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Acutely sprained ankles represent a frequent and common injury among active duty troops in training, and are a significant source of morbidity with respect to days lost to training. Swelling in the form of periarticular edema limits motion, causes pain, prevents wearing of normal foot gear, and slows the healing process. Reduction of edema was attempted in acutely sprained ankles by the use of pulsed electromagnetic energy (Diapulse). In a randomized, prospective, double blind study of 50 grade I and II (no gross instability) sprained ankles, a statistically significant (p < 0.01) decrease in edema was noted following one treatment with Diapulse. The application of this modality in acutely sprained ankles could result in significant decreases in time lost to military training.

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To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.

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Investigation of host-parasite relationships involving the parasitic form of Coccidioides immitis has been difficult because, previously, spherules and endospores have not been grown continuously in tissue culture medium without detectable formation of hyphae. Arthroconidia were harvested from mycelial cultures and inoculated into tissue culture flasks which contained RPMI 1640 medium supplemented with 10% calf serum and N-Tamol (Rohm & Haas Co., Philadelphia, Pa.). Flasks were purged with 5% CO2, sealed, and placed on a reciprocating shaker at 35 degrees C. Hyphae which arose during incubation were removed by filtration. Arthroconidia readily converted to the spherule-endospore form within 12 days. Six days after complete conversion, spherules and endospores were transferred to RPMI 1640 without N-Tamol. The spherule-endospore cycle was maintained in tissue culture medium for 84 days without the formation of detectable hyphae.

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A 54-year-old man with plasma cell myeloma had sustained bleeding develop after prophylactic hip hemiarthroplasty. Routine coagulation studies revealed significant prolongation of the prothrombin time, activated partial thromboplastin time, and thrombin time. Further evaluation showed failure of the activated partial thromboplastin time to correct in a 1:1 mixture with pooled normal plasma, correction of the prolonged thrombin time by addition of protamine, and a normal reptilase time. A purified preparation of the immunoglobulin component of patient plasma produced the same pattern of coagulation abnormalities, suggesting the paraprotein possessed heparin-like anticoagulant activity. This appears to be a rare mechanism of bleeding diathesis in plasma cell myeloma.

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Steroid 21-hydroxylase activity has been demonstrated, previously, in microsome-enriched fractions prepared from a number of human fetal tissues. The finding that this enzyme activity was present in thymus and spleen is suggestive of the possibility that deoxycorticosterone is important in regulation of immunological processes. In the present investigation, we characterized steroid 21-hydroxylase activity in microsome-enriched preparations of guinea pig spleen. The activity of the enzyme was linear with time for 40 min and with protein concentrations up to 4.8 mg X ml-1 incubation mixture. The apparent Km of the enzyme for progesterone was 0.405 microM. Thus, the potential exists for the biosynthesis of DOC from progesterone in the spleen of the guinea pig as well as in the spleen of the human fetus. Therefore, the guinea pig may be an appropriate animal model for the study of the regulation of steroid 21-hydroxylase activity in the spleen as well as a model for the study of the role of this enzyme in immunologic processes.

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Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.

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Murine polymorphonuclear leukocytes (PMN) are stimulated to produce hydrogen peroxide (H2O2) by some unopsonized fungi, including Coccidioides immitis spherules, Candida albicans blastospores, and Saccharomyces sp. blastospores (zymosan). In this communication we have examined the basis for this stimulation by studying the effects of mannan and its primary constituent, D-mannose, on PMN function in vitro, since this polysaccharide is a significant component in many fungal cell walls. Our results show that mannan stimulated H2O2 production and iodination by PMN and enhanced H2O2 production by cells stimulated with zymosan. Moreover, the amount of H2O2 released by PMN was directly related to the concentration of mannan, and mannan obtained commercially or prepared in our laboratory worked equally well. Mannose, on the other hand, inhibited H2O2 release from PMN stimulated with zymosan or mannan and reduced oxygen consumption, carbon dioxide production, superoxide release, and iodination by these cells as well. With respect to specificity of inhibition, 18 different monosaccharides were examined by using 2 different assays for H2O2 release; and only 2-deoxy-D-glucose, a potent glycolytic inhibitor, reduced H2O2 release from zymosan-stimulated PMN. Furthermore, H2O2 release from cells stimulated with phorbol myristate acetate or opsonized Sephadex was not inhibited by mannose whereas H2O2 release was reduced when PMN were stimulated with C. albicans blastospores or C. immitis spherules in the presence of this sugar. From these data we propose that initiation of PMN oxidative metabolic burst in response to some unopsonized fungi occurs through a mannose-specific mechanism.

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The response of human polymorphonuclear leukocytes (PMN) to blastospores and pseudo-hyphae of the opportunistic fungus Candida albicans has been studied in vitro and in vivo. Of the fungicidal mechanisms elucidated thus far, the myeloperoxidase-hydrogen peroxide-halide system appears to be most effective against cells of this fungus. In our studies on the interaction between murine PMN and blastospores, we assayed the release of H2O2 by PMN incubated with viable or killed, unopsonized or opsonized blastospores by using two assay systems, lysis of murine erythrocytes and oxidation of scopoletin. Our results showed that PMN released increasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized killed blastospores, but released decreasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized viable blastospores. The oxidative metabolic burst by PMN in the presence of viable or killed blastospores was also measured by using reduction of nitroblue tetrazolium and chemiluminescence. Viable blastospores stimulated a stronger metabolic burst than killed blastospores, suggesting that PMN respond to live blastospores more vigorously than killed blastospores; however, live blastospores appear to alter or inhibit the release of H2O2 by PMN.

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Spleen cells from mice immunized with a variety of antigens and incubated in vitro with killed spherules of Coccidioides immitis lyse six to eight times more autologous murine erythrocytes than normal spleen cells and spherules. Cellular and biochemical events in this phenomenon were investigated to ascertain its significance. Kinetic studies suggested that hemolysis results from the activation of some immune cells by spherules. The capacity of spherules to activate these cells is rather unusual because, of the inert particles tested, only zymosan A and crude chitin demonstrated comparable activity. Furthermore, although the hemolytic phenomenon occurred in serum-free medium, more lysis was effected by immune cells and opsonized spherules or zymosan A than by immune cells and untreated fungal particles. Sheep, chicken, and human erythrocytes were not lysed in the hemolytic phenomenon; however, hemoglobin in chicken and sheep erythrocytes was oxidized. Both the murine erythrocyte lysis and oxidation of ovine hemoglobin correlated with the reduction of Nitro Blue Tetrazolium by immune cells adherent to spherules, and both phenomena appeared to be mediated by H2O2 released into the medium by activated cells. Spleen cells reactive with spherules could not be depleted by treatment with iron carbonyl, antiimmunoglobulin plus complement, or anti-brain-associated theta plus complement, but they were partially or completely depleted after rosette formation with erythrocytes coated with antibody or murine complement. Using light and electron microscopy, we noted that immune spleens contained more neutrophils than normal spleens, that these neutrophils reduced Nitro Blue Tetrazolium after stimulation with phorbol myristate acetate, and that they were the most prevalent cell type adherent to spherules after incubation in vitro.

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Antibody-dependent cell-mediated cytotoxicity (ADCC) exhibited by peripheral blood leukocytes was used as a criterion for assessing immune competence of groups of noncancerous individuals and of treated and untreated cancer patients. The results show that, as a group, leukocytes of nontreated cancer patients exhibited significantly lower ADCC than that exhibited by leukocytes of noncancerous individuals. However, the ADCC of leukocytes from cancer patients under treatment approximated that of normals. Thus, using ADCC as criterion, the results indicate that as a group, cancer patients under treatment tend to exhibit restored immune competence.

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This communication describes an in vitro system wherein mouse erythrocytes are lysed in the presence of spherules of the fungus Coccidioides immitis and spleen cells from syngeneic mice immunized with a variety of antigens. The antigens include: tobacco mosaic virus in complete Freund's adjuvant (CFA), CFA alone, separate components of CFA, sheep erythrocytes, and allogeneic tumor. Spleen cells from mice sublethally infected with C. immitis are also capable of participating in this response. The lytic phenomenon, which does not require complement, is dependent upon the number of spleen cells per culture, the number of spherules per culture, the time of culture incubation, the amount of antigen injected into the animal and the time after immunization at which spleen cells are recovered. Live spherules or spherules killed with heat, with dimethylsulfoxide, or with formalin were effective participants, together with immune spleen cells, in the lytic reaction.

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