Influence of inoculation site on development of the Lewis lung carcinoma and suppressor cell activity in syngeneic mice.

The growth of the Lewis lung carcinoma (3LL) was studied in syngeneic C57BL/6 mice inoculated sc with similar numbers of tumor cells in either the flank or the hind footpad (fp). After injection of small numbers of 3LL cells, the incidence of tumors was lower in the flank than in the fp. However, after a successful 3LL transplant, tumors in the flank progressed faster than those in the fp, as evidenced by the early metastatic dissemination to the lungs and the shorter survival of the hosts. Local adoptive transfer tests demonstrated the early appearance of suppressor cell activity in spleens from mice bearing tumors in the flank. Adult thymectomy as well as treatement with antithymocyte serum after the tumor transplant inhibited the growth of a flank tumor but did not modify significantly that of an fp tumor. Thus variations in the site of a subcutaneous tumor implant resulted in differences in tumor development that appeared to depend on the characteristics of the immune response elicited by the inoculum.

Primary antibody response against K and D regions of the H-2a and H-2d haplotypes.

The capacity of D and K antigens of the H-2d and H-2a haplotypes to induce primary IgM response was evaluated in host-donor combinations using congenic recombinant strains of mice. Host animals were immunized with an i.p. injection of 2 X 10(7) allogeneic spleen cells. Direct plaque-forming cells appearing in immune spleens were assayed with cells of lymphomas L2 of strain A and L5178Y of strain DBA/2, and mastocytoma P815 of strain DBA/2, as targets. Serum antibodies were titrated for cytolytic activity with the same targets as above, and with spleen cells of strain A mice. Titration of serum hemagglutinins was performed using A erythrocytes. The following results were obtained: (1) H-2Dd antigens were shown to be more potent in stimulating primary IgM antibody response than were H-2Kk and H-2Kd antigens, as measured by both plaque assay and serum antibody titration. (2) The observed dominance of D antigens of the haplotypes under study did not seem to be dependent on the number of antigenic specificities involved. (3) Comparable results were obtained with both lymphoid and nonlymphoid target cells of either normal or malignant origin.

[Relation of fat consumption to colorectal cancer mortality in a Venezuelan population]. / Relación entre el consumo de grasas y la mortalidad por cáncer colorrectal en la población venezolana.

Epidemiologic and experimental studies have pointed to an association between fat intake and colorectal carcinogenesis. In the present work we have studied the correlation between fat intake and mortality caused by colorectal cancer in the venezuelan population. For this purpose, we have calculated the correlation coefficients between the ingestion of total fat, visible fat (vegetable oil, margarine, butter, mayonnaise) as well as non-visible fat (that contained in other foods) and the mortality rate by colorectal cancer with data from nine venezuelan states and geographical regions. The highest lipid consumption and mortality rates were observed in the more developed states. There was a positive and significant correlation between total as well as visible fat consumption and colorectal cancer mortality (r = 0.756 p < 0.02, and r = 0.958, p < 0.001; respectively). In contrast, there was no significant correlation between the consumption of non visible fats and colorectal mortality (r = 0.543, p < 0.05). More than 80% of the visible fats ingested in Venezuela are constituted by vegetable oil and margarine, which contain a high proportion of polyunsaturated fatty acids. Based on the above, is possible to infer that colorectal carcinogenesis in Venezuela is associated to the uptake of unsaturated fat, and that measures leading to the prevention of this disease should be based on the reduction in the consumption of total and unsaturated lipids.

[Changes in the serum lipid fractions of malnourished children with and without clinical infection. Paradoxical hypertriglyceridemia in malnutrition]. / Alteraciones de las fracciones lipídicas en el suero de niños desnutridos con y sin infección clínica. Hipertrigliceridemia paradójica en desnutrición.

We have studied the levels of total triglycerides, total cholesterol and high, low and very low density lipoproteins, in serum from undernourished children and from eutrophic controls matched by age, race and socioeconomical condition with the undernourished group. Malnourished children were classified according to the severity of the nutritional deficiency and according to the presence or absence of associated overt infections. Serum lipids fractions were evaluated by colorimetric procedures and by electrophoretic isolation and elution of the desired lipoprotein followed by colorimetric evaluation of the cholesterol content. Increased levels of total triglycerides and very low density lipoproteins were observed in the undernourished group. The rise in the levels of total triglycerides and very low density lipoproteins was more marked in children with moderate and severe undernutrition as well as in undernourished with associated overt infection. In contrast, as expected, serum levels of total cholesterol, high density and low density lipoproteins were significantly diminished in undernourished children. The depression in the amount of total cholesterol, high density and low density lipoproteins was more marked in the severe forms of undernutrition. Increased levels of total triglycerides and very low density lipoprotein could be determined by a defect in the clearance of these lipid fractions due to depressed activity of lipoprotein lipase.

[Serum levels of prealbumin in undernourished children and controls with or without associated clinical infection]. / Niveles séricos de prealbúmina en niños desnutridos y controles con y sin infección clínica asociada.

Due to its short half life and high tryptophan content, prealbumin has been considered a sensitive indicator of protein and/or energy deficiency. In addition, prealbumin diminishes during the acute phase response elicited by either infection or tissue injury. Serum levels of prealbumin were determined in undernourished children with or without associated clinical infection and in their infected or non-infected controls matched for age, sex, race and socioeconomical conditions. Serum levels of prealbumin were significantly lower in undernourished than in control children without overt infections. On the other hand, the concentrations of serum prealbumin diminished significantly and to similar levels both in undernourished and control children with associated clinical infection as compared with those observed in non infected children belonging to the similar nutritional status. A positive correlation was found between prealbumin level and the Z-scores of weight-for-age, height-for-age and weight-for height in children without associated infections, which disappeared in clinically infected patients. Thus, prealbumin is a marker of undernutrition in the absence of infection and could be an earlier and more sensitive indicator of actual undernutrition caused by the metabolic effects of acute phase cytokines, than the anthropometrical measurements used here.

Differences in the effect of indomethacin and preincubation on the lymphoproliferative response to concanavalin A of spleen cells from low responder C57BL/6 and high responder BALB/c mice.

In vivo treatment with 100 micrograms of indomethacin each 48 h for 2 weeks enhanced the proliferative response to concanavalin A (Con A) of spleen cells from mice of the C57BL/6 (B6) strain, low responder to T cell mitogens, but did not modify the response of spleen cells from mice of the high responder strain BALB/c (C). The enhancing effect of in vivo indomethacin treatment was more marked in cultures of B6 splenocytes stimulated with high, moderately supraoptimal doses of Con A than in cultures stimulated with optimal mitogen doses. Addition of indomethacin to cultures of spleen cells from untreated donors induced greater increase of the lymphoproliferative response of cells from low responder B6 than from high responder C mice. The enhancing effect of indomethacin added in vitro was observed in cultures stimulated by optimal but not by supraoptimal doses of Con A. The addition of indomethacin did not enhance the response of B6 spleen lymphocytes depleted of adherent cells. Preincubation for 24 h prior to mitogen stimulation increased the response to high Con A doses of spleen cells from low responder B6 mice whereas this procedure did not enhance lymphocyte proliferation in cultures of spleen cells from high responder C mice. Supplementation with indomethacin in vitro combined with preincubation induced additive enhancing effects on DNA synthesis by B6 spleen lymphocytes, suggesting that each treatment acts through different mechanism(s). The results indicated that spleen cells from low responder B6 strain mice are more sensitive than cells from high responder C mice to the potentiating effect of indomethacin and preincubation on the proliferative response to Con A. These observations suggest that mechanisms sensitive to indomethacin and to preincubation contribute to the depression of mitogen induced DNA synthesis in low responder B6 mice.

Affinity and distribution of subpopulations of antibody-producing cells in protein-restricted C57BL/6 mice.

To study the effect of protein restriction on the affinity of antibodies produced by plaque-forming cells (PFC), C57BL/6 mice were fed diets containing 4% (R4%), 8% (R8%), or 27% (N) casein for 2 (short-term) or 12 (long-term) weeks and immunized with dinitrophenyl (DNP) bovine gamma-globulin in complete Freund's adjuvant. Affinity was assessed by inhibition of plaque formation in the presence of free hapten. Anti-DNP PFC per 10(7) spleen cells were not diminished in short- and long-term R8% mice, and were increased in the former group at certain times after immunization. Affinity of indirect PFC was increased at Days 14 and 21 after immunization in short-term R8% mice and at Day 7 in R4% mice, and was similar in long-term R8% and N animals. No limitation in the heterogeneity of PFC affinities was observed in the restricted groups. Short-term restricted mice showed a rise of the high-affinity PFC subpopulation. The number of mice with hapten-augmentable PFC was diminished in the short-term R8% group at 7 days after immunization and in long-term restricted mice at 14 days, suggesting depressed levels of auto-anti-idiotypic antibodies in protein restriction.

Polyclonal B-cell response to stimulation with Escherichia coli lipopolysaccharide in dietary protein restriction.

The polyclonal B-cell response to Escherichia coli lipopolysaccharide was studied in C57BL/6 mice maintained after weaning on either a moderate protein-restricted diet with 8% casein or a normal diet. After in vitro or in vivo stimulation with the endotoxin, autoreactive and anti-hapten antibody-producing cells were quantitated by direct plaque assay, using bromelain-treated mouse erythrocytes and trinitrophenylated sheep erythrocytes as targets. Larger numbers of plaque-forming cells were generated in cultures of spleen cells from dietary-restricted than from normal mice stimulated with various doses of lipopolysaccharide. The number of background plaque-forming cells was also higher in nonstimulated spleen cell cultures from restricted animals. After injection of lipopolysaccharide in vivo, the number of cells producing antibodies to bromelain-treated mouse erythrocytes per 10(7) spleen cells was significantly increased in dietary-deficient mice. The results are discussed in relation to the different sensitivities of lymphocyte populations to protein deficiency and to the possible presence of high levels of endogenous polyclonal B-cell activators in the restricted mice.

Enhancement of the in vitro antibody response in dietary protein restriction. Failure in the regulation of antibody synthesis.

We have studied the antibody response in vitro of spleen cells from C57BL/6 mice kept on a protein deficient (D) or a normal diet (N). Short or long term protein restriction initiated after weaning led to increased plaque forming cell (PFC) responses to sheep red blood cells (SRBC), TNP-ficoll and TNP lipopolysaccharide. The influence of dietary restriction on the suppression of the antibody response to SRBC was studied in mixed cultures of antigen sensitized and fresh, non-immune cells from either D or N donors. Addition of pre-sensitized D or N cells to non-immune N spleen cells in a 1:1000 ratio resulted in marked suppression of the PFC response whereas co-cultures of pre-sensitized cells and non-immune D spleen cells did not result in significant suppression. Similarly, non-immune T cells from DF mice exerted a lower suppressor effect than non-immune T cells from N mice. Either dietary restriction or low dose cyclophosphamide treatment of the donors of non-immune spleen cells determined a similar reduction in suppression. It is suggested that nutritional deficiency selectively depletes short-lived suppressor effector lymphocytes which are activated in the presence of antigen-stimulated inducer cells.

Autoallergic states. Tumor immunology. Impaired function of peripheral lymphocytes in systemic lupus erythematosus.

DNA synthesis by peripheral lymphocytes from active non-treated SLE patients and matched normal donors was assessed at various times after stimulation with high and low doses of PHA, PWM and ConA and in non-stimulated control cultures. The time response curve of SLE lymphocytes stimulated with a suboptimal dose of PHA, and with both optimal and suboptimal doses of PWM and ConA was markedly depressed, while their spontaneous DNA synthesis was significantly increased. Preincubation of the cells to remove putative coating antibodies did not revert the response of patients' lymphocytes to normal values. The antibody-induced cell-mediated lysis of 51Cr-labelled chicken results demonstrate that cellular factors contribute to the depressed in vitro responses in active SLE, suggesting a wide defect of lymphocyte populations in this disease.

Influence of protein restriction on lymphoid cell populations characterized by the binding of peanut agglutinin.

Cells binding peanut agglutinin (PNA) were studied in the thymus, spleen, and mesenteric lymph nodes from mice placed in the post weaning period on protein-restricted diets containing 8% (R8%) and 4% (R4%) casein. The proportion of PNA+ thymocytes and the absolute number of total and PNA+ cells in the thymus were significantly diminished in R8% and R4% mice. Larger proportion of PNA+ thymocytes showed weaker fluorescence in R8% and R4% than in normally fed (N) animals. Recovery of PNA+ thymocytes was observed in R8% but not in R4% mice at 8 weeks. The number of total and PNA+ cells was significantly diminished although the proportion of PNA+ cells was not modified in the peripheral lymphoid organs of R8% and R4% mice. Results indicate that protein restriction preferentially affected the immature cortical PNA+ cells in the thymus whereas cell depletion in the peripheral lymphoid organs occurred at the expense of both the PNA+ and PNA- subpopulations.

Influence of the oral administration of excess copper on the immune response.

We have studied the influence of the oral administration of excess copper (Cu) on the immune response. With this aim, mice maintained on standard laboratory diet received 50, 100, 200, or 300 ppm of Cu as copper sulfate in the drinking water during 3 to 10 weeks. Inhibition of the proliferative response to concanavalin A was observed in mice exposed to 100 ppm of Cu for 8 weeks and to 200 ppm of Cu for either 3 or 8 weeks. Conversely, a significant increase in the proliferative response to Escherichia coli lipopolysaccharide (LPS) was observed in mice exposed to 50 or 100 ppm of Cu for 3 weeks. However, the response to LPS was also significantly inhibited following prolonged Cu administration. In contrast, mice exposed to low or high Cu doses during short or long periods showed increased production of autoantibodies directed to bromelain-treated mouse erythrocytes. The DTH response to sheep red blood cells was not modified following short-term administration of 100 ppm of Cu, but was depressed after prolonged exposure to this dose of the metal. Significant inhibition of the DTH response was observed in mice exposed to 300 ppm of Cu for 5 or 10 weeks. Thus, oral administration of excess Cu altered the immune response in a fashion related to the dose and duration of treatment.

Calorie restriction modifies the delayed-type hypersensitivity response to the hapten trinitrobenzenesulfonic acid and to hapten-modified syngeneic spleen cells.

We have studied the influence of different degrees of calorie restriction on the induction and the regulation of the delayed type hypersensitivity (DTH) response to trinitrobenzenesulfonic acid (TNBS) and TNBS-modified spleen cells (TNBS-SC), injected by the sc or the iv route. Immediately after weaning, BALB/c mice were placed on restricted diets for either 2 or 4 weeks and then the DTH response was induced. The results showed that a 37.5% restriction in the food supply significantly depressed the level of the DTH response induced by the sc injection of TNBS-SC. In contrast, a 25% restriction in the food supply was insufficient to depress the response. Calorie restriction did not modify the inhibitory influence of an iv injection of TNBS-SC on the DTH response. However, iv presensitization with free hapten or the simultaneous injection of TNBS-SC by the iv and the sc routes did not significantly depress the DTH response in calorie-restricted mice, indicating a defect in the inhibitory regulation of the DTH response in these dietary groups.

Mitogenic effect of zinc on lymphocytes from strains of mice that are either high or low-responder to T-cell mitogens.

We have studied the in vitro mitogenic effect of ZnCl2 in cultures of lymphocytes from Balb/c or C57BL/6 mice which are high-responder or low-responder to T-cell mitogens respectively. Zn induced proliferation of spleen cells from Balb/c mice cultured without 2-ME. Higher levels of proliferation were observed in cultures with 2-ME. In contrast, Zn only induced proliferation of spleen cells from C57BL/6 mice in the presence of 2-ME. No response to Zn was observed in cultures without 2-ME, of spleen cells from either Balb/c or C57BL/6 mice depleted of plastic adherent cells. However, in cultures with 2-ME, Zn induced proliferation of non-adherent as well as plastic adherent cells from either strain of mice. In cultures without 2-ME, Zn induced proliferation of thymocytes from Balb/c mice, whereas did not show constant mitogenic effect on thymocytes from C57BL/6 mice. In contrast, Zn determined higher levels of proliferation of thymocytes from either strain of mice when cultured with 2-ME. Zn had earlier and stronger mitogenic effect on mature thymocytes of either strain of mice than in total thymocytes, both in cultures with or without 2-ME. However, Zn did not induced proliferation in cultures of immature thymocytes of either strain of mice.

Influence of dietary protein restriction on the delayed-type hypersensitivity response to sheep red blood cells in mice.

Delayed-type hypersensitivity responses to sheep erythrocytes were studied in inbred C57BL/6 and outbred NMRI mice fed either protein-deficient diets containing 8% and 4% casein or a normal diet with 27% casein. Following sensitization with optimal doses of antigen, the magnitude of the response was similar in mice fed the 8% protein and the normal diet. Large numbers of sheep red blood cells which suppressed the delayed hypersensitivity response in normal mice, failed to inhibit this response in animals fed the 8% casein diet. However, the titres of serum haemagglutinins were similar in mice of either dietary group immunized with high doses of antigen. Sensitized spleen cells from deficient mice kept on the 8% casein diet, had lower suppressor capacity than those from normal mice upon transfer into syngeneic hosts. Delayed-type hypersensitivity was significantly depressed in mice fed the 4% protein diet whereas the titres of serum antibodies to sheep erythrocytes were not diminished.

The effects of protein malnutrition on the course of Leishmania mexicana infection in C57Bl/6 mice: nutrition and susceptibility to leishmaniasis.

The course of cutaneous infection with Leishmania mexicana was studied in normally nourished and protein deprived C57Bl/6 mice. Mice fed a normal diet showed self-resolving lesions and produced cellular and humoral responses against the parasite. In contrast, mice fed a protein deficient diet which developed chronic protein calorie deficiency failed to recover from L. mexicana infection. Non-healing protein deprived mice had depressed delayed hypersensitivity response (DHR) and in vitro lymphocyte reactivity to leishmanial antigen. Responses to PHA and Con A were also suppressed. The possible interaction between malnutrition, impairment of the immune response and chronicity of cutaneous leishmaniasis is discussed.

Lymphocytotoxic antibodies in SLE patients and their relatives.

A search for lymphocytotoxic antibodies in 50 SLE patients and in 109 of their blood relatives revealed an incidence of cold-reacting antibodies of 80% and 39% respectively, as compared with 15% in 110 normal blood donors paired for age, sex, and racial origin. The antibodies were also present in 7 of 18 (39%) of the spouses of SLE patients. Lymphocytotoxic antibodies exhibited broad reactivity in all groups, although positive SLE sera showed a tendency to react with a wider variety of lymphocytes and to present higher titers than positive sera from the other groups tested.

Altered immune response during cadmium administration in mice.

C57BL/6 mice were administered 50 or 200 ppm of Cd as CdCl2 in the drinking water for either 3 to 4 (short term) or 9 to 11 (long term) weeks. In other experimental designs, mice were exposed orally to 300 ppm of Cd or injected with 2.5 mg/kg of Cd ip. The proliferative response to the T cell mitogens Con A and PHA was increased in cultures of spleen cells from orally treated mice in most of the experiments performed. After primary immunization with sheep red blood cells, the number of IgM antibody forming cells per 10(7) spleen cells was also moderately higher in mice exposed to 50 or 200 ppm of Cd for short or long term. In contrast, long-term exposure to 300 ppm of Cd depressed the antibody response to SRBC. Administration of ZnCl2 prevented the enhancement of the PFC response in mice orally administered 50 ppm of Cd. The capacity to suppress the antibody response of spleen cells preincubated with sodium periodate was decreased after short-term oral or ip. Cd administration but was completely or partially recovered after long-term exposure to either 50 or 200 ppm of Cd.

Modulatory effect of zinc on the proliferative response of murine spleen cells to polyclonal T cell mitogens.

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnCl2 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of 10(-4) M ZnCl2 inhibited proliferation whereas 10(-5) to 10(-6) M ZnCl2 did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 micrograms/ml of concanavalin A and 10 or 25 microliters/ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnCl2 did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl2 on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10(-5) M 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells.