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Immune responses in vitro. I. Cellular requirements for the immune response by nonprimed and primed spleen cells in vitro.

A cell suspension culture system combined with a procedure which separates most macrophages from lymphoid cells was used to investigate some of the cellular requirements for direct and indirect plaque-forming cell responses by nonprimed and primed mouse spleen cells in vitro. The plaque-forming cell response to heterologous erythrocytes in cultures of nonprimed spleen cells required both macrophages and lymphoid cells for its development. A significant indirect plaque-forming cell response did not develop in cultures of nonprimed spleen cells. In contrast, cultures of separated or macrophage-poor lymphoid cells from primed mice exhibited increasing responses relative to the response of unseparated spleen cells as the interval after priming increased. The cultures of separated lymphoid cells were not entirely free of phagocytic cells. Despite some evidence which suggests that these phagocytic cells had little function in the response, one cannot ascertain whether the lymphoid cells were responding directly to a second contact with antigen or whether the few contaminating phagocytic cells were performing a function essential to the response by the lymphoid cells. Physiologically different populations of cells appear to develop after priming and are able to respond in vitro in a macrophage-poor culture. Some of the properties of these populations suggest that they are "memory cell" pools containing precursors of direct and indirect plaque-forming cells highly susceptible to a second antigenic stimulus.

Animals↗

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro.

The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.

Animals↗

Spontaneously induced suppressor cells in vitro: nonspecific suppression of in vitro antibody formation.

Nonspecific suppressor cells were induced during in vitro culture of normal mouse spleen cells (SPC) using the Marbrook culture system. The suppressor cells inhibited both the primary and secondary antibody-formation responses antigen nonspecifically in vitro, and both IgM- and IgG-responses were inhibited. The supernatants from suppressive precultured cells were not suppressive. The suppressor cells also inhibited the response of allogeneic SPC beyond H-2 compatibility. The induction of the suppressor cells did not require the presence of antigen but required fetal calf serum (FCS) or both FCS and 2-mercaptoethanol (2-ME). The suppressor cells were generated from the nylon-wool adherent, radiation-sensitive T cell population. On the other hand, the suppressor cells were nylon-wool nonadherent, relatively radiation-sensitive T cells. Actively antibody-producing cells were not affected by the suppressor cells. The suppressor cells inhibited the mitogenic responses of normal SPC to phytohemagglutinin-P (PHA), bacterial lipopolysaccharide (LPS) and concanavalin A (Con A). The suppressor cells themselves inhibited the growth of EL4 cells (T-cell leukemia of C57BL/6 mouse origin) and MOPCll cells (B cells, plasmacytoma of BALB/c mouse origin) even at a low effector-to target cell ratio (E:T ratio = 1:1), but did not kill these tumor cells. These results indicate that the target cells of the suppressor cells are both T and B cells, and that the mechanism of action of the suppression is either inhibition of proliferation or inhibition of early events in the course of the immune response.

Animals↗

In vitro antiretroviral activity and in vitro toxicity profile of SPD754, a new deoxycytidine nucleoside reverse transcriptase inhibitor for treatment of human immunodeficiency virus infection.

SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 microM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.

Anti-HIV Agents↗

Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

Alcohol Dehydrogenase↗

In vitro susceptibility to pentavalent antimony in Leishmania infantum strains is not modified during in vitro or in vivo passages but is modified after host treatment with meglumine antimoniate.

BACKGROUND: Leishmaniasis is a common parasitic disease in Southern Europe, caused by Leishmania infantum. The failures of current treatment with pentavalent antimonials are partially attributable to the emergence of antimony-resistant Leishmania strains. This study analyses the in vitro susceptibility to pentavalent antimony of intracellular amastigotes from a range of L. infantum strains, derived from the same infected animal, during in vitro and in vivo passages and after host treatment with meglumine antimoniate. RESULTS: SbV-IC50 values for strains from two distinct isolates from the same host and one stock after two years of culture in NNN medium and posterior passage to hamster were similar (5.0 +/- 0.2; 4.9 +/- 0.2 and 4.4 +/- 0.1 mgSbV/L, respectively). In contrast, a significant difference (P < 0.01, t test) was observed between the mean SbV-IC50 values in the stocks obtained before and after treatment of hosts with meglumine antimoniate (4.7 +/- 0.4 mgSbV/L vs. 7.7 +/- 1.5 mgSbV/L). Drug-resistance after drug pressure in experimentally infected dogs increased over repeated drug administration (6.4 +/- 0.5 mgSbV/L after first treatment vs. 8.6 +/- 1.4 mgSbV/L after the second) (P < 0.01, t test). CONCLUSIONS: These results confirm previous observations on strains from Leishmania/HIV co-infected patients and indicate the effect of the increasing use of antimony derivatives for treatment of canine leishmaniasis in endemic areas on the emergence of Leishmania antimony-resistant strains.

Animals↗

Reactivity of blood with biomaterials in vitro: evaluation of some in vitro test systems for estimation of the blood compatibility of biomaterials.

Several different in vitro tests designed to estimate the degree of compatibility of blood with biomaterials have been examined to determine possible effects of donor or donor-material interaction. Statistical evaluation of data was performed, and adequate data were collected with each different test system to render such evaluation meaningful. Isolated examples of donor and donor-material interaction were detected, but these proved to be minor or insignificant in each case. Data are presented for each of the in vitro test systems examined to indicate the number of replicate determinations required for observed differences to be significant at the 5% level.

Biocompatible Materials↗

In vitro ruminal disappearance of fumonisin B1 and its effects on in vitro dry matter disappearance.

Fumonisins were produced by inoculating corn with Fusarium moniliforme M-1325 and incubating for 5 w. Fumonisin B1 (FB1) concentration determined by high performance liquid chromatography was 5,087 ppm. Ruminal fluid inoculum obtained from 2 ruminally cannulated steers and tubes containing 0, 50 or 100 ppm of FB1 were incubated in vitro with ruminal fluid and artificial saliva for 72 h in a 39 C oscillating incubator. Supernatant was analyzed for FB1 concentration, and in vitro dry matter disappearance (IVDMD) was calculated in the remaining precipitates. There was minimal degradation of FB1 by ruminal microbes (about 10%) irrespective of concentration of FB1 used when incubated for the 72 h. No differences in IVDMD rates, were found between treatments, and the rates were normal, indicating that microbial efficiency was unaffected by the presence of FB1 at diet concentrations up to 100 ppm.

Animal Feed↗

[Studies on the in vitro formation of antibodies. III. Induction of primary and secondary immune response in vitro].

Peritoneal cells from normal, unimmunized mice (female NMRI, 28-32 gr) produced in vitro primary and secondary immune response after induction with the bacteriophage T2 6 hours or 7 day resp. after establishing the cultures. We confirmed the induction of a primary and secondary immunological response in vitro in the very same culture by the following data: 1. In vivo the donor animals were not in contact with the antigen used. We found neither the phage nor its host E. coli B in the gut of 97 mice investigated and no humoral antibodies against T2. The kinetics of humoral antibody production in vivo by different doses of T2 also showed that there are no related or identical antigen structures incorporated in our animals. 2. The T2 neutralizing activity in the culture medium after the first induction had the sedimentation constant of 19.7 +/- 2.3 S (n = 9) but the activity found after the second induction sedimented with 8.1 +/- 0.7 S (n = 10). 3. The primary activity was more sensitive to mercaptoethanol than the secondary. 4. Complement was bound by the complex T2 + neutralizing activity.

Animals↗

[Formulation and in vitro investigation of antibacterial vaginal suppositories. Part 2. In vitro membrane diffusion and microbiologic studies].

After the physical parameters had been determined, the in vitro drug liberation from vaginal suppositories containing 100 mg of antibacterial agent (sulphadimine, chloramphenicol, gentamicin-sulphate) was studied by membrane diffusion and microbiological methods. Among the vehicles available in Hungary the hydrophylic Massa macrogoli was found to be the best for this purpose. Among the lipophilic bases the in vitro drug liberation of the French Suppocire NA product was significantly better (p < 0.05) compared to the other lipophilic bases. This vehicle is recommended by the authors for the topical treatment of vaginitis, as these suppositories have the further advantage that they can easily be produced on a magistral, galenical or industrial scale as well. In the first part of the publication the formulation and some important physical parameters of lipophilic and hydrophilic antibacterial suppositories for vaginal use were described. In the present paper the drug liberation ability of the compositions with proper physical parameters was studied. The published results were obtained from measurements performed 1 week after formulation.

Anti-Infective Agents, Local↗

The use of image analysis to evaluate the development of uterine and oviduct epithelial cells during in vitro culture. A potential quality assurance procedure for in vitro laboratories.

The objective of this study was to establish selection criteria for morphologic assessment of cell quality using a computer image-analysis system. Uterine and oviduct epithelial cells were isolated from the reproductive tracts of cyclic cows using a trypsin solution. Harvested cells were cultured in Tissue Culture Medium-199 with 10% fetal bovine serum and maintained at 37 degrees C with 5% carbon dioxide in air. Subcultures of different confluent monolayers were established using a weak trypsin solution. Morphologic assessment of cell integrity and viability were made visually, with electron microscopy and image analysis. After morphologic assessment, cells were assigned quality scores. Evaluations were conducted on cells during primary culture and following the first and third subpassages. Image-analysis evaluation was conducted on cells using a silicon-intensified target camera and computer-based software. Gray-level density was determined on cells from each flask. There was no difference (P greater than .05) between sample replicates, indicating repeatable measurements were obtained with the image-analysis system. In addition, there was no significant difference in parameters measured between uterine and oviduct cells; therefore, data from both cell types were combined for further statistical comparisons. Gray-level density values for combined uterine and oviduct cells during primary culture and following the first and third subpassages were as follows: 139.7, 154.5, and 173.3, respectively. There was an increase (P less than .05) in gray-level density values with each subpassage of the cell populations. Furthermore, gray level was influenced (P less than .01) by cell quality for combined uterine and oviduct cell populations. These results indicate that epithelial cell quality during in vitro culture may be effectively determined using image analysis. This approach should not be overlooked in establishing quality control measures for uterine and oviduct cells before mammalian embryos are cocultured in vitro.

Animals↗

In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency.

A method for obtaining large numbers of Giardia lamblia cysts in vitro was developed based on modification of earlier methods of in vitro encystation. Maximal numbers of cysts were obtained by growing trophozoites to confluence in TYI-S-33 growth medium containing 0.5 mg/ml of bovine bile, followed by incubation in medium containing 10 mg/ml of bovine bile, at pH 7.8 for 96 h at 37 C. Up to 4 x 10(5) cysts were obtained per milliliter of encystation medium. Cysts thus obtained were similar in structure to those in vivo, were resistant to hypotonic lysis, and reacted with a cyst-specific monoclonal antibody. Further modification of this method by returning the trophozoites to growth medium after 24 hr of exposure to encystation medium resulted in production of cysts that were shown to be viable by fluorogenic dye staining and ability to excyst. This method was scaled up using roller bottles, which resulted in production of up to 1.6 x 10(8) cysts per roller bottle. In addition, of 4 strains tested, the LT strain yielded the highest number of cysts. Of 4 clones of the WB strain, clone A consistently produced the largest number of cysts.

Animals↗

Effects of in vivo and in vitro exposure to bromocriptine on testicular LH receptors and in vitro testosterone production in Syrian hamsters.

The effects of in vivo and in vitro exposure to bromocriptine (CB-154) were studied in testes of Syrian hamsters. In animals treated for two days with CB-154, a decrease in LH receptors (LH-R) was observed, with a greater decrease being measured in hamsters treated for 14 days, when compared with controls. Injection of HCG caused, in hamsters treated with CB-154 for 14 days, up-regulation of LH-R and increased testosterone synthesis in response to HCG administration in vitro. These changes were not observed in the two other groups of animals. When testis fragments were incubated with CB-154, those incubated with a large dose (10 micrograms/ml) had a normal pattern of response to HCG, and those incubated with a small dose (1 ng/ml) had a smaller maximum response. These actions are similar to those observed in men treated with CB-154. It can be therefore concluded that: a) CB-154 has a direct effect on the testes; b) it probably is through modulation of LH-R synthesis; c) Syrian hamsters probably represent the best model for the study of the effects of CB-154 on the testes; and d) the possibility of using CB-154 as an adjuvant of gonadotropin treatment in hypogonadism has to be considered.

Animals↗

Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro.

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.

Animals↗

In vitro sensitivity of human hematopoietic progenitor cells to hyperthermia: critical temperature for cells to survive and its application to in vitro purging.

Normal tissue toxicity is a major concern in cancer therapy. Since the hyperthermic killing of normal human hematopoietic progenitor cells including multilineage progenitor cells (CFU-mix) other than committed granulocyte-macrophage progenitor cells (CFU-GM) is not known, we have studied the thermal sensitivity of various types of human hematopoietic progenitor cells: early and late erythroid progenitors (CFU-E and BFU-E), CFU-GM and CFU-mix using semisolid clonogenic assays. When these progenitor cells were exposed at 42-44 degrees C for 2 h, their thermal sensitivity always appeared to be in the order of CFU-E greater than BFU-E greater than CFU-GM greater than CFU-mix; the CFU-mix was the most tolerant to heat damage. In addition, the survival of human hematopoietic progenitor cells decreased exponentially in exposure time- and temperature-dependent manner. At 44 degrees C human hematopoietic progenitor cells including CFU-mix dropped dramatically in number and therefore were unable to form colonies in vitro after exposure for 2 h. This suggests that 44 degrees C may be the critical temperature for the CFU-mix to survive in vitro. Thus temperatures selected for hyperthermia used to purge residual leukemic cells in remission marrow grafts should be 43 degrees C or lower.

Cell Survival↗

[Study of in vitro OK432-induced IFN-gamma production in patients with gastric and esophageal cancers: comparison with in vitro skin tests].

In the present study, we measured in vitro production of IFN-gamma induced by OK432 in peripheral mononuclear cells (PMC) and investigated the relationship between the skin reaction to Su-Ps, an extract of Streptococcus Pyogenes, and purified protein derivative (PPD) and the productibility of IFN-gamma in patients with gastric and esophageal cancers. PMC isolated with the Ficoll-conray centrifugation technique were adjusted to 10(6) cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum and incubated over 7 days at 37 degrees C in the presence of OK432 at 0.17 KE/ml in microculture plates. IFN-gamma secreted in the supernatants was measured consecutively during the observation period with radioimmunoassay. The results are summarized as follows. 1. The production of IFN-gamma in PMC from patients with negative skin reaction to Su-Ps and PPD was significantly decreased as compared with the value obtained from patients who were positive in these skin tests (p less than 0.001). 2. In vitro production of IFN-gamma induced by OK432 was significantly correlated with the degree of skin tests to Su-Ps and PPD as well (r = 0.48, r = 0.41, p less than 0.01, respectively). Thus, it is concluded that the assay of IFN-gamma produced in PMC cultures is useful to evaluate the immunological status of patients with cancer.

Bacterial Proteins↗

Studies on the synergism of sulbactam and beta-lactam antibiotics under in vitro conditions and in healthy volunteers after intravenous administration. Antibacterial activity in vitro, compatibility and pharmacokinetics of the drugs in combination.

Sulbactam, a new beta-lactam inhibitor, increased the in vitro activity of cefotaxime, mezlocillin and piperacillin against 803 clinical bacterial isolates. The synergism of sulbactam and these antibiotics was particular marked against 467 beta-lactamase positive strains, both aerobic and anaerobic. In the presence of sulbactam the mean minimal inhibitory concentrations (MICs) of the antibiotics against beta-lactamase positive bacteria were greatly reduced: with cefotaxime by 58%, with mezlocillin by 64% and with piperacillin by 70%. Sulbactam alone at low concentrations inhibited the growth of only a few strains (Neisseria spp., Acinetobacter spp.). The inhibitor proved to be very stable in infusion media under a variety of conditions and was compatible in vitro with 14 different beta-lactam antibiotics. The pharmacokinetics profiles of sulbactam and the antibiotics cefotaxime, mezlocillin and piperacillin were similar after infusion to healthy volunteers. The relevant pharmacokinetic parameters of the single substances were essentially unchanged when administered in combination. The general similarity between the pharmacokinetics of sulbactam and of the beta-lactam antibiotics appears to be an essential precondition for the therapeutic success of such a synergistic combination. Thus the physicochemical and pharmacological properties of sulbactam apparently permit flexible dosage in combination with different penicillins or cephalosporins and sulbactam can be administered as non-fixed combination in the clinical treatment of bacterial infections.

Adult↗

In vitro growth of murine T cells. II. Growth of in vitro sensitized cells cytotoxic for alloantigens.

Growth factors (GM), produced by murine lymphoid cells incubated with Concanavalin A, have been used to grow cytotoxic lymphoid cells in culture. C57BL/6 and DBA/2 lymphoid cells were sensitized against each other in primary, secondary, and tertiary in vitro cultures. These sensitized cells were grown in vitro in GM and retained their cytotoxic properties. Cells grew in culture about 10-fold every 5 to 7 days for over 2 months. Initial growth of cytotoxic cells in GM resulted in marked enhancement of specific cytotoxicity that returned to original levels after subsequent subcultures. After five 10-fold cell culture generations some nonspecific cytotoxicity directed against the responding target cell strain appeared in continuous cultures. This technique for growing large numbers of cytotoxic cells may be of value in the development of adoptive immunotherapies.

Animals↗