The Mechanism of Action of Anaphylatoxin. Its Effect on Guinea Pig Mast Cells

In the guinea pig anaphylatoxin produces mast cell damage that is similar to that produced by antigen-antibody reaction but is different from that produced by chemical histamine liberators. This damage is inhibited by iodoacetate, p-chloromercuribenzoate, phenol and cold, but is not inhibited by calcium lack or previous heating of the tissue to 45° C. Mepyramine reduces but does not abolish the contraction of the ileum produced by anaphylatoxin. Previous heating of a sensitized ileum to 45° C., although completely abolishing the anaphylactic response, does not interfere with the contraction induced by anaphylatoxin. Furthermore desensitization to anaphylatoxin does not modify the anaphylactic contraction. The implication of anaphylatoxin with the anaphylactic reaction is discussed. It is concluded: (a) that anaphylatoxin shares with the anaphylactic reaction part of the pathway leading to histamine release; (b) that the mechanism of action of anaphylatoxin is quite different from that of the chemical histamine liberators.     

Studies on Sperm Antigenicity 6. In vivo and in Vitro Cellular Reactivity in Guinea Pigs Sensitized with Fractions of Guinea Pig Spermatozoa

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultracentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes, the lack of sperm accumulation in the epididymis was obvious.     

Studies on Sperm Antigenicity 6. In vivo and in Vitro Cellular Reactivity in Guinea Pigs Sensitized with Fractions of Guinea Pig Spermatozoa

Normal guinea pig spermatozoa cells were homogenized by a French pressure cell. Three soluble and three insoluble fractions were obtained by ultracentrifugation and (emulsified in CFA) were used for guinea pig sensitization. The following were observed: 1) all fractions were immunogenic except one; 2) in vivo and in vitro delayed hypersensitivity was elicited in animals immunized with these fractions; 3) two distinctive histopathologic lesions were observed in the testes of sensitized animals: lesions of orchitis type developed in animals injected with some fractions. Other fractions induced lesions of aspermatogenic type. These results correlated well with delayed hypersensitivity results obtained by in vivo and in vitro tests. Although some other spermatozoal fractions did not cause severe changes in the testes, the lack of sperm accumulation in the epididymis was obvious.     

Guinea Pig Herpes-Like Virus Infection I. Antibody Response and Virus Persistence in Guinea Pigs After Experimental Infection

Guinea pigs, experimentally infected with guinea pig herpes-like virus produced antibodies which were detectable by indirect hemagglutination (IHA), complement-fixation (CF), and neutralization tests. The IHA test appeared to be a more sensitive method than the CF or neutralization test for determining antibody response in guinea pigs immediately after infection, but the IHA method was not suitable for the detection of antibody in animals receiving small doses of virus. Antibody titers obtained by CF tests were generally higher than those obtained by the neutralization test, and they followed the same time course when individual animals were studied serially. Intracardiac inoculation produced the best antibody response in guinea pigs when compared with other routes of infection. Guinea pigs infected by the intraperitoneal, intranasal, or oral route showed rising antibody titers but the levels were low. Infectious virus was isolated from and persisted in all inoculated animals in the presence of antibody regardless of the route of inoculation. Recovery of infectious virus required cultivation or cocultivation of tissue cells containing virus. The administration of antilymphocyte sera delayed the appearance of IHA antibody but had no effect on antibodies determined by the CF- and neutralization tests.     

The Mongolian Gerbil as a Model for Lead Toxicity: I. Studies of Acute Poisoning

Mongolian gerbils fed diets containing lead acetate maintained body weight comparable to gerbils fed the same diet without added lead. Intranuclear lead inclusion bodies in epithelial cells of the proximal convoluted tubules of the kidney were first observed at 4 weeks, and increased in number to about 50 per high power field at 12 weeks. At this time, a corticomedullary area of empty-appearing tubules was prominent. Transmission electron microscopy confirmed the increase in number and size of nuclear lead inclusion over the 12-week period. Cytoplasmic changes observed in proximal tubule cells containing lead inclusions were considered indicative of acute lethal injury. Distinct cytoplasmic fibrillar structures, first apparent at 8 weeks, were present in some proximal tubular lining cells and strongly resembled newly formed intranuclear lead inclusions. After 12 weeks, the total amount of lead present in the gerbil kidney was four to six times greater than that in rat kidney as determined by atomic absorption spectrophotometry. A hypothesis has been formulated that relates the more efficient nephron of the gerbil kidney to the rapid and extensive development of intranuclear inclusion bodies and the greater accumulation of total lead.     

Infection at the Subcellular Level I. Localization of Intravenously Injected Brucellae in the Vacuolar Apparatus of Cells of Guinea Pig Liver

The distribution of Brucella melitensis in various tissues and in subcellular fractions obtained from liver was investigated to evaluate the initial phases of brucellosis in the guinea pig. Fifty minutes after intravenous infection, brucellae were found principally in the blood and liver, with a substantial number recovered from spleen. Fractionation of liver established that most bacteria were found in the mitochondrial plus lysosomal (M + L) fraction; a significant number, however, sedimented in the nuclear (N) fraction. With time, there was a progressive shift of bacteria from the M + L to the N fraction, accompanied by a similar shift in acid phosphatase activities. Isopycnic centrifugation of mixtures of M + L fractions and brucellae permitted complete separation of acid phosphatase-bearing particles from bacteria. Similar experiments with fractions from infected animals showed that viable bacteria were found in both the acid phosphatase and free brucellae regions of the gradient. At 10 min postinfection, 52% of the recovered organisms were in the acid phosphatase region; at 30 min, 65%; at 60 min, 85%; and at 315 min, 79%. Detergent plus sonic treatment of an M + L fraction from the liver of an animal killed 50 min after infection caused most of the bacteria in the acid phosphatase region to shift to the region where free bacteria were found. These data suggested that brucellae sequestered in the liver were located primarily in the vacuolar apparatus of the cells which phagocytized them.     

Chemotherapeutic Activities of Carthami Flos and Its Reversal Effect on Multidrug Resistance in Cancer Cells

Multidrug-resistance (MDR) represents a major cause of failure in cancer chemotherapy. The need for a reduction in MDR by natural-product-based drugs of low toxicity led to the current investigation of applying medicinal herbs in future cancer adjuvant therapy. Carthami Flos (CF), the dried flower of safflower (Carthamus tinctorius L.), is one of the most popular traditional Chinese medicinal herbs used to alleviate pain, increase circulation, and reduce blood-stasis syndrome. The drug resistance index of the total extract of CF in MDR KB-V1 cells and its synergistic effects with other chemotherapeutic agents were studied. SRB cell viability assays were used to quantify growth inhibition after exposure to single drug and in combinations with other chemotherapeutic agents using the median effect principle. The combination indexes were then calculated according to the classic isobologram equation. The results revealed that CF showed a drug resistance index of 0.096. In combination with other chemotherapeutic agents, it enhanced their chemo-sensitivities by 2.8 to 4.0 folds and gave a general synergism in cytotoxic effect. These results indicate that CF could be a potential alternative adjuvant antitumour herbal medicine representing a promising approach to the treatment of some malignant and MDR cancers in the future.     

Lymphopoiesis in Protein Deficiency: Stathmokinetic and Tritiated Thymidine Uptake Studies of the Mesenteric Lymph Node of the Guinea Pig

In view of the depressed immunity in protein malnutrition, an assessment of the lymphoproliferative activity of the constantly stimulated mesenteric lymph node of the guinea pig was undertaken. A significant reduction of this activity was observed in protein deficiency. a) The germinal centers were reduced in number and size; new formation in the medulla was rarely seen. Lymphoid cells showed lowering of mitotic index and mitotic rate and prolongation of mitotic duration and turnover time. Nuclear labeling with ³H-thymidine was focal and localized to the peripheral zone. b) Mitotic activity and nuclear labeling were less pronounced in the outer cortex and medulla and least in the paracortical area. Specific uptake of ³H-thymidine paralleled the low labeling index. c) No appreciable reduction in the number of plasma cells in the medulla was observed. Serum γ-globulin levels were not significantly altered, but albumin levels were consistently reduced. This was suggestive of preferential preservation of plasma cell activity in the malnourished host. d) There were significant lymphopenia and neutropenia with relative increase of neutrophils in the peripheral blood. This might indicate more severe involvement of lymphopoiesis than myelopoiesis in protein malnutrition.     

Studies on the Mode of Action of Diphtheria Toxin V. Protein Metabolism in a Guinea Pig Model Simulating Chronic Diphtheritic Toxemia

An experimental model of "chronic" diphtheria intoxication in the guinea pig was developed. Adult guinea pigs, subjected to a regimen of multiple sublethal doses of purified diphtheria toxin (total of 1.4 minimum lethal doses divided equally in four daily doses), developed a toxemia which terminated in death between 6 and 8 days. During an advanced stage of illness, de novo protein synthesis was assessed by in vivo incorporation of tritiated leucine into tissue proteins. With the exception of pancreas and skeletal muscle, protein synthesis in healthy, control and toxin-treated guinea pigs was comparable. The absence of notable impairment of protein synthesis in this experimental situation is discussed in terms of the mode of action of the diphtheria toxin and the pathophysiology of the disease.     

Nephrotoxicity of Combined Treatment with Cisplatin and Gentamicin in the Guinea Pig: Glomerular Injury Findings

The drugs cisplatin and gentamicin are given consecutively to various cancer patients suffering from infections. Little information exists about the ultrastructural alterations of kidney glomeruli caused by treatment with these drugs. Renal glomeruli of guinea pigs treated with cisplatin alone and in combination with gentamicin were studied by transmission electron microscopy. The findings revealed foci of damage induced by cisplatin and especially by cisplatin/gentamicin in all glomerular components: glomerular capillaries, including their endothelial cells; basement membrane, epithelial podocytes, mesangial cells, and parietal cells of Bowman's capsule. The damage was expressed by endothelial cytoplasmic extrusions into the vascular lumen, thickening and lamination of capillary basement membrane, focal foot process fusion of podocytes, vacuolization in cytoplasm of endothelial cells of epithelial podocytes and of parietal cells, and the presence of lipid bodies and myeloid bodies in all glomerular cell types. Additionally, injurious effects to cytoplasmic organelles such as mitochondria, nuclei, and endoplasmic reticulum were observed. The results indicate that cisplatin alone and in combination with gentamicin is toxic to renal glomerular tissue. Since these drugs were previously found toxic for strial capillaries in the inner ear and since the main glomerular component is the glomerular capillaries, potential vascular damage and vascular complications in different body systems have to be taken into consideration when these drugs are needed in clinical use.     

Immunity to Chlamydial Infections of the Eye II. Studies of Passively Transferred Serum Antibody in Resistance to Infection with Guinea Pig Inclusion Conjunctivitis

Conjunctival infection of guinea pigs by the chlamydial agent of guinea pig inclusion conjunctivitis confers immunity. However, the mechanism of resistance to this intracellular pathogen is not yet defined. In the study reported here, serum immunoglobulin was passively transferred with resultant titers in excess of those known to be associated with immunity. Nonetheless, when the passive transfer recipients were challenged, they acquired infection which was neither delayed nor attenuated. Eye secretion antibody titers appeared and increased only after 11 days of infection in both passive transfer recipients and control groups, suggesting but not proving de novo local synthesis of secretory antibody. This study suggests that cellular or secretory immune mechanisms may predominate in resistance to this infection.     

Inhibitory Effect of MY-1250 on Histamine Release from Rat Peritoneal Mast Cells and Guinea Pig Lung Fragments: The Elucidation of the Mechanism

When the effect of MY-1250 (5, 6-dihydro-7, 8-dimcthyl-4, 5dioxo-4 H-pyrano [3, 2-c] quinolinc-2-carboxylic acid) on histamine release from rat peritoneal mast cells induced by compound 48/80 was studied, MY-1250 caused a significant inhibition of histamine release at concentrations higher than 3 _pM. Furthermore, the compound inhibited not only 45C _a uptakc into the mast cells but also Ca²⁺ release from thc intraccllular Ca store at a concentration of 10 _pM in both cases. By contrast. MY-1250 did not affect either histarnine release from permeabilized mast cells induced by TPA, IP3 and GTPyS or Ca²⁺ release from the endoplasmic reticulum induced by IP3. In the chopped lung preparations, MY-1250 at doses of 10 and 100 _pM caused a significant inhibition in histarnine release from thc pieces of actively scnsitizcd guinea pigs exposed to antigen and simultaneously prevented a decrease in intracellular CAMP contents taking place in association with antigen-antibody reaction. No significant changes were effected by MY-1250 in a-chymotrypsin activity and phospholipasc A2 activity. Also, no antagonistic effects on LTD4 and PAF were observed.     

HLA—D Typing with Homozygous Cells Identified in an American Indigenous Isolate. I. Population Studies

Lymphocytes from six individuals homozygous for their HLA--A, --B, --C and --D loci belonging to an American indigenous group, the Warao, have been used as typing cells to detect HLA--D determinants in 121 donors from the same indigenous isolate and in 71 donors of mixed ethnic origin living in Venezuela. Two determinants responsible for strong proliferation in mixed lymphocyte cultures have thus been identified. Four HLA--A2, B5 cells (r values between 0l72 and 0.57) identify a determinant provisionally called LD5a showing a gene frequency of 0.30 among the Warao and of 0.09 among the population of mixed ethnic origin. The nearest B locus antigen to this new specificity among the Warao is HLA--B5 (r value = 0.20). A second determinant identified by two HLA--A2, B15 sisters (r values of 0.71) shows a gene frequency of 0.15 among the Warao and of 0.03 among the mixed population. The latter is related to Dw8 as shown by results of the VI and VII Histocompatibility Workshops, and is weakly associated (r value of 0.28) to HLA--B15 among the American indigenous population tested.     

Effect of Na+,K+,2Cl- Cotransport Inhibitor Bumetanide on Electrical and Contractile Activity of Smooth Muscle Cells in Guinea Pig Ureter

The effect of Na⁺,K⁺,2Cl^- cotransport inhibitor bumetanide on action potentials and contractions of smooth muscle cells in the ureter of guinea pigs evoked by electrical stimulation was studied by the method of double sucrose bridge. Bumetanide (10-100 M) dose-dependently suppressed action potential and contractions of smooth muscle cells induced by 1-10 M histamine, 10 M mesatone, 5 mM tetraethylammonium, and 100 M sodium nitroprusside. Our findings suggest that test substances modulate Na⁺,K⁺,2Cl^- cotransport in smooth muscle cells.