bluuebunny/crossref_metadata_2025 huggingface dataset:
Legal and ethical issues in the use of anonymous images in pathology teaching and research
Authors: Tranberg H A , Rous B A , Rashbass J
Published: March 2003

The privacy of patients’ health information is of paramount importance. However, it is equally important that medical staff and students have access to photographs and video recordings of real patients for training purposes. Where the patient can be identified from such images, his or her consent is clearly required to both obtain the image and to use it in this way. However, the need for consent, both legally and ethically, is much less convincing where the patient cannot, by the very nature of the image, be identified from it. This is the case for many images used in the teaching of clinical medicine, such as videos taken of laparoscopies, images of internal organs and unlabelled X‐rays.
Molecular heterogeneity in deficiency of complement protein C2 type I
Authors: WANG  , CIRCOLO  , LOKKI  , SHACKELFORD  , WETSEL*  , COLTEN
Published: March 2003

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child’s fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child’s C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT‐PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base‐pair deletion) on one allele and a previously unrecognized two base‐pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up‐regulated appropriately by interferon‐γ and the flanking sequence containing the Bf promoter was normal in this C2‐deficient patient. Moreover, the concentration of Bf protein was normal in the patient’s plasma.
T‐cell receptor Vδ‐Jα rearrangements in human thymocytes: the role of Vδ‐Jα rearrangements in T‐cell receptor‐δ gene deletion
Authors: VERSCHUREN  , WOLVERS‐TETTERO  , BREIT  , VAN DONGEN
Published: March 2003

The differentiation mechanisms that force thymocytes into the T‐cell receptor (TCR)‐αβ or TCR‐γδ lineage are poorly understood, but rearrangement processes in the TCR‐α/δ locus are likely to play an important role. It is assumed that the TCR‐δ gene is deleted prior to Vα‐Jα rearrangements by rearrangement of the so‐called TCR‐δ‐deleting elements δRec and ψJα. However, the TCR‐δ gene can also be deleted via Vδ‐Jα rearrangements. We studied the different TCR‐δ‐deleting rearrangements of Vδ1, δRec, Vδ2 and Vδ3 to Jα gene segments in human thymocytes and peripheral blood using polymerase chain reaction analysis. The Vδ1 gene segment is the most upstream Vδ gene segment tested and appears to rearrange to almost all Jα gene segments. In contrast, the δRec and Vδ2 gene segments only rearrange to the 5′‐located Jα gene segments, thereby preserving an extensive TCR‐α combinatorial diversity, because most Jα gene segments are kept available for subsequent Vα‐Jα rearrangements. Based on our combined data we hypothesize that the different Vδ gene segments and the δRec gene segment play different roles in T‐cell development with regard to TCR‐δ deletion.
In situ study on the pathogenesis and immune reaction of equine herpesvirus type 1 (EHV‐1) infections in mice
Authors: BARTELS  , STEINBACH  , HAHN  , LUDWIG  , BORCHERS
Published: March 2003

The mouse model was used to study the pathogenesis of equine herpesvirus type 1 (EHV‐1) after primary and secondary intranasal infections. Within a few hours after infection, EHV‐1 was found in nasal and olfactorial epithelium and sub‐epithelial cells of the respiratory mucosa, but antigen‐specific immune cells were never detected. Next to the lung, EHV‐1 was transmitted early and directly to the brain, both via the olfactory route and the trigeminal nerve, but traces of degenerative or inflammatory processes were not detected there. In the lung, the immune cells residing or invading the parenchyma did not contain viral DNA or proteins. The primary immune response in the lungs was an alveolar and interstitial inflammation, dominated by the sequential appearance of neutrophils and macrophages, while the number of T and B lymphocytes remained unaltered. Within 24 hr after re‐infection, lymphocytes accumulated around the blood vessels, outnumbering monocytes more than twofold, without neutrophils appearing. The lymphocytes comprised of little more B than T cells and the T cells were predominantly CD8+ cells. Those and B cells infiltrated the parenchyma. These results show the route of virus distribution and demonstrate the lack of antigen‐specific immune cells in the lungs of mice after primary intranasal infection with EHV‐1.
Anti‐imm_unoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice
Authors: HAAK‐FRENDSCHO  , SABAN†  , SHIELDS  , JARDIEU
Published: March 2003

Inhibition of antigen‐specific IgE response has been shown to lead to amelioration of allergic disease symptoms. In an effort to design a therapy aimed at decreasing IgE levels, we reported previously that treatment of mice with an anti‐IgE antibody coincident with the primary antigen imm_unization resulted in significant decreases in antigen‐specific IgE synthesis, without substantially altering IgG levels. In the present study, we employed this mouse model and a surrogate antibody to investigate the capacity of anti‐IgE treatment to block an established IgE response in vivo. Results of these experiments suggest that anti‐IgE treatment concomitant with an antigen boost results in removal of detectable circulating IgE for at least 7 weeks (the duration of the study). Moreover, tissues removed from mice following anti‐IgE treatment failed to release histamine and contract in response to antigen challenge ex vivo. These findings demonstrate that reduction of circulating IgE correlates to an inhibition of tissue mast cell sensitization and mediator release in response to antigen challenge and further supports the concept of anti‐IgE treatment as a promising therapy for the treatment of allergic disease.
Recombinant cholera toxin B subunit is not an effective mucosal adjuvant for oral imm_unization of mice against Helicobacter felis
Authors: BLANCHARD  , LYCKE  , CZINN  , NEDRUD
Published: March 2003

Cholera toxin is a potent oral mucosal adjuvant for enteric imm_unization. Several studies suggest that commercial cholera toxin B subunit (cCTB; purified from holotoxin) may be an effective non‐toxic alternative for oral imm_unization. The present study was performed, using an infectious disease model, to determine if the oral mucosal adjuvanticity of CTB is dependent on contaminating holotoxin. Mice were orally imm_unized with Helicobacter felis sonicate and either cholera holotoxin, cCTB or recombinant cholera toxin B subunit (rCTB). Serum imm_unoglobulin G (IgG) and intestinal imm_unoglobulin A (IgA) antibody responses were determined and the mice were challenged with live H. felis to determine the degree of protective imm_unity induced. All orally imm_unized mice responded with serum IgG antibody titres regardless of the adjuvant used. However, only mice imm_unized with either holotoxin or the cCTB responded with an intestinal mucosal IgA response. Consistent with the production of mucosal antibodies, mice imm_unized with either holotoxin or cCTB as adjuvants were protected from challenge while mice receiving H. felis sonicate and rCTB all became infected. cCTB induced the accumulation of cAMP in mouse thymocytes at a level equal to 0.1% of that induced by holotoxin, whereas rCTB was devoid of any activity. These results indicate that CTB possesses no intrinsic mucosal adjuvant activity when administered orally. Therefore, when used as an oral adjuvant, CTB should also include small, non‐toxic doses of cholera toxin.
Molecular characterization of the putative T‐cell receptor cavity of the superantigen staphylococcal enterotoxin B
Authors: Garcia C. , Briggs C. , Zhang L. , Guan L. , Gabriel J. L. , Rogers T. J.
Published: March 2003

A number of investigators have utilized a variety of methods to identify the structural basis for the interaction of superantigens with the T‐cell receptor β‐chain. The previous studies strongly suggest that a region of the toxin near residues N23, Y61, Y91 and D209 is important for this binding activity. Examination of crystal structure data shows that these residues line the rim of one side of a shallow cavity in the toxin. In an attempt further to define the face of the staphylococcal enterotoxin B (SEB) molecule involved in the interaction with the β‐chain, we have employed a polymerase chain reaction (PCR)‐based, site‐specific mutagenesis method to generate amino acid substitutions of residues on the opposite side of this putative T‐cell receptor interaction cavity. Our results show that Y175 and N179 appear to be involved in the function of this superantigen, since each of several substitutions at this position exhibits a significantly reduced ability to induce T‐cell proliferation. At the same time, mutation of the proximal Y186 does not alter the superantigen activity of SEB. Binding analysis of these mutants shows that class II binding activity is not significantly altered. Analysis of the responding T cells shows that the mutant toxins maintain T‐cell receptor Vβ selectivity. However, responses of T cells bearing the Vβ8.1 allele appear to be particularly diminished. When viewed in the context of other results reported in the literature, our results suggest that the T‐cell receptor interaction site involves SEB residues which ring both the Y175/N179‐side and the N23‐side of a cavity on one side of the toxin molecule.
An allogeneic microenvironment influences the phenotype of intermediate T‐cell receptor cells expanding in MRL‐ lpr/lpr mice
Authors: Tsukahara A. , Iiai T. , Moroda T. , Tada T. , Suzuki S. , Takeda K. , Hatakeyama K. , Abo T.
Published: March 2003

MRL‐lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double‐negative (DN) CD4− CD8−αβT cells expressing a low density of interleukin‐2 receptor β‐chain (IL‐2Rβ). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T‐cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL‐2Rβ. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL‐2Rβ. A cell‐sorting experiment for purified fractions demonstrated that only CD8+ cells reconstituted TCRint cells in scid mice. Namely, DN CD4− CD8− cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.

int, intermediatelpr, MRL‐lpr/lpr miceMNC, mononuclear cellsIL‐2Rβ, interleukin‐2 receptor β‐chain.
Biological activities of C3βc, a novel neutrophil chemoattractant derived from the β‐chain of rat complement C3
Authors: Nakagawa H. , Sunada Y. , Ando Y.
Published: March 2003

Biological activities of C3βc, which is a C‐terminal fragment of the β‐chain of rat complement C3, have been studied by in vivo and in vitro experiments. C3βc was purified as a novel neutrophil chemoattractant from the exudate of the chronic phase of rat carrageenin‐induced inflammation. The purified C3βc induced neutrophil chemotaxis in vivo when C3βc was injected into the preformed air‐pouch on the back of rats. C3βc transiently increased the intracellular free Ca2+ concentration of neutrophils, and enhanced the adhesion of neutrophils to fibrinogen in vitro, suggesting that C3βc has the ability to express an adhesion molecule of rat neutrophils. In addition, C3βc at low concentrations (10−10–10−11 m) stimulated rat macrophages to produce cytokine‐induced neutrophil chemoattractant‐2, a member of the interleukin‐8 family. Furthermore, C3βc enhanced vascular permeability in vivo, which is suppressed by cyproheptadine, suggesting that C3βc may have the characteristics of an anaphylatoxin. Our results suggest that C3βc contributes to oedema formation and neutrophil accumulation at inflammatory sites in rats.

BSA, bovine serum albumin CINC, cytokine‐induced neutrophil chemoattractant DMEM, Dulbecco’s modified Eagle’s minimal essential medium ELISA, enzyme‐linked immunosorbent assay fMLP, N‐formyl‐methionyl‐leucyl‐phenylalanine IL, interleukin LPS, lipopolysaccharide PBS, phosphate‐buffered saline.
Staphylococcal enterotoxin B inhibits the production of interleukin‐4 in a human mast‐cell line HMC‐1
Authors: Ackermann L. , Pelkonen J. , Harvima I. T.
Published: March 2003

Staphylococcal enterotoxins belong to the recently characterized group of immunocytotropic bacterial superantigens that are potent mitogens for human T cells. Superantigens are presented, but without intracellular processing, to T cells by monocyte/macrophages, Langerhans’ cells and keratinocytes via the class II major histocompatibility complex (MHC) molecules. Superantigens have been demonstrated to act as potent inducers of several proinflammatory cytokines in the antigen‐presenting cells such as interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α). As mast cells participate in the pathogenesis of several inflammatory skin disorders such as atopic dermatitis (AD), which is often aggravated by staphylococcal infections, we studied the effect of staphylococcal enterotoxin B (SEB) superantigen on the histamine release and IL‐4 expression in a human mast‐cell line (HMC‐1). Incubation of SEB (50 μg/ml) with HMC‐1 cells for 45 min, could not induce any histamine release. The HMC‐1 cells were also stimulated with various concentrations of SEB (0, 1, 10, 20, 50 μg/ml) for 1, 2, 3 and 4 days. Clear dose‐dependent inhibition of IL‐4 protein production and release was observed on day 4 without any observed effect on cell viability. Compared with unstimulated HMC‐1 cells, after 50 μg/ml SEB stimulation, the IL‐4 mRNA levels decreased steadily in the 2, 6, 18 and 24 hr samples in repeated experiments as measured with the reverse transcription–polymerase chain reaction (RT–PCR) method. In comparison, a biphasic decrease in TNF‐α expression was found. Our results show that in human leukaemic mast cells, superantigen stimulation downregulates the production of IL‐4.

IL, interleukin TNF‐α, tumour necrosis factor‐alpha AD, atopic dermatitis SEB, Staphylococcal enterotoxin B Th, T helper IFN‐γ, interferon‐γ ICAM‐1, intercellular adhesion molecule‐1 RT–PCR, reverse transcription–PCR.
Human/BALB radiation chimera engrafted with splenocytes from patients with idiopathic thrombocytopenic purpura produce human platelet antibodies
Authors: DEKEL B. , MARCUS H. , SHENKMAN B. , SHIMONI A. , CANAAN A. , BERREBI A. , VARON D. , REISNER Y.
Published: March 2003

We have previously shown that lethally irradiated normal strains of mice, radioprotected with severe combined immunodeficient (SCID) bone marrow, can be engrafted with human peripheral blood mononuclear cells (PBMC). The human/mouse radiation chimera can mount marked humoral and cellular responses to recall antigens, as well as primary responses. In the present study, we adoptively transferred splenocytes from patients with chronic immune thrombocytopenic purpura (ITP) into lethally irradiated BALB/c mice, radioprotected with SCID bone marrow. High titres of total human immunoglobulin appeared as early as 2 weeks post‐transplant and declined after 6 weeks, while human anti‐human platelet antibodies were detected 2–8 weeks after the transfer of splenocytes. The immunoglobulin G (IgG) fraction contained antibodies against glycoprotein (GP) IIb/IIIa (CD41) or GPIb/IX (CD42). The human platelet antibodies showed a low level of cross‐reactivity with mouse platelets, and thrombocytopenia in the animals was not observed. Splenocytes from individual ITP patients differed in their capacity to produce either human platelet antibodies or total human immunoglobulin. Furthermore, antibodies produced in the murine system were not always identical to the original antibodies present in the serum of the patients. The study of the serological aspects of autoantibodies against human platelets in an animal model might be useful for the investigation of potential therapeutics in ITP.
Synergistic augmentative effect of interleukin‐10 and CD27/CD70 interactions on B‐cell immunoglobulin synthesis
Authors: NAGUMO H. , AGEMATSU K.
Published: March 2003

Interleukin‐10 (IL‐10) is a potent cytokine that regulates immunoglobulin synthesis by B cells. CD27/CD70 interactions by direct cell‐to‐cell contact are also needed to produce substantial amounts of immunoglobulin. We have investigated the effects of IL‐10 and CD27/CD70 interactions on the immunoglobulin synthesis. In the presence of IL‐10 stimulation, the production of IgG, IgM and IgA was increased synergistically by the addition of CD27 ligand (CD70)‐transfectants in a dose‐dependent manner, which was completely blocked by anti‐CD70 monoclonal antibody. In contrast, CD70‐transfectants additively enhanced the immunoglobulin production in the presence of IL‐2, IL‐4, or IL‐6. The synergistic enhancement of the immunoglobulin production by IL‐10 and CD70‐transfectants was remarkable in highly purified CD27+ B cells, but there was no immunoglobulin production in CD27− B cells. Furthermore, by the addition of CD70‐transfectants, the synthesis of IgG1, IgG2, IgG3 and IgG4 was also enhanced in the presence of IL‐10. On the other hand, IL‐10 diminished CD27 expression in B cells. B‐cell proliferation was augmented by CD70‐transfectants with IL‐10 or IL‐10 plus IL‐2. The addition of IL‐2 further augmented the immunoglobulin production which was synergistically enhanced by IL‐10 and CD27 triggering. Taken together, the co‐operative response to IL‐10 and CD27/CD70 interactions regulates B‐cell immunoglobulin production.
Increased serum IgG1 levels and reduced numbers of B‐1 B cells in DBA/2J mice
Authors: TOCCE* K. , MASTERS† G. , RAGO‡ C. , PRIOR§ L. , SUPPIAH¶ K. , GLYSING‐JENSEN** T. , LEARY S. , RIGGS J.
Published: March 2003

B‐cell heterogeneity studies have historically focused upon BALB/c mice and their derivatives. In contrast, the B cells of DBA/2J mice, a prototype strain for the study of the endogenous minor lymphocyte stimulatory (Mls) viral superantigen Mls‐1a, have not been extensively investigated. DBA/2J B cells, by functioning as Mls‐1a antigen‐presenting cells, influence their own differentiation and diversity by inducing the proliferation and differentiation of specific CD4 T‐cell subsets. In this report, the B cells of DBA/2J and BALB/c mice were compared for their ability to restore B‐cell function in severe combined immunodeficient (SCID) recipients. Although spleen and bone marrow cells from these strains exhibited similar restoration of serum IgM production, the transfer of DBA/2J B cells into SCID mice led to greater IgG1 production. The peritoneal cells of DBA/2J mice consisted of a lower percentage of B‐1 B cells and were less capable of restoring B‐cell function after transfer into SCID recipients. These differences are discussed with respect to the possible role of viral superantigens in influencing B‐lymphocyte diversity.
ICAM‐1 costimulation induces IL‐2 but inhibits IL‐10 production in superantigen‐activated human CD4+ T cells
Authors: LABUDA  , WENDT  , HEDLUND  , DOHLSTEN
Published: March 2003

We have previously reported that costimulatory pathways including B7–CD28 and lymphocyte function‐associated antigen‐3 (LFA‐3)–CD2 shape distinct activation profiles in human CD4+ T cells. We now show that superantigen (SAg), in combination with intracellular adhesion molecule‐1 (ICAM‐1) costimulation drives a proliferative response accompanied by high levels of interleukin‐2 (IL‐2) and moderate levels of interferon‐γ (IFN‐γ) and tumour necrosis factor (TNF). This response profile differs from that observed in B7 or LFA‐3 costimulated T cells because our previous results showed that B7–CD28 costimulation was accompanied by high levels of IL‐2, IFN‐γ and TNF, whereas LFA‐3 was a potent inducer of IFN‐γ and TNF, but had little influence on IL‐2 production. The ICAM‐1‐induced IL‐2 production could efficiently be abrogated with monoclonal antibody (mAb) against ICAM‐1 or LFA‐1, showing that the activation is dependent of a functional ICAM‐1–LFA‐1 pathway. SAg‐induced IL‐2, IFN‐γ and TNF were detected in both CD4+ and CD8+ T cells, whereas production of IL‐10 was restricted to CD4+ T cells. A major finding in the present study was that ICAM‐1 costimulation strongly inhibits IL‐10 production in CD4+ T cells. Our data demonstrate that ICAM‐1 costimulation is sufficient to induce large amounts of IL‐2. The presence of ICAM‐1 results in suppression of IL‐10 production in T helper (Th) cells, which may favour the development of Th1 and not Th2 T cells.
Endogenous IL‐10 regulates IFN‐γ and IL‐5 cytokine production and the granulomatous response in Schistosomiasis mansoni‐infected mice
Authors: BOROS  , WHITFIELD
Published: March 2003

In murine Schistosomiasis mansoni circumovum, granuloma formation is regulated by pro‐ and anti‐inflammatory cytokines. Among the latter, interleukin‐10 (IL‐10) has been shown to regulate the inflammatory response. In this study we examined the role of endogenously produced IL‐10 in T‐helper 1 (Th1)‐ and Th2‐type cytokine production and granuloma formation. The dynamics of IL‐10 production through the course of the infection were different in granuloma versus splenic cells. In the former, production peaked during the early developmental stage (6 weeks of infection) of the granuloma and then declined. In splenocytes production peaked at 12 weeks, before down‐modulation of the granuloma response. In the developing granuloma both macrophages and T cells secreted IL‐10. In anti‐IL‐10 monoclonal antibody (mAb)‐supplemented granuloma cell cultures endogenous IL‐10‐mediated regulation of interferon‐γ (IFN‐γ) was manifest only at 6 weeks; that of IL‐2 continued throughout the infection (6–20 weeks). IL‐4 production was unaffected, but IL‐5 production was regulated at the 6 and 8 weeks time point. Splenocytes showed regulation of IFN‐γ and IL‐2 production at the peak of the granulomatous response (8 weeks). IL‐4 production was not regulated, whereas IL‐5 production was regulated only at 6 weeks. Repeated injections of anti‐IL‐10 mAb given to mice at 6, 12 or 20 weeks of the infection significantly enhanced liver and lung granuloma growth, tissue eosinophilia, and IFN‐γ, IL‐5 production at the early developmental phase (6 weeks) of the lesions. Thus, in schistosome‐infected mice endogenous IL‐10 is shown to regulate Th1‐ and Th2‐type cytokine production and granuloma formation during the early Th0/Th1 phase of the immune response.
An anti‐inflammatory role for interleukin‐11 in established murine collagen‐induced arthritis
Authors: WALMSLEY  , BUTLER*  , MARINOVA‐MUTAFCHIEVA  , FELDMANN
Published: March 2003

Interleukin‐11 (IL‐11) is a cytokine belonging to the IL‐6 family which has both pro‐ and anti‐inflammatory potential. Like IL‐6 it can diminish tumour necrosis factor‐α and IL‐1 production, and augment immunoglobulin synthesis. We have explored the immunomodulatory effects of IL‐11 treatment in mice in a model of inflammatory autoimmune joint disease, collagen‐induced arthritis (CIA). Recombinant human IL‐11 was administered at various doses to DBA/1 mice after the onset of CIA. IL‐11 treatment caused a significant reduction in the clinical severity of established CIA, which was associated with protection from joint damage, as assessed by histology. Although there was a suggestion at high doses of IL‐11 that the anticollagen type II (CII) response may have been augmented, there was no statistically significant effect of IL‐11 treatment on anti‐CII antibody levels. Similarly, the acute‐phase reactant serum amyloid P was only elevated in mice receiving very high doses (50–100 μg/day) of IL‐11. Endogenous IL‐11 was abundantly produced in synovial membrane cultures derived from CII‐immunized mice with active disease, suggesting that, as in rheumatoid arthritis, this cytokine is spontaneously produced in the inflammatory response in CIA. The results presented here demonstrate an anti‐arthritic immunoregulatory role for IL‐11 in murine CIA, and suggest that IL‐11 is a candidate therapeutic molecule for human inflammatory arthritic diseases.
Unresponsiveness of CD4−8+/− thymocytes to lectin stimulation in LEC mutant rats
Authors: SAKAI  , AGUI  , KAICHUN  , OKA  , HISAEDA  , NAGASAWA  , HIMENO  , MATSUMOTO
Published: March 2003

A mutant strain of rat, LEC, shows a novel arrest of T‐cell maturation from CD4+8+ to CD4+8−, but not to CD4−8+ cells in the thymus. The responsible mutant locus is designated the thid, which was acted upon in a recessive manner of inheritance. We found that LEC rat thymocytes failed to respond to interleukin (IL)‐1, IL‐6 and IL‐7 in the presence of the mitogenic lectins, Allo A or concanavalin A (Con A). The unresponsiveness appeared to be due to unresponsiveness to the lectin stimulation rather than because of cytokine stimulation. Normal rat CD4−8+/− (consisting of CD4−8+ and CD4−8− thymocytes), CD4+/−8− (consisting of CD4+8− and CD4−8− thymocytes), and CD4−8− thymocyte subsets normally responded to mitogenic stimulation, while CD4+8+ thymocytes did not. In contrast, all LEC rat CD4−8+/−, CD4+/−8−, CD4−8− and CD4−8+ thymocytes did not respond to the mitogenic stimulation, suggesting that the unresponsiveness of the CD4−8+/− thymocytes seems to be responsible for the unresponsiveness of whole thymocytes in LEC rats. LEC rat CD4−8+/− thymocytes normally expressed Con A receptor (R), lymphocyte function‐associated antigen‐1 (LFA‐1), and CD45, which are thought to be important molecules for lectin stimulation. When backcross rats from (F344×LEC)F1×LEC were examined, the phenotype for the thid mutation correlated with the [3H]thymidine deoxyribose (TdR) incorporation level in response to Con A stimulation; thymocytes from backcross rats showing +/thid phenotype responded to Con A stimulation normally, whereas thymocytes from backcross rats showing thid/thid phenotype showed significantly lower responsiveness compared with +/thid rats. However, in WKAH.C‐thid congenic rat thymocytes that carry the thid mutation, the responsiveness to mitogenic stimulation was comparable to that of normal rat thymocytes. These results suggest that a defect in responsiveness to mitogenic stimulation in LEC rat thymocytes is controlled by multiple genetic loci and the thid locus is one of the important loci for the development of this abnormal phenotype.
Mouse lymphoblasts lose their immunogenicity and susceptibility to specific cytotoxic T lymphocyte lysis during maintenance in culture
Authors: LESHEM  , BRASS
Published: March 2003

This study was undertaken to search for possible mechanisms by which T‐cell lines become non‐immunogenic and refractory to cellular‐mediated lysis during culture. We demonstrate that mouse lymphoblasts (LB) lost their susceptibility to specific cytotoxic T lymphocyte (CTL)‐mediated lysis following culture for more than 5 days in the presence interleukin‐2 (IL‐2), IL‐7 but not IL‐4. In contrast, the cultured lymphoblasts (CLB) were efficiently lysed by specific antibody and C′ and by CTL in the presence of concanavalin A. In addition, CLB did not inhibit cytotoxicity against LB in a cold target competition assay, indicating that CLB and LB differ in the expression of certain surface molecules. Indeed, a significantly lower expression of H‐2D class I antigen, the Fas antigen and the adhesion molecules intracelluar adhesion molecule‐1 (ICAM‐1) and very late activation antigen‐4 (VLA‐4) was observed on the CLB surface. Consequently, CLB could not form conjugates with specific CTL, a prerequisite for CTL‐mediated lysis. In addition, there was a marked decrease in CLB immunogenicity: the cultured cells were unable to stimulate allogeneic spleen cells in mixed lymphocyte culture nor could they induce a cytotoxic response following their injection into allogeneic mice. The reduced immunogenicity enabled the prolonged survival of active CLB in an allogeneic host. We suggest that the extended survival in an allogeneic tumour‐bearing host of cultured, hence weakly immunogenic, anti‐tumour CTL, will enable them the in vivo implementation of their anti‐tumour activity.
Superantigen activation and kinetics of cytokines in the Long–Evans rat
Authors: HUANG  , KOLLER
Published: March 2003

This study was conducted to identify and quantify, over time, selected cytokine responses in Long–Evans rats that were exposed to staphylococcus enterotoxin B (SEB). The kinetics of selected cytokines [interleukin‐2 (IL‐2), IL‐6, interferon‐γ (IFN‐γ) and tumour necrosis factor (TNF)] and phenotype and cell cycle analysis of T lymphocytes were determined in Long–Evans rats administered a single intraperitoneal (i.p.) dose of either 50 μg or 500 μg of SEB. Rats injected with 50 μg SEB had significantly elevated levels of IL‐2, IL‐6 and IFN‐γ in their serum 2 hr post‐injection. IL‐2 serum levels were significantly elevated at 2 hr and returned to near control values by 12 hr while both IL‐6 and IFN‐γ peaked at 6 hr but remained significantly increased at 24 hr post SEB exposure. A 500 μg dose of SEB did not further enhance these cytokine responses. When spleen cells were collected for culture 2 hr after rats were injected i.p. with 50 μg SEB and cocultured with SEB, TNF and IL‐6 levels were significantly increased after 2 hr incubation, while IL‐2 and IL‐6 were significantly elevated at 6 hr. Production of all these cytokines in spleen cell cultures continued to increase over the 24 hr sampled. Peritoneal cells were collected for culture either at 1 hr or 2 hr after injection of either 50 μg or 500 μg of SEB. IL‐6 was significantly increased after 1 hr in culture while TNF was significantly increased by 2 hr regardless of whether the cells were harvested 1 or 2 hr after SEB injection. The greatest response for both IL‐6 and TNF occurred when cells from animals injected with 50 μg SEB were restimulated in vitro with SEB. The peak levels for IL‐6 were at 12 hr post SEB exposure while TNF peaked at 6 hr. The percentage of CD4+ cells was significantly increased at 48 hr and 72 hr post SEB (50 μg) administration while the percentage of CD8+ cells remained similar to control values for the 168‐hr test period. A similar pattern was observed in cell cycling where the CD4+ cells proliferated up to 2 days post SEB injection and then were significantly suppressed at day 3. The CD8+ cells were comparable to control values. These studies demonstrate that the cytokine responses in Long–Evans rats exposed to a superantigen are somewhat similar to those that occur in mice and humans, e.g. a rapid short increase in the production of IFN‐γ and TNF that was accompanied by an increase in the production of IL‐2. Additional responses noted in this species, however, were a marked increase in IL‐6 production, as well as an early increase in the number and cycling of CD4+ cells followed by a down‐regulation of these events. These activities occurred in the absence of notable histopathological alteration of lymphoid organs. The results indicate that the Long–Evans rat is an acceptable animal model to investigate the pathogenesis of superantigen‐induced disease and that IL‐6 may be an active mediator of this process.
CD2 signalling induces phosphorylation of CREB in primary lymphocytes
Authors: GUYOT  , NEWBOUND  , LAIRMORE
Published: March 2003

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor‐1 (ATF‐1) family. We have used a human T‐lymphotropic virus type 1 (HTLV‐1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV‐1 transcription following CD2 but not CD3 receptor cross‐linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor‐mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross‐linking increased cAMP detected by competitive enzyme‐linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross‐linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post‐translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp‐cAMP‐sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross‐linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF‐1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE‐mediated transcription following ligand engagement (e.g. cell‐to‐cell contact).
Expression of heat shock protein 70 blocks thymic differentiation of T cells in transgenic mice
Authors: LEE  , PARK  , KIM  , PARK  , KIM  , JANG  , SEO
Published: March 2003

Heat shock protein 70 (HSP70) is involved not only in protein folding, but also in processes of differentiation and cell‐cycle progression. Recently, HSP70 has been implicated in mediation of functions of some immunosuppressive agents. To study the role of HSP70 in differentiation of haematopoietic cells, we generated transgenic mice using the human inducible hsp70 gene fused to the mouse H‐2K promoter. These mice develop a T‐cell deficiency that is characterized by thymic hypoplasia and a significant reduction in peripheral T cells. The total number of thymocytes is about 100‐fold less than that in normal mice. The majority of the thymocytes are immature T cells that express neither CD4 nor CD8 molecules, indicating that T cells are affected at an early stage of thymic differentiation. Expression of the transgenic HSP70 was detected both in bone marrow cells and in thymocytes. Furthermore, injection of normal bone marrow cells into the T‐cell deficient mice led to the generation of mature T cells indicating that the T‐cell deficiency was caused by the action of HSP70 in T cells. The blockage of differentiation occurred only in T cells, both αβ‐ and γδ‐T‐cell receptor (TCR)‐bearing cells, but not in B cells, granulocytes, and monocytes. The observations suggest that HSP70 may inhibit a cellular process that is essential for the differentiation of early stage T cells. Further experiments using this model system will widen our understanding of HSP70 and its function on a molecular level.
Cross‐talk between γδ T lymphocytes and immune cells in humoral response
Authors: SZCZEPANIK  , NOWAK  , ASKENASE  , PTAK
Published: March 2003

The role of γδ T cells in immunoregulation is largely unknown. In the current study we noted that γδ T cells play a positive role in the humoral response. These positively acting γδ T cells are required for the successful adoptive cell transfer of the humoral response, as well as for in vitro generation of plaque‐forming cells (PFC). The presented results show that γδ T cells cause an increase in interleukin‐10 (IL‐10) production, which partly elucidates the mechanism of action of these cells. However, experiments with cell culture inserts strongly suggest that direct cell–cell contact between immune and γδ H‐2‐compatible regulatory T cells is critical to the exertion of the positive immunoregulatory function of γδ cells. The mechanism of cross‐talk between these two cell populations is still not clear but we regard as most likely that the positively acting γδ T cells may interact with a complex of heat‐shock protein–non‐polymorphic MHC (IB) on the surface of T helper type 2 and/or B cells. This could provide, by direct cell–cell contact, the cognate recognition between γδ T‐cell receptors and heat‐shock protein–MHC that leads to positive internal signalling in the immune cells.
Peptide‐binding motifs of the mixed haplotype Aβz/Aαd major histocompatibility complex class II molecule: a restriction element for auto‐reactive T cells in (NZB×NZW)F1 mice
Authors: MINE*  , KOARADA*  , SAI  , MIYAKE  , KIMOTO
Published: March 2003

We previously showed that the mixed haplotype Aβz/Aαd major histocompatibility complex (MHC) class II molecules function as restricting element for autoreactive T‐cell clones derived from autoimmune prone (NZB×NZW)F1 (B/WF1) mice. Subsequent analysis revealed that some of these Aβz/Aαd‐restricted autoreactive T‐cell clones were pathogenic upon transfer to pre‐autoimmune B/WF1 mice. In this paper, we analysed the peptide‐binding motif of Aβz/Aαd class II molecules. Amino acid‐sequencing analysis of peptides eluted from purified Aβz/Aαd molecules revealed several sequences, including one that corresponds to murine l‐plastin 588–601. Synthetic 18‐mer l‐plastin 588–605 peptide (SMARKIGARVYALPEDLV, as described by the amino acid single letter code) was demonstrated to bind to Aβz/Aαd MHC class II molecules on transfectant B lymphoma cells (TAβz). A competitive binding inhibition assay using truncation peptides revealed the core sequence for binding resides in 591Arg to 601Pro. Binding inhibition assay using substitution peptides, each having substitution to the other 19 residues at positions from 590Ala to 601Pro, revealed four major anchor sites 592Lys (p1), 594Gly (p3), 595Ala (p4), 597Val (p6) and one minor anchor site 600Leu (p9). Positively charged residues are not allowed at p3 and negatively charged residues are not allowed at p4 and p6. Relatively large hydrophobic residues (Leu, Ile) are not tolerated at p3 and p4. Met and Trp are not tolerated at p6. Based on these findings, the characteristics of peptides recognized by autoreactive T cells in B/WF1 mice are discussed.
Prevention of immune dysfunction and vitamin E loss by dehydroepiandrosterone and melatonin supplementation during murine retrovirus infection
Authors: ZHANG  , ARAGHI‐NIKNAM  , LIANG  , INSERRA  , ARDESTANI  , JIANG  , CHOW  , WATSON
Published: March 2003

Female C57BL/6 mice infected with the LP‐BM5 leukaemia retrovirus developed murine acquired immune‐deficiency syndrome (AIDS). Dehydroepiandrosterone (DHEA) and melatonin (MLT) modify immune dysfunction and prevent lipid peroxidation. We investigated whether DHEA and MLT could prevent immune dysfunction, excessive lipid peroxidation, and tissue vitamin E loss induced by retrovirus infection. Retrovirus infection inhibited the release of T helper 1 (Th1) cytokines, stimulated secretion of Th2 cytokines, increased hepatic lipid peroxidation, and induced vitamin E deficiency. Treatment with DHEA or MLT alone, as well as together, largely prevented the reduction of B‐ and T‐cell proliferation as well as of Th1 cytokine secretion caused by retrovirus infection. Supplementation also suppressed the elevated production of Th2 cytokines stimulated by retrovirus infection. DHEA and MLT simultaneously reduced hepatic lipid peroxidation and prevented vitamin E loss. The use of DHEA plus MLT was more effective in preventing retrovirus‐induced immune dysfunction than either DHEA or MLT alone. These results suggest that supplementation with DHEA and MLT may prevent cytokine dysregulation, lipid oxidation and tissue vitamin E loss induced by retrovirus infection. Similarly, hormone supplementation also modified immune function and increased tissue vitamin E levels in uninfected mice.
Granulocyte–macrophage colony‐stimulating factor and interleukin‐3 cause basophil histamine release by a common pathway: downregulation by sodium
Authors: TEDESCHI  , SALMASO  , DI DONATO  , LORINI  , MIADONNA
Published: March 2003

Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐3 (IL‐3) are recognized as enhancers, but not as inducers, of histamine release from normal human basophils. However, when extracellular Na+ is removed IL‐3 acquires the capacity to induce histamine release. The aim of this study was to evaluate whether GM‐CSF can induce basophil histamine release using the same pathway of IL‐3. Leucocyte suspensions from normal human subjects were stimulated with GM‐CSF, IL‐3 and anti‐IgE, and histamine release was evaluated by an automated fluorometric method. In a physiological medium, GM‐CSF (10 ng/ml) and IL‐3 (10 ng/ml) did not provoke histamine release, in spite of an efficient response to anti‐IgE (10 μg/ml). However, when extracellular Na+ was substituted iso‐osmotically with N‐methyl‐d‐glucamine+ or with choline+, GM‐CSF and IL‐3 were able to trigger histamine release from either mixed leucocyte suspensions or purified human basophils. The effect of GM‐CSF on basophil histamine release was dose dependent, with optimal release at a dose of 1 ng/ml after incubation at 37° for 60–120 min. The kinetics of IL‐3‐induced histamine release were similar, whereas anti‐IgE‐induced histamine release was more rapid, being almost maximal after incubation for 30 min. A good correlation was found between GM‐CSF‐induced and IL‐3‐induced histamine release; furthermore, the combined effects of the two cytokines were less than additive, suggesting that they share the same pathways leading to histamine release. When extracellular Na+ concentration was increased from 0 to 140 mm, histamine release induced by GM‐CSF, IL‐3 and anti‐IgE was reduced progressively. In contrast, histamine release induced by these stimuli was upregulated when the concentration of extracellular Ca2+ was increased. These results provide indirect evidence that GM‐CSF and IL‐3 can induce basophil histamine release by a common pathway that is downregulated by Na+.
Importance of intrathymic mixed chimerism for the maintenance of skin allograft tolerance across fully allogeneic antigens in mice
Authors: ETO  , KONG  , UOZUMI  , NAITO  , NOMOTO
Published: March 2003

In B6 (H‐2b) mice that had been given, neonatally, 1×108 B6AKF1 spleen cells intraperitoneally (i.p.), only a moderate prolongation of donor (AKR:H‐2k) skin graft survival was observed. In such B6 mice, no mixed lymphocyte reaction (MLR) to AKR could be detected on day 35 (35 days after birth), but it was clearly evident on day 84. Similarly, neither Vβ6+ (reactive to MTV‐7‐encoded antigens) nor Vβ11+ (reactive to I‐E+MTV‐derived superantigens) T cells were detected on day 35, but both were clearly evident on day 84 in both the thymus and the lymph nodes, thus indicating the breakdown of intrathymic mixed chimerism at the antigen‐presenting cell level. Furthermore, by day 84, all skin grafts from AKR had already been rejected in such B6 mice. In the periphery, however, Vβ6+, but not Vβ11+, T cells were clonally anergic on day 84, based on a stimulation assay with anti‐T‐cell receptor (TCR) monoclonal antibody (mAb), thus suggesting that tolerance to some antigens, but not to others, may be induced by the clonal anergy in fully allogeneic combinations, and that the clonal anergic state may be masked by other proliferative responses. These results therefore indicate the importance of intrathymic mixed chimerism (central tolerance) and the limitations of clonal anergy (peripheral tolerance) in maintaining tolerance across fully allogeneic antigen barriers.
Interleukin‐4 regulation of human monocyte and macrophage interleukin‐10 and interleukin‐12 production. Role of a functional interleukin‐2 receptor γ‐chain
Authors: BONDER  , FINLAY‐JONES  , HART
Published: March 2003

Interleukin‐4 (IL‐4) is the prototypic type 2 immunoregulatory cytokine that can suppress the production of many monocyte and macrophage pro‐inflammatory mediators. In this study we investigated the regulation by IL‐4 of IL‐12 and IL‐10 production. While IL‐4 suppressed lipopolysaccharide (LPS)‐induced IL‐12 and IL‐10 production by human peripheral blood monocytes, IL‐4 suppressed LPS‐induced IL‐12, but not IL‐10, production by synovial fluid mononuclear cells from patients with rheumatoid arthritis. IL‐4 also suppressed IL‐12, but not IL‐10 production, by LPS‐stimulated in vitro monocyte‐derived macrophages. Similarly, IL‐4 cannot suppress LPS‐induced tumour necrosis factor‐α (TNF‐α) production by synovial fluid cells and monocyte‐derived macrophages. The failure of IL‐4 to regulate IL‐10 production is not due to the failure of IL‐4 to suppress TNF‐α, and vice versa. The data suggest that the IL‐4 receptor subunit, γc, is essential for IL‐4 regulation of LPS‐induced IL‐10 production and that a correlation exists between duration of monocyte culture, reduction in γc mRNA in cultured cells and hyporesponsiveness of monocyte‐derived macrophages to IL‐4 for regulation of LPS‐induced IL‐10 production. This study highlights the importance of investigating responses to IL‐4, as a potential therapeutic anti‐inflammatory agent, by cells isolated from inflammatory sites and not by the more easily accessible blood monocytes. This study emphasizes the involvement of signalling from γc in IL‐4 regulation of LPS‐induced IL‐10 production by monocytes and macrophages.