Plasmodium berghei: effect of antithymocyte serum on induction of immunity in the mouse.

Swiss mice, immunized against the malaria parasite Plasmodium berghei, were treated with antithymocyte serum (ATS) at various times before and after challenge with the homologous parasite. Strong sensitivity to ATS treatment was observed at the end of the sensitization period, and especially if administered close in time to the challenge inoculum. Sensitivity to ATS treatment was reduced again within a period of 1 to 2 days after challenge. When a breakthrough of parasitemia after ATS treatment was prevented by chemoprophylaxis, a recovery from the state of immunodepression and reestablishment of immunity occurred within a period of 1 to 2 weeks.     

Short-term tests for transplacentally active carcinogens: Induction of sister-chromatid exchanges in foetal brain,lung and blood-forming cells by procarbazine and cyclophosphamide

The induction of sister-chromatid exchanges (SCEs) by cyclophosphamide (CP) and procarbazine (PC) in mouse granulocyte-macrophage precursor cells (GM cells) and erythroblasts from foetal liver, and cells from foetal brain and foetal lung has been measured. Agents were administered in vivo, and cells explanted into BrdUrd-containing medium for 2 cell cycles in vitro (using specific growth-promoting substances where necessary) to determine SCE frequency. Tissue and cell-type differences in responses were observed, and it is concluded that the in vivo/in vitro transplacental SCE technique is a useful indicator of genotoxic effects of agents which are potential transplacental carcinogens.     

Effect of the joint use of BCG and antithymocyte serum on the growth of a syngeneic tumor in mice

The growth of the syngeneic tumor Acatol in BALB/c mice was retarded if the animals were pretreated with BCG or antilymphocyte serum (ATS). Combined use of BCG and ATS led to a significantly more powerful retardation as compared to the effect produced by each factor alone. Using the adoptive transfer of splenocytes from treated mice it was shown that tumor growth suppression is effected by the cell types other than T lymphocytes and macrophages. It is probable that the effector cells within the given system are K cells and natural killer cells. The results attest to a possibility of search for a two-directional action on the immune system of the tumor host, which would stimulate antitumor effector cells and inhibit the activity of suppressors, particularly that of T suppressors.     

Depression of macrophage function by a factor produced by neoplasms: a merchanism for abrogation of immune surveillance.

The possibility that macrophages mediate surveillance against the development of neoplasms has been reciving increasing support. The acquisition, by neoplastic cells, of the capacity to subvert macrophage function may be an important mechanism by which they escape destruction by the host and become established tumors. Indeed, animals implanted with syngeneic neoplasms developed depressed macrophage migratory ability in vivo and chemotactic responsiveness in virto. It therefore seemed plausible that neoplasms might be capable of producing inhibitors of macrophage function. The present report describes the identification of such a low molecular weight (6,000 to 10,000), heat-stable inhibitor of murine macrophage accumulation in vivo and chemotaxis in vitro. The inhibitor of macrophages was present in four different murine neoplasms, but not present in normal liver, spleen, or inflammatory exudate cells and did not affect PMN chemotaxis in vitro. When given with low numbers of neoplastic cells, the inhibitor increased both the frequency of tumor development and rate of tumor growth. By producing inhibitors of macrophage function, neoplasms may escape initial host surveillance mechanisms.     

Studies on the regulation of granulopoiesis. V. Effect of marrow cells on colony stimulating activity in serum

Serum obtained from irradiated mice lost its colony stimulating activity when incubated with normal marrow cells. The loss of activity appeared to be due to absorption or inactivation of the active factor in the serum rather than to production of an inhibitor by the cells.     

Effect of adrenalectomy and corticosterone on cocaine-induced sensitization in rats.

Effects of adrenalectomy (ADX) and corticosterone (CORT) on the development and expression of sensitization to the locomotor effect of cocaine (COC) were studied in rats. Sensitization was evoked by 5 daily injections of COC (10 mg/kg) and measured after a challenge dose of the drug (10 mg/kg) after a 5-day withdrawal (on day 10 of the experiment). ADX, performed before the start of COC administration, completely blocked the manifestation of COC-induced sensitization. In contrast, ADX performed on animals already sensitized to COC did not affect the sensitized locomotor activity response to a challenge dose of COC (on day 18). Pretreatment with CORT, 10 mg/kg, but not 5 mg/kg, before each of the 5 daily COC injections facilitated the development of COC sensitization, tested after a 5-day withdrawal. When pretreated with CORT alone (10 mg/kg), the challenge dose of COC administered on day 10 induced cross-sensitization to CORT. CORT (10 mg/kg) injected acutely before COC on day 10, potentiated the expression of COC sensitization. When given alone, on day 10 CORT (5-10 mg/kg) induced an increase in the locomotor activity of rats pretreated daily (5 injections) with COC. No drug treatment induced conditioned locomotion, as measured after saline challenge on day 8. Our results indicate that CORT facilitates the development and expression of COC sensitization, while ADX blocks the initiation of the behavioral phenomenon only. Moreover, there takes place cross-sensitization between CORT and COC, which indicates a close relationship between the drug-related mechanism and behavioral sensitization.     

Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts

The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.     

Use of enzyme-immunoassay for oestradiol-17beta and progesterone quantification in canine serum.

The objective of this investigation was to develop and evaluate competitive inhibition-enzyme-immunoassays for canine serum oestradiol-17beta (E(2)) and progesterone (P(4)) quantification. Sera from 56 healthy bitches at various stages of oestrus cycle and pregnancy were tested. For E(2) measurement, each sample (0.4 ml) was extracted with diethyl ether and after solvent evaporation the resultant hormone was reconstituted to one-fifth of the original sample volume in aqueous buffer. Each reconstitute (30 microl) was assayed for E(2) to estimate respective serum concentration. For P(4), each sample (10 microl) was directly assayed without extraction. The classic cyclic hormonal pattern during the oestrus cycle of the bitch was observed. The brief, sharp dominance of E(2) during the follicular phase was followed by the long-lasting dominance of P(4) during the luteal phase (late oestrus, dioestrus or pregnancy). During the anoestrus phase both hormones were found at basal levels, with the exception of E(2) during late anoestrus which appeared to be rising. Both assays had acceptable specificity (cross-reactions < or =10%), precision (coefficient of variation (C.V.) < 7%) and accuracy (E(2) recovery: 97%; P(4) recovery: 104.7%). The sensitivity of E(2) and P(4) assay was 4 pgml(-1) and 0.28 ngml(-1), respectively.     

Effect of serum and serum lipoproteins on testosterone production by adult rat Leydig cells in vitro.

The effect of serum factors other than luteinizing hormone on Leydig cell testosterone secretion was examined using an in vitro bioassay system based on the stimulation of purified adult rat Leydig cells during a 20 h incubation in the presence of a maximal dose of human chorionic gonadotrophin (hCG). Charcoal-extracted serum and testicular interstitial fluid (IF) from normal adult male rats were separated into lipoprotein and lipoprotein-deficient fractions by density ultracentrifugation. Stimulatory bioactivity was found in the lipoprotein fraction of both serum and IF, although the levels of lipoprotein and corresponding bioactivity recovered from IF were significantly lower (25%) than those of serum. There was no difference between the effects of serum lipoproteins on Leydig cell testosterone production stimulated by either hCG or dibutyryl cAMP. In time-course studies, the serum lipoprotein fraction had no effect on hCG-stimulated testosterone production in vitro at 3.0 or 6.0 h, but partially prevented the normal decline in hCG-stimulated testosterone production after 6.0 h. In contrast, unfractionated serum was stimulatory at all time-points. In the absence of hCG, the lipoprotein fraction was stimulatory at both 6.0 and 20 h, although not at 3.0 h. The lipoprotein-deficient protein fraction of serum had no effect on hCG-stimulated testosterone production alone, but significantly enhanced the bioactivity of the lipoprotein fraction, and caused a dose-dependent stimulation of testosterone production in the presence of a constant concentration of serum lipoproteins. Both a stimulatory peak of activity (apparent MW 40-80 kDa), and a large MW (> 100 kDa) inhibitor of testosterone production were identified in serum after fractionation by gel filtration (Sephadex G-100). The data indicate that (i) the stimulatory effect of serum on short-term hCG-stimulated Leydig cell testosterone production in vitro is predominantly due to the serum lipoprotein fraction, possibly by providing additional precursors for testosterone synthesis, (ii) the biological activity of the lipoproteins is influenced by both stimulatory and inhibitory serum proteins in addition to luteinizing hormone, and (iii) that serum lipoproteins may be involved in supporting Leydig cell steroidogenesis in vivo.     

Effect of storage on sperm membrane integrity and other functional characteristics of canine spermatozoa: In vitro bioassay for canine semen

The effect of storage of canine semen on sperm membrane integrity, as determined by the hypoosmotic swelling test, and on other functional characteristics of the canine spermatozoa was evaluated by established procedures. The results of this study indicated that storage of canine semen at a chilling temperature of 5 degrees C for 24 h did not significantly impair the physical and functional characteristics of the canine spermatozoa. The overall mean percentage of motility, hypo-osmotic swelling response, which assessed sperm membrane integrity, acrosome-reacted spermatozoa, acrosomal defects, and the percentage of live spermatozoa, did not significantly differ between the fresh and chilled semen samples. However, storage altered the rate of motility and acrosome reaction. The percentage of acrosome reaction in the canine capacitating medium peaked earlier in chilled than in fresh semen. It is probable that storing semen at 5 degrees C initiated/triggered the acrosome reaction. This did not amount to impairment of functional properties. Significant correlations were observed between hypo-osmotic swelling vs motility (r=0.98, P<0.002); hypo-osmotic swelling vs acrosome reaction (r=0.83, P<0.08); and acrosome reaction vs motility (R=0.89, P<0.04) in the fresh semen, and between hypo-osmotic swelling vs motility (r=0.87, P<0.05) and hypo-osmotic swelling vs acrosome reaction (r=0.56, P<0.05) in the chilled semen. It was concluded: that 1) storage of canine semen at 5 degrees C for 24 h did not significantly impair the physical and functional integrity of the spermatozoa; 2) the significant association between motility or acrosome reaction vs hypo-osmotic swelling indicates their value in assessing sperm viability; and 3) the hypo-osmotic swelling assay could have predictive value in screening out subfertile males with apparently normal spermiograms.     

Immunosuppression by procarbazine. I. Sites of action of the drug and effect on adjuvant arthritis and circulating antibody responses

The effect of procarbazine hydrochloride on adjuvant arthritis and on circulating antibody responses in the rat was studied. Procarbazine had a profoundly depressing effect on both adjuvant arthritis and the circulating antibody response to ovalbumin (OA). In contrast, it had no effect on the immune response to sheep erythrocytes (SRBC) alone; the presence of adjuvant, however, enhanced the response to SRBC, and this enhancement was suppressed by procarbazine. Preinjection with adjuvant enhanced the response to OA given at a different site: procarbazine treatment at the time of adjuvant injection suppressed this enhancement. These results taken in conjunction with histological observations, reinforce the idea that the initial effect of the drug is predominantly on the thymus and thymus-dependent cell populations, though prolonged treatment may also affect the bone marrow. They also give a clue to the mode of action of adjuvant in enhancing immune responsiveness. It is suggested that judicious use of procarbazine may assist fine dissection of various components of the immune response.     

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.