, they have a higher incidence of allergic inflammatory diseases than the latter populations. These observations suggest that there may be general patterns of recent evolutionary adaptation of the human immune system to particular regions and that these adaptations can produce differences in disease susceptibility.

Passive cutaneous anaphylaxis and vasoactive amine challenge in nude (nu/nu) mice. A comparison with nu/+, axenic and young normal animals.

Recent suggestions of a thymic origin or thymo-dependent differentiation of tissue mast cells prompted an investigation in the athymic nude (nu/nu) mouse. The outbred nu/nu examined were found to possess mast cells in at least comparable numbers to the phenotypically normal nu/+. These nu/nu were superior to nu/+ as recipient for mast cell-dependent, long latent period (IgE-type), passive cutaneous anaphylactic (PCA) reactions. A variety of studies performed, including competition with nu/+ serum, thymic restoration, quantitation of nu/+ homocytotropic antibody responses, comparison with axenic or young mice and time-course studies of skin sensitization suggested that the higher PCA titres in nu/nu skin were not entirely due to a lack of competitive inhibition of mast-cell sensitization. The nu/+ used in these studies were in fact poor IgE recipients, but were somewhat more sensitive than nu/nu to hypovolaemic shock induced by histamine and serotonin mixtures. Pretreatment with Bordetella pertussis greatly enhanced the sensitivity of nu/+, axenic, holoxenic and young normal mice to the vasoactive effects of histamine and serotonin, but was somewhat less effective in nu/nu and old mice.

Immunoglobulin classes carrying homocytotropic and passive hemagglutinating antibody activities in the marsupial Setonix brachyurus (the quokka).

On the basis of their physicochemical and antigenic properties, and by analogy with guinea pig immunoglobulins, two antibody classes in the serum of the marsupial Setonix brachyurus have been classified as IgG1 and IgG2 isotypes. The analogy between these marsupial immunoglobulins and those of eutherian (placental) species was extended by an investigation of their biological activities. The electrophoretically slow antibody (IgG2) fixed hemolytic complement, precipitated soluble antigen and predominated early in the response mounted when quokkas were immunized with antigen emulsified in Freund's complete adjuvant (FCA). The more anodic antibody (IgG1) in this marsupial did not fix complement, was inefficient in precipitating antigen and was the predominant antibody synthesized by quokkas immunized with antigen adsorbed to alumina gel. The IgG1 isotype in this marsupial appears to possess both passive hemagglutinating (HA) and homocytotropic antibody (HCA) activities. However, the HCA and IgG1 HA activities did not develop in parallel during the course of the immune response, thus suggesting that only a functional subpopulation of the IgG1 antibodies possess homocytotropic activity.

Genome screen and candidate gene studies in parasitized populations.

The immunoglobulin E (IgE) antibody system is important in the genesis of asthma, but it appears to have originally evolved for defence against parasite infection. In order to study how IgE contributes to asthma, there are advantages in studying parasitized populations. Firstly, the IgE system can be studied when it is operating in a more natural state, and this could allow new insight into basic immune function. Secondly, the genetic susceptibility to produce high levels of IgE is more likely to be expressed, as the most intense IgE responses in nature are those found in the presence of parasitic infection. These more intense IgE responses should facilitate finding new 'asthma genes', assist in investigating how the DNA variations in candidate genes affect gene function and provide the possibility of developing new approaches to the treatment of asthma.

High allergic reactivity in a tropical environment.

The prevalence of atopic disease in tropical populations is often considered to be low, and this has been attributed to an immunological modulating effect of intestinal helminthiasis. We, however, report that the frequency of positive allergic history and skin-test reactivity to groups of major environmental allergens is extremely high (43 and 63% respectively) in children in Caracas, Venezuela (Lat. 10 degrees N). These values were statistically significantly greater than in a group of children with a similar age and sex distribution studied in parallel, but having limited or no contact with a tropical environment (29 and 37% respectively). The two groups differed with respect to the sporadic and light intestinal helminthic infections (such as ascariasis) experienced by the Venezuelans, compared to rare contact in the 'non-tropical' group. This was probably the cause of the increased total serum IgE levels of the former children (369 vs 68 iu/ml), and possibly, therefore, their higher allergic reactivity.