bluuebunny/crossref_metadata_2025 huggingface dataset:
Can pulmonary sclerosing haemangioma be accurately diagnosed by intra‐operative frozen section?
Authors: Chan A C L , Chan J K C
Published: March 2003

Aims:  Pulmonary sclerosing haemangioma is a rare benign tumour with a characteristic variegated histological pattern. In this retrospective study we aimed to identify features that can aid in making a correct diagnosis and avoiding potential pitfalls at the time of intra‐operative frozen section.

Methods and results:  Twenty cases of pulmonary sclerosing haemangioma with intra‐operative frozen section were reviewed. The four major histological patterns (solid, sclerotic, papillary and haemorrhagic) were found in various combinations in the frozen sections. In 17 cases, three or more patterns were present. There could be focal areas mimicking epithelioid haemangioendothelioma or carcinoid tumour. Intra‐operative imprint/scrape cytology served as a helpful adjunct in confirming the cytological blandness, although occasional atypical cells could be present. An intra‐operative frozen section diagnosis of ‘sclerosing haemangioma’ or ‘benign tumour’ was given in 14 cases; the diagnosis was deferred in six cases. Retrospective analysis of the deferred cases showed that a definitive intra‐operative diagnosis could have been made in three, because three or more major histological patterns were present. One case showed a pure papillary pattern at frozen section, mimicking the appearance of papillary adenocarcinoma (primary or secondary), bronchioloalveolar carcinoma, epithelioid mesothelioma or papillary adenoma; two tumours from a patient with multicentric disease showed widespread significant cytological atypia in the tumours raising a serious consideration of malignancy.

Conclusion:  A diagnosis of pulmonary sclerosing haemangioma can be made at intra‐operative frozen sections in most cases based on the tumour circumscription and variegated histological patterns. When only a single histological pattern is identified or when there is significant cytological atypia, distinction from other tumours can be problematic, and the diagnosis is best deferred.
Unusual sclerosing haemangiomas and sclerosing haemangioma‐like lesions, and the value of TTF‐1 in making the diagnosis
Authors: Nicholson A G , Magkou C , Snead D , Vohra H A , Sheppard M N , Goldstraw P , Beddow E , Hansell D M , Travis W D , Corrin B
Published: March 2003

Aims:  Sclerosing haemangiomas typically comprise a mixture of four architectural patterns (papillary, sclerotic, solid and haemorrhagic) and two cell types, eosinophilic cuboidal epithelial lining cells and sheets of rounded cells with either eosinophilic or clear cytoplasm. In most instances, recognition of these architectural and cytological features provides sufficient evidence for diagnosis. This study presents and discusses the histogenesis of four cases where difficulties in diagnosis were encountered, and reports the value of the antibody TTF‐1 in making the diagnosis.

Methods and results:  Four cases with focal areas reminiscent of sclerosing haemangioma were reviewed and immunostained with an antibody panel including antibodies to TTF‐1 and surfactant apoprotein A. Of these, one case was classified as sclerosing haemangioma combined with typical carcinoid, in which there was a mediastinal lymph node metastasis solely comprising the solid component of sclerosing haemangioma. The second was classified as an alveolar adenoma with sclerosing haemangioma‐like areas. In the remaining two cases, diagnosis was confounded by presentation with predominantly cystic masses, the largest 70 mm in diameter. Immunohistochemically, TTF‐1 was of greater value than surfactant apoprotein, in particular in identifying the solid component of sclerosing haemangioma when this was solely present.

Conclusion:  Sclerosing haemangiomas should be considered in the differential diagnosis of cystic pulmonary masses. They may also present histologically as combined tumours and metastasize to mediastinal nodes, indicating an, albeit low, malignant potential. TTF‐1 is a valuable antibody in identifying the presence of a sclerosing haemangioma when typical features are absent.
The value of ‘mesothelium‐associated’ antibodies in distinguishing between metastatic renal cell carcinomas and mesotheliomas
Authors: Osborn M , Pelling N , Walker M M , Fisher C , Nicholson A G
Published: March 2003

Aims: Despite increasing usage of mesothelium‐associated antibodies in diagnosis, a meta‐analysis of studies analysing these antibodies in relation to distinguishing mesothelioma from renal cell carcinoma shows a paucity of published data. Given the clinical importance of elucidating this differential diagnosis, we compared the phenotypes of these two tumours using a panel of antibodies comprising recently described ‘mesothelium‐associated’ antibodies and the more established ‘epithelium‐associated’ antibodies.

Methods and results: We applied an antibody panel comprising calretinin, cytokeratin (CK)5/6, thrombomodulin, carcinoembryonic antigen (CEA), BerEP4 and BCA225 to 37 cases of pleural mesotheliomas and 40 cases of renal cell carcinoma (27 primary tumours and 13 metastatic to the pleura). All mesotheliomas were either purely epithelioid or of mixed type. Cases of renal cell carcinoma were graded and classified as to cell type and architecture. For mesotheliomas, 0% stained for CEA, 16% for BerEP4, 83% for BCA225, 78% for CK5/6, 86% for thrombomodulin and 97% showed nuclear staining for calretinin. For renal cell carcinomas, 0% stained for CEA, 50% for BerEP4, 88% for BCA225, 5% for CK5/6, 32% for thrombomodulin and 10% showed nuclear staining for calretinin.

Conclusion: Calretinin, CK5/6 and BerEP4 appear the most useful antibodies in helping to distinguish between renal cell carcinomas and mesotheliomas, although BerEP4 was not particularly sensitive for renal cell carcinomas. Thrombomodulin was not as specific as the other ‘mesothelium‐associated’ antibodies in this study, reflecting how staining for mesothelium‐associated antibodies varies in carcinomas from different primary sites, and such variations should be taken into account when assessing the differential diagnosis of mesothelioma. In cases where doubt remains over distinguishing metastatic renal cell carcinoma from mesothelioma, data from such a panel should be viewed with caution and assessed in association with clinical, imaging and morphological features.
Variance in the resistance of murine early bone marrow B cells to a deficiency in zinc
Authors: OSATI‐ASHTIANI  , KING  , FRAKER
Published: March 2003

Little is known of the effects of nutritional deficiencies on lymphopoietic processes. Nevertheless, deficiencies in zinc adversely affect imm_une function causing thymic atrophy and lymphopenia in both humans and animals. Previous studies of the effects of zinc deficiency (ZD) on lymphopoiesis in adult mice indicated that a suboptimal intake of zinc caused a 50% or more depletion of the marrow of developing B cells. Thus, interference in the production of lymphocytes by ZD appeared to be a significant factor in the loss of host defence capacity. In the current study three‐colour imm_unofluorescence phenotyping of early bone marrow B lymphocytes (B220+ imm_unoglobulin−) using flow cytometry demonstrated that a 27‐day period of ZD caused a 50–70% decline in pre‐B cells (B220+ CD43− imm_unoglobulin M (IgM)−) for moderate and severely zinc‐deficient mice, respectively. Conversely, early pro‐B cells (B220+ CD43+ 6C3−) and late pro‐B cells (B220+ CD43+ 6C3+) exhibited little or no change in their distribution within the marrow. Indeed, the greater resistance of pro‐B cells resulted in a 50% increase in the proportion of this subset within the B‐cell compartment of the marrow as the deficiency in zinc advanced. Collectively, the data indicate that the B‐cell compartment of the marrow is substantially altered by ZD and the stage specific sensitivity noted among early B cells may be related to chronically elevated levels of glucocorticoids present during ZD or other parameters that affect their survival and resistance to apoptosis.
RETRACTED: Peroxynitrite‐mediated DNA strand breakage activates poly (ADP‐ribose) synthetase and causes cellular energy depletion in carrageenan‐induced pleurisy
Authors: Cuzzocrea  , Caputi  , Zingarelli
Published: March 2003

The aim of the present study was to investigate the role of poly (ADP‐ribose) synthetase in acute local inflammation (carrageenan‐induced pleurisy), where oxyradicals, nitric oxide and peroxynitrite are known to play a crucial role in the inflammatory process. DNA single‐strand breakage and activation of the nuclear enzyme poly (ADP‐ribose) synthetase (PARS) triggers an energy‐consuming, inefficient repair cycle, which contributes to peroxynitrite‐induced cellular injury. Here we investigated whether peroxynitrite production and PARS activation are involved in cytotoxicity in macrophages collected from rats subjected to carrageenan‐induced pleurisy. Macrophages harvested from the pleural cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, and by nitrotyrosine Western blotting at 4 hr after carrageenan injection. Furthermore, carrageenan‐induced pleurisy caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, activation of PARS and reduction of NAD+ cellular levels. In vivo treatment with 3‐aminobenzamide (10 mg/kg intraperitoneally, 1 hr after carrageenin injection) significantly inhibited the decrease in mitochondrial respiration and the activation of PARS and partially restored the cellular level of NAD+. In a separate group of experiments, in vivo pretreatment with NG‐nitro‐L‐arginine methyl ester, a non‐selective inhibitor of nitric oxide (NO) synthesis (10 mg/kg intraperitoneally, 15 min before carrageenan administration), reduced peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration and the loss of cellular levels of NAD+. Our study suggests that formation of peroxynitrite and subsequent activation of PARS may alter macrophage function in inflammatory processes and inhibition of NO and PARS may be a novel pharmacological approach to prevent cell injury in inflammation.
Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin‐4
Authors: ASEA  , STEIN‐STREILEIN
Published: March 2003

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti‐CD3 and anti‐CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non‐specific mechanism. C57BL/6 mice were inoculated with anti‐natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T‐cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin‐4 (IL‐4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T‐cell deficient [β2‐microglobulin β2m‐deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when β2m‐deficient mice were reconstituted with NK T cells. NK‐specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL‐4.
Administration of killed bacteria together with listeriolysin O induces protective imm_unity against Listeria monocytogenes in mice
Authors: XIONG  , TANABE  , OHYA  , MITSUYAMA
Published: March 2003

It is known that only listeriolysin O (LLO)‐producing Listeria monocytogenes strains are able to induce protective imm_unity, but the underlining relationship between LLO produced by virulent strains and generation of protective imm_unity in the infected host remains poorly understood. In the present study, it was found that LLO gene expression was only detected in the mice infected with virulent strain which was able to induce protective imm_unity, while non‐virulent strains or killed bacteria were not able to generate protective imm_unity. When mice were imm_unized with LLO plus killed bacteria in the presence of incomplete Freund’s adjuvant, the protective imm_unity was partially generated, and adoptive transfer experiment confirmed that this protection was antigen specific. Reverse‐transcription polymerase chain reaction revealed that LLO plus killed bacteria induced the expression of interferon‐γ (IFN‐γ) and interleukin‐12 (IL‐12). Our results also showed CD4+ T cells were the principal cells constituting protective imm_unity. Taken together, it may be concluded that LLO produced from virulent strains of L. monocytogenes was essential for the generation of protective imm_unity, and that LLO plus killed bacteria induced IFN‐γ and IL‐12 expression which resulted in the generation of protective imm_unity.
Intestinal IgA plasma cells of the B1 lineage are IL‐5 dependent
Authors: Bao S. , Beagley K. W. , Murray A. M. , Caristo V. , Matthaei K. I. , Young I. G. , Husband A. J.
Published: March 2003

Two lineages of B cells, designated B1 and B2 cells, have been identified based upon their origins, anatomical distribution, cell surface markers, antibody repertoire and self‐replenishing potential. B1 cells are maintained by self‐renewal of cells resident in the peritoneal cavity (PerC) and they utilize a limited repertoire of germline V‐region genes, mostly directed against ubiquitous bacterial antigens such as phosphoryl choline (PC). B2 cells are replenished from bone marrow precursors and use a larger repertoire of immunoglobulin V‐region genes. Whereas most immunoglobulin A (IgA) plasma cells in the intestine derive from B2 lineage precursors in the Peyer’s patch, a subpopulation of Per C‐derived B1 cells populate the intestinal lamina propria where they mature into IgA plasma cells. In previous in vivo studies we have shown that whereas IgA+ B2 cells are interleukin (IL)‐6 dependent, B1 cells are IL‐6 independent. In view of the in vitro evidence that IL‐5 is also involved in IgA expression, in the studies reported here we have used IL‐5‐deficient mice to evaluate the role of IL‐5 in vivo in IgA expression in the gut. The results demonstrate that although total IgA cell numbers are only marginally depressed in IL‐5‐deficient mice, there is a marked selective depletion of IgA+ cells of the B1 lineage in the gut and a corresponding depression in the capacity of these mice to mount an intestinal response to a B1 antigen (PC) but not to a B2 antigen (oralbumin; OVA), reflecting intact B2‐derived IgA cell function but a defect in the B1 cell contribution to IgA responses in IL‐5 deficient mice. Collectively these data demonstrate differential cytokine regulation of subsets of IgA+ cells in the gut in that IgA+ cells of the B2 lineage are IL‐6 dependent but IL‐5 independent, but B1‐derived IgA+ cells are IL‐5 dependent and IL‐6 independent.

PerC, peritoneal cavity OVA, ovalbumin Ars, arsanilic acid HBBS, Hanks balanced salt solution LPL, lamina propria lymphocytes PE, phycoerythrin AOCC, anti‐OVA containing cells IPP, intra‐Peyer’s patch.
Immunolocalization of protease‐activated receptor‐2 in skin: receptor activation stimulates interleukin‐8 secretion by keratinocytes in vitro
Authors: HOU L. , KAPAS S. , CRUCHLEY A. T. , MACEY M. G. , HARRIOTT P. , CHINNI C. , STONE S. R. , HOWELLS G. L.
Published: March 2003

The protease‐activated receptor‐2 (PAR‐2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity‐purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR‐2 was used for an immunohistochemical study of skin. The expression of PAR‐2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR‐2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV‐NH2) caused a dose‐dependent increase in the secretion of the chemokine interleukin‐8 (IL‐8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR‐2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL‐8 secretion by keratinocytes. These data raise the possibility that PAR‐2 may play a role in epidermal homeostasis and inflammatory conditions.
A long‐lasting interferon‐γ response is induced to a single inoculation of antigen‐pulsed dendritic cells
Authors: DILLON  , GRIFFIN  , HART  , WATSON  , BAIRD
Published: March 2003

Vaccines against infectious organisms must produce not only long‐lasting immunity but also the appropriate immune response to clear the infection. Obligate intracellular parasites, such as mycobacteria, require a predominantly cell‐mediated immune response rather than antibody. Presentation of antigen by dendritic cells (DC) has been associated with the development of strong cell‐mediated responses generating the production of interferon‐γ (IFN‐γ). This cytokine has an essential role in the elimination of mycobacteria. Therefore, we investigated both the duration and the nature of the immune response after priming with DC pulsed with mycobacterial antigen and compared this with priming using a conventional adjuvant. We used two strains of mice: C57BL/6, which inherently produces a T‐helper 1 (Th1)‐type response to mycobacterial antigen, and BALB/c, which does not. DC‐enriched cell suspensions, purified DC or cultured bone marrow cells resembling DC (BMAPC) were prepared, pulsed overnight with PPD and injected intravenously (i.v.) into naive mice. Six and 12 weeks later, splenic T lymphocytes from these mice were challenged in vitro with antigen and their proliferative response and cytokine production was determined. Significant antigen‐specific proliferation was observed in all assays on rechallenge with antigen in vitro 6 and 12 weeks after the initial priming with DC. IFN‐γ was detected in both strains but was only antigen specific in the C57BL/6 strain. Purified protein derivative (PPD)‐pulsed BMAPC generated similar responses 6 weeks after priming. Thus, long‐term T‐lymphocyte responses and the production of IFN‐γ can be generated using a single inoculation of PPD‐pulsed DC.
Identification of Streptococcus mutans PAc peptide motif binding with human MHC class II molecules (DRB1*0802, *1101, *1401 and *1405)
Authors: SENPUKU  , YANAGI  , NISIZAWA
Published: March 2003

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A‐region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA‐DR) molecules in the A‐region, we prepared a panel of overlapping synthetic peptides in the second unit of the A‐region, and established that a simple enzyme‐linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR‐crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316–334) peptide bound more strongly to the HLA‐DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6(DRB1*1402 and*1405), which bound strongly to PAc(316–334) peptide, were identified. Moreover, we synthesized glycine‐substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L‐RV‐K‐A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side‐effects, could be designed by combining the multiple binding motif with the B‐cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.
Interactions of dendritic cells with fibronectin and endothelial cells
Authors: JANCIC  , CHULUYAN  , MORELLI  , LARREGINA  , KOLKOWSKI  , SARACCO  , BARBOZA  , LEIVA  , FAINBOIM
Published: March 2003

We studied the phenotypic characteristics of spontaneously migrated skin dendritic cells (sDC) and monocyte‐derived dendritic cells (moDC), generated under different culture conditions, and their interactions with fibronectin (FN) and endothelial cells. Monocyte‐derived dendritic cells were obtained after culturing monocytes with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (800 U/ml) and interleukin‐4 (IL‐4) (500 U/ml) with either 10% fetal bovine serum (FBS) or 10% allogeneic human serum (HS). Regardless of the type of serum used, the majority of moDC expressed human leucocyte antigen‐DR (HLA‐DR) and CD86. On day 5 of incubation, 20–67% of moDC cultured in the presence of HS (HS‐moDC) expressed CD1a, b and c versus 94–97% when cultured in the presence of FBS (FBS‐moDC). DC showed a differential gradient of adhesion to FN: FBS‐moDC>HS‐moDC>sDC≈monocytes. Both FBS‐moDC and HS‐moDC were strongly positive for CD49e (α5‐integrin) and CD29 (β1‐integrin) but negative for CD49d (α4‐integrin). A monoclonal antibody (mAb) against CD49e blocked the adhesion of both types of moDC to FN. Although both FBS‐moDC and HS‐moDC attached to endothelium (a 76% and 63% increase, respectively), only HS‐moDC were able to migrate through non‐activated endothelium. Overall, these results suggest that spontaneously migrated sDC are less adherent to FN than moDC, that HS and FBS induce differences in CD1 expression, that HS‐moDC are less adhesive to FN and endothelial cells but more motile than FBS‐moDC, and that α5β1‐integrin is the molecule involved in moDC adhesion to FN.
The role of γδ T cells in induction of bacterial antigen‐specific protective CD8+ cytotoxic T cells in immune response against the intracellular bacteria Listeria monocytogenes
Authors: NOMURA  , MATSUZAKI  , TAKADA  , HIROMATSU  , NABESHIMA  , NAKAMURA  , KISHIHARA  , NOMOTO
Published: March 2003

The role of T‐cell receptor (TCR) γδ T cells in the induction of protective TCR αβ T cells against infection by the intracellular bacteria Listeria monocytogenes was analysed. We found that depletion of γδ T cells by anti‐TCR δ monoclonal antibody treatment before intravenous immunization of mice with a sublethal dose of viable L. monocytogenes resulted in reduction of protection against secondary challenge infection in the immunized mice. The γδ T‐cell depletion also reduced induction of protective αβ T cells capable of transferring the protection against challenge infection of L. monocytogenes into naive mice. Furthermore, the protective T cells that were affected by the γδ T‐cell depletion were suggested to be CD8+ cytotoxic T cells rather than CD4+ T cells by the following observations. First, induction of cytotoxic T lymphocytes specific to a L. monocytogenes‐derived H‐2Kd‐restricted peptide (listeriolysin O 91–99) was significantly suppressed by γδ T‐cell depletion before immunization. Second, γδ T‐cell depletion did not affect cytokine production and proliferation of T cells from immunized mice in response to in vitro stimulation with heat‐killed Listeria which preferentially stimulates CD4+ T cells. Third, CD8+αβ T cells from control immunized mice transferred protection against infection of L. monocytogenes into naive mice but only a limited degree of protection was transferred by CD8+ T cells from the γδ T‐cell‐depleted immunized mice; and fourth, CD4+αβ T cells from the γδ T‐cell‐depleted mice transferred a similar level of protection as those from the control immunized mice. All these results suggest that γδ T cells participate in establishment of protective immunity against intracellular bacteria by supporting priming of bacterial antigen‐specific CD8+ cytotoxic T cells.
The effects of androstenediol and dehydroepiandrosterone on the course and cytokine profile of tuberculosis in BALB/c mice
Authors: HERNANDEZ‐PANDO  , DE LA LUZ STREBER  , OROZCO  , ARRIAGA  , PAVON  , AL‐NAKHLI  , ROOK
Published: March 2003

Immunity to Mycobacterium tuberculosis requires a T helper 1 (Th1) cytokine balance accompanied by tumour necrosis factor‐α (TNF‐α), and activated macrophages. These facets of the immune response are sensitive to suppression by glucocorticoids (GC), which can reactivate and exacerbate tuberculosis in man and animals. Dehydroepiandrosterone (DHEA) and its derivative, 3β,17β androstenediol (AED), are reported to have antiglucocorticoid properties in vivo. We therefore investigated the effects of predetermined optimal doses of these compounds, on the course of pulmonary tuberculosis in an established model in BALB/c mice in which an early phase of Th1‐mediated response accompanied by adrenal hyperplasia, is followed by a switch to Th2, progressive loss of TNF‐α expression and disease progression. Both compounds were protective, particularly AED which caused a fall in bacterial counts and prolonged survival. These effects correlated with the appearance within 3 days of cellular infiltrates rich in cells expressing interleukin‐2 (IL‐2), IL‐1α and TNF‐α, and with partial suppression of the switch to IL‐4 producing cells that occurred in controls. AED also caused enhanced development of granulomas at 14 days, and persistence of granuloma formation to 120 days, with a corresponding suppression of areas affected by pneumonia. Much of the therapeutic effect of AED and DHEA was obtained by treating for only the first 3 weeks, which is the phase of adrenal hyperplasia. These results suggest that the ratio of GC to anti‐GC steroids may play a role in the pathogenesis of tuberculosis, and further investigation could lead to novel treatment strategies.
Surface and gene expression of immunoglobulin E receptors on mast cells and mast‐cell numbers in interleukin‐4‐gene knockout mice
Authors: CHEN  , LYCKE  , ENERBÄCK
Published: March 2003

We quantified immunoglobulin E (IgE) on peritoneal mast cells of interleukin‐4 (IL‐4)‐gene knockout (−/−) mice and wild‐type (+/+) controls using a cytofluorometric method, and examined the expression of IgE receptors, estimated by quantifying the total binding of IgE on the mast cells of IL‐4 (−/−) mice. The mast cells of IL‐4 (+/+) mice, identified and measured using microscope fluorometry, had a fluorescence intensity five to six times higher than that of non‐mast cells, while the mast cells obtained from IL‐4 (−/−) mice had fluorescence intensities within the range of those of non‐mast cells. Two weeks after an infection with Nippostrongylus brasiliensis, the fluorescence intensity of the mast cells of IL‐4 (+/+) mice increased to a level about twice as high as that before immunization. However, no significant increase after infection was observed in IL‐4 (−/−) mice. Furthermore, the mast cells of IL‐4 (−/−) mice did not bind IgE when incubated with IgE at concentrations that saturated IgE receptors on the mast cells of wild‐type controls, thereby indicating that the expression of IgE receptors on mast cells was impaired in the IL‐4‐deficient mice. Using a reverse transcription–polymerase chain reaction, we found gene expression of all three subunits (α‐, β‐ and γ‐chains) of the IgE receptor in IL‐4 (−/−) like that in IL‐4 (+/+) mice. The results thus suggest that the binding of IgE may be essential to induce the translation of mRNA to IgE‐receptor proteins. We also observed that there were about twice as many peritoneal mast cells in the IL‐4 (−/−) mice as there were in the IL‐4 (+/+) mice, in both immunized and non‐immunized animals. This was unexpected in view of previous findings suggesting that IL‐4, in concert with stem cell factor and IL‐3, stimulates the proliferation and differentiation of mast cells in vitro.
Interleukin‐12‐secreting fibroblasts are more efficient than free recombinant interleukin‐12 in inducing the persistent resistance to Mycobacterium avium complex infection
Authors: Kang  , Chung  , Lim  , Kim  , Kim  , Hwang  , Kim
Published: March 2003

To determine whether the paracrine secretion of interleukin‐12 (IL‐12) can efficiently stimulate the resistance to Mycobacterium avium complex (MAC) infection, 3T3 fibroblasts were stably transfected to secrete IL‐12 (480 U/106 cells/48 hr) and their effect on MAC infection was investigated in genetically susceptible BALB/c mice, compared with that of free recombinant IL‐12 (rIL‐12). Injection with IL‐12‐secreting fibroblasts (3T3‐IL‐12) during intranasal infection with MAC resulted in a significant decrease in the bacterial load of the lung during the entire 10‐week observation period, while rIL‐12 reduced the bacterial load initially, at 2 weeks, but not by 10 weeks postinfection. Lung CD4+ T cells in mice injected with the 3T3‐IL‐12 cells showed a persistent T helper type 1 (Th1) response throughout the 10‐week period. Furthermore, immunization with the 3T3‐IL‐12 cells induced and maintained significantly higher levels of cytotoxic activity and nitric oxide production by lung cells than did rIL‐12 immunization. This work suggests that IL‐12‐secreting fibroblasts may serve as a vehicle for paracrine secretion of IL‐12 for immunotherapy of MAC infection.
Visualizing the T‐cell response elicited by oral administration of soluble protein antigen
Authors: Williamson  , O’malley  , Viney
Published: March 2003

Oral administration of soluble protein antigen induces tolerance, while particulate antigens encountered in the intestine provoke active immunity. Although the events that lead to these distinct outcomes are not yet fully characterized, they may reflect differences at the antigen‐presenting cell (APC) level. The role of dendritic cells (DC) in regulating responses at mucosal sites has remained largely undefined because of the low frequency of DC in mucosal‐associated tissues. In this study we have used the growth factor Flt3‐ligand (Flt3L) to expand DC populations in vivo, in combination with an adoptive transfer system, in order to track antigen‐specific T cells during oral tolerance induction. We observed rapid T‐cell activation, localized particularly in the mucosal tissues, within hours after feeding the soluble protein antigen, ovalbumin (OVA). The response was enhanced in Flt3L‐treated mice, indicating an important role for DC during the inductive phase of tolerance.
C57BL/6 mice are more susceptible to antigen‐induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses
Authors: Morokata T. , Ishikawa J. , Ida K. , Yamada T.
Published: March 2003

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)‐type cytokines interleukin (IL)‐4 and IL‐5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL‐4 and IL‐5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti‐IL‐4 and anti‐IL‐5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL‐4 and IL‐5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2‐type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)‐type responses in the lungs, despite considerable production of IL‐4 and IL‐5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background.
Mice transgenic for a soluble form of murine cytotoxic T lymphocyte antigen 4 are refractory to murine acquired immune deficiency sydrome development
Authors: De Leval L. , Debrus S. , Lane P. , Boniver J. , Moutschen M.
Published: March 2003

Interactions between B and CD4+ T cells are central to the pathogenesis of retrovirus‐induced murine acquired immune deficiency virus (MAIDS). Prompted by previous work showing that treatment with cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) partly inhibited the disease, we studied the course of infection in mice deficient for CD28–B7 interactions (mCTLA4‐Hγ1 transgenic mice). Despite a relative viral load identical to that of non‐transgenic mice, the transgenic mice did not develop any of the major MAIDS symptoms (i.e. lymphoproliferation and immune anergy). The mCTLA4‐Hγ1 did not however, completely inhibit B‐cell activation as indicated by a slight hypergammaglobulinaemia and microscopic blastic transformation. Absence of MAIDS in transgenic mice was associated with much lower levels of both interleukin‐4 and interferon‐γ transcripts following viral infection. These results support the theory that the CD28/B7 costimulatory pathway is a critical determinant to MAIDS development.
Regulation of murine dendritic cell functions in vitro by taurine chloramine, a major product of the neutrophil myeloperoxidase–halide system
Authors: Marcinkiewicz J. , Nowak B. , Grabowska A. , Bobek M. , Petrovska L. , Chain B.
Published: March 2003

Taurine chloramine (TauCl) is a major chloramine generated in activated neutrophils as a result of the reaction of highly toxic hypochlorous acid and taurine, the most abundant free amino acid in cytosol. In this study we have tested the influence of TauCl on the properties of murine dendritic cells (DC), the major cell population involved in the initiation of an adaptive immune response against pathogenic organisms. N418+, MHC II+, B7‐2+ dendritic cells, generated from the mouse bone marrow cells cultured in the presence of granulocyte–macrophage colony‐stimulating factor, were stimulated by interferon‐γ and lipopolysaccharide to produce nitric oxide, reactive oxygen species, interleukin‐6 (IL‐6), tumour necrosis factor‐α, and IL‐12, in the presence of different doses of TauCl. TauCl differently inhibited the generation of these inflammatory mediators in a dose‐dependent manner. Furthermore, TauCl selectively modulated the ability of DC to induce the release IL‐2 and IL‐10 from T cells. These results suggest that neutrophil‐derived mediators, such as TauCl, at a site of inflammation, may affect the functions of sentinel DC and macrophages, and play a role in maintaining the balance between the inflammatory response and the induction of an antigen‐specific immune response.
Lack of activation induced cell death in human T blasts despite CD95L up‐regulation: protection from apoptosis by MEK signalling
Authors: Walker L. S. K. , Mcleod J. D. , Boulougouris G. , Patel Y. I. , Ellwood C. N. , Hall N. D. , Sansom D. M.
Published: March 2003

The generation of effective immunity requires that antigen‐specific T cells are activated, clonally expanded and ultimately eliminated by apoptosis. The involvement of CD95‐mediated apoptosis in T‐cell elimination is well established, but the conditions which regulate the death pathway under normal circumstances are still emerging. Using superantigen‐activated human T cells, we found that whilst T‐cell receptor (TCR) signalling triggered up‐regulation of CD95 ligand (CD95L), the majority of T cells were resistant to apoptosis induction, despite co‐expressing high levels of CD95. Resistance was maintained following direct antibody‐mediated cross‐linking of CD95 and was not confined to early time periods following activation. Our data implicate TCR‐derived signals in protection from apoptosis and reveal a role for the mitogen‐activated protein (MAP) kinase pathway by use of a MAP kinase kinase (MEK) inhibitor. Collectively these data demonstrate that resistance to activation‐induced cell death in human T cells is prolonged rather than transient, is not attributable to a lack of CD95L up‐regulation and is due, at least in part, to signalling via the MEK pathway.
Disparate effects of phorbol esters, CD3 and the costimulatory receptors CD2 and CD28 on RANTES secretion by human T lymphocytes
Authors: Sotsios Y. , Blair P. J. , Westwick J. , Ward S. G.
Published: March 2003

This study has examined the stimuli required for secretion of regulated upon activation, normal T‐cell expressed, presumed secreted (RANTES) from T lymphocytes and found that stimuli such as phorbol 12‐myristate 13‐acetate (PMA), which are unable to support T‐cell proliferation and interleukin‐2 (IL‐2) production, are nevertheless able to elicit strong secretion of RANTES. Conversely, stimuli such as CD2 and CD28 ligation, which are able to support T‐cell proliferation, are unable to elicit RANTES secretion. Coligation of CD3 and CD28 drives T‐cell proliferation to a similar degree as CD2 and CD28 coligation, yet also supports modest RANTES secretion. Furthermore, CD28 ligation enhances the secretion of RANTES stumulated by PMA and this costimulatory effect is abrogated by the phosphoinositide 3‐kinase inhibitor wortmannin. Our data also indicate that the observed effects of PMA on RANTES secretion are probably due to activation of protein kinase C (PKC) isoenzymes, since RANTES secretion was unaffected by the non‐PKC activating 4α‐phorbol ester, whilst the general PKC inhibitor Ro‐32‐0432 inhibits PMA‐stimulated RANTES secretion. Moreover, the effect of PMA appears to be chemokine‐specific because PMA was unable to increase secretion of the related CC chemokine MIP‐1α. Under stimulation conditions where increases in [Ca2+]i occur (e.g. PMA plus ionomycin or CD3 plus CD28 ligation) RANTES secretion can be severely reduced compared with the levels observed in response to the phorbol ester PMA. Hence, whilst PKC‐dependent pathways are sufficient for strong RANTES secretion, a calcium‐dependent factor is activated which negatively regulates RANTES secretion. This correlates well with the observation that ligation of cytolytic T lymphocyte‐associated antigen‐4 (CTLA‐4) (expression of which has been reported to be dependent on a sustained calcium signal), inhibits RANTES secretion induced by CD3/CD28, but has no effect on PMA‐stimulated RANTES secretion.
Alteration in the responsiveness to tumour necrosis factor‐α is crucial for maximal expression of monocyte chemoattractant protein‐1 in human neutrophils
Authors: Yamashiro S. , Kamohara H. , Yoshimura T.
Published: March 2003

We previously reported delayed expression of monocyte chemoattractant protein‐1 (MCP‐1) in human neutrophils cultured with a cytokine‐rich crude supernatant of phytohaemagglutinin‐stimulated peripheral blood mononuclear cells (PHA‐sup). Tumour necrosis factor‐α (TNF‐α) contained in the PHA‐sup played a key role in this event, but there appeared to be another factor(s) in the same supernatant that co‐operated with TNF‐α for maximal MCP‐1 expression. In the present study, we reduced TNF‐α concentrations in the PHA‐sup to minimal levels using anti‐TNF‐α affinity columns (TNF‐depleted‐sup) and investigated the co‐operation between TNF‐α and TNF‐depleted‐sup. Nine hours of preincubation with TNF‐depleted‐sup altered the responsiveness of neutrophils to TNF‐α and enabled TNF‐α to increase the level of MCP‐1 expression to a maximal level within 4 hr. The priming effect was not due to the increased expression of cell‐surface TNF receptors. However, the activation of primed cells by TNF‐α was clearly through TNF receptor–p55. Finally, the activity in the TNF‐depleted‐sup that co‐operated with TNF‐α was eluted at 60 000 MW on high‐performance liquid chromatography–gel filtration. Thus, delayed neutrophil expression of MCP‐1 is regulated by a cytokine‐dependent mechanism that induces neutrophils to enter a ‘mature’ stage.
Analysis of the mechanism for extracellular processing in the presentation of human immunodeficiency virus‐1 envelope protein‐derived peptide to epitope‐specific cytotoxic T lymphocytes
Authors: Nakagawa Y. , Takeshita T. , Berzofsky J. A. , Takahashi H.
Published: March 2003

An immunodominant epitope of human immunodeficiency virus‐1 (HIV‐1) gp160 recognized by Dd class I major histocompatibility complex (MHC) molecule‐restricted, CD8+ cytotoxic T lymphocytes (CTL) was originally identified as a peptide composed of 15 amino acids (P18IIIB: RIQRGPGRAFVTIGK). However, further study has indicated that a 10‐mer peptide, I‐10 (RGPGRAFVTI), within P18IIIB is the minimal‐sized epitope and the trimming step(s) of two carboxyl terminal amino acids (GK) is essential to produce I‐10 from P18IIIB. In the processing, angiotensin‐1‐converting enzyme (ACE), found in sera, plays a central role in generating I‐10. Target cells could be sensitized with I‐10 under conditions where ACE activity in the sera was abrogated. In contrast, in the case of P18IIIB, requiring further processing to delete the C‐terminus of two amino acids in order to act, sensitization of target cells was completely abrogated under the conditions. Pretreatment of target cells with brefeldin A (BFA), preventing the presentation of endogenous antigens from the class I MHC molecule pathway, did not inhibit the presentation of P18IIIB. Moreover, glutaraldehyde‐fixed cells, which can not process native protein, though they could present the exogenously added peptides, were also sensitized by P18IIIB. These results clearly demonstrate that the fine processing to produce I‐10 occurred in the extracellular milieu. Furthermore, our result suggests that the longer P18IIIB can bind to the class I molecules on the cell surface, and then be trimmed by ACE while it is bound. The mechanisms behind the extracellular processing outlined in this paper will offer important information for designing peptide‐based vaccines to elicit MHC molecule‐restricted effectors.
Human endothelial cells are activated by interferon‐γ plus tumour necrosis factor‐α to kill intracellular Pseudomonas aeruginosa
Authors: De Assis M. C. , Da Costa A. O. , Barja‐Fidalgo T. C. , Plotkowski M. C.
Published: March 2003

Proinflammatory cytokines have been shown to activate endothelial cells. To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α) for 24 hr and exposed to P. aeruginosa suspension for 1 hr. Light microscopy showed that activated cells internalized significantly more bacteria than control cells. To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay. In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection. In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower. Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants. HUVEC anti‐P. aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the
l‐arginine analogues aminoguanidine and NG‐monomethyl‐
l‐arginine (
l‐NMMA), but was significantly inhibited by catalase. Our results indicate that HUVEC can be activated by IFN‐γ plus TNF‐α to kill IC P. aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity.
Induction of specific transplantation immunity by oral immunization with allogeneic cells
Authors: Holáň V. , Zajícová A. , Krulová M. , Plšková J. , Frič J. , Filipec M.
Published: March 2003

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon‐γ (IFN‐γ) and significantly less interleukin (IL)‐4 than did cells from control mice. The production of IL‐2 was essentially unchanged and that of IL‐10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second‐set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.
Antigen‐specific proliferation of porcine CD8αα cells to an extracellular bacterial pathogen
Authors: Waters W. R. , Hontecillas R. , Sacco R. E. , Zuckermann F. A. , Harkins K. R. , Bassaganya‐Riera J. , Wannemuehler M. J.
Published: March 2003

A vaccine inducing protective immunity to a spirochaete‐induced colitis of pigs predominantly stimulates expansion of CD8+cellsin vivoand in antigen‐stimulated lymphocyte cultures. CD8+cells, however, are rarely considered necessary for protection against extracellular bacterial pathogens. In the present study, pigs recovering from colitis resulting from experimental infection withBrachyspira (Serpulina) hyodysenteriaehad increased percentages of peripheral blood CD4– CD8+(αα‐expressing) cells compared with non‐infected pigs. CD8αα+cells proliferated in antigen‐stimulated cultures of peripheral blood mononuclear cells fromB. hyodysenteriae‐vaccinated pigs. Proliferating CD8αα+cells consisted of CD4–, CD4+and γδ T‐cell receptor‐positive cells. CD4– CD8αβ+cells from vaccinated or infected pigs did not proliferate uponin vitroantigen stimulation. Of the CD8αα cells that had proliferated, flow cytometric analysis indicated that the majority of the CD4+ CD8+cells were large (i.e. lymphoblasts) whereas the CD4– CD8+cells were predominantly small. Addition of monoclonal antibodies (mAb) specific for either porcine major histocompatibility complex (MHC) class I or class II antigens diminishedB. hyodysenteriae‐specific proliferative responses whereas addition of mAb to porcine MHC II, but not porcine MHC I, reduced the CD8αα response.In vitrodepletion of CD4+cells by flow cytometric cell sorting diminished, but did not completely abrogate, the proliferative response of cells from vaccinated pigs toB. hyodysenteriaeantigen stimulation. These results suggest that CD8αα cells are involved in recovery and possibly protection from a spirochaete‐induced colitis of pigs; yet, this response appears to be partially dependent upon CD4+cells.