Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Sex-dependent effects of non-H-2 loci on lethal GVH reaction induced across an H-2 barrier

We have studied the influence of DBA/2 non-H-2 antigens on the lethal graft-versus-host reaction (GVHR) developed across an H-2 barrier. (DBA/2 x B10.D2)F₁ x B10.D2 (H-2 ^d) backcross (BC) mice were typed for their allelic constitution at nine genetically independent chromosome markers and used as individual cell donors simultaneously for two to three (DBA/2 X B10.D2)F₁ recipients incompatible for DBA/2 non-H-2 antigens alone and two to three (DBA/2 x B10.BR)F₁ recipients incompatible for DBA/2 non-H-2 antigens and H-2^k. The results showed that, when compared with that developed in a control group incompatible for H-2 ^kalone [B10.D2(B10.D2xB10.BR)F₁], the GVHR mortality seen in the presence of an additional incompatibility for DBA/2 non-H-2 antigens [(DBA/2 X B10.BR)F₁recipients] is significantly delayed but only in female mice. An analysis of individual BC donors indicated that this protective effect of DBA/2 non-H-2 antigens correlates with incompatibility for gene(s) linked to the Pgm-1 chromosome marker. In contrast, incompatibility for gene(s) linked to Mod-1 and Es-3 markers accelerates GVHR mortality, but only in male mice. Finally, the results obtained with (DBA/2 x B10.D2)F₁ and (DBA/2 x B10.BR)F₁ recipients were compared; they showed that the intensity of the GVHR developed by cells from individual BC donors against a given set of DBA/2 non-H-2 antigens correlates well with that developed by the same BC donor against the same set of non-H-2 antigens plus H-2^k. We conclude that certain non-H-2 genes (and antigens) can modulate the intensity of the GVHR developed across an H-2 barrier. The number of such genes is probably great; their effects are strong and complex, and can be sex-dependent.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice     

Genetic control of mitogen-induced B-cell hyperproliferation in SM/J mice

Previous studies have shown that B cells from SM/J mice exhibit hyperproliferative responsiveness to several bacterial-derived B-cell mitogens. This hyperresponse trait was found to be under autosomal, polygenic control by non-H-2 genes. We have now estimated the number of genes involved by statistical analysis of the proliferative responses of splenocytes from SM/J and low-responder C57BL/6J strains, and progeny from the (B6 × SM)F₁, F₂ and (F₁ × B6) crosses. The number of loci involved was ascertained using two different statistical approaches. An estimate of two loci was determined using chi-squared statistics. The second approach, based on an additive model in the natural log scale, also pointed to a lower bound of two genes. We conclude that the hyperresponse to B-cell mitogens in SM/J mice is determined by two autosomal genes which are not linked to the H-2 major histocompatibility complex.Abbreviations used in this paper LPS a bacterial lipopolysaccharide - AVIS a mitogenic preparation from Actinomyces viscosus - B6 C57BL/6J mice - ¹²⁵IUdR ¹²⁵Iodo-deoxyuridine     

Interacting genetic factors controlling the antibody response against the H-2.2 specificity

The antibody response against the H-2.2 specificity has been studied in three H-2 ^d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F₁ and (B10.D2 × BALB/c)F₁. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 ^d /H-2 ^k hybrids (B10.BR × B10.D2)F₁ or (B10.BR × DBA/2)F₁ are responders when immunized with C57BL/10 (H-2 ^b ) but not with B10.A(2R) (H-2 ^h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.     

Influence of the H-2 u haplotype on immune function in F1 hybrid mice

Recently we reported that antigen-primed T cells from (H-2 ^u × H-2 ^s)F₁ and (H-2 ^u × H-2 ^q)F₁ mice responded poorly in vitro to antigen in the context of antigen-presenting cells of the non-H-2 ^u parent. It was suggested that this effect might be due to unbalanced expression of parental antigens in the F₁ hybrid with the result that the non-H-2^u A antigens were greatly reduced or absent in these mice. If this were the case, non-H-2^u Ia-A cells might be expected to stimulate a mixed lymphocyte reaction (MLR) when cultured with Fl responder cells. When tested, (SJL × PL)F₁ responder cells reacted strongly to SJL stimulator cells. There was no significant reaction to PL stimulator cells. The use of major histocompatibility complex (MHC) congenic mice showed the stimulatory antigens to be associated with the MHC. The MLR could be blocked significantly by monoclonal A-specific antibody of the appropriate specificity. When a monoclonal antibody reactivewith a private epitope associated with A^s was used to probe for the presence of A^s on the surface of (SJL × PL)F₁ spleen cells, no antigen could be detected, indicating loss or alteration of this antigen. These findings suggest that an alteration of the expression of the parental A^s molecule may be responsible for this phenomenon.Abbreviations used in this paper APC antigen-presenting cells - BSS balanced salt solution - CTL cytotoxic T lymphocyte - IL-2 interleukin-2 - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - T₂ suppressor T lymphocyte     

Tumour-induced altered gastrointestinal steady-states: absence of MHC restriction in the paraneoplastic gastrointestine

Abstract The growth of a number of experimental rodent tumours including the Lewis lung tumour (LLca) progressively compromises the integrity of the host's gastrointestine by inducing cytokinetic alterations in the small bowel resembling those generally defining the intestinal phase of a graft-versus-host reaction (GVHR). To determine whether the induction of this paraneoplastic gastrointestine (PGI) involves, similar to a GVHR, a disparity between the MHC of the donor (LLca tumour) and the recipient (host), PGI development was evaluated in various LLca tumour-bearing murine strains that were either ‘syngeneic’[C57BL/6 and BL/10 (H-2^b)], ‘semisyngeneic’[B6D2F₁ (H-2^bd) and B6C3F₁ (H-2^bk)] or ‘allogeneic’[C3H/HeJ (H-2^k) and DBA/2 (H-2^d)] to the H-2^b LLca tumour. The temporal appearance and magnitude of a PGI developing in either LLca-syngeneic or semi-syngeneic hosts, but not the allogeneic strains, suggested that the mechanism(s) involved in PGI development, like the GVHR, was restricted by the MHC. Subsequent studies using congenic strains [B10.A (H-2^k) and B10.D2/nSn (H-2^d)], however, demonstrated that the mechanism(s) responsible for the PGI was restricted by the non-MHC loci of the C57BL mouse. These observations were supported by the appearance of a LLca-induced PGI in various B10.A congenic strains carrying mutations at the I-A or I-E/I-J loci of the MHC. Not unlike the intestinal phase of a GVHR, development of the PGI required the participation of enhanced mucosal mast cells which were limited in the WCB6F₁ (S1/S1^d) but not the (+/+) murine strains. These observations are discussed in light of the postulated premature migration of immature thymocytes that accompany tumour growth and their ability to non-specifically enhance (or suppress) cell mediated immune reactions in the host.     

Histocompatibility-2 system in wild mice

Six semicongenic lines carrying differentt haplotypes on the background of strain C57BL/10Sn (B10.t strains) and a (B10 ×T/t ⁰) F₁ hybrid were tested against one another in the mixed lymphocyte reaction (MLR) and cell-mediated lymphocytotoxicity (CML) assays. In every instance, the MLR results paralleled those of the CML typing: strain combinations giving a positive result in one assay gave a positive result in the second; combinations in which no response was observed in the MLR assay also failed to kill target cells specifically in the CML assay. Furthermore, the MLR and CML results were concordant with the results of the serological typing of these strains, as reported previously by us. The combined results suggest sharing ofH-2 hyplotypes between B10.t¹² and B10.t³², between B10.t⁶ and B10.t^w1, and between B10.t^w2 and (B10. ×T/t ⁰) F₁. These data support the conclusion, reached in our previous publication, that members of the samet-complementation group, with few exceptions, shareH-2 haplotypes.     

Suppression of murine NK activity induced by Corynebacterium parvum

Summary Formalin-killed Corynebacterium parvum (CP), given at a dose of 0.4–0.7 mg/mouse IV or IP, induced suppressor cells for NK activity in B6C3F₁ mice. The suppressor cells belong to at least two different populations, plastic adherent and nonadherent, and were not depleted by antibodies specific for Thy-1.2, Ia^k, or NK-1.2 surface markers. Administration of p-I:C, an interferon-inducer, to animals 18 h before the assay did not affect the suppressor activity. Hypotonic shock treatment of splenocytes abrogated the in vitro suppressive activity, and subsequent reconstitution of the shock-treated cells with RBC failed to restore the suppressive activity. SJL/J mice, which have low NK activity, exhibited suppressor activity comparable to B6C3F₁ mice following CP treatment, whereas CP-treated BALB/c athymic and euthymic mice showed a lower ability to generate suppressors for NK as compared to B6C3F₁ mice.     

Permeability properties of cell-to-cell channels: Kinetics of fluorescent tracer diffusion through a cell junction

Summary We have analyzed the intracellular and cell-to-cell diffusion kinetics of fluorescent tracers in theChironomus salivary gland. We use this analysis to investigate whether membrane potential-induced changes in junctional permeability are accompanied by changes in cell-to-cell channel selectivity. Tracers of different size and fluorescence wavelength were coinjected into a cell, and the fluorescence was monitored in this cell and an adjacent one. Rate constants,k _j , for cell-to-cell diffusion were derived by compartment model analysis, taking into account (i) cell-to-cell diffusion of the tracers; (ii) their loss from the cells; (iii) their binding (sequestration) to cytoplasmic components; and (iv) their relative mobility to cytoplasm, as determined separately on isolated cells. In cell pairs, we compared a tracer'sk _j with the electrical cell-to-cell conductance,g _j .At cell membrane resting potential, thek _j 's ranged 3.8–9.2×10^–3 sec^–1 for the small carboxyfluorescein (mol wt 376) to about 0.4×10^–3 sec^–1 for a large fluorescein-labeled sugar (mol wt 2327). Cell membrane depolarization reversibly reducedg _j andk _j for a large and a small tracer, all in the same proportion. This suggests that membrane potential controls the number of open channels, rather than their effective pore diameter or selectivity. From the inverse relation between tracer mean diameter and relativek _j we calculate an effective, permeation-limiting diameter of approximately 29 Å for the insect cell-to-cell channel. Intracellular diffusion was faster than cell-to-cell diffusion, and it was not solely dependent on tracer size. Rate constants for intracellular sequestration and loss through nonjunctional membrane were large enough to become rate-limiting for cell-to-cell tracer diffusion at low junctional permeabilities.     

Measurement of Cerebral Glucose Utilization Using Washout After Carotid Injection in the Rat

Abstract: The carotid injection technique, used previously to quantitate the kinetics of blood-brain barrier transport of metabolic substrates, may be modified to analyze the rate of cerebral glucose utilization. A 0.2-ml solution of [¹⁴C]glucose (G_F) and [³H]methylglucose (M), an internal reference, is rapidly injected into the carotid artery, followed by microwave fixation of brain at various times up to 4 min after injection. The brain radioactivity is separated into a fraction containing neutral hexoses (G_F and M) and a fraction containing metabolites of glucose. The G_F/M ratio is related to the rate constant (k₃) of brain glucose utilization by the simple, linear equation: In(G_F/M) = In(G_F°/M°) –k₃t, where G_F°/M°= the brain uptake index of glucose, relative to methylglucose, at 5-15 s after injection, and t= the time after carotid injection, e.g., 1–4 min. It is assumed that (a) the rate of influx due to recirculation of label is minimal during the 4-min circulation period; and (b) the rate constants of glucose efflux (k₂) and methylglucose efflux (k₂*) are identical. Independent estimates of k₂ and k₂* showed these parameters to be identical: k₂= 0.14 + 0.08 min-I; k₂*= 0.14 ± 0.02 min-I. A logarithmic plot of G_F/M ratios versus time was linear (r = 0.99), and was described by the slope k₂= 0.21 ± 0.02 min^?1. Assuming glucose is uniformly distributed in brain, then the glycolytic rate = k₃× brain glucose = (0.21 min^?1) (2.6 μmol g^?1) = 0.55 μmol min^?1 g^?1 for the cortex of the barbiturate-anesthetized rat. These studies provide the basis for a simple method of measurement of regional brain glycolysis that does not require either the use of correction factors, e.g., the lumped constant, or the use of differentially labeled glucose.     

Allostimulatory analysis of a newly-defined and widely-distributed Mls superantigen

We previously noted that Mls^a,c C58/J responder cells proliferated unexpectedly to H-2^k-compatible Mls^a or Mls^c prototypic stimulator cells in a primary mixed lymphocyte reaction. The present investigation was performed to evaluate whether the response of C58/J T cells to these H-2- and Mls-compatible stimulator cells could functionally identify a newly-defined member of the Mls superantigen family through its allostimulatory ability. We observed that C58/J responder cells also proliferated when cultured with H-2-compatible prototypic Mls^null, Mls^b (nonstimulatory), or Mls^a,c splenic stimulator cells. The widely distributed nature of the non-MHC ligand recognized by C58/J T cells is indicated by the finding that 11 of 12 H-2^k inbred mouse strains clearly expressed this specificity. A gradient of stimulatory capacity from low to high across this newly-defined non-MHC difference was detected with splenocytes from these different inbred mouse strains. The Mls^a,c genetic composition of C58/J was confirmed by the observation that crossing C58/J with parental B10.BR (responsive to both Mls^a and Mls^c determinants) generated F₁ progeny that were unresponsive to H-2^k-compatible Mls^a, Mls^c, or Mls^a,c stimulator cells. Like prototypic Mls^a and Mls^c, the non-MHC specificity recognized by C58/J responder cells, termed Mls^f, was particularly sensitive to radiation (versus mitomycin C) treatment of the stimulator cells, was greatly augmented after anti-IgD activation of splenic stimulator cells, was blocked with anti-MHC class II antibody, and was effectively presented by phenotypically normal female but not B cell-defective xid⁺ male CBA/N F₁ stimulator cells. Address correspondence and offprint requests to: J. J. Ryan     

Linkage of isozyme loci in the mouse: Phosphoglucomutase-2 (Pgm-2), mitochondrial NADP malate dehydrogenase (Mod-2), and dipeptidase-1 (Dip-1)

The linkages of the isozyme genes Mod-2, Pgm-2, and Dip-1 have been determined in tests with established linkage group markers among inbred strains of mice. Unique alleles for both Mod-2 and Pgm-2 have been observed in the strain of SM/J. Linkage was determined from backcross progeny of the matings C57BL/6J×(SM/J×C57BL/6J)F₁, (SM/J×SWR/J)F₁×SM/J, and (SM/J×SWR/J)F₁×SJL/J. The gene Mod-2 is on linkage group 1. In a three-point cross of the loci Gpi-1, c, and Mod-2, the c locus was determined to be the middle gene. No double crossovers were observed. Our combined data show the following linkages: Gpi-1 to c, 28.3±3.2%; Gpi-1 to Mod-2, 33.3±3.0%; and c to Mod-2, 4.1±2.8%. The proposed gene order for four markers on LG I is Gpi-1-p-c-Mod-2. The gene Pgm-2 was linked to Gpd-1 (27.0±4.2%) on LGVIII. Two backcrosses segregating for Pgm-2 and b, (SM/J×DBA/2J) F₁×DBA/2J and (SM/J×DBA/2J)F₁×C57BR/cdJ, showed 9.1±4.3% recombination. The proposed gene order on LG VIII is b-Pgm-2-Gpd-1. The genes Pgm-1 and Pgm-2 are not linked (53.4±4.4%). Linkage of the isozyme genes Dip-1 and Id-1 on LG XIII was observed in backcross progeny of the crosses (SJL/J×C57BL/6J)F₁×SJL/J and C57BL/6J×(SM/J×C57BL/6J)F₁. The combined recombination was 23.8±2.8%. Two cases are established where genes whose enzyme products share substrate affinities (Pgm-1 and Pgm-2; Mod-1 and Mod-2) are not linked. Our data generally support the conclusion that functionally or metabolically related isozyme genes are not contiguous on mouse linkage groups.This investigation was supported in part by Public Health Service General Research Support Grant GM-09966 and in part by Public Health Service Training Grant 5T01 HD-00032-07 from the National Institute of Child Health and Human Development, and by Atomic Energy Commission contract AT(30-1)-3671.     

Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: Possible involvement of specific suppressor cell activity

Major histocompatibility complex (MHC)-homozygous Xenopus laevis were rendered tolerant to semi-allogeneic antigens by grafting skins of adult frogs during larval stages (larvally induced tolerance), and this tolerant state was compared with the tolerance induced in early thymectomized frogs by the grafting of semi-allogeneic nonlymphoid thymuses (thymus-reconstituted tolerance). In contrast to a total inability of thymus-reconstituted frogs both to reject skins and to exhibit a mixed leukocyte reaction (MLR) against the semi-allogeneic donor, larvally induced tolerant frogs showed a strong MLR against leukocytes of the tolerizing skin donor (split tolerance). Breakdown of the tolerant state in thymus-reconstituted frogs were easily accomplished by inoculation with syngeneic splenocytes, but this breakdown was extremely difficult to achieve in frogs with larvally induced tolerance. The injection of splenocytes from larvally induced tolerant frogs into normal frogs significantly suppressed semi-allogeneic graft rejection in the latter group; no suppression was obtained when splenocytes from thymus-reconstituted frogs were used. In addition, in the thymectomized frogs, recovery of allograft rejection capacity against the pertinent semi-allogeneic antigens were suppressed by the injection of splenocytes from larvally induced tolerant frogs, with the degree of suppression depending on the splenocyte dose. These results indicate that the larvally induced tolerant state is maintained by specifically induced suppressor cells affecting the in vivo allograft response but not the MLR.     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

Complexation of lithium(I), sodium(I), silver(I) and thallium(I) by 4,7,13,16-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane and 4, 7, 13-trioxa-1,10-diazabicyclo[8.5.5]eicosane in trimethyl phosphate. A potentiometric titration and Li and Na nuclear magnetic resonance study

Complexation of M⁺=Li⁺, Na⁺, Ag⁺ and TI⁺ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C₅) to form the cryptates [M.C211]⁺ and [M.C21C₅]⁺ has been studied in trimethyl phosphate by potentiometric titration and ⁷Li and ²³Na NMR spectroscopy. For [M.C211]⁺ the logarithm of the apparent stability constants, log K (dm³ mol^-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M⁺ =Li⁺, Na⁺, Ag⁺ and TI⁺, respectively; and for [M.C21C₅]⁺ log K (dm³ mol^-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M⁺=Li⁺, Na⁺, Ag⁺ and Tl⁺, respectively. The decomplexation kinetic parameters for [Na.C211]⁺ are: k_d (298.2 K)=6.924±0.50 s^-l, ΔH_d≠=62.2±0.9 kJ mol^-1, and ΔS_d≠= -20.3±2.7 J K^-1 mol^-1; and those for [Li.C21C₅]⁺ are: k_d (298.2 K)=23.3±0.4 s^-1, ΔH_d≠ =61.2±1.1 kJ mol^-1, and ΔS_d≠= -13.6±3.6 J K^-1 mol^-1. Metal ion exchange on [Li.C211]⁺ is in the very slow extreme of the NMR timescale up to 390 K and k_d « 4 s^-1 at 298.2 K, while in contrast exchange on [Na.C21C₅]⁺ is in the fast extreme of the NMR timescale at 298.2 K (k_d≈ 10⁴ s^-1). These data are compared with those obtained in other solvents.     

Kinetic analysis of carrier-mediated ion transport by the charge-pulse technique

Summary The charge-pulse technique has been used previously for the study of quasistationary processes in membranes which required only a moderate time resolution. It is shown here that a time resolution of about 400 nsec may be achieved with this technique and that it may be applied to the kinetic analysis of carrier-mediated ion transport. By this method we have studied the transport of alkali ions through optically black monoolein membranes in the presence of the ion carrier valinomycin. All three relaxation processes that are predicted by theory have been resolved. From the relaxation times and the relaxation amplitudes the rate constants for the association (k _R ) and the dissociation (k _D ) of the ioncarrier complex, as well as the translocation rate constants of the complex (k _MS ) and the free carrier (k _S ) could be obtained. For 1m Rb⁺ at 25° C the values arek _R =3×10⁵ m ^–1 sec^–1,k _D =2×10⁵ sec^–1,k _MS =3×10⁵ sec^–1,k _S =4×10⁴ sec^–1. The activation energies of the single rate constants which have been estimated from experiments at two different temperatures range between 50 and 90 kJ/mol.     

Immunosuppressive activity of murine newborn spleen cells. I. Selective inhibition of in vitro lymphocyte activation.

Cell-mediated immune responses to newborn lymphocyte alloantigens were investiated using mitogen activation, mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML). Spleen cells from 1- to 5-day-old (C57BL/6 × Balb/c) F₁ mice co-cultured with maternal strain (BALB/c) splenocytes did not affect DNA synthesis of maternal strain cells in the presence of concanavalin A or phytohemagglutinin. Newborn cells did inhibit the lipopolysaccharide response of maternal strain lymphocytes and these cells also depressed DNA synthesis when added to MLR cultures of BALB/c and C57BL/6 spleen cells. Newborn cells expressed poor stimulatory capacity in semiallogeneic MLR and also caused marked inhibition of DNA synthesis when added to semiallogeneic MLR containing BALB/c (responder) and CB6F₁ adult splenocytes (stimulator). The suppression of MLR by neonatal cells persisted for the first 2 weeks of life and was associated with a soluble factor released during culture. The suppressive activity was almost completely abrogated after depleting the T-cells from newborn splenocytes. However, these same cells did not interfere with the in vitro generation of cytotoxic lymphocytes in the CML assay. The selective immunosuppressive properties of newborn spleen cells may be important during pregancy by protecting the immunologically alien fetus from rejection by the mother.     

Tcrb-V3+3T-cell deletion and a new mouse mammary tumor provirus,Mtv-44

Genes encoding endogenous superantigens causing Tcrb-V3⁺ T-cell deletion co-segregate with mouse mammary tumor proviruses (Mtv),Mtv-3, Mtv-6, andMtv-13. In additionMtv-1 has been implicated in deletion of these T cells. We have examined levels of Tcrb-V3⁺ T cells andMtv integrations in the following off-spring and their parental strains, [(CBA-T6 × NZW)F₁ × CBA], [(CBA × C3H/He)F₁ × CBA], and [(B10.S (9R) × NOD]F₁ mice. We show that a newMtv (Mtv-44) from NZW mice andMtv-1 from C3H/He mice cosegregate with genes encoding ligands for partial deletion of Tcrb-V3^s+ T cells and that some NOD mice have an additionalMtv (Mtv-45) which is closely linked toMtv-3. Address correspondence and offprint requests to: K. Tomonari.     

Homopoietic histocompatibility (Hh-1) phenotype and the regulation of its expression

Hybrid resistance (HR) is primarily controlled by the genes of the Hemopoietic histocompatibility-1 (Hh-1) locus within the H-2 complex. HR is a consequence of the Hh-1-controlled target determinants in homozygous parental strain mice and their absence in heterozygous F₁ hybrid mice. To examine the mechanism that controls the Hh-1 phenotype, three independent clones of somatic cell hybrids between parental lines EL-4 (C57BL/6 origin, H-2 ^b ) and R1 (C58 origin, H-2 ^k ) were studied. The line EL-4 is Hh-1^b-positive and is subject to HR by H-2 ^b heterozygous F₁ mice, but R1 lacks the Hh-1 ^b allele and is not susceptible to HR. Of the three hybrid clones, F263.2 is Hh-1^b-positive, whereas the other two, F262.2 and F264.2, are Hh-1-negative, as judged by these cells' capacity to compete in vivo with the grafted parental C57BL/6 bone marrow cells in the resistant (C57BL/6 × C3H)F₁ mice. All three clones express the H-2^b and H-2^k class I antigens equally well, are susceptible to activated NK cells to the same extent, and all carry four copies of chromosome 17. However, Southern analysis reveals that clone F263.2 contains three copies of H-2 ^b chromosome and one H-2 ^k , whereas the other two clones carry two copies each of the parental chromosome 17. The results suggest that the relative copy number of specific alleles is the crucial determinanr of the Hh-1 phenotype, and render unlikely both the gene dosage hypothesis and the trans-acting dominant suppression hypothesis to account for the noncodominant expression of the Hh-1 phenotype.