Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

The onset of T_h1 immunity is in part regulated by genetic background.To elucidate the cell type carrying critical factors determiningthe T_h1 response, we employed Rag-2^–/– mice on Leishmaniamajor-susceptible BALB/c and -resistant B10.D2 backgrounds.By using bone marrow (BM) chimeras generated by the transplantationof B10.D2 BM cells into BALB/c-Rag-2^–/– mice, andvice versa, it was shown that hematopoietic cells carry factorsdetermining the disease outcome and T_h1 response against L.major infection. B10.D2-Rag-2^–/– mice reconstitutedwith BALB/c CD4⁺ T cells exhibited a T_h1 response and controlledL. major infection. Wild-type BALB/c mice inoculated with L.major-parasitized B10.D2-Rag-2^–/– splenocytes alsoexhibited a T_h1 response and a mild disease outcome, whereassuch a T_h1 response was not induced when CD11c⁺ dendritic cells(DCs) were depleted from parasitized B10.D2-Rag-2^–/–splenocytes. T_h1 response was reconstituted by the additionof L. major-parasitized B10.D2 DCs but not L. major-parasitizedBALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/–splenocytes. These results indicate that DCs determine the outcomeof the disease upon L. major infection.     

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures

The continuous rat hepatoma cell line H4IIEC3/G^– and rathepatocyte primary cultures (hpc) were compared with regardto their capacity to metabolize structurally different promutagens.The sensitivities of both activation systems were evaluatedby comparing the induction of SCE in H4IIEC3/G^– cellsthemselves with that in V79 cells co-cultured with hpc. Of thesix chemicals tested, aflatoxui B₁ (AFB1), cyclo-phosphamide,dimethylnitrosamine and nitrosomorpholine (NM) were shown tobe inducers of SCE in H4HEC3/G^– cells as well as in V79cells with hepatocyte activation. 7,12-Dimethylbenzanthracenegave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G^–cells whereas benzo[a]pyrene was negative in both systems. Theseresults suggest that H4IIEC3/G^– cells retain metabolicactivities to convert different indirect mutagens into theiractive forms and clearly indicate the presence of liver specificcytochrome P-450-dependent mono-oxygenases. However, freshlyisolated hepatocytes are more efficient in metabolizing thetest compounds. Although hpc provide only external activation,the V79 cells system appears to be more sensitive for the detectionof promutagens.     

CD4+Thy1- thymocytes with a Th-type 2 cytokine response

We have identified a small subset of CD3⁺, CD4⁺, CD8^–thymocytes that do not express Thy1 (CD90). This Thy1^–subset represents 1–3.7% of the total number of thymocytesin a naive mouse. CD4⁺Thy1^– thymocytes express high levelsof CD3, intermediate to high levels of heat-stable antigen (HSA),and low levels of CD25, CD45RB, CD69, CD44 and CD62L. They producehigh titers of IL-4 and no IFN- upon stimulation in vitro, aresponse characteristic of T_h2 cells. In the thymi of mice infectedneonatally with a high dose of the retrovirus Cas-Br-E MuLV,the frequency of CD4⁺Thy1^– cells increased ~10-fold. High-dosevirus infection resulted in decreased HSA and increased CD44expression on CD4⁺Thy1^– cells relative to cells from naivemice. CD4⁺Thy1^– cells from high-dose infected mice alsosecreted IL-4 and not IFN- upon in vitro stimulation. We previouslyreported that infection of newborn mice with a high dose ofmurine retrovirus results in the induction of a non-protectiveanti-viral T_h2 T cell response; CD4⁺Thy1^– thymocytes witha T_h2-like cytokine profile may play a role in determining thecytokine bias of this anti-viral response.     

Aneuploidy in male Indian muntjac cells is limited to the Y2 chromosome

In untreated cell cultures of Indian muntjac (Munticus muntiacusvaginalis; 2n = 6, 7) we observed that spontaneous aneuploidyis limited primarily to the Y₂ chromosome. We therefore treatedthe cells with aneugenic agents to determine if induced aneuploidyfollows the same pattern and, hence, if there are limitationson the generation of aneuploidy or survival of cells lackingcertain chromosomes. Exposing the cells to benomyl (8–100µg/ml for 1 h), caffeine (5 x 10^–5–2x10^–4for 2, 24 and 72 h) and colchicine (2 x 10^–4 and 5 x 10^–5M for 1, 24, 48 and 72 h) resulted in cells primarily aneuploidfor the Y₂ chromosome. The frequency of cells lacking Y₂ wasfar higher than those having an extra Y₂ chromosome. The frequencyof cells aneuploid for all other chromosomes combined was muchlower than that for Y₂. The data imply that the Indian muntjacgenome can tolerate loss of the Y₂ chromosome only and thataneuploidy for other chromosomes might cause lethality. Thismight be because the small number of chromosomes in the genomepredisposes aneuploid cells to an imbalance of genes carryingout the basic activities required for cell division and cellsurvival. Because of the small chromosome number, the largesize of the chromosomes and the ease of distinguishing everychromosome without banding or any other special treatment, e.g.FISH, this system could be useful and convenient in the studyof induction of aneuploidy. This simple and inexpensive systemcan be utilized as a screening system for preliminary studiesdealing with induced aneuploidy. ¹To whom correspondence should be addressed. Tel: +1 702 784 6544; Fax: +1 702 784 1302; Email: vig{at}med.unr.edu     

Effect of gamma radiation at high- and low-dose rate on a novel in vivo mutation assay in mouse intestine

We have previously proposed that an in vivo mutagencity assaycould be based on the detection of mutations affecting the Dlb-1locus in the small intestine of C57BL/6J x SWR F₁ mice. F₁ miceare heterozygous Dlb–1^b/Dlb–1^a and have only a singleallele (Dlb–1^b) which specifies expression of the bindingsite for the lectin Dolichos biflorus agglutinin (DBA) in intestinalepithelia. In whole-mount preparations of small intestine stainedwith a DBA-peroxidase conjugate, mutated stem cells and theirprogeny can be recognized as ribbons of unstained cells againsta stained background. These DBA-negative ribbons can be quantified.The present investigation characterizes the responses of F₁mice to high-and low-dose rate gamma radiation (1.8 and 0.01Gy/min respectively) and shows that the induction of ribbonsis dose dependent in both cases. Dose sparing is evident atthe lower dose rate: 20 ribbons/10⁴ villi can be induced by4 Gy at high-dose rate and by 7.1 Gy at low-dose rate, a dosesparing of 1.76. Age-matched untreated mice had a backgroundlevel of 3.7 ribbons/10⁴ villi. As expected, no ribbons wereobserved in homozygous (Dlb–1^b/Dlb–1^b) C67BL/6Jmice, in which each stem cell has two alleles specifying lectinbinding. The results further validate this system as a sensitivein vivo mutagenicity assay and suggests that the target cellsare capable of repairing radiation damage.     

Altered response of CD4+ T cell subsets to Plasmodium chabaudi chabaudi in B cell-deficient mice

Mice depleted of B cells from birth by treatment with anti-_µantibodies can control but not clear an infection with the malariaparasite Plasmodium chabaudi chabaudi (AS). Splenic CD4⁺ T cellsfrom these mice were unable to mount a significant T_h2 responseto the parasite in vitro as shown by much lower precursor frequenciesof T_h cells for antibody production and of IL-4-producing cellscompared with the response of control-treated mice. CD4⁺ T cellsof the anti-_µ-treated mice which respond to antigens ofP. chabaudi chabaudi maintained a T_h1 phenotype throughout primaryinfection, in contrast to control mice in which a sequentialappearance of T_h1 and T_h2 responses was observed. These datashow that T_h1 responses in anti-_µ-treated mice are sufficientto control parasitemia but not to eliminate an infection. Thedata further suggest that depletion of B cells by treatmentwith anti-_µ; antibodies reduces the generation of theT_h2 subset during a primary response to P. chabaudi chabaudi.     

Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells

Cleistanthin B, one of the toxic constituents of Cleistanthuscollinus, was found to be cytotoxic to normal and tumour cells.In comparison with normal cells, tumour cells were sensitiveto lower doses of toxin. The 50 growth inhibition GI₅₀ valuesfor normal cell lines were from 2 x 10^–5 to 4.7x10^–4M and for tumour cells the values ranged from 1.6x10^–6to 4x10^–5 M. Short exposure 30 min of Chinese hamsterovary CHO cells to cleistanthin B at 1–6 µg/ml resultedin extensive chromatid and isochromatid breaks and gaps. Howeverthere was no significant increase in cell death and DNA strandbreaks in cells treated under the above conditions. CleistanthinB induced micronucleus formation in cultured lymphocytes ina dose-dependent manner. CHO cells treated with high doses ofcleistanthin B showed a decrease in cell viability and a concomitantincrease in DNA strand-breaks. The cell death appears to bedue to apoptosis since nucleosome-like ladders were observedin the treated cells when the DNA was electrophorized in agarosegels. ¹To whom correspondence should be addressed     

T cell subpopulation phenotypes in filarial infections: CD27 negativity defines a population greatly enriched for Th2 cells

Three-color flow cytometric analysis was used to define surfacemarkers which identify the T_h2-type CD4⁺ cells responsible forthe eosinophilia and elevated serum IgE typical of tissue invasivehelminth infections. A group of six mAba to well known cellsurface markers were screened for differential expression onCD4⁺ CD45RO⁺ lymphocytes from normal individuals (NL; n = 6)and filaria-infected patients (PT; n = 10). The majority ofmarkers were expressed equally by both groups, but the CD4⁺CD45RO⁺ cells in the PTs showed significantly higher levelsof expression of HLA-DR than those of NLs (P = 0.014). ThisCD4⁺ HLA-DR⁺ subpopulation was then studied further for itsexpression of an additional 10 activation and adhesion molecules.CD27 showed a trend for lower intensities of expression on PTCD4⁺ HLA-DR⁺ cells than on those of NLs. Analysis of the serumfrom both NLs and PTs revealed that PTs had significantly higherlevels of soluble CD27 and CD25 (IL-2R) In the serum than NLs(P < 0.01 and P = 0.022 respectively) indicating a generalstate of Immune activation and differentiation. Functional analysisof the CD4⁺ HLA-DR⁺ and the CD4⁺ CD27^– subpopulatlonsrevealed that the CD4⁺ HLA-DR⁺ cells produced significantlyhigher levels of IL-5 than the CD4⁺ HLA-DR^– cells (P <0.04), and the CD4⁺ CD27^– cells produced significantlyhigher levels of both IL-4 and IL-5 than the CD4⁺ CD27^–cells (P <0.05 and P <0.001 respectively). Thus, whilethe CD4⁺ CD27^– and CD27⁺ subpopulatlons contain T_h1 andT_h0 cells, only the CD4⁺ CD27^– population contains theT_h2 cells (producing both IL-4 and IL-5).     

Detection of 6-thioguanine-resistant human peripheral blood lymphocytes using 5-bromodeoxyuridine labeling in combination with immunocytochemical staining

Measurement of frequencies of 6-thioguanine-resistant (TG^r)human peripheral lymphocytes may contribute to quantitativegenetic risk assessment in occupationally or environmentallyexposed human populations. A simple procedure for the detectionof TG^r human peripheral blood lymphocytes was developed in ourlaboratory, using whole blood culturing and 5-bromodeoxyuridine(BrdU) labeling in combination with immunocytochemical staining.Modifications of the procedure designed to reduce the falsepositive effects of spontaneously cycling lymphocytes (phenocopies),and to optimize duration of BrdU labeling and the culturingperiod, were evaluated. A standard procedure was developed whichapplied 24 h cold storage of the diluted heparinized blood (1:10,v:v in RPMI 1640 medium) at 4°C to reduce the effect ofspontaneously cycling lymphocytes, and whole blood culturingin RPMI 1640 complete medium with stimulation of T lymphocytesusing phytohemagglutinin (PHA), selection of TG^r lymphocytesby adding TG to a final concentration of 2 x 10^–4 M and,after 24 h of incubation, labeling of TG^r lymphocytes with 2.5x 10^–5 M BrdU during 16 h. Using this standard procedure,frequencies of TG^r cells in 45 healthy Individuals (aged 21–64)were observed to range from 0.3 to 229.8 x 10^–6, witha mean variant frequency VF (±SD) of 136 x 10^–6(±35.8). After exclusion of the one extremely high valueof 229.8 x 10^–6, mean VF was 8.7 x 10^–6 (±14.1).A significant inverse correlation was found between logVF andthe labeling index of control cultures (LI_c), indicating thatcultures with low LI_c tend to yield higher VF. No effect ofdonor sex or smoking behavior was observed, but logVF significantlyincreased with age; a 2.4% increase per year was found. Afteradjustment for the effect of LI_c on VF and after exclusion oflymphocyte cultures with LI_c <10% which are considered tobe less reliable, the positive correlation between age and VFstill existed. In addition, an inverse correlation was observedbetween age and LI_c. Even after adjustment for the effects ofLI_c and age, however, no statistically significant effect ofsmoking behavior on VF was found.     

Pregnancy: Activation of protein kinase C is required for oxytocin-induced contractility in human pregnant myometrium

Intracellular mediators regulating the initiation of parturitionare not fully understood. This study was designed to determinethe possible mechanism of oxytocin-induced uterine contractilityduring labour. In-vitro isometric contraction studies were performedwith longitudinal strips of human pregnant myometrium in thepresence and absence of the protein kinase C inhibitors, staurosporineand RO 31–8220, and the tyrosine kinase inhibitor, genistein.Phospholipase D activity was measured by employing the transphosphatidylationreaction. Staurosporine significantly reduced oxytocin-stimulatedcontractile activity with mean activity reduced by >50% followingthe addition of 10^–6M staurosporine (P < 0.01), whileaddition of 10^–5 M resulted in a measured mean contractileactivity of –10% of the control (P < 0.001, n = 5).Similarly, uterine activity was minimal with oxytocin applicationfollowing incubation with RO 31–8220, mean contractileactivity being reduced by -40% by the addition of 10^–7M RO 31–8220 (P < 0.05) and by –87% by the additionof either 10^–6 or 10^–5 M (P < 0.01, n = 3). Conversely,addition of genistein (10^–7 and 10^–6 M) had littleeffect on oxytocin-induced contractions, although at a higherconcentration (10^–5 M) a significant reduction in oxytocin-inducedcontractile activity was observed (P < 0.01). Oxytocin evokedphospholipase D activation in a concentration- and time-dependentmanner in cultured human pregnant myometrial cells (n = 4).These results indicate that activation of protein kinase C andtyrosine kinase are involved in the regulation of oxytocin-mediatedmyometrial contractile activity and that a coupled phospholipaseD/phosphatidate phosphohydrolase pathway may play a role inthe sustained stimulation of myometrial activity during labour.     

Arachidonic acid and its lipoxygenase metabolites stimulate prolactin release in superfused pituitary cells

The direct effect of leukotrienes and other lipoxygenase productson prolactin release has been assessed. Arachidonic acid andits lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoicacid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetraenoic acid(15-HETE) stimulated the release of prolactin in superlusedrat pituitary cells in a dose-dependent manner. Leukotrienes(LT) A₄, B₄, C₄ and E₄ provoked a very marked biphasic and dose-dependentsecretion of prolactin from superfused cells. Maximal effectswere achieved with leukotrienes at a concentration of 3 x 10^–11to 3 x 10^–10 M but LTD₄ did not affect peptide releaseunder these conditions. The metabolites were more potent thanarachidonic acid in affecting hormone secretion. Pukes of 4mioutes duration of these fatty acids may even elicit a morepronounced response thao thyrotrophin-releasing hormone (TRH).Nordihydroguaiaretic acid (NDGA 10^10–6 M), a lipoxygenaseinhibitor, prevented the effect of arachidonic acid on peptidesecretion. Repeated TRH (10^–7 M) administration to pituitarycells led to a reduction in cell response, which may also beobserved in cells pre-treated with pulsatile 5-HETE or 15-HETE.These data support previous findings that arachidonic acid andits lipoxygenase metabolites may play a role in the secretorymechanism of prolactin release in pituitary cells.     

Anergy in vivo: down-regulation of antigen-specific CD4+ Th1 but not Th2 cytokine responses

Efficient immunologic tolerance, defined as antlgen-speclflcunresponslveness, can be peripherally induced by the l.v. Injectionof syngenelc splenocytes coupled with antigen using ethylenecarbodilmlde (ECDI). We have previously reported that unresponslvenessinduced via l.v. Injection of syngenelc splenocytes coupledwith intact, UV-lnactlvated Theiler's murine encephalomyelitisvirus (TMEV-SP) resulted in ‘split tolerance’. Bothvtrus-speclflc delayed-type hypersensltlvlty and lgG2a levelswere inhibited, whereas lgG1 levels were increased when comparedwith sham tolerized controls. In the present report we demonstratethat tolerance induced by l.v. Injection of TMEV-coupled splenocytesresulted in antigen-specific inhibition of T cell proliferation,as well as IL-2 and IFN- production in response to both wholeTMEV and the immunodomlnant viral epitope. Additionally, toleranceinduction resulted in abrogation of T_h1 -derived [IL-2, IFN-and LT/tumor necrosis factor-ß (TNF-ß)]cytokine mRNA expression in response to In vitro stimulationwith UV-inactlvated TMEV as determined by reverse transcrlptasepolymerase chain reaction. In contrast, expression of T_h2-derived(IL-4, IL-6 and IL-10) cytokine mRNA was not affected in tolerizedmice. Tolerance functioned directly at the level of CD4⁺ T_h1cells at both the induction and effector limbs as depletionof CD8⁺ T cells both prior to in vivo tolerizatlon or in vitroculture had no effect on inhibition of T_h1-specific responses.The mechanism of In vivo tolerance induction appeared to beanergy of CD4⁺ T_h1 cells since IL-2, IFN- and LT/TNF-ßmRNA expression as well as virus-specific prollferatlve responsescould be restored by addition of rlL-2 to In vitro culturesof tolerant, CD4⁺ T_h1 populations. These results suggest thatin vivo ‘split tolerance’ Induced by l.v. Injectionof ECDI-flxed, antigen-coupled splenocytes involves anergy ofTMEV-speclflc, CD4⁺ T_h1 lymphocytes and concomitant primingof T_h2 cells. The induction of antlgen-speclflc, in vivo anergyhas important implications in the design of therapeutic strategiesfor immunopathologic diseases mediated by T_h1 lymphocytes, especiallyT cell-mediated autoimmune disorders.     

Role of T-cell-associated lymphocyte function-associated antigen-1 in the pathogenesis of experimental colitis

The ß₂ integrin lymphocyte function-associated antigen-1(LFA-1; CD11a/CD18) is important for lymphocyte traffickingand activation as well as recruitment to sites of tissue inflammation.The objective of this study was to assess the role of ‘T-cell-associated’LFA-1 in the pathogenesis of chronic colitis in vivo. Transferof CD4⁺CD25^– T cells isolated from wild-type (wt) miceinto immunodeficient recipients [recombinase-activating gene-1-deficient(RAG-1^–/–)] produced moderate to severe colitis,whereas RAG-1^–/– mice injected with CD11a-deficient(CD11a^–/–; LFA-1^–/–) donor T cells displayedminimal macroscopic and histological evidence of colitis. Surfaceexpression of L-selectin, ₄, ₄ß₇ and chemokine receptor-7were similar for wt and CD11a^–/– donor T cells.Attenuated disease in the CD11a^–/– RAG-1^–/–animals was associated with decreased numbers of CD4⁺ T cellsin the mesenteric lymph nodes (MLNs), spleen and intestinallamina propria (LP). In addition, significant reductions inT_h1 cytokines were observed following ex vivo stimulation ofmononuclear cells obtained from the MLNs and colonic LP. Interestingly,mononuclear cells obtained from the spleens of CD11a^–/– RAG-1^–/– exhibited enhanced pro-inflammatory cytokineproduction compared with splenocytes obtained from wt RAG-1^–/–colitic mice. Taken together, our data suggest that T-cell-associatedCD11a (LFA-1) expression plays a dual role in the initiationof chronic gut inflammation by facilitating naive T-cell priming/activationand expansion within MLNs and by augmenting pro-inflammatorycytokine production following secondary stimulation by antigen-presentingcells in the colonic interstitium.     

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy

We have demonstrated the presence of epidermal growth factor(EGF) and its receptors in human non-gestational corpora lutea.To determine further the characteristics of EGF receptor binding,we examined 30 human corpora lutea throughout the luteal phaseand during pregnancy. Scatchard plots of EGF binding in 29 ofthe 30 corpora lutea were curvilinear, suggesting negative co-operativity.The mean ± SE of the association constant K_a was (0.9± 0.2) x 10⁹ 1/mol, the dissociation constant K_d was(2.2 ± 0.3) x10^–9 mol/1 and the number of bindingsites (Rt) was (15.8 ± 2.1) x10^–19 mol/µgprotein for non-gestational corpora lutea. The K_d increasedsignificantly in late pregnancies compared to early pregnancies(P = < 0.005), while Rt was significantly higher in termpregnancies than in either early pregnancy (P < 0.01) orthe menstrual cycle (P < 0.001). Corpora lutea atretica (n= 2) and ovarian stroma (n = 6) did not show any EGF bindingactivity. Our findings demonstrate the presence of specificEGF receptors in human corpora lutea of both the menstrual cycleand pregnancy. The changes in EGF binding parameters in earlypregnancy suggest that there may be a relationship between therole of EGF and ovarian steroidogenesis.     

Differential effects of cordycepin on the induction of sister-chromatid exchange and chromatid breaks in BALB/Mo mouse lymphocytes treated with mitomycin C

BALB/Mo mice lymphocytes, carrying endogenous Moloney murineleukaemia virus (M-MuLV), show significantly higher in vitrobaseline frequencies of sister chromatid exchange (SCE) thanlymphocytes from control (M-MuLV free) BALB/c mice. In vitrotreatment of lymphocytes with cordycepin (10 µg/ml), anantiviral substance, lowers the level of SCE in BALB/Mo cellsto the same value of BALB/c cells. The drug also reduces thehigher sensitivity of BALB/Mo compared to BALB/c lymphocytesto the induction of SCE by mitomycin C (MMC) administered eitherin vitro (3 x 10^–8, 10^–7 M) or in vivo (0.3, 3 mg/kg).BALB/Mo lymphocytes treated in vivo with a high dose of MMC(10 mg/kg) show reduced susceptibility to the induction of SCEbut increased frequencies of chromatid breaks and micronuclei.In this situation, cordycepin increases the level of SCE inBALB/Mo lymphocytes to exactly the level seen in BALB/c cells,but it does not affect the frequency of chromosomal aberrations.Since cordycepin is known to inhibit poly(A) synthesis, thusblocking RNA maturation, it is suggested that M-MuLV proviralintegration is not the sole factor responsible for the alteredsusceptibility of BALB/Mo lymphocytes to SCE induction, butthat it is most likely viral gene expression that is neededfor this effect to occur. On the contrary, the high susceptibilityof BALB/Mo lymphocytes to the induction of chromatid aberrationsby a high dose of MMC administered in vivo seems to be independentof viral maturation. ²To whom correspondence should be addressed     

A deficiency for nucleotide excision repair strongly potentiates the mutagenic effectiveness of methyl bromide in Drosophila

A DNA repair assay measuring hypermutability response in theabsence of nucleotide excision repair (NER) was employed tostudy the impact of a deficiency in NER on the induction offorward mutations (X-chromosomal recessive lethals) by methylbromide (MeBr) in Drosophila melanogaster. Postmeiotic malegerm-cell stages reacted with MeBr were introduced in eitherNER competent oocytes (exr⁺) or in cells from the NER^–strain mus-201. The high average M_exr^–/M_exr⁺ hypemutabilityratio of 8.3 determined for MeBr is similar to the M_exr^–/M_exr⁺indices found for other monofunctional alkylating agents withhigh Swain—Scott s values, such as methyl methanesulphonateand dimethyl sulphate. It is concluded that the genotoxic profileof methyl bromide is in keeping with those of high s-value alkylatingagents but it seems incompatible with a methylating agent producingsubstantial amounts of O⁶methylguanine. ¹To whom correspondence should be addressed     

Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge

DNA vaccination offers the advantages of viral gene expressionwithin host cells without the risks of infectious virus. Likeviral vaccines, DNA vaccines encoding internal influenza virusproteins can induce immunity to conserved epitopes and so maydefend the host against a broad range of viral variants. CD8⁺cytotoxic T lymphocytes (CTL) have been described as essentialeffectors in protection by influenza nucleoprotein (NP), althougha lesser role of CD4⁺ cells has been reported. We immunizedmice with plasmids encoding influenza virus NP and matrix (M).NP + M DNA allowed B6 mice to survive otherwise lethal challengeinfection, but did not protect B6-ß₂m(–/–)mice defective in CD8⁺ CTL. However, this does not prove CTLare required, because ß₂m(–/–) mice have multipleimmune abnormalities. We used acute T cell depletion in vivoto identify effectors critical for defense against challengeinfection. Since lung lymphocytes are relevant to virus clearance,surface phenotypes and cytolytic activity of lung lymphocyteswere analyzed in depleted animals, along with lethal challengestudies. Depletion of either CD4⁺ or CD8⁺ T cells in NP + MDNA-immunized BALB/c mice during the challenge period did notsignificantly decrease survival, while simultaneous depletionof CD4⁺ and CD8⁺ cells or depletion of all CD90⁺ cells completelyabrogated survival. We conclude that T cell immunity inducedby NP + M DNA vaccination is responsible for immune defense,but CD8⁺ T cells are not essential in the active response tothis vaccination. Either CD4⁺ or CD8⁺ T cells can promote survivaland recovery in the absence of the other subset.     

Concentration-dependent effects of a selective estrogen receptor modulator raloxifene on proliferation and apoptosis in human uterine leiomyoma cells cultured in vitro

BACKGROUND: This study was conducted to elucidate the effects of raloxifeneon proliferation and apoptosis in cultured human uterine leiomyomacells. METHODS: The monolayer cultures were treated with graded concentrations(10^–9, 10^–8 and 10^–7 M) of raloxifeneand 10^–7 M 17-estradiol (E₂). Cell viability, percentageof proliferating cell nuclear antigen (PCNA)-positive cells,percentage of terminal deoxynucleotidyl transferase-mediated2'-deoxyuridine 5'-triphosphate nick-end labelling (TUNEL)-positivecells and the expression of PCNA and Bcl-2 proteins were assessedby 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl)-2H-tetrazolium assay, immunocytochemistry, TUNEL assay and western blotanalysis, respectively. RESULTS: Compared with untreated cultures, the number of viable culturedcells, percentage of PCNA-positive cells and PCNA protein expressionwere significantly decreased by treatment with 10^–9 Mraloxifene, but increased by treatment with either 10^–8 Mor 10^–7 M raloxifene. In contrast, the percentageof TUNEL-positive cells was significantly increased and Bcl-2protein expression was significantly decreased by treatmentwith 10^–9 M raloxifene, whereas they were not affectedby treatment with either 10^–8 or 10^–7 M raloxifene. CONCLUSIONS: In cultured leiomyoma cells, low concentration (10^–9 M)of raloxifene may inhibit the growth of leiomyoma cells, whereashigh concentrations (10^–8 M, 10^–7 M) ofraloxifene may promote their growth.     

Metabolic activation of the genotoxic environmental contaminants 2- and 3-nitrofluoranthene in variants of Salmonella typhimurium TA98

The mutagenic environmental pollutants 2-nitrofluoran-thene(2-NFA) and 3-nitrofluoranthene (3-NFA), labelled with ³H and¹⁴C respectively, were incubated with Salmonella typhimuriumstrain TA98, its nitroreductase-deficient variant TA98NR andits 0-acetytransferase-deficient variant TA98/1,8-DNP₆, to investigatethe activity of these metabolic pathways under conditions approximatingthose of the Ames assay, hence their contribution to mutagenicpotency. 2-Aminofluoranthene (2-AFA) was the major metaboliteof 2-NFA (4 µM) in all three TA98 variants, isolated byreverse-phase HPLC and identified by UV-vis and NMR spectroscopyand mass spectrometry. 2-AFA was formed more slowly in TA98NR(65 pmol/h/ml resting phase bacterial broth, 1 to 2x10⁹ bacteria/ml)than in TA98 (295 pmol/h/ml) or TA98/1,8-DNP₆ (82 pmol/h/ml).2-Acetamidofluoranthene (2-AAFA) was also identified in incubationswith TA98 (80 pmol/h/ml), TA98NR (21 pmol/ h/ml), and TA98/1,8-DNP6(8 pmol/h/ml). 3-Aminofluoran-thene (3-AFA, confirmed by UV-visand NMR spectroscopy and mass spectrometry) was formed by allthree variants from 3-NFA (4 µM): TA98, 1.76 nmol/h/ml;TA98NR, 0.55 nmol/h/ml; TA98/1,8-DNP₆, 2.93 nmol/h/ml. 3-Acetamidofluoranthene(3-AAFA) was not detected in any of the variants. 3-AFA and3-AAFA were less mutagenic than 3-NFA, and required S9 for activation.Mutagenicity of 3-NFA relative to initial nitroreduction ratewas similar in TA98 and in TA98NR, but almost 10-fold lowerin TA98/ 1,8-DNP₆; hence 0-acetylation considerably enhancesthe mutagenicity of reduction products of 3-NFA. Mutagenicityof 2-NFA relative to initial nitroreduction rate was similarin TA98 and in TA98/1,8-DNP₆; the bacterial genotoxicity of2-NFA is therefore largely independent of O-acetyltrans-feraseactivity. Ratios of mutagenicity to nitroreduction rate weresimilar in TA98 for 2-NFA and 3-NFA; differences in the potencyof these isomers arise primarily from their respective suitabilitiesas substrates for nitroreductase enzymes. ⁴whom correspondence should be addressed     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.